The Roles Of GLUT-1 And HIF-1? In Radioresistance Of Head And Neck Carcinoma

Head and neck squamous cell carcinoma is one of the common malignant tumors,the mobidity ranks sixth of all the malignant tumors,in the world the number of new cases is more than 500,000 each year.Some of head and neck squamous cell carcinoma have the characteristics of occult,aggressive,easy to relapse and metastasis,and poor prognosis.Although in recent 40 years the therapeutic modalities are being advanced,the life qualities of patients have been greatly improved,5-year survival rate of some head and neck squamous cell carcinoma patients have not been significantly improved.Therefore,further study of the pathogenesis of head and neck squamous cell carcinoma and searching for new methods of treatment have important scientific significance and clinical transformation value.Otto Warburg and his team found that the tumor tissue compared to normal tissue in aerobic conditions,glucose consumption and lactic acid production are increased,the metabolism of malignant tumors perform aerobic glycolysis,it is called Warburg effect.Therefore,blocking this glycolytic pathway may become a new method of tumors therapy.Glucose transporter protein?GLUT?is an important energy transporter that mediates the Warburg effect.Glucose transporter is a protein that mediates the transmembrane transport of glucose,which is the main reason for the increase of glucose metabolism in malignant tumor cells.GLUT-1 is a representative protein of the GLUT family and is widely expressed in whole body tissue cells.GLUT-1 is overexpressed in many malignant tumors to meet the energy needs of malignant tumor cells.The level of GLUT-1 expression was associated with prognosis,TNM stage,tumor differentiation,metastasis and recurrence of the malignant tumors.Our preliminary study found that GLUT-1 overexpression was associated with lymph node metastasis,progression and poor prognosis of the head and neck cancer.Thus,inhibition of GLUT-1 expression may be a new method for the treatment of malignant tumors.Hypoxia is a common phenomenon of solid tumors.Hypoxia inducible factor-1?HIF-1?is an important hypoxia-inducible factor.It has been found that high expression of HIF-la was associated with radioresistance of esophageal cancer,Sarcoma,lungcancer,liver cancer and other malignant tumors,and the overexpresion of HIF-1?promoted cancer cells to be more invasive and to its distant metastasis.Our previous studies and other studies had also shown that the expression level of HIF-la is associated with lymph node metastasis,T stage,prognosis and radiation resistance of laryngeal squamous cell carcinoma.Thus,inhibition of HIF-la expression is also a new method of malignant tumors treatment.It was found that GLUT-1 and HIF-la overexpression were associated with radiation resistance of malignant tumor,and inhibition of GLUT-1 or HIF-1? expression could improve the curative effect of malignant tumor.In our previous study we found that antisense oligodeoxynucleotides?AS-ODN?could inhibit GLUT-1 expression of Hep-2 laryngeal cancer cell line and inhibit glucose uptake and proliferation of Hep-2 cells,further study found overexpression of GLUT-1 was associated with radiation resistance of laryngeal carcinoma,and inhibition of GLUT-1 expression by AS-ODN enhances the radiosensitivity of laryngeal carcinoma.Some studies have also found that inhibition of GLUT-1 or HIF-la expression can improve the radiosensitivity of malignant tumors.But there is no reports of simultaneous inhibition of GLUT-1 and HIF-la expression to enhance the radiosensitivity of laryngeal cancer.The Warburg effect has been demonstrated by the clinical application of positron emission tomography-computed tomography?PET/CT?,the most widely used developer is 2'-deoxy-2'-[¹⁸F]Fluoro-D-glucose,(¹⁸F-FDG),it is an analogue of glucose.Its working principle is that the ¹⁸F-FDG is administered by GLUT into the cells after intravenous injection,and phosphorylated to ¹⁸F-FDG-6-phosphate by hexokinase?HK?,which is no further metabolized,deposited in the cell,in the PET image showed a high concentration of polymer metabolism.PET/CT has been widely used in the early diagnosis of malignant tumors,the evalution of treatment efficience,and monitoring of malignant tumor recurrence,metastasis and residue after treatment,etc.,and some researches found that the maximum standard uptake?SUVmax?was associated with prognosis,recurrence,metastasis of some malignant tumors.This study was to investigate the expression of GLUT-1 and HIF-la of laryngeal and hypopharyngeal carcinoma patients who took ¹⁸F-FDG PET/CT before operation and to explore relationship between the expression of GLUT-1,HIF-la,SUVmax and