| Purpose: To study the killing effect of sequential treatment of m-THPC-based PDT followed by platinum-based chemotherapy on Hep-2 cells in vitro.Materials & Methods: In this study,the cell line was Hep-2 cell,the photosensitizer in PDT treatment was m-THPC,the light source was 650 nm laser,and the chemotherapy drug was CDDP and 1,3,5,7-tetramethyl-8-(4-pyridyl)-4,4'-difluorobora-dia-zaindacene labeled Platinum conjugate(BODIPY-Pt).The gradients of m-THPC concentration,light intensity and CDDP concentration were set,and the concentration of m-THPC(0.5 ?M),light intensity(2 J/cm~2)and CDDP concentration(5?M)were determined by MTT assays.The study was divided into four groups: 1)Control group;2)PDT treatment group;3)CDDP chemotherapy group;4)PDT+CDDP sequential treatment group.The changes of cellular activities in each group were studied by MTT assays.Apoptosis,necrosis and total cell deaths were analyzed by flow cytometry.The expressions of apoptotic markers(Bax,Bcl-2),autophagic markers(ATG-7,LC3(LC3-I,LC3-II),and immune markers PD-L1 were observed by Western blot.Hep-2 cells in each group were stained with Hoechst 33258 to observe the changes of cellular nuclei.BODIPY-Pt and mitochondrial fluorometric probe Mito Tracker? Red were used to assess the distribution of drugs in Hep-2 cells and the changes in mitochondrial content in each group.The expressions of PD-L1 in each group was detected by reverse transcription quantitative polymerase chain reaction(RT-q PCR).The formation of autophagosomes in Hep-2 cells was observed under transmission electron microscope.Results: MTT results showed that m-THPC had no significant effect on the survival rate of Hep-2 cells in the absence of light,and the survival rate of Hep-2 cells decreased with the increase of m-THPC concentration when the light intensity was constant.When the light intensity is 2 J/cm~2,the IC50 of m-THPC is 0.74?M.2 J/cm~2 light intensity and 0.5 m-THPC concentration were used in the study,at which time the cellular survival rate decreased by about 30%.The IC50 of CDDP was 29.01?M,and 5?M CDDP concentration was used in the study,at which time the cell survival rate decreased by about 30%.MTT results showed that the PDT treatment followed by CDDP significantly reduced the survival rate of Hep-2 cells compared with the monotherapy group.Flow cytometry results showed that PDT mainly cause cell necrosis,while CDDP mainly lead to cell apoptosis.Hep-2 cells necrosis rate of sequential treatment group was significantly higher than that of CDDP group,with no significant difference with PDT group.The apoptosis rate of sequential treatment group was significantly higher than that of PDT group,but there were no significant differences with CDDP group.Total cell death of sequential treatment group was significantly higher than that of single treatment group.Western blot results showed that the expression of Bax of Hep-2 cells in the sequential treatment group was significantly higher than that in the single treatment group,the expression of Bcl-2 was significantly lower than that in the PDT group,the expression of ATG-7 was significantly lower than that in the single treatment group,the LC3-II / LC3-I ratio was significantly lower than that in the PDT group,the ratio was significantly higher than that in the CDDP group,and the expression of PD-L1 was significantly lower than that in the CDDP group.The results of Hoechst 33258 staining showed that the majority of Hep-2 cell nuclei in the control group were normal with only about 4% of the cells had uniform hyperchromatic staining.The rate of Hep-2 nucleus irregularity and hyperchromatin in PDT+CDDP sequential treatment group,CDDP group and PDT group was about 80%,38% and 35%,respectively.The results of confocal laser showed that sequential therapy decreased mitochondrial content and drug retention.The results of transmission electron microscope showed that the autophagy of Hep-2 cells was more obvious in sequential treatment group than that in the single treatment group.Conclusion: The sequential treatment of m-THPC based PDT followed by CDDP based chemotherapy synergistically killed Hep-2 cells through the apoptotic,necrotic,autophagic,immune and mitochondrial pathways. |
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