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Ethanolic Neem (Azadirachta indica) Leaf Extract Prevents Growth of MCF-7 and HeLa Cells and Potentiates the Therapeutic Index of Cisplatin

Ethanolic Neem (Azadirachta indica) Leaf Extract Prevents Growth of MCF-7 and HeLa Cells and... Hindawi Publishing Corporation Journal of Oncology Volume 2014, Article ID 321754, 10 pages http://dx.doi.org/10.1155/2014/321754 Research Article Ethanolic Neem (Azadirachta indica) Leaf Extract Prevents Growth of MCF-7 and HeLa Cells and Potentiates the Therapeutic Index of Cisplatin 1 1 1 1 Chhavi Sharma, Andrea J. Vas, Payal Goala, Taher M. Gheewala, 2 1 Tahir A. Rizvi, and Arif Hussain Department of Biotechnology, Manipal University, Dubai International Academic City, P.O. Box 345050, Dubai, UAE Department of Microbiology andImmunology,Faculty of Medicine andHealthScience,UAE University,P.O.Box 17666,AlAin,UAE Correspondence should be addressed to Arif Hussain; dr.ariu fh ssain@yahoo.co.in Received 5 September 2013; Revised 13 November 2013; Accepted 15 November 2013; Published 30 January 2014 Academic Editor: Akira Hara Copyright © 2014 Chhavi Sharma et al. This 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. The present study was designed to gain insight into the antiproliferative activity of ethanolic neem leaves extract (ENLE) alone or in combination with cisplatin by cell viability assay on human breast (MCF-7) and cervical (HeLa) cancer cells. Nuclear morphological examination and cell cycle analysis were performed to determine the mode of cell death. Further, to identify its molecular targets, the expression of genes involved in apoptosis, cell cycle progression, and drug metabolism was analyzed by RT-PCR. Treatment of MCF-7, HeLa, and normal cells with ENLE differentially suppressed the growth of cancer cells in a dose- and time-dependent manner through apoptosis. Additionally, lower dose combinations of ENLE with cisplatin resulted in synergistic growth inhibition of these cells compared to the individual drugs (combination index<1). ENLE significantly modulated the expression of bax, cyclin D1, and cytochrome P450 monooxygenases (CYP 1A1 and CYP 1A2) in a time-dependent manner in these cells. Conclusively, these results emphasize the chemopreventive ability of neem alone or in combination with chemotherapeutic treatment to reduce the cytotoxic effects on normal cells, while potentiating their efficacy at lower doses. us, Th neem may be a prospective therapeutic agent to combat gynecological cancers. 1. Introduction their antioxidant properties by decreasing TNF-𝛼 ,increasing IFN-𝛾 , and modulating antioxidant enzymes such as glu- Therapeutic properties of neem ( Azadirachta indica)have tathione S-transferase (GST) and certain hepatic cytochrome been recognized since ancient times and have been extensive- P450-dependent monooxygenases [7–13]. It induces apopto- ly used in ayurveda, unani, and homoeopathic medicine [1]. sis via both the intrinsic and extrinsic pathways and induces Many compounds such as limonoids, azadirone, azadiracht- cell cycle arrest via p53-dependent p21 accumulation and in, and flavonoids, having therapeutic potential, have been downregulation of the cell cycle regulatory proteins cyclin isolated from various parts of neem tree and have been eval- B, cyclin D1, p53, and proliferating cell nuclear antigen uated for their pharmacological actions and plausible medic- (PCNA) [11, 14–16]. Interestingly, when used in conjunc- inal applications along with their safety evaluation. Recent tion with chemotherapeutic drugs like cyclophosphamide, studies have shown that neem possesses anti-inflammatory, cisplatin, 5-fluorouracil, or with radiotherapy, it potentiates antiarthritic, antipyretic, hypoglycemic, antigastric ulcer, their antitumor eect ff s by activating proapoptotic signaling antifungal, antibacterial, and antitumor activities [2–6]. and negating survival signaling along with attenuating their The antineoplastic properties of neem are gaining side effects [ 14, 17–20]. Notably, cisplatin, the rfi st member attention due to its cancer preventive, tumor-suppressive, of a class of platinum-containing anticancer drugs, is widely antiproliferative, apoptosis-inducing, antiangiogenic, and used for treatment of solid malignancies. Cisplatin has a immunomodulatory effects via several molecular mecha- number of side-effects that can limit its use: nephrotoxicity, nisms [6, 7]. Neem or its derivatives have been shown to exert nausea and vomiting, ototoxicity (hearing loss), electrolyte 2 Journal of Oncology disturbance, and hemolytic anemia, etc. Also, the majority of 570 nm [23]. The effect of ENLE on growth inhibition was cancer patients eventually develop cisplatin-resistant disease assessed as percent cell viability and was calculated as (OD necessitating combination therapy approach using multiple of the drug-treated sample/OD of the nontreated sample) chemotherapeutic agents or combining with chemopreven- × 100, considering that the colorimetric signal is directly tive agents [21, 22]. proportional to the number of viable cells. eTh EC (50% Based on the facts mentioned above, the present study effective concentration) values were calculated from the dose- aims to evaluate the chemopreventive potential of ethanolic response curves. neem leaves extract (ENLE) alone or concurrently with cisplatin on human breast (MCF-7) and cervical (HeLa) 2.4. Calculation of Combination Effects of Cisplatin and ENLE. Calculations of combination eeff cts were expressed as a cancer cells, with the objective of studying its antiproliferative activity on cancer cells while decreasing the cytotoxic effects combination index (CI) as described previously [23]. CI analysis provides qualitative information on the nature of on normal cells. Also, the molecular targets of ENLE were drug interaction, and CI, a numerical value, was calculated delineated to elucidate its in vitro anticancer effects. according to the following equation: 2. Material and Methods C C A,𝑥 B,𝑥 CI= + , (1) IC IC 2.1. Cell Culture. The human breast cancer cell line, MCF-7, 𝑥, A 𝑥, B and human cervical carcinoma cell line, HeLa were main- where C and C are, respectively, the concentrations of A,𝑥 B,𝑥 tained in DMEM (Sigma, USA) supplemented with 10% drugs A and B used in combination to achieve𝑥 %drugeeff ct. fetalbovineserum (FBS)(Sigma, USA)and 100x Pen-strep IC and IC are the concentrations for single agents to 𝑥, A 𝑥, B (Sigma, USA) in a humidified atmosphere of 5% CO in ∘ achieve the same effect. A CI value <1, =1, or>1represents, air at 37 C. Lymphocytes were isolated from healthy non- respectively, synergy, additivity, or antagonism of cisplatin smoking donors using HiSep Media (HiMedia, India) as per and ENLE, respectively. the manufacturer’s instructions [23] and were maintained in RPMI media (Sigma, USA). 2.5. Detection of Apoptosis in MCF-7 and HeLa Cells aeft r 2.2. Preparation of Drug Solutions. 5% ethanolic neem leaves Treatment with ENLE extract (ENLE) was prepared as described previously by Sub- 2.5.1. Microscopic Examination. Morphological changes of apriya and coworkers (2005) with slight modicfi ations [ 24]. MCF-7and HeLa cells were noted on treatment with ENLE Brieyfl , 2.5 g of fresh mature neem leaves was ground to a n fi e at different concentrations (50, 200, and 500 𝜇 g/mL) and paste in 50 mL of 100% ethanol and the slurry was air-dried time-points (48 and 72 h for MCF-7 and 24 and 48 h for in a shaking incubator at 37 C with intermittently stirring HeLa) using normal inverted microscope (Labomed, USA) at 2 h and then left overnight. The powder obtained was (Figures 2(a) and 2(b)). The untreated cells were used as weighed and resuspended in dimethyl sulphoxide (DMSO) negative control. (Sigma, USA) to prepare a stock solution of 80 mg/mL which was filtered through 0.2 𝜇 m filter. Further dilutions were prepared in DMEM to require concentrations between 10 2.5.2. Nuclear Morphological Studies. Apoptosis induction and 500𝜇 g/mL for treatment of MCF-7 cells, HeLa cells, and aer ft treatment of MCF-7 and HeLa cells with ENLE at their respective EC concentrations and varying time-points lymphocytes. A stock solution of 3.3 mM of cisplatin (Cadila Phar- (350𝜇 g/mL for 0, 48, and 72 h in MCF-7 and 175𝜇 g/mL for 0, maceuticals Ltd., India) was used to make