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Linderalactone Suppresses Pancreatic Cancer Development In Vitro and In Vivo via Negatively Regulating PI3K/AKT Signaling Pathway

Linderalactone Suppresses Pancreatic Cancer Development In Vitro and In Vivo via Negatively... Hindawi Journal of Oncology Volume 2022, Article ID 8675096, 12 pages https://doi.org/10.1155/2022/8675096 Research Article Linderalactone Suppresses Pancreatic Cancer Development In Vitro and In Vivo via Negatively Regulating PI3K/AKT Signaling Pathway 1,2,3 1 1 1 1 Dongchao Xu , Mengyao Tian, Wangyang Chen, Ying Bian, Xiaofeng Xia, 1,2,3 1,2,3 1,2,3 1,2,3 Qiang Liu, Liyun Zheng, Xiaofeng Zhang , and Hongzhang Shen Department of Gastroenterology, A liated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hangzhou 310000, China Hangzhou Institute of Digestive Diseases, Hangzhou 310000, China Key Laboratory of Integrated Traditional Chinese and Western Medicine for Biliary and Pancreatic Diseases of Zhejiang Province, Hangzhou 310000, China Correspondence should be addressed to Xiaofeng Zhang; zxf837@tom.com and Hongzhang Shen; sakshen@126.com Received 5 January 2022; Revised 2 March 2022; Accepted 30 June 2022; Published 4 August 2022 Academic Editor: Ashok Pandurangan Copyright © 2022 Dongchao Xu et al. …is 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. Linderalactone is one of the main extracts of Linderae Radix, which is widely used in traditional Chinese medicine. …ere have been few studies on the antitumor e‹ect of linderalactone in the past. In this study, we explored the anti-pancreatic cancer activity of linderalactone in vitro and in vivo. …e results showed that linderalactone inhibited the proliferation of pancreatic cancer cells in a time- and dose-dependent manner. Cell migration and invasion were signi‘cantly inhibited by linderalactone. …e cell cycle was arrested in the G2/M phase, and the expression levels of cell cycle-associated proteins changed signi‘cantly with linder- alactone treatment. In addition, linderalactone induced cell apoptosis and altered the expression of apoptotic markers, such as caspase 3 and PARP1. Mechanistically, linderalactone suppressed the PI3K/AKT signaling pathway by downregulating the phosphorylation of PI3K and AKT. …e xenograft study results were consistent with the in vitro results, and there was no obvious chemical toxicity. …us, our research demonstrated that linderalactone exhibits antitumor activity against pancreatic cancer and may be developed as a potential anti-pancreatic cancer agent in the future. However, adverse side e‹ects and drug resistance signi‘- 1. Introduction cantly limit their clinical application [6]. …erefore, there is …e incidence of pancreatic cancer, a highly fatal solid tu- an urgent need to develop novel antitumor agents. mor, has increased in recent years, and the mortality rate is Natural plant products are now receiving much attention extremely high: only 10% of patients survive for more than 5 for the treatment of tumors. For example, the combination of paclitaxel, a secondary metabolite puri‘ed from yew, with years [1, 2]. Due to the lack of e‹ective screening methods, more than 90% of patients have metastatic disease upon gemcitabine has become a typical treatment for pancreatic diagnosis, which is the main reason for the high mortality of cancer [7, 8]. Here, we explored a compound extracted from pancreatic cancer [3]. For most patients with distant me- Linderae Radix that has the potential to treat pancreatic tastases, chemotherapy is the only treatment strategy, but the cancer. Linderae Radix has been used for hundreds of years currently used chemotherapeutics have a very limited ability as a traditional Chinese herbal plant with tremendous to prolong patient survival [4]. Currently, chemotherapy medicinal properties. It is widely used in the treatment of drugs, such as gemcitabine, erlotinib, nab-paclitaxel, and 5- gastrointestinal diseases, mainly for abdominal distension Ÿuorouracil, are widely used to treat pancreatic cancer [5]. and pain, nausea, and vomiting. Although unprecedented 2 Journal of Oncology accomplishments have been achieved with modern medi- 2.4. Colony Formation Assay. Pancreatic cancer cells were cine, Linderae Radix is used in clinical practice because it seeded into six-well plates at a density of 1 × 10 cells/well. exerts analgesic and anti-inflammatory effects with an ex- 1ree days later, linderalactone was added to the medium at cellent curative effect and few side effects [9]. varying concentrations and the cells were cultured for 24 h. Linderalactone is a hydroxylated biphenyl compound After 7 days, the cells were washed with PBS (Solarbio, China) isolated from Linderae Radix that has anti-inflammatory and fixed with 4% paraformaldehyde (PFA, Beyotime, China) properties [10, 11]. Previous studies have found that lin- for 30 min. Subsequently, 1% crystal violet solution (Beyotime, deralactone has value in the treatment of lung cancer [12]. In China) was used to stain the colonies. After 10 min, the plates light of the extensive role of Linderae Radix in digestive were washed with pure water for further counting and analysis. system diseases and its antitumor effect in lung cancer, we have a keen interest in linderalactone and thus explored 2.5. Wound-Healing Assay. Pancreatic cancer cells (1 × 10 ) whether it inhibits the activity of pancreatic cancer. In this were seeded into six-well plates and cultured to a confluence study, we showed the cytotoxicity of linderalactone in greater than 90%. 1e medium was replaced with a serum- pancreatic cancer in vitro and its inhibitory effect on tumor free medium for 24 h to eliminate the effect of cell prolif- progression in vivo. 1is study revealed for the first time that eration on the experimental results. 1e confluent mono- linderalactone plays a role in pancreatic cancer by inhibiting layer of cells was scraped with a 10 μL sterile pipette tip, and the PI3K/AKT signaling pathway. the cells were then incubated with a serum-free medium containing varying concentrations of linderalactone. 1e 2. Materials and Methods scratch width was recorded using phase-contrast micros- copy at 0 h and 24 h. 1e migration distance was calculated 2.1. Cell Culture and Reagents. Four pancreatic cancer cell using ImageJ software (National Institutes of Health, USA). lines, ASPC-1, BXPC-3, CFPAC-1, and SW-1990 were obtained from American Type Culture Collection (ATCC, 2.6. Transwell Assay. Transwell assays were used to evaluate USA). All cell lines were cultured in the RPMI-1640 medium migration and invasion. For migration, pancreatic cancer cells (Sigma, USA) supplemented with 10% fetal bovine serum (5 ×10 ) were mixed with varying concentrations of linder- (Gibco, USA) and a 1% penicillin and streptomycin mixture alactone in a serum-free medium. Transwell cell culture (Solarbio, China). 1e cells were placed in a cell incubator ° chambers (Falcon, USA) with 8.0 μm Transparent PET (1ermo Scientific, USA) with 37 C and 5% CO . 1e Lu- Membranes were used. Two hundred microliters of a cell- ciferase Mycoplasma Detection Kit (TransGen Biotech, medium mixture was placed in the upper chamber, and China) was used to confirm that the cells were free of 600 μL of medium containing 20% FBS was placed in the mycoplasma contamination. Linderalactone (Chengdu lower chamber as a cell migration chemoattractant. After 24 h Herbpurify, China) was dissolved in dimethyl sulfoxide of incubation in the incubator, the cells in the upper chamber (DMSO, Sigma, USA). were removed with cotton swabs and the cells in the lower chamber were fixed in 4% PFA for 30 min at room tem- perature and stained with 1% crystal violet solution for 20 min 2.2. Cell Viability. 