Regulation of Chloride Intracellular Channel Protein 1 and Caspase -3 mRNA Expression by Hydroethanolic Extract of Aegle marmelos Fruit Human Breast Cancer Cell Line-MCF-7

Introduction: Cancer is the second leading cause of death all over the world where among all types of cancer breast cancer is said to be the leading cancer followed by lung cancer. The aim of this study is to find the regulation of chloride intracellular channel protein 1 and caspase -3 mRNA expression by hydroethanolic extract of Aegle marmelos fruit human breast cancer cell line-MCF-7. Materials and methods: MCF-7 cells were collected from NCCS Pune, India. It is stored in Dubecos Modified Eagle's Medium. The Aegle marmelos fruit was collected from the herbal department and its extract was prepared. The extract of Aegle marmelosis used in treating MCF-7 cells at different dosages in in vitro. Isolation of total RNA from MCF-7 cells. The cells will be mixed with total RNA Original Research Article Dhivya et al.; JPRI, 33(47B): 586-593, 2021; Article no.JPRI.74426 587 isolation reagent, sonicated and RNA will be isolated as per the standard method. c-DNA conversion and real time polymerase chain reaction. The c-DNA will be synthesized using reverse transcription by commercially available (RT-PCR) kit. Two microlitres of c-DNA will be used for amplification of clic-1 and caspase-3 using gene specific primers by commercially available RTPCR kit (SyBr kit) and comparative CT method will be used to see the expression of genes. Untreated MCF-7 cells were compared with MCF-7 cells treated with various concentrations of the extract (10, 20 and 40ug). The statistical data’s were collected from the SPSS software version 21. Result: The given extract inhibits the proliferation of MCF-7 cells therefore said to have antiproliferative activity. Different doses of extract were tested (200ug-500ug) out of which 400ug of extract were preferred. Conclusion: The given plant extract has anti proliferative properties and hence can be used as a drug to treat breast cancer.

isolation reagent, sonicated and RNA will be isolated as per the standard method. c-DNA

INTRODUCTION
Cancer is an uncontrollable proliferation of cells which leads to tumor formation and spreads from one organ to another. Cancer is the main cause of death and acts as an important barrier of increasing lifespan [1]. Generally a cancer cell grow more and then break down from original mass of cells and then travels through blood and lymph systems and attaches to other organ and again starts its abnormal growth cycle. This process of leaving an organ and going to other organ and continuing its abnormal growth cycle is called metastasis [2]. Such kind of tumor spreads all over the body is called malignant tumor. For example if the cancerous cells of breast spread to the bone that doesn't mean that the person is suffering from bone cancer it is said to be metastatic breast cancer to bone and if the cancer starts from bones it is said to be bone cancer. Cancer is caused due to exposure to chemicals or toxic substances, ionizing radiation, pathogens and through genes. Chemical exposure to nickel, cadmium, benzene, cigarette smoking (etc) leads to cancer [3]. HBV, EBV, hepatitis B and C (etc) are some of the pathogens which cause cancer. Ionizing radiations like UV rays, alpha, beta, gamma rays and X rays can cause cancer. There are specific kinds of cancer related to human genes like breast cancer cells, ovarian cancer, colorectal cancer, skin cancer, prostate cancer and melanoma cancer. In some people there is a high immune response which controls or eliminates cells that can become cancerous cells [4]. Other situations like obesity, lack of exercise, chronic inflammation and hormones lead to cancer. Lump in breast and armpit, nipple pain, bleeding from nipple or any secretions from nipple and redness around nipple are the symptoms of breast cancer. Change in eating habits, sore throat for a long time, continuous cough with bleeding, difficulty in swallowing, persistent fatigue and vomiting are some of the common symptoms of cancer. Based on the stage, tumor location and health of patients, cancer can be treated by surgery, ionizing radiation or chemotherapy [5]. They have more side effects which affect their health and these methods of cure are more expensive due to which people prefer alternate methods.
Ayurveda is an old traditional medicinal system followed in India. Aegle marmelosis one of the most used ayurvedic medicines used in India [6,7,8]. It is also called bael fruit, japanese bitter fruit, wood apple (etc) and is available in the Indian subcontinent, South Asia[9] [2]. [9]. It contains chemicals like tannins, flavonoids, coumarins which reduce swelling and proliferation of cells. Aegle marmeloscontains antimicrobial , antidiarrhoeal, antiviral, radio protective, chemo preventive, antipyretic, ulcer healing, antigenotoxic, diuretic, antifertility which plays an important role in curing diseases [4,10]. Aegle marmelosis also used in the treatment of asthma, eye infections, used as snake bite antidote, antiemetic drug , treating constipation, pepticulcer, antigenotoxic drug (etc)[9][11] [2].. The root of the plant possesses astringent activity as a home remedy to cure ear problems. The root is dipped in neem oil and lighted and the oil dripped from the burning end is an effective medicine for ear problems. The astrigment extract of root combines with the antiseptic properties of neem which is helpful in curing ear infection, pus discharge and chronic inflammation [12]. The juice of the leaves is also used in treatment of ear infection [9]. The powdered leaf extract is effective to cure leprosy. The oil extract from the seeds was said to be a very effective fungal infection. The ethanolic extract of the plant is very effective against Curvularialunata, Aspergillus niger and Rhizopus nodulans. The bark of the tree is very effective in preventing dandruff, hairfall and scaly skin of the scalp[12].