drug dilutions of 24, and 48 h in HeLa cells) was evaluated by the nuclear mor- phological changes associated with it using propidium iodide varying concentrations (1–200𝜇 M) in complete medium. staining (Figures 3(a) and 3(b))[22]. Briefly, ∼10 cells/mL cells were seeded on glass coverslips and incubated overnight 2.3. Cell Viability Assay. The effect of ENLE and its combina- in complete medium at 37 C. Further, cells were treated with tion with cisplatin, a chemotherapeutic agent, on the viability ENLE at its EC for above mentioned time periods. At the of MCF-7, HeLa, and lymphocytes was determined by 3- end of the desired time interval, cells were fixed in a mixture [4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide of acetone: methanol (1 : 1) at−20 Cfor 10min, washed with (MTT) assay. eTh cells were plated in triplicate at a density of 1X PBS (pH 7.4) twice, and stained with propidium iodide ∼1× 10 cells/well in 200𝜇 L of complete culture medium con- (10 mg/mL in PBS) for 30 s in dark at RT. eTh coverslips were taining 10–500𝜇 g/mL concentrations of ENLE alone for 48 thoroughly washed with PBS and placed upturned onto a and72 hand24and48 hforMCF-7andHeLa,respectively,or glass slide with mounting media (DPX). Slides were viewed a combination of ENLE (N1M and N2M = 50 and 100𝜇 g/mL; at 515 nm under the Progress Fluorescent Microscope (Olym- N1H and N2H = 10 and 50𝜇 g/mL) with cisplatin (C1M and pus, USA). The images were captured at 40x magnification. C2M = 1 and 10𝜇 M in MCF-7; C1H and C2H = 1 and 5𝜇 M) for 48 and 24 h, respectively, for MCF-7 and HeLa in 96- well microtiter plates. Aeft r incubation for specified times at 2.5.3. Quantification of Apoptotic Cells by Flow Cytome- 37 Cinahumidiefi dincubator,MTT (5mg/mLinPBS)was try. Cell cycle analysis of ENLE-treated MCF-7 and HeLa added to each well and incubated for 2 h. eTh absorbance cells was performed by flow cytometry as described earlier was recorded on a microplate reader at the wavelength of (Figure 4)[23]. After treatment of synchronous cultures of Journal of Oncology 3 110 110 60 60 50 50 0 50 100 150 200 250 300 350 400 450 500 550 0 50 100 150 200 250 300 350 400 450 500 550 ENLE concentration (𝜇 g/mL) ENLE concentration (𝜇 g/mL) 48 h 24 h 72 h 48 h (a) (b) 0 50 100 150 200 250 300 350 400 450 500 550 ENLE concentration (𝜇 g/mL) (c) Figure 1: Differential cytotoxic effect of ENLE on MCF-7, HeLa, and lymphocytes. (a, b) MCF-7 and HeLa cells treated with ENLE at varying concentrations (10–500𝜇 g/mL), resulting in dose- and time-dependent growth inhibition. eTh EC forMCF-7 andHeLacells wasfound to be 350𝜇 g/mL at 72 h and 175𝜇 g/mL at 48 h, respectively. However, lymphocytes did not show significant growth inhibition when treated with similar concentrations of ENLE for 24 h (c). Values are means + SD of three independent experiments. Each value with ENLE treatment differs from the control value ( 𝑃<0.05 ). MCF-7and HeLa cellswithENLEattheir respective EC and HeLa cells was carried out as per the manufacturer’s concentrations at various time-points (350𝜇 g/mL for 0, 48, instructions (GenElute Mammalian Genomic Total RNA Kit, and 72 h in MCF-7 and 175𝜇 g/mL for 0, 24, and 48 h in Sigma, USA) at various time intervals. Further, total RNA HeLa cells), both adherent and floating cells were harvested, was subjected to first strand synthesis as per manufacturer’s washed with phosphate buffered saline (PBS, pH 7.2), and protocol (ProtoScript M-MuLV Taq RT-PCR Kit, New Eng- fixed with ice-cold absolute ethanol at −20 Covernight.Cells land Biolabs, USA) followed by PCR using gene-specific were then washed with PBS prior to resuspending in a buffer primers [23, 25–28].𝛽 -Actin was taken as an internal control. containing PI (50 mg/mL), 0.1% sodium citrate, 0.1% Triton The PCR cycle was as follows: initial denaturation at 95 C X-100, and 100 mg/mL of RNase A. The cells were analyzed for 5 min, followed by 35 amplification cycles (denaturation ∘ ∘ using Flow cytometry (Beckman Coulter flow Cytometer at 94 Cfor 30s; annealingat55 Cfor 𝛽 -actin, CYP 1A1, ∘ ∘ FC500, CXP Version 2.2). eTh data was analyzed using the and CYP 1A2, 56 Cfor Bax, and54 Cfor cyclin D1;and ∘ ∘ Beckman Coulter KALUZA 1.1 analysis software. extension at 72 C for 45 s), with na fi l extension at 72 Cfor 7 min. Amplified products were visualized on a 2% agarose 2.6. Expression Analysis of Various Genes Targeted by ENLE. gel containing ethidium bromide. Reverse transcription PCR was used to detect the expression of Bax, cyclin D1, CYP 1A1, and CYP 1A2 in response to 3. Statistical Analysis treatment with ENLE at EC for varying time intervals (350𝜇 g/mL for 0, 48, and 72 h in MCF-7 and 175𝜇 g/mL for All data are expressed as means± SD of at least 3 experiments. 0, 24, and 48 h in HeLa cells) (Figures 5(a) and 5(b)). Total Fisher’s exact test was adopted for statistical evaluation of the RNA extraction from untreated and ENLE-treated MCF-7 results. Signicfi ant differences were established at 𝑃<0.05 . Cell viability (%) Cell viability (%) Cell viability (%) 4 Journal of Oncology 48 h 72 h 50 𝜇 g/mL 200 𝜇 g/mL 500 𝜇 g/mL (a) 24 h 48 h 50 𝜇 g/mL 200 𝜇 g/mL 500 𝜇 g/mL (b) Figure 2: ENLE-induced morphological changes in MCF-7 (a) and HeLa cells (b) at varying concentrations and time-points. 50, 200, and 500𝜇 g/mL ENLE-treated (a) MCF-7 (for 48 and 72 h) and (b) HeLa cells (24 and 48 h) showed dose- and time-dependent increase in the morphological changes associated with cell death via apoptosis compared to the untreated cells (magnification 100x). 4. Results MCF-7 cells, the EC was observed at 350𝜇 g/mL aer ft 72 h treatment with ENLE, whereas in HeLa cells, it was found to 4.1.ENLEShows SelectiveCytotoxic Eeff cts towardsMCF- be 175𝜇 g/mL in 48 h (Figures 1(a) and 1(b)). 7 and HeLa Cells. The antiproliferative eeff cts of different Notably, to assess if ENLE possesses a safe cytotoxic concentrations of ENLE on MCF-7 cells, HeLa cells, and profile, MTT assay was performed on lymphocytes isolated lymphocyteswereevaluated by theMTT assay. MCF-7and from a healthy nonsmoker adult at similar doses of ENLE (10– HeLa cells treated with increasing concentrations of ENLE 500𝜇 g/mL) (Figure 1(c)). No significant effect on cell viability ranging from 10 to 500𝜇 g/mL showed a dose- and time- wasobservedaeft rtreatmentwithENLEfor24hatthesecon- dependent increase in cell death (Figures 1(a) and 1(b)). In centrations, thereby proving the fact that chemopreventive Journal of Oncology 5 0 h 48 h 72 h (a) 0 h 24 h 48 h (b) Figure 3: The nuclear morphological changes induced by ENLE treatment at various time intervals on (a) MCF-7 (for 0, 48, and 72 h) and (b) HeLa (for 0, 24, and 48 h) cells. Untreated MCF-7 and HeLa cells (0 h) showed large and prominent nuclei, indicating no significant characteristics of apoptosis (white arrows). On the other hand, ENLE treatment of these cells at their respective EC induced time-dependent increase in nuclear morphological changes characteristic of apoptotic cells such as nuclear condensation and fragmentation (green arrows), nuclear marginalization (yellow arrows), and appearance of apoptotic bodies (pink arrows) (magnification 400x). agents like neem can specially target the cancer cells an increase in duration of ENLE exposure, there was a cumu- (Figure 1(c)). This property of neem can be utilized for the lative accrual of the said features consistent with apoptosis in both of the cell lines (Figures 3(a) and 3(b)). purposeofcancertreatment becauseofits safety profile. 4.2.3. Eeff ct of ENLE on the Cell Cycle Distribution. The effect 4.2. ENLE Induces Cell Death via Apoptosis in MCF-7 and of ENLE treatment on the cell cycle distribution of MCF- HeLa Cells 7 and HeLa cells was determined by flow cytometry after treatment of these cells with ENLE at their respective EC 4.2.1. Morphological Changes Induced by ENLE on MCF-7 concentrations for 48 and 72 h for MCF-7 and, 24 and 48 h andHeLaCells. ENLE-treated MCF-7 (for 48 and 72 h) and for HeLa cells. eTh untreated cells (0 h) showed appropriate HeLa (for 24 and 48 h) cells at the concentrations 50, 200, distribution of cells in the different phases of cell cycle and 500𝜇 g/mL were observed under an inverted microscope (Figure 4), while in the case of ENLE-treated cells, there was and their morphological characteristics were noted. In com- a significant time-dependent increase in the number of cells parison to untreated cells, ENLE-treated cells showed typical in the sub-G phase of the cell cycle (17 and 30% for MCF- features of cell death at the morphological level such as 7 after 48 and 72 h and 15 and 29% for HeLa after 24 and rounding off of cells, cell shrinkage, and detachment from the 48 h treatment) (Figure 4). This confirms that ENLE induces substrate which accumulated in a dose- and time-dependent apoptotic cell death in these cells. manner, thus indicating that ENLE induces cell death by apoptosis in these cells (Figures 2(a) and 2(b)). 