EthanolicNeem (Azadirachtaindica) Leaf Extract Prevents Growth of MCF-7 and HeLa Cells and Potentiates the Therapeutic Index of Cisplatin

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. Thus, neem may be a prospective therapeutic agent to combat gynecological cancers.

1. Introduction

Therapeutic properties of neem (Azadirachta indica) have been recognized since ancient times and have been extensively used in ayurveda, unani, and homoeopathic medicine [1]. Many compounds such as limonoids, azadirone, azadirachtin, and flavonoids, having therapeutic potential, have been isolated from various parts of neem tree and have been evaluated for their pharmacological actions and plausible medicinal applications along with their safety evaluation. Recent studies have shown that neem possesses anti-inflammatory, antiarthritic, antipyretic, hypoglycemic, antigastric ulcer, antifungal, antibacterial, and antitumor activities [2–6].

The antineoplastic properties of neem are gaining attention due to its cancer preventive, tumor-suppressive, antiproliferative, apoptosis-inducing, antiangiogenic, and immunomodulatory effects via several molecular mechanisms [6, 7]. Neem or its derivatives have been shown to exert their antioxidant properties by decreasing TNF-α, increasing IFN-γ, and modulating antioxidant enzymes such as glutathione S-transferase (GST) and certain hepatic cytochrome P450-dependent monooxygenases [7–13]. It induces apoptosis via both the intrinsic and extrinsic pathways and induces cell cycle arrest via p53-dependent p21 accumulation and downregulation of the cell cycle regulatory proteins cyclin B, cyclin D1, p53, and proliferating cell nuclear antigen (PCNA) [11, 14–16]. Interestingly, when used in conjunction with chemotherapeutic drugs like cyclophosphamide, cisplatin, 5-fluorouracil, or with radiotherapy, it potentiates their antitumor effects by activating proapoptotic signaling and negating survival signaling along with attenuating their side effects [14, 17–20]. Notably, cisplatin, the first member of a class of platinum-containing anticancer drugs, is widely used for treatment of solid malignancies. Cisplatin has a number of side-effects that can limit its use: nephrotoxicity, nausea and vomiting, ototoxicity (hearing loss), electrolyte disturbance, and hemolytic anemia, etc. Also, the majority of cancer patients eventually develop cisplatin-resistant disease necessitating combination therapy approach using multiple chemotherapeutic agents or combining with chemopreventive agents [21, 22].

Based on the facts mentioned above, the present study aims to evaluate the chemopreventive potential of ethanolic neem leaves extract (ENLE) alone or concurrently with cisplatin on human breast (MCF-7) and cervical (HeLa) cancer cells, with the objective of studying its antiproliferative activity on cancer cells while decreasing the cytotoxic effects on normal cells. Also, the molecular targets of ENLE were delineated to elucidate its in vitro anticancer effects.

2. Material and Methods2.1. Cell Culture

The human breast cancer cell line, MCF-7, and human cervical carcinoma cell line, HeLa were maintained in DMEM (Sigma, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma, USA) and 100x Pen-strep (Sigma, USA) in a humidified atmosphere of 5% CO2 in air at 37°C. Lymphocytes were isolated from healthy non-smoking donors using HiSep Media (HiMedia, India) as per the manufacturer’s instructions [23] and were maintained in RPMI media (Sigma, USA).

2.2. Preparation of Drug Solutions

5% ethanolic neem leaves extract (ENLE) was prepared as described previously by Subapriya and coworkers (2005) with slight modifications [24]. Briefly, 2.5 g of fresh mature neem leaves was ground to a fine paste in 50 mL of 100% ethanol and the slurry was air-dried in a shaking incubator at 37°C with intermittently stirring at 2 h and then left overnight. The powder obtained was weighed and resuspended in dimethyl sulphoxide (DMSO) (Sigma, USA) to prepare a stock solution of 80 mg/mL which was filtered through 0.2 μm filter. Further dilutions were prepared in DMEM to require concentrations between 10 and 500 μg/mL for treatment of MCF-7 cells, HeLa cells, and lymphocytes.

A stock solution of 3.3 mM of cisplatin (Cadila Pharmaceuticals Ltd., India) was used to make drug dilutions of varying concentrations (1–200 μM) in complete medium.

2.3. Cell Viability Assay

The effect of ENLE and its combination with cisplatin, a chemotherapeutic agent, on the viability of MCF-7, HeLa, and lymphocytes was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide (MTT) assay. The cells were plated in triplicate at a density of ~1 × 104 cells/well in 200 μL of complete culture medium containing 10–500 μg/mL concentrations of ENLE alone for 48 and 72 h and 24 and 48 h for MCF-7 and HeLa, respectively, or a combination of ENLE (N1M and N2M = 50 and 100 μg/mL; N1H and N2H = 10 and 50 μg/mL) with cisplatin (C1M and C2M = 1 and 10 μM in MCF-7; C1H and C2H = 1 and 5 μM) for 48 and 24 h, respectively, for MCF-7 and HeLa in 96-well microtiter plates. After incubation for specified times at 37°C in a humidified incubator, MTT (5 mg/mL in PBS) was added to each well and incubated for 2 h. The absorbance was recorded on a microplate reader at the wavelength of 570 nm [23]. The effect of ENLE on growth inhibition was assessed as percent cell viability and was calculated as (OD of the drug-treated sample/OD of the nontreated sample) × 100, considering that the colorimetric signal is directly proportional to the number of viable cells. The EC50 (50% effective concentration) values were calculated from the dose-response curves.

