Effect Of Shikonin On Cervical Caski Cells And Expression Of HPV E6/E7

Background: Cervical cancer is one of the most common tumors in women,which seriously threatens women’s physical and mental health.The research of rational and effective therapeutic drugs is of great significance.High-risk human papillomavirus infection has been proved to be the most important pathogeny.The mechanism is related to the overexpression of viral oncogenes E6 and E7 that interfere with the cell cycle and inhibit apoptosis leading to malignant proliferation of cells.The replication,transcription and translation of E6 and E7 genes are important molecular targets against HPV and HPV-related cervical cancer.Shikonin,as the main active component of Lithospermum erythrorhizon,has been found to surpress the proliferation of cervical cancer Hela cells(HPV18)and induce its apoptosis,which is relevant to the pathways of caspase、ERK、P13K、ATK、mTOR、ROS/p38.Its effect has not been reported on the expression of E6/E7 mRNA and protein and the effect on HPV16 cervical cancer cells recently.Objective: To study the effects of shikonin on the proliferation,apoptosis and the expression of E6/E7 mRNA and protein in Caski cells with HPV16 infected.Methods: Caski cells were cultured in vitro and tested for HPV gene.Caski cells were treated with different concentrations of shikonin(1.25~160 μmol/L).The cell viability was measured by MTT assay,and flow cytometry was used to evaluate the apoptosis rate.DAPI staining was performed to observe the apoptosis phenomenon.The expression of E6 and E7 mRNA were detected by qRT-PCR.Western blot was carried out to detect the expression of E6 and E7 proteins.Results: Gene detection showed that this cell only contains HPV16 gene.The best time to inhibit proliferation of cancer cells by MTT assay is 24 h,the optimal concentration is 5~40 μmol/L.Under these conditions,the inhibitory effect of shikonin on Caski cells showed concentration-dependent relationship(P<0.05).The apoptosis rate rised gradually with the increase of drug concentration(P<0.001).Typical apoptosis morphology including nuclear fragmentation and pyknosis were observed with DAPI staining.The E6/E7 mRNA expression gradually decreased with increasing drug concentration,which were statistically significant(P<0.001).The relative expression of E6/ E7 protein decreased with the drug concentration by western blot(P<0.001).Conclusion: Shikonin can induce the apoptosis and inhibit the proliferation of HPV16 type cervical cancer Caski cells.The mechanism may be related to the decrease of E6 and E7 mRNA and protein expression in cancer cells.