4.3. ENLE Treatment Signicfi antly Modulates the Expression of Bax, Cyclin D1, CYP 1A1, and CYP 1A2. With the target of 4.2.2. Nuclear Morphological Changes Induced by ENLE on determining the eeff ctor genes involved in ENLE-mediated MCF-7 and HeLa Cells. ENLE-induced nuclear morphologi- cellular responses in MCF-7 and HeLa cells, the expression calchanges characteristic of typicalcellundergoingapoptosis of Bax, cyclin D1, CYP 1A1, and CYP 1A2 was analyzed before were studied in MCF-7 and HeLa cells at their respective and after treatment with ENLE (48 and 72 h treatment for EC at various time-points. Untreated MCF-7 and HeLa MCF-7 cells and 24 and 48 h treatment for HeLa cells).𝛽 - cells appeared uniform in chromatin density with an intact Actin was used as an internal control for comparison of nucleus. However, treatment of MCF-7 cells with ENLE for 48 samples. and 72 h resulted in apoptosis-associated nuclear morpholog- The aberrant expression of cyclin D1, a key player in ical changes like chromatin condensation and fragmentation the progression of the cells from G1 to S phase, has been alongwithappearanceofapoptoticbodies(Figure 3(a)). Also, associated with the deregulated cell cycle control in many HeLa cells treated with ENLE showed similar changes in humancancers.Itwas foundtobeoverexpressed in both the addition to chromatin marginalization (Figure 3(b)). With untreated MCF-7 and HeLa cells (Figures 5(a) and 5(b)). As 6 Journal of Oncology 120 cisplatin was evaluated to potentiate the chemotherapeutic index of cisplatin. The effect of concurrent treatment of MCF-7 and HeLa cells with different sub-lethal concentrations of cisplatin and ENLE for 48 and 24 h, respectively, was analyzed by cell viability assay. It was observed that 1𝜇 M of cisplatin (C1) used in combination with 50 (N1) and 100𝜇 g/mL (N2) ENLE 60 resulted in a significant decrease in cell viability (82 and 71%, resp.) of MCF-7 cells as compared to either of the compounds alone (93.1% for C1 and 96 and 85% for N1 and N2) (Figure 6(a)). In HeLa cells, the combination of 1𝜇 Mof cisplatin (C1) with 10 (N3) and 50𝜇 g/mL (N4) resulted in 61.7 and 60% (for C1N3 and C1N4) significant decrease in cell viability while individual drugs decreased the cell viability by 94.1% for C1 and 84 and 77.3% with N3 and N4, respectively 0 h 48 h 72 h 0 h 24 h 48 h (Figure 6(b)). Also, treatment of MCF-7 and HeLa cells with 5𝜇 M of cisplatin (C2) combined with N1 and N2 and N3 MCF-7 HeLa andN4, respectively,resultedinsynergistic decrease in cell viability (73 and 65% for MCF-7; 51.0 and 52.2% for HeLa) G -G phase G -M phase 0 1 as compared to individual doses (C2 = 87.7% and 81.8% for Sub-G S phase MCF-7 and HeLa) (Figures 6(a) and 6(b)). Combinational Figure 4: ENLE induces apoptosis in MCF-7 and HeLa cells as indices (CI) were calculated and CI were found to be less than analyzed by flow cytometry. Untreated MCF-7 and HeLa cells 1 indicating a synergistic interaction between the two drugs at showed normal distribution of cells in various phases of cell cycle, the doses used for both MCF-7 and HeLa cells. whereas when treated with ENLE at their respective EC doses, there was a significant increase in the number of cells in the sub- 5. Discussion G phase of the cell cycle with increasing time of treatment (48 and 72 hforMCF-7and24and48 hforHeLacells).eTh histogramshows Regardless of recent advances in the prevention and detection %analysisofcells in thedieff rentphasesofthe cell cyclefroma of cancer and development of newer treatment modalities, representative experiment (out of three individual experiments). cancer still as remains one of the most dreadful diseases due to the limitations of available treatment strategies [29, 30]. Research is under way to identify pharmacologically safe showninFigures 5(a) and 5(b), a significant, time-dependent chemopreventive agents that can suppress the carcinogenesis inhibitory effect of ENLE was observed on the expression of process at various stages along with enhancing the thera- cyclin D1 in both of the cell lines compared to untreated cells. peutic effects of conventional cancer therapy by tapping the Bax, the rfi st identified proapoptotic member of the Bcl- potential of combinational approaches utilizing one or more 2 protein family, plays a major role in inducing apoptosis. In synthetic or natural phytochemicals along with an eeff ctive drug such as chemotherapy [18, 23, 31, 32]. both untreated MCF-7 and HeLa cells, the expression of Bax was found to be low which significantly increased in ENLE- The present study focused on the antiproliferative proper- treated MCF-7 and HeLa cells in a time-dependent manner ties of neem as a biosafe chemopreventive agent. It was found that treatment of MCF-7 and HeLa cells with ethanolic neem in comparison to the untreated cells (Figures 5(a) and 5(b)). CYP 1A1 and CYP 1A2 are the members of the cyto- leaf extract (ENLE) inhibited the growth of these cells in a chrome P450 enzyme superfamily which act as drug metabo- dose- and time-dependent manner (Figures 1(a) and 1(b)). The EC (effective concentration, the dose which reduces lizing enzymes and lead to the accumulation reactive oxygen species forming ultimate carcinogens that are toxic to the the viability of cells by 50%) of ENLE was found to be cell and thereby leading to tumorigenesis. Expression of CYP 350𝜇 g/mL on MCF-7 cells and 175𝜇 g/mL on HeLa cells aeft r 1A1 and CYP 1A2 was detected in untreated MCF-7 and 72 and 48 h treatment, respectively. Notably, there was no HeLa cells (Figures 5(a) and 5(b)). However, in comparison significant effect of ENLE on the viability of lymphocytes pointing to its selective cytotoxicity towards the cancer cells to the untreated cells, ENLE treatment resulted in significant downregulation of these genes in both cancer cell lines and, thus, it provides a rationale for development of neem as (Figures 5(a) and 5(b)). a biosafe chemopreventive agent (Figure 1(c)). These results areinlinewithother studieswhich also showed that neem 4.4. ENLE and Cisplatin Infusion Act Synergistically to Inhibit and its derivatives inhibit growth of various cancer cells such the Growth of MCF-7 and HeLa Cells. Since currently avail- as prostate cancer, leukemia cells, head-and-neck squamous able chemotherapeutic drugs are associated with nonspecific cell carcinoma cells, human choriocarcinoma cells, murine cytotoxicity towards normal cells as well as development of Ehrlich’s carcinoma (EC), melanoma cells and exhibited only chemoresistance, a combinational treatment with the natural weak or no cytotoxic effect on normal cells [ 33–38]. dietary agents may serve as a better approach towards cancer Since carcinogenesis is associated with imbalances in treatment. In the present study, a combination of ENLE and theantiapoptotic andproapoptoticmechanismsleading to Cell cycle distribution (%) Journal of Oncology 7 1 2 3 4 1 2 3 4 Cyclin D1 Bax CYP 1A1 CYP 1A2 𝛽 -Actin (a) (b) Figure 5: ENLE-treated MCF-7 (a) and HeLa (b) cells at their respective EC doses (48 h and 72 h for MCF-7 cells and, 24 and 48 h for HeLa cells) show a significant decrease in the expression of cyclin D1, CYP 1A 1, and CYP 1A2 but a significant upregulation in the expression of bax in a time-dependent manner compared to untreated cells.𝛽 -Actin wasusedasaninternalcontrol.Lanes 1–4represent untreatedcells,cells treated with ENLE at their particular time of treatments, and negative control for RT-PCR, respectively. 