2?,4?-Dihydroxy-6?-methoxy-3?,5?-dimethylchalcone and its amino acid-conjugated derivatives induce G0/G1 cell cycle arrest and apoptosis via BAX/BCL2 ratio upregulation and in silico insight in SiHa cell lines

We modified the chemical structure of 2 ',4 '-dihydroxy-6 '-methoxy-3 ',5 '-dimethylchalcone (DMC, 1), a phyto-chemical found in the seed of Syzygium nervosum A.Cunn. ex DC., by conjugation with the amino acid L-alanine (compound 3a) or L-valine (compound 3b) to enhance anticancer activity and water solubility. Compounds 3a and 3b had antiproliferative activity in human cervical cancer cell lines (C-33A, SiHa and HeLa), with half -maximal inhibitory concentrations (IC50) of 7.56 +/- 0.27 and 8.24 +/- 0.14 mu M, respectively in SiHa cells; these values were approximately two-fold greater than DMC. We investigated the biological activities of compounds 3a and 3b based on a wound healing assay, a cell cycle assay and messenger RNA (mRNA) expression analysis to determine the possible mechanism of anticancer activity. Compounds 3a and 3b inhibited SiHa cell migration in the wound healing assay. After treatment with compounds 3a and 3b, there was an increase in SiHa cells in the G1 phase, indicative of cell cycle arrest. Moreover, compound 3a showed potential anticancer activity by upregulating TP53 and CDKN1A that resulted in upregulation of BAX and downregulation of CDK2 and BCL2, leading to apoptosis and cell cycle arrest. The BAX/BCL2 expression ratio was increased after treatment with compound 3a via the intrinsic apoptotic pathway. In silico molecular dynamics simulation and binding free energy calculation shed light on how these DMC derivatives interact with the HPV16 E6 protein, a viral onco-protein associated with cervical cancer. Our findings suggest that compound 3a is a potential candidate for anti -cervical cancer drug development.