[Ca(2+) is an important mediator of nanosecond steep pulse-induced apoptosis in human ovarian cancer SKOV3 cells].

OBJECTIVE: To explore the role of Ca(2+) in nanosecond steep pulse (NSP)-induced apoptosis of human ovarian carcinoma cell line SKOV3 in vitro. METHODS: The early apoptotic rate of SKOV3 cells treated with NSP was detected by Annexin V/PI double staining and flow cytometry. MTT assay was used to detect the viability of the cells pretreated with BAPTA-AM (0, 25, 50 and 100 µmol/L) chelation for 1 h to increase the intracellular free Ca(2+) prior to NSP exposure, and the cell morphological changes and caspase 12 expression were detected using Hoechst 33342 staining and Western blotting, respectively. RESULTS: Flow cytometry showed that NSP induced early apoptosis of SKOV3 cells, and the optimal effect was achieved with the treatment parameter configuration of field strength of 90 kV/cm, pulse width of 100 ns, frequency of 1 Hz, and exposure time of 30 s. The highest early apoptotic rate and necrosis rate was (60.31∓5.67)% and (1.35∓0.39)%, respectively. Pretreatment with BAPTA-AM chelation prior to NSP exposure significantly increased the cell viability (P<0.05), and resulted also in lowered apoptosis rate and decreased expression of caspase 12 (P<0.05). CONCLUSION: NSP can induce apoptosis in SKOV3 cells. Increased intracellular free Ca(2+) functions as an important mediator in NSP-induced cell apoptosis, which may also involve Ca(2+)-mediated endo- plasmic reticulum pathway.

The effect of high frequency steep pulsed electric fields on in vitro and in vivo antitumor efficiency of ovarian cancer cell line skov3 and potential use in electrochemotherapy.

BACKGROUND: Patients received electrochemotherapy often associated with unpleasant sensations mainly result from low-frequency electric pulse induced muscle contractions. Increasing the repetition frequency of electric pulse can reduce unpleasant sensations. However, due to the specificity of SPEF, frequency related antitumor efficiency need to be further clarified. The aim of this study was to compare in vitro cytotoxic and in vivo antitumor effect on ovarian cancer cell line SKOV3 by SPEF with different repetition frequencies. Explore potential benefits of using high frequency SPEF in order to be exploitable in electrochemotherapy. METHODS: For in vitro experiment, SKOV3 cell suspensions were exposed to SPEF with gradient increased frequencies (1, 60, 1000, 5000 Hz) and electric field intensity (50, 100, 150, 200, 250, 300, 350, 400 V/cm) respectively. For in vivo test, SKOV3 subcutaneous implanted tumor in BALB/c nude mice (nu/nu) were exposure to SPEF with gradient increased frequencies (1, 60, 1000, 5000 Hz) and fixed electric field intensity (250 V/cm) (7 mice for each frequency and 7 for control). Antitumor efficiency was performed by in vitro cytotoxic assay and in vivo tumor growth inhibition rate, supplemented by histological and TEM observations. Data were analyzed using one-way ANOVA followed by the comparisons of multiple groups. RESULTS: SPEF with a given frequency and appropriate electric field intensity could achieve similar cytotoxicity until reached a plateau of maximum cytotoxicity (approx. 100%). SPEF with different frequencies had significant antitumor efficiency in comparison to the control group (P < 0.05). However, there was no difference in tumor responses among test groups (P > 0.05). Histological and TEM observations demonstrated obvious cell damages in response to SPEF exposure. Furthermore, SPEF with 5 kHz could induce apoptosis under TEM observations both in vitro and in vivo. CONCLUSION: SPEF with high frequency could also achieve similar antitumor efficiency which can be used to reduce unpleasant sensations in tumor electrical treatment. Our research proposed potential applications of using high frequency SPEF in clinical cancer treatment.

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.

The stress reaction and its molecular events: splicing variants.

The growth of cells and tissues is regulated by stress. When body is injured, it manifests a large spectrum of metabolic, endocrine, and immune alterations, which is named stress reaction. Among them, the production of growth factors may play a critical role. For osteoblasts and myoblasts, IGF-I has been shown to be involved in the process of cells in response to overloads. There are two splicing forms, one is IGF-Ea, the other is the IGF-IEb in the rodents and corresponds to IGF-IEc in humans. The latter is markedly up-regulated in response to overloads. Therefore, it has been named mechanogrowth factor. The link between the mechanical stimulus and the gene expression represents a new and important area in cell science. Understanding the process of splicing in IGF-I helps one to investigate the mechanotransduction of cells in response to mechanical stimulation at molecular level.