100 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30 20 20 10 10 0 0 C1 C2 N1 N2 C1N1 C1N2 C2N1 C2N2 C1 C2 N3 N4 C1N3 C1N4 C2N3 C2N4 (a) (b) Figure 6: Simultaneous treatment of MCF-7 (a) and HeLa cells (b) with sublethal doses of cisplatin (C1 and C2) and ENLE (N1 and N2 for MCF-7 and N3 and N4 for HeLa) was found to induce synergistic decrease in viability of these cells (combination index (CI< 1)). Each value is a ratio of the level in the treated cells to that in the untreated control cells. Values are means± SD of 3 independent experiments. Each value with cisplatin and ENLE treatment differs from the control value ( 𝑃<0.05 ). rapid and uncontrolled proliferation of cancer cells, there- cells (Figures 3(a) and 3(b)). These changes which are the fore, inducing cell death is an important aspect in cancer hallmarks of apoptosis accumulated in the ENLE-treated cells prevention and therapy [39–41]. For this reason, the mode of in a time-dependent manner (Figures 3(a) and 3(b)). cell death induced by ENLE in MCF-7 and HeLa cells was Further, these results were verified by cell cycle analysis analyzed by changes in the cellular and nuclear morphology. of MCF-7 and HeLa cells with or without ENLE treatment. MCF-7 and HeLa cells treated with various concentrations Treatmentofthese cellsattheir respective EC concen- (50, 200, and 500𝜇 g/mL) of ENLE for 48 and 72 h and 24 and trations for 48 and 72 h (MCF-7) and 24 and 48 h (HeLa) 48 h, respectively, were examined microscopically. ENLE- correspondingly resulted in increased proportion of cells treated cells showed distinct features such as rounding o,ff in the sub-G phase of the cell cycle in a time-dependent cell shrinkage, and detachment from the matrix, which are manner compared to the untreated cells (Figure 4). These the typical characteristics of cells undergoing programmed results conclusively prove that ENLE induces cell death cell death (apoptosis) compared to untreated cells in which in these cells mediated by the apoptotic pathway which these morphological changes were absent (Figures 2(a) and are in agreement with previous studies that demonstrated 2(b)). Also, ENLE-treated MCF-7 and HeLa cells (at EC apoptosis induction through various mechanisms such as doses) showed discernible variations in the nuclear morphol- inhibiting PI3 K/Akt pathway, decrease in Bcl-2/Bax ratio ogy of these cells, namely, formation of apoptotic bodies, with increased expression of Apaf-1 and caspase-3, and cleav- nuclear condensation, fragmentation, and marginalization age of poly (ADP-ribose) polymerase was the mode of cell in comparison to uniform and intact nuclei of untreated death induced by neem or its derivatives such as nimbolide in Cell viability (%) Cell viability (%) 8 Journal of Oncology various cancers [6, 11, 14–16, 35–37, 42]. u Th s, the activation both of the cancer cell lines (MCF-7 and HeLa cells) along of apoptosisisbelievedtobeacritical therapeutictargetfor with the normal cells (data not shown). In context of the above mentioned facts, this is the rfi st chemoprevention-based therapies. report analyzing the combined effect of ENLE and cisplat- ENLE-induced anticancer effects were then correlated in on MCF-7 and HeLa cells. It was observed that sub- with the modulation of gene expression of various effector lethal doses of ENLE and cisplatin in various combinations molecules involved in cell cycle regulation, apoptosis, and (C1N1, C1N2, C2N1, and C2N2 for MCF-7 and C1N3, C1N4, drug metabolism. Cyclin D1, an important cell cycle regu- C2N3 and C2N4 for HeLa cells) showed enhanced growth lator, is frequently overexpressed in several human cancers inhibitory effects in comparison to the individual doses as including breast and cervical [43, 44]. It was observed that reflected in the CI less than 1 indicating a synergistic interac- untreated MCF-7 and HeLa cells showed a high expression of tion between these drugs at the doses used (Figures 6(a) and cyclin D1, which was significantly downregulated in a time- 6(b)). Veeraraghavan and coworkers (2011) have also shown dependent manner in ENLE-treated cells (Figures 5(a) and that neem induced radiosensitization radiotherapy [19]. Also, 5(b)). eTh se results are consistent with previous studies in neem leaf preparation (NLP) has been shown to prevent which the antiproliferative action of neem and its bioactive the cyclophosphamide, cisplatin, and 5-fluorouracilinduced components was associated with the downregulation of cyclin hematological complications [14, 18]. eTh refore, combina- D1 expressionincancercells [15, 16, 37]. tions of chemopreventive agents with chemotherapeutic Apoptosisistightly regulatedbyanumber of gene prod- drugs may have immense prospects for development of ucts that promote or block cell death at different stages. Bax, a therapeutic strategies to overcome chemotherapy associated proapoptotic gene, commits the cell to undergo programmed resistance and side-effects in human cancers by synergistic cell death in response to a wide range of cytotoxic stimuli [45]. crosstalk between two probable therapies. The untreated MCF-7 and HeLa cells showed low expression of Bax (Figures 5(a) and 5(b)). However, on treatment of these 6. Conclusion cells with ENLE at their respective EC doses, there was a It can be inferred from the present study that neem alone or its significant increase in the Bax gene expression in a time- infusion with cisplatin exhibits antineoplastic eeff cts in breast dependent manner, accounting for the apoptosis-inducing and cervical cancers by inducing apoptosis and modulation of activity of ENLE (Figures 5(a) and 5(b)). eTh se results are in expression of eeff ctor molecules. Thereby, this study provides concordance with previous studies which found that neem a rationale for extensive research and development work on and its component nimbolide upregulate Bax expression in neem for its better therapeutic utilization in cancer preven- human prostrate and colon cancer cells thus proving the tion and treatment. potential of ENLE to induce apoptosis at the molecular level [36, 37, 46, 47]. Conflict of Interests CYP 1A1 and CYP 1A2, members of cytochrome P450 enzyme superfamily, are involved in the oxidative metabolism eTh authors declare that there is no conflict of interests of endogenous compounds, such as steroids and fatty acids, regarding the publication of this paper. andinthe metabolism of foreignchemicals such as drugs, carcinogens, and other environmental pollutants. Increases in their expression have been linked to a higher risk of Acknowledgments malignancies [48]. This is the rfi st study in which the effect The authors are grateful to Dr. Kota Reddy, Academic of ENLE on modulation of expression of CYP 1A1 and President, and Dr. Firdos Alam Khan, Chairperson, Depart- CYP1A2 wasanalyzed. ENLE treatmentresultedinsigni-fi ment of Biotechnology, Manipal University, Dubai, for their cant decrease in their expression in a time-dependent manner constant support and encouragement. as compared to untreated cells which showed relatively higher expression of these genes. This indicates neem can prevent or revert carcinogen induced accumulation of reactive oxygen References metabolites which play a pivotal role in carcinogenesis [49]. [1] K. Biswas,I.Chattopadhyay,R.K.Banerjee, andU.Bandyopad- Other studies have shown similar results in which quercetin, hyay, “Biological activities and medicinal properties of neem azadirachtin, and nimbolide exhibited free radical scaveng- (Azadirachta indica),” Current Science,vol.82, no.11, pp.1336– ing activity by downregulation of CYP 1A1 and 1A2 [50– 1345, 2002. 54]. CYPs have also been correlated with bioactivation or [2] U. Bandyopadhyay, K. Biswas, A. Sengupta et al., “Clinical inactivation of both carcinogens and anticancer drugs and studies on the effect of Neem ( Azadirachta indica)barkextract thus modulation of their expression may be key determinants on gastric secretion and gastroduodenal ulcer,” Life Sciences,vol. of cancer therapy [55, 56]. 75,no. 24,pp. 2867–2878, 2004. Conventional cancer treatments such as chemotherapy [3] B.Sultana,F.Anwar,and R. Przybylski,“Antioxidantactivityof are associated with several cytotoxic effects; hence it was phenolic components present in barks of Azadirachta indica, postulated that these drugs, when combined at lower dose Terminalia arjuna, Acacia nilotica, and Eugenia jambolana with chemopreventive agents such as neem, can minimize the Lam. trees,” Food Chemistry, vol. 104, no. 3, pp. 1106–1114, 2007. cytotoxicity while potentiating the therapeutic index [57]. It [4] P.E.Ebong,I.J.Atangwho, E. U. Eyong, andG.E.Egbung, “eTh was found that cisplatin had nonspecific cytotoxicity towards antidiabetic efficacy of combined extracts from two continental Journal of Oncology 9 plants: Azadirachta indica (A. Juss) (Neem) and Vernonia [17] D. Ghosh, A. Bose, E. Haque, and R. Baral, “Neem (Azadirachta amygdalina (Del.) (African Bitter Leaf),” The American Journal indica) leaf preparation prevents leukocyte apoptosis medi- of Biochemistry and Biotechnology,vol.4,no. 3, pp.239–244, ated by cisplatin plus 5-fluorouracil treatment in swiss mice,” 2008. Chemotherapy,vol.55, no.3,pp. 137–144, 2009. [5] S.Mahapatra,C.Y.F.Young,M.Kohli et al., “Antiangiogenic [18] D. Ezz-Din, M. S. Gabry, A. R. H. Farrag, and A. E. 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Ethanolic Neem (Azadirachta indica) Leaf Extract Prevents Growth of MCF-7 and HeLa Cells and Potentiates the Therapeutic Index of Cisplatin

