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Autophagy Regulated by Gain of Function Mutant p53 Enhances Proteasomal Inhibitor-Mediated Cell Death through Induction of ROS and ERK in Lung Cancer Cells

Autophagy Regulated by Gain of Function Mutant p53 Enhances Proteasomal Inhibitor-Mediated Cell... Hindawi Journal of Oncology Volume 2019, Article ID 6164807, 17 pages https://doi.org/10.1155/2019/6164807 Research Article Autophagy Regulated by Gain of Function Mutant p53 Enhances Proteasomal Inhibitor-Mediated Cell Death through Induction of ROS and ERK in Lung Cancer Cells Heena Saini, Ifrah Hakeem, Sudeshna Mukherjee , Shibasish Chowdhury , and Rajdeep Chowdhury Department of Biological Sciences, Pilani Campus, BITS, Pilani, Rajasthan , India Correspondence should be addressed to Rajdeep Chowdhury; rajdeep.chowdhury@pilani.bits-pilani.ac.in Received 16 July 2018; Revised 23 October 2018; Accepted 13 December 2018; Published 6 January 2019 Academic Editor: George P. Studzinski Copyright © 2019 Heena Saini et al. is Th is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mutations inp53, especially gainoffunction(GOF) mutations, arehighly frequent inlungcancers and are knowntofacilitate tumor aggressiveness. Yet, the links between mutant GOF-p53 and lung cancers are not well established. In the present study, we set to examine how we can better sensitize resistant GOF-p53 lung cancer cells through modulation of cellular protein degradation machineries, proteasome and autophagy. H1299 p53 null lung cancer cells were stably transfected with R273H mutant GOF-p53 or wild-type (wt) p53 or empty vectors. eTh presence of R273H-P53 conferred the cancer cells with drug resistance not only against the widely used chemotherapeutic agents like cisplatin (CDDP) or 5-flurouracil (5-FU) but also against potent alternative modes of therapy like proteasomal inhibition. er Th efore, there is an urgent need for new strategies that can overcome GOF-p53 induced drug resistance and prolong patient survival following failure of standard therapies. We observed that the proteasomal inhibitor, peptide aldehyde N-acetyl-leu-leu-norleucinal (commonly termed as ALLN), caused an activation of cellular homeostatic machinery, autophagy in R273H-P53 cells. Interestingly, inhibition of autophagy by chloroquine (CQ) alone or in combination with ALLN failed to induce enhanced cell death in the R273H-P53 cells; however, in contrast, an activation of autophagy by serum starvation or rapamycin increased sensitivity of cells to ALLN-induced cytotoxicity. An activated autophagy was associated with increased ROS and ERK signaling and an inhibition of either ROS or ERK signaling resulted in reduced cytotoxicity. Furthermore, inhibition of GOF-p53 was found to enhance autophagy resulting in increased cell death. Our findings provide novel insights pertaining to mechanisms by which a GOF-p53 harboring lung cancer cell is better sensitized, which can lead to the development of advanced therapy against resistant lung cancer cells. 1. Introduction p53’s tumor-suppressive function but also in certain instances can endow mutant proteins with neomorphic properties Non-small cell lung carcinoma (NSCLC) is a collective term described as mutant GOF-p53 which can contribute actively for a group of lung cancers which affects both smokers and to various stages of tumor progression and to increased nonsmokers. It represents approximately 85% of all lung resistance to chemotherapy. In this regard, the central DNA- cancers. In India, more than one million new cases arise every binding domain of p53 spans the most conserved region composed of a vast number of these missense mutations year with a burgeoning incidence of NSCLC reported annu- ally. In more than 50% of NSCLC patients, p53 is arguably and among these the hot spot residues occur with unusually high frequency [2–4]. p53 missense mutations in the hot the most frequent target for genetic alterations associated spot region can generally be classified as DNA contact (or with poor prognosis and relatively more chemoresistance [1]. Accumulating evidences show that a vast majority of classI)mutants,likeR273H-p53,whichnormally makedirect contact with target DNA sequences and conformational (or p53 mutations are missense that results in production of a stable, full-length mutated protein carrying only single class II) mutants, like R175H-p53, which disrupt the structure amino acid substitution. These mutations not only annul of the p53 protein partially or completely, thus altering 2 Journal of Oncology its function [5, 6]. R175H-P53 and R273H-P53, being the cells, which acted as a prodeath mechanism. Furthermore, most frequently occurring GOF mutations in cancer cells, we observed that GOF-p53 serves to mitigate cell death were observed to induce resistance to chemotherapeutic induced by autophagy in the lung cancer H1299 cells. Our study provides novel insights into modes of sensitization agents in multiple cancer cell types [7, 8]. Interestingly, few reports suggest that, unlike wild-type (wt) p53, mutant p53 of resistant NSCLC cells harboring GOF-R273H mutant can escape MDM-2-dependent proteasomal degradation and P53. hence accumulate stimulating the oncogenic effect [9]. us, Th how to effectively promote degradation of GOF-p53 and 2. Materials and Methods sensitize cancer cells, thus reducing drug resistance, is an .. Chemicals and Reagents. ALLN (#sc-221236), pitfi hrin- important question to be investigated. Autophagy is a well-established self-degradation process alpha (PFT-𝛼 , #sc-45050), and rapamycin (Rapa, #sc-3504A) were purchased from Santa Cruz Biotechnology. CDDP that degrades and recycles numerous intracellular cytoplas- 󸀠 󸀠 (#232120) was obtained from MERCK. 2 ,7 -dichloroflu- mic constituents to maintain homeostasis. However, in a cancerous state, autophagy is known to play a paradoxical orescin diacetate (DCFDA, # D6883), monodansylcadav- erine (MDC, # D4008), CQ (#C6528), and propidium role by either activating cell death and inhibiting tumor progression or promoting cell survival and later stages of iodide (PI; #P4864) were purchased from Sigma; N-Acetyl- cancer progression [10]. Substantial evidences show that L-cysteine (NAC, #47866) and 3-(4, 5-dimethylthiazol-2- yl)-2,5-di-phenyltetrazolium bromide (MTT, #33611) were dieff rent forms of autophagy, for example, macroautophagy and chaperone-mediated autophagy, are implicated in the obtained from SRL. Geneticin (G418, # 10131-035), FITC depletion of stable, mutant p53 isoforms [11]. Functional conjugated Annexin V (#A13199), Annexin V binding buffer (#V13246), and LysoTracker Green DND-26 (# L7526) were involvement of mutant p53 in the regulation of autophagy and in turn being regulated by the cellular degradation system procured from Thermo Fisher Scientific. Lipofectamine 3000 was from Invitrogen (#L3000-001). The MAPK inhibitor, in cancer cells and identification of associated molecular U0126, and gene specific primary and secondary antibod- mechanisms governing it are still incompletely understood. Deciphering the details of these interactions can provide clues ies were obtained from Cell Signaling Technology (CST, USA). pCMV-Neo-Bam p53 wt (Addgene Plasmid #16434), to appropriate sensitization of resistant cancer cells. A growing number of studies suggest that both intra- pCMV-Neo-Bam p53 R273H (Addgene Plasmid #16439), and cellular degradation pathways, for example, ubiquitin pro- pCMV-Neo-Bam Empty Vector (Addgene Plasmid # 16440 ) were a gift from Bert Vogelstein. teasome pathway (UPS), and autophagy are mechanisti- cally and functionally linked such that blockage to either one can lead to upregulation of the other in a way that .. Cell Culture. The non-small cell lung carcinoma cell line, remains yet to be clarified [12]. For example, proteasomal H1299, was a kind gift from Dr. Sanjeev Das (NII, New Delhi). inhibition can enhance the load of misfolded proteins and MCF-7 and MDA-MB-468 cells were procured from NCCS (Pune, India); HCT116 (p53 null) cells were obtained from can trigger autophagy as a compensatory mechanism for their degradation [13]. However, autophagy, serving as an Wogan Lab (MIT, USA); HT-29 and SW480 cells were a gift essential mechanism to cope with cellular stresses, may from Dr. Susanta Roychoudhury, IICB-Kolkata. Cells were directly contribute to survival of cancer cells exposed to cultured at 37 C, 5% CO in Dulbecco’s modified minimal proteasomal inhibitors and, hence, in consequence, might essential medium supplemented with 10% fetal bovine serum reduce effectiveness of therapy. Therefore, an inhibition of and 1% penicillin-streptomycin mixture [16]. Cells were autophagic u fl x after induction of prosurvival autophagy grown to 70-80% confluency prior to treatments. Trypsin- has often been utilized as a strategy to sensitize multiple EDTA solution (0.05%) was used for detachment of cells. cancer cell types [14]. Based on these findings, clinical trials Stable transfected cells were maintained in 600 𝜇 g/ml of are currently ongoing investigating autophagy inhibition G418. in conjunction with anticancer therapies, like bortezomib. However, conversely, an overactivation of autophagy can also .. Preparation of Cell Lines Stably Expressing GOF Mutant act as a bona fide death inducer or death effector, upstream and wt-p. P53 null H1299 cells were cultured in six-well of other death pathways, like apoptosis [15]. Based on the plate and transfected with either 2𝜇 gof pCMV-Neo-Bam above considerations, there might be physiological situations p53-wt or pCMV-Neo-Bam p53-R273H or pCMV-Neo-Bam in which autophagy protects death or, in contrary, kills Empty Vector (EV) purified plasmid with Lipofectamine cancer cells that would have otherwise manifested prolonged 3000 according to manufacturer’s instructions. Around 24 survival. Because autophagy plays an important roles in cell h after transfection, the cells were transferred to a 10 cm fate, it is therefore critical to understand the mechanism dish and selected for transfection positivity by geneticin by which autophagy interacts with and aec ff ts cell survival (600𝜇 g/ml) selection. Transfected cells were maintained for or death machinery, which might allow new avenues to several days under G418 pressure. Nonsurviving cells were effectively sensitize resistant cancer cell types. washed off with PBS and fresh media with G418 was added. Taking this into consideration, in this study, we exposed This was done till the time cell colonies were obtained. GOF-R273H-P53 cells to proteasomal inhibitor ALLN Random colonies were selected and allowed to grow in a and observed its effect on autophagy. A ROS-dependent new culture dish under G418 pressure. Cells were grown autophagy was found to be induced in the R273H-P53 till confluency and then stable transfection of wild-type or Journal of Oncology 3 R273H-P53 vector was conrfi med by immunoblotting against with drug for 48 h. Coverslips were then withdrawn and p53 antibody. washed with PBS and cells were xfi ed in formaldehyde for 10 min at room temperature. Coverslips were mounted .. Analysis of Cytotoxicity. In vitro cytotoxicity was per- on a glass slide with antifade DAPI. Nuclear morphology formed using 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltet- was further observed by u fl orescence microscopy (Olympus, razolium bromide (MTT) assay, following methods previ- Japan). ously described in Chowdhury et al’s work [17]. Briefly, H1299 (null P53/EV), H1299/wt (wt P53), and H1299/R273H . . Annexin V/Propidium Iodide (PI) Staining. Cells were (R273H P53) cells were cultured in 96-well plates. Aeft r seeded on 6 cm dishes at a density of 5 × 10 cells and overnight culture of cells, they were treated with specific thereafter exposed to various treatments. After that the cells drugs for a stipulated period of time. Following that, MTT were harvested, washed with PBS, and resuspended in 500 was added to each well and incubated for 4 h. Viable cells 𝜇 L of 1X binding buffer. Following that, 4 𝜇 l of Annexin V- form formazan crystals with MTT, which were solubilized FITC and 10𝜇 l of PI were added. Cells were then incubated in DMSO, and readings were obtained at 570 nm with a in the dark for 20 min and samples were then acquired using differential lfi ter of 630 nm using Multiskan GO microplate flow cytometer (Cytoflex, Beckmann Coulter) [17]. CytExpert spectrophotometer. Percentage of viable cells was calcu- software was used to analyze the acquired data. To detect both lated using the following formula: viability (%) = (mean early and late apoptotic cells, the percentage of cells in lower absorbance value of drug-treated cells)/(mean absorbance and upper right (LR and UR) quadrant representative of only value of the control) x 100. Annexin V and both Annexin V-PI positive cells, respectively, were counted. Percentage of apoptotic cells is represented .. Caspase Assay. For measurement of caspase