The Molecular Mechanisum Of Semiconductor Laser On Apoptosis Of Multi-drug Resistance MG-63 Cells Lines

Objective: To find the molecular mechanism of Apoptosis of multi-drug resistance cells lines (MDR-MG-63) induced by semiconductor laser and provide theoretical basis for its clinical application.Methods: Cultured MDR-MG-63 cells (1.0×109·L-1) were treated with 532nm semiconductor laser at various irradiation dosage of 30 J·cm-2,60 J·cm-2,90 J·cm-2,120J·cm-2 respectively. To find the inhibitory effect of 532nm semiconductor laser on MDR-MG-63 cells, The cell viability was detected by methylthiazolyl tetrazolium (MTT) assay. The morphologic changed of cells were observed under invert microscope and fluorescence microscope (Hoechst 33258). To detect the cell early apoptosis rate, we observed with Annexin-V–FITC labeling in FCM analysis which can divided the early apoptosis cell, late apoptosis cells and dead cells into different group. To shed light on apoptosis pathway, the protein espressions of caspase-8,caspase-9 and caspase-3 were assayed by Western blotting. Previous studies suggested that the reactive oxygen species (ROS), which acted as signaling intermediates play an important role in mitochondrial dysfunction. We detected ROS and mitochondrial membrance potencial after staining DCFH-DA and Rhodamine 123, by flow cytometry and confocal micrograph at 120J/cm~2 for 0min, 30min, 60min, 90min, 120min, 150min and 180min; the content of calcium was detected by confocal microscopy at 120J/cm~2 for 0min, 60min, 90min, 120min, 150min and 180min; the protein espressions of p53, Bcl-2 and Bax were assayed by Western blotting.Results: Compared with control group, 532nm semiconductor laser at 60J/cm~2-120 J/cm~2 was capable of inhibiting MDR-MG-63 cells proliferation in a dose dependent manner (P<0.05), but laser at 30J/cm~2 has no inhibiting effect on cells (P>0.05). The morphologic changed of characteristic apotosis of cells observed under invert microscope and fluorescence microscope treated with laser at 60J/cm~2-120 J/cm~2, namely, cell detachment,membrane blebbing, cell shrinkage with a condensed cytoplasm, and cesicle formation, etc, while, above events doesn't appear in the group of laser at 30J/cm~2. The early rate assay with Annexin-V labeling in FCM analysis shows that laser at 60J/cm~2-120 J/cm~2 could effective induce early apoptosis and necrosis (P<0.05) in a dose dependent manner, laser at 30J/cm~2 has no above function (P>0.05). At the same time,the expression of caspase-9 and caspase-3 are both increased similary whithin the range of 60J/cm~2-120 J/cm~2 of laser (P<0.05), but that of caspase-8 has no alert (P>0.05), and still, laser at 30J/cm~2 has no any effect on expression of these three apoptotic protein (P>0.05). After treated with laser at 60 J/cm~2-120 J/cm~2, the ROS of MDR-MG-63 cells sharply increased within 30min (P<0.05), peaking at 120min, the mitochondrial membrance potencial shows gradully decreased in fore 90min (P>0.05), and sharply decreasd therafter (P<0.05). The results indicate that the level of calcium on cells increased markly from 150min after laser treated (P<0.05). As result of western blot show the expressions of Bax/Bcl-2,P53 protein were both increased in a dose depenendent manner treated with laser at 60 J/cm~2-120 J/cm~2 (P<0.05)。conclusions: 532nm semiconductor laser at 60J/cm~2-120 J/cm~2 was capable of inhibiting proliferation and inducing apoptosis of MDR-MG-63 cells. 532nm semiconductor laser at 60J/cm~2-120 J/cm~2 can markly increase the ROS on MDR-MG-63 cells in a very short time, activate the mitochondrial apoptotic pathway,induce the expressions of apoptotic relating protein Bax/Bcl-2,P53, and lead to overload of calcaim ion on MDR-MG-63 cells at last.The series events was closed related and feedback to each other,and play an important role on the effect of proliferation inhibiting apoptotic inducing on MDR-MG-63 cells .