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Copyright © 2014 Chhavi Sharma et al. This 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.
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Hindawi Publishing Corporation Journal of Oncology Volume 2014, Article ID 321754, 10 pages http://dx.doi.org/10.1155/2014/321754 Research Article Ethanolic Neem (Azadirachta indica) Leaf Extract Prevents Growth of MCF-7 and HeLa Cells and Potentiates the Therapeutic Index of Cisplatin 1 1 1 1 Chhavi Sharma, Andrea J. Vas, Payal Goala, Taher M. Gheewala, 2 1 Tahir A. Rizvi, and Arif Hussain Department of Biotechnology, Manipal University, Dubai International Academic City, P.O. Box 345050, Dubai, UAE Department of Microbiology andImmunology,Faculty of Medicine andHealthScience,UAE University,P.O.Box 17666,AlAin,UAE Correspondence should be addressed to Arif Hussain; dr.ariu fh ssain@yahoo.co.in Received 5 September 2013; Revised 13 November 2013; Accepted 15 November 2013; Published 30 January 2014 Academic Editor: Akira Hara Copyright © 2014 Chhavi Sharma et al. This 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. The present study was designed to gain insight into the antiproliferative activity of ethanolic neem leaves extract (ENLE) alone or in combination with cisplatin by cell viability assay on human breast (MCF-7) and cervical (HeLa) cancer cells. Nuclear morphological examination and cell cycle analysis were performed to determine the mode of cell death. Further, to identify its molecular targets, the expression of genes involved in apoptosis, cell cycle progression, and drug metabolism was analyzed by RT-PCR. Treatment of MCF-7, HeLa, and normal cells with ENLE differentially suppressed the growth of cancer cells in a dose- and time-dependent manner through apoptosis. Additionally, lower dose combinations of ENLE with cisplatin resulted in synergistic growth inhibition of these cells compared to the individual drugs (combination index<1). ENLE significantly modulated the expression of bax, cyclin D1, and cytochrome P450 monooxygenases (CYP 1A1 and CYP 1A2) in a time-dependent manner in these cells. Conclusively, these results emphasize the chemopreventive ability of neem alone or in combination with chemotherapeutic treatment to reduce the cytotoxic effects on normal cells, while potentiating their efficacy at lower doses. us, Th neem may be a prospective therapeutic agent to combat gynecological cancers. 1. Introduction their antioxidant properties by decreasing TNF-𝛼 ,increasing IFN-𝛾 , and modulating antioxidant enzymes such as glu- Therapeutic properties of neem ( Azadirachta indica)have tathione S-transferase (GST) and certain hepatic cytochrome been recognized since ancient times and have been extensive- P450-dependent monooxygenases [7–13]. It induces apopto- ly used in ayurveda, unani, and homoeopathic medicine [1]. sis via both the intrinsic and extrinsic pathways and induces Many compounds such as limonoids, azadirone, azadiracht- cell cycle arrest via p53-dependent p21 accumulation and in, and flavonoids, having therapeutic potential, have been downregulation of the cell cycle regulatory proteins cyclin isolated from various parts of neem tree and have been eval- B, cyclin D1, p53, and proliferating cell nuclear antigen uated for their pharmacological actions and plausible medic- (PCNA) [11, 14–16]. Interestingly, when used in conjunc- inal applications along with their safety evaluation. Recent tion with chemotherapeutic drugs like cyclophosphamide, studies have shown that neem possesses anti-inflammatory, cisplatin, 5-fluorouracil, or with radiotherapy, it potentiates antiarthritic, antipyretic, hypoglycemic, antigastric ulcer, their antitumor eect ff s by activating proapoptotic signaling antifungal, antibacterial, and antitumor activities [2–6]. and negating survival signaling along with attenuating their The antineoplastic properties of neem are gaining side effects [ 14, 17–20]. Notably, cisplatin, the rfi st member attention due to its cancer preventive, tumor-suppressive, of a class of platinum-containing anticancer drugs, is widely antiproliferative, apoptosis-inducing, antiangiogenic, and used for treatment of solid malignancies. Cisplatin has a immunomodulatory effects via several molecular mecha- number of side-effects that can limit its use: nephrotoxicity, nisms [6, 7]. Neem or its derivatives have been shown to exert nausea and vomiting, ototoxicity (hearing loss), electrolyte 2 Journal of Oncology disturbance, and hemolytic anemia, etc. Also, the majority of 570 nm [23]. The effect of ENLE on growth inhibition was cancer patients eventually develop cisplatin-resistant disease assessed as percent cell viability and was calculated as (OD necessitating combination therapy approach using multiple of the drug-treated sample/OD of the nontreated sample) chemotherapeutic agents or combining with chemopreven- × 100, considering that the colorimetric signal is directly tive agents [21, 22]. proportional to the number of viable cells. eTh EC (50% Based on the facts mentioned above, the present study effective concentration) values were calculated from the dose- aims to evaluate the chemopreventive potential of ethanolic response curves. neem leaves extract (ENLE) alone or concurrently with cisplatin on human breast (MCF-7) and cervical (HeLa) 2.4. Calculation of Combination Effects of Cisplatin and ENLE. Calculations of combination eeff cts were expressed as a cancer cells, with the objective of studying its antiproliferative activity on cancer cells while decreasing the cytotoxic effects combination index (CI) as described previously [23]. CI analysis provides qualitative information on the nature of on normal cells. Also, the molecular targets of ENLE were drug interaction, and CI, a numerical value, was calculated delineated to elucidate its in vitro anticancer effects. according to the following equation: 2. Material and Methods C C A,𝑥 B,𝑥 CI= + , (1) IC IC 2.1. Cell Culture. The human breast cancer cell line, MCF-7, 𝑥, A 𝑥, B and human cervical carcinoma cell line, HeLa were main- where C and C are, respectively, the concentrations of A,𝑥 B,𝑥 tained in DMEM (Sigma, USA) supplemented with 10% drugs A and B used in combination to achieve𝑥 %drugeeff ct. fetalbovineserum (FBS)(Sigma, USA)and 100x Pen-strep IC and IC are the concentrations for single agents to 𝑥, A 𝑥, B (Sigma, USA) in a humidified atmosphere of 5% CO in ∘ achieve the same effect. A CI value <1, =1, or>1represents, air at 37 C. Lymphocytes were isolated from healthy non- respectively, synergy, additivity, or antagonism of cisplatin smoking donors using HiSep Media (HiMedia, India) as per and ENLE, respectively. the manufacturer’s instructions [23] and were maintained in RPMI media (Sigma, USA). 2.5. Detection of Apoptosis in MCF-7 and HeLa Cells aeft r 2.2. Preparation of Drug Solutions. 