Cell Line and Cell Culture
MCF-7 cells will be obtained from NCSS, Pune, India and grown in DMEM (Dubecos Modified Eagles Medium) and after 80% of confluency of the cells it will be passed to T-25 culture flask. Different doses of plant extract will be extracted, will be treated and kept for 24 hours and then the cells will be trizinised and used in various parameters.

Cell Viability by MTT Assay
The cell viability was seen using purple foramen crystals. The MCF-7 Human breast cancer cell line was treated with different concentration of Bael fruit extract for about 48 hours. Then at the end 100ul of 0.5mg/ml MTT solution were added to concentrated solution and were incubated at 37 degree celsius for an hour in vitro. The foramen crystal was dissolved in dimethyl sulfoxide and was incubated in dark for a hour. Then the intensity of the color developed was seen using a microELISA plate reader at 570um.

Gene Expression Analysis by Real Time PCR
Each sample was submerged in 2ml Trizol for RNA extraction and stored at -80 degree celsius until further process. cDNA was synthesized by performing on 2ug RNA in a 10ul sample volume using reverse transcriptase. Then 2ug RNA is used in amplification of clic-1 and caspase-3 using genes specific primers by commercially available RT-PCR kit (SyBr kit) and comparative CT method will be used to see the expression of genes. The study was carried out blue lab in Saveetha Dental College.

Statistical Analysis
The statistical analysis followed is one way analysis variance (ANOVA) and Duncan's multiple range tests with computerbased software to find the significance of individual variations. The significance was considered at p<0.05.

RESULT
Cell viability was studied by MTT assay. And the result showed that there was maximum inhibition of cancer cells at 300 and 400 μg/ml suggesting the cytotoxic effect (p <0.05) (Fig. 1). Effect of Aegle marmelos fruit extract on CLIC-1 mRNA expression in MCF-7 cells. The result showed that extract has significantly decreased the CLIC-1 mRNA expression in MCF-7 cancer cells and caspase 3 mRNA expression (p <0.05) (Fig. 2  and 3). 400 μg/ml concentration of the extract is preferred from the different doses of extract since less concentration have very less effects on cancer cells and more concentration of the extract leads to diarrhoea, bowel movements (etc).

DISCUSSION
Aegle marmelosbelong to the family of Rutaceae. Bael is a slow growing, tough, medium sized subtropical tree and the tree produces fruits once in a year. According to ayurveda it is a healing tree as all its parts cure all kinds of diseases and are edible in nature. The bael fruit pulp also contains vitamin A, C, thiamine, riboflavin, niacin and minerals suchas calcium, phosphorus (etc). Unripe fruits aid digestion and stomach irritation and are used in the treatment of diarrhoea and dysentery (etc) [38]. Sweet drink is prepared from bael which cures bacillary dysentery. It is also used in treating rheumatism and gout. The Aegle marmelos leaf extract is said to be very effective against various tumor lines including breast cancer cell lines MCF-7. The Aegle marmelosextract is found to have anti inflammatory and anti proliferative activity [39] [4]. The bael leaf is also said to inhibit the growth of breast cancer cell lines (MCF-7), leukemia K562, T Lymphoid Jurkat, Melanoma colo38, erythroleukemia HEL. The roots of the plant prevent dysentery, fever and the leaves reduce bowel movements, bleeding piles and diarrhea [8] [40]. The leaves extract of the plant can be used in treating kidney problems and the paste of ground roots in bael in butter is used in treating insomnia. The pulp of the fruit has detergent properties, therefore can be used as a herbal soap for allergic patients. Aegle marmelos contain marmenol, marmelin, marmesin, marmelosin (etc) which is used as an anticancer drug where the effect is mediated through inhibition of cell proliferation of cancer cells [6] [41]. The present study proved that the given extract contains anti-inflammatory and antiproliferative properties and therefore can be used in the treatment of breast cancer.
In the present study, Aegle marmelosfruit extract has effects on MCF-7 breast cancer cell lines and showed anti-inflammatory and antiproliferative activity. The activity has been proven only fruit extract, further studies required to confirm which part of the plant has potent antiinflammatory and antiproliferative activity.

CONCLUSION
From this study it has been proved that Aegle marmeloshas effects on MCF-7 breast cancer cell lines. It showed anti-inflammatory and anti proliferative activity and therefore can be used as a drug to treat breast cancer.

CONSENT
It is not applicable.