1e Cell Counting Kit-8 (CCK-8, Bimake, at room temperature. After the cells were dried, the number of USA) was used to detect cell viability. In brief, pancreatic stained cells was counted. For the invasion assays, all the steps cancer cells were plated at 3000 cells per well in 96-well were the same as those for the migration assays, except that plates and treated with varying concentrations of linder- the 8.0 μm transparent PET membranes were coated with a alactone (0, 30, 40, 50, 60, 70, 80, 90, and 100 μM) for 0.5 mg/mL basement membrane matrix (Corning, USA). different periods of time (0, 24, and 48 h). Ten microliters of CCK-8 solution were added to each well, and the plates (Corning, USA) were incubated at 37 C for 2 h. 1e per- 2.7. Cell Cycle Analysis. A cell cycle analysis kit was pur- centage of surviving cells was calculated by measuring the chased from Beyotime (China). In brief, pancreatic cancer absorbance at an optical density of 450 nm using a micro- 4 cells (9 ×10 per well) were seeded in 6-well plates. After plate reader (1ermo Fisher, USA). 1e IC50 value was overnight culture, the cells were treated with varying con- determined based on a linear regression curve. centrations of linderalactone. After 24 h of treatment, the cells were collected and fixed with prechilled 70% alcohol for 12 h. 1en, the cells were washed with PBS; resuspended in a PBS 2.3. EdU Assay. 1e EdU Cell Proliferation Kit with Alexa mixture containing 0.05% Triton X-100, 0.1 mg/mL DNase- Fluor 488 was purchased from Beyotime (China). All steps free RNase A, and 25 μg/mL PI; and incubated for 30 min at were conducted in accordance with the manufacturer’s 37 C in the dark. A flow cytometer (BD Biosciences, USA) was instructions. In brief, pancreatic cancer cells were treated used to detect light scatter and red fluorescence at an exci- with varying concentrations of linderalactone, and then, tation wavelength of 488 nm. EdU was added and incubated with the cells for one-tenth of the cell doubling time (BXPC-3, 5 h; CFPAC-1, 3 h). 1e cells were incubated with Hoechst stain for 20 min to vi- 2.8. Cell Apoptosis Analysis. 1e FITC Annexin V/Dead Cell sualize the nucleus and then examined under a fluorescence Apoptosis Kit was purchased from Invitrogen (USA). microscope (Nikon, Japan). Pancreatic cancer cells (9 ×10 per well) were treated with Journal of Oncology 3 varying concentrations of linderalactone for 24 h. 1e cells of treatment with linderalactone, all tumor cell lines un- were then washed with prechilled PBS and centrifuged, and derwent quantitative and morphological changes. As shown the supernatant was removed. 1e cells were resuspended in in Figure 1(a), the cells lost their unique morphology, annexin binding buffer, and 5 μL of FITC annexin V and appeared round, and showed worse adherence to the well. 1 μL of 100 μg/mL PI working solution were added to each Figure 2 shows the chemical structure of linderalactone. 100 μL aliquot of cell suspension. After incubating the cells at Given the inhibitory effect of linderalactone on pancreatic room temperature for 15 min, annexin binding buffer was cancer cells, we performed CCK-8 assays to detect the ac- added, and the stained cells were analyzed by flow cytometry tivity of cells treated with varying concentrations of lin- with fluorescence emission measured at 530 nm. deralactone for different periods of time. 1e results are shown in Figure 1(c). Linderalactone inhibited pancreatic cancer activity in a time- and dose-dependent manner. 2.9. Western Blotting. Western blotting was performed as previously described [13]. 1e cells were collected and lysed in RIPA lysis buffer (Fude, China) with protease inhibitors 3.2. Linderalactone Inhibits the Proliferation of Pancreatic (1ermo Scientific, USA). Protein (50 μg) was separated by Cancer Cells. Replicative immortality is one of the hallmarks SDS-PAGE (Sangon, China) and transferred to PVDF of cancer [14]. Cell proliferation is accompanied by DNA membranes (Millipore, USA). After blocking with 3% BSA replication. We performed EdU assays to determine the (Solarbio, China), the membranes were incubated with pri- effect of linderalactone on cell proliferation. 1e results mary antibodies overnight. 1e next day, the membranes showed that (Figures 2(a)–2(d), Supplementary Figures 1(a)–1(d)) the population of EdU-positive cells were washed with TBST (Sangon, China) and incubated with the secondary antibody (Promega, USA) for 90 min at room decreased as the concentration of linderalactone increased temperature. Enhanced chemiluminescence detection (Fude, compared with the control. Colony formation experiments China) was used to detect the target protein. Primary anti- were conducted to evaluate the long-term effects of lin- bodies against the following proteins were used: p-PI3K, deralactone on pancreatic cancer cells. 1e number and size PI3K, p-AKT, AKT, PCNA, Ki-67, caspase 3, PARP1, Bcl-2, of colonies were significantly reduced by treatment with Bax, cyclin A2, cyclin B1, cyclin D1, and cyclin E1. All an- linderalactone (Figures 2(e) and 2(f)). 1ese results indi- tibodies were purchased from Abcam (USA). cated that linderalactone can inhibit the proliferative po- tential of pancreatic cancer. 2.10. Xenograft Experiments. Four- to six-week-old BALB/c nude mice were purchased from Shanghai SLAC Laboratory 3.3. Linderalactone Inhibits the Migration and Invasion of Animal Company (China). 1e protocol was approved Pancreatic Cancer Cells. 1e systemic metastasis of tumors according to the ethical standards of the Institutional Animal often underlies the inability to cure the disease [15]. We used Care and Use Committee of Zhejiang Chinese Medical Uni- wound-healing and Transwell experiments to evaluate the migration ability of cells. In addition, Transwell assays with versity and complied with the Regulations for the Adminis- tration of Affairs Concerning Experimental Animals (approved Matrigel were used to evaluate cell invasion ability. To re- by the State Council of China, No. SYXK (Zhejiang) 2018- duce the influence of cell proliferation on the migration 0012). 1e pancreatic cancer cell line BXPC-3 was injected into results in the scratch assays, we first starved the cells with a the right abdomen of each nude mouse. After two weeks, 18 serum-free medium for 24 h; thus, the results only reflected nude mice with tumors of the same size were selected into three the effects of linderalactone. As shown in Figures 3(a) and groups. Normal saline or low-dose (25 mg/kg) or high-dose 3(b), the migration distance of cells treated with linder- (50 mg/kg) linderalactone was injected every three days, and alactone was significantly reduced and the distance migrated tumor weight and volume were measured. After three weeks, by cells in the 60 μM group was shorter than that by those in the 30 μM group. Transwell experiments were used to detect the mice were sacrificed, and the tumors were removed. 