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Introduction

Cervical cancer one of the most frequently detected cancers in women around the globe and ranks third among all the cancers diagnosed in women. Approximately 0.5 million patients are diagnosed for this lethal type of cancer that is almost 9% of all the new cancer cases diagnosed annually (1). Although, the currently used treatment options such as chemotherapy, radical hysterectomy and radiotherapy have shown promising outcomes, approximately 0.3 millions deaths still occur due to cervical cancer annually (2). For the early stage cervical cancers mainly involves office-based ablative therapies. Cold-knife conization or electroconization is performed in most of the patients to exclude invasive disease. Furthermore, surgery followed by chemotherapy is recommended for patients with advanced stage cervical cancer (2). However the chemotherapeutic agents exhibit adverse effect that compromise the health of the patients and as such Therefore, to there is an urgent need to identify novel molecules that could prove efficient in the treatment of cervical cancer with minimal side effects (3).

The molecules derived from plants are considered prospective anticancerous agents and are therefore being screened for their anticancer activity every now and then. Bergenin is considered as one of the rare acylated iridoid glucosides isolated from some species of Bergenia (4,5). It has been reported to exhibit a diversity of pharmacological properties. Recently it was found to exhibit significant pharmacological activities such as anticancer (6). However, the anticancer activity of bergenin against cervical cancer cells has not been evaluated so far. In the present study we evaluated the anticancer effects of bergenin against cervical cancer cell line HeLa and normal cervical cell line HCerEpiC. In this study we report that bergenin inhibits the proliferation of cervical HeLa cancer cells. The anticancer effects are mainly due to the induction of apoptosis and cell cycle arrest in the G0/G1 phase. Moreover, bergenin exhibited the capacity to inhibit the migration of the HeLa cells. The effects of bergenin were also evaluated on the STAT3 signalling pathway (7). It was observed that bergenin inhibited the phosphorylation of STAT3 proteins. Taken together, this study reveals that bergenin is an important candidate for the treatment of cervical cancer.

Materials and methods

Chemicals, reagents and culture conditions

All the chemicals and reagents that were used in the present study were obtained from Sigma-Aldrich, Merck KGaA, Darmstadt, Germany. Bergenin (95% purity by HPLC, CAS no.: 477-90-7) was obtained from Sigma-Aldrich, Merck KGaA. Primary and secondary antibodies were purchased from Santa Cruz Biotechnology Inc., Dallas, TX, USA. Human cervical cancer HeLa and non-cancerous human cervical epithelial cells (HCerEpiC) cell lines were purchased from Type Culture Collection of Chinese Academy of Sciences, Shanghai, China. The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, penicillin and streptomycin (100 U/ml each) and maintained in a humidified atmosphere containing 5% CO2.

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay for cell viability

The cell viability was assessed by MTT assay. Briefly, the HeLa and HCerEpiC cells were cultured in a 96-well plates at the density of 5×103 cells/well at 37°C for 24 h. The cells were incubated for one night and then the medium was removed and replaced with a new medium with bergenin (Dissolved in 10% DMSO) at different concentrations (0–200 µM) for a time period of 24 h at 37°C. The untreated cells were treated with 10% DMSO. Thereafter, an MTT solution of the concentration of 0.5 mg/ml for the last 4 h of incubation and finally the absorbance was measured at 570 nm.

Apoptosis assays: Cervical cancer HeLa cells were seeded at the density of 2×105 cells/well in 6-well plates. The cells were then administrated with 0, 7.5, 15 and 30 µM of bergenin and incubated for a time period of 24 h. DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride) staining was carried by incubating the cells in 6-well plates with DAPI. The cells were then washed with PBS, fixed in formaldehyde (10%) and then again washed with PBS. The DAPI stained cells were then examined by fluorescence microscope (10 fields). For estimation of apoptotic cell populations, similar procedure was carried out except for the cells were stained with Annexin V/PI staining and analysed by flow cytometer.

Analysis of cell cycle

In order to estimate the number of cells in phase of the cell cycle, the bergenin treated HeLa cervical cancer cells were harvested and washed with PBS. Thereafter the cells were fixed with ethanol (70%) for approximately an hour and then washed again by PBS. The cells were finally resuspended in solution of propidium iodide (PI) (50 µl/ml) and RNase1 (250 µg/ml). This was followed by incubation for a period of 30 min at room temperature and final investigation under a fluorescence-activated cell sorting cater-plus cytometer using 10,000 cells/group.

