The Expression And Clinical Significance Of Hypoxia Induced Factor 1α And Glucose Transporters 1 In Lung Cancer Tissues

Objective: Rapid proliferation is one of the important features of malignant tumors. During the development of tumors, the excessive proliferation of tumor cells results in constant and relative hypoxia in tumor microenvironment. Hypoxia can trigger tumors producing a series of protective reaction in the stress lest tumor cells be injured or killed in hypoxia environment. Some experimental studies indicated that hypoxia cells existed in almost all kinds of solid tumors, and their numbers was associated with histological phenotype and growth rate of tumor and generally the number of hypoxia cells increased as the tumor size increase. Recent studies have revealed that there might be hypoxia induced factor1(HIF1) in tumor cells, which plays a central role in hypoxia environment for tumor survival and a regulatory role for hemeoxygenase, glucose transporters, glycolytic enzymes, angiogenesis factor, EPO and P53, etc. HIF1 could induce agiogenesis in tumor and affect cell metabolism to meet rapid proliferation of tumor cells. HIF1 is a heterodimers and is consisted of HIF1αand HIF1β. Studies showed that HIF1αdepends predominantly on HIF1βin HIF1 family. With the decrease of oxygen concentration around it, the expression of HIF1αmay increase, and furthermore, the increase of its expression shows in multiple levels, both in transcription and protein level, mainly in the latter. HIF1βsubunit is necessary in HIF1 in spite of it being less dependent on hypoxia because both of them displayed regulatory function as long as they were combined with the hypoxic responsive element of downstream factor or enzyme regulated by themselves through getting together and making adaptive deformation. Zhong et al detected the expression of HIF1αin several different tumors with HIF1αmonoclonal antibody method and found over-expression in 13 different tumors, such as colorectal cancer, mammary cancer, carcinoma of kidney, lung cancer, skin carcinoma , ophoro-cancer and carcinoma of prostate, etc. The expression of HF1 is increased in 90% of mammary cancer, lung cancer and prostate cancer. However, it was not detected in the normal tissues adjacent to tumors. The increased expression of HIF1αwas found in less than 1/3 of primary tumor and more than two thirds of remote metastastic foci in mammary cancer. At the same time, increased expression of HIF1αcould also be found in the earlier malignant lesions, such as intraductal carcinoma in situ, suggestting that HIF1αmight be used as a putative reference indicator for the early diagnosis of malignant tumors. Glucose transporters (GLUT s) are the main transporters of glucose transmembrane transportation in mammal cells. Compared with the normal cells, the uptake and metabolism of glucose in malignant cells are all significantly increased. Accordingly, glucose transporter, in charge of glucose uptake, was highly expressed in malignancy tissues. Up to now, 13 members were found in GLUT s family. GLUT 1 was much closely related to glucose transportation in tumor cells. Many studies showed the highest positive expression rate of GLUT 1 was seen in malignant tumors followed by dysplasia or borderline tumors and the positive expression rate in normal tissues or benign tumors was lower. The investigation of GLUT1 expression is clinically of great significance in early diagnosis, treatment and the identification of behavior of malignant tumors. Immunocytochemical study of exudates smear with polyclonal GLUT1 antibody in 31 malignant ovarian neoplastic patients and 25 individuals with benign diseases indicated that 29 cases in the malignant tumor patients were positive for GLUT1(93.5%), while that in the non-tumor patients was only 20%(20 cases). Weak positive staining of mesothelial cells could be seen in 5 cases. Among the 5 cases, 3 came from ascitic fluid of liver cirrhosis. The results showed that the expression of GLUT1 was of differential value in the diagnosis of the malignant tumors.There have been several separate studies on significance of HIF1αand GLUT 1 on the carcinogenesis and progression of malignant tumors. Few works have been done on the possible relationship between HIF1αand Glut1 in malignant tumor and no work has been seen on the putative effects of the expression of HIF1αand GLUT 1 on drug resistance in malignant tumor. Chemotherapy is one of the main treatments