Wet-lab validation of In Silico predicted anticancer potential of selected compounds of curry leaf (Murraya koenigii L.)

Abstract

Cancer is one among the most prevailing human diseases and nearly 0.1 billion people in the world are living with different types of cancers. Four most common cancers are lung, breast, bowel and prostate cancers. Chemotherapy, radiation therapy, surgery, bone marrow transplantation and immunotherapy are the major treatment strategies for cancer. Severe side effects of chemotherapy including fatigue, hair loss, nausea and bleeding, demand the finding of plant based molecules for cancer treatment. Several phytocompounds are identified to have therapeutic potential against multiple human diseases. Molecular docking of these ligand compounds with the target proteins is an efficient strategy for the initial large-scale screening to reveal the drug potential of the phytocompounds. Molecular docking studies on the antioxidant fractions identified through LC-MS/MS on curry leaf extracts have revealed their anticancer properties. The drug potential of the molecules identified through in silico analysis has to be confirmed through subsequent steps such as in vitro analyses on cancer cell lines and in vivo analyses in mouse tumour models. Hence, the study entitled ‘Wet-lab validation of in silico predicted anticancer potential of selected compounds of Curry leaf (Murraya koenigii L.)’ was undertaken at the Department of Plant Biotechnology, CoA, Thrissur, during 2019 to 2022, with the objective of evaluating the anticancer potential of selected curry leaf (Murraya koenigii) compounds by in vitro and in vivo studies using different cancer cell lines and mouse tumour models. Three compounds viz. Doxylamine succinate salt, L-histidinol dihydrochloride and Pheniramine maleate salt were tested in vitro on murine cancer cell lines EAC and DLA and human cancer cell lines, MCF-7 (breast cancer) and HCT-116 (colorectal cancer). The compound which performed better was carried forward for in vivo study in mouse mammary tumour model. DMBA was used to induce cancer in Swiss albino mice. There were six animal groups such as normal, control, vehicle control, standard, drug-low dose and drug-high dose and each group had six mice. After six weeks, all the animals were sacrificed, blood and serum collected for blood, liver function and renal function tests and tissues of mammary pad, liver and kidney collected for histopathology analysis. From the mammary pad tissue, RNA was isolated and used for cDNA isolation. The II qRT-PCR analysis was done using the cDNA to study the expression of ER-𝛼1, Bcl-2, cMyc and Pin 1 genes, with reference to the house-keeping gene 𝛽 െactin. Trypan blue assay conducted on murine cell lines has exhibited no significant cytotoxicity from the drugs. MTT assay on the cell lines MCF-7 and HCT-116 grown in DMEM showed that PMS efficiently inhibited the proliferation of MCF-7 cells. Hence, PMS was selected for further validation in mouse breast tumour model. The histopathology analysis of the mammary pad, liver and kidney of the mice showed that PMSHD group showed better histo-architecture than that of control group. The results of the blood, liver and renal function tests, analyzed by two way ANOVA, showed a significant reduction in the WBC count (standard, PMSLD and PMSHD groups), SGOT level (standard and PMSLD), SGPT level (standard, PMSLD and PMSHD) and ALT level (standard, PMSLD and PMSHD), in comparison with control group. The qRT-PCR results indicated a reduced expression in all the genes in the drug treated mice compared to that of control. Overall results obtained from the in vitro, in vivo and gene expression studies indicated that Pheniramine maleate salt could be the potential candidate for anti-cancer drug. Other biological properties of Pheniramine maleate salt such as anti-inflammatory and antioxidant properties may be further validated in the same way. The molecular mechanisms involved in the downstream signaling pathways may also be explored in the future.