Investigation of cell signaling pathways responsible for anticancer effect of in house synthesized small molecules

Abstract

Distinct cellular expression of various proteins, due to various endo/exogenous stresses, contribute to the carcinogenic characteristics of cells. Targeting such dysregulated proteins is a more effective method of killing cancer cells with lesser toxicity to normal cells. Topoisomerase IIand#945;, which maintains DNA topology, is highly expressed in many malignancies, including breast cancer. Similarly, over-activation of the EGFR has been associated with a number of cancers, and it interferes with other cell signaling pathways leading to cancer initiation and development. Identifying the biological targets and anticancer mechanisms of drug candidates require multidirectional preclinical screening. The validation of downstream signaling and associated functional proteins in-vitro aids in the development of anticancer therapies with better biological activity and the overcoming of cancer cell resistance. The present work explores detailed biological evaluation of two novel compounds in cancer cell lines.Compound 4h has shown promising anticancer activity against the breast cancer cells MCF-7 and MDA-MB-231, where it also targeted stemness of these cancer cells s evident in inhibition of mammosphere formation upon treatment. Compound 4hinhibited cell cycle regulatory proteins such as cyclins and CDKs, causing G2/M phase cell cycle arrest and also downregulated oncogenic signaling involving STAT3, PSTAT-3, AKT, P-AKT, and P-NF-kand#946;and#945;. In silico and in-vitro enzyme assay showed that compound 4h selectively inhibited topoisomerase IIand#945;, leading to DNA damage as observed by comet formation and and#947;H2AX immunofluorescent staining. Interestingly compound 4h is only cytostatic in MDA-MB-231 while it is cytotoxic to MCF-7 cells. Detailed study using overexpression of mutant p53 in MCF-7 cells showed that the cytotoxic effect of the compound is due to wild type p53 in MCF-7 cells. Thus compound 4h targets multiple signaling cascades along with topoisomerase IIand#945; inhibition, leading to cytotox