Study Of Photodynamic Inhibitory Action On Human Breast Cancer Cell Line MCF-7 With CPD4 And Its Mechanisms

Objective:Photodynamic therapy(PDT) is a form of photochemotherapy requiring the simultaneous presence of a photosensitiser,activating light of the proper wavelength and molecular oxygen in order to produce a localised therapeutic effect thought to be due to singlet oxygen generation.Photosensitizer is the core of photodynamic therapy. Chlorophyll Derivative 4(CPD4),a newly developed photosensitizer extracted from excrementum bombysis has the properties of ideal photosensitizer such as chemical purity,significant absorption at longer wavelength.However,it has not been reported to treat breast cancer by photodynamic therapy with CPD4,and the photobiological mechanisms of photodynamic therapy with CPD4 need to be further investigated.The objective was to study the photodynamic inhibitory action and the mechanism of photodynamic therapy with on MCF-7 cells with CPD4.The study will provide a foundation for in vivo study and clinical application.Methods:1.Distribution and binding characteristic of CPD4 in MCF-7 cells:Fluorescence Spectrometry was applied to detect the binding characteristic of CPD4 in MCF-7 cells.Laser scanning confocal fluorescence microscope was applied to detect the distribution and subcellular localization of CPD4.2.The photodynamic inhibitory effect of CPD4 on MCF-7:MCF-7 cells were cultured and divided into 4 groups:blank control group,laser alone group,(670 nm laser 10J/cm~2 for 3min) photosensitizer alone group,added with CPD4 for 2h,and PDT group,added with different concentration of CPD4(2.5,2.0,1.5,1.0,0.5 ug/ml) and undergoing laser irradiation.Dye exclusion method was applied to detect the death rate MCF-7 cells.3.Study on the mechanisms of photodynamic inhibitory action on MCF-7 by CPD4-PDT:Light microscopy was used to observe cellular morphology after CPD4-PDT.Apoptotic ratio and the characteristics of cell death were analyzed by Annexin V-FITC /PI double labelled staining assay.Then mitochondria membrane potential(MMP) was analyzed by flow cytometry with labeling of Rhodamine-123 and the expressions of bcl-2 and bax proteins were detected by western-blot.Results:1.Configuration fluorescence spectrum of CPD4 in MCF-7 cell was the same as it in PBS and RPMI-1640.But fluorescence peak appeared red shift.The fluorescence of the photosensitizer and that of the probe were observed in the cytoplasm and in the peri-nuclear region,mainly in the mitochondrial of MCF-7 cells.2.The results showed that the death rate of MCF-7 cells in CPD4-PDT group increased along with the increase of CPD4 concentration,but CPD4 alone or laser radiation alone had no notable effect on MCF-7 cells.3.It showed the shrinkage,round-up and membrane integrity of treated cells under light microscopy.The apoptotic rate of MCF-7 cells after CPD4-PDT significantly increased to 41.2%±1.41,and it was higher than that of the controls (p＜0.01);Mean fluorescent intensity of Rhodamine-123 after CPD4-PDT obviously dropped and it was lower than that of the controls(p＜0.05),which indicated that CPD4-PDT decreased mitochondria membrane potential;The expressions of bcl-2 proteins in CPD4-PDT group were lower than those of the control,and the expressions of bax proteins in CPD4-PDT group were higer than those of the control.The ratio of bax to bcl-2 obviously increased in CPD4-PDT group than that in the controls,which suggested The expressions of bcl-2 and bax proteins play an important role in the process of CPD4-PDT.Conclusions:1.CPD4 can bind with MCF-7 cells.CPD4 is localized in the cytoplasm,mainly in the mitochondrial of MCF-7 cells2.The photodynamic inhibitory effect of CPD4-PDT on MCF-7 ceils increases with CPD4 concentration.3.CPD4-PDT can notably trigger MCF-7 cells into apoptosis,which suggests that apoptosis in MCF-7 cells induced by CPD4-PDT contributes to the photodynamic inhibitory effect on breast cancer cells. 4.CPD4-PDTcan decrease MMP.So CPD4 may trigger apoptosis through mito chondria-initiated apoptosis pathway.5.CPD4-PDT can downregulate the expressions of bcl-2 proteins,upregulate the expressions of bax proteins and increase the ratio of bax to bcl-2,which may be one of the mechanisms of apoptosis triggered by CPD4-PDT.