Effect Of Nanosecond Pulsed Electric Fields On Treatment Of Human Melanoma Xenograft In Nude Mice And Its Mechanisms

The treatment model of malignant tumor has already entered a newstage of muti-modality therapy, in which pulsed electric fields owingunique strongpoint in non-thermal effect show a prefect application. Thenon-thermal effect mainly include three categories:（1） ReversibleElectrical Breakdown （REB） which occurs electroporation of the plasmamembrane after exposing to pulses of0.1⁵kV/cm amplitude and0.1²0ms duration;（2） Irreversible Electrical Breakdown （IREB） which result innecrosis at higher field strength or pulse duration than electroporationeffect;（3） Intracellular Electromanipulation （IEM） which induce apoptosisafter exposing to pulses of10³50kV/cm amplitude and1³00ns durationthat happen to the change of structure and function after acting on plasmamembrane、internal organelles membrane and nuclear membrane.The deficiency of traditional cancer therapies which killing cells orinhibiting the proliferation is that this is only remission but not a cure.Apoptosis, however, can do it by following the procedure and causing tumor to shrink or disappear, as well as reducing adverse effect of thesurrounding tissue cells. Thus, nanosecond pulsed electric fields （nsPEFs）has become a hotspot in research of cancer treatment at home and abroadbecause of inducing cancer celll apoptosis mainly. The present andprevious studies demonstrate that nsPEFs have got good anti-tumor effectson mouse fibrosarcoma tumors、pancreatic cancer xenografts in nude miceand B16-F10melanoma in SKH-1mice and so on. We know that differentparameters of pulsed electric fields are different on antitumor effects ofvaried tumors. Here we choosed200ns in pulse duration basing on300nsin pulse caps on non-thermal effect while using the pulse amplitude,number and frequency （40kV/cm,1000pulses,1Hz） and focused onhuman melanoma A375cell xenograft in nude mice to investigate the effectof nanosecond pulsed electric fields （nsPEFs） on subcutaneousallotransplanted tumor in nude mice, as well as to explore its possibleanticancer mechanisms. Objective: To investigate the effect of nanosecond pulsed electricfields（nsPEFs） on treatment of human melanoma A375cell xenograft innude mice.Methods: Subcutaneous allotransplanted tumor model of human melanoma A375cell in nude mice were randomly divided into long-term group andcontrol group, exposed to the pulse parameters （40kV/cm in amplitude,200ns in duration,1000pulses and at a pulse frequency of1pulse/s）.General tumor changes, tumor growth and survival time of long-term groupand control group after a single nsPEFs treatment were monitored.Results:1.Tumor of long-term group turned grey and firm without bleedingspots minutes after a single treatment, the tumor volume started to appearsmall for3or4days after treatment in long-term group,6mice melanomaof which were completely gone within two weeks after treatment and didnot recur until natural death,4mice melanoma of which shrank ordisappeared in different extent on average within12.8days and appearedlocal recurrence on average within30.4days after treatment.2.The tumor volume was significantly smaller in long-term group thancontrol group （P<0.01）. The inhibition ratio of tumor were42.67%on the4thday and then99.22%on the32thday after treatment in long-term group.As compared with the long-term group, control tumors in all mice grewfaster and larger.3.On the36thday after nsPEFs treatment, the survival rates of mice inlong-term group and control group were100%and0, respectively. Themean survival time was significantly longer in long-term group（60.6±6.9d）than in control group（17.7±5.4d）（P<0.01）. The increase ratio of life span reached up to242%.Conclusion: A single nsPEFs （40kV/cm,200ns,1000pulses,1Hz）treatment of subcutaneous allotransplanted melanoma mice could gaintherapeutic results. Objective: To investigate the mechanism of nanosecond Pulsed ElectricFields （nsPEFs） on human melanoma A375cell xenograft in nude mice.Methods：Subcutaneous allotransplanted tumor model of human melanomaA375cell in nude mice were randomly divided into short-term group（divided into two groups again:2h group and4d group） and control group,exposed to the pulse parameters （40kV/cm in amplitude,200ns in duration,1000pulses and at a pulse frequency of1pulse/s）.1. After a single nsPEFs treatment, tumor morphology was observedwith HE staining.2.The cell apoptosis was detected by DNA agarose gelelectrophoresis and terminaI deoxynucleotidyl transferase-mediated dUTPnick end-labeling （TUNEL） assay, the expression of Bax and Bcl-2proteinwas detected by Western blot and SP immunohistochemistry.3.The expression of microvessel density （MVD）、vascular endothelialgrowth factor （VEGF） and proliferating cell nuclear antigen （PCNA） were detected by SP immunohistochemistry.Results:1.HE stain showed that necrosis was not obvious in short-term groupon the2thday after a single nsPEFs treatment, and there were areas ofnecrosis in short-term group on the4^thday after a single nsPEFs treatment.The morphological characteristics of cancer cells in control group wereactive, density and shaped obviously with deeply stained nuclei and mitoses.2.（1） On the4^thday after a single nsPEFs treatment, gel electrophoresis of short-term group revealed that DNA extracted from tumor cellshad appeared ladder electrophoresis bands, because of being completelydegraded into tiny fragments like different multiple of100bp or200bp.There were no discrete bands formed as a result of entire DNA in controlgroup.（2） On the4^thday after a single nsPEFs treatment, TUNEL pictures ofshort-term group showed that the expression of nucleus of cancer cellsstaining brown is visible in short-term group more than in control group,the apoptosis rate of tumor cells was significantly higher in short-termgroup than in control group （0.5467±0.0401vs0.0241±0.0045, P<0.01）.（3） On the4^thday after a single nsPEFs treatment, the results ofwestern blot showed that the expression of Bax was significantly higher in4d group than in control group （2.68±0.08vs1.43±0.13, P<0.01） and the expression of Bcl-2was significantly lower in short-term group than incontrol group （1.46±0.05vs2.63±0.18, P<0.01）. The results of SPimmunohistochemistry showed that the expression of Bax being visible inthe cytoplasm of cancer cells and being stained brown was significantlyhigher in short-term group than in control group （0.54±0.01vs0.22±0.07,P<0.01）. However, the protein of Bcl-2was visible in the cytoplasm ofcancer cells and stained brown, its expression was significantly lower in4dgroup than in control group （0.17±0.05vs0.41±0.09, P<0.01）.3.On the4^thday after a single nsPEFs treatment, the results of SPimmunohistochemistry showed that microvascular in control group weremostly irregular looking like lumen, stricture or expansion, point ortree-in-bud, and so on. However, microvascular in short-term group weremostly blocked. Immunohistochemistry showed that VEGF protein andPCNA protein being stained brown were respectively located in cytoplasmand nucleus, the expression of MVD, VEGF protein and PCNA protein inshort-term group were significantly less than those in control group（26.13±2.29、0.3293±0.1155、0.3462±0.0388vs57.14±10.84、0.8190±0.0900、0.6048±0.0834, P<0.01）.Conclusion: A single nsPEFs （40kV/cm,200ns,1000pulses,1Hz） canreduce blood flow in short time, decrease the angiogenesis and proliferativeactivity and induce cell apoptosis under the Bax and Bcl-2gene regulationmechanism.