1e tumor volume was calculated as 0.5 × lengths × width . the migration ability of cells in 3D space, and the results (Figures 3(c) and 3(d)) were consistent with the scratch assay results. As the concentration of linderalactone increased, the 2.11. Statistical Analyses. All experiments were repeated at migration ability decreased. Cell invasiveness was evaluated least three times to ensure the reliability of the results. Data using Transwell assays with Matrigel (Figures 3(e) and 3(f)). were analyzed using SPSS 12.0, and the results are expressed In general, linderalactone not only inhibited proliferation as the mean± standard error. An unpaired Student’s t-test but also showed effective antimetastatic activity. All the was used to determine statistical significance, which was results shown in Figure 3 were from experiments performed indicated by p< 0.05. with BXPC-3 cells, and these experiments were repeated in CFPAC-1 cells (Supplementary Figure 2). 3. Results 3.1. Linderalactone Inhibits the Viability of Pancreatic Cancer 3.4. Linderalactone Arrests the Cell Cycle and Induces Cells. To determine the effect of linderalactone on pan- Apoptosis. Our previous research found that linderalactone inhibits the proliferation of pancreatic cancer cells, so we creatic cancer, we treated four pancreatic cancer cell lines with increasing concentrations of linderalactone. After 72 h used flow cytometry to detect potential changes in the cell 4 Journal of Oncology 0 M 40 M 80 M (a) (b) ASPC-1 BXPC-3 CFPAC-1 SW 1990 150 150 150 100 100 100 100 50 50 50 50 0 0 0 0 0 20406080 100 0 20 40 60 80 100 020 40 60 80 100 020 40 60 80 100 Magnolol (M) Magnolol (M) Magnolol (M) Magnolol (M) 24 h 24 h 24 h 24 h 48 h 48 h 48 h 48 h 72 h 72 h 72 h 72 h (c) Figure 1: Linderalactone inhibits the viability of pancreatic cancer cells. (a) Morphological changes of pancreatic cancer cells exposed to LL. (b) 1e chemical structure of LL. (c) CCK8 assay detected the viability of pancreatic cancer cells under varying concentrations and time of LL treatment. Scale bar � 200 μm. cycle. 1e results are shown in Figures 4(a) and 4(b). After Bcl-2, PARP1, and caspase 3 protein levels and the increase treatment with linderalactone, the number of cells in the G2/ in Bax and cleaved PARP1 levels indicated the occurrence of M phase increased significantly compared with that in the apoptosis. control group. Western blotting was used to detect the expression of cyclin A2, cyclin B1, cyclin D1, and cyclin E1. 1e protein levels of cyclin B1, which marks the G2/M phase, 3.5. Linderalactone Exhibits Antitumor Activity by Inhibiting the PI3K/AKT Signaling Pathway. We next tried to elucidate increased significantly, while those of cyclin A2, cyclin D1, how linderalactone inhibits pancreatic cancer activity. It is and cyclin E1, which marks the G1 or S phase, decreased, confirming the cell cycle results obtained by flow cytometry well known that pancreatic cancer is a malignant tumor driven by oncogenes. Numerous studies have found that (Figure 4(e)). Cell cycle arrest is often accompanied by apoptosis, so we subsequently detected apoptosis by flow genes such as K-ras, Smad, and Stat are active in pancreatic cancer. We evaluated the changes in a number of signaling cytometry, and the results were consistent with our ex- pectations (Figures 4(c) and 4(d)): linderalactone induced pathway components, including PI3K/AKT, before and after linderalactone treatment. Significant changes in the PI3K/ apoptosis. 1e expression levels of Bcl-2, Bax, PARP1, and caspase 3 were determined (Figure 4(f)); the decrease in AKT signaling pathway were observed after treatment viable cell (%) SW-1990 CFPAC-1 BXPC-3 ASPC-1 viable cell (%) viable cell (%) viable cell (%) Journal of Oncology 5 DAPI EdU Merge DAPI EdU Merge (a) (b) BXPC-3 CFPAC-1 60 40 ** ** ** 0 0 LL (M) 030 60 LL (M) 0 30 60 (c) (d) BXPC-3 0 M 30 M 60 M ** ** LL (M) 0 30 60 CFPAC-1 ** ** LL (M) 030 60 (e) (f) Figure 2: Linderalactone inhibits the proliferation of pancreatic cancer cells. (a, b) EdU staining assay detected the proliferation of BXPC-3 and CFPAC-1 cell lines under varying concentrations of LL treatment after 24 hours. (c, d) Quantification of EdU stain: the percentage of EdU positive cells to the total number of cells. (e) Clone formation assay to detect the effect of LL on BXPC-3 and CFPAC-1 cell lines. ∗ ∗∗ (f) Quantification of clone formation. Scale bar � 200 μm. p< 0.05; p< 0.01. (Figures 5(a) and 5(b)). Previous studies have found that that linderalactone functions in this way [16, 17]. To confirm inhibiting the PI3K/AKT signaling pathway can effectively our conjecture, we used YS-49, a PI3K/AKT activator [18]. inhibit the function of pancreatic cancer; thus, we speculated As shown in Figures 5(c) and 5(d), linderalactone alone CFPAC-1 BXPC-3 0 M 60 M 30 M EdU positive cell rate (%) 60 M 30 M 0 M Number of Clone Formation Number of Clone Formation EdU positive cell rate (%) 6 Journal of Oncology BXPC-3 0 M 30 M 60 M ** ** LL (M) 0 30 60 (a) (b) BXPC-3 0 M 30 M 60 M ** ** LL (M) 0 30 60 (c) (d) BXPC-3 0 M 30 M 60 M ** ** LL (M) 0 30 60 (e) (f) Figure 3: Linderalactone inhibits the migration and invasion of pancreatic cancer cells. (a) Wound-healing assay detects the migration ability of BXPC-3 under varying concentrations of LL after 24 hours. (b) Quantification of wound-healing assay: the area of cell migration in 24 hours accounts for the percentage of the wound area in 0 hours. (c) 1e transwell assay detected the migration ability of cells under varying concentrations of LL treatment after 24 hours. (d) Quantification of transwell assay: percentage of the compound treatment group in the control group. (e) 1e transwell assay with Matrigel to detect cell invasion ability under varying concentrations of LL treatment after 24 hours. (f) Quantification of transwell assay: percentage of the compound treatment group in the control group. Scale bar � 200 μm. ∗ ∗∗ p< 0.05; p< 0.01. decreased the levels of p-PI3K and p-AKT, while YS-49 3.6. Linderalactone Inhibits Tumor Progression In Vivo. alone did not affect the phosphorylation of these proteins. To explore the effect of linderalactone on tumor growth in When the two compounds were combined, YS-49 reversed vivo, a nude mouse pancreatic tumor model was con- the inhibitory effect of linderalactone on p-PI3K and p-AKT. structed. Physiological saline or low-dose (25 mg/kg) lin- None of the evaluated treatments caused changes in total deralactone or high-dose (50 mg/kg) linderalactone was PI3K and AKT levels. In the colony formation assays, injected into the abdominal cavity of the mice once every cotreatment with YS-49 effectively reversed the antitumor three days, and tumor volume and weight were recorded. 1e experimental results revealed that the tumors of mice effects of linderalactone. 