Relationship Between HIF-1 Alpha And Angiogenesis, Chemotherapy Response And The Mechanism In Esophageal Squamous Cell Carcinoma

In recent years, a mounting body of evidence has demonstrated that a hypoxic microenvironment is common phenomena of many tumor types. Hypoxia, as a stimulating factor, can activate a series of genes responsing to hypoxia. It changes the transcription activity of many genes which induce many biological changes. Strong evidence has accumulated that hypoxia plays a pivotal role in tumor angiogenesis, progression and acquired treatment resistance. In the process, hypoxia-inducible factor 1 (HIF-1) plays a vital role in response to hypoxia. But the mechanism which may be compromised are not fully understood. Transcription factor HIF-1, as a key determinant in hypoxic microenvironment, has become one of the most popular subjects in recent.HIF-1, as Transcrettion factor, widespreadly presents in human and mammalian cells under hypoxia. It is a member of the basic helix-loop-helix Per, AhR, and Sim (bHLH-PAS) family. HIF-1 is composed of two subunits of HIF-1αand HIF-1β. The activity and stability of HIF-1, is decided by HIF-1α, a subnnit regulated by O₂. HIF-1αplays a crucial role in tumor cell adaptation to the hypoxic microenvironment through transcriptional regulation of its target genes. Therefore, it is of great importance to study the effects of HIF-1αin tumor growth, invasion, metastasis and therapy.Esophageal squamous cell carcinoma(ESCC) is one of the leading cancers in China. Is there the molecular mechanism of hypoxia response in it? At present, studies on the expression of HIF-1αare limited. The detail study about relationship between HIF-1αand angiogenesis, chemotherapy response in ESCC are seldom seen. Especially studies on relationship between HIF-1αand chemotherapy resistance and its mechanism in ESCC are not found.In order to study the relationship between HIF-1αand angiogenesis in ESCC, expression of HIF-1αon mRNA and protein levels in ESCC tissue was investigated respectively by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. The expressions of VEGF and CD34 were detected using immunohistochemistry. The relationship between HIF-1αand clinical pathological factors,VEGF, and microvessel density (MVD) was analyzed. In order to investigate whether HIF-1αinduces angiogenesis through regulating the expression of VEGF, Cobalt chloride (COC12) was used to establish hypoxia model with EC9706 cell line to mimic hypoxic microenvironment of tumors, and RT-PCR, immunocytochemistry and Western-blot were used to observe the expression of HIF-1αand VEGF. In addition, RNA interference(RNAi) was used to silence HIF-1αgene expression. VEGF expression after silencing HIF-1αgene was observed in EC9706 cells under hypoxia. The regulation effect of HIF-1αon angiogenesis of ESCC under hypoxia and its mechanism were studied, and evaluate whether HIF-1αcould be used as a novel, tumor-specific target for anti-angiogenesis therapy in ESCC.In order to investigate the relationship between HIF-1αand chemotherapeutic resistance and its mechanism in ESCC, immunohistochemistry and 3-(4,5-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) methods were used. Semi-quantative RT-PCR, immunocytochemistry, RNA interference were used to study the mechanism of chemotherapeutic resistance induced by HIF-1αunder hypoxia from aspects of multidrug resistance, apoptosis and cell cycle. In addition, RNA interference was used to investigate whether it could reverse the chemotherapeutic resistance of ESCC in vitro. All these studies would offer a theorectical foundation for enhancing therapeutic level by targeting HIF-1αas a breaking point in ESCC. Partâ… : HIF-1αexpression in ESCC and its mechanism induced by hypoxiaMethods1. The expression of HIF-1αin 46 cases of ESCC tissue and 20 cases of normal mucosa of esophagus was measured on mRNA and protein level by RT-PCR and immunohistochemistry.2. To establish a hypoxia model of EC9706 cells by a chemical hypoxia inducer (Cobalt chloride, COCl₂).3. The expression of HIF-1αin EC9706 cells under hypoxia on mRNA and protein level was measured by RT-PCR immunocytochemistry and Western-blot.4. Statistical analysis: The SPSS11.0 Statistical Package was used for all analyses. Values were expressed as means±SD. Data were analyzed by t test, ANOVA and x² test. The standard of significant level wasα=0.05.Results:1. The mRNA expressions of HIF-1αwas detected in 48 cases of ESCC and 20 cases of normal mucosal tissue of esophagus. But the level in tumor tissue(range:0.32～1.62; mean: 1.17±0.25)was significantly higher than that in corresponding normal tissue(range: 0.18～0.58; mean 0.35±0. 