Effects Of Autophagy In Curcumin-mediated Photodynamic Therapy On Epithelialmesenchymal Transition Of Lung Cancer Cells

BackgroundLung cancer is one of the most prevalent malignant tumors with high morbidity and mortality worldwide,which is a serious threat to human health.Although diagnostic and therapeutical techniques have been developing in recent years,lung cancer is one of the major disease burdens of human society.Aggressive local invasion and distant metastasis are the leading cause of death by lung cancer.Photodynamic therapy(PDT)is a physical factor therapy with wide application prospect.Due to its advantages of high selectivity in tumor sites and tolerable side effects,it is a promising therapy to deal with various solid tumors.It activates photosensitizers and produces cytotoxic reactive oxygen species(ROS)to inhibit invasion and metastasis of cancer.However,PDT also has limitations,such as increasing PDT-mediated resistance and poor effects of repeated treatments.Therefore,it is of great scientific significance to further clarify the antitumor mechanism of PDT,explore new therapeutic targets and develop possible combination therapy strategies.Epithelial-mesenchymal Transition(EMT)has been considered to play a key role in tumor metastasis such as lung cancer.In the early stage of metastasis,cancer cells progress through EMT,a process during which epithelial cells obtain semblable properties of mesenchymal cells with altered adhesive properties and motility and thereby achieve increased migratory probability to invade surrounding tissues.It has been reported that PDT represses the EMT process by influencing relevant signaling pathways,and inhibits the migration and invasion of tumor cells.Additionally,many reports have shown that PDT activates autophagy by producing ROS through a variety of signaling pathways.Curcumin(CUR)is extracted from turmeric,a traditional Chinese medicine.It has been received immense attention over the past decades because of various pharmacological properties,including anticancer,anti-inflammatory,anti-oxidation,antimetastatic and with fewer adverse effects.Furthermore,curcumin is a natural photosensitizer,and curcumin-mediated photodynamic therapy(CUR-PDT)has exhibited remarkable efficiency against various cancers.However,the effect of CUR-PDT on EMT and autophagy induction in lung cancer cells needs to be further confirmed.Previous studies have shown that autophagy plays a double-edged sword role in the tumorigenesis and cancer progression,which may inhibit tumor during cancer progression and also enable tumor cells to survive under stress.Recent studies suggest that autophagy can promote or inhibit EMT in lung cancer metastasis.On one hand,activated autophagy can inhibit EMT of lung cancer cells,and inhibition of autophagy can promote the EMT process of lung cancer.On the other hand,autophagy can also promote EMT in the process of lung cancer metastasis,and inhibition of autophagy can further inhibit EMT.Based on the important role of autophagy in tumor invasion and metastasis,in-depth exploration of the role and related mechanism of PDT-induced autophagy in EMT of lung cancer cells will provide new ideas and theoretical basis for the treatment of lung cancer.Therefore,this study combined physical therapy with traditional Chinese medicine to explore the role of curcumin-mediated photodynamic therapy(CUR-PDT)-induced autophagy in the EMT process of lung cancer,in order to find new potential therapeutic targets and explore better combination therapies for improving the anti-tumor effect of PDT.Methods1.To determine the intervention conditions of CUR-PDT on lung cancer cells(1)CKK-8 was used to detect the effects of different curcumin concentrations on the viability of lung cancer cells at different incubation times.(2)The intracellular curcumin of lung cancer cells at different incubation times were observed by inverted fluorescence microscope.(3)The effects of different laser energy densities on the viability of lung cancer cells were detected by CCK-8.2.Effects of CUR-PDT on EMT,migration and invasion of lung cancer cells(1)The expression levels of EMT-related proteins E-cadherin,N-cadherin,Vimentin and Snail of human lung cancer A549 cells and SPCA1cells after CUR-PDT were detected by Western Blot.(2)The effects of CUR-PDT on the migration of human lung cancer A549 cells and SPCA1 cells were observed by wound healing assays.(3)The effects of CUR-PDT on the migration and invasion of human lung cancer A549 cells and SPCA1 cells were observed by Transwell assays.3.CUR-PDT regulates autophagy of lung cancer cells(1)The expression levels of autophagy related proteins LC3Ⅱ/LC3Ⅰ,p62 and beclin1 in A549 and SPCA1 cells after CUR-PDT were detected by Western Blot.(2)LC3 fluorescence spots and co-location of LC3 and Lamp2 in lung cancer cells after CUR-PDT were observed by immunofluorescence.(3)The ultrastructure of autophagy in lung cancer cells after CUR-PDT was observed by transmission electron microscopy(TEM).4.The role of CUR-PDT induced autophagy in the regulation of EMT in lung cancer(1)The expression levels of EMT-related proteins and autophagy related proteins after CUR-PDT combined with autophagy inhibitor Chloroquine(CQ)were detected by Western Blot.(2)The effects of CUR-PDT combined with CQ on the migration ability of lung cancer cells were observed by wound healing assays.(3)The effects of CUR-PDT combined with CQ on the migration and invasion ability of lung cancer cells were observed by Transwell assays.Results1.The curcumin concentration of 20μM,incubation time of 12 h and laser energy density of 2.4 J/cm~2 were selected for the following experiments.2.CUR-PDT inhibited EMT,migration and invasion of lung cancer cells(1)CUR-PDT significantly increased the expression of epithelial marker E-cadherin protein,and decreased the expressions of mesenchymal markers N-cadherin,Vimentin and Snail in A549 and SPCA1 cells.(2)CUR-PDT significantly inhibited the motility of A549 and SPCA1cells after wounding.(3)CUR-PDT significantly decreased the number of migrated and invaded both in A549 and SPCA1 cells.3.CUR-PDT promoted autophagy in lung cancer cells(1)CUR-PDT significantly up-regulated the expression of LC3-II and beclin1,and down-regulated the expression of p62,which was most obvious at 24 h after PDT.(2)The LC3 fluorescence spots of lung cancer cells after CUR-PDT were most obvious at 24 h after PDT.The co-location fluorescence of LC3and Lamp-2 in lung cancer cells after CUR-PDT treatment was more obvious than that in other groups.(3)The significantly increased ultrastructure of autophagosomes were observed in lung cancer cells after CUR-PDT by TEM.4.Inhibition of CUR-PDT induced autophagy significantly enhanced the effect of CUR-PDT inhibiting EMT,migration and invasion of lung cancer cells(1)CUR-PDT combined with CQ further significantly up-regulated epithelial marker E‐cadherin,and significantly down-regulated mesenchymal markers N-cadherin,Vimentin and Snail.(2)CUR-PDT combined with CQ further inhibited the migration of lung cancer cells after wounding.(3)CUR-PDT combined with CQ significantly decreased the number of migrated and invaded human lung cancer cells.Conclusions(1)Curcumin-mediated PDT inhibited EMT,migration and invasion of lung cancer cells.(2)CUR-PDT induced autophagy in lung cancer cells.(3)Inhibition of CUR-PDT induced autophagy further inhibited EMT,migration and invasion of lung cancer cells,suggesting that autophagy played a positive role in the process of CUR-PDT inhibiting EMT in lung cancer cells.(4)CUR-PDT combined with autophagy inhibitor improved the anti-tumor effect of PDT,and provided a theoretical basis for seeking new combined therapies.