1erefore, we confirmed that lin- deralactone inhibits the tumor activity of pancreatic cancer treated with linderalactone were generally smaller than those by inhibiting PI3K/AKT. of mice in the control group and the therapeutic effect of 24 h 24 h 24 h 0 h Cell Invasion Rate (%) Cell Migration Rate (%) Wound healing percentage Journal of Oncology 7 BXPC-3 LL 0 M LL 30 M LL 60 M 600 600 500 500 400 400 300 300 200 200 100 100 0 0 0 020 40 60 80 100 120 020 40 60 80 100 120 020 40 60 80 100 120 LL (M) 0 30 60 Channels (PI-A) Channels (PI-A) Channels (PI-A) CFPAC-1 200 50 0 0 0 030 60 90 120 150 030 60 90 120 150 030 60 90 120 150 Channels (PI-A) Channels (PI-A) Channels (PI-A) LL (M) 0 30 60 Debris Dip G2 Debris Dip G2 Debris Dip G2 G2/M Aggregates Dip S Aggregates Dip S Aggregates Dip S Dip G1 Dip G1 Dip G1 G0/G1 (a) (b) BXPC-3 LL 0 M LL 30 M LL 60 M 60 ** 5 5 10 ** 10 10 4 4 10 10 10 3 3 20 10 10 10 2 2 2 10 10 10 0 0 0 LL (M) 0 30 60 2 3 4 5 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 CFPAC-1 5 5 5 10 ** 10 10 ** 4 4 10 10 3 3 3 10 10 2 2 10 10 0 0 2 3 4 5 2 3 4 5 2 3 4 5 LL (M) 0 30 60 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 (c) (d) BXPC-3 CFPAC-1 BXPC-3 CFPAC-1 LL (M) 0 30 60 0 30 60 LL (M) 0 30 60 0 30 60 PARP-1 Cyclin D1 cl-PARP-1 Cyclin B1 Casepase 3 ACTB ACTB (e) (f) Figure 4: Linderalactone blocks the cell cycle and induces apoptosis. (a) Flow cytometry using PI staining to detect the cycle distribution of BXPC-3 and CFPAC-1 cell lines treated with varying concentrations of LL for 24 hours. (b) 1e percentage of cell cycle distribution for BXPC-3 and CFPAC-1. (c) Annexin V/PI staining to detect the apoptotic death of BXPC-3 and CFPAC-1. (d) 1e percentage of cell ∗ ∗∗ apoptosis for BXPC-3 and CFPAC-1. (e, f) Western blot analysis of key cell cycle and cell apoptosis markers. p< 0.05; p< 0.01. CFPAC-1 BXPC-3 Number Number CFPAC-1 BXPC-3 Number Number Number Number Cell Cycle Phase (%) Cell Cycle Phase (%) Cell Apoptosis Rate (%) Cell Apoptosis Rate (%) 8 Journal of Oncology BXPC-3 BXPC-3 CFPAC-1 CFPAC-1 LL (M) 0 30 60 LL (M) 0 30 60 LL (M) 0 30 60 LL (M) 0 30 60 p-PI3K t-PI3K p-PI3K t-PI3K p-AKT t-AKT p-AKT t-AKT ACTB ACTB ACTB ACTB (a) (b) BXPC-3 BXPC-3 LL (60 M) + + LL (60 M) + + − − − − + + + + YS-49 (30 M) −− YS-49 (30 M) −− p-PI3K t-PI3K p-AKT t-AKT ACTB ACTB (c) CFPAC-1 CFPAC-1 + + + + LL (60 M) − − LL (60 M) − − YS-49 (30 M) −− + + YS-49 (30 M) −− + + p-PI3K t-PI3K p-AKT t-AKT ACTB ACTB (d) Control LL YS-49 LL+YS-49 (e) Figure 5: Continued. CFPAC-1 BXPC-3 Journal of Oncology 9 BXPC-3 ns ns ** CFPAC-1 ns ns ** (f) Figure 5: Linderalactone exhibits antitumor activity by inhibiting the PI3K/AKT signaling pathway. (a, b) Western blot analysis of phosphorylated PI3K and phosphorylated AKT, total PI3K, and total AKT expression in BXPC-3 and CFPAC-1 cells after treatment with varying concentrations of LL. (c, d) After LL or YS-49 treatment, the expression of phosphorylated PI3K and phosphorylated AKT and total PI3K and total AKT in BXPC-3 and CFPAC-1 cells. (e) After LL or YS-49 treatment, the clone formation assay detects the proliferation ∗ ∗∗ ability of BXPC-3 and CFPAC-1. (f) Quantification of clone formation. p< 0.05; p< 0.01. Control 25 mg/kg 50 mg/kg (a) Control 25 mg/kg 50 mg/kg (b) Figure 6: Continued. Number of Clone Formation Number of Clone Formation Control Control LL LL YS-49 YS-49 LL+YS-49 LL+YS-49 10 Journal of Oncology 2000 24 1500 22 ns ns ** 500 18 0 16 0369 12 15 18 21 0 3 6 9 12151821 Days Days control control low(25 mg/kg) low (25 mg/kg) high(50 mg/kg) high (50 mg/kg) (c) Control 25 mg/kg 50 mg/kg (d) Figure 6: Linderalactone inhibits tumor progression in vivo. (a) Images of the control group and low-dose (LL 25 mg/kg) and high-dose (LL 50 mg/kg) tumor-bearing mice. (b) Quantitative volume of the tumor progression: the tumor volume was measured every three days for three weeks after the injection of LL. (c) Quantitative mice weight: the body weight of the mice was measured every three days for three ∗ ∗∗ weeks after the injection of LL. (d) Immunohistochemical detection of PCNA and Ki-67 expression level in tumor cells. p< 0.05; p< 0.01. high-dose linderalactone was better than that of low-dose increasingly elucidated, as studies have shown that they linderalactone (Figures 6(a) and 6(b)). In addition, trends in induce apoptosis, inhibit proliferation, and increase chemical sensitivity [19]. Previous studies have shown the body weight of mice treated with linderalactone were similar to those of mice in the control group (Figure 6(c)), that lutein can reduce the levels of K-ras and AKT in with no significant difference, indicating that linderalactone mouse colon cancer and induce cell cycle arrest [20]. does not exert significant toxicity. 1e immunohisto- Lycopene can be used as a preventive agent because it chemistry results (Figure 6(d)) showed that PCNA and Ki- decreases MMP-7 expression and inhibits the invasion of 67 expressions were lower in the experimental group than in HT-29 human colon cancer cells [21]. Procyanidin the control group. In general, the antitumor activity of therapy exerts antiproliferative and anti-invasive effects in linderalactone in vivo was consistent with that in vitro. pancreatic cancer cells and is an effective chemo- preventive agent for this disease [22]. Linderae Radix has been used in East Asian countries 4. Discussion such as China and Japan for centuries as a folk medicine without any obvious toxicity. Linderae Radix has anti-in- Although some progress has been made in prolonging patient survival, adverse reactions to chemotherapy drugs flammatory and antioxidant effects in the treatment of al- used to treat pancreatic cancer have seriously affected coholic liver injury and hyperlipidemia [23–26]. In this patients’ quality of life. Phytochemicals are generally study, we explored the antitumor effects of linderalactone, considered to have minor adverse effects and great po- an extract of Linderae Radix, on pancreatic cancer. We used tential in the treatment of tumors. Phytochemicals are a four pancreatic cancer cell lines and mouse tumor-bearing class of biological secondary metabolites that are abun- models for in vitro and in vivo studies. 1e antitumor activity dant in fruits, grains, and vegetables. In recent years, the of linderalactone was evaluated, and its mechanism of action role of phytochemicals as anticancer agents has been was investigated [27–30]. Ki-67 PCNA Tumer volume (mm ) Mouse Weight (g) Journal of Oncology 11 1e inhibition of proliferation and metastasis and the Conflicts of Interest induction of cell cycle arrest are effective means of 1e authors declare no conflicts of interest. treating tumors. Here, our study found that linder- alactone effectively inhibited the proliferation of pan- creatic cancer cells both in the short and long term. Authors’ Contributions Invasion and migration are hallmarks of cancer cell Dongchao Xu, Mengyao Tian, and Wangyang Chen con- metastasis, and our research showed that linderalactone tributed equally. All authors agreed to submit this effectively inhibited the migration and invasion of pan- manuscript. creatic cancer cells, thereby inhibiting metastasis. Flow cytometry analyses revealed that pancreatic cancer cells arrested the G2/M phase of the cell cycle in response to Acknowledgments linderalactone treatment and apoptosis occurred. Cell 1e work was supported by the Zhejiang Medical and Health cycle arrest in the G2/M phase stops tetraploid cells from Science and Technology Plan (Grant Nos. WKJ-ZJ-2136 and dividing. Numerous drugs that induce tumor cell arrest in 2018PY037), Hangzhou Medical and Health Science and the G2/M phase have been used for tumor treatment. 1e Technology Plan (Grant Nos. 2016ZD01, OO20190610, G2/M checkpoint is one of the key targets of tumor A20200737, and A20200174), Hangzhou Major Science and treatment. 1e cell cycle is regulated by cyclin B1. Our Technology Projects (Grant No. 202004A14), and the results showed that cyclin B1 expression decreased, Zhejiang Charity Plan Project (Grant No. LGF21H310004). confirming our flow cytometry results. In addition, our apoptosis analyses and western blot experiments proved that linderalactone induced the apoptosis of pancreatic Supplementary Materials cancer cells by activating the apoptosis pathway. It will Supplementary Figure 1: linderalactone inhibits the prolif- perhaps be more appropriate to further explore the effects eration of pancreatic cancer cells. Supplementary Figure 2: of linderalactone on pancreatic cancer metastasis in an linderalactone inhibits the migration and invasion of pan- orthotopic mouse model. creatic cancer cells. (Supplementary Materials) Pancreatic cancer is known to be driven by gene mutations. Most pancreatic cancers have TP53 gene mutations leading to the loss of TP53. 1is leads to the upregulation of the PI3K/AKT References signaling pathway. 1e PI3K/Akt pathway is one of the most [1] R. L. Siegel, K. D. Miller, H. E. Fuchs, and A. 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Linderalactone Suppresses Pancreatic Cancer Development In Vitro and In Vivo via Negatively Regulating PI3K/AKT Signaling Pathway

