| OBJECTIVE:Cancer is a generic term for a large group of diseases that can affect any part of the body and induce abnormal metastasis which are the major cause of death from cancer. WHO has reported that cancer was a leading cause of death worldwide and accounted for7.6million deaths (around13%of all deaths) in2008, and deaths from cancer worldwide are projected to continue to rise to over11million in2030. A new technology using high pulsed electric field has emerged from military application into biology and medicine by applying nanosecond pulse electric field (nsPEF) as a means to inhibit cancer. In this research, we want to study the anti-tumor effect of nsPEF and its possible molecular mechanism via in vitro and in vivo experiments, from therapeutic targets of the hallmarks of cancer:inducing apoptosis, inhibiting proliferation, and inactivating invasion and metastasis.MATERIALS AND METHODS:Pancreatic carcinoma (PANC-1) cells were prepared for in vitro experiment, and treated by nsPEF in different intensity (0,20,40, and60kV/cm). After incubation for0, 0.5,1,2,24,48and72hours, cell viability was determined by counting viable cells and cell inhibition rate was measured by CCK-8assay. Cell apoptosis was identified by DNA ladder assay, cell TUNEL, cell TEM and flow cytometry. Cell cycle was analyzed by flow cytometry. Cell migration and invasion abilities were detected by Transwell assay. Apoptosis relative proteins, NF-κB pathway proteins, and MMP-9were ascertained by Western-blot. In addition, HepG2cells were injected into nude mice to perform in vivo experiment. Tumor growth was observed per week, and tumor was exposed to nsPEF when its volume reached about lmm3. Nuclear magnetic resonance imaging (MRI) of tumor-bearing mouse was performed to observe in vivo tumor size. On6.5weeks after tumor implant, all mice were sacrificed to compare tumor volume. Tumor hematoxylin and eosin (H&E), TdT-dUTP Terminal Nick-end Labeling (TUNEL), and transmission electron microscopy (TEM) were performed to explore the possible mechanisms of tumor shrinkage.RESULTS:NsPEF could significantly decrease cancer cell number and viability in vitro. It induced cell apoptosis, expressed by forming DNA fragmentation, increasing TUNEL positive cells, presenting mitochondria degeneration and apoptotic body. With increased intensity of nsPEF, cells presented from early apoptosis to late apoptosis and even cell necrosis, along with high expression of caspase-3and low expression of anti-apoptotic proteins such as Mcl-1, Bcl-2. NsPEF could inhibit cancer cell proliferation and stop cell cycle at G2phase, which might be by inhibiting NF-κB signal pathway. NsPEF depresses the metastasis and invasion of cancer cells by inhibiting the activation of NF-κB signal pathway and expression of MMP9in vitro. In addition, our in vivo experiments via tumor MRI and visual comparison provided vigorous evidences that nsPEF significantly inhibited tumor growth. Tumor cell shrinkage, vessels damage, and leucocytes infiltration were observed by H&E stain. Tumor cell apoptosis was found by tumor TUNEL and TEM.CONCLUSION:NsPEF has the biological effects to decrease cancer cell number and viability in vitro, which is expressed by not only cells apoptosis, but also the inhibition of cell proliferation. NsPEF induces apoptosis in cancer cells via dependent-mitochondria intrinsic apoptotic pathway in vitro. NsPEF inhibits cell proliferation in cancer cells via suppressing NF-κB signal pathway to negatively regulate cell cycle in vitro. NsPEF also decreases migration and invasion ability of cancer cells via suppressing NF-κB signal pathway to reduce MMP-9expression. NsPEF exerts the powerful anti-tumor functions via inducing cell apoptosis and destructing tumor microenvironment in vivo. In short, nsPEF inhibits tumor growth in vitro and in vivo via inducing apoptosis, inhibiting proliferation, and decreasing invasion and metastasis, which will provide a novel and effective therapeutic strategy for cancers. |
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