Girinimbine Inhibits the Proliferation of Human Ovarian Cancer Cells In Vitro via the Phosphatidylinositol-3-Kinase (PI3K)/Akt and the Mammalian Target of Rapamycin (mTOR) and Wnt/β-Catenin Signaling Pathways.

BACKGROUND Worldwide, ovarian cancer is increasing in prevalence and has a high mortality rate. Girinimbine is a carbazole alkaloid isolated from Murraya koenigii (the curry tree) and is used in Chinese herbal medicine. The aim of this study was to evaluate the effects of girinimbine on cell proliferation, cell migration, and apoptosis in human ovarian cancer cells in vitro. MATERIAL AND METHODS A human ovarian cancer cell line panel, which included SKOV3 cells and the SV40 immortalized normal human ovarian cell line, were treated with increasing doses of girinimbine. Cell proliferation was evaluated using the MTT assay. Confocal immunofluorescence using 4',6-diamidino-2-phenylindole (DAPI), Annexin-V, and propidium iodide (PI) were used to measure cell apoptosis. Cell migration and invasion were determined by transwell assays. Protein expression was determined by Western blot. RESULTS Girinimbine inhibited SKOV3 ovarian cancer cell proliferation in a dose-dependent manner. The half maximal inhibitory concentration (IC50) of grinimbine was 15 µM for the SKOV3 cells, and 120 µM for the SV40 cells. Grinimbine treatment resulted in apoptosis of SKOV3 cells, from 2.2% in untreated cells to 58.8% at a dose of 30 µM, which was associated with an increase in the Bax/Bcl-2 ratio. Girinimbine inhibited cell migration and invasion of the SKOV3 cancer cells in vitro and inhibited the PI3K/AKT/mTOR and Wnt/β-catenin signaling pathways. CONCLUSIONS Girinimbine, a carbazole alkaloid used in Chinese herbal medicine, inhibited the proliferation and cell migration of human ovarian cancer cells in vitro, in a dose-dependent manner, via the PI3K/Akt/mTOR and Wnt/β-catenin signaling pathways.

Background

Worldwide, in 2016, according to data from the World Health Organization (WHO) more than 14 million patients were diagnosed with cancer and approximately eight million people died from cancer [1]. Of all the gynecologic cancers, ovarian cancer is responsible for significant mortality in women [2]. Ovarian cancer is usually diagnosed at an advanced stage, as there are few symptoms in the early stage, and ovarian cancer is difficult to treat and has a poor prognosis [3]. However, ovarian cancer can initially respond well to the first-line chemotherapy, but relapses are frequent and are mostly associated with the development of the chemoresistance [4]. Despite recent advances in cancer research, there are no reliable markers for the diagnosis of early ovarian cancer [5]. Also, currently used chemotherapeutic agents for the treatment of ovarian cancer are associated with adverse clinical effects [6].

Recent studies on the use of natural herbal-derived anticancer agents have shown that natural products could prove promising as anticancer agents with fewer side effects [7]. Among the natural agents studied, plant-derived secondary metabolites have been shown to exhibit potent anticancer activity [8]. Alkaloids of herbal origin have been reported to exhibit a wide range of bioactivities, which include antimicrobial and anticancer activities [9].

Girinimbine is a carbazole alkaloid isolated from Murraya koenigii (the curry tree) and is used in Chinese herbal medicine [10,11] and can be synthesized in the laboratory [12,13]. Although girinimbine has been reported to exhibit anticancer effects against several types of cancer cells [14,15], the anticancer activity of girinimbine has not been previously studied for ovarian cancer cells. Also, the possible mechanisms responsible for the anticancer activity of girinimbine have not been explored.

Therefore, the aim of this study was to evaluate the effects of girinimbine on cell proliferation, cell migration, and apoptosis in a panel of human ovarian cancer cells in vitro.

Material and Methods

Cell lines and culture conditions

Ovarian cancer cell lines, including PA-1, CAOV-3, SW-626, TOV-112D, SKOV3, OVACAR-3, and the SV40 immortalized normal human ovarian cell line, were obtained from American Type Culture Collection (ATCC). All cell lines were maintained in Dulbecco s modified Eagle s medium (DMEM) containing 10% fetal bovine serum (FBS), antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin), and 2 mM glutamine. The cells were cultured in a CO2 incubator (Thermo Scientific) at 37°C with 98% humidity and 5% CO2.