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Hindawi Journal of Oncology Volume 2022, Article ID 8675096, 12 pages https://doi.org/10.1155/2022/8675096 Research Article Linderalactone Suppresses Pancreatic Cancer Development In Vitro and In Vivo via Negatively Regulating PI3K/AKT Signaling Pathway 1,2,3 1 1 1 1 Dongchao Xu , Mengyao Tian, Wangyang Chen, Ying Bian, Xiaofeng Xia, 1,2,3 1,2,3 1,2,3 1,2,3 Qiang Liu, Liyun Zheng, Xiaofeng Zhang , and Hongzhang Shen Department of Gastroenterology, A liated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hangzhou 310000, China Hangzhou Institute of Digestive Diseases, Hangzhou 310000, China Key Laboratory of Integrated Traditional Chinese and Western Medicine for Biliary and Pancreatic Diseases of Zhejiang Province, Hangzhou 310000, China Correspondence should be addressed to Xiaofeng Zhang; zxf837@tom.com and Hongzhang Shen; sakshen@126.com Received 5 January 2022; Revised 2 March 2022; Accepted 30 June 2022; Published 4 August 2022 Academic Editor: Ashok Pandurangan Copyright © 2022 Dongchao Xu et al. …is 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. Linderalactone is one of the main extracts of Linderae Radix, which is widely used in traditional Chinese medicine. …ere have been few studies on the antitumor e‹ect of linderalactone in the past. In this study, we explored the anti-pancreatic cancer activity of linderalactone in vitro and in vivo. …e results showed that linderalactone inhibited the proliferation of pancreatic cancer cells in a time- and dose-dependent manner. Cell migration and invasion were signi‘cantly inhibited by linderalactone. …e cell cycle was arrested in the G2/M phase, and the expression levels of cell cycle-associated proteins changed signi‘cantly with linder- alactone treatment. In addition, linderalactone induced cell apoptosis and altered the expression of apoptotic markers, such as caspase 3 and PARP1. Mechanistically, linderalactone suppressed the PI3K/AKT signaling pathway by downregulating the phosphorylation of PI3K and AKT. …e xenograft study results were consistent with the in vitro results, and there was no obvious chemical toxicity. …us, our research demonstrated that linderalactone exhibits antitumor activity against pancreatic cancer and may be developed as a potential anti-pancreatic cancer agent in the future. However, adverse side e‹ects and drug resistance signi‘- 1. Introduction cantly limit their clinical application [6]. …erefore, there is …e incidence of pancreatic cancer, a highly fatal solid tu- an urgent need to develop novel antitumor agents. mor, has increased in recent years, and the mortality rate is Natural plant products are now receiving much attention extremely high: only 10% of patients survive for more than 5 for the treatment of tumors. For example, the combination of paclitaxel, a secondary metabolite puri‘ed from yew, with years [1, 2]. Due to the lack of e‹ective screening methods, more than 90% of patients have metastatic disease upon gemcitabine has become a typical treatment for pancreatic diagnosis, which is the main reason for the high mortality of cancer [7, 8]. Here, we explored a compound extracted from pancreatic cancer [3]. For most patients with distant me- Linderae Radix that has the potential to treat pancreatic tastases, chemotherapy is the only treatment strategy, but the cancer. Linderae Radix has been used for hundreds of years currently used chemotherapeutics have a very limited ability as a traditional Chinese herbal plant with tremendous to prolong patient survival [4]. Currently, chemotherapy medicinal properties. It is widely used in the treatment of drugs, such as gemcitabine, erlotinib, nab-paclitaxel, and 5- gastrointestinal diseases, mainly for abdominal distension Ÿuorouracil, are widely used to treat pancreatic cancer [5]. and pain, nausea, and vomiting. Although unprecedented 2 Journal of Oncology accomplishments have been achieved with modern medi- 2.4. Colony Formation Assay. Pancreatic cancer cells were cine, Linderae Radix is used in clinical practice because it seeded into six-well plates at a density of 1 × 10 cells/well. exerts analgesic and anti-inflammatory effects with an ex- 1ree days later, linderalactone was added to the medium at cellent curative effect and few side effects [9]. varying concentrations and the cells were cultured for 24 h. Linderalactone is a hydroxylated biphenyl compound After 7 days, the cells were washed with PBS (Solarbio, China) isolated from Linderae Radix that has anti-inflammatory and fixed with 4% paraformaldehyde (PFA, Beyotime, China) properties [10, 11]. Previous studies have found that lin- for 30 min. Subsequently, 1% crystal violet solution (Beyotime, deralactone has value in the treatment of lung cancer [12]. In China) was used to stain the colonies. After 10 min, the plates light of the extensive role of Linderae Radix in digestive were washed with pure water for further counting and analysis. system diseases and its antitumor effect in lung cancer, we have a keen interest in linderalactone and thus explored 2.5. Wound-Healing Assay. Pancreatic cancer cells (1 × 10 ) whether it inhibits the activity of pancreatic cancer. In this were seeded into six-well plates and cultured to a confluence study, we showed the cytotoxicity of linderalactone in greater than 90%. 1e medium was replaced with a serum- pancreatic cancer in vitro and its inhibitory effect on tumor free medium for 24 h to eliminate the effect of cell prolif- progression in vivo. 1is study revealed for the first time that eration on the experimental results. 1e confluent mono- linderalactone plays a role in pancreatic cancer by inhibiting layer of cells was scraped with a 10 μL sterile pipette tip, and the PI3K/AKT signaling pathway. the cells were then incubated with a serum-free medium containing varying concentrations of linderalactone. 1e 2. Materials and Methods scratch width was recorded using phase-contrast micros- copy at 0 h and 24 h. 1e migration distance was calculated 2.1. Cell Culture and Reagents. Four pancreatic cancer cell using ImageJ software (National Institutes of Health, USA). lines, ASPC-1, BXPC-3, CFPAC-1, and SW-1990 were obtained from American Type Culture Collection (ATCC, 2.6. Transwell Assay. Transwell assays were used to evaluate USA). All cell lines were cultured in the RPMI-1640 medium migration and invasion. For migration, pancreatic cancer cells (Sigma, USA) supplemented with 10% fetal bovine serum (5 ×10 ) were mixed with varying concentrations of linder- (Gibco, USA) and a 1% penicillin and streptomycin mixture alactone in a serum-free medium. Transwell cell culture (Solarbio, China). 1e cells were placed in a cell incubator ° chambers (Falcon, USA) with 8.0 μm Transparent PET (1ermo Scientific, USA) with 37 C and 5% CO . 