Effct Of Hypoxia On Phenotype Of Tongue Squamous Cell Carcinoma Cells Line Tca8113 And Their Mechanisms

Background:The oxygen supply to cells and tissues is pivotal in maintaining their function and integrity.Rapid growth of tumors often results in the development of a hypoxic microenvironment.Therefore,signal of hypoxic microenvironment has become an important factor that affects the biological characteristics of solid tumor.Under hypoxic microenvironment,the tumors present more progressive phenotype in abnormal neovascularization,the invasion,metastasis and resistance to chemo- and radiotherapies.In recent years,many researches focus on the effect of hypoxia on the biological characteristics of tumor,and the possible mechanisms.New strategy of tumor treatment based on hypoxia has shown a good future.During adaptation of tumor to the hypoxic microenvironment,a wide variety of tumor- and host-derived factors are regulated.Among those factors,hypoxia-inducible factor-1 alpha(HIF-1α) might be a central transcriptional regulator of tumor microenvironment.A lot of studies have proved that these signal pathways such as HIF-1αpathway are playing an important role in the regulation of tumor's malignant phenotype.However,very few reports are regarding the expression of HIF-1αin oral carcinoma and tongue squamous cell carcinoma cells line Tca8113,and their mechanisms by which to adapt the hypoxic microenvironment.Over-expression of HIF-1αwidely presents in the solid tumor and it has been proved as a key transcription factor to adapt to the hypoxic microenvironment.HIF-1 is a heterodimeric transcription factor composed of a strictly regulatedαsubunit and a constitutiveβsubunit(HIF-1β/ARNT).The stability and function as a transcription fcctor of HIF-1α,is regulated by level of O₂.Under hypoxic condition,protein degradation of HIF-1αis reduced and it is allowed to translocate into the nucleus where it dimerizes with ARNT.HIF-1 binds to hypoxia-responsive elements(HRE) located in the promoter and enhancer regions of hypoxia-regulated genes,causing their transactivation.According to the studies up to today,HIF-1αcan regulate more than 100 genes,which are involved in adaptive responses to hypoxia,to regulate many biological behaviors of cell,such as the process of glycolytic enzymes,growth factors and vasoactive peptides,angiogenesis factors,and genes involved in apoptosis, invasion and proliferation.Since different kind of tumor has high heterogeneity,the effects of HIF-1αand its possible mechanisms may be quite different.Carcinoma of the oral tongue is the most common malignancy of the oral cavity. Over the past three decades,the incidence of all oral cancers has increased in china. The most important reasons that caused failure in treatment to carcinoma of the oral tongue are invasion,metastasis and resistance to chemo- and radiotherapies.And hypoxic microenvironment is one of the key affective factors.It was proved that the hypoxic microenvironment was present in human oral tumor,and high levels of HIF-1αin human squamous cell carcinomas seemed correlated with tumor resistance to radio- and chemotherapy.Nonetheless,its effects on the phenotype of human tongue squamous cell carcinoma cells under hypoxic condition have not yet been sufficiently studied.Recent studies have discovered that Adrenomedullin(ADM) involves in regulating of biological characteristics of tumor,such as proliferation and apoptosis.Also it may be an important hypoxic regulated gene involved in hypoxic adaption of tumor cells.It was reported that under hypoxic conditions,the expression of HIF-1αand ADM are both up-regulated.Our study also proved that hypoxia will obviously increase the content of HIF-1αprotein and ADM mRNA in the tongue squamous cell carcinoma cells line Tca8113.However,there is no report regarding the relationship of them,and whether HIF-1αwill cause more