Responses Of Human Cancer Cell HepG2 To Nanoseconds Pulsed Electric Field (nsPEF) In Vitro

Pulsed electric fields is a new approach for cancer treatment, which have some advantages over traditional means, such as chemotreatment and radiation therapy. How human liver cancer cell HepG2 response to nanosecond pulsed electric field (nsPEF) with high strength of 10KV/cm, durations of 500ns and frequency of 1HZ had been studied, which was related to cell apoptosis, mitochondria membrane potential(ΔΨm), electroporation, gap junctional intercellular communication(GJIC). Furthermore, the feasibility of application of nsPEF in cancer treatment was analyzed. Annexin-V and propidium iodide (PI) were used as the ?uorescent markers for the detection of cell apoptosis under ?ow cytometry. Result showed that HepG2 cells which were exposed to nsPEf for 20s were induced remarkable apoptosis(11.81%), compared with cells in control group(1.66%) without nsPEF treatment. Trypan Blue exclusion experiment indicated that cell viability of HepG2 cells in pulsed group(73.1%) was significantly lower than that of control group(94.6).Fluorospectrophotometer combined with mitochondrial membrane potential assay kit with JC-1 was applied to detection how mitochondria membrane potential(ΔΨm) changed after nsPEF treatment. Result indicated thatΔΨm had been decreasing for at least 4 hours, of which the first half hour after exposed to nsPEF was the sharpest decline scale that indicated early apoptosis happened in HepG2 cells, simultaneously, little change ofΔΨm of HepG2 cells happened in non-treatment group.Propidium iodide (PI) was utilized as the fluorescence marker in detection whether nanosecond pulses caused conventional electroporation under inverted fluorescence microscope. Fluorescence images of cells showed that there was no fluorescence of PI until 3min after nsPEF treatment, however, the density of fluorescence became massive 8min after nsPEF was taken away and lasted for at least 20min, which indicated a long-last damage on cell membrane from nsPEF. With the results of cell apoptosis, it could be concluded that irreversible electrical breakdown happened in HepG2 cells after exposed to nsPEF.Fluorescence recovery after photobleaching(FRAP) with 5 , 6-CFDA as the fluorescence marker under laser scanning confocal microscope was used for real time detection the changes of gap junctional intercellular communication(GJIC) between HegG2 cells. A significantly higher ratio of fluorescence recovery(46%) in nsPEF treatment group than that of non-treatment group(10%), which indicated a low function level of GJIC in HepG2 cancer cells. Therefore, nsPEF can induced the recovery of GJIC function.