MTT cell viability assay

The viability of ovarian cancer cells was determined by the 3-(4,5-dimethylimidazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, the cultured ovarian cancer cells were seeded at the density of 1.5×104 cells 96-well microtiter plates and treated with varying concentrations of girinimbine (98% purity) for 24 h at 37°C. Then, 20 μl of MTT solution (2.5 mg/ml) was added to all the wells. The absorbance was measured at 570 nm was using an enzyme-linked immunoassay (ELISA) plate reader.

Immunofluorescence apoptosis assay

The ovarian cancer cells were seeded in 6-well plates (2×105 cells per well). The cells were then stained with the blue nuclear fluorescent stain 4′,6-diamidino-2-phenylindole (DAPI) (5 μg/ml) for 20 min at room temperature to detect cell apoptosis by fluorescence microscopy, as previously described [16]. The percentage of the apoptotic cells was determined using a fluorescein isothiocyanate (FITC), Annexin-V, and propidium iodide (PI) apoptosis detection kit, according to the manufacturer’s instructions (Beijing Biosea Biotechnology Co. Ltd., Beijing, China).

Transwell cell migration and invasion assay

The migration of the ovarian cancer cells was assessed, as previously described, using a transwell assay [17]. Briefly, the cells were seeded at a density of 2×105 cells/mL and incubated for 24 h at 37°C. Then, 200 ml cell suspensions were added into the upper part of the assay chamber, and complete medium was placed into the bottom wells. After 24 h of culture, the cells in the upper chambers were removed and cells migrated through the chambers were fixed with methyl alcohol, followed by staining with crystal violet. Finally, the number of migrating cells was determined by counting the cells using an inverted microscope, at a magnification of ×200 and using ten different fields.

Western blot

The ovarian cancer cells were harvested and lysed in lysis buffer. The protein extracts were incubated at 99°C for 15 min in the presence of loading buffer, followed by separation of the cell extracts using a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The samples were then placed onto polyvinylidene fluoride (PVDF) membranes and blocked using 5% skimmed milk powder. Membranes were incubated with primary antibodies for 24 h at 4°C, followed by incubation with horseradish peroxidase (HRP)-linked biotinylated secondary antibodies at 1: 1,000 dilution for 2 h. The membranes were washed with phosphate-buffered saline (PBS), followed by visualization of the immunoreactive bands using the ECL-PLUS Kit, according to the manufacturer’s guidelines. The immune complex development was carried out using an enhanced chemiluminescence (ECL) detection kit according and the bands were analyzed using the Gel Doc 2000 imaging system.

Statistical analysis

The experiments were performed in triplicate, and the data were presented as the mean of the three replicates ± the standard deviation (SD). Student’s t-test, for comparison between two samples, and one-way analysis of variance (ANOVA) was followed by Tukey’s test, for comparison between more than two samples. Statistical analysis was performed using GraphPad Prism software version 7. The values were considered as statistically significant at p<0.05.

Results

Girinimbine inhibited the proliferation of ovarian cancer cells in vitro

The antiproliferative effect of girinimbine (Figure 1A) was assessed in six ovarian cancer cell lines, PA-1, CAOV-3, SW-626, TOV-112D, SKOV3, OVACAR-3 and the SV40 immortalized normal human ovarian cell line. Cell survival and proliferation were evaluated by the MTT assay. The results of the MTT assay showed that girinimbine treatment resulted in a dose-dependent antiproliferative effect on all the ovarian cancer cell lines. The minimal inhibitory concentration (MIC) of girinimbine treatment of the ovarian cancer cell lines ranged from between 15–60 μM. The lowest MIC of 15 μM was found with girinimbine treatment of the SKOV3 ovarian cancer cell line, while the highest MIC was found with girinimbine treatment of the PA-1 cancer cell line (Table 1; Figure 1B). However, girinimbine showed minimum cytotoxicity against the normal SV40 cells, with a MIC of 120 μM (Figure 1C). Given that the lowest MIC following girinimbine treatment was for the SKOV3 ovarian cancer cells, further experiments using girinimbine were performed only on the SKOV3 ovarian cancer cells.

