Abstract
Colon cancer stem cells have been attributed to poor prognosis, therapeutic resistance and aggressive nature of the malignancy. Recent reports associated CD44v6 expression with relapse, metastasis and reduced 5-year survival of colon cancer patients, thereby making it a potential therapeutic target. Thus, in this study, comprehensive prediction and screening of CD44v6 against 1674 lead compounds was conducted. Silibinin was identified as a potential compound targeting CD44v6. Inorder to substantiate these findings, the cytotoxic effect of 5FU, Silibinin and 5FU+ Silibinin was assessed on human colon carcinoma cell line HCT116 derived CD44+ subpopulation. 5FU+ Silibinin inhibited cell proliferation of CD44+ subpopulation at lower concentration than Silibinin standalone. Further, corresponding to CD44v6 knockdown cells, 5FU+ Silibinin treatment significantly decreased CD44v6, Nanog, CTNNB1 and CDKN2A expression whereas increased E-cadherin expression in HCT116 derived CD44+ cells. Moreover, synergistic effect of these drugs suppressed sphere formation, inhibited cell migration, triggered PARP cleavage and perturbation in mitochondrial membrane potential, thereby activating intrinsic apoptotic pathways and induced autophagic cell death. Importantly, 5FU+ Silibinin could inhibit PI3K/MAPK dual activation and arrest the cell cycle at G0/G1 phase. Thus, our study suggests that inhibition of CD44v6 attenuates stemness of colon cancer stem cells and holds a prospect of potent therapeutic target.
Highlights
Colon cancer stem cells have been attributed to poor prognosis, therapeutic resistance and aggressive nature of the malignancy
Docking results suggested that Silibinin could be identified as potential antagonist that binds around the binding loop of HA with different interacting residues as well as H-bonds
Enhancement of docking results by molecular dynamic simulation analysis showed that interaction of Silibinin resulted in ligand induced conformational changes in amino acid residues located in variant v6 insertion region
Summary
Colon cancer stem cells have been attributed to poor prognosis, therapeutic resistance and aggressive nature of the malignancy. The expression of standard isoform (CD44s) has been more extensively studied, the variant isoforms (CD44v) are reported to have an indispensable role in cancer progression and development[8,9] Amongst these isoforms, CD44v6 has been characterized as a functional marker which has been associated with tumor growth, metastasis, recurrence, poor prognosis and reduced 5-year survival of colon cancer patients, thereby indicating the imperative significance of this CSC marker as an effective therapeutic target[9,10,11]. We sought to investigate the role of identified potential drug compounds on cancer stem-like CD44+ cells from the human colon carcinoma cell line HCT116 in order to explore the impact of drug based suppression of CD44v6 on molecular and functional characteristics such as anchorage independent growth potential, migration, expression of vital stemness and EMT markers, cell cycle regulation, induction of apoptotic and autophagic mechanisms and various downstream signaling pathways. An in-depth analysis of CD44v6 with these compounds would thereby provide newer avenues for development of CSC-targeted therapies in future
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