for malignant tumor. Multidrug-resistance is the great headache in the chemotherapeutic practice. The phenotype of MDR can be divided into natural resistance and acquired induced resistance. The mechanism already known for the drug resistance involves the increase of P-Glycoprotein, multi-drug resistance correlating protein(MRP) and lung relating protein, decreased activity of topoisomerase II (Topo-II), the raise of Glutathione-s-transerase(GST-Ï€)and prototype Glutathione(GSH ) as well as the changes in the biochemical features of tumor, membrane ionic channel and protein kinase (PKC), and so on. But the exact mechanism of the drug resistance is unknown. Lung cancer is one of malignancies seriously threatening peoples health. Up to 60%-80% of patients with lung cancer were in the late stage when they were clinically diagnosed. Comprehensive treatment including radiotherapy and chemotherapy is needed. Accordingly, the study of biological characters of lung cancer is very important for formulating individual therapy protocol. To explore significance of HIF1αand GLUT1 expression in lung cancer, the effects of HIF1 αand GLUT1 expression on multi-drug resistance and study the possible relationship between the expression of HIF1αand that of GLUT1, we analyzed the expression of HIF1α, GLUT 1, MRP1, GST-Ï€at protein level by immunohistochemical method in 42 cases of lung cancer as well as esophageal cancer, breast cancer, and colon cancer tissues. The expression of HIF1αand GLUT1 at mRNA level was further studied with RT-PCR in lung cancer and tissue adjacent to cancerand with timely fluorescence quantitative PCR in experimental lung adenocarcinoma in mice. At the same time, the correlation between the expression of MRP1, GST-Ï€and that of HIF1αand Glut1 was analyzed. Methods:1.Expression of HIF-1α, GLUT1 in lung cancer was studied in paraffin embedded specimens from the Department of Pathology, Second Affiliated Hospital , Hebei Medical University and the People's Hspital of Hebei Province from January, 2003 to March, 2004. Among the 42 cases of lung cancer studied, 28 were males and 14 females, aged from 41 to 72 years old (with an average of 54.7). Pathologically, 2 cases were small cell carcinoma, 21 cases squamous carcinoma, 16 cases adenocarcinoma, and 3 cases alveolar carcinoma. Among the 39 cases of esophageal carcinoma studied, 30 were males and 9 females, with an average of 60.6 in age. In addition, the cases studied also included 39 breast cancer cases (all female, 49.8 years old in mean), 36 cases of colon carcinoma (22 males, 17 females, averaging 63.6 years old). All these specimens used were from the patients without any chemotherapeutic treatment. The pathological significance of the expression of GLUT1, GST-Ï€, MRP1 was analyzed. The immunohistochemical results for HIF-1α,GLUT1,GST-Ï€and MRP1 protein were reviewed by 3 specialists and the immunoreactivity evaluated as follows: -,no staining; +,nuclear staining in less than 1% of cells;++, nuclear staining in 1—10% of cells and/or with weak cytoplasmic staining;+++, nuclear staining in 10-50% of cells and/or with distinct cytoplasmic staining;++++, nuclear staining in more than 50% of cells and/or with distinct cytoplasmic staining. 2. Expression of HIF-1αmRNA, GLUT1 mRNA, MRP1 mRNA and GST-Ï€mRNA and their relationship in lung cancer Thirty six fresh specimen of lung cancer tissue and 12 specimen of tissue adjacent to cancer from hospitals affiliated to Hebei Medical University were collected. All these cases were without any chemotherapeutic treatment. Among the 36 cases of lung cancer studied, 32were males and 14 females, aging from 41 to 72 years old, with an average of 60.6 years old. Pathologically, 16 cases were squamous carcinoma and 20 cases adenocarcinoma. We used TRIZOL Reagent to extract RNA from the tissues and used the RT-PCR Kit to carry out the RT-PCR procedures. The products of the amplification were took for the agarose gel eletrophoresis and the results of the caraphoresis were photographed and analyzed by the computer. The gray degree (IOD) of the eletrophoresis straps were recorded to caculate the odds with that of the GAPDH. Compared the expression of HIF-1α, GLUT1, MRP1, and GST-Ï€in different types of the lung cancer, such as that with different degrees of invasion, that with or not with lymphatic metastasis and remote metastasis. Also we studied their correlation with the expressions of HIF-1α, GLUT1, MRP1, and GST-Ï€. So