Preliminary Study Of Autophagy And Apoptosis In Photodynamic Killing Of Gastric Cancer Cell Lines

Objective: To investigate the preliminary mechanism of autophagy and apoptosis occurring in gastric cancer cells killed by Photodynamic Therapy(PDT)and the possible role of autophagy and Nrf2,and to provide a theoretical support in the understanding of the mechanism of gastric cancer death after PDT and combination therapy.Methods:(1)The proliferation rate of MKN45 cells and AGS cells under different light doses and different combinations of photosensitizer concentrations were determined using the experimental method of Cell Counting Kit-8,and the optimal light dose and photosensitizer concentration at IC50 were selected as the subsequent experimental intervention conditions.(2)Different groups were set:Control group(C group),PDT 6 h group,PDT 12 h group,PDT 24 h group,and images of different groups were taken under the inverted fluorescence microscope after PDT intervention according to their groups to observe the changes of cell morphology at different time periods before and after PDT.(3)Different groups were set up: Control group(group C),PDT 12 h group and PDT 24 h group,and the changes of cell morphology and the production of apoptotic vesicles and autophagic vesicles were observed by transmission electron microscopy before and after the PDT intervention according to their groups.(4)Different groups were set up: Control group(C group),photosensitizer group(P group),laser light group(L group),PDT 6 h group,PDT 12 h group,PDT 24 h group,and the expression of autophagy-related proteins Beclin-1,ATG5,LC3 B,p62 in each group was detected by Western blotting technique;the expression of mitochondrial apoptosis-related proteins Bax,Bcl2 and endoplasmic reticulum apoptosis-related protein CHOP were detected by Western blotting;the expression of anti-oxidative stress protein Nrf2 was detected by Western blotting.Results:(1)The proliferation inhibition of AGS and MKN45 cells by PDT showed a dependence of light dose and photosensitizer concentration.(2)As observed by inverted fluorescence microscopy,compared with the Control group,gastric cancer cells after PDT showed an increase in the number of cell death,a decrease in the number of adherent cells,an increase in cell spacing,an increase in intracellular and cell membrane outgrowth vesicles,and a gradual nucleus consolidation and disintegration as time progressed.(3)The formation of intracellular apoptotic vesicles and autophagic vesicles was observed in the PDT group compared with normal cells by transmission electron microscopy,and the number of mitochondria decreased,the number of intracellular and membrane effervescent vesicles increased,the nucleus gradually disintegrated and the cell membrane ruptured with time progression.(4)Changes in autophagy-related protein expression after PDT: MKN45: Beclin-1expression increased,and was most significant at a 6 h time point.ATG5 expression increased,most significantly at 6 h.LC3 B expression increased during a 24 h time point,but not statistically significant at other time points(P > 0.05).p62 expression increased,most significantly at a 6 h time point.AGS: Beclin-1 expression increased,most significant at 6 h.The expression of ATG5 was not statistically significant(P>0.05).Additionally,LC3 B expression increased,most significantly at a 12 h time point.Moreover,p62 expression increased,most significantly at 24 h.(5)Changes in apoptosis-related protein expression after PDT: MKN45: Bcl2 expression decreased with time at 6 h,12 h,and 24 h,Bax expression increased,most significantly at 6h,CHOP expression increased,most significantly at 6 h;AGS: Bcl2 expression decreased with time at 6 h,12 h,24 h,Bax expression increased with time,CHOP expression increased with time.(6)Changes of oxidative stress pathway-related protein Nrf2 after PDT: MKN45: Nrf2 expression increased at 6 h and 12 h,most significant at 6 h;AGS: Nrf2 expression increased,at a 6 h time point.Conclusion:(1)The killing and destruction of gastric cancer cells by PDT is photosensitizer dose and light energy dependent.(2)PDT can induce autophagy in gastric cancer cells,and autophagy and Nrf2 may be the mechanism of anti-PDT treatment in gastric cancer cells in the early stage of PDT.(3)PDT can induce Endoplasmic reticulum apoptosis and mitochondrial apoptosis in gastric cancer cells.