1e Lu- Membranes were used. Two hundred microliters of a cell- ciferase Mycoplasma Detection Kit (TransGen Biotech, medium mixture was placed in the upper chamber, and China) was used to confirm that the cells were free of 600 μL of medium containing 20% FBS was placed in the mycoplasma contamination. Linderalactone (Chengdu lower chamber as a cell migration chemoattractant. After 24 h Herbpurify, China) was dissolved in dimethyl sulfoxide of incubation in the incubator, the cells in the upper chamber (DMSO, Sigma, USA). were removed with cotton swabs and the cells in the lower chamber were fixed in 4% PFA for 30 min at room tem- perature and stained with 1% crystal violet solution for 20 min 2.2. Cell Viability. 1e Cell Counting Kit-8 (CCK-8, Bimake, at room temperature. After the cells were dried, the number of USA) was used to detect cell viability. In brief, pancreatic stained cells was counted. For the invasion assays, all the steps cancer cells were plated at 3000 cells per well in 96-well were the same as those for the migration assays, except that plates and treated with varying concentrations of linder- the 8.0 μm transparent PET membranes were coated with a alactone (0, 30, 40, 50, 60, 70, 80, 90, and 100 μM) for 0.5 mg/mL basement membrane matrix (Corning, USA). different periods of time (0, 24, and 48 h). Ten microliters of CCK-8 solution were added to each well, and the plates (Corning, USA) were incubated at 37 C for 2 h. 1e per- 2.7. Cell Cycle Analysis. A cell cycle analysis kit was pur- centage of surviving cells was calculated by measuring the chased from Beyotime (China). In brief, pancreatic cancer absorbance at an optical density of 450 nm using a micro- 4 cells (9 ×10 per well) were seeded in 6-well plates. After plate reader (1ermo Fisher, USA). 1e IC50 value was overnight culture, the cells were treated with varying con- determined based on a linear regression curve. centrations of linderalactone. After 24 h of treatment, the cells were collected and fixed with prechilled 70% alcohol for 12 h. 1en, the cells were washed with PBS; resuspended in a PBS 2.3. EdU Assay. 1e EdU Cell Proliferation Kit with Alexa mixture containing 0.05% Triton X-100, 0.1 mg/mL DNase- Fluor 488 was purchased from Beyotime (China). All steps free RNase A, and 25 μg/mL PI; and incubated for 30 min at were conducted in accordance with the manufacturer’s 37 C in the dark. A flow cytometer (BD Biosciences, USA) was instructions. In brief, pancreatic cancer cells were treated used to detect light scatter and red fluorescence at an exci- with varying concentrations of linderalactone, and then, tation wavelength of 488 nm. EdU was added and incubated with the cells for one-tenth of the cell doubling time (BXPC-3, 5 h; CFPAC-1, 3 h). 1e cells were incubated with Hoechst stain for 20 min to vi- 2.8. Cell Apoptosis Analysis. 1e FITC Annexin V/Dead Cell sualize the nucleus and then examined under a fluorescence Apoptosis Kit was purchased from Invitrogen (USA). microscope (Nikon, Japan). Pancreatic cancer cells (9 ×10 per well) were treated with Journal of Oncology 3 varying concentrations of linderalactone for 24 h. 1e cells of treatment with linderalactone, all tumor cell lines un- were then washed with prechilled PBS and centrifuged, and derwent quantitative and morphological changes. As shown the supernatant was removed. 1e cells were resuspended in in Figure 1(a), the cells lost their unique morphology, annexin binding buffer, and 5 μL of FITC annexin V and appeared round, and showed worse adherence to the well. 1 μL of 100 μg/mL PI working solution were added to each Figure 2 shows the chemical structure of linderalactone. 100 μL aliquot of cell suspension. After incubating the cells at Given the inhibitory effect of linderalactone on pancreatic room temperature for 15 min, annexin binding buffer was cancer cells, we performed CCK-8 assays to detect the ac- added, and the stained cells were analyzed by flow cytometry tivity of cells treated with varying concentrations of lin- with fluorescence emission measured at 530 nm. deralactone for different periods of time. 1e results are shown in Figure 1(c). Linderalactone inhibited pancreatic cancer activity in a time- and dose-dependent manner. 2.9. Western Blotting. Western blotting was performed as previously described [13]. 1e cells were collected and lysed in RIPA lysis buffer (Fude, China) with protease inhibitors 3.2. Linderalactone Inhibits the Proliferation of Pancreatic (1ermo Scientific, USA). Protein (50 μg) was separated by Cancer Cells. Replicative immortality is one of the hallmarks SDS-PAGE (Sangon, China) and transferred to PVDF of cancer [14]. Cell proliferation is accompanied by DNA membranes (Millipore, USA). After blocking with 3% BSA replication. We performed EdU assays to determine the (Solarbio, China), the membranes were incubated with pri- effect of linderalactone on cell proliferation. 1e results mary antibodies overnight. 1e next day, the membranes showed that (Figures 2(a)–2(d), Supplementary Figures 1(a)–1(d)) the population of EdU-positive cells were washed with TBST (Sangon, China) and incubated with the secondary antibody (Promega, USA) for 90 min at room decreased as the concentration of linderalactone increased temperature. Enhanced chemiluminescence detection (Fude, compared with the control. Colony formation experiments China) was used to detect the target protein. Primary anti- were conducted to evaluate the long-term effects of lin- bodies against the following proteins were used: p-PI3K, deralactone on pancreatic cancer cells. 1e number and size PI3K, p-AKT, AKT, PCNA, Ki-67, caspase 3, PARP1, Bcl-2, of colonies were significantly reduced by treatment with Bax, cyclin A2, cyclin B1, cyclin D1, and cyclin E1. All an- linderalactone (Figures 2(e) and 2(f)). 1ese results indi- tibodies were purchased from Abcam (USA). cated that linderalactone can inhibit the proliferative po- tential of pancreatic cancer. 2.10. Xenograft Experiments. Four- to six-week-old BALB/c nude mice were purchased from Shanghai SLAC Laboratory 3.3. Linderalactone Inhibits the Migration and Invasion of Animal Company (China). 1e protocol was approved Pancreatic Cancer Cells. 1e systemic metastasis of tumors according to the ethical standards of the Institutional Animal often underlies the inability to cure the disease [15]. We used Care and Use Committee of Zhejiang Chinese Medical Uni- wound-healing and Transwell experiments to evaluate the migration ability of cells. In addition, Transwell assays with versity and complied with the Regulations for the Adminis- tration of Affairs Concerning Experimental Animals (approved Matrigel were used to evaluate cell invasion ability. To re- by the State Council of China, No. SYXK (Zhejiang) 2018- duce the influence of cell proliferation on the migration 0012). 1e pancreatic cancer cell line BXPC-3 was injected into results in the scratch assays, we first starved the cells with a the right abdomen of each nude mouse. After two weeks, 18 serum-free medium for 24 h; thus, the results only reflected nude mice with tumors of the same size were selected into three the effects of linderalactone. As shown in Figures 3(a) and groups. Normal saline or low-dose (25 mg/kg) or high-dose 3(b), the migration distance of cells treated with linder- (50 mg/kg) linderalactone was injected every three days, and alactone was significantly reduced and the distance migrated tumor weight and volume were measured. After three weeks, by cells in the 60 μM group was shorter than that by those in the 30 μM group. Transwell experiments were used to detect the mice were sacrificed, and the tumors were removed. 