Anti-Proliferative Effect of Hydroethanolic Leaf Extract of Citrullus colocynthis (L) on Retinoblastoma Cell Line

Introduction: Citrullus colocynthis has many pharmacological, biological, and therapeutic properties making it a very important medicinal plant. The cucurbitacin produced/derived from the plant play an important role in anticancer activities. The retinoblastoma Y-79 cell lines are studied in attachment culture. A proper study of these cells can pave the way for control of its proliferative property. Cell viability was assayed using a modified colorimetric technique that is based on the ability of the live cells to convert MTT, a tetrazolium compound into purple formazan crystals. There was an effect on the proliferation and gene expressions of the retinoblastoma Y-79 cell line. Bioactive extracts of these plants are being majorly used as a potential chemopreventive element as an alternative approach for cancer progression management. Materials and Methods: Human retinoblastoma cell line (Y-79)cell line was purchased from the Original Research Article Nair et al.; JPRI, 33(59B): 679-686, 2021; Article no.JPRI.78832 680 National Centre for Cell Sciences (NCCS), Pune, India. Cell viability was assayed using a modified colorimetric technique that is based on the ability of live cells to convert MTT. Results: The experimental study showed antiproliferative effects against the retinoblastoma Y-79 cells. The experimental study showed the effect on cell viability, Bcl2 mRNA expression and BclxL mRNA expression in Y-79 cells. Conclusion: The hydroethanolic leaf extract obtained from the Citrullus colocynthis exhibited an antiproliferative effect on the retinoblastoma Y-79 cell line by affecting the expression of the Bcl2 mRNA and BclxL mRNA and hindering the cell viability.


INTRODUCTION
Citrullus colocynthis, a perennial plant, has the ability to propagate both by vegetative and generative means. Citrullus colocynthis in general is used in the treatment of various diseases like diabetes, asthma, leprosy, jaundice, constipation, bronchitis, joint pain, cancer, and mantitis [1]. Very little is known regarding the other parts of the plant. Many studies have reported antioxidant and antibacterial activities of the leaf extract of Citrullus colocynthis. Leaf extracts give cucurbitacin derivatives and their glycosides which have been studied the most. The cucurbitacins induce arrest in growth and apoptosis in colorectal cancer cells targeting the JAK/STAT3 signaling pathway [2]. The cucurbitacin glycosides extracted from Citrullus colocynthis leaves had an anti-proliferative effect on human breast cancer cell growth which reveals that cucurbitacin glycosides cause both cell cycle arrest and apoptosis due to its pleiotropic effect on cells [3][4]. The leaf extract of Citrullus colocynthis has the highest antioxidant potency, The extract showed free-radicalscavenging and antioxidant abilities to its maximum [5-6]. This study research will help in bringing forward the anticancer activity of leaf extract of Citrullus colocynthis on these retinoblastoma Y-79 cell lines. Citrullus colocynthis contains many useful properties along with antioxidation, it's also a useful anti-inflammatory source that helps in preventing subsequent harm or damage to the adjacent cells [19]. The apoptosis effect induced by the cucurbitacin glycoside combination causes loss of membrane polarity followed by translocation of membrane [20,21]. Hence this research will shed light on the antiproliferative effect of Citrullus colocynthis leaf extract on retinoblastoma cell lines.

Cell Lines and Cell Culture
Human retinoblastoma cell line (Y-79)cell line was purchased from the National Centre for Cell Sciences (NCCS), Pune, India. Cells were cultured in DMEM medium (Thermo Fisher Scientific, CA, USA) containing 10% fetal bovine serum (Thermo Fisher Scientific, CA, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, CA, USA) at 37°C with 5% CO 2 .

Cell Viability by MTT Assay
Cell viability was assayed using a modified colorimetric technique that is based on the ability of live cells to convert MTT, a tetrazolium compound into purple formazan crystals by mitochondrial reductases (Mosmann, 1983). Briefly, the cells (1 ×10 4 /well) were exposed to different concentrations of Citrullus colocynthis leaf extract (100-500µg/ml) with Y-79 cells for 48 h. At the end of the treatment, 100 µl of 0.5 mg/ml MTT solution was added to each well and incubated at 37 •C for an hour. Then the formazan crystals formed were dissolved in dimethyl sulfoxide (100 µl ) and incubated in the dark for an hour. Then the intensity of the color developed was assayed using a Micro ELISA plate reader at 570 nm. The number of viable cells was expressed as the percentage of control cells cultured in serum-free medium. Cell viability in the control medium without any treatment was represented as 100%. The cell viability is calculated using the formula: % cell viability = [A570 nm of treated cells/A570 nm of control cells] × 100.

Statistical Analysis
The obtained data were analyzed statistically by one-way analysis of variance (ANOVA) and Duncan's multiple range test with a computerbased software (Graph Pad Prism version 5) to analyze the significance of individual variations among the control and experimental groups. The significance was considered at p<0.05 level in Duncan's test The anticancer effect of Citrullus colocynthis has been via a variety of pathways which include apoptotic pathway, anti-inflammatory traits, and antioxidant pathway, etc [19]. Bioactive extracts of plants are being majorly considered as potential chemopreventive elements as an alternative approach for cancer progression management [22]. Treatment with AMF and its constituent for 24 hours improved the cell motility and also the keratinocyte expression drastically in the cell lines in the experiment conducted by Azmi et al. [23]. Similarly, the expression of the genes Bcl2 mRNA and BclxL mRNA in the Y-79 cells were affected along with the cell viability [24][25][26]. In this study, the extract obtained from the leaves were used to treat the cells. In the study conducted by Tehila Tannin-spitz et al.,

RESULTS AND DISCUSSION
The cucurbitacins obtained exhibited inhibition of growth of the cell line [20] which was exhibited by the inhibition of growth in the present study in the retinoblastoma cells. Many derivatives from different plants such as taxol from the Pacific yew tree [27] combrestastian from theSouth African bush willow, and Campthothecin obtained from the Chinese 'Happy Tree' have shown worldwide use in drugs employed in chemotherapy [28]. Herbal medicine is becoming more and more attentive as it has shown many potential cancer therapeutic and preventive agents. In a cross-sectional study, a great number of people were taking herbal medicine for cancer treatment [29]. Citrullus colocynthis has shown many therapeutic effects in treating many diseases. It has many pharmacological activities like antidiabetic, antineoplastic, antiallergic, etc. According to the study by Gerald J Chaer in his experimental research, stated that when retinoblastoma cells are treated with specific agent inducing partial neuronal or glial-like morphology, they begin to express other specific markers by 'maturation' and start losing inappropriate markers [18, 30,18]. this ability can be hindered by the use of the cucurbitacins derivative which hinders the expression of mRNA in retinoblastoma cells, extracted from the leaves of Citrullus colocynthis (L) [31][32][33][34][35]. The present study had studied a small proportion of the sample. A much more and bigger scale study can help understand the effects even better and also help for other related issues.

CONCLUSION
The hydroethanolic leaf extract obtained from the Citrullus colocynthis exhibited an antiproliferative effect on the retinoblastoma Y-79 cell line by affecting the expression of the Bcl2 mRNA and BclxL mRNA and hindering the cell viability. Hence, it can be a great scope for using the extract into medicinal properties with further studies to improve herbal medicine for cancer over expensive drugs.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.