Anti-proliferative activities of Byrsocarpus coccineus using ovarian cancer cell lines

Background: Ovarian cancer is one of the most lethal tumors of gynecologic malignancies, due to lack of early detection, and a high rate of metastasis. The standard treatment is surgery and cytotoxic chemotherapy, but due to its cost and side effects, medicinal plants are widely used in developing countries. Byrsocarpus coccineus plant preparation, has been administered to patients traditionally in the management of tumour in Nigeria. In this study, we investigated the anti-proliferative effects of B. coccineus ethanol leaf extract against OVCAR3 and SW626 ovarian cancer cell lines. After treatment of the two cell lines with the extracts, analyses were carried out to determine inhibition of proliferation and expression of cell cycle markers, pro-apoptotic and anti-apoptotic markers. Results: Results showed that B. coccineus ethanol leaf extract, significantly inhibited cell migration and colony formation in OVCAR3 and SW626 treated cells in a dose-dependent manner. Results also show that B. coccineus ethanol leaf extract modulated the expression of p53 gene, cell cycle progression, anti-apoptotic and pro-apoptotic genes. Conclusions: These results suggest that B. coccineus have anti-proliferative properties and could induce apoptosis. Further investigation will be carried out to isolate bioactive compounds for the treatment of ovarian cancer. coccineus inhibit cell migration in OVCAR3 cell lines the assay not to the after incubating for and two cell untreated cells, cell migration after incubating for h, 48 h and 72h two cell lines. B. coccineus was able to inhibit colony formation in the two cell lines after treating for 10 days. coccineus significantly inhibited cell migration and colony formation in the two cell lines. one-way analysis of variance (ANOVA) and unpaired t- tests. standard errors of means (±SEM). P values less than 0.05 were statistically significant.


Background
Ovarian cancer is the fifth most aggressive and lethal cancer worldwide in women [1], due to lack of early detection and a high rate of metastasis [2]. Epithelial ovarian carcinomas make up to 90% of malignant ovarian cancer and it is the most aggressive [3]. In 2019, 22,530 new ovarian cancer cases and 13,980 deaths due to ovarian cancer were estimated to occur in the United States [4]. The standard treatment for ovarian cancer is surgery and 3 chemotherapy [5], but relapse occurs in most women [1].
Due to poor prognosis, cost and side effect of treatment, phytochemicals have become a growing source of alternate medicine in Africa. Byrsocarpus coccineus plant extract preparation is administered to patients traditionally in the management of cancer in Africa. B. coccineus known as 'Amuje wewe or Ado kanti-kanti' is a scandent shrub and it is indigenous to Nigeria (West Africa). Studies have shown that the plant has antiplasmodia [6], antimicrobial [7], and anti-diarrhea activity [8]. Fractions of B. coccineus have been reported to modulate cytochrome P450 (CYP) enzyme activity, cytokine production and anti-proliferation in colon cancer cell line [9]. Studies have also shown that the plant extract has cytotoxic activity against human breast and prostate carcinoma cell lines [10]. Activation of p53 (a tumor suppressor protein) signaling pathway inhibits cancer cell proliferation by cell cycle arrest and induction of apoptosis through the intrinsic and extrinsic pathway [11]. The p53 protein, is a key regulator of apoptosis and has been implicated in the development of ovarian cancer [1].
Therefore, the study is to justify the folkloric use as anti-tumour plant and propose a mechanism/pathway of action of the extracts by investigating p53 involvement in cell cycle arrest and induction of apoptosis [11].

Cell proliferation assay
The anti-proliferation effects of B. coccineus ethanol leaf extract against OVCAR3 and SW626, were determined by treating the two cell lines with different concentrations of the extract. The cells were treated for 24 h, 48 h and 72 h and DMSO was used as control. The effect of the extract on cell viability was most effective dose dependently in the two cell lines, after 48 h when compared to the control. The optimal IC50 values were determined to be OVCAR3 446.5 µg/mL and SW626 486.94 µg/mL. The results indicate that B. coccineus ethanol leaf extract inhibits the proliferation of OVCAR3 and SW626 (Table 1).

Live/Dead cell staining
Cytotoxic effect of B. coccineus ethanol leaf extract was confirmed, after treating OVCAR3 and SW626, by live/dead staining assay. The live nuclei are stained blue, whereas the dead nuclei or detached cells are stained green. Images of both cell lines (untreated and treated) taken using immunofluorescence microscope at x4 showed a high degree of dead nuclei in the treated cells, after incubating for 48 h when compared to untreated cells ( Fig. 1).