activity, 2.5 through bar diagram. × 10 cells/well were seeded in 6-well plate and exposed to IC dose of CDDP for 48 h. Caspase-3 colorimetric 50 .. Monodansylcadaverine (MDC) Staining of Autophagic protease assay kit (Invitrogen) was used to measure caspase-3 Vacuoles. MDC, an autophagolysosomal marker, was used activity following procedure described elsewhere [17]. Briefly, to analyze autophagy induction. Cells were grown over protein was extracted using RIPA buffer; concentration was coverslips and the following day drug treatment was made. determined by Bradford assay and then equal amount (60 The cells were then incubated in the dark for 10 min with 𝜇 g) of protein was added to microtiter plates with caspase- 0.05 mM MDC at 37 C. Thereaer ft , the coverslips with 3 substrate (Ac-DEVD-pNA). The concentration of the p- cells were washed and mounted with antifade DAPI. MDC nitroaniline (pNA) released from the substrate was calculated punctate dots were analyzed under uo fl rescence microscope. from the absorbance values at 405 nm. For uo fl rimetric measurements, cells were grown in 6-well plate. After treatment, cells were labelled with MDC for 10 .. Analysis of DNA Fragmentation. Apoptosis was evalu- min followed by PBS wash and then collected in 10 mM ated by fragmented genomic DNA forming DNA ladders Tris-HCl (pH 8.0) containing 0.1% Triton X-100 [20, 21]. (short fragments of∼200 base pairs) on agarose gel [18]. To Intracellular MDC was assessed by uo fl rescence photometry analyze DNA ladder formation, null P53 and R273H-P53 cells (excitation 380 nm and emission 525 nm) on a microplate were seeded in 6 cm dishes at a density of 5× 10 cells/plate reader (Fluoroskan Ascent). Change in MDC uo fl rescence and treated with CDDP for 48 h. DNA was extracted using with respect to control is expressed as fold change. Invitrogen apoptotic DNA ladder detection kit and ladder formation was analyzed on 1% agarose gel. A DNA marker .. LysoTracker Green (LTG) Staining of Acidic Vesicles. was run parallel to the samples. LTG constituting a fluorophore linked to a weak base is a uo fl rescent acidotropic probe, used for labelling and tracking . . RNA Isolation and Real-Time PCR (RT-PCR). Total RNA acidic organelles in live cells. Cells were cultured overnight was isolated using TRIzol reagent. GeneSure First Strand on coverslips and then exposed to different concentration of cDNA Synthesis kit was used for complementary DNA ALLN for 48 h. After treatment, the media was removed, (cDNA) synthesis. Templates were amplified using gene- the cells were washed with PBS, and thereaer ft LTG was specific primers for ABCB1 taking GAPDH as housekeeping added (0.05𝜇 M). Cells were then incubated for 20 min in control and detected using SYBR Green Supermix in CFX CO incubator. Fluorescence intensity of LTG was observed Connect RT-PCR System (Bio-Rad) [16]. The primers used under a u fl orescence microscope and compared to untreated andtheirsequencesaregiven asfollows:GAPDH,forward5 - control. 󸀠 󸀠 GCA CCG TCA AGG CTG AGA AC-3 and reverse 5 -TGG 󸀠 󸀠 TGA AGA CGC CAG TGG A-3 ;ABCB1,forward 5 -GGG .. Measurement of Intracellular ROS. Cells were seeded at 󸀠 󸀠 ATG GTC AGT GTT GAT GGA-3 and reverse 5 -GCT ATC adensity of 9x10 in 96-well plates and exposed to treatments. GTG GTG GCA AAC AAT A-3 . The relative RNA expression NAC, a ROS scavenger (5 mM), was added 1 h prior to was calculated using Pfa’ffls method [19]. treatments. After treatment, the cells were washed with PBS and then incubated in 100𝜇 lof working solution (10𝜇 M) of 󸀠 ∘ . DAPI Staining of Nucleus. 4 -6-Diamidino-2-phenylin- DCFDA at 37 C for 30 min. Fluorescence was measured using dole (DAPI) stain was used to detect apoptotic nuclei [18]. a microplate reader (Fluoroskan Ascent) at 485 nm excitation Cells grown on coverslips were either untreated or treated and 530 nm emission [18]. 4 Journal of Oncology .. Immunoblotting. Immunoblotting was performed fol- null P53 cells or EV stable transfected cells (Figure 1(a)(i), lowing protocols described elsewhere [22]. Cells were grown Supplementary Figure 1.b, and Supplementary Figure 1.c); in 10 cm dishes. Following treatment for specific time, cells the sensitivity of EV cells to cisplatin was comparable to were lysed in RIPA buffer (Sigma-Aldrich), and the protein null cells. We hence compared caspase-3 activity upon concentration was estimated using Bradford reagent. Then, CDDP treatment between H1299 null P53 (IC ∼ 15𝜇 M) 5X gel loading dye was added to the lysates followed by and R273H-P53 (IC ∼ 30𝜇 M) cells through ELISA based heat denaturation (100 Cfor 10 min). Proteins were then method. As expected, R273H-P53 cells showed significantly loaded in denaturing polyacrylamide gels and transferred decreased enzymatic activity compared to null P53 cell to polyvinylidene u fl oride membranes. Skimmed milk (5%) type (Figure 1(b)). Similar results were obtained in DNA was used for blocking. The blots were then probed or fragmentation study (Figure 1(c)). Since R273H-P53 cells reprobed with specific primary antibodies and detected using showed a marked increase in resistance compared to null enhanced chemiluminescence detection on ChemiDoc (Bio- or wt-P53 cell types, we further confirmed it by analyzing Rad) [14]. The primary antibodies used were as follows: mRNA expression of ABCB1, which showed a substantially Atg-5 (CST; D1G9), Atg-3 (CST; 3415), LC3-II (CST; D11), increased expression upon CDDP treatment in R273H-P53 phospho-MAPK (ERK) (CST; Rabbit mAb #4370), and p53 stable transfected cells compared to control (Figure 1(d)). (SCBT; DO-1).𝛽 -actin (SCBT; sc69879) was used as loading Based on the above experimental evidences, it is clear that control. The secondary antibodies used were horseradish R273H-P53 cells show resistance to CDDP, compared to null peroxide-conjugated goat anti-rabbit and goat anti-mouse or wt-P53 cells. However, to analyze that the resistance is IgG at dilution of 1:10,000 and 1:20,000, respectively. The a generalized phenomenon across multiple drug types or expression levels were densitometrically quantified using is purely specific to CDDP, we evaluated cross resistance ImageJ software and normalized to the control. of R273H-P53 cells to other conventionally used anticancer drugs like 5-FU or methotrexate (data not shown). As evident .. Statistical Analysis. The obtained data were analyzed from Figure 1(e), R273H-P53 cells were less sensitive to 5- using GraphPad Prism sowa ft re version 5.0. Effect of treat- FU as well. Collectively, these observations suggest that null- ment in comparison to control was statistically determined P53 cells acquire drug-resistant characteristics upon stable using one-way or two-way ANOVA. The Bonferroni method transfection of R273H-P53 vector in NSCLC cells. was used to analyze multiple comparisons. Throughout the text, the representative images are of experiments done in .. P Positive Cells Are More Sensitive to Proteasomal multiples. Data are represented in mean± SEM. The symbols Inhibition. Targeting the proteasomal degradation pathway in parenthesis denote the following:∗ compared to control, is increasingly getting recognized as a promising strategy # compared to ALLN or R273H, not significant (ns) p > for cancer therapy [24–26]. Wt-p53 protein is primarily ∗/# ∗∗/## ∗∗∗/### degraded by the UPS pathway; however, mutations in p53 0.05; p≤0.05; p≤0.01; p≤0.001. might stabilize this protein and inhibit MDM2 interaction, thereby preventing degradation [27–29]. However, there are 3. Results and Discussion reports suggesting that several “hot-spot” p53 mutants like R175H, R248W, or R273H remain sensitive to ubiquitin- .. R H-P Cells Exhibit Increased Drug Resistance Com- pared to Null or wt-p Cells. In NSCLC patients, p53 mediated degradation [30]. We assumed that proteasomal status shows no prognostic significance in the absence of degradation of GOF-p53 might be context-dependent and adjuvant chemotherapy; however, after undergoing treatment shows increased bias towards the nature of inhibitor used. with cisplatin, a reduced disease-free interval and overall We used ALLN, a well-known proteasome inhibitor that is survival are seen bearing a GOF-p53 protein [23]. Given known to induce apoptosis by virtue of accumulated protein the importance of GOF-p53 in NSCLCs, in this study, we response. ALLN was chosen over the widely used proteaso- prepared a stable transfected GOF-R273H-P53 NSCLC cell mal inhibitor, bortezomib, because the eeff cts of ALLN are model aimed at understanding modus operandi of resistance relatively less explored and the use of bortezomib has recently and develop an effective strategy for sensitizing GOF p53 been challenged by severe adverse side effects and resistance cells, which is elusive till date. P53 null H1299 cell line was [31]. Exposure of ALLN (5 and 10 𝜇 M) for 48 h showed more cell deaths in wt-P53/R273H-P53 transfected cells than stable transfected with empty vector (EV), wt-P53, or R273H- P53. Stable transfection was conrfi med by immunoblotting null (Figure 2(a)) or EV transfected cells (Supplementary against p53 antibody (Supplementary Figure 1.a). The cells Figure 2). Similar results were obtained when cells were were then exposed to varying concentrations of CDDP (a treated with another proteasomal inhibitor, MG132 (data not standard chemotherapy for NSCLC patients) for 48 h. Simi- shown). Interestingly, R273H-P53 cells in comparison to wt- larly other cancer cell types possessing wt-P53 (MCF7), null- p53 showed decreased sensitivity to proteasomal inhibition as P53 (HCT116), or R273H-P53 (MDA-MB-468, HT-29, and well (Figures 2(a) and 2(b)) as evident from cell viability assay SW480) were also exposed to various doses of CDDP. Inter- or apoptosis assay by Annexin V/PI staining. Importantly, estingly, in all the cell types studied, GOF-p53 cells showed unlike response to conventionally used drugs like cisplatin, significantly decreased sensitivity to CDDP compared to both wt-P53 and R273H-P53 cells were more sensitive than wt-P53 harboring cells demonstrating resistance (Figures the parental null or EV cells, suggesting that the presence of p53 protein provides a therapeutic advantage to target- 1(a)(i)–1(a)(iii)). Importantly, R273H-P53 cells showed less sensitivity to CDDP compared to either parental H1299 ing strategies based on interference of protein degradation Journal of Oncology 5 120 120 100 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ 100 100 ### 80 ### ∗∗∗ 80 80 ∗∗∗ ### ### 60 60 40 40 20 20 0 0 0 MCF-7 MDA-MB-468 05 15 35 5 M HCT116-/- HT-29 SW-480 R273H-P53 0 M 0 M Null-P53 35 M 35 M WT-P53 15 M 55 M 15 M 55 M (i) (ii) (iii) (a) CDDP CDDP Marker Cntrl (LD) (IC50) 2.5 2.5 ∗∗ 2.0 2.0 ns 1.5 1.5 1.0 1.0 ns 0.5 0.5 0.0 0.0 Null-P53 R273H-P53 Null-P53 R273H-P53 Control Control IC-50 IC-50 (b) (c) (d) ∗∗∗ ∗∗∗ Null-P53 R273H-P53 0 M 130 M 70 M 170 uM 110 M (e) Figure 1: GOF-R273H-p53 mutation imparts drug resistance. (a) Cell lines with varied p53 status were exposed to dieff rent doses of cisplatin. Cell viability was analyzed after 48 h of treatment through MTT assay. Untreated samples were taken as control. ∗ and # indicate significant dieff rence compared to untreated cells and R273H-P53 cells, respectively. (b) Fold change in caspase-3 enzyme activity was measured following CDDP treatment at IC-50 dose for 48 h in H1299 (null-P53) and H1299-R273H-P53 stable transfected cells. Level of caspase-3 activity in untreated control was taken as arbitrary unit “1.” (c) DNA fragmentation was measured on agarose gel. Null-P53 and R273H-P53 cells were treated with CDDP at low dose (LD) and IC-50 dose for 48 h; DNA was extracted and run on an agarose gel. A DNA marker was run alongside the samples. (d) Real-time PCR showing expression of ABCB1 mRNA levels upon exposure of null-P53 and R273H-P53 cells to CDDP (IC- 50) for 48 h. (e) Null-P53 and R273H-P53 cells were given different doses of 5-FU and cross resistance was analyzed through MTT assay. ∗ indicates significant difference compared to untreated cells. % Viability Caspase-3 Enzyme activity % Viability % Viability R273H-P53 Null-P53 Fold Change in ABCB1 expression % Viability 6 Journal of Oncology ### ∗∗∗ ## Cntrl 5 10 M Cntrl 5 10 M WT-P53 Null-P53 R273H-P53 WT-P53 R273H-P53 (a) (b) Cntrl 5M 10 M (c) Figure 2: Proteasomal inhibitor (ALLN) induces apoptosis. (a) Null-P53, wt-P53, and R273H-P53 cells were exposed to varying doses of ALLN. Aeft r 48 h of exposure, cell viability was measured through MTT assay. Untreated samples were taken as control. ∗ and # indicate significant dieff rence compared to Null-P53 and R273H-P53 cells, respectively. (b) Percentage of apoptotic cells was measured through flow cytometry using Annexin V/PI staining and compared between wt-P53 and R273H-P53 cells upon ALLN treatment. (c) R273H-P53 cells were either untreated or treated with ALLN for 48 h and then stained with DAPI. Nuclear fragmentation or condensation aeft r