Inhibition of Proliferation in Ovarian Cancer Cell Line (PA‐1) by the Action of Green Compound “Betanin”

Ovarian carcinoma has a cure rate of 30% which makes it deadlier than any other disease. There are a number of genetic and epigenetic changes that lead to ovarian carcinoma cell transformation. Chemoprevention of cancer through application of natural compounds is the need of present generation as other methods are rigorous and have many side effects. Betanin, a compound from Beta vulgaris extract is used in present study to check its potential for inhibition of (PA-1) cancer cell proliferation. Determination of IC50 values through MTT assay was carried out, in addition measurement of mitochondrial membrane potential (MMP), effect of reactive oxygen species (ROS) generation, and induction of apoptosis in ovarian cancer cells through betanin was also observed. Results have shown betanin as a potential candidate for inhibition of ovarian cancer cell proliferation and it can be taken up as a serious compound for further studies for its application in cancer cure.


Introduction
Ovarian cancer (OC) is one of the most lethal cancer among women. It is ranked 4th for all the deadly diseases in women. Data from 2019 Cancer statistics have shown a total of 14,170 deaths in 22,240 women suffering from OC [1]. Early diagnosis of ovarian cancer (OC) is very difficult since symptoms are seen only at the later stages. Women from age 20 to 55 are largely affected. More than 60% of patients with OC have shown symptoms of recurrence even after surgery. With the progression of cancer's various stages, the 5-year rate of survival decreases eventually. Major treatments for cancer include radiotherapy, chemotherapy, and surgery. Out of all the treatment techniques for cancer, chemotherapeutics is the most favored one. Depending on the stage of OC, the chemotherapeutic agents are being selected. At various levels, the cancer cells are affected by chemical agents and through apoptotic program induction the cell is destroyed. Rapidly growing cancer cells as well as normal cells are targeted by

Cell Culture Maintenance
Human ovarian (PA-1) cancer cells were obtained from the cell repository, National Centre for Cell Sciences (NCCS), Pune, India. For cell line maintenance, DMEM was used and 10% of FBS (fetal bovine serum) was used as a supplement. To prevent any bacterial contamination in the medium, streptomycin (100 μg/ml) and penicillin (100 U/ ml) were added. Cell lines were then incubated with 5% CO 2 in a humidified condition at 37 °C.

MTT Assay
Ten milliliters of PBS was used to dissolve 50 mg of MTT dye to make stock solution. Using a 0.45-micron filter, it was filtered after 1 min of vortexing. To prevent the exposure of light, aluminum foil was used to wrap the apparatus as MTT is light sensitive. Until further use, it was stored at 4 °C. To determine betanin cytotoxicity on PA-1 cells, Mosmann method (1983) [25] was used. Firstly, hemocytometer was used to count the harvested viable PA-1 cells and DMEM medium was used further. In 96 well plates 1 × 10 4 cells/ml of density was seeded in each well and to allow attachment it was incubated for 24 h. After treating the PA-1 cells with control, each well was applied with different concentrations of betanin (10-60 μg/ml). At 37 °C, PA-1 cells were incubated for 24 h in a humidified CO 2 (5%) and air (95%) incubator. After the incubation period is over, the drug containing cells were washed with fresh medium and each well was added with MTT dye (5 mg/ml in PBS) followed by another 4 h of incubation at 37 °C. The concentrated DMSO (100 μl) was used to dissolve the precipitated purple formazan and using multi-well plate reader the viability of cell was measured at absorbance 540 nm. The results were represented as percentage stable cells in comparison to the control. The values for half maximal inhibitory concentration (IC50) were calculated and the optimum doses were analyzed at different time period. From the dose responsive curve of betanin, the values of IC50 were determined, where 50% cytotoxicity inhibition was compared to control cells. All experiments were performed in triplicates to minimize errors.

Statistical Analysis
The values were obtained using mean ± SD. One-way analysis of variance (ANOVA) and Duncan's multiple range test (DMRT) were used for statistical comparisons on Statistical Product and Service Solutions (SPSS) software (version 12.0, SPSS Inc. Chicago; http:// www. spss. com). The values were taken as statistically significant when p values come under 0.05.

ROS Generation
Using the method given by Pereira et al. (1999) [26], ROS values were generated. PA-1 cells were added in 6-well plates (2 × 10 6 cells/well) for 24 h before exposing them to betanin (40 μg/well), and untreated cells were maintained at 37 °C (5% CO 2 ). Overnight grown PA-1 cells were then treated with betanin (40 μg/well) for 24 h. After being exposed to betanin, PBS (1%) is used to wash PA-1 cells and loaded with DCFH-DA (25 μM) in DMEM at 37 °C for 30 min. DMEM was then used to wash the treated cells, and fluorescence was recorded for every 5 min for the next 30 min (emission 535 nm, excitation 485 nm) through spectrofluorometry at 37 °C. Increase in ROS was calculated by mean slope per minute and normalized to the control which is unexposed to betanin.