Cell migration
The effect of B. coccineus ethanol leaf extract on cell migration of OVCAR3 and SW626 cells was investigated using the scratch assay. This result showed that B. coccineus extract was able to inhibit cell migration in the two ovarian cancer cell lines (Fig. 2). In the baseline control the average gap distance was 69.25µm. After scratching and incubating OVCAR3 and SW626 cells for 24 h, 48h and 72 h, there was no significant difference in the gap distance between treated cells when compared to the 0 h, but there the gap closed completely in the untreated cells when compared to the control. It could be inferred from the results, that B. coccineus ethanol leaf was able to inhibit cell migration in OVCAR3 and SW626.

Colony formation
To investigate the effect of B. coccineus on colony formation, a clonogenic assay was carried out using OVCAR3 and SW626 cells. After treating the two cell lines and incubating for 10 days, the results showed that B. coccineus could inhibit colony formation in OVCAR3 and SW626 cells, when compared to the control groups (Fig. 3).

Flow cytometry
Annexin V/PI apoptosis detection 5 Annexin V/PI was used to determine apoptosis in OVCAR3 and SW626 cells, after treatment with B. coccineus 48h, staining with PI and analysed using the flow cytometer.
The flow cytometer data analysis shows that apoptotic cells were found more in the right lower (early apoptotic) quadrant (Q3) in the treated cells when compared to the control (Fig 4). This implies that B.coccineus was able to induce apoptosis in the two cell lines.

Immunomodulatory properties of B. coccineus on OVCAR3 and SW626 cells
Tumor necrosis factor alpha (TNF-α) and IL-10 are multifunctional inflammatory cytokine which are involved in apoptosis and cell survival. The result shows that TNF-α was significantly up-regulated in OVCAR3 and SW626 treated cells. IL-10 was significantly upregulated in OVCAR3 cells, but the extract had no significant effect on SW626 treated cells ( Table 2).

B. coccineus induced the regulation of pro-and anti-apoptotic protein OVCAR3 and SW626 cells
To corroborate the anti-proliferation effects of B. coccineus ethanol leaf extract against OVCAR3 and SW626 treated cells, the anti-and pro-apoptotic protein markers were analysed using western blot after treating the two cell lines with known concentration of the extract. This study shows that B. coccineus extract has anti-proliferative properties after treating for 48 h and 72 h by reducing cell viability significantly. This is corroborated by previous studies which show that B. coccineus has anti-proliferative activities using breast cancer cell lines and prostate cancer cell lines [10].
B. coccineus extract was shown in this study, to inhibit cell migration in OVCAR3 and SW626 cell lines using the scratch assay [13]. In the treated cells it was observed that cells did not migrate to the gap after incubating for 24 h, 48 h and 72h in two cell lines but in the untreated cells, cell migration to the gap was observed after incubating for 24 h, 48 h and 72h in two cell lines. These results show that B. coccineus was able to inhibit colony formation in the two cell lines after treating for 10 days. B. coccineus significantly inhibited cell migration and colony formation in the two cell lines.

Apoptosis is important in the prevention of proliferation of cancer cells. Studies have
shown that most cytotoxic agents with anti-proliferative activity can regulate apoptosis [14]. The flow cytometry result indicates that B. coccineus induced apoptosis in OVCAR3 and SW626 ovarian cancer cell lines.
Apoptosis is one of the important functions of p53 and it leads to disruption of tumour progression [14]. The tumour suppressor gene (p53), is important in cell cycle progression and apoptosis. Up-regulation of p53 leads to induction of apoptosis [11]. Up-regulation of p53 by B. coccineus probably resulted in the up-regulation of pro-apoptotic markers (BAX, BID and BAD) and the down-regulation of anti-apoptotic markers (BCL-2, and MCL-1) as shown in this study. Caspase-3 (aspartate-specific cysteine proteases) and PARP genes were also up-regulated after treating the two cell lines with B. coccineus.
p53 regulates apoptosis through the extrinsic pathway, by up-regulating BID which inhibits anti-apoptotic proteins (BcL-2, BcL-XL and MCL-1) and intrinsic pathway by up-regulating 8 BAX (See Addtional file 1). p53 also activates BAD which result in the release of BAX and its translocation to the mitochondria. BAX increases the permeability of the outer mitochondria membrane which leads to an efflux of cytochrome c. Cytochrome c then binds to Aparf-1 and initiates the initiator caspase 9 and this in turn initiates executioner caspases, such as caspase 3, 6 and 7. Caspase-3 cleaves PARP which leads to DNA fragmentation and then apoptosis [15]. Up-regulation of caspase-3 and PARP in the treated cells of the two cell lines further confirms the induction of apoptosis.
Activation of p53 in OVCAR3 and SW626 cells treated with B. coccineus, arrest cell cycle by inducing the activation of p21 (See Addtional file 1). Result shows that p21 was upregulated and cyclin D and CDK4 were significantly down regulated in OVCAR3 and SW626 cell lines. p21, a CDK family inhibitor inhibits the cell cycle at the G1 to S phase by binding to cyclin D/CDK4 and cyclin E/Cdk2 thereby preventing its progression [11]. The binding of p21 to the CDK4 and CDK2 results in their down regulation and this prevents the phosphorylation of pRb and leads to pRb forming a complex with E2F1.This results in inhibition of DNA replication and arrest of cell cycle [16]. Studies have shown that p21 can independently regulate pro-apoptotic proteins like BAX and anti-apoptotic protein like BcL-2 without the expression of p53 [17].
TNF and IL-10, are multifunctional cytokines produced by the immune system and they have pro-and anti-cancer effect [18]. TNF-α was significantly up-regulated in OVCAR3 and SW626 treated cell lines when compared to untreated cells. Studies have shown that TNFα induces apoptosis through the extrinsic pathway by binding to TNFR which recruits the adaptor protein TNF receptor-associated death domain (TRADD) and Fas-associated death domain protein (FADD). This adopter proteins then transmit signals from TNFR to activate the caspase which leads to apoptosis (19). TNF-α also induces apoptosis through the intrinsic pathway by cleaving of BcL-2 from BID which induces apoptosis via the intrinsic pathway (20). Accumulation of p53 has been reported in some cells which TNF-α has been shown to induce apoptosis, implicating p53 in TNF-α mediated apoptosis (21).