progressive phenotype in tumor cells mediated by regulating the expression of ADM.In the present study,we are using tongue squamous cell carcinoma cells line Tca8113,and using siRNA_HIF-1α to study the effects of HIF-1αto proliferation,apoptosis and invasion,as well as study the expression of ADM regulated by HIF-1α.We would like to discover the expression of HIF-1αin the tongue squamous cell carcinoma cells line Tca8113,and the new regulative pathway of HIF-1α,with a view to find an efficient target to work out more reasonable treatment strategy,target to hypoxic characteristic.Sectionâ… : Influence of hypoxia on the biological characteristics of tongue squamous cell carcinoma cells line Tca8113 and the expression of HIF-1α/ADMObjectiveTo observe the hypoxia- induced changes of biological characteristics in the tongue squamous cell carcinoma cells line Tca8113 and to investigate the expression of HIF-1αand ADM in Tca8113 cells.Meterial and Methods1.Cell culture:Tongue squamous cell carcinoma cells line(Tca8113 cells) were routinely cultured in RPMI1640 containing 10%foetal bovine serum(FBS),penicillin (100 units/ml),and streptomycin(100μg/ml) at 37℃in a humidified air atmosphere containing 5%carbon dioxide.For hypoxic culture,Tca8113 cells were cultured at 37℃in 94%N₂,5%CO₂ and 1%O₂ for 4h,24h or 48h when growing to 80% confluence.2.Real-time RT-PCR analysis for HIF-1α:The HIF-1αmRNA expression was assayed quantitatively by real-time reverse transcriptionpolymerase chain reaction (real-time RT-PCR).Tca8113 cells were incubated under normoxic or hypoxic condition for 4h or 24h and harvested,total RNA was extracted by TRIZOL according to the manufacturer's instructions.Synthesis of first-strand cDNA was carried out with Revert Aid^TM First Strand cDNA Synthesis Kit.The relative amount of PCR product was calculated as threshold cycle(CT value) of the sample divided by that of humanβ-actin.3.Western blot analysis:Tca8113 cells were incubated under normoxic or hypoxic condition and cellular protein extracts were prepared.HIF-1αexpression induced by hypoxia was measured on protein level by Western blot.4.Real-time RT-PCR analysis for ADM mRNA:Tca8113 cells(2×10⁵) were incubated under hypoxic condition for 2h,4h,8h,or 24h and harvested.Total RNA was extracted by TRIZOL.The expression of ADM mRNA was assayed quantitatively by real-time PCR.5.Cell proliferation assay:The Tca8113 cells were incubated overnight at a density of 6,000 cells/well in 96-well plates.Then Tca8113 cells were incubated under hypoxic condition for 6h,12h,24h,48h,or 72h respectively.20μl of MTT was added each well and color development was measured on a microplate reader at 570 nm.6.Cell cycle and apoptosis analysis:Tca8113 cells were plated at a concentration of 2×10⁵ cells per well in six-well plates.Growing to 80%confluence,the cells were incubated under hypoxic condition for 4h,24h or 48h and stained with PI.DNA content was detected on a FACS Calibur.After incubated under hypoxic condition for 4h,24h or 48h,Tca8113 cells were collected and labeled with Annexin V-biotin followed by PI.Annexin V-PI were measured by FACS Calibur and analyzed with the Modfit Software.7.Adhesion assay by CytoMatrix^TM cell adhesion kit:Tca8113 cells were collected after incubated under hypoxic condition for 4h or 24h.1×10⁵ cells were added each well of CytoMatrix^TM cell adhesion kit.Absorbance was measured at A490 nm.8.Invasion assay by cell invasion assay kit:The Tca8113 cells(3×10⁵ cells) were added into the transwell inserts and were incubated for 24 hours under hypoxic condition.After incubation,the invaded cells were counted in five fields for each filter under a light microscope at 20×magnification.Results1.Proliferation of Tca8113 cells were inhibited by hypoxia.The proliferation of Tca8113 cells was inhibited significantly after the cells were cultured under hypoxic condition