| Objective:Our research group has recently synthesized a new and low-cost photosensitizer (PS), chlorophyllin f, with complete proprietary intellectual property rights. However, its photodynamic characters and effects, as well as its mechanisms against bladder cancer cells are still unknown. In this study, we aimed to investigate the preparation and the photodynamic characters of chlorophyllin f, and clarify its photodynamic effects and potential mechanisms of phototoxicity in human bladder cancer cells.Methods:l.We first obtained crude chlorophyll material which was extracted from traditional Chinese herb Excrementum bombycis. Then the crude chlorophyll was undergone basic hydrolysis, separation, extraction and purification, and we harvested chlorophyllin e6. Chlorophyllin e6 was then converted into chlorophyllin f under certain procedures.2. The purity of the sample was detected by high pressure liquid chromatograph, the relative molecular weight of chlorophyllin f was examined by mass spectrometry, and the absorption spectrum and peak was analysed by fluorescence spectrophotometer. In addition,1,3-diphenylisobenzofuran (DPBF), a sensitive probe of reactive oxygen species (ROS), was used to detect the 1O2 produced in the system in which 5mM DPBF was first mixed with 2mM chlorophyllin f and then irradiated by a 40mW filtered 405nm red laser from a projector for different times of 15,30,45,60, and 75 seconds.3.5637 and T24 bladder cancer cells were grown into exponential phase in vitro. Afterwards the cells were incubated with different concentrations of chlorophyllin f and then irradiated with 650 nm laser light. The controls included cells treated with chlorophyllin f but without laser light, cells exposed to light without chlorophyllin f as well as cells with neither laser light nor chlorophyllin f. Photocytotoxicity was monitored with the Cell Counting Kit-8 assay. 4.5637 and T24 cells were first co-incubated with chlorophyllin f for 4h and then the Mito-Tracker Green probe was used to label mitochondria, the CLSM excited by 400nm and 488nm dual channels was applied to reveal chlorophyllin f intracellular localization.5. The morphological changes of the f-PDT-treated cell 5637 and T24 cells after DAPI staining were observed under fluorescence microscopy.6. Flow cytometry was used to analyze the apoptosis rates of 5637 and T24 cells after chlorophyllin f-mediated-PDT.7. The Apoptosis Antibody Array was used to detect the expression of apoptosis related proteins after the cancer cells were treated by chlorophyllin f-mediated photodynamic therapy, including bad, bax, BID, BIM, Bcl-2, Bcl-w, cytochrome C, Caspase-8, Caspase-3, XIAP and SMAC, so that we clarify the possible mechanisms of cell apoptosis.8.5637 and T24 cells were first co-incubated with chlorophyllin f for 2h, and then the Lyso-Tracker Green probe was used to label lysosomes, the CLSM excited by 400nm and 504nm dual channels was applied to reveal chlorophyllin f intracellular localization.9. Western blot, and staining with Cyto-ID(?)utophagy Detection dye, MDC and AO staining were performed to assess autophagy.10. TEM was used to evaluate the autophagical morphological changes of the f-PDT-treated cells.11. The role of autophagy was examined by measuring cell viability and apoptosis in both cell lines pretreated with the autophagy inhibitor 3-methyladenine (3-MA).Results:1. The purity of chlorophyllin f was 90.19%, and its relative molecular weight was 539.3. The absorption peaks of chlorophyllin f were 653.5nm,599.Onm, 501.5nm and 398.5nm, with the highest ones in 398.5nm and 653.5nm. What is more, the fluorescence intensity of DPBF in chlorophyllin f solutions decreased with the light irradiation from 15 to 75 seconds. The degradation rate is proportional to the 1O2 yield.2. Chlorophyllin f exhibited significant photocytotoxicity in both 5637 and T24 cells. In 5637 cells, with 4J/cm2 light dose, the growth inhibition rates of lug/ml, 2ug/ml and 4ug/ml chlorophyllin f mediated photodynamic effect were 33.92%, 57.38% and 84.88%; while with 2J/cm2 light dose, the growth inhibition rates were 25.4%,51.21% and 70.09% respectively. In T24 cells, with 4J/cm2 light dose, the growth inhibition rates of 2.5,5 and 10 ug/ml chlorophyllin mediated