5% ethanolic neem leaves Treatment with ENLE extract (ENLE) was prepared as described previously by Sub- 2.5.1. Microscopic Examination. Morphological changes of apriya and coworkers (2005) with slight modicfi ations [ 24]. MCF-7and HeLa cells were noted on treatment with ENLE Brieyfl , 2.5 g of fresh mature neem leaves was ground to a n fi e at different concentrations (50, 200, and 500 𝜇 g/mL) and paste in 50 mL of 100% ethanol and the slurry was air-dried time-points (48 and 72 h for MCF-7 and 24 and 48 h for in a shaking incubator at 37 C with intermittently stirring HeLa) using normal inverted microscope (Labomed, USA) at 2 h and then left overnight. The powder obtained was (Figures 2(a) and 2(b)). The untreated cells were used as weighed and resuspended in dimethyl sulphoxide (DMSO) negative control. (Sigma, USA) to prepare a stock solution of 80 mg/mL which was filtered through 0.2 𝜇 m filter. Further dilutions were prepared in DMEM to require concentrations between 10 2.5.2. Nuclear Morphological Studies. Apoptosis induction and 500𝜇 g/mL for treatment of MCF-7 cells, HeLa cells, and aer ft treatment of MCF-7 and HeLa cells with ENLE at their respective EC concentrations and varying time-points lymphocytes. A stock solution of 3.3 mM of cisplatin (Cadila Phar- (350𝜇 g/mL for 0, 48, and 72 h in MCF-7 and 175𝜇 g/mL for 0, maceuticals Ltd., India) was used to make drug dilutions of 24, and 48 h in HeLa cells) was evaluated by the nuclear mor- phological changes associated with it using propidium iodide varying concentrations (1–200𝜇 M) in complete medium. staining (Figures 3(a) and 3(b))[22]. Briefly, ∼10 cells/mL cells were seeded on glass coverslips and incubated overnight 2.3. Cell Viability Assay. The effect of ENLE and its combina- in complete medium at 37 C. Further, cells were treated with tion with cisplatin, a chemotherapeutic agent, on the viability ENLE at its EC for above mentioned time periods. At the of MCF-7, HeLa, and lymphocytes was determined by 3- end of the desired time interval, cells were fixed in a mixture [4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide of acetone: methanol (1 : 1) at−20 Cfor 10min, washed with (MTT) assay. eTh cells were plated in triplicate at a density of 1X PBS (pH 7.4) twice, and stained with propidium iodide ∼1× 10 cells/well in 200𝜇 L of complete culture medium con- (10 mg/mL in PBS) for 30 s in dark at RT. eTh coverslips were taining 10–500𝜇 g/mL concentrations of ENLE alone for 48 thoroughly washed with PBS and placed upturned onto a and72 hand24and48 hforMCF-7andHeLa,respectively,or glass slide with mounting media (DPX). Slides were viewed a combination of ENLE (N1M and N2M = 50 and 100𝜇 g/mL; at 515 nm under the Progress Fluorescent Microscope (Olym- N1H and N2H = 10 and 50𝜇 g/mL) with cisplatin (C1M and pus, USA). The images were captured at 40x magnification. C2M = 1 and 10𝜇 M in MCF-7; C1H and C2H = 1 and 5𝜇 M) for 48 and 24 h, respectively, for MCF-7 and HeLa in 96- well microtiter plates. Aeft r incubation for specified times at 2.5.3. Quantification of Apoptotic Cells by Flow Cytome- 37 Cinahumidiefi dincubator,MTT (5mg/mLinPBS)was try. Cell cycle analysis of ENLE-treated MCF-7 and HeLa added to each well and incubated for 2 h. eTh absorbance cells was performed by flow cytometry as described earlier was recorded on a microplate reader at the wavelength of (Figure 4)[23]. After treatment of synchronous cultures of Journal of Oncology 3 110 110 60 60 50 50 0 50 100 150 200 250 300 350 400 450 500 550 0 50 100 150 200 250 300 350 400 450 500 550 ENLE concentration (𝜇 g/mL) ENLE concentration (𝜇 g/mL) 48 h 24 h 72 h 48 h (a) (b) 0 50 100 150 200 250 300 350 400 450 500 550 ENLE concentration (𝜇 g/mL) (c) Figure 1: Differential cytotoxic effect of ENLE on MCF-7, HeLa, and lymphocytes. (a, b) MCF-7 and HeLa cells treated with ENLE at varying concentrations (10–500𝜇 g/mL), resulting in dose- and time-dependent growth inhibition. eTh EC forMCF-7 andHeLacells wasfound to be 350𝜇 g/mL at 72 h and 175𝜇 g/mL at 48 h, respectively. However, lymphocytes did not show significant growth inhibition when treated with similar concentrations of ENLE for 24 h (c). Values are means + SD of three independent experiments. Each value with ENLE treatment differs from the control value ( 𝑃<0.05 ). MCF-7and HeLa cellswithENLEattheir respective EC and HeLa cells was carried out as per the manufacturer’s concentrations at various time-points (350𝜇 g/mL for 0, 48, instructions (GenElute Mammalian Genomic Total RNA Kit, and 72 h in MCF-7 and 175𝜇 g/mL for 0, 24, and 48 h in Sigma, USA) at various time intervals. Further, total RNA HeLa cells), both adherent and floating cells were harvested, was subjected to first strand synthesis as per manufacturer’s washed with phosphate buffered saline (PBS, pH 7.2), and protocol (ProtoScript M-MuLV Taq RT-PCR Kit, New Eng- fixed with ice-cold absolute ethanol at −20 Covernight.Cells land Biolabs, USA) followed by PCR using gene-specific were then washed with PBS prior to resuspending in a buffer primers [23, 25–28].𝛽 -Actin was taken as an internal control. containing PI (50 mg/mL), 0.1% sodium citrate, 0.1% Triton The PCR cycle was as follows: initial denaturation at 95 C X-100, and 100 mg/mL of RNase A. The cells were analyzed for 5 min, followed by 35 amplification cycles (denaturation ∘ ∘ using Flow cytometry (Beckman Coulter flow Cytometer at 94 Cfor 30s; annealingat55 Cfor 𝛽 -actin, CYP 1A1, ∘ ∘ FC500, CXP Version 2.2). eTh data was analyzed using the and CYP 1A2, 56 Cfor Bax, and54 Cfor cyclin D1;and ∘ ∘ Beckman Coulter KALUZA 1.1 analysis software. extension at 72 C for 45 s), with na fi l extension at 72 Cfor 7 min. Amplified products were visualized on a 2% agarose 2.6. Expression Analysis of Various Genes Targeted by ENLE. gel containing ethidium bromide. Reverse transcription PCR was used to detect the expression of Bax, cyclin D1, CYP 1A1, and CYP 1A2 in response to 3. Statistical Analysis treatment with ENLE at EC for varying time intervals (350𝜇 g/mL for 0, 48, and 72 h in MCF-7 and 175𝜇 g/mL for All data are expressed as means± SD of at least 3 experiments. 0, 24, and 48 h in HeLa cells) (Figures 5(a) and 5(b)). Total Fisher’s exact test was adopted for statistical evaluation of the RNA extraction from untreated and ENLE-treated MCF-7 results. Signicfi ant differences were established at 𝑃<0.05 . Cell viability (%) Cell viability (%) Cell viability (%) 4 Journal of Oncology 48 h 72 h 50 𝜇 g/mL 200 𝜇 g/mL 500 𝜇 g/mL (a) 24 h 48 h 50 𝜇 g/mL 200 𝜇 g/mL 500 𝜇 g/mL (b) Figure 2: ENLE-induced morphological changes in MCF-7 (a) and HeLa cells (b) at varying concentrations and time-points. 50, 200, and 500𝜇 g/mL ENLE-treated (a) MCF-7 (for 48 and 72 h) and (b) HeLa cells (24 and 48 h) showed dose- and time-dependent increase in the morphological changes associated with cell death via apoptosis compared to the untreated cells (magnification 100x). 4. Results MCF-7 cells, the EC was observed at 350𝜇 g/mL aer ft 72 h treatment with ENLE, whereas in HeLa cells, it was found to 4.1.ENLEShows SelectiveCytotoxic Eeff cts towardsMCF- be 175𝜇 g/mL in 48 h (Figures 1(a) and 1(b)). 7 and HeLa Cells. The antiproliferative eeff cts of different Notably, to assess if ENLE possesses a safe cytotoxic concentrations of ENLE on MCF-7 cells, HeLa cells, and profile, MTT assay was performed on lymphocytes isolated lymphocyteswereevaluated by theMTT assay. MCF-7and from a healthy nonsmoker adult at similar doses of ENLE (10– HeLa cells treated with increasing concentrations of ENLE 500𝜇 g/mL) (Figure 1(c)). No significant effect on cell viability ranging from 10 to 500𝜇 g/mL showed a dose- and time- wasobservedaeft rtreatmentwithENLEfor24hatthesecon- dependent increase in cell death (Figures 1(a) and 1(b)). In centrations, thereby proving the fact that chemopreventive Journal of Oncology 5 0 h 48 h 72 h (a) 0 h 24 h 48 h (b) Figure 3: The nuclear morphological changes induced by ENLE treatment at various time intervals on (a) MCF-7 (for 0, 48, and 72 h) and (b) HeLa (for 0, 24, and 48 h) cells. Untreated MCF-7 and HeLa cells (0 h) showed large and prominent nuclei, indicating no significant characteristics of apoptosis (white arrows). On the other hand, ENLE treatment of these cells at their respective EC induced time-dependent increase in nuclear morphological changes characteristic of apoptotic cells such as nuclear condensation and fragmentation (green arrows), nuclear marginalization (yellow arrows), and appearance of apoptotic bodies (pink arrows) (magnification 400x). agents like neem can specially target the cancer cells an increase in duration of ENLE exposure, there was a cumu- (Figure 1(c)). This property of neem can be utilized for the lative accrual of the said features consistent with apoptosis in both of the cell lines (Figures 3(a) and 3(b)). purposeofcancertreatment becauseofits safety profile. 