The Objective Evaluation On Hypoxic Microeviroment And Effects Of Hypoxia Correction On Chemotherapy Resistance In Laryngeal Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the6th mostcommon malignant tumor worldwide. Every year, neopathy number exceeds500thousands. Its clinicopathologic characters involve insidious onset, stronginvasiveness, and liability to recurrence and metastasis with poor prognosis.Although improvement in therapeutic strategies has been made in the past2to3decades, the overall five-year survival rate remains almost unchanged.Therapeutic resistance and recurrence are pending problems in HNSCC. It iscritical to explore the mechanism and effective detection method in order toselect the best therapeutic strategy.As we all known, microenvironment plays an important role in tumorgenesis, invasion, and metastasis. Hypoxia, which is caused by the imbalancebetween malignant proliferation of tumor cells and blood supply in localtumor tissue is closely related with cancer therapeutic resistance andrecurrence. During this course, some gene transcription activities are alteredand their productions facilitate tumor cells the ability to adapt to hypoxicmicroenvironment. Simultaneously, they promote tumor growth, angiogenesis,metabasis and radiochemotheraputic resistance. Hypoxia-inducible factor1(HIF-1) which is composed of HIF-1α and HIF-1β subunits, is a key regulatorto hypoxia. The activity of HIF-1is determined by expression of the HIF-1αsubunit. Under normoxia, HIF-1α is degraded quickly but under hypoxia itsstability is enhanced which could up-regulate more than100genes expression.As the two main promotion factors of angiogenesis and glycolysis, the roles ofvascular endothelial growth factor (VEGF) and glucose transporter protein(Glut-1) should not be underestimated. Therapeutic resistance and recurrencemay be overcomed by blocking up these factors. As hypoxia could induce tumor cell to produce protective proteins whichdirectly affect the therapeutic effect and prognosis, accurate detection andeffective improvement to tumor hypoxic microenvironment are imperative. Bynow, the degree of hypoxia in tumor tissue can be evaluated by endogenoushypoxic markers, such as glucose transporter, and modern imaging technology,such as magnetic resonance imaging (MRI) and positron emission tomography(PET). With regard to the improvement of hypoxia in tumor tissue, there aremany methods as following:1. directly or indirectly increase oxygen contentthrough high concentration of oxygen inhalation or hemoglobin concentrationand its oxygen carrying capacity elevation;2. enhance lethal effect of X-ray tohypoxic cell by different methods;3. apply radiosensitizer to hypoxic cell;4.use hypoxic cell selective agents;5. thermotherapy;6. gene therapy. Althoughhypoxia can be corrected by these methods, imaging results show a poorcoincidence with the expression of endogenous hypoxic markers. It revealsthat normalization of local oxygen partial pressure (PO₂) cannot represent thethorough improvement of hypoxia in tumor cells. There may exist atime-window between recovery of PO₂in tumor tissue and normalization ofhypoxic metabolism within the tumor cells.Based on all above questions, we performed this investigation to lookinto the regulation of Glut-1and VEGF by HIF-1α in laryngeal carcinoma andverify the clinical implications of HIF-1α expression in laryngeal carcinoma.The dynamic alteration of HIF-1α, Glut-1and VEGF in tumor tissue after PO₂normalization was detected in vitro and in vivo, and the influence of hypoxiacorrection on chemotherapy resistance was explored. This study was dividedinto five parts:Part one: Regulation of Glut-1and VEGF by HIF-1α under hypoxia andits impact on clinicopathologic characters in human laryngeal squamouscell carcinomaObjective: To evaluate the role of HIF-1α on regulation of Glut-1andVEGF under hypoxia and its impacts on clinicopathologic features inlaryngeal squamous cell carcinoma (LSCC). Moreover, the relationship between hypoxia and expression of these three proteins was verified in vitro. Itsuggested that hypoxia played an active role in up-regulating the expression ofHIF-1α and its target genes in HNSCC.Methods:(1) Surgical specimens from laryngeal cancer patients wereselected to detect the expression of HIF-1α, Glut-1and VEGF byimmunohistochemical method and their relationships with clinicopathologiccharacteristics were evaluated.(2) Human laryngeal squamous carcinomaHep-2cells and HIF-RNAi-Hep-2cells were cultured in RPMI1640mediumand cell proliferation was detected by MTT assay.(3) Hep-2cells andHIF-RNAi-Hep-2cells were cultured in RPMI1640medium under differentPO₂and the mRNA expression of HIF-1α, Glut-1and VEGF was detected byRT-PCR technique.