The Effect Of Nanosecond Pulsed Electric Fields Combined With Gemcitabine In The Proliferation,Apoptosis And Invasion Of Human Tongue Squamous Cancer Cal-27Cells

Objective:Through study the effects of Nanosecond Pulsed Electric Fields (nsPEFs) combined with low concentration of gemcitabine in the cell proliferation, apoptosis and invasion of human tongue cancer Cal-27cells in vitro, To investigate the antitumor effect of best combination therapy with nsPEFs and chemotherapy drug in human tongue cancer. The aims of this study were to provide some experimental and theoretical basis of nsPEFs in the clinical application and basic research for head and neck cancer.Methods:In this study we used the human tongue cancer Cal-27cells to investigate the cell proliferation, apoptosis and invasion in vitro. Experiment was divided into four groups:blank control group, gemcitabine group, nsPEFs group and the group of gemcitabine combined with nsPEFs. MTT assay was used to explore the inhibitory proliferation effect of gemcitabine at different time points (24h,48h and72h) and different drug concentrations (0.01μg/ml,0.1μg/ml,1.0μg/ml,10μg/ml,100μg/ml). Through MTT assay we also to investigate the inhibitory proliferation effect of different field intensity nsPEFs (0kV/cm,10kV/cm,30kV/cm and60kV/cm) combined with low concentration of gemcitabine; The colony-formation ability of Cal-27cells was studied by colony-formation assay; The apoptosis rate was analyzed with Annexin-V-FITC/PI double-staining assay; The morphological changes before and after treatment with nsPEF and gemcitabine in Cal-27cells were observed using transmission electron microscope (TEM); Cell invasion was measured using Transwell migration assay and wound healing assay.Result:1) MTT assay showed that the proliferation of Cal-27was significantly (P<0.05) inhibited by gemcitabine in a dose-and time-dependent manner; the inhibition of cell proliferation was increased with the extension of time drug action and drug concentration increased. At24h,48h,72h the inhibitory rate of0.01μg/ml gemcitabine were7%,10%,38%, and all of the inhibitory rates showed less than50%inhibition. Lower inhibitory concentration0.01μg/ml was used to combine with different field strength of nsPEFs in Cal-27cells. The inhibitory ability showed a time-and field strength-dependent. At the combination treatment of60kV/cm and0.01μg/ml gemcitabine group, the inhibitory rate was79%,85%and92%at24h,48h and72h, respectively. The results indicated nsPEFs and0.01μg/ml of gemcitabine synergistically inhibited proliferation of human tongue cancer Cal-27cells (SQ>1, P<0.01)2) Cell clone formation assay revealed that0.01μg/ml gemcitabine combined with nsPEFs (10,30,60kV/cm) have a synergistic effect on the inhibition of colony formation in human tongue cancer Cal-27cells (SQ>1, P<0.01).3) Annexin-V-FITC/PI double-staining assay showed that0.01μg/ml gemcitabine combined with nsPEFs (10,30,60kV/cm) have a synergistic effect in early apoptosis and late apoptosis rate in human tongue cancer Cal-27cells. Under the same reaction time conditions, with increasing of electric field strength the capacity to induce apoptosis increased (SQ>1, P<0.01).4) Transmission electron microscopy results showed that0.01μg/ml gemcitabine combined with nsPEFs (10,30,60kV/cm) have significantly induced typical apoptotic morphological changes, including orange cell shrinkage with condensation, Chromatin condensation, Mitochondrial vacuolization degeneration and apoptotic bodies.5) The cell invasion assay showed that0.01μg/ml gemcitabine combined with nsPEFs (10,30,60kV/cm) have significantly inhibited in vitro invasion of human tongue cancer Cal-27cells, obviously reduced the rate of cancer cells invasion through basement membrane and the invasion inhibition rate was increased. There was significant difference between each group (SQ<1, P<0.01).6) The wound healing assay showed that0.01μg/ml gemcitabine combined with nsPEFs (10,30,60kV/cm) have significantly inhibited cell migration of human tongue cancer Cal-27cells, cell migration is significantly reduced in comparison with untreated cells.Conclusion:1) Gemcitabine could effectively inhibit the proliferation of human tongue cancer Cal-27cells; the inhibition of cell proliferation was in a dose-and time-dependent manner.2) NsPEFs and0.01μg/ml of gemcitabine synergistically inhibit cell proliferation, cell colony formation and induce cell apoptosis in the human tongue cancer Cal-27cells.3) NsPEFs combined with0.01μg/ml of gemcitabine significantly inhibited cell invasion and migration of human tongue cancer Cal-27cells.