treatment is marked with white arrows. The scale bar represents 100 𝜇 m. (Figure 2(a)). Nuclear staining by DAPI in R273H cells show- of enhanced autophagy was observed with ALLN treatment ing fragmented nucleus, as depicted in Figure 2(c), further (Figure 3(a)). However, an increase in LC3B-II protein levels confirmed cell sensitization on ALLN treatment. Overall, can be resultant of increased autophagy or an inhibition of the above results highlight the importance of targeting the n fi al step of autophagosome-lysosomal fusion [33]. Hence, protein degradation machinery in p53 positive cells, though to confirm autophagic ux, fl we checked for the changes in an adjuvant therapy might be essential for R273H-P53 cells LC3B-II protein levels with or without the lysomotropic as they are more resistant than wt-p53 cells to cell death by agent, CQ. An increased expression of LC3B-II was observed protein overload. in ALLN+CQ treated samples when compared to only independent treatments indicative of enhanced autophagic .. Proteasomal Inhibitor, ALLN, Induces Autophagy in GOF u fl x with ALLN (Figure 3(b)). Autophagy induction was further validated through MDC staining; MDC preferentially R H-P Cells. Two major pathways of degradation main- tain protein homeostasis, the UPS, responsible for degrading accumulates in acidic autophagic vacuoles. MDC uo fl res- majority of proteins including many short-lived, denatured, cence, represented by the green punctate dots, increased with or, in general, damaged proteins, and autophagy, which, ALLN treatment in a dose-dependent manner, suggesting by contrast, is mostly responsible for degradation of long- an increase in autophagy (Figure 3(c)). Furthermore, an lived proteins [32]. Although UPS and autophagy were increase in the number of lysosomes is oen ft associated initially considered to be largely disconnected pathways, with increased autophagy. We observed an increase in recent advances in understanding of UPS and autophagy LTG staining with ALLN (Figure 3(d)). The above results have highlighted a strong connection between them. To aresupportiveofthe fact that proteasomal inhibition by examine the effect of ALLN on autophagy, R273H-P53 cells ALLN in R273H-p53 cells activates autophagy. However, were treated with 10 𝜇 M ALLN for different time points in tumor cells, autophagy can act as both a prodeath and LC3B-II (marker for autophagy) protein expression was or prosurvival mechanism in a context-dependent manner analyzed. An increase in LC3B-II expression levels indicative [34]. % Viability % Apoptotic cells Journal of Oncology 7 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ 60 6 ∗∗ 4 LC3B-II LC3B-II B-actin B-actin Cntrl CQ ALLN ALLN+CQ Cntrl 3 24 48 h (a) (b) ∗∗∗ Cntrl 5 10 M Cntrl 5M10M (c) (d) Figure 3: ALLN induces autophagy in GOF R273H-P53 cells. (a) R273H-P53 cells were treated with 10 𝜇 M ALLN for different time points and expression of LC3B-II was analyzed through immunoblotting. (b) Immunoblot analysis showing expression of LC3B-II upon 48 h of exposure to CQ, ALLN, and ALLN+CQ in R273H-P53 cells. (c) Fluorescent microscopic images of MDC uo fl rescence in ALLN-treated (0, 5, and 10𝜇 M; 48 h) R273H-P53 cells. eTh scale bar represents 100 𝜇 m. (d) R273H-P53 cells were treated with ALLN for 48 h and then stained with LTG dye. Green dots representing the lysosomes were counted and represented as bar diagram. The scale bar represents 100 𝜇 m. .. GOF-R H-p Cells Are Sensitive to Autophagy Induc- accumulation of acidic vesicles, thereby blocking autophagic tion. Enhanced autophagy has recently been implicated in u fl x, and can act as a potent strategy to sensitize R273H- multiple studies facilitating cancer cell survival under physi- P53 cells. We initially checked for cytotoxicity inducing ological stresses. Hence, we hypothesized that a combination property of CQ alone. CQ even at 50 𝜇 Mwas not able treatment with ALLN (being an autophagy inducer) with to impart any significant cytotoxic effect on R273H-P53 CQ (an agent that inhibits lysosomal function) might lead to cells as analyzed by MTT assay and also Annexin V/PI Fold Change in LC3B-II expression 10MC 5M ntrl Fold Change in lysosome number Fold Change in LC3B-II expression 8 Journal of Oncology staining (Figures 4(a) and 4(b)). Since CQ is a late-stage with or without NAC pretreatment. The above experiments autophagy inhibitor, we thought of exploring the eeff ct of an also confirm that ALLN-induced cytotoxicity is not only early-stage autophagy inhibitor, 3-methyl adenine (3-MA). autophagy-dependent but is also regulated by an increase in ROS levels. Interestingly, R273H-P53 cells when pretreated Interestingly, on the contrary, 3-MA treatment was able to sensitize R273H-P53 cells. As depicted in Supplementary with NAC resulted in a decrease in autophagic marker Figure 3, only 3-MA treatment reduced cell viability to less expression, Atg-3 or LC3B-II, suggesting that, in this context, ROS is upstream to autophagy and a quenching of ROS than 50% in R273H-P53 cells. However, as we analyzed the expression of autophagic markers upon 3-MA exposure, reduces autophagy-induced cell death as well (Figures 5(e)(i), we noticed an increased expression of LC3B-II and Atg-5 5(e)(ii), and 5(e)(iii)). proteins levels (Figures 4(c)(i) and 4(c)(ii)). We assumed that 3-MA might have autophagy inducing effects as well. .. Sensitization of GOF-p Is Mediated by ERK along with ROS Accumulation. Previous reports show that mitogen Indeed, in corroboration to above, there are previous reports suggesting that prolonged 3-MA treatment can inhibit PI3K- activated protein kinase (MAPK) signaling pathways are able class-I, in turn inhibiting mTOR and activating autophagy to modulate autophagy and determine cell fate [39]. There [35]. However, results obtained with 3-MA provided us with a are also a growing number of reports stating that, in certain hint that, rather than autophagy inhibition, these cells might conditions, the MAPK-ERK can promote cell death [40, 41]. be more sensitive to autophagy induction. Accordingly, the Interestingly, an increase in phospho-ERK indicative of its effect of rapamycin (Rapa), a widely used mTOR inhibitor, activation was observed in cells treated with ALLN, which and serum starvation (SS), which are known to elicit an went down with autophagy inhibition by CQ (Figure 6(a)). autophagic response, was investigated in R273H-P53 cells [36, To confirm the role of ERK in cell death, U0126, a widely 37]. Interestingly, R273H-P53 cells treated with autophagy used selective inhibitor of the upstream MAP kinase pathway, inducers like Rapa or exposed to SS alone or in combination was used and cell viability was measured in presence of with ALLN had significantly enhanced cytotoxic effects when ALLN or ALLN plus SS. Importantly, inhibition of the ERK compared to autophagy inhibition with CQ alongside ALLN pathway by U0126 significantly reduced cell death induced (Figures 4(d)–4(h)). In fact, autophagy inhibition with CQ by ALLN alone or ALLN plus autophagy induction. This was found to moderately decrease the sensitivity of cells to implicates that ERK signaling acts as a prodeath mecha- ALLN (Figure 4(i) and Supplementary Figure 4). Hence, in nism (Figure 6(b)). ROS, such as hydrogen peroxide, are this study, we suggest that although physiologically relevant reported to regulate ERK phosphorylation as ERK-specific levels of autophagy are required for cellular homeosta- phosphatases are sensitive to ROS. Hence, ROS-mediated sis maintenance, enhanced autophagy can in turn induce prolonged ERK activation might be a crucial mechanism autophagy-dependent cell death. Excessive autophagy has regulating cell death [42]. Interestingly, a significant decrease been previously observed in association with various forms of in phospho-ERK levels was observed when ALLN-treated cell death and the term “autophagic cell death” was originally cells were predisposed to NAC (Figures 6(c)(i) and 6(c)(ii)). introduced to describe cell death associated with autophagy. We also checked for the reverse effect, that is, ROS production However, evidences associating autophagy to cell death in upon ERK inhibition. R273H-P53 cells were pretreated with these reports were more circumstantial, and the nature of U0126 for 2 h prior to addition of ALLN or ALLN plus such death occurring in cancer cells remains poorly defined. autophagy inducers. Interestingly, a drastic decline in level We were hence interested in analyzing the modus operandi of of ROS was observed with ERK inhibition (Figure 6(d)). cell death observed upon autophagy enhancement. Collectively, the above findings highlight that R273H-P53 cell sensitization is regulated by ROS-autophagy-ERK signaling .. ALLN-Induced Autophagy Facilitates Cell Sensitization by loop upon ALLN or ALLN plus autophagy induction. Regulating ROS Levels. Generation of ROS through oxidative stress is known to cause cell death; however, the role of oxida- . . Induction of Prodeath Autophagy in R H-P Cells Is a tive stress in autophagy-induced cell death is relatively unex- p-Dependent Process. Although there are many links that plored. Conventionally, autophagy serves as a bueff r system to connect autophagy with p53, molecular crosstalk between control the level of ROS in cells and reduce their toxic effects them is still incompletely understood [43]. One of the [38]. However, the connection of ROS with autophagy in our earliest studies describing the relationship between mutant experimental context was found to be different. Intracellular p53 and autophagy states that the subcellular localization ROS levels measured by DCFDA dye followed by uo fl rimetric of mutp53 is the major determinant of autophagy and it is conventionally accepted that p53 inhibits autophagy [44]. On analysis showed a prominent increase with ALLN treatment in R273H-P53 cells (Figure 5(a)). A profound increase in ROS the other hand, in many cells, it is observed that prolonged levels was observed upon ALLN treatment in SS condition inhibition of the proteasome leads to its autophagy-mediated degradation of p53, suggesting that autophagy in turn can (Figure 5(a)). Interestingly, pretreatment of cells with the ROS scavenger-NAC resulted in substantial increase in cell regulate the stability of p53 protein [11]. In this study, we viability with only ALLN, ALLN plus SS, or ALLN plus inhibited p53 with a well-known p53 inhibitor, pifithrin- 𝛼 Rapa treated cells, suggesting a direct positive correlation of (PFT-𝛼 ) (Figure 7(a)). Interestingly, a significantly decreased increase in ROS with cell death (Figure 5(b)). Figures 5(c) cell viability was obtained with PFT-𝛼 plus ALLN treatment and 5(d) represent the status of ROS level and cell viability compared to only ALLN (Figure 7(b)). This suggests that on Rapa or SS exposure. The experiment was performed GOF-R273H-p53 inhibition had a positive impact on cell Journal of Oncology 9 2.0 1.5 1.0 0.5 0 0.0 0 10 50 M Null-P53 WT-P53 R273H-P53 0uM 10uM 50uM (a) (b) ∗∗∗ 3.0 25 2.5 ∗∗ ∗∗ 20 2.0 15 1.5 10 1.0 5 0.5 0.0 LC3B-II Atg-5 B-actin B-actin Cntrl CQ 3-MA ALLN Cntrl CQ 3-MA ALLN (i) (ii) (c) ### ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ Cntrl Rapa SS SS+Rapa Cntrl ALLN SS SS+ALLN (d) (e) Figure 4: Continued. % Viability Fold Change in % Viability LC3B-II expression % Viability % Apoptotic cells Fold Change in Atg-5 expression 10 Journal of Oncology 100 ### ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ### Cntrl ALLN Rapa ALLN+Rapa Cntrl ALLN 3-MA ALLN+3-MA (f) (g) ### 80 ∗∗∗ ∗∗∗ Cntrl ALLN SS Rapa ALLN+SS ALLN+Rapa Cntrl CQ ALLN ALLN+CQ (h) (i) Figure 4: Autophagy induction promotes cell death in R273H-P53 cells. (a) Null-P53, wt-P53, and R273H-P53 cells were exposed to varying doses of CQ. After 48 h of exposure, cell viability was measured through MTT assay. (b) CQ-treated R273H-P53 cells were analyzed for apoptosis induction through Annexin V/PI staining followed by flow cytometry. Fold change is represented through bar diagram. (c) R273H- P53 cells were exposed to CQ (10𝜇 M), 3-MA (5 mM), and ALLN (10𝜇 M) for 48 h and immunoblot analysis was performed for LC3B-II and Atg-5.