(4) The expression of HIF-1α, Glut-1and VEGF proteinin Hep-2cells and HIF-RNAi-Hep-2cells under different PO₂were detectedby immunocytochemical method and Western blot technique.Results:(1) Clinical immunohistochemistry results showed that out ofthe35surgical specimens,16were positive for HIF-1α (positive rate45.71%),16for Glut-1(positive rate45.71%), and19for VEGF (positive rate54.29%).Among16HIF-1α-positive specimens,11were positively stained for Glut-1and14for VEGF. Positive staining of HIF-1α was mainly located in thenecrosis of cancer nest and gland epithelial cells. Immunoreaction of Glut-1and VEGF was seen extensively in cancer nest. It is demonstrated that thereexisted significant correlation of protein expression between HIF-1α andGlut-1or VEGF in laryngeal carcinoma (P<0.05). The expression of HIF-1αand VEGF protein was closely related with pathologic grading of the primarytumor and cervical lymphnode metastasis, while the expression of Glut-1wasrelated with cervical lymphnode metastasis (P<0.05).(2) MTT results showedthat the proliferation of Hep-2cells was increased in hypoxic condition andthe difference was remarkable when compared with the cell proliferation innormoxic condition (P<0.05). The proliferation rate of Hep-2cells wasdecreased when HIF was silenced (P<0.05).(3) RT-PCR result showed thatmRNA levels of HIF-1α remained stable in hypoxic condition, while mRNA levels of Glut-1and VEGF were elevated over time and reached to the peak at36h. After HIF-1α gene was silenced, the mRNA expression of Glut-1andVEGF were significantly decreased (P<0.05).(4) Immunocytochemicalmeasurement and Western blot showed that the expressions of HIF-1α, Glut-1and VEGF protein in Hep-2cells in hypoxic condition were higher than thosein normoxic condition. The expression of HIF-1α protein was located innucleus or cytoplasm, the expression of Glut-1protein was located incytomembrane or cytoplasm, and the expression of VEGF protein was locatedin the cytoplasm. Within36h, the expressions of these proteins were intime-dependent manner (P<0.05). But at48h, the protein expressions weredecreased. Moreover, HIF-1α could up-regulate the expression of Glut-1andVEGF protein (rGlut-1=0.937, P <0.05; rVEGF=0.909, P <0.05).Conclusions: The expression of HIF-1α is intimately related to theexpression of Glut-1and VEGF. HIF-1α may play a major role inup-regulating the expression of Glut-1and VEGF, and thus promotes tumorangiogenesis, invasion, and metastasis of the laryngeal carcinoma.Part two: The impact of reoxygeonation on the expression of endogenoushypoxic markers in vitroObjective: The proliferation of Hep-2cell was detected afterreoxygeonation and the impact of hypoxic microenvironment alteration on theexpression of endogenous hypoxic markers was demonstrated.Methods:(1) The Hep-2cells proliferation under36h of hypoxia andreoxygeonation at different time (36+6h,36+9h,36+12h,36+24h) weredetected by MTT assay.(2) The expression of HIF-1α, Glut-1and VEGFprotein in Hep-2cells at different time after reoxygeonation were detected byimmunocytochemical method and Western blot technique.(3) The mRNAlevel of HIF-1α, Glut-1and VEGF in Hep-2cells after reoxygeonation wasdetected by RT-PCR technique.Results:(1) MTT assay results showed that the cell proliferation whichwas increased after36h of hypoxia was maintained after6h and9h ofreoxygenation. While after12h and24h of reoxygeonation, the cell proliferation was decreased.(2) Immunocytochemical results showed thatafter6h of reoxygeonation, the protein expression of HIF-1α, Glut-1andVEGF was descended to normoxic level. And western blot result wasconsistent with immunocytochemical results.(3) RT-PCR results showed thatmRNA levels of HIF-1α remained stable under hypoxia (P>0.05). But themRNA level of Glut-1and VEGF was gradually increased within36h ofhypoxia (P<0.05). When Hep-2cells were cultured in normoxic conditionagain for about6h, the mRNA level of Glut-1and VEGF were approximatelydecreased to normoxic level (P>0.05).Conclusions: The proliferation rate of Hep-2cell was slow down afterreoxygeonation. The expression of HIF-1α, Glut-1and VEGF protein inHep-2cells was increased under hypoxia. After9h of reoxygeonation, theprotein expression of HIF-1α, Glut-1and VEGF were gradually descended tothe levels under normoxia. The mRNA expression of HIF-1α was stable underhypoxia. The regulation of hypoxia on mRNA expression of Glut-1and VEGFwas at transcriptional level. When hypoxia was corrected, the mRNA level ofGlut-1and VEGF were gradually decreased to the levels under normoxiccontition.Part Three: Establishment of xenograft model with Hep-2cells in nudemice and objective evaluation by¹⁸F-FDG PET imagingObjective: To establish xenograft model with Hep-2cells in nude miceand to study tumor imaging by¹⁸F-FDG PET technique. The tumor-bearinganimals were handled with different disposals before experiment and werescanned at different time. Then tumor metabolism state was observed andoptimized condition of tumor imaging was established. It could providefavorable detection method for human LSCC xenograft in animal model．