Preliminary Study On Deuteporfin-mediated Photodynamic Therapy Induction Of Apoptosis And Autophagy In HPV18~+ Human Cervical Cancer Hela Cells

Human papillomavirus(HPV)can cause various types of epithelial diseases,and low-risk HPV(such as HPV 6 and 11)can cause condyloma acuminata(CA)and recurrent respiratory papillomatasis(RRP).High-risk HPV(such as HPV 16 and 18)can cause anal genital and oropharyngeal squamous cell carcinoma(SCCs).It is estimated that approximately 34,000 new HPV-related cancers are diagnosed,more than 4,000 die of cervical cancer,and approximately 1,100 die of anal cancer each year in the United States.In China,the incidence of cervical cancer is increasing year by year,and the patient population is also younger.The mortality rate of malignant tumors in young women aged 15 to 44 is ranked third in cervical cancer.It has been very difficult to cure HPV clinically so far.Photodynamic therapy(PDT)brings the dawn of treatment for HPV infection-related diseases.PDT can treat patients with high-risk HPV-positive and persistent infection for more than one year as well as patients with CIN I and II.Photodynamic therapy(PDT)is an anticancer therapy that relies on the selective photosensitizing of tumor cells,followed by visible light irradiation at the appropriate wavelength.The end result is formation of reactive oxygen species(ROS)that can cause photodamage to the cell.The interaction between light,cell or tissue molecular oxygen and PS gets the photodynamic reaction.Deuteporfin is an innovative porphyrin photodynamic drug independently developed in China.However,the mechanisms of deuteporfin induced target cell death have not been understood.So,a preliminary study was undertaken for the purpose of understanding the mechanisms of deuteporfin induced target cell apoptosis and autophagy.The main result are as follows:1、The killing effect of deuteporfin-PDT on cervical cancer Hela cellsThe cultured cells were dicided into 4 group:control group,deuteporfin group,LED group and deuteporfin-PDT group.After incubating with different concentrations of deuteporfin in the dark,the cells were irradiated through a series of light doses.The results of CCK-8 method indicated that deuteporfin-PDT significantly inhibited the growth of Hela cells.Under the same laser dose,the cell survival rate is significantly different with different same concentration.The growth inhibition rate of Hela cells was significantly increased with increasing concentration of deuteporfin.Then,we chose the concentration of deuteporfin at 0.4μmol/L and light dose at 9 J/cm~2 for the subsequent experiments.The morphology of the hela cells at different time points after deuteporfin-PDT treatmented at observed using an inverted fluorescence microscope.Hela cells treated with deuteporfin-PDT.After 6 h,both live cells and dead cells were present.At 24 h,the cells were pyknosis,and the reflectance was poor.The above results indicate that deuteporfin-PDT can significantly inhibit the activity of human cervical cancer Hela cells,and its inhibitory effect is deuteporfin concentration-light dose-dependent.The cells are pyknotic and have poor reflectivity.The above results indicate that deuteporfin-PDT can significantly inhibit the activity of human cervical cancer Hela cells,and its inhibitory effect is deuteporfin concentration-light dose-dependent.2、Deuteporfin-PDT induces cell apoptosisTo explore whether deuteporfin-PDT induces apoptosis in Hela.The morphological changes of PDT were observed by Hochst33342 staining microscopy.It was found that Hela cells showed nucleus pyknosis and apoptotic bodies formed 24 h after deuteporfin-PDT.Apoptosis was detected by Annexin V/PI staining-flow cytometry.The apoptosis rate of deuteporfin-PDT group was significantly different from that of the control group.These results indicate that deuteporfin-mediated photodynamic induction of apoptosis in Hela cells.3、Deuteporfin-PDT induces autophagyThe autophagic vacuoles of the cells were detected by MDC staining.The autophagosomes were observed by transmission electron microscopy.The expressions of autophagy-related proteins LC-3,Beclin-1 and P62 were detected by Western blot.Under the fluorescence microscope,deuteporfin-PDT was observed to cause accumulation of autophagic vesicles.The ultrastructure of the cells was observed under transmission electron microscopy.A large number of vesicular-like proteins or organelles were observed in the cytoplasm,and fused with lysosomes to form typical autophagosomes.The WB results showed that autophagy marker proteins LC3 and Beclin-1 were increased after photodynamic therapy,while P62 production decreased,suggesting autophagy.4、Deuteporfin-PDT induces ER stressAfter endoplasmic reticulum staining,the target of deuteporfin subcellular organelle was observed by confocal microscopy.It was found that the photosensitizer deuteporfin was targeted to the endoplasmic reticulum.Western blot was used to detect the expression of endoplasmic reticulum stress marker GRP78 and CHOP in Hela cells treated with deuteporfin-PDT.It was observed that the protein expression level of GRP78 and CHOP was up-regulated in a time-dependent manner.It is suggested that deuteporfin-PDT induces endoplasmic reticulum stress in Hela cells.