∗ indicates significant difference to untreated control. (d) Cell viability in R273H-P53 cells was measured through MTT assay aeft r 48 h of treatments at various combinations, (d) Rapa (500 nM), Rapa + SS, (e) SS, ALLN+SS, (f) Rapa, ALLN+Rapa, (g) 3-MA, 3-MA+ALLN and represented in the form of bar diagram. (h) Apoptosis was analyzed through flow cytometry using Annexin V/PI staining in R273H-P53 cells, aeft r treatment with various autophagy inducers. Percentage of apoptotic cells is represented through bar diagram. (i) Cell viability was measured through MTTassay inR273H-P53 cells aeft r autophagy inhibitioninpresence orabsence of ALLN. ∗ indicates significant dier ff ence compared to untreated control, while # indicates significant dier ff ence compared to ALLN-treated cells. sensitization. We were hence curious to analyze the effects of transcriptional and translational control. While the aspects PFT-𝛼 on autophagy levels as well. Interestingly, pretreatment of protein degradation have been largely overlooked, it of PFT-𝛼 followed by ALLN exposure caused an increase is a natural way by which cells clean up proteins that are in Atg-5 levels and a decrease in sequestrosome p62 levels, redundant or have been misfolded or damaged [45, 46]. indicating an enhancement of autophagy (Figures 7(c)(i) and Inhibition of this protein degradation machinery has been 7(c)(ii)). Enhanced autophagy induction on ALLN plus PFT- useful previously for treatment of autoimmune diseases and 𝛼 treatment was further confirmed by MDC staining which cancer [26, 47]. Especially in particular types of cancer, where showed an additive effect as well (Figure 7(d)). Furthermore, protein production is much higher than normal, primarily an increased ROS accumulation was observed with ALLN to meet their overproliferative or secretary demand, an plus PFT-𝛼 treatment (Figures 8(a) and 8(b)). ROS scav- inhibition of proteasomes can cause proteins to pile up, enger, NAC, successfully reversed the effect substantiating eventually killing the cancer cell [48, 49]. There are already a ROS-dependent phenomenon. Further, as evident from proteasome inhibitors like bortezomib in the clinics for Figure 8(c), U0126 pretreatment followed by ALLN plus treating Kahler’s disease (multiple myeloma). However, the PFT-𝛼 exposure increased the cell viability and decreased proteasomal inhibitors not only affect the proteasome but accumulated ROS (Figure 8(d)). A signicfi antly increased also can significantly alter the functioning of another cellular phospho-ERK level was also observed with ALLN plus PFT- homeostatic machinery like autophagy. The latter is primarily 𝛼 treatment (Figure 8(e)). Taken together, we postulate that devoted towards maintaining cellular balance of organelles, GOF-p53 inhibition facilitates cell sensitization by upregulat- proteins, and other macromolecules. For example, cancer ing autophagy and by enhancing ROS and ERK activation. cells that produce excess unfolded proteins generate high endoplasmic reticulum stress leading to protein removal via either the proteasome or autophagy, suggesting that both 4. Conclusion pathways may be specifically exploited therapeutically. This is further relevant because proteasome inhibition, as in our For decades, majority of previous studies have focused on understanding protein synthesis, particularly their study, with ALLN leads to compensatory upregulation of % Viability % Apoptotic cells % Viability % Viability Journal of Oncology 11 ### ### 2.5 ∗∗∗ ### 2.0 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ 1.5 ∗∗∗ ∗∗∗ 1.0 ∗∗∗ 0.5 0.0 Cntrl ALLN ALLN+ ALLN+ Cntrl ALLN ALLN+ ALLN+ SS Rapa SS Rapa (-) NAC (-) NAC (+) NAC (+) NAC (a) (b) 2.5 2.0 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ 1.5 ∗∗∗ ∗∗ ∗∗ 1.0 0.5 0.0 0 Cntrl SS Rapa Cntrl SS Rapa (-) NAC (-) NAC (+) NAC (+) NAC (c) (d) 1.5 ### ∗∗ ∗∗∗ ∗∗∗ 60 ∗∗∗ 4 50 1.0 40 ### ### ∗∗∗ ∗∗∗ 30 ## 0.5 ns 0 0.0 0 LC3B-II Atg-3 LC3B-II B-actin B-actin B-actin (i) (ii) (iii) (e) Figure 5: ALLN-induced autophagy sensitizes R273H-P53 cells by increasing ROS levels. (a) R273H-P53 cells were exposed to ALLN, ALLN+SS, and ALLN+Rapa for 48 h. NAC (5 mM) was applied 1 h prior to treatment wherever mentioned. Fold change in ROS levels is represented through bars; untreated control was taken as arbitrary unit “1.” (b) MTT assay was performed to analyze cell viability following exposure of R273H-P53 cells to ALLN and other autophagy inducers; data is represented through bar diagram. (c) R273H-P53 cells were exposed to serum starved media or Rapa (500 nM) for 48 h. Fold change in ROS levels is represented. (d) MTT assay was performed to check cell viability following exposure of R273H-P53 cells to autophagy inducers with/without NAC.∗ indicates significant difference compared to untreated control, while # indicates significant dieff rence compared to ALLN-treated cells. (e)(i)/(ii)/(iii) R273H-P53 cells were treated with ALLN or ALLN plus SS for 48 h. NAC was given 1 h prior to treatment wherever mentioned. Immunoblotting was thereaer ft performed for LC3B-II and Atg-3.∗ indicates significant difference compared to untreated control, while # indicates significant dieff rence compared to ALLN-treated cells. Fold Change in LC3B-II expression Relative change in Relative change in DCFDA fluorescence DCFDA fluorescence Cntrl ALLN ALLN+ NAC Fold Change in Atg-3 expression Cntrl % Viability % Viability ALLN ALLN+ NAC Fold Change in LC3B-II expression Cntrl ALLN ALLN+ SS ALLN+SS+ NAC 12 Journal of Oncology ### ### ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ 80 ∗∗∗ ∗∗∗ ## 60 ∗∗ ∗∗∗ 0 0 Cntrl SS ALLN ALLN+SS Ph ERK (-) U0126 (+) U0126 B-actin Cntrl CQ ALLN ALLN+CQ (a) (b) ∗∗∗ ∗∗∗ ∗∗∗ ### ∗∗∗ ### ∗∗ Ph ERK Ph ERK B-actin B-actin Cntrl ALLN ALLN+ ALLN+SS+ Cntrl ALLN ALLN+NAC SS NAC (i) (ii) (c) ### ### 2.5 ∗∗∗ ∗∗∗ 2.0 ∗∗∗ ∗∗∗ 1.5 ∗∗∗ ∗∗∗ 1.0 0.5 0.0 Cntrl SS ALLN ALLN+SS (-) U0126 (+) U0126 (d) Figure 6: Sensitization of R273H-P53 cells is mediated by ERK signaling and ROS. (a) Immunoblot analysis representing ph-ERK expression upon ALLN and/or CQ treatment in R273H-P53 cells. (b) MTT assay was performed to analyze ALLN and/or serum starvation induced cell death in presence or absence of ERK inhibitor, U0126, given 2 h prior to treatment. Percentage viability is represented in the form of a bar diagram. (c) Immunoblot analysis was performed for ph-ERK expression upon ALLN and/or serum starvation in R273H-P53 cells. NAC was given 1 h prior to treatment wherever mentioned. (d) DCFDA u fl orimetric assay measuring intracellular levels of ROS aer ft treatment of R273H-P53 cells with or without U0126, given 2 h prior to ALLN and/or serum starvation for 48 h.∗ indicates significant difference compared to untreated control, while # indicates significant dieff rence compared to ALLN-treated cells. Fold Change in Fold Change in Ph ERK expression Ph-ERK expression Relative change in DCFDA fluorescence % Viability Fold Change in Ph ERK expression Journal of Oncology 13 ∗∗∗ ∗∗∗ ### ∗∗∗ ∗∗ 0 0 Cntrl PFT- ALLN ALLN+ PFT- P53 B-actin Cntrl PFT- ALLN ALLN+ PFT- (a) (b) ### 2.0 ∗∗ ### 1.5 ∗∗∗ ∗∗∗ ∗∗∗ 1.0 0.5 0 0.0 Atg-5 P62 B-actin B-actin Cntrl PFT- ALLN ALLN+ Cntrl PFT- ALLN ALLN+ PFT- PFT- (i) (ii) (c) ∗∗∗ ∗∗ Cntrl PFT- ALLN ALLN+ PFT- (d) Figure 7: P53 regulates induction of autophagy in R273H-P53 cells. (a) Immunoblot analysis depicting P53 expression upon ALLN treatment with or without 30 min of prior PFT-𝛼 exposure. (b) MTT assay was performed after ALLN and/or PFT- 𝛼 treatment for 48 h and percentage viability represented in the form of a bar diagram. (c) R273H-P53 cells were exposed to ALLN and/or PFT-𝛼 for 48 h and autophagy was checked through immunoblot analysis of Atg-5 (i) and P62 (ii). (d) MDC uo fl rescence assay was conducted aer ft R273H-P53 cells were exposed to ALLN or PFT-𝛼 or in combination for 48 h.∗ indicates significant difference compared to untreated control, while # indicates significant dier ff ence compared to ALLN-treated cells. “@” indicates significant dier ff ence compared to PFT- 𝛼 -treated cells. Fold Change in Fold Change in Atg-5 expression P53 expression Fold Change in MDC fluorescence Fold Change in % Viability P62 expression 14 Journal of Oncology 2.0 $$$ ∗∗∗ ∗∗∗ 100 $$$ 1.5 ∗∗∗ ### $$$ $$$ ∗∗∗ 1.0 0.5 0.0 Cntrl PFT- ALLN ALLN+ Cntrl PFT- ALLN ALLN+ PFT- PFT- (-) NAC (-) NAC (+) NAC (+) NAC (a) (b) 2.0 ### ∗∗∗ ∗∗∗ 1.5 ### ∗∗∗ ∗∗∗ 1.0 0.5 0.0 Cntrl PFT- ALLN ALLN+ Cntrl PFT- ALLN ALLN+ PFT- PFT- (-) U0126 (-) U0126 (+) U0126 (+) U0126 (c) (d) ∗∗∗ Ph ERK B-actin Cntrl PFT- ALLN ALLN+ PFT- (e) Figure 8: R273H-P53 cells show ROS and ERK accumulation aer ft PFT-𝛼 treatment. ((a) and (b)) R273H-P53 cells were exposed to ALLN and/or PFT-𝛼 (30 min prior to ALLN treatment) with and without NAC (1h prior to ALLN treatment) for 48 h and then cell viability and ROS levels were measured through (a) MTT assay and (b) DCFDA u fl orimetric assay. ((c) and (d)) Cell viability and ROS levels were analyzed with/without 2 h prior treatment of U0126 and the result is shown in the form of a bar diagram. (e) Immunoblot analysis showing ph-ERK expression on ALLN and/or PFT-𝛼 treatment in R273H-P53 cells.∗ indicates significant difference compared to untreated control and # indicates significant dier ff ence compared to ALLN-treated cells, while $$$ indicates the statistical dier ff ence compared to minus(-) NAC. % Viability % Viability Fold Change in Ph ERK expression Relative change in Relative change in DCFDA fluorescence DCFDA fluorescence Journal of Oncology 15 autophagy for clearance of proteins, which may provide Autophagy ALLN survival advantage to cells. Given the intense interest in targeting proteasomal degradation and the promise they hold for a multitude of cancer types, it is therefore important to precisely understand the consequences of autophagy GOF-p53 ERK ROS induction after inhibition of proteasomal degradation as a cancer therapeutic strategy. In this study, we have demonstrated that cells transfected with GOF mutant p53 show resistance to not only conventional drugs like CDDP or 5-FU but also to ALLN when compared to wt-P53. There was Cell Death induction of autophagy upon ALLN treatment as well. Since ALLN induces autophagy and the latter may be a tumor survival response, we initially hypothesized that blocking Figure 9: Schematic representation of the probable mechanism autophagy with CQ, along with ALLN, may initiate cell involved in proteasomal inhibitor, ALLN induced cell death in R273H-P53 cells. death or apoptosis. However, despite the proven benefits of lysomotropic agents in cancer clinics in conjunction to autophagy inducers or drugs, in our study, CQ rather than enhancing ALLN-induced cytotoxicity reduced its sensitivity PFT-𝛼 :Pitfihrin-alpha in GOF-R273H-P53 cells.Hence,from these studies, we CQ: Chloroquine concluded that, in this context, autophagy does not serve as ROS: Reactive oxygen species a mechanism that prolongs cell survival. MDC: Monodansylcadaverine It is increasingly getting recognized that the well-con- LC3B-II: Microtubule-associated protein light chain served autophagic machinery may be essential for cell death, 3-II at least in certain settings. However, it is controversially SS: Serum starvation discussed in literature whether cells truly die “by autophagy” Rapa: Rapamycin. or in dying cells autophagy is just a bystander or programmed mechanism facilitating apoptosis [50]. Few studies in the past have indeed provided evidence that cells can possess a Data Availability novel death mechanism that may depend on autophagy [51– The data used to support the findings of this study are 53]. However, the nature of stimulus leading to autophagy- available from the corresponding author upon request. dependent cell death has remained poorly defined till date. In this study, we showed that the R273H harboring P53 cells can be better sensitized to proteasomal inhibition by ALLN, Disclosure by enhancement of autophagy rather than its inhibition. Though autophagy is predominantly thought to play an An initial part of this work was presented as poster in house important protective role in sustaining homeostasis of cancer conference, BITS International Conference on Life Science cells supporting their proliferation, we provide evidences of Research & Its Interface with Engineering and Allied Sciences autophagy as a death promoter in the resistant lung cancer (LSRIEAS 2018). cells. This death was characterized by an enhanced ROS and ERK signaling. We further prove that inhibition of GOF mutant p53 can enhance cell death in the lung cancer Conflicts of Interest cells. Figure 9 schematically represents the summary of our The authors declare that there are no conflicts of interest. findings. Currently, there are very few literatures available which identify molecular mechanisms where autophagy is a death enhancer. This signifies the importance of our study. Authors’ Contributions However, more broadly, it still remains to be investigated whether the cell death observed was autosis (that represents Heena Saini, Ifrah Hakeem, and Sudeshna Mukherjee per- a subtype of autophagic cell death) or whether a bona fide formed and analyzed the experiments. Rajdeep Chowdhury, cell death by autophagy also requires the core apoptotic Shibasish Chowdhury, and Heena Saini designed the study machinery. Together, our results reveal novel mechanism plan and wrote the manuscript. through which mutant p53 harboring lung cancer cells can be sensitized by exploiting the crosstalk between the cellular homeostatic protein degradation machineries. Acknowledgments The authors thank DBT (BT/PR/8799/MED/30/1067/2013) Abbreviations for providing funding support to Rajdeep Chowdhury for conducting the experiments. 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Autophagy Regulated by Gain of Function Mutant p53 Enhances Proteasomal Inhibitor-Mediated Cell Death through Induction of ROS and ERK in Lung Cancer Cells