Measurement of Mitochondrial Membrane Potential (MMP)
Method given by Bhosle et al. (2005) [27] was used to measure MMP. For the assay, stock solution was made using 10 mg Rh-123 dye dissolved in 1 ml PBS buffer while working solution was made by taking 4 μL of the stock solution and dissolved into 0.96 ml PBS. PA-1 cells were exposed to betanin of different concentrations (40 μg/ml) and were placed in a 6-well plate with a cover slip. The cells are kept under incubation for 15 min once they are stained with Rh-123. PBS was used to wash and fix the cells. At 535 nm wavelength, the intensity of fluorescence was measured and the percentage of PA-1 cells reflecting pathological changes was calculated.

Induction of Apoptosis by Betanin
The method proposed by Baskić et al. (2006) [28] was used to analyze the apoptotic cell death using fluorescence microscopy. For staining, 200 μL of dye mixture was prepared by using 100 μL/mg EB and 100 μL/mg AO and dissolving it in 1% PBS. A density of 5 × 10 4 cells/well of PA-1 cells were added in a 6-well plate and given 24 h of incubation. The cells were detached after 24-h treatment with betanin (40 μg/ml) and then washed using cold PBS. Furthermore, staining was performed using the mixture of AO (100 μg/ml)/EB (100 μg/ml) at a ratio 1:1 for 5 min at room temperature. At a magnification of 40×, the stained cells were observed using fluorescence microscope. After the treatment finished, the cells were collected and washed with PBS up to three times.
The number of cells showing feature of apoptosis was counted as a function of the total number of cells present in the field.

Results and Discussion
While strategies for early OC diagnosis have yet to be developed, dietary compound chemoprevention might be an advisable strategy for preventing ovarian cancer. Previous studies have reported that betanin inhibited the cell growth in ovarian cancer [12]. In this research, betanin have more cytotoxicity to ovarian cancer cell line PA-1 cells.

MTT Assay
The PA-1 cancer cell lines were treated with various concentration of betanin (10-60 μg/ml) for 24 h, and the results are expressed as a % of the control value in presenting as a cell cytotoxicity ratio for PA-1 cells using MTT assay (Fig. 1). The mitochondrial dehydrogenase enzyme of viable cell reduces MTT (3-(4,5-dimethylthiozol-2-yl)-2,5diphenyltetrazoliumbromide) and a visible color change from yellow to purple was observed in the product formed. A NAD(P)H-dependent reductase was present in viable cells that reduce the reagent MTT to formazan, thus giving a deep purple color. Solubilizing solution was then used to dissolve formazan crystals and by plate reader 500-600 nm of absorbance was measured. Data were presented as mean ± SD asterisks indicate statistically different experiments compared to control. For PA-1, IC50 was found to be about 40 μg/ml. Results have shown that when betanin dose was increased the cell viability was decreased gradually.
The results of MTT assays can be further correlated with the photomicrographic representation of changes in morphology of PA-1 cells like detachment, shrinkage, membrane blebbing, and distortion in shape induced by betanin treatment (40 μg/ml for 24 h) as compared with control (Fig. 2). Normal intact cell morphology was seen in control cells, whereas morphological changes attributed to non-viability of cells were observed in betanin-treated cells

ROS Generation
PA-1 cells were exposed to betanin (40 μg/ml) for 24 h and DCFH-DA was used for staining. Through fluorescence microscope (Labomed, USA), the digital images were obtained. DCFH-DA assay was used to measure intracellular ROS level. DCFH-DA is cell permeable, lipophilic compound. DCFH-DA is deacetylated to DCF in the cytoplasm by cellular esterases [20]. A fluorescent molecule is obtained by oxidation of Fig. 1 Anti-proliferative effects of betanin on PA-1 cells DCF by various radicals such as hydroxyl, alkoxyl, peroxyl, carbonate, and nitrate (emission 485 nm, excitation 530 nm). One thing to note is that DCF was not oxidized by superoxide radical or hydrogen peroxide. Control cells (green fluorescence) and a bright DCF florescence was observed in PA-1 cells treated with betanin (40 μg/ml) (Fig. 3), which shows intracellular ROS production induced by betanin in PA-1 cells. Studies have reported that higher concentration of ROS molecules production leads to the activation of death processes like apoptosis, while at lower concentration it induces the pathways related to cell survival [29]. p53, a tumor suppressor protein get activated by ROS when at low concentration [29]. p53 is important in controlling various pathways such as induction of DNA repair, cell cycle arrest, stress responses of cells, and cell survival processes [30]. Other studies have shown ROS molecules such as H 2 O 2 induces apoptosis in different cell clines by increasing not only p53 but also Bax, Noxa, and Puma and have also shown phosphorylation of p53 [31][32][33][34].

Measurement of MMP
Studies have reported the dysfunctional mitochondria is one of the main recurring characteristics of cancer cells where the activity of NADH dehydrogenase and Complex I's protein content are significantly reduced and when cancer cells experience hypoxic conditions release of ROS is favored. ROS production is enhanced when MMP is increased which in turn increases tumorigenicity [35,36]. For MMP characterization, "Rh-123" a laser dye was used as a specific probe because of its specificity for localizing mitochondria in living cell. Photo luminescent quenching occurs when Rh123 enters the mitochondrial matrix and MMP depletion accompanied by fluorescence emission can be observed when Rh123 exits mitochondria. MMP description is indicated by high value of fluorescence. Decrease in MMP indicated by a gradual decrease of green fluorescence in cells treated with betanin (40 μg/ml) which was analyzed by fluorescent microscope (Labomed, USA) at 40× magnification (Fig. 4). The image shows Rh accumulated control cells, and betanin (40 μg/ml) treated cells had no Rh accumulation.

