INTRODUCTION:

Ovarian cancer has been known as the second most common gynaecological cancer and also deadliest^[1]. Due to the lack of specific symptoms and the tendency of the tumor cells for early peritoneal dissemination, nearly 75% of patients are found at advanced stage^[2]. Almost all ovarian cancers are asymptomatic and hence nearly 60% of patients are at stage III and IV at time of diagnosis^[3].Currently, therapies include primary cytoreductive surgery in addition with chemotherapy that includes paclitaxel with platinum^[4]. High dose of chemotherapeutic drugs may damage ovarian tissues as a consequence of the compound’s side effects^[5]. Survival rates are also reduced due to the development of drug resistance, which thereby limits future treatment. Therefore, discovery and development of novel therapeutic compounds are urgently in need for the survival and treatment of patients with ovarian cancer.

Natural compounds derived from food have been investigated as new sources of chemotherapy drugs. These agents act in a manner similar to the conventional chemotherapy drugs by disrupting the cell cycle or inducing apoptosis^[6;7]. Apoptosis is a sequential process of programmed cell death that is involved in many cellular processes including tumor suppression. Hence, development of natural and less toxic anti-cancer agents that promote apoptosis in tumor cells has been intensively investigated.

Naringin is a citrus bioflavonoid that has been investigated to be an antioxidant, anti-inflammatory, and also as an anticancer compound^[8;9]. Naringin has been documented to suppress various tumors like breast, and melanoma^[10]It has also been reported that naringin could inhibit the metastasis of human glioblastoma cells via inactivation of p38 signaling pathway^[9]. There were also reports showed that naringin could suppress PI3K/AKT pathway, c-Myc pathway and c-Srcpathways^{[8;9;11;12;13]}

In this study, we determined the effect of naringin on cell proliferation inhibition in PA-1 ovarian cancer cell line. Furthermore, to establish the anticancer mechanism of naringin, we determined the expression levels of apoptosis related molecules, which are strongly associated with the anti-proliferative effect of the compound in ovarian cancer.

MATERIALS AND METHODS:

Chemicals:

Naringin, trypan blue solution, dimethyl sulfoxide (DMSO) were obtained from Sigma. Penicillin, streptomycin, fetal bovine serum (FBS), Dulbecco’s modified Eagle medium (DMEM) were purchased from Gibco. For immunoblotting, anti-Bax, anti-Bcl-2, anti-Caspase-3, anti-β-actin and secondary anti-mouse antibodies were purchased from Santa Cruz Biotechnology, Inc.

Cell culture:

Human ovarian cancer cell line PA-1 was obtained from National Centre for Cell Science (NCCS), Pune, India and maintained in DMEM supplemented with 10% FBS and antibiotics. Cells were incubated at 37⁰C and 5% CO₂.

Cell viability assay:

Cell viability following naringin treatment was estimated by Trypan blue exclusion assay. Briefly, cells were cultured in 6-well plates 2.5 x 10⁵ cells per well and treated with increasing concentrations of naringin (0-100μM) for 24, 48 and 72hrs. Following that, cells were trypsinized and Trypan blue reagent was added to count the number of viable cells by hemocytometer to calculate viability. Data from the naringin-treated samples are normalized to the untreated samples.

Western blotting:

PA-1 cells were cultured in T25 flasks and treated with 10, 20 and 30μM of naringin for 48 hrs. After treatment, cell lysates were obtained and centrifuged to remove cell debris. 50μg of proteins were heated to 95⁰C for 5 minutes and separated on polyacrylamide gels. Proteins were then transferred to nitrocellulose membranes (BioRad) and blocked for an hour at room temperature with 5% bovine serum albumin (HiMedia). Following that, membranes were incubated with specific primary antibody solutions (anti-Bax, anti-Bcl-2, anti-β-actin and anti-Caspase-3) overnight and washed with 1X TBST thrice and then incubated with secondary anti-mouse antibody solutions for an hour. Followed by three washes with 1XTBST, protein bands were developed by enhanced chemiluminescence staining captured on (Thermofischer) and quantified with densitometric images.