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Hindawi Journal of Oncology Volume 2019, Article ID 6164807, 17 pages https://doi.org/10.1155/2019/6164807 Research Article Autophagy Regulated by Gain of Function Mutant p53 Enhances Proteasomal Inhibitor-Mediated Cell Death through Induction of ROS and ERK in Lung Cancer Cells Heena Saini, Ifrah Hakeem, Sudeshna Mukherjee , Shibasish Chowdhury , and Rajdeep Chowdhury Department of Biological Sciences, Pilani Campus, BITS, Pilani, Rajasthan , India Correspondence should be addressed to Rajdeep Chowdhury; rajdeep.chowdhury@pilani.bits-pilani.ac.in Received 16 July 2018; Revised 23 October 2018; Accepted 13 December 2018; Published 6 January 2019 Academic Editor: George P. Studzinski Copyright © 2019 Heena Saini et al. is Th is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mutations inp53, especially gainoffunction(GOF) mutations, arehighly frequent inlungcancers and are knowntofacilitate tumor aggressiveness. Yet, the links between mutant GOF-p53 and lung cancers are not well established. In the present study, we set to examine how we can better sensitize resistant GOF-p53 lung cancer cells through modulation of cellular protein degradation machineries, proteasome and autophagy. H1299 p53 null lung cancer cells were stably transfected with R273H mutant GOF-p53 or wild-type (wt) p53 or empty vectors. eTh presence of R273H-P53 conferred the cancer cells with drug resistance not only against the widely used chemotherapeutic agents like cisplatin (CDDP) or 5-flurouracil (5-FU) but also against potent alternative modes of therapy like proteasomal inhibition. er Th efore, there is an urgent need for new strategies that can overcome GOF-p53 induced drug resistance and prolong patient survival following failure of standard therapies. We observed that the proteasomal inhibitor, peptide aldehyde N-acetyl-leu-leu-norleucinal (commonly termed as ALLN), caused an activation of cellular homeostatic machinery, autophagy in R273H-P53 cells. Interestingly, inhibition of autophagy by chloroquine (CQ) alone or in combination with ALLN failed to induce enhanced cell death in the R273H-P53 cells; however, in contrast, an activation of autophagy by serum starvation or rapamycin increased sensitivity of cells to ALLN-induced cytotoxicity. An activated autophagy was associated with increased ROS and ERK signaling and an inhibition of either ROS or ERK signaling resulted in reduced cytotoxicity. Furthermore, inhibition of GOF-p53 was found to enhance autophagy resulting in increased cell death. Our findings provide novel insights pertaining to mechanisms by which a GOF-p53 harboring lung cancer cell is better sensitized, which can lead to the development of advanced therapy against resistant lung cancer cells. 1. Introduction p53’s tumor-suppressive function but also in certain instances can endow mutant proteins with neomorphic properties Non-small cell lung carcinoma (NSCLC) is a collective term described as mutant GOF-p53 which can contribute actively for a group of lung cancers which affects both smokers and to various stages of tumor progression and to increased nonsmokers. It represents approximately 85% of all lung resistance to chemotherapy. In this regard, the central DNA- cancers. In India, more than one million new cases arise every binding domain of p53 spans the most conserved region composed of a vast number of these missense mutations year with a burgeoning incidence of NSCLC reported annu- ally. In more than 50% of NSCLC patients, p53 is arguably and among these the hot spot residues occur with unusually high frequency [2–4]. p53 missense mutations in the hot the most frequent target for genetic alterations associated spot region can generally be classified as DNA contact (or with poor prognosis and relatively more chemoresistance [1]. Accumulating evidences show that a vast majority of classI)mutants,likeR273H-p53,whichnormally makedirect contact with target DNA sequences and conformational (or p53 mutations are missense that results in production of a stable, full-length mutated protein carrying only single class II) mutants, like R175H-p53, which disrupt the structure amino acid substitution. These mutations not only annul of the p53 protein partially or completely, thus altering 2 Journal of Oncology its function [5, 6]. R175H-P53 and R273H-P53, being the cells, which acted as a prodeath mechanism. Furthermore, most frequently occurring GOF mutations in cancer cells, we observed that GOF-p53 serves to mitigate cell death were observed to induce resistance to chemotherapeutic induced by autophagy in the lung cancer H1299 cells. Our study provides novel insights into modes of sensitization agents in multiple cancer cell types [7, 8]. Interestingly, few reports suggest that, unlike wild-type (wt) p53, mutant p53 of resistant NSCLC cells harboring GOF-R273H mutant can escape MDM-2-dependent proteasomal degradation and P53. hence accumulate stimulating the oncogenic effect [9]. us, Th how to effectively promote degradation of GOF-p53 and 2. Materials and Methods sensitize cancer cells, thus reducing drug resistance, is an .. Chemicals and Reagents. ALLN (#sc-221236), pitfi hrin- important question to be investigated. Autophagy is a well-established self-degradation process alpha (PFT-𝛼 , #sc-45050), and rapamycin (Rapa, #sc-3504A) were purchased from Santa Cruz Biotechnology. CDDP that degrades and recycles numerous intracellular cytoplas- 󸀠 󸀠 (#232120) was obtained from MERCK. 2 ,7 -dichloroflu- mic constituents to maintain homeostasis. However, in a cancerous state, autophagy is known to play a paradoxical orescin diacetate (DCFDA, # D6883), monodansylcadav- erine (MDC, # D4008), CQ (#C6528), and propidium role by either activating cell death and inhibiting tumor progression or promoting cell survival and later stages of iodide (PI; #P4864) were purchased from Sigma; N-Acetyl- cancer progression [10]. Substantial evidences show that L-cysteine (NAC, #47866) and 3-(4, 5-dimethylthiazol-2- yl)-2,5-di-phenyltetrazolium bromide (MTT, #33611) were dieff rent forms of autophagy, for example, macroautophagy and chaperone-mediated autophagy, are implicated in the obtained from SRL. Geneticin (G418, # 10131-035), FITC depletion of stable, mutant p53 isoforms [11]. Functional conjugated Annexin V (#A13199), Annexin V binding buffer (#V13246), and LysoTracker Green DND-26 (# L7526) were involvement of mutant p53 in the regulation of autophagy and in turn being regulated by the cellular degradation system procured from Thermo Fisher Scientific. Lipofectamine 3000 was from Invitrogen (#L3000-001). The MAPK inhibitor, in cancer cells and identification of associated molecular U0126, and gene specific primary and secondary antibod- mechanisms governing it are still incompletely understood. Deciphering the details of these interactions can provide clues ies were obtained from Cell Signaling Technology (CST, USA). pCMV-Neo-Bam p53 wt (Addgene Plasmid #16434), to appropriate sensitization of resistant cancer cells. A growing number of studies suggest that both intra- pCMV-Neo-Bam p53 R273H (Addgene Plasmid #16439), and cellular degradation pathways, for example, ubiquitin pro- pCMV-Neo-Bam Empty Vector (Addgene Plasmid # 16440 ) were a gift from Bert Vogelstein. teasome pathway (UPS), and autophagy are mechanisti- cally and functionally linked such that blockage to either one can lead to upregulation of the other in a way that .. Cell Culture. The non-small cell lung carcinoma cell line, remains yet to be clarified [12]. For example, proteasomal H1299, was a kind gift from Dr. Sanjeev Das (NII, New Delhi). inhibition can enhance the load of misfolded proteins and MCF-7 and MDA-MB-468 cells were procured from NCCS (Pune, India); HCT116 (p53 null) cells were obtained from can trigger autophagy as a compensatory mechanism for their degradation [13]. However, autophagy, serving as an Wogan Lab (MIT, USA); HT-29 and SW480 cells were a gift essential mechanism to cope with cellular stresses, may from Dr. Susanta Roychoudhury, IICB-Kolkata. Cells were directly contribute to survival of cancer cells exposed to cultured at 37 C, 5% CO in Dulbecco’s modified minimal proteasomal inhibitors and, hence, in consequence, might essential medium supplemented with 10% fetal bovine serum reduce effectiveness of therapy. Therefore, an inhibition of and 1% penicillin-streptomycin mixture [16]. Cells were autophagic u fl x after induction of prosurvival autophagy grown to 70-80% confluency prior to treatments. Trypsin- has often been utilized as a strategy to sensitize multiple EDTA solution (0.05%) was used for detachment of cells. cancer cell types [14]. Based on these findings, clinical trials Stable transfected cells were maintained in 600 𝜇 g/ml of are currently ongoing investigating autophagy inhibition G418. in conjunction with anticancer therapies, like bortezomib. However, conversely, an overactivation of autophagy can also .. Preparation of Cell Lines Stably Expressing GOF Mutant act as a bona fide death inducer or death effector, upstream and wt-p. P53 null H1299 cells were cultured in six-well of other death pathways, like apoptosis [15]. Based on the plate and transfected with either 2𝜇 gof pCMV-Neo-Bam above considerations, there might be physiological situations p53-wt or pCMV-Neo-Bam p53-R273H or pCMV-Neo-Bam in which autophagy protects death or, in contrary, kills Empty Vector (EV) purified plasmid with Lipofectamine cancer cells that would have otherwise manifested prolonged 3000 according to manufacturer’s instructions. Around 24 survival. Because autophagy plays an important roles in cell h after transfection, the cells were transferred to a 10 cm fate, it is therefore critical to understand the mechanism dish and selected for transfection positivity by geneticin by which autophagy interacts with and aec ff ts cell survival (600𝜇 g/ml) selection. Transfected cells were maintained for or death machinery, which might allow new avenues to several days under G418 pressure. Nonsurviving cells were effectively sensitize resistant cancer cell types. washed off with PBS and fresh media with G418 was added. Taking this into consideration, in this study, we exposed This was done till the time cell colonies were obtained. GOF-R273H-P53 cells to proteasomal inhibitor ALLN Random colonies were selected and allowed to grow in a and observed its effect on autophagy. A ROS-dependent new culture dish under G418 pressure. Cells were grown autophagy was found to be induced in the R273H-P53 till confluency and then stable transfection of wild-type or Journal of Oncology 3 R273H-P53 vector was conrfi med by immunoblotting against with drug for 48 h. Coverslips were then withdrawn and p53 antibody. washed with PBS and cells were xfi ed in formaldehyde for 10 min at room temperature. Coverslips were mounted .. Analysis of Cytotoxicity. In vitro cytotoxicity was per- on a glass slide with antifade DAPI. Nuclear morphology formed using 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltet- was further observed by u fl orescence microscopy (Olympus, razolium bromide (MTT) assay, following methods previ- Japan). ously described in Chowdhury et al’s work [17]. Briefly, H1299 (null P53/EV), H1299/wt (wt P53), and H1299/R273H . . Annexin V/Propidium Iodide (PI) Staining. Cells were (R273H P53) cells were cultured in 96-well plates. Aeft r seeded on 6 cm dishes at a density of 5 × 10 cells and overnight culture of cells, they were treated with specific thereafter exposed to various treatments. After that the cells drugs for a stipulated period of time. Following that, MTT were harvested, washed with PBS, and resuspended in 500 was added to each well and incubated for 4 h. Viable cells 𝜇 L of 1X binding buffer. Following that, 4 𝜇 l of Annexin V- form formazan crystals with MTT, which were solubilized FITC and 10𝜇 l of PI were added. Cells were then incubated in DMSO, and readings were obtained at 570 nm with a in the dark for 20 min and samples were then acquired using differential lfi ter of 630 nm using Multiskan GO microplate flow cytometer (Cytoflex, Beckmann Coulter) [17]. CytExpert spectrophotometer. Percentage of viable cells was calcu- software was used to analyze the acquired data. To detect both lated using the following formula: viability (%) = (mean early and late apoptotic cells, the percentage of cells in lower absorbance value of drug-treated cells)/(mean absorbance and upper right (LR and UR) quadrant representative of only value of the control) x 100. Annexin V and both Annexin V-PI positive cells, respectively, were counted. Percentage of apoptotic cells is represented .. Caspase Assay. For measurement of caspase activity, 2.5 through bar diagram. × 10 cells/well