clinicopathological factors,prognosis of the patients.Futhermore we studied the mechanism of radioresistance by simultaneous inhibition of GLUT-1 and HIF-la induced by AS-ODN in vivo and tried to evaluate the radiosensitivity with SUVmax of microPET/CT.Part I The relationship between GLUT-1 and HIF-la expression¹⁸F-FDG uptake and prognosis of patients of laryngeal and hypopharyngeal carcinomaPurpose:To investigate the relationship between GLUT-1 and HIF-la expression and ¹⁸F-FDG uptake and clinicopathological factors and prognosis of laryngeal and hypopharyngeal carcinoma patients.Method:The expression of GLUT-1 and HIF-la protein in 55 cases of laryngeal and hypopharyngeal carcinoma were examined by immunohistochemistry.The relationship between GLUT-1 and HIF-1? expression and SUVmax and clinicopathological andprognosis of patients with laryngeal and hypopharyngeal carcinoma were analyzed.Results:The univariate analyses revealed a significantrelationship between treatment approaches?p=0.004?,tumor size?p=0.002?and survival rate of head and neck squamous cell carcinoma patients?laryngeal carcinoma and hypopharyngeal carcinoma?,however,SUVmax?p=0.306?,Glut-1 expression?p=0.919?,HIF-la expression?p=0.083?,age?p=0.929?,clinical stage?p=0.249?,lymph node metastasis?p=0.924?and tumor differentiation?p=0.875?were not correlated with survival rate.HIF-laexpression was correlated with tumor size?r=0.351,p=0.009?.The positive rates of Glut-1 and HIF-la in head and neck squamous cell carcinoma were 49.1%and 56.4%,respectively.Glut-1 and HIF-la were not expressed in vocal cord polyps.The expression of Glut-1 and HIF-la in head and neck squamous cell carcinoma was significantly higher than that in vocal cord polyp?p<0.05?.Glut-1 expression was correlated with HIF-laexpression?r=0.571,p<0.001?and SUVmax?r = 0.495,p<0.001?,HIF-la expression was not correlated with SUVmax?p=0.199?.The multivariate analysis demonstrated that treatment approaches?p=0.025?,tumor size?p=0.008?,Glut-1 expression?p=0.032?andHIF-la expression?p=0.032?were correlated with survival rate of head and neck squamous cell carcinoma patients.Conclusion:1.The expression of Glut-1 and HIF-la in head and neck squamous cell carcinoma was significantly higher than that in vocal cord polyp.The expression of HIF-la was correlated with tumor size.2.The expression of Glut-1 was correlated with HIF-la expression and SUVmax,the expression of HIF-la was not correlated with SUVmax.3.Cox proportional hazards model multivariate analysis found that treatment approaches,tumor size,Glut-1 and HIF-la expression was correlated with prognosis of head and neck squamous cell carcinoma patients.Part II In vivo study of effect of simultaneous inhibition of GLUT-1 and HIF-la by antisense oligodeoxynucleotides on radiosensitivity of laryngeal carcinomaPurpose:In vivo study the mechanism of improving the radiosensitivity of laryngeal carcinoma by combined inhibition of GLUT-1 and HIF-la expression with AS-ODN.Method:In this study,we conducted a 2³ factorial design,nude mice were subcutaneously implanted with Hep-2 and Tu212 laryngeal cancer cell,we selected 24 nude mice of Hep-2 and Tu212 cells respectively?3 in each group?,euthanasia were administered 8 days after treatment.Nude mice were treated with AS-ODN GLUT-1 and AS-ODN HIF-la and radiotherapy 3 factors?AS-ODN100?g or O?g,radiotherapy 10Gy or OGy?,tumor volume,tumor weight,microvessel density?MVD?,apoptosis index?AI?and necrosis were analyzed to evaluate the effect of AS-ODN GLUT-1 and AS-ODN HIF-la and radiotherapy on laryngeal cancer.Results:GLUT-1 AS-ODNs or HIF-la AS-ODNs or 10Gy X-ray irradiation could not significantly decrease the volume of the xenografts?p>0.05?.10Gy X-ray irradiation could decrease the weights of Hep-2 and Tu212 cell xenografts 8 days after treatment.There was synergistic interaction effect of three treatments combined GLUT-1 AS-ODNs and HIF-la AS-ODNs and lOGy X-ray-irradiation on increasing apoptosis in Hep-2 cells 8 days after treatment?p=0.018?,GLUT-1 AS-ODNs combined with HIF-la AS-ODNs,GLUT-1 AS-ODNs combined with 10Gy X-ray irradiation had synergistic interaction effects on increasing apoptosis in Tu212 cells xenografts 8 days after treatment?P<0.001,p=0.047,respectively?.There was synergistic interaction effect of three treatments combined GLUT-1 AS-ODNs and HIF-1? AS-ODNs and 10Gy X-ray-irradiation on decreasing MVD in Tu212 cells 8 days after treatment?p=0.045?,GLUT-1 AS-ODNs combined with HIF-la AS-ODNs,GLUT-1 AS-ODNs combined with 10Gy X-ray-irradiation,and HIF-la AS-ODNs combined with lOGy X-ray-irradiation had synergistic interaction effects on decreasing MVD in Hep-2 cells 8 days after treatment?P=0.015,0.002,0.026,respectively?.There was synergistic interaction