Research Of Apoptosis Induced By Icaritin In Human Cervical Cancer Cells And Its Mechanism

Cervical cancers are mostly caused by persistent infection by human papillomavirus?HPV?,and the major types include cervical squamous cell carcinomas and cervical adenocarcinoma.Although early and mid-stage patients can be treated by surgery with optimal prognosis,treatment outcomes of late-stage and relapsed paptients are sub-optimal,and novel therapies and drugs are urgently needed.Past research has revealed that cancer cells share some rewired biochemical and metabolic pathways,which may become their vulnerabilities to selectively attack them.Both clinical practice and experimental studies have shown that cancer cells are sensitive to additional ROS and raising ROS may be a promissing approache to develop anticancer drugs.Extracts of Epimedium plants have long been used in China to treat various malfunctions.Many studies have shown that one of the bioactive compounds,icaritin,has potent anticancer activity against most types of cancer but the mechanisms of action remain to be clarified.It was reported that icaritin triggered ROS generation in some cancer cell lines,but it was not clear if ROS played any role in the anticancer toxicity of icaritin.In this study,we analyzed the anticancer potency of icaritin in human cervical cancer cells,focusing on the roles of oxidative DNA damage induced by ROS.Results of MTT assay showed that icaritin potently suppressed the viability of both HeLa and SiHa cells but not the non-cancerous CCD?1095Sk and HEK293cells,indicating that icaritin selectively killed cancer cells.3-hour icaritin treatment dramatically increased cellular ROS levels in HeLa and SiHa cells and blocking ROS by NAC significantly decreased the cytotoxicity,suggesting that icaritin-induced ROS were responsible for icaritin's cancer cell toxicity.Subsequent studies showed that after treatment by icaritin,the number of DNA breaks,?H2AX and 53BP1 positive cells,levels of CDC25C-pS216,activated caspases 3/9 and Bax increased significantly but levels of CDK1-pT14,Bcl-2 and XIAP decreased,and flow cytometry analysis showed induction of G₂/M cell cycle arrest and apoptosis.All of these were blocked by pretreatment with NAC,indicating that icaritin-induced ROS promoted DNA damage response which triggered G2/M cell cycle arrest and apoptosis.qRT-PCR and Western blot analyses showed no change in the expression and protein levels of HPV E6 and E7,suggesting that icaritin's cancer cell toxicity was unrelated to E6 and E7 regulation.Next,icaritin-induced changes in cellular proteomics were studied by isotop-labeled tandem mass spectrometry.A total of 716 proteins were quantitatively compared,698 of them?97.5%?were identified with less than 20%deviations in paired samples,indicating good accuracy.The biggest cellular process involved was cell cycle progression?32.8%?and the biggest cellular space involved was the cytosol?42%?,followed by organelles?25.9%?;finally,6 proteins were involved in apoptosis control,among them,icaritin dose-dependently down-regulated Bcl-2 and XIAP,while up-regulating Bax,cleaved caspase 3 and 9.Finally,computer-assisted virtual analyses were applied to examine the drug properties and toxicology of icaritin.The results showed that icaritin likely possesses good water solubility and intestinal absorption,and plasma protein binding could be less than 90%,potentially with excellent blood-brain-barrier penetration.Icaritin was assumed to have low mutagenicity and carcinogenicity and good ADMET.