were seeded in 6-well plate and exposed to IC dose of CDDP for 48 h. Caspase-3 colorimetric 50 .. Monodansylcadaverine (MDC) Staining of Autophagic protease assay kit (Invitrogen) was used to measure caspase-3 Vacuoles. MDC, an autophagolysosomal marker, was used activity following procedure described elsewhere [17]. Briefly, to analyze autophagy induction. Cells were grown over protein was extracted using RIPA buffer; concentration was coverslips and the following day drug treatment was made. determined by Bradford assay and then equal amount (60 The cells were then incubated in the dark for 10 min with 𝜇 g) of protein was added to microtiter plates with caspase- 0.05 mM MDC at 37 C. Thereaer ft , the coverslips with 3 substrate (Ac-DEVD-pNA). The concentration of the p- cells were washed and mounted with antifade DAPI. MDC nitroaniline (pNA) released from the substrate was calculated punctate dots were analyzed under uo fl rescence microscope. from the absorbance values at 405 nm. For uo fl rimetric measurements, cells were grown in 6-well plate. After treatment, cells were labelled with MDC for 10 .. Analysis of DNA Fragmentation. Apoptosis was evalu- min followed by PBS wash and then collected in 10 mM ated by fragmented genomic DNA forming DNA ladders Tris-HCl (pH 8.0) containing 0.1% Triton X-100 [20, 21]. (short fragments of∼200 base pairs) on agarose gel [18]. To Intracellular MDC was assessed by uo fl rescence photometry analyze DNA ladder formation, null P53 and R273H-P53 cells (excitation 380 nm and emission 525 nm) on a microplate were seeded in 6 cm dishes at a density of 5× 10 cells/plate reader (Fluoroskan Ascent). Change in MDC uo fl rescence and treated with CDDP for 48 h. DNA was extracted using with respect to control is expressed as fold change. Invitrogen apoptotic DNA ladder detection kit and ladder formation was analyzed on 1% agarose gel. A DNA marker .. LysoTracker Green (LTG) Staining of Acidic Vesicles. was run parallel to the samples. LTG constituting a fluorophore linked to a weak base is a uo fl rescent acidotropic probe, used for labelling and tracking . . RNA Isolation and Real-Time PCR (RT-PCR). Total RNA acidic organelles in live cells. Cells were cultured overnight was isolated using TRIzol reagent. GeneSure First Strand on coverslips and then exposed to different concentration of cDNA Synthesis kit was used for complementary DNA ALLN for 48 h. After treatment, the media was removed, (cDNA) synthesis. Templates were amplified using gene- the cells were washed with PBS, and thereaer ft LTG was specific primers for ABCB1 taking GAPDH as housekeeping added (0.05𝜇 M). Cells were then incubated for 20 min in control and detected using SYBR Green Supermix in CFX CO incubator. Fluorescence intensity of LTG was observed Connect RT-PCR System (Bio-Rad) [16]. The primers used under a u fl orescence microscope and compared to untreated andtheirsequencesaregiven asfollows:GAPDH,forward5 - control. 󸀠 󸀠 GCA CCG TCA AGG CTG AGA AC-3 and reverse 5 -TGG 󸀠 󸀠 TGA AGA CGC CAG TGG A-3 ;ABCB1,forward 5 -GGG .. Measurement of Intracellular ROS. Cells were seeded at 󸀠 󸀠 ATG GTC AGT GTT GAT GGA-3 and reverse 5 -GCT ATC adensity of 9x10 in 96-well plates and exposed to treatments. GTG GTG GCA AAC AAT A-3 . The relative RNA expression NAC, a ROS scavenger (5 mM), was added 1 h prior to was calculated using Pfa’ffls method [19]. treatments. After treatment, the cells were washed with PBS and then incubated in 100𝜇 lof working solution (10𝜇 M) of 󸀠 ∘ . DAPI Staining of Nucleus. 4 -6-Diamidino-2-phenylin- DCFDA at 37 C for 30 min. Fluorescence was measured using dole (DAPI) stain was used to detect apoptotic nuclei [18]. a microplate reader (Fluoroskan Ascent) at 485 nm excitation Cells grown on coverslips were either untreated or treated and 530 nm emission [18]. 4 Journal of Oncology .. Immunoblotting. Immunoblotting was performed fol- null P53 cells or EV stable transfected cells (Figure 1(a)(i), lowing protocols described elsewhere [22]. Cells were grown Supplementary Figure 1.b, and Supplementary Figure 1.c); in 10 cm dishes. Following treatment for specific time, cells the sensitivity of EV cells to cisplatin was comparable to were lysed in RIPA buffer (Sigma-Aldrich), and the protein null cells. We hence compared caspase-3 activity upon concentration was estimated using Bradford reagent. Then, CDDP treatment between H1299 null P53 (IC ∼ 15𝜇 M) 5X gel loading dye was added to the lysates followed by and R273H-P53 (IC ∼ 30𝜇 M) cells through ELISA based heat denaturation (100 Cfor 10 min). Proteins were then method. As expected, R273H-P53 cells showed significantly loaded in denaturing polyacrylamide gels and transferred decreased enzymatic activity compared to null P53 cell to polyvinylidene u fl oride membranes. Skimmed milk (5%) type (Figure 1(b)). Similar results were obtained in DNA was used for blocking. The blots were then probed or fragmentation study (Figure 1(c)). Since R273H-P53 cells reprobed with specific primary antibodies and detected using showed a marked increase in resistance compared to null enhanced chemiluminescence detection on ChemiDoc (Bio- or wt-P53 cell types, we further confirmed it by analyzing Rad) [14]. The primary antibodies used were as follows: mRNA expression of ABCB1, which showed a substantially Atg-5 (CST; D1G9), Atg-3 (CST; 3415), LC3-II (CST; D11), increased expression upon CDDP treatment in R273H-P53 phospho-MAPK (ERK) (CST; Rabbit mAb #4370), and p53 stable transfected cells compared to control (Figure 1(d)). (SCBT; DO-1).𝛽 -actin (SCBT; sc69879) was used as loading Based on the above experimental evidences, it is clear that control. The secondary antibodies used were horseradish R273H-P53 cells show resistance to CDDP, compared to null peroxide-conjugated goat anti-rabbit and goat anti-mouse or wt-P53 cells. However, to analyze that the resistance is IgG at dilution of 1:10,000 and 1:20,000, respectively. The a generalized phenomenon across multiple drug types or expression levels were densitometrically quantified using is purely specific to CDDP, we evaluated cross resistance ImageJ software and normalized to the control. of R273H-P53 cells to other conventionally used anticancer drugs like 5-FU or methotrexate (data not shown). As evident .. Statistical Analysis. The obtained data were analyzed from Figure 1(e), R273H-P53 cells were less sensitive to 5- using GraphPad Prism sowa ft re version 5.0. Effect of treat- FU as well. Collectively, these observations suggest that null- ment in comparison to control was statistically determined P53 cells acquire drug-resistant characteristics upon stable using one-way or two-way ANOVA. The Bonferroni method transfection of R273H-P53 vector in NSCLC cells. was used to analyze multiple comparisons. Throughout the text, the representative images are of experiments done in .. P Positive Cells Are More Sensitive to Proteasomal multiples. Data are represented in mean± SEM. The symbols Inhibition. Targeting the proteasomal degradation pathway in parenthesis denote the following:∗ compared to control, is increasingly getting recognized as a promising strategy # compared to ALLN or R273H, not significant (ns) p > for cancer therapy [24–26]. Wt-p53 protein is primarily ∗/# ∗∗/## ∗∗∗/### degraded by the UPS pathway; however, mutations in p53 0.05; p≤0.05; p≤0.01; p≤0.001. might stabilize this protein and inhibit MDM2 interaction, thereby preventing degradation [27–29]. However, there are 3. Results and Discussion reports suggesting that several “hot-spot” p53 mutants like R175H, R248W, or R273H remain sensitive to ubiquitin- .. R H-P Cells Exhibit Increased Drug Resistance Com- pared to Null or wt-p Cells. In NSCLC patients, p53 mediated degradation [30]. We assumed that proteasomal status shows no prognostic significance in the absence of degradation of GOF-p53 might be context-dependent and adjuvant chemotherapy; however, after undergoing treatment shows increased bias towards the nature of inhibitor used. with cisplatin, a reduced disease-free interval and overall We used ALLN, a well-known proteasome inhibitor that is survival are seen bearing a GOF-p53 protein [23]. Given known to induce apoptosis by virtue of accumulated protein the importance of GOF-p53 in NSCLCs, in this study, we response. ALLN was chosen over the widely used proteaso- prepared a stable transfected GOF-R273H-P53 NSCLC cell mal inhibitor, bortezomib, because the eeff cts of ALLN are model aimed at understanding modus operandi of resistance relatively less explored and the use of bortezomib has recently and develop an effective strategy for sensitizing GOF p53 been challenged by severe adverse side effects and resistance cells, which is elusive till date. P53 null H1299 cell line was [31]. Exposure of ALLN (5 and 10 𝜇 M) for 48 h showed more cell deaths in wt-P53/R273H-P53 transfected cells than stable transfected with empty vector (EV), wt-P53, or R273H- P53. Stable transfection was conrfi med by immunoblotting null (Figure 2(a)) or EV transfected cells (Supplementary against p53 antibody (Supplementary Figure 1.a). The cells Figure 2). Similar results were obtained when cells were were then exposed to varying concentrations of CDDP (a treated with another proteasomal inhibitor, MG132 (data not standard chemotherapy for NSCLC patients) for 48 h. Simi- shown). Interestingly, R273H-P53 cells in comparison to wt- larly other cancer cell types possessing wt-P53 (MCF7), null- p53 showed decreased sensitivity to proteasomal inhibition as P53 (HCT116), or R273H-P53 (MDA-MB-468, HT-29, and well (Figures 2(a) and 2(b)) as evident from cell viability assay SW480) were also exposed to various doses of CDDP. Inter- or apoptosis assay by Annexin V/PI staining. Importantly, estingly, in all the cell types studied, GOF-p53 cells showed unlike response to conventionally used drugs like cisplatin, significantly decreased sensitivity to CDDP compared to both wt-P53 and R273H-P53 cells were more sensitive than wt-P53 harboring cells demonstrating resistance (Figures the parental null or EV cells, suggesting that the presence of p53 protein provides a therapeutic advantage to target- 1(a)(i)–1(a)(iii)). Importantly, R273H-P53 cells showed less sensitivity to CDDP compared to either parental H1299 ing strategies based on interference of protein degradation Journal of Oncology 5 120 120 100 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ 100 100 ### 80 ### ∗∗∗ 80 80 ∗∗∗ ### ### 60 60 40 40 20 20 0 0 0 MCF-7 MDA-MB-468 05 15 35 5 M HCT116-/- HT-29 SW-480 R273H-P53 0 M 0 M Null-P53 35 M 35 M WT-P53 15 M 55 M 15 M 55 M (i) (ii) (iii) (a) CDDP CDDP Marker Cntrl (LD) (IC50) 2.5 2.5 ∗∗ 2.0 2.0 ns 1.5 1.5 1.0 1.0 ns 0.5 0.5 0.0 0.0 Null-P53 R273H-P53 Null-P53 R273H-P53 Control Control IC-50 IC-50 (b) (c) (d) ∗∗∗ ∗∗∗ Null-P53 R273H-P53 0 M 130 M 70 M 170 uM 110 M (e) Figure 1: GOF-R273H-p53 mutation imparts drug resistance. (a) Cell lines with varied p53 status were exposed to dieff rent doses of cisplatin. Cell viability was analyzed after 48 h of treatment through MTT assay. Untreated samples were taken as control. ∗ and # indicate significant dieff rence compared to untreated cells and R273H-P53 cells, respectively. (b) Fold change in caspase-3 enzyme activity was measured following CDDP treatment at IC-50 dose for 48 h in H1299 (null-P53) and H1299-R273H-P53 stable transfected cells. Level of caspase-3 activity in untreated control was taken as arbitrary unit “1.” (c) DNA fragmentation was measured on agarose gel. Null-P53 and R273H-P53 cells were treated with CDDP at low dose (LD) and IC-50 dose for 48 h; DNA was extracted and run on an agarose gel. A DNA marker was run alongside the samples. (d) Real-time PCR showing expression of ABCB1 mRNA levels upon exposure of null-P53 and R273H-P53 cells to CDDP (IC- 50) for 48 h. (e) Null-P53 and R273H-P53 cells were given different doses of 5-FU and cross resistance was analyzed through MTT assay. ∗ indicates significant difference compared to untreated cells. % Viability Caspase-3 Enzyme activity % Viability % Viability R273H-P53 Null-P53 Fold Change in ABCB1 expression % Viability 6 Journal of Oncology ### ∗∗∗ ## Cntrl 5 10 M Cntrl 5 10 M WT-P53 Null-P53 R273H-P53 WT-P53 R273H-P53 (a) (b) Cntrl 5M 10 M (c) Figure 2: Proteasomal inhibitor (ALLN) induces apoptosis. (a) Null-P53, wt-P53, and R273H-P53 cells were exposed to varying doses of ALLN. Aeft r 48 h of exposure, cell viability was measured through MTT assay. Untreated samples were taken as control. ∗ and # indicate significant dieff rence compared to Null-P53 and R273H-P53 cells, respectively. (b) Percentage of apoptotic cells was measured through flow cytometry using Annexin V/PI staining and compared between wt-P53 and R273H-P53 cells upon ALLN treatment. (c) R273H-P53 cells were either untreated or treated with ALLN for 48 h and then stained with DAPI. Nuclear fragmentation or condensation aeft r treatment is marked with white arrows. The scale bar