photodynamic effect were 34.03%,60.11% and 86.51%, while with 1J/cm2 light dose, the growth inhibition rates were 31.79%,53.83% and 72.89% respectively. Treatment with chlorophyllin for laser light alone did not induce cytotoxicity.3. The CLSM revealed that the red fluorescence stimulated by 400nm laser light emitted by chlorophyllin f and green fluorescence stimulated by 488nm emitted by Mito-Tracker Green probe distributed almost the same.4. In the 5637 and T24 cells which were treated by f-PDT and stained by DAPI, the typical apoptotic morphagical features including nuclear with irregular edges, karyopycnosis, nuclear dissolution, nuclear fragmentation, the increased nucleosomes and other debris, can be observed under fluorescence microscope; while these could not be found in the control cells.5 With 4ug/ml and 4J/cm2 light dose, the apoptosis rate of 5637 cells after chlorophyllin f-mediated-PDT was50.61±1.66%, the apoptosis rate of the control group was 4.05±0.12%. With 10 ug/ml and 4J/cm2 light dose, the apoptosis rate of T24 cells after f-PDT was 54.3±1.32%, and the apoptosis rate of the cell control group was 11.31±0.71%. Both apoptosis rates in f-PDT groups were significantly higher than control groups (P<0.01).6 In 5637 cells, the expression of bad, BID, BIM, cytochrome C, Caspase-3 and SMAC showed an increase of different degrees in PDT treated group, while Bcl-2, Bcl-w and XIAP protein decreased. And in T24 cells, the expression of bad, bax, BID, BIM, cytochrome C, Caspase-8, Caspase-3 and SMAC rised in different degrees in PDT treated group, while Bcl-2, Bcl-w and XIAP protein decreased.7 The CLSM showed that the red fluorescence stimulated by 400nm laser light emitted by chlorophyllin f and green fluorescence stimulated by 504nm emitted by Lyso-Tracker Green probe were almost indentical in 5637 and T24 cells.8 The expression of Beclinl protein and the proportion of LC3-II:LC3-I in both 5637 and T24 cell lines significantly increased after f-PDT.9 Autophagy, characterized by an increase in the formation of Cyto-ID(?)utophagy Detection dye-labeled autophagosomes, MDC fluorescent dye-labeled late-stage autophagosomes and acridine orange-labeled autolysosomes, was observed in f-PDT-treated cells.10.5637 and T24 cells treated by chlorophyllin f-mediated-PDT, were then observed by TEM, and it revealed double-membrane autophagosome structures 1 hour after f-PDT. But the control groups did not show the same changes.11 The CCK-8 assay showed that the growth inhibition rates of f-PDT with 3-MA group, f-PDT group and 3-MA group in 5637 cells were 93.52%,83.15% and 6.73% respectively; while in T24 cells, the growth inhibition rates were 95.51%,86.19% and 5.78% respectively.12 There was also a significant increase in apoptotic cell death in the f-PDT+3-MA group compared with the f-PDT alone group in both the two cell lines(P<0.05),Conclusion:1.The purity of chlorophyllin f that we prepared is high, and its optical properties meet ideal photosensitizer requirements.2, The photodynamic effects of chlorophyllin f combine with 650nm laser light on 5637 and T24 cells are significant, and depend on the concentration of chlorophyllin f and the laser dose.3. Chlorophyllin f could locate and also function at both mitochondria and lysosome. Chlorophyllin f-mediated-PDT first induces autophagy, and then apoptosis which may be one of the mechanisms against 5637and T24 cells.4. In f-PDT-treated 5637 and T24 cells, there were a significant increase in the expressions of bad, bax, BID and BIM, with a notable decrease in those of Bcl-2 and Bcl-w. These changes could trigger cytochrome C, XIAP and SMAC in mitochondria, and they were released into cytoplasm which then activated Caspase-3 and other Caspases. The executioner caspases then induced apoptosis of bladder cancer cells.5. Autophagy seems to play a tumour-promoting role in the treatment of f-PDT for bladder cancer cells, and more importantly, its inhibition could enhance f-PDT-mediated apoptotic cell death. These results suggest that chlorophyllin f is a new, effective photosensitizer and that the combination of f-PDT with autophagy inhibitors may be an attractive therapeutic strategy against human non-muscle invasive bladder cancer. |
PDF Full Text Request