1e tumor volume was calculated as 0.5 × lengths × width . the migration ability of cells in 3D space, and the results (Figures 3(c) and 3(d)) were consistent with the scratch assay results. As the concentration of linderalactone increased, the 2.11. Statistical Analyses. All experiments were repeated at migration ability decreased. Cell invasiveness was evaluated least three times to ensure the reliability of the results. Data using Transwell assays with Matrigel (Figures 3(e) and 3(f)). were analyzed using SPSS 12.0, and the results are expressed In general, linderalactone not only inhibited proliferation as the mean± standard error. An unpaired Student’s t-test but also showed effective antimetastatic activity. All the was used to determine statistical significance, which was results shown in Figure 3 were from experiments performed indicated by p< 0.05. with BXPC-3 cells, and these experiments were repeated in CFPAC-1 cells (Supplementary Figure 2). 3. Results 3.1. Linderalactone Inhibits the Viability of Pancreatic Cancer 3.4. Linderalactone Arrests the Cell Cycle and Induces Cells. To determine the effect of linderalactone on pan- Apoptosis. Our previous research found that linderalactone inhibits the proliferation of pancreatic cancer cells, so we creatic cancer, we treated four pancreatic cancer cell lines with increasing concentrations of linderalactone. After 72 h used flow cytometry to detect potential changes in the cell 4 Journal of Oncology 0 M 40 M 80 M (a) (b) ASPC-1 BXPC-3 CFPAC-1 SW 1990 150 150 150 100 100 100 100 50 50 50 50 0 0 0 0 0 20406080 100 0 20 40 60 80 100 020 40 60 80 100 020 40 60 80 100 Magnolol (M) Magnolol (M) Magnolol (M) Magnolol (M) 24 h 24 h 24 h 24 h 48 h 48 h 48 h 48 h 72 h 72 h 72 h 72 h (c) Figure 1: Linderalactone inhibits the viability of pancreatic cancer cells. (a) Morphological changes of pancreatic cancer cells exposed to LL. (b) 1e chemical structure of LL. (c) CCK8 assay detected the viability of pancreatic cancer cells under varying concentrations and time of LL treatment. Scale bar � 200 μm. cycle. 1e results are shown in Figures 4(a) and 4(b). After Bcl-2, PARP1, and caspase 3 protein levels and the increase treatment with linderalactone, the number of cells in the G2/ in Bax and cleaved PARP1 levels indicated the occurrence of M phase increased significantly compared with that in the apoptosis. control group. Western blotting was used to detect the expression of cyclin A2, cyclin B1, cyclin D1, and cyclin E1. 1e protein levels of cyclin B1, which marks the G2/M phase, 3.5. Linderalactone Exhibits Antitumor Activity by Inhibiting the PI3K/AKT Signaling Pathway. We next tried to elucidate increased significantly, while those of cyclin A2, cyclin D1, how linderalactone inhibits pancreatic cancer activity. It is and cyclin E1, which marks the G1 or S phase, decreased, confirming the cell cycle results obtained by flow cytometry well known that pancreatic cancer is a malignant tumor driven by oncogenes. Numerous studies have found that (Figure 4(e)). Cell cycle arrest is often accompanied by apoptosis, so we subsequently detected apoptosis by flow genes such as K-ras, Smad, and Stat are active in pancreatic cancer. We evaluated the changes in a number of signaling cytometry, and the results were consistent with our ex- pectations (Figures 4(c) and 4(d)): linderalactone induced pathway components, including PI3K/AKT, before and after linderalactone treatment. Significant changes in the PI3K/ apoptosis. 1e expression levels of Bcl-2, Bax, PARP1, and caspase 3 were determined (Figure 4(f)); the decrease in AKT signaling pathway were observed after treatment viable cell (%) SW-1990 CFPAC-1 BXPC-3 ASPC-1 viable cell (%) viable cell (%) viable cell (%) Journal of Oncology 5 DAPI EdU Merge DAPI EdU Merge (a) (b) BXPC-3 CFPAC-1 60 40 ** ** ** 0 0 LL (M) 030 60 LL (M) 0 30 60 (c) (d) BXPC-3 0 M 30 M 60 M ** ** LL (M) 0 30 60 CFPAC-1 ** ** LL (M) 030 60 (e) (f) Figure 2: Linderalactone inhibits the proliferation of pancreatic cancer cells. (a, b) EdU staining assay detected the proliferation of BXPC-3 and CFPAC-1 cell lines under varying concentrations of LL treatment after 24 hours. (c, d) Quantification of EdU stain: the percentage of EdU positive cells to the total number of cells. (e) Clone formation assay to detect the effect of LL on BXPC-3 and CFPAC-1 cell lines. ∗ ∗∗ (f) Quantification of clone formation. Scale bar � 200 μm. p< 0.05; p< 0.01. (Figures 5(a) and 5(b)). Previous studies have found that that linderalactone functions in this way [16, 17]. To confirm inhibiting the PI3K/AKT signaling pathway can effectively our conjecture, we used YS-49, a PI3K/AKT activator [18]. inhibit the function of pancreatic cancer; thus, we speculated As shown in Figures 5(c) and 5(d), linderalactone alone CFPAC-1 BXPC-3 0 M 60 M 30 M EdU positive cell rate (%) 60 M 30 M 0 M Number of Clone Formation Number of Clone Formation EdU positive cell rate (%) 6 Journal of Oncology BXPC-3 0 M 30 M 60 M ** ** LL (M) 0 30 60 (a) (b) BXPC-3 0 M 30 M 60 M ** ** LL (M) 0 30 60 (c) (d) BXPC-3 0 M 30 M 60 M ** ** LL (M) 0 30 60 (e) (f) Figure 3: Linderalactone inhibits the migration and invasion of pancreatic cancer cells. (a) Wound-healing assay detects the migration ability of BXPC-3 under varying concentrations of LL after 24 hours. (b) Quantification of wound-healing assay: the area of cell migration in 24 hours accounts for the percentage of the wound area in 0 hours. (c) 1e transwell assay detected the migration ability of cells under varying concentrations of LL treatment after 24 hours. (d) Quantification of transwell assay: percentage of the compound treatment group in the control group. (e) 1e transwell assay with Matrigel to detect cell invasion ability under varying concentrations of LL treatment after 24 hours. (f) Quantification of transwell assay: percentage of the compound treatment group in the control group. Scale bar � 200 μm. ∗ ∗∗ p< 0.05; p< 0.01. decreased the levels of p-PI3K and p-AKT, while YS-49 3.6. Linderalactone Inhibits Tumor Progression In Vivo. alone did not affect the phosphorylation of these proteins. To explore the effect of linderalactone on tumor growth in When the two compounds were combined, YS-49 reversed vivo, a nude mouse pancreatic tumor model was con- the inhibitory effect of linderalactone on p-PI3K and p-AKT. structed. Physiological saline or low-dose (25 mg/kg) lin- None of the evaluated treatments caused changes in total deralactone or high-dose (50 mg/kg) linderalactone was PI3K and AKT levels. In the colony formation assays, injected into the abdominal cavity of the mice once every cotreatment with YS-49 effectively reversed the antitumor three days, and tumor volume and weight were recorded. 1e experimental results revealed that the tumors of mice effects of linderalactone. 