Effect Of Nitric Oxide On In Vitro Radiochemosensitivity Of Squamous Cell Carcinoma Of Tongue

Squamous cell carcinoma(SCC)in oral and maxillofacial regions is the top ten malignant tumors in the whole body,whose mortality is the first in oral and maxillofacial malignant neoplasms.Although the great success in tumor treatment,it is still a fetal disease,with increasing morbidity and mortality.New approaches in treatment of SCC are urgently needed.To avoid the complication of defect in appearance and function after radical surgery,radiotherapy and chemotherapy has been considered as one of the best treatment method in some tumor by AJCC.Preoperative chemoradiotherapy has the potential to decrease tumor size,down-grade the tumor size,and enabling a greater chance of obtaining a tumor-free surgical margin.As an important bioactive molecule,the antitumor properties of nitric oxide(NO)has gained much attention.It involved in a large variety of pathophysiological processes,regulate circulation,nervous system and immunological system,such as blood vessel dilatation,vasopermeability,platelet adhension and collection,signal transmission and host defence.It involved in tumor pathology,and plays an important and complicated role on tumor biology.NO has several dose-dependent effect.In physiological condition,to normal cells,low level NO has important function in signal transduction,which protect cells.But in pathological status,depending on the concentration,time and location,NO has different function,promote tumor growth or antitumors.Persist low level NO can damage DNA,cause gene mutation,induce canceration and metastasis,and accelerate tumor growth through promoting tumor vessles.High concentration of NO has antitumor effect through cytotoxicity and apoptotic cell death.Endogenous NO mainly catalysed by NOS(nitric oxide sythase),and iNOS is the key enzyme in catalyzing arginine to form NO.iNOS has important role in oncogenisis,growth,metastasis and treatment.During the process of precancerious lesions developed to oral mucosa squamous cell carcinoma,the expression of iNOS gradually increased,which suggest that high level of NO is the necessary condition for the growth of SCC.This work investigated the cytotoxic and radiosensitising effect of exogenous nitric oxide(NO)donor SNP(Sodium nitroprusside)on human tongue squamous cell carcinoma cell line Tca8113,and the induction of apoptosis by SNP and radiation.But the use of nitric oxide donor is clinically limited by several serious side effect when administrated systemically.To avoid the side effect,basic on the former investigation,iNOS gene was transferred to Tca8113 cell lines by lipofectarnine,and the proliferation effect of iNOS gene on Tca8113 cells were tested.The redistribution of cell cycle and proliferation index(PI)were analyzed by flow cytometry.The third section is to observe the radiochemosensitivity of iNOS gene on Tca8113 cells and the mechanisms. Section 1 Nitric Oxide and Ionizing Radiation Synergistically Promote Apoptosis and Growth inhibition of squamous cell carcinoma of tongueTo investigate the cytotoxic and radiosensitising effect of SNP(Sodium nitroprusside)on human tongue squamous cell carcinoma cell line Tca8113,and the enhanced apoptotic effect of SNP combined with radiation.SNP,an exogenous nitric oxide(NO)donor,was used to assess it's cytotoxic and radiosensitizing potency by MTT assay.The changes of cell cycle by different dosage of SNP was analysed by flow cytometry.The apoptotic cell death was detected by morphology of HE staining and Hoechest 33258 staining,DNA agarose gel electrophoresis and Annexin V/PI assay.The expression of easpase-3 and the percentage of activated caspase-3 in living cells was detected by western blot and flow cytometry respectively.The result showed SNP had a remarkable proliferation inhibitating effect on Tca8113 cells in various concentration(0.1,0.5,1.0,2.0,3.0,4.0,5.0 mmol / L),which is time and concentration dependent.ID₅₀was 2mmol/L.The cell cycle was arrested in G₂/M phase,accompanied by decrease in the S phase cells(P<0.05).After being exposed to various concentration of SNP(0.5,1.0,2.0 mmol/ L),radiosensitising effect was observed.The radiosensitising power was 2.56 for 0.5 mmol / L and 4.97 for 2.0 mmol / L.The apoptosis cell death was verified by DNA fragmentation and the early apoptotic cell detection by Annexin-V/PI,also a series of morphological changes such as cell shrinkage,nuclear condensation and nuclear fragment also showed apoptotic cell death.The expression and upregulation of caspase-3 and activated caspase-3 in SNP group and SNP combined with radiation also were detected.It suggested that exogenous NO has a proliferation inhibition and apoptosis induction effect on Tca8113 cells in a concentration and time-dependent manner,and has radiosensitizing effect by apoptotic cell death.Caspase-3 may be involved in the apoptotic process induced by SNP. Section 2 Transfer of Inducible Nitric Oxide Synthase Gene on Cell Proliferation in Tca8113 Cell LinesTo investigate the effect of transfer of inducible nitric oxide synthase(iNOS)gene on cell proliferation in Tca8113 cell lines.pCMV-iNOS plasmid,designed for high-level expression of iNOS gene,was transfected into Tca8113 cells mediated by lipofectamine,and the tranfected cells were named as pCMV-iNOS-Tca.Empty vector tranfered TcaS113 cells were named as pCMV-Tca.The protein expression of transfected gene was verified by Western blot.Nitric oxide concentratin was detected by Griess reaction.Also the morphological changes were observed by microscope.To observe the proliferation effect of iNOS gene on Tca8113 cells,cell growth curves were tested by MTT assay.The redistribution of cell cycle and proliferation index(PI)were analyzed by flow cytometry.The results showed,Compared with the pCMV-Tca and Tca8113 cells,the iNOS protein expression upregulated in pCMV-iNOS-Tca cells,and the concentration of NO during various times is much higher in pCMV-iNOS-Tca cells than the other two groups,pCMV-iNOS-Tca cells grew rapidly during the third,fouth and fitch day(P<0.05).The cell cycle was arrested in G₂ / M phase in pCMV-iNOS-Tca cells,which the distribution of G₂ / M was higher than the other two groups(P<0.05).PI in the pCMV-iNOS-Tca cells,pCMV-Tca and Tca8113 cells was 78.71%,54.29%and 39.18%,respectively.It suggested that iNOS gene transfection can upregulate the expression of iNOS protein,and increase the output of nitric oxide,it also make the cells grow rapidly and arrested in G₂ / M phase. Section 3 Effect of iNOS gene on in vitro Radiochemosensitivity of Tca8113 CellsTo observe the radiochemosensitivity of iNOS gene on Tca8113 Cells and the mechanisms, cells before and after iNOS gene transfection were treated with cisplatin,5-fluorouracil(5-FU), bleocin and radiation therapy.The sensitivity were tested by MTT assay.The expression of p53 and p21 were also detected by western blot.The results showed:compared with the Tca8113 cells,the pCMV-iNOS-Tca cells showed distinct sensitivity to cisplatin,bleocin and radiation.The sensitivity increased 4,2-fold to cisplatin,1.7-fold to bleocin and 1.6-fold to radiation(caeulated by IC₅₀)compared with Tca8113 cells.However,the sensitivity to 5-FU did not change.iNOS gene remarkable upregulate the expression of p53,but not for p21.It suggested that iNOS gene can enhance the cell sensitivity to cisplatin,bleocin and radiation.These function of iNOS gene may be associated with the G₂/M phase arrest and the expression of p52 and p21.