Cell migration assay

The cell migration potential of bergenin treated cervical cancer HeLa cells was investigated by wound healing assay. Briefly 5×104 cells/well were seeded in 96-well plates. Afterwards the plates were incubated overnight at 37°C to allow the cells to adhere. Then a wound was scratched using a sterile pipette tip after the cells reached confluence. The cells were then washed with PBS to clear the detached cells. The cells were monitored after 20 h interval and photographed.

Western blotting analysis

Total protein from untreated and bergenin treated HeLa cervical cancer cells was isolated in RIPA lysis buffer and the protein concentrations were determined by BCA assay. Equal volumes of the proteins from each sample were run on SDS PAGE. This was followed by then transference to a polyvinylidene fluoride membrane. Afterwards, blocking was done with 5% non-fat milk followed by an incubation at RT for 1 h. The membranes were then subjected to treatment either specific primary antibody (STAT3; cat. no. sc-293151, p-STAT3 (Ser 727); cat. no. sc-8001-R, p-STAT (Tyr705); cat. no. sc-7993-R, Bax; cat. no. sc-20067, Bcl-2; sc-509, Actin; sc-58673 purchased from Santa Cruz Biotechnology Inc.) at 4°C for 20 h. Thereafter, washing in washing buffer was carried out and then the membranes were incubated with secondery antibody (mouse monoclonal secondary antibody conjugated to Horseradish Peroxidase, cat. no. sc-2357) for 1 h. The protein bands were then visualised by an ECL Advanced Western Blot Detection kit.

Statistical analysis

The experiments were carried out thrice and the values represent mean of the three replicates ± SD. Student's test (For comparisons between two groups) and One way ANOVA followed by Tukey's test (for comparison between more than two samples) was used for statistical analysis using GraphPad prism software 7. The values were considered significant at *P<0.01, *P<0.001, *P<0.0001.

Results

Bergenin decreases the viability of HeLa cervical cancer cells

The effects of bergenin on cell viability were assessed by MTT assay. The HeLa cells were subjected to bergenin (Fig. 1) treatment at varied concentrations (0, 7.5, 15 and 30 µM). The outcomes of MTT assay showed that bergenin exhibited significant antiproliferative effects on the HeLa cells and the antiproliferative effects were found to be concentration dependent (Fig. 2). It was found that the IC50 of bergenin against HeLa cervical cancer cells was 15 µM as compared to its IC50 of 75 µM against non-cancerous HCerEpiC cells. These results unequivocally show that bergenin selectively exerts anticancer effects on cervical cancer cells.

Figure 1. Chemical structure of Bergenin.

Figure 2. Effect of indicated concentrations of bergenin on cell viability of cervical cancer HeLa and non-cancerous HCerEpiC cells. The experiments were carried out in triplicates and expressed as mean ± SD. The values were considered significant at *P<0.01, **P<0.01, ***P<0.001 (HeLa vs. HCerEpiC cells).

Bergenin induces apoptosis in HeLa cervical cancer cells

In this study we assessed that if bergenin triggers apoptosis in HeLa cervical cancer cells. The HeLa cervical cancer cells were first treated with bergenin at different concentrations, and then subjected to DAPI staining and finally observed under fluorescence microscope. It was observed that bergenin induced apoptosis in HeLa cervical cancer cells as evident from the increased number of cells with white colour nuclei (Fig. 3). The results of Annexin V/PI further revealed that the apoptotic cell populations increased from 4.12% in control to 62.16% at 30 µM concentration (Fig. 4). To further confirm the apoptosis at molecular level, we determined the expression of Bax and Bcl-2 proteins. The results showed that bergenin treatment increased the expression of Bax and decreased the expression of Bcl-2 in a concentration dependent manner (Fig. 5).

Figure 3. Bergenin induces apoptosis in HeLa cervical cancer cells at indicated concentrations. The experiments were carried out in triplicates (Magnification, ×200).

Figure 4. Estimation of apoptotic HeLa cell populations by Annexin V/PI staining at indicated concentrations of bergenin. The experiments were carried out in triplicates.

Figure 5. Effect of bergenin at indicated concentrations on the expression of Bax and Bcl-2 proteins in HeLa cells as shown in the western blot. The experiments were carried out in triplicates. The values were considered significant at *P<0.01, **P<0.01, ***P<0.001 vs. untreated control.

Bergenin causes G0/G1 cell cycle arrest in HeLa cervical cancer cells

The distribution of HeLa cervical cancer cells in the different cell cycle phases after treatment with bergenin at varied concentrations was determined by flow cytometery. The results showed that bergenin lead to the accumulation of HeLa cervical cancer cells in G0/G1 phase of the cell cycle and ultimately prompting G0/G1 cell cycle arrest (Fig. 6).

Figure 6. Bergenin triggers G0/G1 cell cycle arrest in HeLa cervical cancer cells at indicated concentrations. The experiments were carried out in triplicates.