for 48h or 72h compared with the normoxic control(0.391±0.051 vs 0.537±0.046,P<0.01;0.255±0.031 vs 0.852±0.078,P<0.001).2.Hypoxia induces G₁ phase cell cycle arrest in Tca8113 cells.The cells in G₀/G₁ phase were increased significantly under hypoxic condition for 24h or 48h(73.37±4.01 vs 66.59±5.25,P<0.05;87.62±6.54 vs 61.88±4.38,P<0.01).3.Apoptosis can be induced by hypoxia.No significant increase in the percentage of apoptotic cells was found after the Tca8113 cells were cultured under hypoxic condition for 4h or 24h when compared with normoxic incubated cells.4.The adhesion and invasion potency of Tca8113 cells was stimulated by hypoxia. Adhesion of Tca8113 cells to Matrigel-coated cell culture plates in vitro was increased significantly under hypoxic condition for 4h or 24h((126.0±9.3)%,(128.0±10.8)% vs(100.0±8.7)%,P<0.05).When the cells were cultured under hypoxic condition for 24h,the invasion potency of Tca8113 cells was stimulated significantly compared with the normoxic control.((133.0±11.6)%vs(100.0±9.2)%,P<0.05).5.Hypoxia induced the accumulation of HIF-1αprotein.mRNA and protein was stably expressed in Tca8113 cells under normoxic condition analysed by real-time RT-PCR and Western blot.Exposed to the hypoxic environment for 4h or 24h,no significant increase in HIF-1αmRNA transcript was observed.However,hypoxia induced a rapid and sustained accumulation of HIF-1αprotein in Tca8113 cells up to 24 hours. 6.The expression of ADM mRNA in Tca8113 cells was induced by hypoxia.The expression of ADM mRNA increases when incubated under hypoxic condition up to 24h.Conclusion1.The study focused on the changes of biological characteristics of Tca8113 cells under hypoxic condition.It was proved that chronic hypoxia(for 48h) inhibited the proliferation and induced apoptosis of Tca8113 cells.The adhesion and invasion potency were stimulated and G₁ phase cell cycle arrest was induced by hypoxia in Tca8113 cells.2.Hypoxia had no effect on the transcription of HIF-1αmRNA but caused a rapid and sustained accumulation of HIF-1αprotein in Tca8113 cells.It was suggested that the hypoxia induced post -transcription regulation of HIF-1αexpression.3.We firstly found that the expression of ADM mRNA was induced by hypoxia in Tca8113 cells.HIF- 1αmight play a role in the up-regulation of ADM.Section II The effect of HIF-1αgene silence on the expression of ADM and the phenotype of Tca8113 cellsObjectiveTo investigate the inhibitory effects of RNA interference(RNAi) mediated by siRNA_HIF-1α on expression of HIF-1αand ADM and the regulation of HIF-1α/ADM pathway in the proliferation and invasion of the tongue squamous cell carcinoma cells line Tca8113.Meterial and Methods1.siRNA transfection:Tca8113 cells were plated at a concentration of 2×10⁵ cells per well in six-well plates.Growing to 40-50%confluence,the cells were transfected with the siRNA_HIF-1α(50nM) premixed with the Lipofectamine^TM 2000.2.The assay of potency of the siRNA_HIF-1α to block HIF-1αexpression:Transfected Tca8113 cells were incubated under normoxic or hypoxic condition for 24h.HIF-1αexpression was measured on mRNA level by real-time PCR and protein level by Western blot.3.Immunofluorescence assay:The Tca8113 cells(8×10⁴ in 1ml culture medium) were plated onto chamber slides and allowed to attach overnight.When growing to 40%confluence,the cells were transfected with 50 nM siRNA_HIF-1α for 24h and incubated at 1%oxygen for 24h.The samples were incubated with mouse anti-human HIF-1αmonoclonal antibodies.Expression of HIF-1αwas detected with fluorescence microscopy.4.Cell proliferation assay:For cell proliferation assay of 50nM siRNA_HIF-1α transfected Tca8113 cells,transfected cells were incubated under normoxic or hypoxic condition for 24h,48h,or 72h respectively.Then cell proliferation was analyzed by MTT colorimetric assay.20μl of MTT was added each well and color development was measured on a microplate reader at 570nm.5.Analysis of cell cycle and apoptosis of transfected Tca8113 cells:For analysing the cell cycle,the transfected Tca8113 cells were incubated under hypoxic condition for 24 of 48h.Propidium iodine was added to the cells for 30 minutes in the dark prior to FACS analysis.Transfected Tca8113 cells were incubated under normoxic or hypoxic condition for 24h.After incubation with Annexin V-FlITC antibody and PI in the dark,the cells were analysed by a flow cytometer. 