we can explore the mechanism in the carcinogenesis and the progression of the lung cancer and in the multidrug resistance and the relationship of those four with the biological properties of lung cancer. We detected HIF-1αmRNA and GLUT1mRNA gene expression in the cancer tissues of experimental lung adenocarcinoma in mouse by the method fluorescence quantitative analysis. The experimental lung adenocarcinoma model was established in T 793 genetically pure mouse with LA795 lung adenocarcinoma cell inoculation. The experimental mice were killed 25 days after the experiment. Tumors were token out for morphology and quantitative PCR analysis, and metastatic tumor counting in dissecting microscope. Low metastasis groups were defined as tumor nodes ≤20. Middle metastasis groups were defined as tumor nodes 20-100 while high metastasis groups were defined as tumor nodes exceeding 100. The expression of HIF-1αmRNA and GLUT1mRNA was studied by real time PCR techniques of quantitative fluorescence. We used TRIZOL reagent to extract RNA from the tissues. Integrity of RNA was evaluated by formaldehyde degeneration caraphoresis and OD was determined at260nm and 280nm. RT-PCR was carried out by means of better integrity and 1.8～2.0 OD of RNA. The primer, probe, plasmid used were from Shanghai Biological Engineering Corporation. Recombinant plasmid was diluted to 10⁶,10⁵,10⁴,10³,and 102 copies/μl. The recombinant plasmid was added to fluorescent PCR amplifying system, which was regarded as standard specimen, and it was amplified with cDNA of specimens together under setting negative control. ABI Prism? 7000 software analysis system can automatically draw a standard curve after putting in initial copy number of standard specimens in each tube and setting fluorescent threshod.. Initial copy number of each specimen was obtained from the standard curve in accordance with amplification tube circular domain of unmeasured samples. Result:1. The expression of HIF-1α,GLUT1, MRP1, GST-Ï€in lung cancer as well as some other cancers . The positive expression rate of HIF-1α, GLUT1, MRP1 and GST-Ï€in lung cancer was 83.3% (35/42), 85.7% (36/42), 57.1% (24/42) and 54.8% (23/42) respectively. The positive expression rate of HIF-1α, GLUT1, MRP1 , GST-Ï€in esophageal cancer, colon cancer and breast cancer was 84.6%, 82.1%, 56.4%, 74.4%; 77.8%, 72.2%, 61.1%, 75.0% and 58.9%, 66.7%, 51.3%, 53.8% respectively. Compared with the expression of HIF-1a and GLUT1 in lung cancer tissues of T₁ and T₂, there was markedly increased in the T₃ and T₄ (P<0.01), whereas there was no higher expression of MRP1 and GST-Ï€. The expression of HIF-1a and GLUT1 with lymphatic metastasis in lung cancer was higher than those without lymphatic metastasis (P<0.05), whereas there was no higher expression of MRP1, and the expression of GST-Ï€had no obvious change in both group(p>0.05). Significant correlation between the expression of HIF-1α, GLUT1 and GST-Ï€(P<0.01) was seen in lung cancer. And there was correlation with MRP1(p<0.05).The expression of HIF1αand GLUT1 in lungcancer, esophageal cancer and breast cancer was positive correlated. There was no correlation between the expression of HIF-1α, MRP1 and GST-Ï€in esophageal cancer and breast cancer (p>0.05). There was a positive correlation between the expression of HIF-1α,MRP1 and GST-Ï€in colon cancer(P<0.05). 2. The expression of HIF-1αmRNA, GLUT1mRNA, MRP1mRNA, GST-Ï€mRNA in lung cancer and their significance The expression of HIF-1αmRNA, GLUT1mRNA, MRP1mRNA and GST-Ï€mRNA was significantly higher in lung cancer (36 cases) than that of the tissues adjacent to cancer (P<0.01).The expressions of HIF-1αmRNA in cases of stage T₁, T₂, T₃ and T₄ were 0.3849, 0.4942, 0.6747 and 0.8516, respectively. The analysis of variance indicated that the expression of HIF-1αmRNA in T₃ and T₄ was higher(p<0.01 or <0.05); There was no difference between T₁ and T₂ (p>0.05); The expression of HIF-1αmRNA in cases with lymphatic metastasis in N₁ and N₂ was higher than that without lymphatic metastasis in N₀(p<0.05). The expressions of GLUT1mRNA in cases of stage T₁, T₂, T₃ and T-4 were 0.9491, 1.3034, 1.9281 and 2.2583 respectively. The analysis of variance indicated that the expression of GLUT₁mRNA in T₃ and T₄ is higher(p<0.01 or <0.05); There was no difference between T₁ and T₂ (p>0.05); There was no difference between T₃ and T₄ (p>0.05);The expression of GLUT1mRNA with lymphatic metastasis in N₁ and N₂ was higher than that without