1erefore, we confirmed that lin- deralactone inhibits the tumor activity of pancreatic cancer treated with linderalactone were generally smaller than those by inhibiting PI3K/AKT. of mice in the control group and the therapeutic effect of 24 h 24 h 24 h 0 h Cell Invasion Rate (%) Cell Migration Rate (%) Wound healing percentage Journal of Oncology 7 BXPC-3 LL 0 M LL 30 M LL 60 M 600 600 500 500 400 400 300 300 200 200 100 100 0 0 0 020 40 60 80 100 120 020 40 60 80 100 120 020 40 60 80 100 120 LL (M) 0 30 60 Channels (PI-A) Channels (PI-A) Channels (PI-A) CFPAC-1 200 50 0 0 0 030 60 90 120 150 030 60 90 120 150 030 60 90 120 150 Channels (PI-A) Channels (PI-A) Channels (PI-A) LL (M) 0 30 60 Debris Dip G2 Debris Dip G2 Debris Dip G2 G2/M Aggregates Dip S Aggregates Dip S Aggregates Dip S Dip G1 Dip G1 Dip G1 G0/G1 (a) (b) BXPC-3 LL 0 M LL 30 M LL 60 M 60 ** 5 5 10 ** 10 10 4 4 10 10 10 3 3 20 10 10 10 2 2 2 10 10 10 0 0 0 LL (M) 0 30 60 2 3 4 5 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 CFPAC-1 5 5 5 10 ** 10 10 ** 4 4 10 10 3 3 3 10 10 2 2 10 10 0 0 2 3 4 5 2 3 4 5 2 3 4 5 LL (M) 0 30 60 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 (c) (d) BXPC-3 CFPAC-1 BXPC-3 CFPAC-1 LL (M) 0 30 60 0 30 60 LL (M) 0 30 60 0 30 60 PARP-1 Cyclin D1 cl-PARP-1 Cyclin B1 Casepase 3 ACTB ACTB (e) (f) Figure 4: Linderalactone blocks the cell cycle and induces apoptosis. (a) Flow cytometry using PI staining to detect the cycle distribution of BXPC-3 and CFPAC-1 cell lines treated with varying concentrations of LL for 24 hours. (b) 1e percentage of cell cycle distribution for BXPC-3 and CFPAC-1. (c) Annexin V/PI staining to detect the apoptotic death of BXPC-3 and CFPAC-1. (d) 1e percentage of cell ∗ ∗∗ apoptosis for BXPC-3 and CFPAC-1. (e, f) Western blot analysis of key cell cycle and cell apoptosis markers. p< 0.05; p< 0.01. CFPAC-1 BXPC-3 Number Number CFPAC-1 BXPC-3 Number Number Number Number Cell Cycle Phase (%) Cell Cycle Phase (%) Cell Apoptosis Rate (%) Cell Apoptosis Rate (%) 8 Journal of Oncology BXPC-3 BXPC-3 CFPAC-1 CFPAC-1 LL (M) 0 30 60 LL (M) 0 30 60 LL (M) 0 30 60 LL (M) 0 30 60 p-PI3K t-PI3K p-PI3K t-PI3K p-AKT t-AKT p-AKT t-AKT ACTB ACTB ACTB ACTB (a) (b) BXPC-3 BXPC-3 LL (60 M) + + LL (60 M) + + − − − − + + + + YS-49 (30 M) −− YS-49 (30 M) −− p-PI3K t-PI3K p-AKT t-AKT ACTB ACTB (c) CFPAC-1 CFPAC-1 + + + + LL (60 M) − − LL (60 M) − − YS-49 (30 M) −− + + YS-49 (30 M) −− + + p-PI3K t-PI3K p-AKT t-AKT ACTB ACTB (d) Control LL YS-49 LL+YS-49 (e) Figure 5: Continued. CFPAC-1 BXPC-3 Journal of Oncology 9 BXPC-3 ns ns ** CFPAC-1 ns ns ** (f) Figure 5: Linderalactone exhibits antitumor activity by inhibiting the PI3K/AKT signaling pathway. (a, b) Western blot analysis of phosphorylated PI3K and phosphorylated AKT, total PI3K, and total AKT expression in BXPC-3 and CFPAC-1 cells after treatment with varying concentrations of LL. (c, d) After LL or YS-49 treatment, the expression of phosphorylated PI3K and phosphorylated AKT and total PI3K and total AKT in BXPC-3 and CFPAC-1 cells. (e) After LL or YS-49 treatment, the clone formation assay detects the proliferation ∗ ∗∗ ability of BXPC-3 and CFPAC-1. (f) Quantification of clone formation. p< 0.05; p< 0.01. Control 25 mg/kg 50 mg/kg (a) Control 25 mg/kg 50 mg/kg (b) Figure 6: Continued. Number of Clone Formation Number of Clone Formation Control Control LL LL YS-49 YS-49 LL+YS-49 LL+YS-49 10 Journal of Oncology 2000 24 1500 22 ns ns ** 500 18 0 16 0369 12 15 18 21 0 3 6 9 12151821 Days Days control control low(25 mg/kg) low (25 mg/kg) high(50 mg/kg) high (50 mg/kg) (c) Control 25 mg/kg 50 mg/kg (d) Figure 6: Linderalactone inhibits tumor progression in vivo. (a) Images of the control group and low-dose (LL 25 mg/kg) and high-dose (LL 50 mg/kg) tumor-bearing mice. (b) Quantitative volume of the tumor progression: the tumor volume was measured every three days for three weeks after the injection of LL. (c) Quantitative mice weight: the body weight of the mice was measured every three days for three ∗ ∗∗ weeks after the injection of LL. (d) Immunohistochemical detection of PCNA and Ki-67 expression level in tumor cells. p< 0.05; p< 0.01. high-dose linderalactone was better than that of low-dose increasingly elucidated, as studies have shown that they linderalactone (Figures 6(a) and 6(b)). In addition, trends in induce apoptosis, inhibit proliferation, and increase chemical sensitivity [19]. Previous studies have shown the body weight of mice treated with linderalactone were similar to those of mice in the control group (Figure 6(c)), that lutein can reduce the levels of K-ras and AKT in with no significant difference, indicating that linderalactone mouse colon cancer and induce cell cycle arrest [20]. does not exert significant toxicity. 1e immunohisto- Lycopene can be used as a preventive agent because it chemistry results (Figure 6(d)) showed that PCNA and Ki- decreases MMP-7 expression and inhibits the invasion of 67 expressions were lower in the experimental group than in HT-29 human colon cancer cells [21]. Procyanidin the control group. In general, the antitumor activity of therapy exerts antiproliferative and anti-invasive effects in linderalactone in vivo was consistent with that in vitro. pancreatic cancer cells and is an effective chemo- preventive agent for this disease [22]. Linderae Radix has been used in East Asian countries 4. Discussion such as China and Japan for centuries as a folk medicine without any obvious toxicity. Linderae Radix has anti-in- Although some progress has been made in prolonging patient survival, adverse reactions to chemotherapy drugs flammatory and antioxidant effects in the treatment of al- used to treat pancreatic cancer have seriously affected coholic liver injury and hyperlipidemia [23–26]. In this patients’ quality of life. Phytochemicals are generally study, we explored the antitumor effects of linderalactone, considered to have minor adverse effects and great po- an extract of Linderae Radix, on pancreatic cancer. We used tential in the treatment of tumors. Phytochemicals are a four pancreatic cancer cell lines and mouse tumor-bearing class of biological secondary metabolites that are abun- models for in vitro and in vivo studies. 1e antitumor activity dant in fruits, grains, and vegetables. In recent years, the of linderalactone was evaluated, and its mechanism of action role of phytochemicals as anticancer agents has been was investigated [27–30]. Ki-67 PCNA Tumer volume (mm ) Mouse Weight (g) Journal of Oncology 11 1e inhibition of proliferation and metastasis and the Conflicts of Interest induction of cell cycle arrest are effective means of 1e authors declare no conflicts of interest. treating tumors. Here, our study found that linder- alactone effectively inhibited the proliferation of pan- creatic cancer cells both in the short and long term. Authors’ Contributions Invasion and migration are hallmarks of cancer cell Dongchao Xu, Mengyao Tian, and Wangyang Chen con- metastasis, and our research showed that linderalactone tributed equally. All authors agreed to submit this effectively inhibited the migration and invasion of pan- manuscript. creatic cancer cells, thereby inhibiting metastasis. Flow cytometry analyses revealed that pancreatic cancer cells arrested the G2/M phase of the cell cycle in response to Acknowledgments linderalactone treatment and apoptosis occurred. Cell 1e work was supported by the Zhejiang Medical and Health cycle arrest in the G2/M phase stops tetraploid cells from Science and Technology Plan (Grant Nos. WKJ-ZJ-2136 and dividing. Numerous drugs that induce tumor cell arrest in 2018PY037), Hangzhou Medical and Health Science and the G2/M phase have been used for tumor treatment. 1e Technology Plan (Grant Nos. 2016ZD01, OO20190610, G2/M checkpoint is one of the key targets of tumor A20200737, and A20200174), Hangzhou Major Science and treatment. 1e cell cycle is regulated by cyclin B1. Our Technology Projects (Grant No. 202004A14), and the results showed that cyclin B1 expression decreased, Zhejiang Charity Plan Project (Grant No. LGF21H310004). confirming our flow cytometry results. 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Journal of OncologyHindawi Publishing Corporation

Published: Aug 4, 2022

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