6.Adhesion assay of siRNA_HIF-1α transfected Tca8113 cells:The transfected cells were incubated under hypoxic condition for 24h.1×10⁵ cells were added each well of CytoMatrix^TM cell adhesion kit.Absorbance was measured at A490 nm.7.Invasion assay of siRNA_HIF-1α transfected Tca8113 cells:The siRNA_HIF-1α transfected Tca8113 cells(3×10⁵ cells) were added into the transwell inserts and were incubated for 24 hours under normoxic or hypoxic condition.After incubation, the invaded cells were counted in five fields for each filter under a light microscope at 20×magnification.8.Expression of ADM mRNA in siRNA_HIF-1α transfected Tca8113 cells:The transfected Tca8113 cells were incubated under hypoxic condition for 24h.Total RNA was extracted by TRIZOL.The expression of ADM mRNA was assayed quantitatively by real-time PCR.Results1.HIF-1αexpression was suppressed by siRNA_HIF-1α:Reduction of HIF-1αmRNA expression by approximately 90%was found in siRNA_HIF-1α treated cells,compared to siRNA_Irr treated cells or mock transfection control in both normoxia and hypoxia (P<0.01).Likewise,the results of western blot and immunofluorescence showed that hypoxia-induced HIF-1αprotein was significantly suppressed in Tca8113 cells treated with siRNA_HIF-1α in comparison to mock transfection controls.2.Knockdown of HIF-1αinhibits proliferation of Tca8113 cells:In the first 24 hours,the proliferation of Tca8113 cells treated with siRNA_HIF-1α was not inhibited significantly compared with the mock transfected control under normoxic condition. However,the proliferation was inhibited significantly under hypoxic condition (0.252±0.029 vs 0.322±0.031,P<0.05).At 48h and 72h,siRNA_HIF-1α significantly inhibited the proliferation of Tca8113 cells under hypoxic and normoxic condition compared to the mock transfected control(P<0.01).3.Apoptosis is induced by siRNA_HIF-1α in Tca8113 cells:The apoptosis of siRNA_HIF-1α transfected Tca8113 cells significant increases as compared to mock transfected control when incubated under normoxic or hypoxic condition for 24h ((12.15±1.02)%vs(4.68±0.56)%;(17.77±1.22)%vs(6.43±0.55)%,P<0.01).4.Treated with siRNA_HIF-1α inhibited adhesion and invasion of Tca8113 cells:The adhesion of siRNA_HIF-1α treated Tca8113 cells was decreased to 74%and 63.6%of mock transfected control and the invasion of siRNA_HIF-1α treated Tca8113 cells was decreased to 65%and 55.8%of mock transfected control when incubated under hypoxic condition for 24h.5.The expression of ADM mRNA induced by hypoxia was inhibited by siRNA_HIF-1α:Exposed to the hypoxic environment for 24h,no significant increase in ADM mRNA transcript was observed in siRNA_HIF-1α treated Tca8113 cells.Conclusion1.Down-regualtion of HIF-1αmediated by siRNA can inhibit the proliferation, induced the apoptosis of Tca8113 cells and inhibited adhesion and invasion of Tca8113 cells.It was suggested that HIF-1αplayed an important role in the regulation of phenotype of Tca8113 cells.2.We firstly found that the expression of ADM mRNA induced by hypoxia cound be inhibited completely by siRNA_HIF-1α.It was suggested that HIF-1αregulated the phenotype of Tca8113 cells to adapt the hypoxic condition mediated by regulating the expression of ADM.3.It is important to discover the mechanism of HIF-1α/ADM pathway to Tca8113 cells on hypoxic adaption for finding the new treatment strategy target hypoxia.