lymphatic metastasis in N₀(p<0.05). The expressions of MRP1mRNA with in cases of stage T₁, T₂, T₃ and T₄ were 0.5918, 0.6177, 0.8207 and 0.8103, respectively. The analysis of variance indicated that there was no difference among them( p>0.05); And they went up with the decrease of TNM, but this tide had not been decided by statistic analysis ; The expressions of MRP1mRNA in N₀ and N_1-2 were 06591,0.8496.The expression of MRP1mRNA with lymphatic metastasis in N₁ and N-2 was higher than that without lymphatic metastasis in N₀(p<0.01).The expressions of GST-Ï€mRNA with in cases of stage T₁, T₂, T₃ and T)4 were 1.0851,1.2526,1.3059 and 1.3195, respectively. The analysis of variance indicated that there was no difference among them( p>0.05); And they went up with the decrease of TNM, but this tide had not been decided by statistic analysis ; The expressions of GST-Ï€mRNA in N0 and N_1-2 were 1.0571,1.03874.The expression of GST-Ï€mRNA with lymphatic metastasis in N1 and N₂ was higher than that without lymphatic metastasis in N0(p<0.01). The expression of HIF-1αmRNA and GLUT1mRNA increased as the increase of TNM in lung cancer. Though the expression of MRP1mRNA and GST-Ï€mRNA increased in as TNM stage, no statistical significance could seen. The expression in groups with lymphatic metastasis was higher than that without lymphatic metastasis. Positive correlation between the expression of HIF-1αmRNA and GLUT1 mRNA was found in the lung cancer cases (r=0.60,P<0.01). And the expression of MRP1mRNA, GST-Ï€mRNA and HIF-1αmRNA was also positively correlated (r₁=0.4043,r₂=0.3958, P<0.05). 3. The quantitative analysis of expression of GLUT1mRNA and HIF-1αmRNA in experimental lung adenocarcinoma There was significant correlation between the expression of GLUT1 mRNA and HIF-1αmRNA and metastasis in experimental LA795 lung adenocarcinoma in mice. Among the 35 mice bearing T 739, 2 mice died while the others survived the experiment days. The metastasis node numbers were 194 at most, 0 at least. In the high metastasis group ( 11 mice), the mean lung metastasis node number was 138.45±18.16. In the middle metastasis group(14 mice), the mean lung metastasis node number was 59.14±7.53. In the low metastasis group( 8 mice), the mean lung metastasis nod number was 11.50±2.90. The results of real-time quantitative PCR indicated that the expression of GLUT1 mRNA and HIF-1αmRNA in experiment groups was higher than the control group(P<0.01or P <0.05).The expression of GLUT1mRNA in high metastasis groups was higher than that in middle and low metastasis groups (P<0.01 or <0.05 ). And that in middle metastasis group was higher than in low metastasis group (P<0.05). The expression of HIF-1αmRNA with high metastasis was more than that in middle and low metastasis groups (P<0.01 or <0.05 ). And there was no obvious difference between middle and low metastasis groups. There was positive correlation between the expression of GLUT1mRNA and HIF-1αmRNA and metastasis node numbers in lung cancer (r1=0.57,r2=0.38), and there is a positive correlation between the expression of HIF-1αmRNA and GLUT1mRNA(r=0.56). Conclusion: 1.High expression of HIF1αand GLUT1 at protein level could be seen in human lung cancer. The positive expression rates of HIF1 αand GLUT1 were different in cases with different clinical pathological features. Compared with the expression of HIF-1a and Glut1 in T1 and T2, there was markedly increase in the T3 and T4 (P<0.05) cases and the expression of MRP1 and GST-Ï€was also higher in the latter (P<0.05). The expression of HIF-1a and Glut1 in cases with lymphatic metastasis in lung cancer was higher than those without lymphatic metastasis (P<0.05).The expression of HIF-1α, GLUT1 and GST-Ï€was positively correlated in lung cancer (P<0.01). The expression of HIF1αand GLUT1 could also be seen in esophageal cancer , colon cancer and breast cancer and the expression of HIF1αand GLUT1 was positively correlated. There was no correlation between the expression of HIF-1α, MRP1 and GST-Ï€in esophageal cancer and breast cancer. There was a positive correlation between the expression of HIF-1α, MRP1 and GST-Ï€in colon cancer. 2. The expression of HIF-1αmRNA, GLUT1mRNA, MRP1mRNA and GST-Ï€mRNA in lung cancer was significantly higher than that in the tissues adjacent to cancer. The expression of HIF-1αmRNA and GLUT1 mRNA increased with the increase of TNM. Though the expression of MRP1 mRNA and GST-Ï€mRNA increased in as TNM stage increase, no...