RESULTS:

Naringin inhibits the proliferation of PA-1 ovarian cancer cells:

Trypan blue assay was used for the confirmation of the anti-proliferative effect of naringin in PA-1 cells. The result suggest that proliferation and growth rates of PA-1 cells were significantly suppressed upon naringin treatment.

Figure 1. Effect of Naringin on viability of PA-1 cells. Cells were treated with different concentrations of Naringin (0-100µM) for 48 hrs and analyzed using Trypan blue assay. Data are mean ± SD of triplicate determinations.

Figure 2. PA-1 cells were treated with different concentrations of Naringin (10µM, 20µM,30µM) for 48hrs. Cell lysates were analyzed by western blot for apoptosis-related protein expression: Bcl-2, Bax, cleaved caspase-3 and β-actin. Results were expressed as the Mean±SD. ^**p<0.01, compared with control.

Naringin triggers apoptosis in PA-1 ovarian cancer cells:

Studies have reported the cytotoxic effects of naringin against numerous types of cancer cells, yet the mechanisms are not completely understood. Therefore, we examined the dose-dependent effects of naringin on PA-1 ovarian cancer cells. PA-1 cells were treated with doses of 10, 20 and 30μM of naringin for 48hrs. To further explore, whether naringin could initiate apoptotic cell death in ovarian cancer, Western blot analyses was used to analyse the changes in the expressions of critical apoptotic markers (Fig 2).

We found that treatment with naringin increased the expression of pro-apoptotic protein Bax and the executioner caspase-3, whereas decreased the expression of anti-apoptotic protein Bcl-2. These findings suggest that naringin promoted the activation of intrinsic apoptotic pathway and the potential signaling cascades altered are to be explored.

DISCUSSION:

Many researchers have investigated micro-phytochemicals in the diet, and plants are potential agents for the prophylactic and/or in the treatment of various cancers^[14]. Flavonoids, which are abundant in vegetables, fruits, and medicinal herbs, have antimicrobial and antioxidant effects^[15].Naringin is one of the most abundant flavonoids with anti-tumor activity in various cancers like, breast, bladder and colon^{[11;12;16;17]}. Although the anti-cancer effects have been studied in various cancers, induction of apoptosis in ovarian cancer is limited.

In the present study, we confirm the anti-cancer effect of naringin in PA-1 ovarian cancer cell line. The results of this study support our hypothesis that naringin reduces progression of ovarian cancer cells by blocking their proliferation and inducing apoptosis through altering the pro- and anti-apoptotic protein expressions.

We examined the anti-proliferative effect of naringin using Trypan blue assay. In the analysis, we found a profound cytotoxic effect by a dose-dependent manner for 48h. Our results suggested that naringin inhibited cancer cell growth and proliferation by the anti-proliferative assays and therefore we analysed the mode of cell death.

Cell death may occur in various forms depending on the response to stimuli and different stress. Therefore, we evaluated whether naringin could induce apoptotic cell death in PA-1 cells. Apoptosis is a classical pathway programmed in the cells to undergo a regulated form of cell death upon stimulation of numerous signaling cascades. This process depicts a key mechanism of action mediated by chemotherapeutic drugs to fight cancer cells^[18]. Tumor cells can obtain resistance to apoptosis by increase expression or activation of anti-apoptotic proteins like Bcl-2 or by the decreased expression or mutation of pro-apoptotic proteins like Bax. Bax oligomers assemble within the mitochondrial outer membrane that leads to cytochrome C release into cytosol, which consequently activates caspase-9 that triggers caspase-3 activation which executes apoptosis^[19;20]. Upon naringin treatment, we found that Bax and caspase-3 expressions were increased whereas Bcl-2 expression was decreased in a dose-dependent manner. Naringin has been studied to induce apoptosis in various cancers like, hepatocellular and cervical^[21;22]. Therefore, our study reveals that naringin might a potential therapeutic agent targeting Bax/Bcl-2 and could be effective in ovarian cancer treatment.

CONFLICTS OF INTEREST:

The authors declare that there are no conflicts of interest.

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Received on 14.11.2019 Modified on 28.12.2019

Research J. Pharm. and Tech 2020; 13(9):4051-4054.

DOI: 10.5958/0974-360X.2020.00716.7