Mechanism Of Dihydroartemisinin Combined With 5-ALA Photodynamic Therapy In The Treatment Of High-risk HPV Infected Cervical Cancer

BackgroundWith the global incidence of malignant tumors increasing year by year,cervical cancer has ranked second among the common malignant tumors of female reproductive system at home and abroad,and its morbidity and mortality rank fourth among all female malignant tumors[1].High-risk human papillomavirus(HR-HPV)infection has been proved to be highly associated with cervical cancer,cervical intraepithelial neoplasia and oropharyngeal carcinoma[2,3].As a promising therapy,5-aminolevulinic acid-mediated photodynamic therapy(ALA-PDT)has been used in the treatment of a variety of HR-HPV infection-related diseases,such as persistent cervical HR-HPV infection and cervical cancer,cervical intraepithelial neoplasia(CIN),esophageal squamous cell carcinoma,skin cancer and condyloma acuminatum[4].However,some preclinical studies found that PDT can induce the activation of some tumor resistance pathways(such as NF-?B signaling pathway),which may leads to tumor resistance[5].Dihydroartemisinin(DHA)is one of artemisinin derivatives,which has good antitumor activity and has inhibitory effect on cervical cancer,pancreatic cancer,leukemia,osteosarcoma,lung cancer and other cells[4,6-8].Current studies have shown that DHA can inhibit NF-?B signaling pathway and thereby inhibit tumor cells proliferation[9].In addition,studies have shown that DHA has inhibitory effect on HeLa cells,and can also enhance the sensitivity of HeLa and SiHa cells to chemotherapy[10,11].Therefore,we speculate that DHA may be one of the measures to overcome the tumor resistance of ALA-PDT in the treatment of HPV infection related cervical diseases.Prior to this study,there was no study on the combination of ALA-PDT and DHA to improve the clinical efficacy of cervical cancer.Therefore,the purpose of this study is to explore the efficacy and mechanism of ALA-PDT combined with DHA in the treatment of HR-HPV-infected cervical cancer in vitro and in vivo.Methods1)Effects of ALA-PDT combined with DHA on the activity and proliferation of HeLa cells.HeLa cells were stimulated by different concentrations of DHA solution and different intensity of light energy.The cell activity was determined by CCK-8 method,and the treatment condition with the closest cell survival rate of 50%(IC50)was selected as the conditional parameter of the follow-up experiment.Set up groups:Control group,ALA-PDT group,DHA group,ALA-PDT+DHA group.Cell viability and clone formation were detected by CCK-8 method and clone formation test.2)Effects of ALA-PDT combined with DHA on ROS production and apoptosis of HeLa cells.The production of ROS in each group was detected by immunofluorescence,and the apoptosis level of cells in each group was detected by flow cytometry.3)The regulatory mechanism of ALA-PDT combined with DHA treatment on HeLa cells.The mechanism of PDT regulating anti-tumor immunity was analyzed by bioinformatics,and NF-?B/HIF-1?/VEGF pathways were detected by Western blotting.4)Effect of ALA-PDT combined with DHA on subcutaneous transplanted tumor of HeLa cells in nude mice.The subcutaneous tumorigenesis model of cervical cancer HeLa cells in nude mice was established.The tumor proliferation was observed and the expression level of ki67 in paraffin-embedded tumor samples was detected by immunohistochemical method.Results1)ALA-PDT inhibited the cell activity of HeLa cells in a dose-dependent manner.The treatment condition that the cell survival rate was closest to 50%was treated with 0.25mM ALA for 24 hours,and then cultured for 24 hours after the cells were irradiated with 1.2 J/cm2 of energy.2)DHA inhibited the cell activity of HeLa cells in a concentration-dependent manner.The treatment condition that the cell survival rate was closest to 50%was cultured with 30 ?M DHA for 24 hours.3)DHA combined with ALA-PDT inhibits the activity,proliferation and colony formation ability of HeLa cells.4)ALA-PDT combined with DHA significantly increased the production of ROS and promoted apoptosis in HeLa cells.5)Bioinformatics analysis showed that PDT alone induced macrophages to activate the expression of certain cancer-promoting signaling pathways.6)DHA inhibits NF-?B/HIF-1?/VEGF pathway activated by ALA-PDT.7)In vivo experiments showed that DHA could enhance the inhibitory effect of ALA-PDT on the proliferation of cervical cancer cells in nude mice.ConclusionsALA-PDT can reduce the survival rate and long-term proliferation of HeLa cells,and promote the production of ROS and induce apoptosis.Combination of DHA can enhance the above effects of ALA-PDT.DHA inhibits NF-?B/HIF-1?/VEGF pathway activated by ALA to enhance anti-tumor immunity.In vivo experiment of subcutaneous tumor in nude mice also confirmed that DHA can effectively enhance the efficacy of ALA-PDT in the treatment of cervical cancer infected with HR-HPV.