Induction of Apoptosis by Betanin
In recent years, researchers have come out with various studies to find out the effect of betanin in inducing extrinsic and intrinsic apoptosis pathways in cells [20,24,37,38]. Based on the various studies done, a detailed diagram showing how betanin induces extrinsic and intrinsic apoptosis pathways in cells is shown in Fig. 5.
To check the apoptosis in PA-1 cells induced by betanin, dual staining AO/EB method was used. AO is used to stain PA-1 because of its permeability to viable/nonviable cells. It emits green fluorescence if intercalated with double stranded nucleic acid (DNA) or red fluorescence if bound to single stranded nucleic acid (RNA). When treated with betanin, PA-1 cells lost membrane integrity and the nonviable cells have taken up EB (non-permeable to cells) because of which red fluorescence was emitted due to intercalation into DNA. So, because of the stained nuclei, the chromatin condensation and fluorescence emission, cells can be distinguished into four different viable cells, are those with well-organized nuclear structure emitting green fluorescence. In early apoptotic cells, the membrane is still intact but the DNA starts to cleave which is in a state of perinuclear chromatin condensation, these can be distinguished by bright green fragments or patches. Late apoptotic cells have red or orange fluorescence with fragmented or condensed chromatin. Nuclei of necrotic cells can be identified by uniform distribution of red or orange fluorescence with no chromatin condensation. PA-1 control cells, and betanin (40 μg/ml) treated cells for 24 h were stained with dual dye AO/EB and then analyzed by fluorescence microscopy (Fig. 6). In the microscopy images, living cells have shown clear green nucleus, late apoptotic cells have shown chromatin fragmentation or condensation orange-stained nucleus, while early apoptotic cells have shown fragmented or condensed form of yellow color nucleus with chromatin and necrotic cells have shown uniform red stained cell nuclei.

Conclusion and Future Prospects
While treating cancer with chemotherapy, there are chances that the cancer cells might develop drug resistance. Treating cancer by combining multiple drugs which include natural products and dietary supplements found to be effective than traditional chemotherapeutic drugs along with less adverse effects [39]. Cancer chemoprevention can be defined as the use of natural and pharmacologic agents to prevent the activation of various procarcinogens, thereby inhibiting the initiation, progression, and promotion of tumor. From last decade, studies have increased in finding potential of natural compounds mainly from traditional medicines against cancer including colon cancer, breast cancer, and lung cancer. Chemotherapeutic agents like docetaxel, gemcitabine, oxaliplatin, and cisplatin have been used in combination with natural compounds. Choice of these drugs in these studies has been based on their side effects like gemcitabine causes hematopoiesis suppression and cisplatin causes nephrotoxicity [40][41][42].
Studies have further observed the mechanisms that result in the antioxidant, antiinflammatory, antiproliferative, proapoptotic, and free radical-scavenging properties of natural compounds whose application lead to a marked increase in Caspase-9, Caspase-3, Caspase-7, BAX activities whereas PARP and BCL2 activities are significantly decreased thereby causing apoptosis by inducing the expression of apoptotic proteins like phosphorylated p53, FAS, TRAILR4, bad and altering MMP ultimately affecting tumor environment [20,24]. Effects of betanin on PA-1 cell line have been analyzed in this study. Betanin, a natural pigment, is widely used as food colorants which are water soluble and a good source of nitrogenous chromo alkaloids whose pharmacokinetic effects in human cell lines were demonstrated elsewhere [37,38,43]. Besides being a part of food colorant, a nutraceutical role is also played by natural pigments like betanins, thus avoiding the hazards posed by chemical and synthetic colorants. Unlike other natural pigments, the pharmacological properties as well as the mechanism of action of betanins are still been studied on various cancer cells. In this study, the betanin's anti proliferative effects on ovarian cancer PA-1 cell line have been studied. From the results, it was found that a time and dose dependent inhibition in growth of PA-1 cells is induced betanin with IC 50 of 40 μM. The use of betanin in the current study has showed typical characteristics of apoptosis like laddering of DNA, chromatin condensation membrane blebbing and decrease in membrane potential. But further studies are required to understand the mechanisms that are involved in altering the integrity of the mitochondrial membrane under betanin treatment in PA-1 cells. This research manifests the importance of betanins against ovarian cancer in women. Biochemical alterations exhibited by betanins are reflected in structural changes of the cell that is undergoing apoptosis.
Thus, these findings of betanin's anticancer effects, in ovarian cancer, add further value to the nutritional characteristics of various other fruits of Beta vulgaris and further studies in human trials and animal models are required to determine the effectiveness of this natural compound as an agent to treat and prevent ovarian cancer.