| Objective: BEAS-2B lung bronchial epithelial cell line was selected as a control to compare the differences in cellular uptake and localization of photosensitizers in different lung cancer cell lines(lung adenocarcinoma A549,lung squamous carcinoma H520,lung small cell carcinoma H446).To further investigate the differences in the death pathways of HPD-mediated photodynamic therapy in different types of lung cancer cells.Methods: A standard curve of HPD fluorescence values was plotted using fluorescence values measured at different concentrations of HPD.Four types of cells were incubated with 5 μg/m L of HPD for different times and then the fluorescence intensity was measured by multifunctional enzyme marker.Cell uptake was further quantified by flow cytometry to compare the differences in HPD uptake within the four cell lines.Comparative intracellular HPD uptake and distribution were observed using laser scanning confocal microscopy.The four cell lines were incubated with 5 μg/m L of HPD for 24 h,irradiated with a 630 nm laser at a power density of 50 m W/cm2 and cell viability was measured by the CCK8 method.Intracellular reactive oxygen species(ROS)levels were detected after group treatment and apoptosis was detected by Annexin V-FITC/PI double staining.The expression levels of BCL-2,BAX,Caspase 3,ATG5 and LC3 B proteins were detected by western blotting.Results:(1)The results of the intracellular uptake of HPD localization showed that the uptake of HPD by the four cells increased with time,but the accumulation rate of HPD differed.The results of HPD uptake by cells at 24 h and 48 h showed that the mean fluorescence intensity within the four cells was statistically different(F=199.0,P<0.0001;F=71.15,P=0.0006),with the mean fluorescence intensity within A549 cells being higher than that of H446,BEAS-2B and H520 cells.The mean intracellular fluorescence intensity was not statistically different between H446 and BEAS-2B cells(P=0.2724;P=0.9683,both >0.05),and was higher than that of H520(P<0.05).The four cell lines showed intracellular red fluorescence signals of varying intensities,and all of them were distributed in a punctiform manner in the cytoplasm.(2)CCK8 results showed that the viability of BEAS-2B,A549,H520 and H446 cells was reduced after HPD-PDT treatment and there were differences in cell survival rates(F=117.3,P<0.0001).(3)The results of ROS assay showed that HPD-PDT induced an increase in intracellular ROS production compared with the control group(P<0.05),and the level of ROS was H520>A549>H446>BEAS-2B.(4)The results of Annexin V-FITC/PI double staining assay showed that among the cell lines examined,the control and HPD-PDT groups apoptosis rate was statistically different(P<0.05);and the difference between the four HPD-PDT groups was also statistically significant(P<0.05),with apoptosis rates of H520>A549>H446>BEAS-2.(5)Western blotting results showed that HPD-PDT resulted in loss of Bcl-2 protein,upregulation of BAX and Caspase 3 protein expression,and induced autophagy,as evidenced by increased expression of autophagy-related proteins ATG5 and LC3 B in all cells tested.However,apoptosis-inducing proteins and autophagy proteins were statistically different in these four cell types.Conclusion: In vitro experiments showed differences in HPD uptake between different types of lung cancer cell lines,with lung adenocarcinoma A549 cells having the strongest HPD uptake,bronchial epithelial BEAS-2B and lung small cell carcinoma H446 cells the next strongest,and H520 cells the weakest.Their intracellular localization was located in the cytoplasm and was punctate.The sensitivity of the four cell types to the HPDmediated 630 nm laser was variable,with H520 > A549 > H446 > BEAS-2B.HPD-PDT kills cancer cells by inducing ROS production,leading to apoptosis,necrosis and autophagy.the ability of HPD-PDT to induce apoptosis and autophagy varies between lung cancer types. |
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