Figure 1

The chemical structure of girinimbine and the effects of girinimbine treatment on viability and proliferation of SKOV3 ovarian cancer cells and normal SV40 cells, evaluated by the MTT assay. (A) The chemical structure of girinimbine. (B) The effect of girinimbine treatment on the viability and proliferation of SKOV3 ovarian cancer cells, evaluated by the MTT assay. (C) The effect of girinimbine treatment on the viability and proliferation of the normal ovarian SV40 cells, evaluated by the MTT assay. The experiments were performed in triplicate. The mean ± standard deviation (SD) are used. (p<0.05).

Table 1

Antiproliferative effects of Girinimbine against the ovarian cancer cell lines (expressed as IC50) as determined by MTT assay.

S. No	Cell line	IC50 (μM)
1	PA-1	60
2	Caov-3	30
3	SW-626	30
4	TOV-112D	60
5	SK-OV-3	15
6	OVACAR-3	30
7	SV40	120

Girinimbine induced apoptosis in ovarian cancer cells

Previous studies have indicated that plant-derived alkaloids can induce apoptosis in cancer cells. Therefore, the SKOV3 ovarian cancer cells were treated with increasing concentrations of girinimbine, followed by fluorescence cell nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI). Girinimbine induced apoptosis in the SKOV3 ovarian cancer cells in a dose-dependent manner (Figure 2). The results of Annexin-V and propidium iodide (PI) staining showed that the apoptotic cell populations increased from 2.2% in the SV40 immortalized normal human ovarian cells to 58.8% in the SKOV3 ovarian cancer cells at a dose of 30 μM (Figure 3). Also, girinimbine treatment upregulated the expression of Bax which was also associated with the downregulation of Bcl-2, further confirming the apoptotic cell death of the SKOV3 ovarian cancer cells (Figure 4).

Figure 2

Cell nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) showing the effects of girinimbine treatment on apoptosis in the SKOV3 human ovarian carcinoma cells. The experiments were performed in triplicate. The mean ± standard deviation (SD) are used. (p <0.05).

Figure 3

Estimation of the percentage of SKOV3 human ovarian carcinoma cells undergoing apoptosis shown by Annexin-V and propidium iodide (PI) staining. The experiments were performed in triplicate.

Figure 4

The effects of girinimbine on the expression of Bax and Bcl-2 evaluated by western blot. The experiments were performed in triplicate.

Girinimbine inhibited PI3K/Akt/mTOR and Wnt/β-catenin signaling pathways

The effect of girinimbine treatment was also investigated on the PI3K/AKT/mTOR and Wnt/β-catenin signaling pathways in the SKOV3 ovarian cancer cells at increasing concentrations of girinimbine (0, 7.5, 15, and 30 μM). For the PI3K/AKT/mTOR pathway, girinimbine treatment of SKOV3 cells increased the expression of p-AKT, p-PI3K and p-mTOR, while the expression of AKT, PI3K and mTOR remained unaltered (Figure 5). For the Wnt/β-catenin pathway, girinimbine treatment of SKOV3 inhibited the expression of β-catenin, c-Myc, CDK4, and cyclin D1 (Figure 6)

Figure 5

The effects of girinimbine on the PI3K/AKT/mTOR signaling cascade evaluated by western blot. The experiments were performed in triplicate.

Figure 6

The effect of girinimbine on the Wnt/β-catenin signaling pathway cascade evaluated by western blot. The experiments were performed in triplicate.

Girinimbine inhibited the migration and invasion of SKOV3 ovarian cancer cells

The effect of girinimbine treatment of the SKOV3 ovarian cancer cells was also assessed on the cell migration and invasion ability at half maximal inhibitory concentration (IC50) of grinimbine of 15 μM. The results showed that girinimbine could inhibit the migration of the SKOV3 ovarian cancer cells (Figure 7). Similar effects were also observed on the cell invasion of the SKOV3 ovarian cancer cells (Figure 8).

Figure 7

The effects of girinimbine on cell migration of the SKOV3 human ovarian carcinoma cells evaluated by the transwell assay. The experiments were performed in triplicate. The mean ± standard deviation (SD) are used. (p<0.05).

