| Objective: Cervical cancer is one of the most common gynecological malignant tumors worldwide,for which chemotherapeutic strategies are limited due to their non-specific cytotoxicity and drug resistance.The natural product thymoquinone(TQ)has been reported to target a vast number of signaling pathways for carcinogenesis in different cancers,and hence is regarded as a promising anticancer molecule.Inhibition of epithelial to mesenchymal transition(EMT)mediators is an important approach in anticancer researchs.In this study,TQ was used to treat the cervical cancer cell lines CaSki and SiHa to investigate its effects on migration and invasiveness of cells as well as cell proliferation and apoptosis.The effects of TQ on EMT-associated genes/ proteins have been also studied as EMT process is related to cancer metastasis.Thus,our study aimed to provide new targets of TQ and generate new ideas for the clinical treatment of cervical cancer.Methods:(1)Cell viability was assayed by CCK-8 analysis.For this,CaSki and SiHa cervical cancer cell lines were treated with different dosage of TQ(final concentration 0μM/ mL,1μM/mL,5μM/ mL,10μM/ mL,20μM/ mL,40μM/ mL)for different time periods(12h,36 h,24h and 48h).(2)A real time cell analyzer was used for the real time analysis of CaSki and SiHa cervical cancer cell migration,invasion and growth index,after treated with different dosage of TQ(final concentration 0μM/ mL,5μM/ mL,10μM/ mL).(3)Flow cytometry(FWC)was used to check the cellular apoptosis,after CaSki and SiHa cervical cancer cell treated with different doses of TQ(0μM/ mL,5μM/ mL,10μM/ mL).(4)RNAs were extracted from CaSki and SiHa cervical cancer cells after TQ treatment(0μM/ mL,5μM/ mL,10μM/ mL),and RT-qPCR was performed to analyze the mRNA level expression of pro-apoptotic/ anti-apoptotic genes and EMT-associated genes.(5)Proteins were extracted from CaSki and SiHa cervical cancer cells after TQ treatment(0μM/ mL,5μM/ mL,10μM/ mL),and Western blotting was performed to analyze the protein level expression of genes.(6)Luciferase reporter assay was used to check whether Twist1 and Zeb1 are the direct target of TQ after CaSki and SiHa cervical cancer cell treated at different doage of TQ(0μM/ mL,1μM/ mL,2μM/ mL,4μM/ mL,8μM/ mL,16μM/ mL).(7)Gene methylation assay was used check Twist1 promoter methylation status,after CaSki and SiHa cervical cancer cell treated with TQ(0μM/ mL,5μM/ mL)for 24 h.Results:(1)CCK-8 results demonstrated that TQ has dose-dependent cytotoxic effects on both of CaSki and SiHa cervical cancer cell lines.(2)Real time cell analysis showed that TQ inhibits the migration and invasive characteristtics in both of CaSki and SiHa cervical cancer cell lines.(3)Flow cytometry(FWC)showed that,TQ also increased cellular apoptosis in some extent.(4)qPCR results suggested that,at the mRNA level,TQ treatment inhibited the expression of Twist1,Zeb1 expression,and increased E-Cadherin expression.TQ also affected Snail1,Slug,Vimentin and MMP-9 in CaSki cells,but not in Si Ha cells.N-Cadherin expression was found unaffected.Bax and Bcl-2 were remained unaffected too.(5)Western Blot results demonstrated,at the protein levels,TQ treatment inhibits the expression of Twist1,Zeb1 expression,and increased E-Cadherin expression,while PARP,Caspase-3,Caspase-9 were unaffected.(6)Luciferase reporter assay showed that TQ decreases the Twist1 and Zeb1 promoter activities respectively,as the Relative Light Units(RLU)was decreased with the increase of TQ dosage,indicating that Twist1 and Zeb1 might be the direct targets of TQ.(7)The proximal promoter methylation of Twist1 gene was found slightly increased by TQ treatment.Conclusion: Our findings suggest that TQ markedly inhibited the proliferation of CaSki and SiHa cervical cancer cells in a time-dependent and dose-dependent manner,and suppressed the migration and invasion of these cancer cells.Targeting EMT-TFs,like Twist1 and Zeb1,might be the possible mechanism of action of TQ in controlling metastasis in cervical cancer.The cell apoptosis rate of cervical cancer cells was found to be increased by TQ,but the mechanism is not clear.This study indicates that TQ is a possible chemotherapeutic agent against cervical cancer,however for the further development and establishment of TQ as a clinical drug,clinical investigations are necessary. |
