Effects of irreversible electroporation on cervical cancer cell lines in vitro.

The effects of irreversible electroporation (IRE) on the proliferation, migration, invasion and adhesion of human cervical cancer cell lines HeLa and SiHa were investigated in the present study. HeLa and SiHa cells were divided into a treatment group and control group. The treatment group cells were exposed to electric pulses at 16 pulses, 1 Hz frequency for 100 µsec with 1,000 V/cm strength. Cellular proliferation was determined 24 h after treatment using a Cell Counting Kit­8 (CCK­8) assay and carboxyfluorescein diacetate­succinimidyl ester (CFDA­SE) labeling assay. The different phases of the cell cycle were detected using flow cytometry. Wound healing, Transwell invasion and Matrigel adhesion assays were performed to evaluate the migration, invasion and adhesion abilities of HeLa and SiHa cells. The expression levels of metastasis­associated proteins were determined by western blot analysis. CCK­8 and CFSE labeling assays indicated that the inhibition of cellular proliferation occurs in cells treated with IRE. Additionally, cell cycle progression was arrested at the G1/S phase. A western blot analysis indicated that the expression levels of p53 and p21 proteins were increased, whilst those of cyclin­dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA) proteins were decreased. However, wound healing, invasion and adhesion assays indicated that cellular migration, invasion and adhesion abilities were not significantly altered following exposure to IRE. IRE was not observed to promote the migration, invasion or adhesion capacity of HeLa and SiHa cells. However, IRE may inhibit the capacity of cells to proliferate and their progression through the cell cycle in vitro. Preliminary evidence suggests that the underlying mechanism involves increased expression levels of p53 and p21 and decreased expression levels of CDK2 and PCNA.

Picosecond pulsed electric fields induce apoptosis in a cervical cancer xenograft.

The aim of the present study was to evaluate the efficacy of picosecond pulsed electric fields (psPEF) on a cervical cancer xenograft. Human cervical cancer xenografts were established in nude mice by transplantation of HeLa cells, and the tumors were then treated with psPEF. The histological changes were observed by hematoxylin­eosin staining and transmission electron microscopy. The rate of tumor cell apoptosis was determined using a terminal deoxynucleotidyl­transferase­mediated dUTP nick end labeling assay. The mitochondrial transmembrane potential of the tumor cells was detected by laser scanning confocal microscopy, and the activity of caspase­3, ­8, ­9 and ­12 was determined. The inhibitory rate seven days post­psPEF treatment was also calculated. The results showed that exposure to psPEF led to an increased rate of apoptosis, collapse of mitochondrial transmembrane potential, and activation of caspases. The inhibitory rate was 9.11% at day 7. The results of the present study indicate that psPEF may induce apoptosis in a cervical cancer xenograft through the endoplasmic reticulum stress and caspase­dependent signaling pathways.

Mechanisms for steep pulse irreversible electroporation technology to kill human large cell lung cancer cells L9981.

To explore the mechanisms for steep pulse irreversible electroporation technology to kill the lung cancer cell L9981. The apoptosis, cells mitochondrial membrane potential, internal PH changes and the intra-cellular calcium ions concentration were detected after steep pulses acted on the human large cell lung cancer cell L9981. Apoptosis test results indicated that cancer cells mainly experienced necrosis and apoptosis. Along with the increase of electric parameters, the proportion of the necrotic cells increased rapidly; the detection of cells mitochondrial membrane potential indicated that membrane potential occurred depolarization. Steep pulse can cause cancer cells to produce death and apoptosis .The PH value indicated that intracellular PH level down jumped. Internal PH became more acidic and led to cell death. The detection of intra-cellular calcium ions concentration showed that the number of free calcium significantly increased, and this change had killing effects on cell death and apoptosis. Steep pulse could induce cell apoptosis.

Picosecond pulsed electric fields induce apoptosis in HeLa cells via the endoplasmic reticulum stress and caspase-dependent signaling pathways.

The non-invasive treatment of tumors with preserved fertility holds great promise. The application of pulsed electric field (PEF) is a new biomedical engineering technique for tumor therapy. Picosecond pulsed electric fields (psPEF) can be transferred to target deep tissue non-invasively and precisely; however, research of the biological effects of psPEF on cells is limited. Electric theory predicts that when the pulse duration decreases to nanoseconds and picoseconds, it will mainly affect organelles and lead to intracellular electromanipulations. Previous studies have shown that psPEF targets the mitochondria and induces apoptosis through a mitochondrial-mediated pathway in HeLa cells. The endoplasmic reticulum is also involved in the intrinsic pathways of apoptosis. In the present study, HeLa cells were exposed to psPEF to investigate the underlying mechanisms of apoptosis. MTT assay demonstrated that psPEF displayed strong growth inhibitory effects on HeLa cells. Treatment with psPEF led to marked cell apoptosis and cell cycle arrest at the G2/M phase. In addition, psPEF affected the phosphorylation levels of endoplasmic reticulum sensors and upregulated the expression of glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94) and CCAAT enhancer-binding protein (C/EBP) homologous protein (CHOP). These changes were accompanied by the elevation of intracellular Ca2+ concentrations. Furthermore, the activation of caspase-12, -9 and -3, led to the release of cytochrome c, as well as the upregulation of Bax and the downregulation of Bcl-2, as observed in the HeLa cells. Taken together, these data suggest that psPEF is an efficient apoptosis-inducing agent for HeLa cells, which exerts its effects, at least partially, via the endoplasmic reticulum stress and caspase-dependent signaling pathways.