Methods:(1) Xenograft model with Hep-2cells was established in nudemice. Tumor formation rate and growth curve were recorded and graphed.(2)The tumor-bearing animals were handled with different disposals beforeexperiment and were scanned by¹⁸F-FDG PET at different time. Then theimage quality was compared with each other. Results:(1) Animal xenograft model of LSCC was successfullyestablished and tumor formation rate reached100%.(2)¹⁸F-FDG PET result:no imaging of tumor was observed in "no fasting and warm-keeping disposal"group; poor image quality was achieved in "fasting but no warm-keepingdisposal" group; good image quality was acquired in "fasting andwarm-keeping disposal" group and imaging rate was100%. Differentscanning start time has little effect on image quality.Conclusions: Clinical¹⁸F-FDG PET was practicable for the study ofLSCC xenograft model in nude mice. Tumor ranging from0.8cm to1.5cm indiameter was more appropriate for PET imaging. Animal handling, includingfasting and warming, had direct influence on the image quality. The initiatingtime of scanning had no effect on PET image quality.Part Four: Objective evaluation of the effect of carbogen inhalation onhypoxia improvement in LSCC xenograftsObjective: To observe the imaging of tumor before and after carbogeninhalation by¹⁸F-FDG PET technique, and to detect the protein expression intumor by immunohistochemical technique. The difference was analyzed inorder to verify the time-window existence between PO₂normalization intumor tissue and hypoxic metabolism recovery in tumor cell.Methods:(1) Xenograft model with LSCC Hep-2cells was establishedand gross observation on tumor was conducted including tumor formation rateand tumor growth curve.(2) The imaging of tumor tissue before and aftercarbogen inhalation was observed by¹⁸F-FDG PET technique.(3) Animalswere randomly divided into two groups: one group was given carbogeninhalation and the other group was given air-breathing. The expression ofHIF-1α, Glut-1and VEGF protein in tumor tissue was detected byimmunohistochemical method and Western blot method.Results:(1) Animal xenograft model of LSCC was successfullyestablished and tumor formation rate reached to100%.(2)¹⁸F-FDG PETresults: the rate of tumor imaging was100%and the image quality of tumorwas good; after carbogen inhalation, the image of tumor became blurry. The difference was significant (P<0.05).(3) Immunohistochemical results andWestern blot results showed: after carbogen inhalation, the expression ofHIF-1α, Glut-1and VEGF protein remained high level in tumor tissue andthere was no remarkable difference in experiment group and control group(P>0.05).Conclusions: Carbogen inhalation can effectively reduce the hypoxiaand glycolysis metabolism in tumor tissue. There existed a time-windowbetween PO₂normalization in tumor tissue and thorough correction ofhypoxia within tumor cell.Part Five: The impact of hypoxia improvement on LSCC chemotherapyObjective: To evaluate the effect of hypoxia improvement in Hep-2cellon cisplatin-induced apoptosis.Methods:(1) Hep-2cells and HIF-RNAi-Hep-2cells were cultured innormoxic, hypoxic and reoxygeonation condition. The inhibition of cisplatinon cell proliferation was evaluated by MTT assay.(2) The influence ofcisplatin on cell cycle and apoptosis were detected by flow cytometry (FCM).Results:(1) MTT results showed: the inhibition of cisplatin on cellproliferation was reduced by hypoxia. When cells were cultured in normoxicmicroenviroment again, the cisplatin effect on Hep-2cell proliferation wasgradually recovered to its former level. After HIF gene was silenced, theinhibition of cisplatin on Hep-2cell proliferation was increased apparently,but was still interfered partly by hypoxia.(2) FCM results showed: hypoxiacould induce cell cycle distribution change. G₀/G₁period was extended andthe ratio of S phase was decreased (P<0.05), which led to cell apoptosisdecrease and enhancement of chemotherapeutic resistance. Afterreoxygeonation, cell apoptosis induced by cisplatin was increased significantly(P<0.05). HIF-RNAi-Hep-2cells under hypoxia also showed certainresistance to apoptosis but the sensitivity to cisplatin was higher than that ofHep-2cells. When cells were returned from hypoxic condition to normoxiccondition for some time, the apoptosis induced by cisplatin was increasedsignificantly (P<0.05). Conclusions: Hypoxia could induce Hep-2cell G0/G1arrest, apoptosisrate decrease. When cells were returned to normoxic condition again, thesensitivity of Hep-2cell to cisplatin was improved obviously. After HIF-1αgene knockout, the sensitivity of Hep-2cells to chemotherapy was increased,but this effect was still influenced by hypoxic microenvironment. Only whenthe hypoxia in tumor cells was thoroughly improved, the cytotoxicity ofcisplatin could be fulfilled.