represents 100 𝜇 m. (Figure 2(a)). Nuclear staining by DAPI in R273H cells show- of enhanced autophagy was observed with ALLN treatment ing fragmented nucleus, as depicted in Figure 2(c), further (Figure 3(a)). However, an increase in LC3B-II protein levels confirmed cell sensitization on ALLN treatment. Overall, can be resultant of increased autophagy or an inhibition of the above results highlight the importance of targeting the n fi al step of autophagosome-lysosomal fusion [33]. Hence, protein degradation machinery in p53 positive cells, though to confirm autophagic ux, fl we checked for the changes in an adjuvant therapy might be essential for R273H-P53 cells LC3B-II protein levels with or without the lysomotropic as they are more resistant than wt-p53 cells to cell death by agent, CQ. An increased expression of LC3B-II was observed protein overload. in ALLN+CQ treated samples when compared to only independent treatments indicative of enhanced autophagic .. Proteasomal Inhibitor, ALLN, Induces Autophagy in GOF u fl x with ALLN (Figure 3(b)). Autophagy induction was further validated through MDC staining; MDC preferentially R H-P Cells. Two major pathways of degradation main- tain protein homeostasis, the UPS, responsible for degrading accumulates in acidic autophagic vacuoles. MDC uo fl res- majority of proteins including many short-lived, denatured, cence, represented by the green punctate dots, increased with or, in general, damaged proteins, and autophagy, which, ALLN treatment in a dose-dependent manner, suggesting by contrast, is mostly responsible for degradation of long- an increase in autophagy (Figure 3(c)). Furthermore, an lived proteins [32]. Although UPS and autophagy were increase in the number of lysosomes is oen ft associated initially considered to be largely disconnected pathways, with increased autophagy. We observed an increase in recent advances in understanding of UPS and autophagy LTG staining with ALLN (Figure 3(d)). The above results have highlighted a strong connection between them. To aresupportiveofthe fact that proteasomal inhibition by examine the effect of ALLN on autophagy, R273H-P53 cells ALLN in R273H-p53 cells activates autophagy. However, were treated with 10 𝜇 M ALLN for different time points in tumor cells, autophagy can act as both a prodeath and LC3B-II (marker for autophagy) protein expression was or prosurvival mechanism in a context-dependent manner analyzed. An increase in LC3B-II expression levels indicative [34]. % Viability % Apoptotic cells Journal of Oncology 7 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ 60 6 ∗∗ 4 LC3B-II LC3B-II B-actin B-actin Cntrl CQ ALLN ALLN+CQ Cntrl 3 24 48 h (a) (b) ∗∗∗ Cntrl 5 10 M Cntrl 5M10M (c) (d) Figure 3: ALLN induces autophagy in GOF R273H-P53 cells. (a) R273H-P53 cells were treated with 10 𝜇 M ALLN for different time points and expression of LC3B-II was analyzed through immunoblotting. (b) Immunoblot analysis showing expression of LC3B-II upon 48 h of exposure to CQ, ALLN, and ALLN+CQ in R273H-P53 cells. (c) Fluorescent microscopic images of MDC uo fl rescence in ALLN-treated (0, 5, and 10𝜇 M; 48 h) R273H-P53 cells. eTh scale bar represents 100 𝜇 m. (d) R273H-P53 cells were treated with ALLN for 48 h and then stained with LTG dye. Green dots representing the lysosomes were counted and represented as bar diagram. The scale bar represents 100 𝜇 m. .. GOF-R H-p Cells Are Sensitive to Autophagy Induc- accumulation of acidic vesicles, thereby blocking autophagic tion. Enhanced autophagy has recently been implicated in u fl x, and can act as a potent strategy to sensitize R273H- multiple studies facilitating cancer cell survival under physi- P53 cells. We initially checked for cytotoxicity inducing ological stresses. Hence, we hypothesized that a combination property of CQ alone. CQ even at 50 𝜇 Mwas not able treatment with ALLN (being an autophagy inducer) with to impart any significant cytotoxic effect on R273H-P53 CQ (an agent that inhibits lysosomal function) might lead to cells as analyzed by MTT assay and also Annexin V/PI Fold Change in LC3B-II expression 10MC 5M ntrl Fold Change in lysosome number Fold Change in LC3B-II expression 8 Journal of Oncology staining (Figures 4(a) and 4(b)). Since CQ is a late-stage with or without NAC pretreatment. The above experiments autophagy inhibitor, we thought of exploring the eeff ct of an also confirm that ALLN-induced cytotoxicity is not only early-stage autophagy inhibitor, 3-methyl adenine (3-MA). autophagy-dependent but is also regulated by an increase in ROS levels. Interestingly, R273H-P53 cells when pretreated Interestingly, on the contrary, 3-MA treatment was able to sensitize R273H-P53 cells. As depicted in Supplementary with NAC resulted in a decrease in autophagic marker Figure 3, only 3-MA treatment reduced cell viability to less expression, Atg-3 or LC3B-II, suggesting that, in this context, ROS is upstream to autophagy and a quenching of ROS than 50% in R273H-P53 cells. However, as we analyzed the expression of autophagic markers upon 3-MA exposure, reduces autophagy-induced cell death as well (Figures 5(e)(i), we noticed an increased expression of LC3B-II and Atg-5 5(e)(ii), and 5(e)(iii)). proteins levels (Figures 4(c)(i) and 4(c)(ii)). We assumed that 3-MA might have autophagy inducing effects as well. .. Sensitization of GOF-p Is Mediated by ERK along with ROS Accumulation. Previous reports show that mitogen Indeed, in corroboration to above, there are previous reports suggesting that prolonged 3-MA treatment can inhibit PI3K- activated protein kinase (MAPK) signaling pathways are able class-I, in turn inhibiting mTOR and activating autophagy to modulate autophagy and determine cell fate [39]. There [35]. However, results obtained with 3-MA provided us with a are also a growing number of reports stating that, in certain hint that, rather than autophagy inhibition, these cells might conditions, the MAPK-ERK can promote cell death [40, 41]. be more sensitive to autophagy induction. Accordingly, the Interestingly, an increase in phospho-ERK indicative of its effect of rapamycin (Rapa), a widely used mTOR inhibitor, activation was observed in cells treated with ALLN, which and serum starvation (SS), which are known to elicit an went down with autophagy inhibition by CQ (Figure 6(a)). autophagic response, was investigated in R273H-P53 cells [36, To confirm the role of ERK in cell death, U0126, a widely 37]. Interestingly, R273H-P53 cells treated with autophagy used selective inhibitor of the upstream MAP kinase pathway, inducers like Rapa or exposed to SS alone or in combination was used and cell viability was measured in presence of with ALLN had significantly enhanced cytotoxic effects when ALLN or ALLN plus SS. Importantly, inhibition of the ERK compared to autophagy inhibition with CQ alongside ALLN pathway by U0126 significantly reduced cell death induced (Figures 4(d)–4(h)). In fact, autophagy inhibition with CQ by ALLN alone or ALLN plus autophagy induction. This was found to moderately decrease the sensitivity of cells to implicates that ERK signaling acts as a prodeath mecha- ALLN (Figure 4(i) and Supplementary Figure 4). Hence, in nism (Figure 6(b)). ROS, such as hydrogen peroxide, are this study, we suggest that although physiologically relevant reported to regulate ERK phosphorylation as ERK-specific levels of autophagy are required for cellular homeosta- phosphatases are sensitive to ROS. Hence, ROS-mediated sis maintenance, enhanced autophagy can in turn induce prolonged ERK activation might be a crucial mechanism autophagy-dependent cell death. Excessive autophagy has regulating cell death [42]. Interestingly, a significant decrease been previously observed in association with various forms of in phospho-ERK levels was observed when ALLN-treated cell death and the term “autophagic cell death” was originally cells were predisposed to NAC (Figures 6(c)(i) and 6(c)(ii)). introduced to describe cell death associated with autophagy. We also checked for the reverse effect, that is, ROS production However, evidences associating autophagy to cell death in upon ERK inhibition. R273H-P53 cells were pretreated with these reports were more circumstantial, and the nature of U0126 for 2 h prior to addition of ALLN or ALLN plus such death occurring in cancer cells remains poorly defined. autophagy inducers. Interestingly, a drastic decline in level We were hence interested in analyzing the modus operandi of of ROS was observed with ERK inhibition (Figure 6(d)). cell death observed upon autophagy enhancement. Collectively, the above findings highlight that R273H-P53 cell sensitization is regulated by ROS-autophagy-ERK signaling .. ALLN-Induced Autophagy Facilitates Cell Sensitization by loop upon ALLN or ALLN plus autophagy induction. Regulating ROS Levels. Generation of ROS through oxidative stress is known to cause cell death; however, the role of oxida- . . Induction of Prodeath Autophagy in R H-P Cells Is a tive stress in autophagy-induced cell death is relatively unex- p-Dependent Process. Although there are many links that plored. Conventionally, autophagy serves as a bueff r system to connect autophagy with p53, molecular crosstalk between control the level of ROS in cells and reduce their toxic effects them is still incompletely understood [43]. One of the [38]. However, the connection of ROS with autophagy in our earliest studies describing the relationship between mutant experimental context was found to be different. Intracellular p53 and autophagy states that the subcellular localization ROS levels measured by DCFDA dye followed by uo fl rimetric of mutp53 is the major determinant of autophagy and it is conventionally accepted that p53 inhibits autophagy [44]. On analysis showed a prominent increase with ALLN treatment in R273H-P53 cells (Figure 5(a)). A profound increase in ROS the other hand, in many cells, it is observed that prolonged levels was observed upon ALLN treatment in SS condition inhibition of the proteasome leads to its autophagy-mediated degradation of p53, suggesting that autophagy in turn can (Figure 5(a)). Interestingly, pretreatment of cells with the ROS scavenger-NAC resulted in substantial increase in cell regulate the stability of p53 protein [11]. In this study, we viability with only ALLN, ALLN plus SS, or ALLN plus inhibited p53 with a well-known p53 inhibitor, pifithrin- 𝛼 Rapa treated cells, suggesting a direct positive correlation of (PFT-𝛼 ) (Figure 7(a)). Interestingly, a significantly decreased increase in ROS with cell death (Figure 5(b)). Figures 5(c) cell viability was obtained with PFT-𝛼 plus ALLN treatment and 5(d) represent the status of ROS level and cell viability compared to only ALLN (Figure 7(b)). This suggests that on Rapa or SS exposure. The experiment was performed GOF-R273H-p53 inhibition had a positive impact on cell Journal of Oncology 9 2.0 1.5 1.0 0.5 0 0.0 0 10 50 M Null-P53 WT-P53 R273H-P53 0uM 10uM 50uM (a) (b) ∗∗∗ 3.0 25 2.5 ∗∗ ∗∗ 20 2.0 15 1.5 10 1.0 5 0.5 0.0 LC3B-II Atg-5 B-actin B-actin Cntrl CQ 3-MA ALLN Cntrl CQ 3-MA ALLN (i) (ii) (c) ### ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ Cntrl Rapa SS SS+Rapa Cntrl ALLN SS SS+ALLN (d) (e) Figure 4: Continued. % Viability Fold Change in % Viability LC3B-II expression % Viability % Apoptotic cells Fold Change in Atg-5 expression 10 Journal of Oncology 100 ### ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ### Cntrl ALLN Rapa ALLN+Rapa Cntrl ALLN 3-MA ALLN+3-MA (f) (g) ### 80 ∗∗∗ ∗∗∗ Cntrl ALLN SS Rapa ALLN+SS ALLN+Rapa Cntrl CQ ALLN ALLN+CQ (h) (i) Figure 4: Autophagy induction promotes cell death in R273H-P53 cells. (a) Null-P53, wt-P53, and R273H-P53 cells were exposed to varying doses of CQ. After 48 h of exposure, cell viability was measured through MTT assay. (b) CQ-treated R273H-P53 cells were analyzed for apoptosis induction through Annexin V/PI staining followed by flow cytometry. Fold change is represented through bar diagram. (c) R273H- P53 cells were exposed to CQ (10𝜇 M), 3-MA (5 mM), and ALLN (10𝜇 M) for 48 h and immunoblot analysis was performed for LC3B-II and Atg-5.