Mechanisms Of Autophagy And Apoptosis In The Treatment Of Condyloma Acuminatum With 5-aminolevulinic Acid Photodynamic Therapy

BackgroundPrevious studies have suggested an effective role for 5-aminolevulinic acid photodynamic therapy(ALA-PDT)against various cancers.However,more and more reports have focused on the effectiveness of ALA-PDT in the treatment of human papillomavirus(HPV)-related diseases,such as genital condyloma acuminata and cervical intraepithelial neoplasia(CIN).There is increasing evidence that autophagy and apoptosis play important roles in PDT therapy;however,the mechanisms underlying these functions are complicated and warrant further research.Objective1.To explore the effect of ALA-PDT on HPV viral loads during the treatment of condyloma acuminatum2.HeLa cells were exposed to ALA-PDT in vitro to further demonstrate the underlying mechanisms that how ALA-PDT could reduce HPV viral loads during the treatment of condyloma acuminata and analyze the possible signaling pathways of autophagy and apoptosis in ALA-PDT.3.To explore the specific mechanisms of the autophagy degradation inhibitor hydroxychloroquine(HCQ)on autophagy and HeLa cell proliferation when combined with ALA-PDT.Methods1.The clinical efficacy of ALA-PDT therapy in the treatment of condyloma acuminatumWe initially investigated 111 patients suffering from genital condyloma acuminatum.HPV viral loads detected before and after three or six rounds of ALA-PDT treatment were compared during this procedure;a significant difference was noted1.1 Effect of ALA-PDT on HPV viral load before and after treatment of patientsHPV viral loads were dynamically detected before and after ALA-PDT.The changes of viral loads were analyzed in pretherapy,three and six rounds of PDT treatment.1.2 Effect of ALA-PDT on high risk(HR)and low risk(LR)HPV infectionHPV infection was divided into HR and LR types.The changes of viral loads before and after treatment of ALA-PDT in each subgroup were analyzed1.3 Effect of ALA-PDT on HPV load in patients with cervical condyloma acuminatumTo further explore the ALA-PDT effect on condyloma acuminata in special lesion,the changes of HPV viral loads in cervical infection were analyzed.These findings may be clinically useful for the improved treatment of HR-HPV infection and carcinogenesis.2.The mechanisms of autophagy and apoptosis in HeLa cell proliferation with ALA-PDT treatmentIn order to explore the mechanisms of ALA-PDT in reducing HPV viral loads,HeLa cells(HPV 18)were further exposed to ALA-PDT in vitro.Western Blot,fluorescence and flow cytology were used to detect the expression levels of autophagy and apoptosis and the possible signaling pathways.3.Effect and mechanisms of autophagy degradation inhibitor HCQ on ALA-PDT in cell inhibitionFirstly,cell proliferation rate was detected under time at 3 h,6 h,12 h and 24 h after ALA-PDT treatment with HCQ incubation by the cell counting kit-8(CCK-8)assay.Based on the results,we selected one of the time points for further experiments.The reactive oxygen species(ROS)levels were detected under flow cytometry and the autophagosomes were observed by a fluorescence microscope.Western Blot was used to analyse the Ras/Raf/MEK/ERK and PI3K/AKT/mTOR pathways expression combined with HCQ treatmentResults1.ALA-PDT could significantly reduce viral loads before and after treatment in patients with condyloma acuminatumALA-PDT could significantly reduce HPV viral loads and had therapeutic effect on both HR-and LR-HPV infection.As for patients with cervical condyloma acuminatum,ALA-PDT showed a healing effect as well.The results indicated that ALA-PDT can effectively reduce the viral loads and reduce the recurrence rate.It may also serve as a promising strategy for the treatment of precancerous or carcinogenesis lesions caused by HPV infection.2.ALA-PDT inhibited HeLa cell proliferation by inducing autophagy and apoptosisALA-PDT suppressed cell viability in a dose-and time-dependent manner.Evaluation of autophagy revealed unregulated expression of LC3? and p62.ALA-PDT also induced caspase-3 and cleaved caspase-3 expression.Western Blot analysis showed increased phosphorylation of Ras/Raf/MEK/ERK and downregulation of the expression of phosphorylated PI3K/AKT/mTOR pathways after treatment.ALA-PDT inhibited HeLa cells proliferation and may effectively reduce HPV viral load via autophagy and apoptosis by regulating the above signaling pathways.3.HCQ further strengthened the cytotoxic effect of ALA-PDTHCQ could significantly enhance the ALA-PDT effect in HeLa cells.CCK-8 analysis indicated that HCQ treatment resulted in a significant decrease in cell viability under ALA-PDT at different time gradients.Moreover,significant ROS activation was documented in HeLa cells after exposure to ALA-PDT+HCQ.Western Blot showed upregulated expression of autophagy related gene(Atg)Beclin-1.The phosphorylation levels of PI3K/AKT/mTOR were further decreased while the phosphorylation levels of Ras/Raf/MEK/ERK seemed no change.ConclusionsIn summary,this study demonstrates that ALA-PDT can significantly reduce HPV viral loads in patients with condyloma acuminata and is an effective approach for LR-and HR-HPV infection as well as cervical infection.Dynamic HPV viral loads quantitative monitoring can aid in the evaluation of therapeutic effects and lead to better treatment outcomes.In vitro experiments showed that there should be an optimal dose of ALA and laser intensity for treatment.And the results have implications for exploring the best dose effect and resistance mechanisms of PDT in clinical application.Molecular detection confirmed that autophagy was activated through PI3K/AKT/mTOR and Ras/Raf/MEK/ERK signaling pathways regulation,with apoptosis simultaneously activated.Since previous study demonstrated that ALA-PDT can suppress E6/E7 expression effectively,we conclude that ALA-PDT reduces HPV viral loads via autophagy and apoptosis and cell cycle inhibition by regulating E6/E7 proteins.Autophagy degradation inhibitor HCQ functioned to activate ROS levels of ALA-PDT to enhance the observed effect.The upregulated expression of Beclin-1 and downregulated expression of PI3K/AKT pathway could fiirther induce autophagy related cell death.Based on the above,these findings suggest strategies for the improvement of PDT efficacy in patients and inspired us to explore drugs that could reduce treatment resistance.