Experimental Studies On Apoptosis Of A375 Cells Of Xenografted Tumor Induced By Nanosecond Pulsed Electric Fild And It's Mechanism

Previous studies have showed that different parameters pulsed electrical fields have different effects on malignant tumor: electroporation therapy based on reversible electrical breakdown, energy controllable steep pulse therapy based on irreversible electrical breakdown , and intra-cellular effects characterized by inducing apoptosis. Intra-cellular effects is that cells, exposed to high intensity(>10kv/cm) nanosecond pulsed electric field, appeared absolutely different changes, as compared with microsecond steep pulsed electric field, inducing reversible electrical breakdown, and energy controllable steep pulse, resulting in irreversible electrical breakdown. It characterized by destruction of nuclear membrane, generation of micronuclei, increase in expression of gene, release of calcium, change of cell cycle, and so on. nsPEF can penetrated into the cell membrane for its high-frequency, and stimulated the changes of tunica intima(nucleus, mitochondria, endoplasmic reticulum) and cell signaling transmission, inducing apoptosis. Our studies have showed that , in cells of human ovarian cancer (SKOV3), nsPEF had an effect on apoptosis in vitro. In this paper we used nude mice bearing human melanoma cell (A375), observed the effect of nsPEF on cell structure and function in vivo, and investigated its mechanism. PART I:EFFECT OF NSPEF ON GROWTH INHIBITION AND APOPTOSIS IN HUMAN MELANOMA CELL XENOGRAFTObjective:To assess the effects of nsPEF on growth inhibition by calculating the volume of tumor, to observe the nsPEF-induced apoptosis with electron microscope.Methods:Human melanoma, succeeded in being transplanted into nude mice, were exposed to electric pulse (20KV/cm, 300ns,1hz) for 5 minutes, treated every two days for three times.Measured the tumor volume in treated group and control group, respectively, and then calculated the inhibitory rate , observed the change of tumor ultra-structure.Results:the tumor volume in treated group was significantly smaller than that in control group, and classical morphology of apoptosis was observed with electron microscope in treated group.Conclusion:NsPEF had a significant effect on growth inhibition, and might induce apoptosis in human melanoma in vivo. PART II:EXPERIMENTAL STUDIES ON APOPTOSIS OF A375 CELLS BY NANOSECOND PULSED ELECTRIC FILD AND IT'S MECHANISM.Objective:To observe the effect of nanosecond pulsed electric field on apoptosis, expression of caspase-3 and intracellular concentration of calcium.Methods:Human melanoma transplanted into nude mice were exposed to pulsed field (20KV/cm, 300ns, 1hz) for 5 minutes, treated every two days for three times. Assessed the expression of caspase-3 by immunoflorecence; Investigated the caspase-3 mRNA expression by RT-PCR; Detected the intracellular concentration of calcium with fluorospectrophtometrer; Obsevering the apoptosis of A375 cells with TUNEL.Results:The expressions of caspase-3 and caspase-3 mRNA in treated group increased significantly as compared with control group. The concentration of calcium raised remarkably.Obsevering the apoptosis of A375 cells with TUNEL.Conclusion:Nanosecond pulsed electric field could induce apoptosis in human melanoma cells, increase the expression of caspase-3 and caspase-3 mRNA,the way in apoptosis maybe with the increasing of Ca2+.