∗ indicates significant difference to untreated control. (d) Cell viability in R273H-P53 cells was measured through MTT assay aeft r 48 h of treatments at various combinations, (d) Rapa (500 nM), Rapa + SS, (e) SS, ALLN+SS, (f) Rapa, ALLN+Rapa, (g) 3-MA, 3-MA+ALLN and represented in the form of bar diagram. (h) Apoptosis was analyzed through flow cytometry using Annexin V/PI staining in R273H-P53 cells, aeft r treatment with various autophagy inducers. Percentage of apoptotic cells is represented through bar diagram. (i) Cell viability was measured through MTTassay inR273H-P53 cells aeft r autophagy inhibitioninpresence orabsence of ALLN. ∗ indicates significant dier ff ence compared to untreated control, while # indicates significant dier ff ence compared to ALLN-treated cells. sensitization. We were hence curious to analyze the effects of transcriptional and translational control. While the aspects PFT-𝛼 on autophagy levels as well. Interestingly, pretreatment of protein degradation have been largely overlooked, it of PFT-𝛼 followed by ALLN exposure caused an increase is a natural way by which cells clean up proteins that are in Atg-5 levels and a decrease in sequestrosome p62 levels, redundant or have been misfolded or damaged [45, 46]. indicating an enhancement of autophagy (Figures 7(c)(i) and Inhibition of this protein degradation machinery has been 7(c)(ii)). Enhanced autophagy induction on ALLN plus PFT- useful previously for treatment of autoimmune diseases and 𝛼 treatment was further confirmed by MDC staining which cancer [26, 47]. Especially in particular types of cancer, where showed an additive effect as well (Figure 7(d)). Furthermore, protein production is much higher than normal, primarily an increased ROS accumulation was observed with ALLN to meet their overproliferative or secretary demand, an plus PFT-𝛼 treatment (Figures 8(a) and 8(b)). ROS scav- inhibition of proteasomes can cause proteins to pile up, enger, NAC, successfully reversed the effect substantiating eventually killing the cancer cell [48, 49]. There are already a ROS-dependent phenomenon. Further, as evident from proteasome inhibitors like bortezomib in the clinics for Figure 8(c), U0126 pretreatment followed by ALLN plus treating Kahler’s disease (multiple myeloma). However, the PFT-𝛼 exposure increased the cell viability and decreased proteasomal inhibitors not only affect the proteasome but accumulated ROS (Figure 8(d)). A signicfi antly increased also can significantly alter the functioning of another cellular phospho-ERK level was also observed with ALLN plus PFT- homeostatic machinery like autophagy. The latter is primarily 𝛼 treatment (Figure 8(e)). Taken together, we postulate that devoted towards maintaining cellular balance of organelles, GOF-p53 inhibition facilitates cell sensitization by upregulat- proteins, and other macromolecules. For example, cancer ing autophagy and by enhancing ROS and ERK activation. cells that produce excess unfolded proteins generate high endoplasmic reticulum stress leading to protein removal via either the proteasome or autophagy, suggesting that both 4. Conclusion pathways may be specifically exploited therapeutically. This is further relevant because proteasome inhibition, as in our For decades, majority of previous studies have focused on understanding protein synthesis, particularly their study, with ALLN leads to compensatory upregulation of % Viability % Apoptotic cells % Viability % Viability Journal of Oncology 11 ### ### 2.5 ∗∗∗ ### 2.0 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ 1.5 ∗∗∗ ∗∗∗ 1.0 ∗∗∗ 0.5 0.0 Cntrl ALLN ALLN+ ALLN+ Cntrl ALLN ALLN+ ALLN+ SS Rapa SS Rapa (-) NAC (-) NAC (+) NAC (+) NAC (a) (b) 2.5 2.0 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ 1.5 ∗∗∗ ∗∗ ∗∗ 1.0 0.5 0.0 0 Cntrl SS Rapa Cntrl SS Rapa (-) NAC (-) NAC (+) NAC (+) NAC (c) (d) 1.5 ### ∗∗ ∗∗∗ ∗∗∗ 60 ∗∗∗ 4 50 1.0 40 ### ### ∗∗∗ ∗∗∗ 30 ## 0.5 ns 0 0.0 0 LC3B-II Atg-3 LC3B-II B-actin B-actin B-actin (i) (ii) (iii) (e) Figure 5: ALLN-induced autophagy sensitizes R273H-P53 cells by increasing ROS levels. (a) R273H-P53 cells were exposed to ALLN, ALLN+SS, and ALLN+Rapa for 48 h. NAC (5 mM) was applied 1 h prior to treatment wherever mentioned. Fold change in ROS levels is represented through bars; untreated control was taken as arbitrary unit “1.” (b) MTT assay was performed to analyze cell viability following exposure of R273H-P53 cells to ALLN and other autophagy inducers; data is represented through bar diagram. (c) R273H-P53 cells were exposed to serum starved media or Rapa (500 nM) for 48 h. Fold change in ROS levels is represented. (d) MTT assay was performed to check cell viability following exposure of R273H-P53 cells to autophagy inducers with/without NAC.∗ indicates significant difference compared to untreated control, while # indicates significant dieff rence compared to ALLN-treated cells. (e)(i)/(ii)/(iii) R273H-P53 cells were treated with ALLN or ALLN plus SS for 48 h. NAC was given 1 h prior to treatment wherever mentioned. Immunoblotting was thereaer ft performed for LC3B-II and Atg-3.∗ indicates significant difference compared to untreated control, while # indicates significant dieff rence compared to ALLN-treated cells. Fold Change in LC3B-II expression Relative change in Relative change in DCFDA fluorescence DCFDA fluorescence Cntrl ALLN ALLN+ NAC Fold Change in Atg-3 expression Cntrl % Viability % Viability ALLN ALLN+ NAC Fold Change in LC3B-II expression Cntrl ALLN ALLN+ SS ALLN+SS+ NAC 12 Journal of Oncology ### ### ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ 80 ∗∗∗ ∗∗∗ ## 60 ∗∗ ∗∗∗ 0 0 Cntrl SS ALLN ALLN+SS Ph ERK (-) U0126 (+) U0126 B-actin Cntrl CQ ALLN ALLN+CQ (a) (b) ∗∗∗ ∗∗∗ ∗∗∗ ### ∗∗∗ ### ∗∗ Ph ERK Ph ERK B-actin B-actin Cntrl ALLN ALLN+ ALLN+SS+ Cntrl ALLN ALLN+NAC SS NAC (i) (ii) (c) ### ### 2.5 ∗∗∗ ∗∗∗ 2.0 ∗∗∗ ∗∗∗ 1.5 ∗∗∗ ∗∗∗ 1.0 0.5 0.0 Cntrl SS ALLN ALLN+SS (-) U0126 (+) U0126 (d) Figure 6: Sensitization of R273H-P53 cells is mediated by ERK signaling and ROS. (a) Immunoblot analysis representing ph-ERK expression upon ALLN and/or CQ treatment in R273H-P53 cells. (b) MTT assay was performed to analyze ALLN and/or serum starvation induced cell death in presence or absence of ERK inhibitor, U0126, given 2 h prior to treatment. Percentage viability is represented in the form of a bar diagram. (c) Immunoblot analysis was performed for ph-ERK expression upon ALLN and/or serum starvation in R273H-P53 cells. NAC was given 1 h prior to treatment wherever mentioned. (d) DCFDA u fl orimetric assay measuring intracellular levels of ROS aer ft treatment of R273H-P53 cells with or without U0126, given 2 h prior to ALLN and/or serum starvation for 48 h.∗ indicates significant difference compared to untreated control, while # indicates significant dieff rence compared to ALLN-treated cells. Fold Change in Fold Change in Ph ERK expression Ph-ERK expression Relative change in DCFDA fluorescence % Viability Fold Change in Ph ERK expression Journal of Oncology 13 ∗∗∗ ∗∗∗ ### ∗∗∗ ∗∗ 0 0 Cntrl PFT- ALLN ALLN+ PFT- P53 B-actin Cntrl PFT- ALLN ALLN+ PFT- (a) (b) ### 2.0 ∗∗ ### 1.5 ∗∗∗ ∗∗∗ ∗∗∗ 1.0 0.5 0 0.0 Atg-5 P62 B-actin B-actin Cntrl PFT- ALLN ALLN+ Cntrl PFT- ALLN ALLN+ PFT- PFT- (i) (ii) (c) ∗∗∗ ∗∗ Cntrl PFT- ALLN ALLN+ PFT- (d) Figure 7: P53 regulates induction of autophagy in R273H-P53 cells. (a) Immunoblot analysis depicting P53 expression upon ALLN treatment with or without 30 min of prior PFT-𝛼 exposure. (b) MTT assay was performed after ALLN and/or PFT- 𝛼 treatment for 48 h and percentage viability represented in the form of a bar diagram. (c) R273H-P53 cells were exposed to ALLN and/or PFT-𝛼 for 48 h and autophagy was checked through immunoblot analysis of Atg-5 (i) and P62 (ii). (d) MDC uo fl rescence assay was conducted aer ft R273H-P53 cells were exposed to ALLN or PFT-𝛼 or in combination for 48 h.∗ indicates significant difference compared to untreated control, while # indicates significant dier ff ence compared to ALLN-treated cells. “@” indicates significant dier ff ence compared to PFT- 𝛼 -treated cells. Fold Change in Fold Change in Atg-5 expression P53 expression Fold Change in MDC fluorescence Fold Change in % Viability P62 expression 14 Journal of Oncology 2.0 $$$ ∗∗∗ ∗∗∗ 100 $$$ 1.5 ∗∗∗ ### $$$ $$$ ∗∗∗ 1.0 0.5 0.0 Cntrl PFT- ALLN ALLN+ Cntrl PFT- ALLN ALLN+ PFT- PFT- (-) NAC (-) NAC (+) NAC (+) NAC (a) (b) 2.0 ### ∗∗∗ ∗∗∗ 1.5 ### ∗∗∗ ∗∗∗ 1.0 0.5 0.0 Cntrl PFT- ALLN ALLN+ Cntrl PFT- ALLN ALLN+ PFT- PFT- (-) U0126 (-) U0126 (+) U0126 (+) U0126 (c) (d) ∗∗∗ Ph ERK B-actin Cntrl PFT- ALLN ALLN+ PFT- (e) Figure 8: R273H-P53 cells show ROS and ERK accumulation aer ft PFT-𝛼 treatment. ((a) and (b)) R273H-P53 cells were exposed to ALLN and/or PFT-𝛼 (30 min prior to ALLN treatment) with and without NAC (1h prior to ALLN treatment) for 48 h and then cell viability and ROS levels were measured through (a) MTT assay and (b) DCFDA u fl orimetric assay. ((c) and (d)) Cell viability and ROS levels were analyzed with/without 2 h prior treatment of U0126 and the result is shown in the form of a bar diagram. (e) Immunoblot analysis showing ph-ERK expression on ALLN and/or PFT-𝛼 treatment in R273H-P53 cells.∗ indicates significant difference compared to untreated control and # indicates significant dier ff ence compared to ALLN-treated cells, while $$$ indicates the statistical dier ff ence compared to minus(-) NAC. % Viability % Viability Fold Change in Ph ERK expression Relative change in Relative change in DCFDA fluorescence DCFDA fluorescence Journal of Oncology 15 autophagy for clearance of proteins, which may provide Autophagy ALLN survival advantage to cells. Given the intense interest in targeting proteasomal degradation and the promise they hold for a multitude of cancer types, it is therefore important to precisely understand the consequences of autophagy GOF-p53 ERK ROS induction after inhibition of proteasomal degradation as a cancer therapeutic strategy. In this study, we have demonstrated that cells transfected with GOF mutant p53 show resistance to not only conventional drugs like CDDP or 5-FU but also to ALLN when compared to wt-P53. There was Cell Death induction of autophagy upon ALLN treatment as well. Since ALLN induces autophagy and the latter may be a tumor survival response, we initially hypothesized that blocking Figure 9: Schematic representation of the probable mechanism autophagy with CQ, along with ALLN, may initiate cell involved in proteasomal inhibitor, ALLN induced cell death in R273H-P53 cells. death or apoptosis. However, despite the proven benefits of lysomotropic agents in cancer clinics in conjunction to autophagy inducers or drugs, in our study, CQ rather than enhancing ALLN-induced cytotoxicity reduced its sensitivity PFT-𝛼 :Pitfihrin-alpha in GOF-R273H-P53 cells.Hence,from these studies, we CQ: Chloroquine concluded that, in this context, autophagy does not serve as ROS: Reactive oxygen species a mechanism that prolongs cell survival. MDC: Monodansylcadaverine It is increasingly getting recognized that the well-con- LC3B-II: Microtubule-associated protein light chain served autophagic machinery may be essential for cell death, 3-II at least in certain settings. However, it is controversially SS: Serum starvation discussed in literature whether cells truly die “by autophagy” Rapa: Rapamycin. or in dying cells autophagy is just a bystander or programmed mechanism facilitating apoptosis [50]. Few studies in the past have indeed provided evidence that cells can possess a Data Availability novel death mechanism that may depend on autophagy [51– The data used to support the findings of this study are 53]. However, the nature of stimulus leading to autophagy- available from the corresponding author upon request. dependent cell death has remained poorly defined till date. In this study, we showed that the R273H harboring P53 cells can be better sensitized to proteasomal inhibition by ALLN, Disclosure by enhancement of autophagy rather than its inhibition. Though autophagy is predominantly thought to play an An initial part of this work was presented as poster in house important protective role in sustaining homeostasis of cancer conference, BITS International Conference on Life Science cells supporting their proliferation, we provide evidences of Research & Its Interface with Engineering and Allied Sciences autophagy as a death promoter in the resistant lung cancer (LSRIEAS 2018). cells. This death was characterized by an enhanced ROS and ERK signaling. We further prove that inhibition of GOF mutant p53 can enhance cell death in the lung cancer Conflicts of Interest cells. Figure 9 schematically represents the summary of our The authors declare that there are no conflicts of interest. findings. Currently, there are very few literatures available which identify molecular mechanisms where autophagy is a death enhancer. This signifies the importance of our study. Authors’ Contributions However, more broadly, it still remains to be investigated whether the cell death observed was autosis (that represents Heena Saini, Ifrah Hakeem, and Sudeshna Mukherjee per- a subtype of autophagic cell death) or whether a bona fide formed and analyzed the experiments. Rajdeep Chowdhury, cell death by autophagy also requires the core apoptotic Shibasish Chowdhury, and Heena Saini designed the study machinery. Together, our results reveal novel mechanism plan and wrote the manuscript. through which mutant p53 harboring lung cancer cells can be sensitized by exploiting the crosstalk between the cellular homeostatic protein degradation machineries. Acknowledgments The authors thank DBT (BT/PR/8799/MED/30/1067/2013) Abbreviations for providing funding support to Rajdeep Chowdhury for conducting the experiments. 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