| ObjectivesTongue is the most common primary site of one of Human squamous cell carcinoma the best invasion, and is prone to early metastasis. Human tongue squamous carcinoma has the trend of incidence increasing in these days,the early transfer rate of it is 37% to 85% or so. So the prevebtion and treatment of tongue squamous cell carcinoma and providing the study to the oretial basis is important.Tumor angiogenesis is an effective method of treating cancer, targeted inhibition of the expression of tumor angiogenesis factor is the research focus. The experiments under hypoxic conditions with different doses of iNOS inhibitor 1400W for different time to observe the effects of the iNOS activity and on the expression of iNOS, MMP-9 and the relevance between them to Human tongue squamous cell carcinoma cell lines CAL-27, to explore the inhibition of iNOS expression, the possibility of preventive and treatment of tongue squamous cell carcinoma.Methods1.Under normal culture conditions in vitro ,the experimental groups were used with the concentration of 50,100,200,400μmol/l of 1400W on the CAL-27 in the same conditions,and designed the cell without using 1400W as the control group,24,48,72,96h after the determination of nitrate reductase respectively in each group cell culture supernatant NO content level,and use the RT-PCR detection of each group in the iNOS and MMP-9 mRNA expression changes.After pretest,determined the best concentration is 200μmol/l,the best time is 72h.2.Under hypoxia (5%CO2,95%N2) in vitro were treated with concentration 50,100,200,400μmol/l of 1400W on the same conditions the role of CAL-27 and the cell design without 1400W as control group 72h after the determination of nitrate reductase respectively the group of CAL-27 cell culture supernatant NO levels,and using RT-PCR methond to detect the group of CAL-27,iNOS and MMP-9 mRNA expression changes.Results 1. The supermatant NO content of the test results showed that cultured in the laboratory under routine conditions,the role of different concentrations of 1400W on the cells, cells of the experimental group, NO release was inhibited, with 50,100,200, 400μmol/l 1400W cell for 72h, cell culture supernatant levels of NO were 36.36±1.4,33.23±1.5,21.59±3.0,29.55±2.2μmol/l. Same time point and the experimental group compared with the control group there were significant differences (P<0.05), 1400W on CAL-27 cells in each experimental group inhibition of NO release in the experimental range may increase with dose and time of the extension were phase to reduce relationship; which in hypoxia,50,100,200,400μmol/l 1400W the same cells 72h after treatment, cell culture supernatant NO levels were 28.81±2.3,21.71±2.9,17.19±2.7,18.67±2.6μmol/l, Compared to control group,the levels of NO in the supernatants was decreased(P<0.05), and were progressingly reduced with the increasing of the time and the dose.2. RT-PCR resnlts showed that:(1) Hypoxia in vitro culture conditions(5%CO2,95%N2), after 200μmol/l 1400W role in cell 0,24,48,72,96h,iNOS mRNA amount of expression were 0.269±0.009,0.201±0.010,0.147±0.008,0.135±0.007,0.146±0.008 (with MMP-9, iNOSmRNA with GAPDH as the ratio of MMP-9, iNOSmRNA expression of the relative content); MMP-9mRNA expression were 1.26±0.04,1.09±0.08,0.69±0.03,0.40±0.03,0.59±0.02. Different concentrations of 1400W inhibited iNOSmRNA expression (P<0.05), MMP-9mRNA expression (P<0.05).(2) Hypoxia in vitro culture conditions (5% CO2,95% N2), the control group without 1400W,50μM group, 100μM group,200μM group,400μM group of the cell after 72h, iNOSmRNA expression were 0.248±0.008,0.210±0.010,0.171±0.011,0.135±0.007,0.140±0.009; MMP-9mRNA expression were 1.05±0.02,1.36±0.03,0.91±0.03,0.40±0.03,0.62±0.05. Different concentrations of 1400W inhibited cell iNOSmRNA expression (P<0.05), MMP-9mRNA expression (P<0.05).ConclusionsSelective iNOS inhibitor 1400W significantly inhibited the growth of the laboratory culture of human tongue cancer cell line CAL-27, because it was a direct killer cells and had inhibition of MMP-9 expression, that implyed the 1400W can inhibit CAL-27's growth and metastasis. Clinical use of selective inhibitors for the treatment of tongue squamous cell carcinoma of iNOS provides an experimental and theoretical basis.
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