12)(P＜0.05). High level HIF-1αmRNA (more than 1.17) was detected in 24 out of 46 tumor cases, The high expression rate was 52.17%.2. HIF-1αimmunoreactivity was recognized in both cytoplasm and nuclei. The positive expression rate of HIF-1αwas 41.31%(19/46) in tumor samples. And it was negative expression in normal tissue.3. The mRNA expression level of HIF-1αwas positively correlated with its protein expression(P＜0.05).4. The mRNA expression level of HIF-1αwas not associated with histological grades, lympha node metastasis, infutration depth, orclinical stage(P＞0.05).5. HIF-1αprotein expression was positively correlated with infiltration depth and lympha node metastasis(P＜0.05). 6. Results of immunocytochemistry: HIF-1αimmunoreactivity of EC9706 cells in normoxia was negative, but strongly stained in plasmid/nuclear of EC9706 cells induced by chemical hypoxic condition.7. Results ofWestem-blot: HIF-1αprotein expression detected in EC9706 cells under hypoxia condition for 4h and it was stable expressied till 8h hypoxia. HIF-1αprotein expression significantly decreased when EC9706 cells were reoxygenated for 4h after being under hypoxia for 8h.8. Whereas HIF-la mRNA expression did not change before and after hypoxiaPartâ…¡: Relationship between HIF-1 alpha and angiogenesis and itsmechanism in ESCCMethods1. The angiogenenic profile was assessed using VEGF and CD34. The expression of VEGF and CD34 in above 46 cases of ESCC tissue and 20 cases of normal mucosa of esophagus were measured by immunohistochemistry. MVD was calculated according to immunohistochemical staining of the adhesion molecule CD34 of the endothelial cells.2. A hypoxia model of EC9706 cells was established by a chemical hypoxia inducer (Cobalt chloride, COCl₂).3. RNA-interference targeting HIF-1αwas used to silence the expression of HIF-1αin human EC9706 cell line. The effect of HIF-1αgene silencing was measured by RT-PCR.4. The expression level of HIF-1αinduced by hypoxia after RNAi were measured by Western-blot and immuncytochemistry.5. The expression of VEGF in EC9706 cells, which were under normaxia or under hypoxia or after RNAi under hypoxia, was measured respectively by immunocytochemistry and Semi-quantitative RT-PCR.6. Statistical analysis: The SPSS11.0 Statistical Package was used for all analyses. Values were expressed as means±SD. Data were analyzed by t test, ANOVA and x² test. The standard of significant level wasα＝0.05. Results:1. VEGF expression was detected in cytoplasm. The positive rate was 58.70%, which was significantly higher than that (4.34%) in normal mucosa (P＜0.05).2. MVD in the ESCC tissue ranged from 10 to 101, median 46.12±7.64.3. The expression of VEGF in ESCC was closely related to tumor infiltration depth (P＜0.05) and lymph node metastasis(P＜0.05). MVD was closely related to tumor infiltration depth (P＜0.05), lymph node metastasis(P＜0.05), and clinical stage(P＜0.05).4. The mRNA expression level of HIF-1αwas not associated with VEGF expression or MVD(P＞0.05). Whereas HIF-1αprotein expression was positively correlated with VEGF expression(P＜0.05) and MVD(P＜0.05).5. HIF-1αmRNA expression was obviously knocted down by small interferencing RNA(siRNA). In addition, HIF-1αprotein expression induced by hypoxia in EC9706 cells was inhibited effectively by RNAi (P＜0.05).6. The expression of VEGF on mRNA and protein level was significantly increased under hypoxia condition(P＜0.05). The expression of VEGF on mRNA and protein level under hypoxia was greatly down-regulated by small interferencing RNA(siRNA) targeting HIF-1α(P＜0.05).Partâ…¢: Relationship between HIF-1αand chemotherapy resistance and its mechanism in ESCCMethods1. Platixal in combination with cisplatin was used in 48 