The Study On The Role And Mechanism Of DJ-1/ROS/AMPK Induced Autophagy In CPX Against Cervical Cancer

Background:Cervical cancer is one of the most common gynecological malignancies and the fourth most lethal tumor.And the incidence is second only to breast cancer in gynecological malignancies in China.About 500,000 new cases and 200,000 deaths of cervical cancer are reported globally every year,with China ranking at the forefront of both case and death.In recent years,with the development of cancer diagnosis technology and the application of the HPV vaccine,the incidence and mortality of cervical cancer decreased.But the incidence of cervical cancer is becoming younger,which has seriously affected the physical and mental health of Chinese women.At present,the emergence of targeted drugs for blocking the occurrence and development of cervical cancer.It is very significant to improve the survival rate and quality of life of patients with cervical cancer.The advantages of targeting drugs have targeting killing and fewer side effects.Therefore,the new targeted therapy of research has provided new methods and strategies for cervical cancer.Ciclopirox ethanolamine(CPX)is a lipophilic iron chelator with a broad spectrum of antifungal activity.Recently,CPX is sensitive to colorectal cancer,breast cancer,liver cancer,and it exited its anticancer activity by inhibiting cell proliferation,inducing apoptosis,and inhibiting cell migration and invasion.However,the specific mechanism of CPX is not distinct yet,and further investigation is needed.Method:Firstly,CPX had anti-cervical cancer activity in vitro experiments.Next,cellular and molecular biological methods confirm that CPX could induce autophagy and promote autophagy flux in cervical cancer.Then studies were perfomerd to reveal the molecular mechanism of CPX promoting autophagy in cervical cancer.Finally,clarifing the role of autophagy in the inhibition of cervical cancer by CPX.The main research methods include conventional cell culture technology,exogenous gene introduction or gene silencing technology.Conventional molecular and cytological research methods,including CCK8 assay,Cell clone formation assay,Flow cytometry,qPCR,Western blot and Immunofluorescence,etc.Electron microscopy technology and Biostatistical analysis method.Result:1.CCK-8 assay and flow cytometry assay found that CPX did not have strong lethal effect on cervical cancer cells,while plate clone formation assay and EDU assay showed that CPX significantly inhibited the proliferation of cervical cancer cells;2.The results of flow cytometry cell cycle assay further indicated that CPX inhibited the proliferation of cervical cancer cells;3.Western blotting and immunofluorescence assay showed that CPX induced autophagy and promoted autophagy flow in cervical cancer cells;4.CPX can down-regulate the expression level of DJ-1 in cervical cancer.At the same time,Sidj-1 was used to silence DJ-1 in cervical cancer cells,and the results of western blot showed that the down-regulation of DJ-1 enhanced autophagy in cervical cancer cells;5.Flow cytometry to detect ROS showed that CPX induced the accumulation of ROS in cervical cancer cells.In addition,the Western blotting experiment showed that NAC combined with CPX treatment could inhibit the autophagy activity of cervical cancer cells;6.Autophagy induced by CPX in cervical cancer cells is mediated through the DJ-1/ROS/AMPK signaling pathway.The results of the CCK-8 assay,plate clone formation assay,and EDU assay indicated that inhibition of autophagy could enhance the Anti-cervical cancer effect of CPX.Conclusion:Our study showed that DJ-1 plays a vital role in CPX-mediated protective autophagy of cervical cancer cells.Also,by taking advantage of the characteristic of CPX in inducing protective autophagy in cervical cancer,the combination of autophagy inhibitors can enhance the anticancer activity of commonly used chemotherapy drugs in cervical cancer.