| Objective:To investigate the photodynamic effects in vitro of new photosensitizer B-CPD5 to breast cancer cell MCF-7, observe the associated effect with selective cyclooxygenase-2 inhibitor NS398 and explore the related mechanism in order to provide basal confirm for clinical application of photodynamic therapy on breast cancer chest wall recurrence.Methods:MCF-7 cells were cultured and divided groups, added with different concentration of new photosensitizer B-CPD5, then lighted with 650nm wave length laser meter and observed the time-concentration effect of the drug by MTT. Laser scanning confocal fluorescence microscope was applied to collect the fluorescence of the photosensitizer and the probes and to image the cells. The application of flow cytometry analysed the inhibitory rate of cell and the change of mitochondria membrane potential of each group and the expressions of COX-2 protein were detected by western-blot, the releasing level of Vascular endothelial growth factor (VEGF) and Prostaglandin E2 (PGE2) were detected by ELISA to explore the mechanism of the photodynamic effects on MCF-7 cell line.Results:1. The fluorescence of the photosensitizer and that of the probes were observed in the cell membrane and cytoplasm and in the peri-nuclear region, mainly in the mitochondria of the MCF-7 cell.2. Used MTT colorimetric to screen the effective concentration of B-CPD5 and laser light energy density, which demonstrated that the new photosensitizer B-CPD5 could inhibit MCF-7 cells proliferation. Under the same laser light energy density, the inhibition rate increased along with the photosensitizer concentration (there were statistical significance between PDT group 0.5,1 and 2 but not statistical significance between PDT group 2,4 and 8), time extension; while under the same photosensitizer concentration, the inhibition rate increased along with the increase of light energy density (there were statistical significance between energy density 1.2J/cm2 and 2.4J/cm2,3.6J/cm2 but not statistical significance between 2.4J/cm2 and 3.6J/cm2). Three concentrations(0.5μg/ml,1μg/ml,2μg/ml) and two laser light energy density (1.2J/cm2 and 2.4J/cm2) were chosen. 3. Normal MCF-7 cells possessed the characteristics:polygon shape, adherence growth, high breeding speed; had integrated cell membrane and limpid borderline. After be treated with photodynamic therapy, along with the increasing photosensitizer concentration and light energy density, cell morphology appeared several changes: cell borderline became round, vacuole generated in cytoplasm, cell membrane disrupted, cells were solved and dead.4. The analysis of apoptosis with flow cytometry presentated that cell apoptosis was more obviously along with increasing photosensitizer concentration. After statistical analyses by using SPSS 16.0 software, we found that under the same photosensitizer B-CPD5 concentration, the rate of inhibition increased along with the increasing light energy density. Under the same light energy density, there was significant difference during different photosensitizer B-CPD5 concentration groups.5. The analysis of mitochondria membrane potential with flow cytometry showed that there were not significant difference between Control group, Only photosensitizer group and Only light group (P>0.05), while there was significant difference for the fluorescence intensity between PDT group and the former three groups.6. The western blots results demonstrated that after using PDT to deal with the MCF-7 cells followed 24h cultivation, the expression of COX-2 protein obviously up-regulated, while the expression of COX-2 protein decreased after PDT treatment followed immediately co-cultivation with selective cyclooxygenase-2 inhibitor NS398.7. The detection of VEGF and PGE2 with ELISA showed that the VEGF and PGE2 releasing level after PDT treatment were more higher than Control group, Only photosensitizer group and Only light group, while the VEGF and PGE2 releasing level decreased after PDT treatment followed immediately co-cultivation with selective cyclooxygenase-2 inhibitor NS398.Conclusions:1. The fluorescence of the photosensitizer and that of the probes were observed in the cell membrane and cytoplasm and in the peri-nuclear region, mainly in the mitochondria of the MCF-7 cell.2. PDT has a significant killing effect on human breast caner cell MCF-7 in vitro, and its relative inhibitory rate appears to be correlated with the dose of photosensitizer and light energy density.3. The expression of COX-2 protein obviously up-regulated after PDT treatment, which could be inhibited by co-cultivation with selective cyclooxygenase-2 inhibitor NS398.4. After the B-CPD5 photodynamic therapy, the VEGF and PGE2 releasing level were increasing, which could be inhibited by co-cultivation with selective cyclooxygenase-2 inhibitor NS398. |
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