patients with ESCC at advanced stage.The chemotherapy response was evaluated.2. HIF-1αprotein in the specimens from the 48 patients was measured by immunohistochemistry, and the relation of HIF-1αand chemotherapy response was analyzed.3. RNA-interference targeting HIF-1αwas used to silence the expression of HIF-1αin human EC9706 cell line.4. Growth inhibition rates of EC9706 cells treated with cisplatian/platixal were measured by MTS colorimetric assay under normoxic or hypoxic condition after RNA interference under hypoxia.5. The expressions of MRP1, Bcl-2 and Bax were measured respectively by RT-PCR, immunocytochemistry in EC9706 cells under normia, hypoxia and after RNA interference under hypoxia.6. The apoptosis rate of EC9706 cells treated by platixal under hypoxia before and after RNAi was studied by TUNEL assay and Annexin V/PI double staining, respectively.8. Cell cycles of EC9706 cells before and after RNAi under hypoxia were analyzed with flow cytometry.10. Statistical analysis: The SPSS Statistical Package was used for all analyses. Association between the variables were tested by analysis of variance and x² test, t test. The standard of significant level wasα=0.05.Results:1. Of 48 specimens 21 (43.75%) had positive expression of HIF-1αprotein. Among 21 cases with positive expression of HIF-1α, there was none complete response case and 5 cases of partial responses, and the response rate was 23.81%; whereas in 27 eases with negative expression of HIF-1α, there were 8 cases of complete responses and 11 cases of partial responses, and the response rate was 70.37%. There was significant difference between two groups (P＜0.05).2. After being treated by cisplatin (10μg/ml, 5μg/ml, 2μg/ml), the growth inhibition rates of EC9706 cells under normoxia were significant higher than those under hypoxia(P＜0.05). After being treated by platixal (100μg/ml, 50μg/ml, 20μg/ml) the growth inhibition rates of EC9706 cells under normoxia were significantly higher than those under hypoxia (P＜0.05) .3. In the untransfected group, transfected with control siRNA group and transfected with HIF-1αsiRNA group treatedby cisplatin (10μg/ml, 5μg/ml, 2μg/ml) under hypoxia, respectively, the growth inhibition rates of EC9706 cells transfected with HIF-1αsiRNA were significantly higher than those in the untransfected group and transfected with control siRNA group (P＜0.05). In the untransfected group, transfected with control siRNA group and transfected with HIF-1αsiRNA group treated with platixal (100μg/ml, 50μg/ml, 20μ/ml) under hypoxia respectively, the growth inhibition rates of cells transfected with HIF-1αsiRNA were significantly higher than those in the untransfected group and transfected with control siRNA group (P＜0.05).4. Expression of MRP1 on mRNA and protein level was significantly increased in EC9706 cells after hypoxia (P＜0.05). MRP1 expression on mRNA and protein level in cells transfected with HIF-1αsiRNA was significantly lower than those in untransfected group and transfected with control siRNA group under hypoxia (P<0.05).5. TUNEL detection revealed that after being treated with platixal (50μg/ml)under hypoxia, the apoptosis rate in HIF-1αsiRNA group(38.63%) was significantly higher than that in untransfected group (12.54%), and the transfected with control siRNA group (13.69%) (P＜0.05).6. Flow cytometer(Annexin V FITC/PI) revealed that after being treated with platixal (50μg/ml) under hypoxia, the apoptosis rate in the transfected with HIF-1αsiRNA group (36.57±3.36)% was significantly higher than that in untransfected group (13.54±2.61)%, and the transfected with control siRNA group (15.61±2.42)% (P＜0.05).7. There was not significant difference of Bcl-2 expression on mRNA and protein level between normoxia group and hypoxia group (P＞0.05), as well as between the transfected with HIF-1αsiRNA group, the transfected with control siRNA group and the untransfected group (P＜0.05) .8. The expression of Bax on mRNA and protein level was significantly decreased in EC9706 cells after hypoxia(P＜0.05). Bax expression on mRNA and protein level in cells transfected with HIF-1αsiRNA was significantly higher than those in untransfected group and transfected with control