IL-10 which has both immunosuppressive and anti-angiogenesis effects has been shown in
previous studies to stimulate TNF and other molecules of the immune system (22). IL-10 was significantly up-regulated in OVCAR3 treated cells, but not in SW626 treated cells when compared to the control in this studies. IL-10 expression inhibits angiogenesis by down-regulating some molecules of the immune system like TNF-α which are needed for angiogenesis. This suggests that IL-10 did not play a major role in inducing apoptosis in ethanol leaf extract ranging from 0.020 mg/ml -5 mg/ml, DMSO was added to control and media to blank, the plates were incubated for 48 h and 72 h time points. 20µL of 5 mg/ml MTT solution was then added to each well and incubated for 2 h, 100µL of DMSO was added to dissolve the formazan crystals, and the optical density was read at 570 nm using a micro-plate reader (Spectramax M5, Molecular devices, Sunnyvale, CA) [12].

Live/Dead cell staining
Live/Dead cell staining was carried out as per manufacturer's protocol (NucGreen and NucBlue molecular probes by life technology), by seeding 100,000 cells/well for 24 h, treating with plant extract for 48 h and adding Nuc blue for live cells and Nuc green for dead cells. Images were taken using immunofluorescence microscope at x4.

Annexin V/PI apoptosis detection
FITC annexin V apoptosis detection kit with PI (Biolegend, San Diego, CA) was used to investigate if plant extract could induce cell apoptosis in OVCAR3 and SW626 ovarian cancer cell lines. OVCAR3 and SW626 cells were seeded and treated with plant extract for 48 h, after which the cells were trypsinized with 0.25% trypsin. The hemocytometer (Countess II FL, Life Technology) was used to count the cells and 0.5 × 10 7 cells/ml were added to 5 μL of FITC Annexin V and 10 μL of Propidium Iodide solution. The mixture was vortexed gently and kept in the dark 30min at room temperature then FACS buffer (400 μL) was added to each sample tube. The flow cytometer with guava easy Cyte HT (EMD Millipore, Brillarica) was used to analyze 50,000 cells [11].

Cell Migration Assay
To investigate the effect of plant extract on OVCAR3 and SW626 ovarian cancer cell line migration, 40,000 cells/well were seeded, in 24well plate and place in the incubator for 24h. Cells were treated with extract and a monolayer of the cells was scratched with a 10 μl plastic pipette tip to create a uniform wound. The scratch width was observed after 24 h, 48 h and 72 h of incubation under a phase-contrast microscope ((EVOS XL Core), at ×10 magnification and photographs were taken [13].

Clonogenic assay
Clonogenic or colony formation assay is based on the cells ability to form a colony from one cell. The effect of the plant extract on OVCAR3 and SW626 colony formation was investigated to determine. 1600 cells/well were seeded in 24 well plate and place in the incubator for 24h. Cells were treated with extract and incubated at 37 °C for10 days.
Colonies were fixed with methanol for 20 min, stained with 0.1% crystal violet, and visualize using a phase-contrast light microscopy at ×4, formation of 50 cells and above will be regarded as a colony [13].

Western blot analysis
Western blotting was employed to analyze pro-and anti-apoptotic protein levels. Seeded RT-PCR analysis of fold change expression relative to control in OVCAR3 and SW626 treated cells