| Objective This study aims to investigate the proliferation inhibition and discuss its possibility mechanism of photodynamic therapy(PDT)mediated by hematoporphyrin derivative(HPD)on small cell lung cancer H446 cells and bronchial epithelial BEAS-2B cells and provides experimental basis and theoretical support for the application of photodynamic therapy in the clinical treatment of lung cancer.Methods During logarithmic phase of cultivated cells,small cell lung cancer H446 cells and bronchial epithelial BEAS-2B cells were respectively divided into control group,simple photosensitizer group,simple laser irradiation group and HPD-PDT group.Cells were incubated with different concentrations of HPD(0,5,10,12,15,20 ug/ml)for 4h,and then illuminated by 630 nanometer laser with different energy densities(0,25,50,75,100m W/cm~2)for 2 minutes.The cell survival rate of small cell lung cancer H446 cells and bronchial epithelial BEAS-2B cells were observed with CCK8 assay.Hoechst 33258staining was used to stain each group of cells and observe cell changes.The cell apoptosis of small cell lung cancer H446 cells and bronchial epithelial BEAS-2B cells were detected by Annexin V-FITC and propodium double staining method.The expression levels of apoptosis-related factors Bax,Bcl-2 and Caspase-9 m RNA in each group of H446 cells and BEAS-2B cells were detected by Real-time reverse transcription-polymerase chain reaction(RT-q PCR).Results(1)CCK8 revealed that incubation with hematoporphyrin derivatives alone or 630nm laser irradiation had no obvious inhibitory effects on small cell lung cancer H446 cells and bronchial epithelial BEAS-2B cells.HPD-PDT has inhibitory effects on H446 cells and BEAS-2B cells,and the inhibitory effect on small cell lung cancer H446 cells is more obvious.In the two cell lines,the cell viability decreased significantly,when the laser power density reached 50 m W/cm~2.And the difference was statistically significant compared with the control group(P<0.05).The inhibitory effect is limited to increase when the power density exceeded 50 m W/cm~2.The cell viability of the 15 ug/ml and 20 ug/ml PDT groups was significantly lower than that of the control group,and the difference was statistically significant(P<0.05).Select the laser power density of 50 m W/cm~2 and the HPD concentration of 15 ug/ml as the conditions in subsequent experiments.(2)The results of Hoechst 33258 staining showed that there were a large number of apoptotic cells in the HPD-PDT group of both small cell lung cancer H446 cells and bronchial epithelial BEAS-2B cells.With the increase of HPD concentration,the cell density was reduced and apoptotic cells increased.The apoptotic cells presented chromatin condensation and hyperchromatic nuclei.Compared with bronchial epithelial BEAS-2B cells,small cell lung cancer H446 cells have more apoptosis and significantly lower cell density.There are no obvious apoptotic cells in control group,simple photosensitizer group,simple laser irradiation group.(3)The results of flow cytometry assay revealed that the apoptosis rates of HPD-PDT group of small cell lung cancer H446 cells and bronchial epithelial BEAS-2B cells were(46.73±1.38)%and(28.56±1.80)%respectively.Compared with the black control group,the simple photosensitizer group and the laser irradiation group,the differences were statistically significant(P<0.05).(4)The RT-PCR examination showed that compared with the control group,the expression levels of Bax and Caspase-9 m RNA of HPD-PDT groups in small cell lung cancer H446 cells and bronchial epithelial BEAS-2B cells were up-regulated,while the expression levels of Bcl-2 m RNA were down-regulated.The differences between the HPD-PDT groups and the control group were statistically significant(P<0.05).Conclusion HPD-PDT has proliferation inhibitory effects on human small cell lung cancer H446 cells and human bronchial epithelial BEAS-2B cells.The best concentration of photosensitizer for HPD-PDT is 15 ug/ml and the best laser power density is 50 m W/cm~2.The inhibitory effect on small cell lung cancer H446 cells is more obvious.The apoptosis may be related to up-regulating the expression levels of Bax and Caspase-9 m RNA and down-regulating the expression levels of Bcl-2 m RNA. |