Bergenin inhibits cell migration of HeLa cervical cancer cells

The effects of bergenin at IC50 were determined on the migration of HeLa cervical cancer cells by wound healing assay. The results showed that bergenin significantly inhibited the migration of the HeLa cervical cancer cells (Fig. 7).

Figure 7. Effect of 15 µM concentration of Bergenin on cell migration of HeLa cervical cancer cells as indicated wound healing assay. The experiments were carried out in three biological replicates (magnification, ×100).

Bergenin inhibits STAT3 signalling pathway

STAT3 signalling pathway has been found to be involved the progression and tumorigenisis of different types of cancers (7). In the present study, the effect of bergenin was also investigated on STAT3 signalling pathway. It was observed that bergenin inhibited the phosphorylation of STAT3 proteins in a concentration dependent manner and also decreased the expression of STAT3 (Fig. 8).

Figure 8. Effect of indicated concentration of bergenin of the STAT3 signalling pathway as shown in the western blot. The experiments were carried out in three biological replicates. The values were considered significant at *P<0.01, **P<0.01, ***P<0.001 vs. untreated control.

Discussion

Cervical cancer is one of the lethal and frequently detected cancers around the world. In the recent past the incidence of cervical has increased significantly and is expected to increase in future (8,9). The current treatment strategies are limited and are associated with lot of side effects (6,10). Therefore, there is an urgent need to look for strong and novel therapeutic targets to curb the growing incidence of the cervical cancer. Over the years plant derived secondary metabolites have attained considerable attention as bioactive molecules. They have been shown to exhibit anticancer activity against a range of cancer types (11). In this connection, the present study was carried out to investigate the anticancer effects of bergenin against cervical HeLa cancer cells. The results showed that bergenin exhibits considerable anticancer activity with an IC50 of 15 µM against HeLa cervical cancer cells as compared to IC50 of 75 µM against normal HCerEpiC cervical cells, suggesting lower toxicity of bergenin against the normal cells. To further unveil the reasons behind the anticancer effects of bergenin we carried out DAPI staining and it was observed that bergenin exerted anticancer effects via induction of apoptosis. Moreover, the apoptotic effects of bergenin were concentration dependent and the apoptotic cell populations increased with increased concentration of the bergenin as obvious from the Annexin V/PI staining. Apoptosis includes a form of cell death by which programmed series of actions lead to exclusion of cells from the body without release of any harmful chemicals. It an important mechanism by which several of the chemotherapeutic drugs exert their anti-proliferative molecules (12). The results of the present study are well supported with previous studies wherein plant derived molecules have been reported to trigger apoptosis in cancer cells (11). Next to understand if the bergenin-induced apoptosis follows mitochondrial pathway, we estimated the expression of Bax and Bcl-2 proteins. The results of the western blotting revealed that the expression of Bax was increased and that of Bcl-2 was decreased in response to the bergenin treatment. Another important mechanism that has been reported to contribute to the anticancer effects of many well known drugs is the cell cycle arrest (13). Some anticancer drugs halt the progression of the cells from one phase of the cell cycle to other by targeting specific proteins leading to the accumulation of the cancer cells at a particular stage. Arrest of the cell cycle prevents the cancer cell to develop into tumours and to spread to other parts of the body (14). Consistent with this, we observed that bergenin caused G0/G1 cell cycle arrest of HeLa cervical cancer cells in a concentration dependent manner. Anticancer agents that inhibit the migration of the cancer cells have been reported to of importance as they may efficiently inhibit the metastasis of the cancer cells (15). In the present study we also observed that bergenin could efficiently inhibit the migration of HeLa cancer cells. Earlier it was reported that many anticancer molecules target STAT3 signalling pathway in cancer cells (16). It was observed that bergenin enhanced decreased the expression of STAT3 indicating that the anticancer effects of bergenin may in part be due to inhibition of STAT3 signalling pathway.

In conclusion bergenin shows considerable anticancer effects against human cervical cancer cells. The anticancer activity is mainly due to the induction of apoptosis, cell cycle arrest and inhibition of STAT3 signalling pathway. The results of the present study indicate that bergenin may prove to be a promising lead molecule for the treatment of cervical cancer and deserves in vivo evaluation.

Acknowledgements

Not applicable.

Funding

No funding was received.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

XS and TZ were involved in drafting the manuscript. XS designed the study. XS, MX, KL, WH and HY collected and analyzed the data. XS, MX and TZ interpreted the data and collected the fund for this study and gave final approval of the version to be published. All authors reviewed the initial manuscript and revised it critically for important intellectual content.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References