Study On The Mechanism Of Tumor Cell Apoptosis Induced By Curcumin Mediated Photodynamic Therapy

ObjectiveTo explore the apoptosis and the mechanism of MCF-7 and MCF-7/ADM cells treated with curcumin mediated photodynamic therapy,and to investigate the antitumor activity of curcumin pH sensitive liposomes combined with photodynamic therapy.Aim to provide valuable theoretical basis for the clinical application of curcumin mediated photodynamic therapy,and to provide some basis and ideas for overcoming tumor multi-drug resistance.Methods1.HPLC was used to investigate and verify the analytical method of curcumin,which provided the analytical basis for subsequent experiments.2.MTT assay was served to evaluate the cytotoxic effect of curcumin mediated with photodynamic therapy on MCF-7 and MCF-7/ADM cells.The ability of curcumin mediated with photodynamic therapy in inducing apoptosis was evaluated by Annexin V-FITC/PI apoptosis assay.And immunofluorescence experiment was applied to investigate the change of morphological,F-actin and cell nucleus induced by curcumin mediated with photodynamic therapy.3.The apoptosis mechanism of curcumin mediated with photodynamic therapy was studied by detecting the levels of reactive oxygen species,mitochondrial membrane potential and some relative apoptotic proteins.4.Thin film ultrasonic method was used to prepare curcumin pH sensitive liposomes.We optimized the prescription process and preparation process through the study of particle size,PDI,encapsulation rate,drug loading and release degree.Antitumor activity of curcumin pH sensitive liposomes combined with photodynamic therapy in vitro was tested by MTT assay and Annexin V-FITC/PI staining.Results1.The HPLC method for curcumin was established and met the methodological requirements,which was suitable for the analysis of curcumin.2.(1)MTT results showed that curcumin mediated with photodynamic therapy significantly enhanced the cytotoxicity of MCF-7 and MCF-7/ADM cells,the survival rate was positively correlated with the concentration of curcumin.Meanwhile,the cytotoxic effects of curcumin mediated with photodynamic therapy of two cell lines were basically the same under the concentration range and light conditions.(2)The apoptosis rates of MCF-7 and MCF-7/ADM cells were determined by flow cytometry.The apoptosis rate of curcumin mediated with photodynamic therapy group were(56.80 ± 7.64)%and(57.07 ± 5.89)%,it was statistically significant(P<0.001)when compared to the control group.After treatment with curcumin mediated with PDT,cell rounding,shrinkage significantly,F-actin was loose arrangement,the nucleus becomes small.Experimental results indicated that,curcumin mediated with photodynamic therapy induced MCF-7 and MCF-7/ADM apoptosis,and F-actin and cell nuclear morphology to some extent undermined at the same time.3.(1)The level of reactive oxygen species in curcumin mediated with photodynamic therapy treatment group was increasing compared to the control group in the treatment time(2,4,6,8,10,12 h)and 6 h was maximum,which suggested that curcumin mediated with photodynamic therapy induced apoptosis by increasing intracellular reactive oxygen species in MCF-7 and MCF-7/ADM cells.(2)The mitochondrial membrane potential of MCF-7 and MCF-7/ADM cells was detected with JC-1 dye.The red fluorescence showed that the mitochondrial membrane potential was high and the cells were in a normal state.Green fluorescence indicates that cells are in apoptosis,and the relative ratio of red-green fluorescence is usually used to measure the depolarization ratio of mitochondria with the decrease of mitochondrial membrane potential.The red fluorescence/green fluorescence of curcumin mediated with photodynamic therapy group were(1.14 ± 3.68)%and(1.27±2.94)%,which was statistically significant(P<0.001)when compared with control group.It shows that curcumin mediated with photodynamic therapy can significantly reduce the mitochondrial membrane potential of cells and induce apoptosis.(3)The Western blot experiment showed that the expressions of P-gp,Cyt-c,Caspase-3/7/9 and PARP in MCF-7 and MCF-7/ADM cells were shearing after the treatment with CUR mediated with photodynamic therapy for 24 h.Compared with the control group,P-gp,cytoplasm Cyt-c,cleaved caspase-3/7/9 and cleaved PARP were up-regulation,mitochodria Cyt-c was down-regulation.The result showed that the endogenous apoptosis signaling pathways was mainly by activating mitochondrial/cytochrome c in MCF-7 and MCF-7/ADM cells.Meanwhile,curcumin mediated with photodynamic therapy to overcome MCF-7/ADM multidrug resistance by down-regulating the expression of P-gp.4.curcumin pH sensitive liposomes was successfully preparaed through single factors investigation.The optimized conditions were as following:drug/lipid was l:405 cholesteryl hemisuccinate was 8 mg,cholesteryl was 5 mg,containing with 0.1%tween-80,while the ultrasonic power was 92 W,the ultrasonic/interval time was 20s/5s and the ultrasonic time was 4 min.In this condiction,the average particle size and photodynamic therapy of curcumic pH sensitive liposomes was(94.34 ± 1.14)nm and(0.219± 0.008),the encapsulation efficiency and the drug loading capacity was(100.89 d:5.19)%and(1.33±0.03)%,and showed a good sensitivity to acidic microenvironment,the released ratio of curcumin under the eondiction of 5.5 and 6.5 were(84.27± 3.74)%and(90.41 ± 1.87)%,separately.While there seems no release of curcuruin at pH 7.4.MTT results and cell apoptosis showed that curcumin pH sensitive liposomes combinated with photodynamic therapy significantly display antitomor activity against MCF-7 and MCF-7/ADM cells.ConclusionThe results of this study indicated that curcumin mediated with photodynamic therapy produced reactive oxygen species to induce tumor cell apoptosis,as following:curcumin treatment with photodynamic therapy increase overall reactive oxygen species levels in cells,which lead to a decrease in mitochondrial membrane potential,increased membrane permeability,while cytochrome c from the mitochondrial matrix was released into the cytoplasm,and that apaf-1 and caspase-9 were binding to apoptotic complex.This apoptotic complex hydrolyzed and activated downstream caspase-3/7 to performed cell apoptosis,damaged DNA repair enzyme PARP,and caused cell chromatin condensation and nuclear fragmentation,which are accompanied by loose cytoskeleton protein arrangement,round and shriveled cells and other apoptotic characteristics.In addition,curcumin pH sensitive liposomes was prepared in this study not only improved the water solubility and stability,but also had good in vitro anti-tumor activity in combination with photodynamic therapy.