Extracts derived from a traditional Chinese herbal formula triggers necroptosis in ectocervical Ect1/E6E7 cells through activation of RIP1 kinase

Abstract

Ethnopharmacological relevanceAs one of the most common female malignant tumors mainly infected by human papillomavirus (HPV) worldwide, cervical cancer is widely distributed in about 90% developing countries. An in-hospital preparation derived from a traditional Chinese herbal formula, Youdujing (YDJ), has been developed and clinically used for more than 20 years in our hospital for treating multiple diseases caused by HPV infection, such as cervical precancerous lesions, recurrent condyloma acuminata, fla t warts, etc. However, few investigations on the effect and mechanism of YDJ extract on treating and preventing HPV infection induced cervical cancer have been reported. Aim of the studyPrevious reports showed that YDJ extract is effective in triggering human cervical cancer cells (ectocervical Ect1/E6E7) death in a necrotic manner. Herein, we aim to investigate the anti-proliferation effects and potential mechanisms of YDJ extract in inducing necroptosis in ectocervical Ect1/E6E7 cells. Materials and methodsThe high-performance liquid chromatography (HPLC) fingerprint method was firstly used for better quality control of the chemical components in YDJ extract. MTT assay and flow cytometer were applied for evaluating cytotoxicity and necroptosis induced by YDJ extract in ectocervical Ect1/E6E7 cells. Besides, Western blotting, receptor-interacting protein serine-threonine kinase 1 (RIP1) inhibitor (necrostatin-1), and RIP1 shRNA and pCDNA transfection assays were employed for investigation on the underlying mechanisms and validation the role of RIP1 in YDJ extract induced necroptosis. ResultsYDJ extract induced necroptosis in ectocervical Ect1/E6E7 cells both in time- and concentration-dependent manners, without affecting activation of caspases and elevation of intracellular reactive oxygen species (ROS) level. Moreover, a selective increasing in RIP1expression was observed in YDJ extract treated ectocervical Ect1/E6E7 cells. The induction effect of necroptosis by YDJ extract was partially blocked by the addition of RIP1 inhibitor (necrostatin-1). Co-immunoprecipitation assay demonstrated that the treatment of YDJ extract in ectocervical Ect1/E6E7 cells promoted the combination of RIP1 with RIP3 and MLKL to form necrosome, which facilitates the process of necroptosis. ConclusionsTaken together, YDJ preparation displays an effective ability of inducing necroptosis in cervical cancer cells through activation of RIP1 kinase. However, although the treatment efficacy and potential mechanisms of YDJ extract in vivo remain unclear and need further investigation, it is believed that YDJ extract has the great potential to be used as a starting point to develop more potent agent for treating or preventing cervical cancer and other proliferative diseases.