Intense picosecond pulsed electric fields induce apoptosis through a mitochondrial-mediated pathway in HeLa cells.

The application of pulsed electric fields (PEF) is emerging as a new technique for tumor therapy. Picosecond pulsed electric fields (psPEF) can be transferred to target deep tissue non-invasively and precisely, but the research of the biological effects of psPEF on cells is limited. Electric theory predicts that intense psPEF will target mitochondria and lead to changes in transmembrane potential, therefore, it is hypothesized that it can induce mitochondrial-mediated apoptosis. HeLa cells were exposed to psPEF in this study to investigate this hypothesis. MTT assay demonstrated that intense psPEF significantly inhibited the proliferation of HeLa cells in a dose-dependent manner. Typical characteristics of apoptosis in HeLa cells were observed, using transmission electron microscopy. Loss of mitochondrial transmembrane potential was explored using laser scanning confocal microscopy with Rhodamine-123 (Rh123) staining. Furthermore, the mitochondrial apoptotic events were also confirmed by western blot analysis for the release of cytochrome C and apoptosis-inducing factor from mitochondria into the cytosol. In addition, activation of caspase-3, caspase-9, upregulation of Bax, p53 and downregulation of Bcl-2 were observed in HeLa cells also indicating apoptosis. Taken together, these results demonstrate that intense psPEF induce cell apoptosis through a mitochondrial-mediated pathway.

Steep pulsed electric fields modulate cell apoptosis through the change of intracellular calcium concentration.

A steep electric pulsed field with low intensity (150-250V/cm) and relative long time (10 min) was applied to adherent liver cancer cell line SMMC-7721 and the liver cell line HL-7702. Results showed that the electric field with intensity of 200 and 250V/cm could trigger cell apoptosis, whereas the SMMC-7721 cell was more sensitive to the electric stimulation than the HL-7702 cell. Laser Scanning Confocal Microscope (LSCM) was used to measuring the real-time change of cytosolic free Ca(2+) concentration. When cells were exposed electric pulses with 100V/cm intensity for 10 min, there was no significant change of intracellular calcium concentration. With the intensity increased to 200 and 250V/cm, intracellular calcium concentration decreased significantly. Results demonstrated the relationship between the apoptosis and change of intracellular calcium concentration. And the steep electric pulsed field can be used to the cancer therapy.

[Impacts of steep pulsed electric fields on lymphatic capillaries in VX2 implanted breast cancer in rabbits].

BACKGROUND & OBJECTIVE: Electrochemotherapy mediated by electric pulse has become a multidisciplinary biomedical engineering technique in modern medical science. Its main mechanisms are enhancing the diffusion of chemotherapeutic drugs, antibodies, or genes into the inner part of tumor cells mediated by membrane-electropermeabilization caused by electric pulse. Our previous studies confirmed that steep pulsed electric field (SPEF) could irreversibly cause membrane electropermeabilization, and lead to death of tumor cells. This study was to explore the acute killing effects of SPEF on lymphatic capillaries in VX2 implanted breast cancer in rabbits. METHODS: Tumor model of VX2 implanted breast cancer was successfully established in rabbits. Isosulfan blue staining, 5'-AMP-ALPase enzymohistochemical double staining, and electron microscopy was used to observe the morphologic changes of local lymphatic capillaries around cancer tissues exposed to SPEF. RESULTS: After exposed to SPEF, no lymphatic vessels were found with isosulfan blue staining, only blurred structures were observed; enzymohistochemistry showed no positively stained lymphatic vessels, only fragmental structures around cancer tissues were observed; integrity and continuity of lymphatic endothelium were destroyed under transmission electron microscope. CONCLUSION: SPEF has the potential to destroy lymphatic capillaries around VX2 implanted breast cancer, and can decrease the possibility of post-treatment lymphatic metastasis.

[Pathological studies of tissue damage in experimental rabbit liver cancer induced by energy controllable steep pulses].

OBJECTIVE: To evaluate the efficacy of energy controllable steep pulses (ECSP) in the treatment of rabbit VX2 cancer implanted in livers. METHODS: A tumor model was successfully established using 30 rabbits. ECSP were applied to liver cancer in half of these rabbits and the rest were used as controls. After exposure to ECSP, tissues were obtained and subjected by routine HE and transmission electron microscopic (TEM) observation. The survival time of the animals and the statuses of each group were recorded. RESULTS: From pathological observations, ECSP showed effectively destructive action compared with that of the unexposed group. A clear borderline can be seen between necrotic cancer and its surrounding normal tissue. Irreversible cell changes were present under TEM. The survival periods of the experimental and control group were 83.1 days and 39.0 days respectively, and there was a significant difference between the two groups (Z = -2.943, P < 0.01). CONCLUSION: ECSP can effectively treat rabbit VX2 cancer implanted in the liver; also it is safe for its surrounding normal tissues. ECSP can be a useful method for local treatment of liver cancer.