The Mechanism Of Anti-proliferation Effect Of Cold Atmospheric-pressure Plasma On HepG2 Hepatoma Cells

In recent years, the bio-application of cold atmospheric pressure plasma is becoming a hot spot of present research both at home and abroad. It has been reported that cold plasma has been used in inactivation of bacteria, inhibition of tumor cell proliferation and promotion blood coagulation. Researchers paid more attentions on the new plasma jet devices design, while the mechanism of its biological affects remains unclear. The present research is to clarify the molecular mechanism on the anti proliferation effects of plasma on the cancer cells from the perspective of cytobiology and biochemistry. The major results are as follows:1. At the beginning, the discharge characteristic and the emission spectra of the plasma plume device were investigated, and the parameters and the pulsed power supply were optimized. Then, the plasmid pAHC25 DNA was used to study the effect of plasma plume on DNA conformation changes. We found that under certain conditions, treatments of plasmid pAHC25 by the plasma plume could change the plasmid from supercoiled to linearized or open circular form. Different conformational plasmids were purified and used as the template for PCR amplification. The results showed that the plasma treatment under proper conditions did not affect the completeness of genes in the plasmid, which may have potential application in the plasma-mediated genetic transformation technique.2. MTT assay was used to detect the anti-proliferation effect of plasma treatment on HepG2, B16, PC3, MDA-MB-231 and L02 cell lines. The results showed that the plasma treatment could inhibit cell proliferation of these cell lines, and the EC 50 values were obviously different for each cell line. We also found that the anti-proliferation effect on L02 normal cell line was much weaker than that on HepG2 cells.3. The Annexinâ…¤/PI binding assay and Hoechst 33342 staining method were used toâ…¢ detect the apoptosis in HepG2 cells. DCFH probe and Griess assay were used to detect the changes of the intracellular reactive oxygen species (ROS), nitric oxide (NO) level, and JC-1 probe was used to measure the mitochondrial membrane potential. The activation of caspase 3, caspase 8, caspase 9, and the Bcl-2/Bax expression were also investigated. The results showed that the plasma treatment could induce apoptosis in HepG2 cells in both time and dose dependent manner. Cells became shrinkage and chromatin condensed. Then the mitochondrial pathway was activated, the apoptotic process was accompanied by the loss of mitochondrial membrane potential, increase in intracellular ROS and NO level, activation of caspase 3 and caspase 9, and inactivation of caspase 8. In addition, the plasma treatment also increased the expression of the Bax and suppressed that of Bcl-2 at both mRNA level and protein level.4. HepG2 cells were stained by PI and analyzed by flow cytometry. The expression of genes and proteins related to cell cycle was analyzed by RT-PCR and western-blot. The results showed that, the plasma treatment could cause apoptosis of HepG2 cells associated with cell cycle arrest at G2/M phase. The G2/M phase arrest was accompanied with down-regulation of cyclin Bland CDC2, and up-regulation of p21 and p53 in both mRNA level and protein level.