effect of three treatments combined GLUT-1 AS-ODNs and HIF-la AS-ODNs and 10Gy X-ray-irradiation on increasing necrosis in Hep-2 cells and Tu212 cells 8 days after treatment?P<0.001,P=0.004,respectively?.In the current study we found that GLUT-1 and HIF-la mRNA expression,GLUT-1 and HIF-1? protein expression revealed a significant linear correlation?p<0.05?.Conclusion:1.Combined inhibition of GLUT-1 and HIF-la could improve the radiosensitivity of laryngeal carcinoma,the mechanism may be that AS-ODN GLUT-1 and AS-ODN HIF-la could assist radiotherapy to increase the apoptosis and necrosis of laryngeal carcinoma cells as well as reduce micro vascular density of laryngeal carcinoma.2.The expression of GLUT-1 and HIF-la mRNA and protein were significantly correlated,AS-ODN HIF-la could decrease GLUT-1 mRNA and protein expression.Part III In vivo evaluation of radiosensitivity of laryngeal carcinoma xenografts using micro ¹⁸F-FDG PET/CTPurpose:¹⁸F-FDG PET/CT plays an important role in the detection and staging and choice of treatment regimen of the malignant tumors.However,whether ¹⁸F-FDG PET/CT can evaluate the therapeutic effect of malignant tumor is still controversial.The mechanism of increased uptake of ¹⁸F-FDG in malignant tumor cells was unclear.In this study we explore the radiosensitization by combined inhibition of GLUT-1 and HIF-1?expression of laryngeal carcinoma xenografts 12 days after treatment and explored the role of ¹⁸F-FDG PET/CT in evaluating the radiosensitivity of laryngeal carcinoma xenografts,and to explore the correlation between HIF-la and GLUT-1 expression and ¹⁸F-FDG uptake of the xenografts.Method:In this study,we conducted a 2³ factorial design,nude mice were subcutaneously implanted with Hep-2 and Tu212 laryngeal cancer cell,we selected 24 nude mice of Hep-2 and Tu212 cells respectively?3 in each group?.Before treatment ¹⁸F-FDG PET/CT were taken,and then nude mice were treated with AS-ODN GLUT-1 and AS-ODN HIF-la and radiotherapy 3 factors?AS-ODN100?g or ?g,radiotherapy 10Gy or OGy?,¹⁸F-FDG PET/CT examination was performed on 8th day and 12th day after treatment,and euthanasia were administered 12 days after treatment.The volume,tumor volume,microvessel density,apoptosis rate and necrosis rate of transplanted tumor were measured to evaluate the radiosensitization effect of AS-ODN.Three micro PET/CT examinations were performed before and after treatment The correlation between GLUT-1 and HIF-1? expression and the uptake of ¹⁸F-FDG were analyzed and we analysed whether ¹⁸F-FDG PET/CT can assess the radiosensitivity of laryngeal carcinoma.Results:GLUT-1 AS-ODNs or HIF-la AS-ODNs or lOGy X-ray irradiation could not significantly decrease the volume of the xenografts?p>0.05?.10Gy X-ray irradiation could decrease the weights of Hep-2 cell xenografts 12 days after treatment?p=0.022?,GLUT-1 AS-ODNs could decrease the weights of Tu212 cell xenografts 12 days after treatment?p=0.006?.There was synergistic interaction effect of three treatments combined GLUT-1 AS-ODNs and HIF-la AS-ODNs and 10Gy X-ray-irradiation on increasing apoptosis in Tu212 cells 12 days after treatment?p<0.001?,GLUT-1 AS-ODNs combined with HIF-la AS-ODNs,GLUT-1 AS-ODNs combined with 10Gy X-ray irradiation had synergistic interaction effects on increasing apoptosis in Hep-2 cells xenografts 12 days after treatment?P<0.001,P<0.001,respectively?.There was synergistic interaction effect of three treatments combined GLUT-1 AS-ODNs and HIF-la AS-ODNs and 10Gy X-ray-irradiation on decreasing MVD in Hep-2 cells and Tu212 cells 12 days after treatment?P<0.001,P<0.001,respectively?and on increasing necrosis in Hep-2 cells and Tu212 cells 12 days after treatment?P<0.001,P=0.041,respectively?.In the current study we found that GLUT-1 and HIF-la mRNA and protein expression revealed a significant linear correlation?p<0.05?,however,GLUT-1 or HIF-la expression did not correlate significantly with SUVmaxT/N2.SUVmaxT/N did not show statistical correlation with therapeutic effect?necrosis,apoptosis?.Conclusion:1.AS-ODN GLUT-1 and AS-ODN HIF-la and radiotherapy can synergistically increase the apoptosis,necrosis and decrease the microvessel density of laryngeal carcinoma xenografts 12 days after treatment,and inhibit GLUT-1 and HIF-la expression can enhance the radiosensitivity of laryngeal carcinoma.2.There was a linear correlation between GLUT-1 and HIF-la mRNA and protein expression in laryngeal carcinoma xenograftsr 12 days after treatment.3.The variation of ¹⁸F-FDG microPET/CT SUVmaxT/N before and after treatment can not be used as a means to evaluate the therapeutic effect of laryngeal carcinoma xenografts.Whether SUVmaxT/N and ? SUVmaxT/N are indicators of radiosensitivity remains to be studied.