siRNA group under hypoxia(P＜0.05).9. The results of flow cytometry showed that the cells in G₁-phase, S-phase were (69.05±1.30)% and (23.72±1.30)%, respectively during normoxia. The cells in G₁-phase, S-phase was (78.29±2.70)% and (11.94±2.70)%, respectively during hypoxia. An increase of G₁-phase and a decrease of S-phase was observed in EC9706 cells in response to hypoxia(P＜0.05); the cells in G₁-phase, S-phase of cells in HIF-1αsiRNA transfection group was (70.48±1.91)% and (21.86±1.90)%, respectively after hypoxia for 8h, whereas the cells in G₁-phase, S-phase of cells in transfected with control siRNA group was (77.86±2.31)% and (12.07±1.90)% respectively, under the same hypoxia. Compared with untransfected group and transfected with control siRNA group, the cells in G₁-phase decreased and the cells in S-phase increased significantly(P＜0.05).Conclusions1. The mRNA expression of HIF-1αin ESCC tissue is Significantly higher than that in corresponding normal mucosa. The level of HIF-1αmRNA expression is not correlated with clinicopathological features, VEGF expression or MVD.2. There is positive correlation between the level of HIF-1αmRNA expression and the positive rate of HIF-1αprotein expression. HIF-1αprotein expression in ESCC tissue is correlated with lymph node metastasis and invasion depth. HIF-1αprotein expression is correlated with VEGF expression and MVD, which indicats that HIF-1αprotein expression in ESCC tissue is correlated with angiogenesis.3. The expression of HIF-1αprotein was associated with chemotherapy response. The patients with positive expression of HIF-1αprotein have lower chemotherapeutic efficacy, suggesting that HIF-1αprotein can be used as a new indicator to predict chemotherapy response.4. Chemical hypoxia inducer (Cobalt chloride, COCl₂) establishs a hypoxia model in vitro to mimic hypoxia environment in tumor, and overexpression of HIF-1αprotein in EC9706 cells can be induced, while the expression of HIF-1αmRNA does not change after hypoxia. It indicates that hypoxia mainly affects the expression HIF-1αon protein level. Therefore, it is more important pratice to measure HIF-1αexpression on protein in the treatment of ESCC.5. RNA interference targeting HIF-1αmakes a specificity of HIF-1αsilencing. 6. The overexpression of VEGF regulated by HIF-1αcould be attenuated markedly by HIF-1αsiRNA treatment. Therefore, to silence the expression of HIF-1αmay inhibit the angiogenesis, which offers an avenue of anti-angiogenesis gene therapy for ESCC.7. EC9706 cells under hypoxia is resistant to cisplatin and taxol, the two different chemotherapeutic drugs. Whereas RNAi silencing HIF-1αgene can reverse the resistance to the two drugs.8. HIF-1αcan upregulate the expression of MRP1, which may involve in the chemotherapeutic resistance of EC9706 cells under hypoxia.9. Hypoxia-mediated down-regulation of Bax in EC9706 cells occurs via HIF-1α-dependent mechanism and contributes to chemoresistance.10. HIF-1αis involved in the cell cycle arrest in EC9706 cells in response to hypoxia which may be another therapeutic resistance mechanism under hypoxia.11. The overexpression of HIF-1αinduces angiogenesis and resistance to varied chemotherapeutic drugs. Therefore, HIF-1αcan be used as a novel, tumor-specific target for anticancer therapy in ESCC. This would offer one new way and a theoretic foundation to target therapy for ESCC.Original points:1. The expression of HIF-1αon mRNA and protein level in ESCC was systematically studied. The results indicated that HIF-1αprotein expression can be regarded as a new index to predict chemotherapeutic response in ESCC.2. RNAi and chemical mimic hypoxia were used to further explore the expression of VEGF on mRNA and protein level. The results indicated that HIF-1αprotein was involved in angiogenesis through upregulating VEGF expression.3. The complex mechanism of cell resistance to chemotherapeutic drugs under hypoxia in ESCC was explored systematically. The results indicated that HIF-1αprotein was involved in chemoresistance under hypoxia by by inducing cell cycle arrest in G₁ phrase and up-regulation the expression of MRP1 and down-regulation the expression of Bax which play a role in apoptosis.