2.4. Calculation of Combination Effects of Cisplatin and ENLE

Calculations of combination effects were expressed as a combination index (CI) as described previously [23]. CI analysis provides qualitative information on the nature of drug interaction, and CI, a numerical value, was calculated according to the following equation: where and are, respectively, the concentrations of drugs A and B used in combination to achieve drug effect. and are the concentrations for single agents to achieve the same effect. A CI value <1, =1, or >1 represents, respectively, synergy, additivity, or antagonism of cisplatin and ENLE, respectively.

2.5. Detection of Apoptosis in MCF-7 and HeLa Cells after Treatment with ENLE2.5.1. Microscopic Examination

Morphological changes of MCF-7and HeLa cells were noted on treatment with ENLE at different concentrations (50, 200, and 500 μg/mL) and time-points (48 and 72 h for MCF-7 and 24 and 48 h for HeLa) using normal inverted microscope (Labomed, USA) (Figures 2(a) and 2(b)). The untreated cells were used as negative control.

2.5.2. Nuclear Morphological Studies

Apoptosis induction after treatment of MCF-7 and HeLa cells with ENLE at their respective EC50 concentrations and varying time-points (350 μg/mL for 0, 48, and 72 h in MCF-7 and 175 μg/mL for 0, 24, and 48 h in HeLa cells) was evaluated by the nuclear morphological changes associated with it using propidium iodide staining (Figures 3(a) and 3(b)) [22]. Briefly, ~106 cells/mL cells were seeded on glass coverslips and incubated overnight in complete medium at 37°C. Further, cells were treated with ENLE at its EC50 for above mentioned time periods. At the end of the desired time interval, cells were fixed in a mixture of acetone: methanol (1 : 1) at −20°C for 10 min, washed with 1X PBS (pH 7.4) twice, and stained with propidium iodide (10 mg/mL in PBS) for 30 s in dark at RT. The coverslips were thoroughly washed with PBS and placed upturned onto a glass slide with mounting media (DPX). Slides were viewed at 515 nm under the Progress Fluorescent Microscope (Olympus, USA). The images were captured at 40x magnification.

2.5.3. Quantification of Apoptotic Cells by Flow Cytometry

Cell cycle analysis of ENLE-treated MCF-7 and HeLa cells was performed by flow cytometry as described earlier (Figure 4) [23]. After treatment of synchronous cultures of MCF-7 and HeLa cells with ENLE at their respective EC50 concentrations at various time-points (350 μg/mL for 0, 48, and 72 h in MCF-7 and 175 μg/mL for 0, 24, and 48 h in HeLa cells), both adherent and floating cells were harvested, washed with phosphate buffered saline (PBS, pH 7.2), and fixed with ice-cold absolute ethanol at −20°C overnight. Cells were then washed with PBS prior to resuspending in a buffer containing PI (50 mg/mL), 0.1% sodium citrate, 0.1% Triton X-100, and 100 mg/mL of RNase A. The cells were analyzed using Flow cytometry (Beckman Coulter flow Cytometer FC500, CXP Version 2.2). The data was analyzed using the Beckman Coulter KALUZA 1.1 analysis software.

2.6. Expression Analysis of Various Genes Targeted by ENLE

Reverse transcription PCR was used to detect the expression of Bax, cyclin D1, CYP 1A1, and CYP 1A2 in response to treatment with ENLE at EC50 for varying time intervals (350 μg/mL for 0, 48, and 72 h in MCF-7 and 175 μg/mL for 0, 24, and 48 h in HeLa cells) (Figures 5(a) and 5(b)). Total RNA extraction from untreated and ENLE-treated MCF-7 and HeLa cells was carried out as per the manufacturer’s instructions (GenElute Mammalian Genomic Total RNA Kit, Sigma, USA) at various time intervals. Further, total RNA was subjected to first strand synthesis as per manufacturer’s protocol (ProtoScript M-MuLV Taq RT-PCR Kit, New England Biolabs, USA) followed by PCR using gene-specific primers [23, 25–28]. β-Actin was taken as an internal control. The PCR cycle was as follows: initial denaturation at 95°C for 5 min, followed by 35 amplification cycles (denaturation at 94°C for 30 s; annealing at 55°C for β-actin, CYP 1A1, and CYP 1A2, 56°C for Bax, and 54°C for cyclin D1; and extension at 72°C for 45 s), with final extension at 72°C for 7 min. Amplified products were visualized on a 2% agarose gel containing ethidium bromide.

3. Statistical Analysis

All data are expressed as means ± SD of at least 3 experiments. Fisher’s exact test was adopted for statistical evaluation of the results. Significant differences were established at .

4. Results 4.1. ENLE Shows Selective Cytotoxic Effects towards MCF-7 and HeLa Cells

The antiproliferative effects of different concentrations of ENLE on MCF-7 cells, HeLa cells, and lymphocytes were evaluated by the MTT assay. MCF-7 and HeLa cells treated with increasing concentrations of ENLE ranging from 10 to 500 μg/mL showed a dose- and time-dependent increase in cell death (Figures 1(a) and 1(b)). In MCF-7 cells, the EC50 was observed at 350 μg/mL after 72 h treatment with ENLE, whereas in HeLa cells, it was found to be 175 μg/mL in 48 h (Figures 1(a) and 1(b)).