Mechanism Of 5-aminolevulinic Acid Photodynamic Therapy In Promoting Endoplasmic Reticulum Stress In The Treatment Of High-risk Human Papillomavirus Infection

Background5-aminolevulinic acid photodynamic therapy(ALA-PDT),as a promising treatment,has been clinically used to treat a variety of diseases related to high-risk human papillomavirus(HR-HPV)infection,such as condyloma acuminatum,Bowenoid papulosis,persistent HR-HPV infection of the cervix,and cervical intraepithelial neoplasia.The mechanism of ALA-PDT for HR-HPV infection-related diseases is also one of the research hotspots.Previous studies have shown that the subcellular localization of photosensitizers is closely related to the signaling pathways and cell death induced by PDT.5-aminoketovaleric acid,as a precursor of the strong photosensitizer protoporphyrin IX(PpIX),mainly enters the mitochondria after entering the cell,and the formed Pp? is also mainly localized in the mitochondria.So at present the research on the mechanism of ALA-PDT mainly focuses on its impact on mitochondria.There are few reports on whether ALA-PAT can affect the endoplasmic reticulum and trigger endoplasmic reticulum stress(ERS).Previous research of our team showed that ALA-PDT might reduce the HR-HPV virus load in patients with condyloma acuminatum by regulating apoptosis and autophagy.This study investigated the effects of ALA-PDT on autophagy and apoptotic of HeLa cells(HR-HPV infected cells)and related signaling pathways from the perspective of ERS-related pathways and aimed at providing new theoretical supports for the application of PDT in the clinical treatment of HR-HPV infection-related diseases.Methods1.HeLa cells were treated with different concentrations of ALA and different intensities of light energy.Cell viability was determined by the CCK-8 method,expressed as mean±standard deviation,and calculated as:cell viability=([ODexperiment-ODblank]/[ODcontrol-ODblank])X 100%).The treatment conditions with the cell viability closest to 50%were selected as the condition parameters for the next experiments.Set up groups with different conditions:Control group,ALA group,Light group,ALA-PDT group.Morphological images of different groups of cells were taken under an inverted phase contrast microscope(10 X 10).2.Immunofluorescence was used to detect the production of reactive oxygen species(ROS)in four groups.Western blot and quantitative analysis were used to detect the expression levels of ERS marker proteins CHOP and Grp78 and classical signaling pathways of UPR(PERK-eIF2 ?-ATF4,IRE1 and ATF6)and the expressions of these proteins after pretreatment with the ROS inhibitor NAC.Flow cytometry was used to detect the apoptosic level of each group.Western blot was used to detect the specific apoptosis marker of endoplasmic reticulum-caspase12.3.Western blot,confocal and immunofluorescence methods were used to evaluate the levels of autophagy.4.Flow cytometry was used to detect the changes of the concentration of Ca2+in the cytoplasm after ALA-PDT and after ALA-PDT combined with the pretreatments of ERS inhibitor 4-PBA or the intracellular calcium chelator BAPTA-AM.Western blot was used to detect the activity levels of CamKK ? and AMPK and the expression of LC3 after ALA-PDT and after ALA-PDT combined with the inhibitors(STO-609 or Com.C)or Ca2+chelator BAPTA-AM and ERS inhibitor 4-PBA respectively.6.Flow cytometry was used to detect the change of apoptosis rate of Hela cells after treatment with ALA-PDT combined with autophagy inhibitor 3-MA.Results1)ALA-PDT inhibited the cell viability of HeLa cells,and the cell viability was in ALA concentration and irradiation dose-dependent manner.in ALA and irradiation doses.The treatment condition with the closest cell survival rate to 50%was 0.25 mmol/L ALA for 24 h,and then irradiated HeLa cells with 1.2 J/cm2 of energy for 24 h.In the low-dose ALA-PDT group(0.25 mM,0.6 J/cm2),the interval of HeLa cells was increased,and the connection was disappeared.And in the higher-dose ALA-PDT group(0.25 mM,1.2 J/cm2 or 2.4 J/cm2)HeLa cells atrophied,the interval increased,the adhesion was poor,and cell debris floated.2)ROS was increased and ERS was occured,and the ERS marker proteins GRP78,CHOP,and UPR classic signaling pathways(PERK-eIF2 ?-ATF4,IRE1,and ATF6)were activated in HeLa cells after ALA-PDT;inhibition of production of ROS could reduce the expression of ERS marker proteins CHOP and Grp78.ALA-PDT could induce apoptosis and increase the cleavage of caspase12 in HeLa cells,indicating that ALA-PDT-induced apoptosis included endoplasmic reticulum-specific apoptosis.3)ALA-PDT increased the expression of LC3 ?/? and Beclin-1 proteins and the number of autophagic lysosomes in HeLa cells.4)ALA-PDT increased the cytosolic Ca2+ concentration.Pretreatment with ERS inhibitor 4-PBA or Ca2+chelator BAPTA-AM reduced the level of cytosolic Ca2+.After ALA-PDT,ERS could activate autophagy through Ca2+-CamKK ?-AMPK pathway,and suppression of ERS or chelating Ca2+ in cytoplasm or inhibition of CamKK? or AMPK could inhibit the production of autophagy.5)Inhibition of autophagy could increase the apoptosis rate of HeLa cells after ALA-PDT.ConclusionsALA-PDT could induce ERS in HeLa cells,which was based on the occurrence of oxidative stress.ERS could not only induce endoplasmic reticulum specific apoptosis,but also activate autophagy through Ca2+-CamKK ?-AMPK pathway.Inhibition of autophagy could increase the apoptosis rate of HeLa cells after ALA-PDT,suggesting that autophagy may be one of the mechanisms of PDT resistance;The Ca2+-CamKK?-AMPK pathway and autophagy may be targets to improve the killing effect of ALA-PDT.