4.2.3. Eeff ct of ENLE on the Cell Cycle Distribution. The effect 4.2. ENLE Induces Cell Death via Apoptosis in MCF-7 and of ENLE treatment on the cell cycle distribution of MCF- HeLa Cells 7 and HeLa cells was determined by flow cytometry after treatment of these cells with ENLE at their respective EC 4.2.1. Morphological Changes Induced by ENLE on MCF-7 concentrations for 48 and 72 h for MCF-7 and, 24 and 48 h andHeLaCells. ENLE-treated MCF-7 (for 48 and 72 h) and for HeLa cells. eTh untreated cells (0 h) showed appropriate HeLa (for 24 and 48 h) cells at the concentrations 50, 200, distribution of cells in the different phases of cell cycle and 500𝜇 g/mL were observed under an inverted microscope (Figure 4), while in the case of ENLE-treated cells, there was and their morphological characteristics were noted. In com- a significant time-dependent increase in the number of cells parison to untreated cells, ENLE-treated cells showed typical in the sub-G phase of the cell cycle (17 and 30% for MCF- features of cell death at the morphological level such as 7 after 48 and 72 h and 15 and 29% for HeLa after 24 and rounding off of cells, cell shrinkage, and detachment from the 48 h treatment) (Figure 4). This confirms that ENLE induces substrate which accumulated in a dose- and time-dependent apoptotic cell death in these cells. manner, thus indicating that ENLE induces cell death by apoptosis in these cells (Figures 2(a) and 2(b)). 4.3. ENLE Treatment Signicfi antly Modulates the Expression of Bax, Cyclin D1, CYP 1A1, and CYP 1A2. With the target of 4.2.2. Nuclear Morphological Changes Induced by ENLE on determining the eeff ctor genes involved in ENLE-mediated MCF-7 and HeLa Cells. ENLE-induced nuclear morphologi- cellular responses in MCF-7 and HeLa cells, the expression calchanges characteristic of typicalcellundergoingapoptosis of Bax, cyclin D1, CYP 1A1, and CYP 1A2 was analyzed before were studied in MCF-7 and HeLa cells at their respective and after treatment with ENLE (48 and 72 h treatment for EC at various time-points. Untreated MCF-7 and HeLa MCF-7 cells and 24 and 48 h treatment for HeLa cells).𝛽 - cells appeared uniform in chromatin density with an intact Actin was used as an internal control for comparison of nucleus. However, treatment of MCF-7 cells with ENLE for 48 samples. and 72 h resulted in apoptosis-associated nuclear morpholog- The aberrant expression of cyclin D1, a key player in ical changes like chromatin condensation and fragmentation the progression of the cells from G1 to S phase, has been alongwithappearanceofapoptoticbodies(Figure 3(a)). Also, associated with the deregulated cell cycle control in many HeLa cells treated with ENLE showed similar changes in humancancers.Itwas foundtobeoverexpressed in both the addition to chromatin marginalization (Figure 3(b)). With untreated MCF-7 and HeLa cells (Figures 5(a) and 5(b)). As 6 Journal of Oncology 120 cisplatin was evaluated to potentiate the chemotherapeutic index of cisplatin. The effect of concurrent treatment of MCF-7 and HeLa cells with different sub-lethal concentrations of cisplatin and ENLE for 48 and 24 h, respectively, was analyzed by cell viability assay. It was observed that 1𝜇 M of cisplatin (C1) used in combination with 50 (N1) and 100𝜇 g/mL (N2) ENLE 60 resulted in a significant decrease in cell viability (82 and 71%, resp.) of MCF-7 cells as compared to either of the compounds alone (93.1% for C1 and 96 and 85% for N1 and N2) (Figure 6(a)). In HeLa cells, the combination of 1𝜇 Mof cisplatin (C1) with 10 (N3) and 50𝜇 g/mL (N4) resulted in 61.7 and 60% (for C1N3 and C1N4) significant decrease in cell viability while individual drugs decreased the cell viability by 94.1% for C1 and 84 and 77.3% with N3 and N4, respectively 0 h 48 h 72 h 0 h 24 h 48 h (Figure 6(b)). Also, treatment of MCF-7 and HeLa cells with 5𝜇 M of cisplatin (C2) combined with N1 and N2 and N3 MCF-7 HeLa andN4, respectively,resultedinsynergistic decrease in cell viability (73 and 65% for MCF-7; 51.0 and 52.2% for HeLa) G -G phase G -M phase 0 1 as compared to individual doses (C2 = 87.7% and 81.8% for Sub-G S phase MCF-7 and HeLa) (Figures 6(a) and 6(b)). Combinational Figure 4: ENLE induces apoptosis in MCF-7 and HeLa cells as indices (CI) were calculated and CI were found to be less than analyzed by flow cytometry. Untreated MCF-7 and HeLa cells 1 indicating a synergistic interaction between the two drugs at showed normal distribution of cells in various phases of cell cycle, the doses used for both MCF-7 and HeLa cells. whereas when treated with ENLE at their respective EC doses, there was a significant increase in the number of cells in the sub- 5. Discussion G phase of the cell cycle with increasing time of treatment (48 and 72 hforMCF-7and24and48 hforHeLacells).eTh histogramshows Regardless of recent advances in the prevention and detection %analysisofcells in thedieff rentphasesofthe cell cyclefroma of cancer and development of newer treatment modalities, representative experiment (out of three individual experiments). cancer still as remains one of the most dreadful diseases due to the limitations of available treatment strategies [29, 30]. Research is under way to identify pharmacologically safe showninFigures 5(a) and 5(b), a significant, time-dependent chemopreventive agents that can suppress the carcinogenesis inhibitory effect of ENLE was observed on the expression of process at various stages along with enhancing the thera- cyclin D1 in both of the cell lines compared to untreated cells. peutic effects of conventional cancer therapy by tapping the Bax, the rfi st identified proapoptotic member of the Bcl- potential of combinational approaches utilizing one or more 2 protein family, plays a major role in inducing apoptosis. In synthetic or natural phytochemicals along with an eeff ctive drug such as chemotherapy [18, 23, 31, 32]. both untreated MCF-7 and HeLa cells, the expression of Bax was found to be low which significantly increased in ENLE- The present study focused on the antiproliferative proper- treated MCF-7 and HeLa cells in a time-dependent manner ties of neem as a biosafe chemopreventive agent. It was found that treatment of MCF-7 and HeLa cells with ethanolic neem in comparison to the untreated cells (Figures 5(a) and 5(b)). CYP 1A1 and CYP 1A2 are the members of the cyto- leaf extract (ENLE) inhibited the growth of these cells in a chrome P450 enzyme superfamily which act as drug metabo- dose- and time-dependent manner (Figures 1(a) and 1(b)). The EC (effective concentration, the dose which reduces lizing enzymes and lead to the accumulation reactive oxygen species forming ultimate carcinogens that are toxic to the the viability of cells by 50%) of ENLE was found to be cell and thereby leading to tumorigenesis. Expression of CYP 350𝜇 g/mL on MCF-7 cells and 175𝜇 g/mL on HeLa cells aeft r 1A1 and CYP 1A2 was detected in untreated MCF-7 and 72 and 48 h treatment, respectively. Notably, there was no HeLa cells (Figures 5(a) and 5(b)). However, in comparison significant effect of ENLE on the viability of lymphocytes pointing to its selective cytotoxicity towards the cancer cells to the untreated cells, ENLE treatment resulted in significant downregulation of these genes in both cancer cell lines and, thus, it provides a rationale for development of neem as (Figures 5(a) and 5(b)). a biosafe chemopreventive agent (Figure 1(c)). These results areinlinewithother studieswhich also showed that neem 4.4. ENLE and Cisplatin Infusion Act Synergistically to Inhibit and its derivatives inhibit growth of various cancer cells such the Growth of MCF-7 and HeLa Cells. Since currently avail- as prostate cancer, leukemia cells, head-and-neck squamous able chemotherapeutic drugs are associated with nonspecific cell carcinoma cells, human choriocarcinoma cells, murine cytotoxicity towards normal cells as well as development of Ehrlich’s carcinoma (EC), melanoma cells and exhibited only chemoresistance, a combinational treatment with the natural weak or no cytotoxic effect on normal cells [ 33–38]. dietary agents may serve as a better approach towards cancer Since carcinogenesis is associated with imbalances in treatment. In the present study, a combination of ENLE and theantiapoptotic andproapoptoticmechanismsleading to Cell cycle distribution (%) Journal of Oncology 7 1 2 3 4 1 2 3 4 Cyclin D1 Bax CYP 1A1 CYP 1A2 𝛽 -Actin (a) (b) Figure 5: ENLE-treated MCF-7 (a) and HeLa (b) cells at their respective EC doses (48 h and 72 h for MCF-7 cells and, 24 and 48 h for HeLa cells) show a significant decrease in the expression of cyclin D1, CYP 1A 1, and CYP 1A2 but a significant upregulation in the expression of bax in a time-dependent manner compared to untreated cells.