Figure 8

The effects of girinimbine on cell invasion of the SKOV3 human ovarian carcinoma cells evaluated by the transwell assay. The experiments were performed in triplicate. The mean ± standard deviation (SD) were used. (p<0.05).

Discussion

Worldwide, ovarian cancer remains a gynecological cancer with a high mortality rate [2]. The incidence of ovarian cancer is increasing, and current treatment methods are ineffective in the majority of patients [3]. Chemotherapeutic drugs currently used for the treatment of ovarian cancer have several adverse effects [4]. Therefore, both in vitro and in vivo studies should continue to explore new treatment approaches for women with ovarian cancer.

Girinimbine is a carbazole alkaloid isolated from Murraya koenigii and is used in Chinese herbal medicine. The aim of this in vitro study was to evaluate the anticancer activity of girinimbine, against a panel of ovarian cancer cell lines, including PA-1, CAOV-3, SW-626, TOV-112D, SKOV3, OVACAR-3, and the SV40 immortalized normal human ovarian cell line. The findings of this study showed that girinimbine could inhibit the proliferation of all the ovarian cancer cell lines with minimal cytotoxicity against the normal cell line. The minimal inhibitory concentration (MIC) of girinimbine treatment of the ovarian cancer cell lines ranged from between 15–60 μM. The lowest MIC of 15 μM was found with girinimbine treatment of the SKOV3 ovarian cancer cell line, which explains why the main focus of the study was on the SKOV3 ovarian cancer cells.

The findings of this study are supported by those of previously published studies where girinimbine has been reported to inhibit the cancer cell proliferation and induce cell apoptosis in vitro, including for the HCT-15 colon cancer cell line [18]. In the present study, the effects of girinimbine treatment on apoptosis of SKOV3 ovarian cancer cells was investigated by immunofluorescence using 4′,6-diamidino-2-phenylindole (DAPI), Annexin-V, and propidium iodide (PI). The findings showed that girinimbine could trigger apoptosis in SKOV3 ovarian cancer cells and the degree of apoptosis on girinimbine treatment was dose-dependent.

Apoptosis is an important molecular mechanism that involves the death of malignant cells, and may also prevent the development of drug resistance in cancer cells [19]. Therefore, molecules that cause apoptosis of cancer cells might be potential candidates for the development of anticancer chemotherapy. The findings of the present study are also supported by previous studies reporting that the alkaloid girinimbine induced apoptosis in the K562 myelogenous leukemia cell line [20]. Girinimbine has also been previously reported to trigger apoptosis in lung cancer cells in vitro [21].

In the present study, girinimbine treatment was also shown to inhibit both the PI3K/AKT/mTOR and Wnt/β-catenin signaling pathways in SKOV3 ovarian cancer cells. Previously published studies have shown that both of these pathways are activated in several types of cancer [22]. The PI3K/AKT/mTOR and Wnt/β-catenin signaling pathways are regarded as important therapeutic targets for the treatment of cancer, as recent studies have shown that components of these pathways frequently undergo gene amplification, mutation, and translocation in several types of cancer, and drugs that can deactivate components of these pathways might prove to be beneficial in the management of cancers in patients [23,24].

The findings of this study, showing that girinimbine had inhibitory effects on the PI3K/AKT/mTOR and Wnt/β-catenin signaling pathways, suggest that girinimbine could prove to be an important molecule for the treatment of ovarian cancer. Also, anticancer agents that can inhibit the migration and invasion of cancer cells may have the capacity prevent the metastasis of cancer in vivo [23]. In the present study, girinimbine inhibited the migration and invasion of the SKOV3 ovarian cancer cells. Therefore girinimbine requires further investigation to determine whether it can be developed into a lead molecule for the treatment of ovarian cancer. However, further in vitro studies are required, including studies and more cell lines, combined with future in vivo studies.

Conclusions

The findings of this in vitro study showed that girinimbine, an alkaloid used in Chinese herbal medicine, inhibited the proliferation of ovarian cancer cell lines in a dose-dependent manner. The effects of girinimbine were found to be mainly due to the induction of apoptosis and cell cycle arrest due to the inhibition of the PI3K/AKT/mTOR and Wnt/β-catenin signaling pathways. These findings suggest that girinimbine requires further studies to evaluate its role as a potential anticancer agent, including in vivo studies and synthesis of more efficient derivates using organic chemistry methods.