𝛽 -Actin wasusedasaninternalcontrol.Lanes 1–4represent untreatedcells,cells treated with ENLE at their particular time of treatments, and negative control for RT-PCR, respectively. 100 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30 20 20 10 10 0 0 C1 C2 N1 N2 C1N1 C1N2 C2N1 C2N2 C1 C2 N3 N4 C1N3 C1N4 C2N3 C2N4 (a) (b) Figure 6: Simultaneous treatment of MCF-7 (a) and HeLa cells (b) with sublethal doses of cisplatin (C1 and C2) and ENLE (N1 and N2 for MCF-7 and N3 and N4 for HeLa) was found to induce synergistic decrease in viability of these cells (combination index (CI< 1)). Each value is a ratio of the level in the treated cells to that in the untreated control cells. Values are means± SD of 3 independent experiments. Each value with cisplatin and ENLE treatment differs from the control value ( 𝑃<0.05 ). rapid and uncontrolled proliferation of cancer cells, there- cells (Figures 3(a) and 3(b)). These changes which are the fore, inducing cell death is an important aspect in cancer hallmarks of apoptosis accumulated in the ENLE-treated cells prevention and therapy [39–41]. For this reason, the mode of in a time-dependent manner (Figures 3(a) and 3(b)). cell death induced by ENLE in MCF-7 and HeLa cells was Further, these results were verified by cell cycle analysis analyzed by changes in the cellular and nuclear morphology. of MCF-7 and HeLa cells with or without ENLE treatment. MCF-7 and HeLa cells treated with various concentrations Treatmentofthese cellsattheir respective EC concen- (50, 200, and 500𝜇 g/mL) of ENLE for 48 and 72 h and 24 and trations for 48 and 72 h (MCF-7) and 24 and 48 h (HeLa) 48 h, respectively, were examined microscopically. ENLE- correspondingly resulted in increased proportion of cells treated cells showed distinct features such as rounding o,ff in the sub-G phase of the cell cycle in a time-dependent cell shrinkage, and detachment from the matrix, which are manner compared to the untreated cells (Figure 4). These the typical characteristics of cells undergoing programmed results conclusively prove that ENLE induces cell death cell death (apoptosis) compared to untreated cells in which in these cells mediated by the apoptotic pathway which these morphological changes were absent (Figures 2(a) and are in agreement with previous studies that demonstrated 2(b)). Also, ENLE-treated MCF-7 and HeLa cells (at EC apoptosis induction through various mechanisms such as doses) showed discernible variations in the nuclear morphol- inhibiting PI3 K/Akt pathway, decrease in Bcl-2/Bax ratio ogy of these cells, namely, formation of apoptotic bodies, with increased expression of Apaf-1 and caspase-3, and cleav- nuclear condensation, fragmentation, and marginalization age of poly (ADP-ribose) polymerase was the mode of cell in comparison to uniform and intact nuclei of untreated death induced by neem or its derivatives such as nimbolide in Cell viability (%) Cell viability (%) 8 Journal of Oncology various cancers [6, 11, 14–16, 35–37, 42]. u Th s, the activation both of the cancer cell lines (MCF-7 and HeLa cells) along of apoptosisisbelievedtobeacritical therapeutictargetfor with the normal cells (data not shown). In context of the above mentioned facts, this is the rfi st chemoprevention-based therapies. report analyzing the combined effect of ENLE and cisplat- ENLE-induced anticancer effects were then correlated in on MCF-7 and HeLa cells. It was observed that sub- with the modulation of gene expression of various effector lethal doses of ENLE and cisplatin in various combinations molecules involved in cell cycle regulation, apoptosis, and (C1N1, C1N2, C2N1, and C2N2 for MCF-7 and C1N3, C1N4, drug metabolism. Cyclin D1, an important cell cycle regu- C2N3 and C2N4 for HeLa cells) showed enhanced growth lator, is frequently overexpressed in several human cancers inhibitory effects in comparison to the individual doses as including breast and cervical [43, 44]. It was observed that reflected in the CI less than 1 indicating a synergistic interac- untreated MCF-7 and HeLa cells showed a high expression of tion between these drugs at the doses used (Figures 6(a) and cyclin D1, which was significantly downregulated in a time- 6(b)). Veeraraghavan and coworkers (2011) have also shown dependent manner in ENLE-treated cells (Figures 5(a) and that neem induced radiosensitization radiotherapy [19]. Also, 5(b)). eTh se results are consistent with previous studies in neem leaf preparation (NLP) has been shown to prevent which the antiproliferative action of neem and its bioactive the cyclophosphamide, cisplatin, and 5-fluorouracilinduced components was associated with the downregulation of cyclin hematological complications [14, 18]. eTh refore, combina- D1 expressionincancercells [15, 16, 37]. tions of chemopreventive agents with chemotherapeutic Apoptosisistightly regulatedbyanumber of gene prod- drugs may have immense prospects for development of ucts that promote or block cell death at different stages. Bax, a therapeutic strategies to overcome chemotherapy associated proapoptotic gene, commits the cell to undergo programmed resistance and side-effects in human cancers by synergistic cell death in response to a wide range of cytotoxic stimuli [45]. crosstalk between two probable therapies. The untreated MCF-7 and HeLa cells showed low expression of Bax (Figures 5(a) and 5(b)). However, on treatment of these 6. Conclusion cells with ENLE at their respective EC doses, there was a It can be inferred from the present study that neem alone or its significant increase in the Bax gene expression in a time- infusion with cisplatin exhibits antineoplastic eeff cts in breast dependent manner, accounting for the apoptosis-inducing and cervical cancers by inducing apoptosis and modulation of activity of ENLE (Figures 5(a) and 5(b)). eTh se results are in expression of eeff ctor molecules. Thereby, this study provides concordance with previous studies which found that neem a rationale for extensive research and development work on and its component nimbolide upregulate Bax expression in neem for its better therapeutic utilization in cancer preven- human prostrate and colon cancer cells thus proving the tion and treatment. potential of ENLE to induce apoptosis at the molecular level [36, 37, 46, 47]. Conflict of Interests CYP 1A1 and CYP 1A2, members of cytochrome P450 enzyme superfamily, are involved in the oxidative metabolism eTh authors declare that there is no conflict of interests of endogenous compounds, such as steroids and fatty acids, regarding the publication of this paper. andinthe metabolism of foreignchemicals such as drugs, carcinogens, and other environmental pollutants. Increases in their expression have been linked to a higher risk of Acknowledgments malignancies [48]. This is the rfi st study in which the effect The authors are grateful to Dr. Kota Reddy, Academic of ENLE on modulation of expression of CYP 1A1 and President, and Dr. Firdos Alam Khan, Chairperson, Depart- CYP1A2 wasanalyzed. ENLE treatmentresultedinsigni-fi ment of Biotechnology, Manipal University, Dubai, for their cant decrease in their expression in a time-dependent manner constant support and encouragement. as compared to untreated cells which showed relatively higher expression of these genes. This indicates neem can prevent or revert carcinogen induced accumulation of reactive oxygen References metabolites which play a pivotal role in carcinogenesis [49]. [1] K. Biswas,I.Chattopadhyay,R.K.Banerjee, andU.Bandyopad- Other studies have shown similar results in which quercetin, hyay, “Biological activities and medicinal properties of neem azadirachtin, and nimbolide exhibited free radical scaveng- (Azadirachta indica),” Current Science,vol.82, no.11, pp.1336– ing activity by downregulation of CYP 1A1 and 1A2 [50– 1345, 2002. 54]. CYPs have also been correlated with bioactivation or [2] U. Bandyopadhyay, K. Biswas, A. 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