| BackgroundLung cancer(LC) is a form of malignancies which threatens the human life and health,and its mortality is among the top of the malignant tumor in worldwide.Meanwhile its morbidity and mortality rate tends to increase gradually annually.Non-small cell lung cancer (NSCLC) is in the majority, which accounts for 80% to 85%.The treatments are comprehensive therapies which include surgery,radiotherapy,chemotherapy and molecular targed therapy.The etiology of NSCLC is still not definitive and its pathogenesis is the result of multiple factors.Previous research has shown that the pathogenesis of NSCLC is related to environmental factors such as air pollution,smoking and genetic factors.In recent years,the scholars are discussing the development mechanism of NSCLC from different perspective.The cancer cells of NSCLC are characterized by enhanced proliferation than normal cells,and this characteristic is found to correlate with glucose uptake and glycolysis.Glucose uptake and glycolysis result in enhanced cell invasion,metastasis,malignant transformation and resistance to radiotherapy and chemotherapy,so that the conventional tumor treatment is difficult to achieve the ideal effect.Under normal physiological conditions,Glucose transport protein(GLUT),that is a kind of embedded in the membrane transport carrier protein of glucose,can efficiently transport glucose.Glucose transport protein 1 (GLUT1) is the glucose transporter protein,most widely distributed in the body,which is the main limit of cell metabolism of sugar,excessive expressed in almost all the tumor tissue.Excessive expression of GLUT1 is responsible for the high efficient uptake and utilization of glucose in ischemia and hypoxia tumor cells,by gaining more energy supply to maintain rapid growth of tumor cells.A recent study showed that overexpression of GLUT1,one of the key factors involved in the regulation of aerobic glycolysis,is closely related to aggressive and vigorous growth of NSCLC accompanied by increasing glucose metabolism.Curcumin is a kind of plant polyphenols,which extract from the roots of curcuma,such as turmeric?curcuma aromatica?curcuma zedoary. It has multiple biological effects such as antibacterial,antiviral,antioxidant and anti-inflammatory.Adams et al found more than one hundred kinds of curcumin derivatives on the basis of the molecular structure of curcumin,most of them have the mechanism of antitumor. Among them the effect of Diphenyl difluoroketone(EF-24) is most significant.Study found that EF-24 has obvious inhibitory effect in most malignancy cell lines,such as gastric cancer,colon cancer,liver cancer,prostate cancer,breast cancer and ovarian cancer et al,and the effect is at least more than 10 times of curcumin.EF-24 can directly inhibit the transcriptional activity of HIF-1 alpha in the treatment of prostate cancer and breast cancer, and increase sugar transporters and sugar glycolysis enzyme genes regulated by HIF-1 alpha with the purpose of using glycolytic pathway during hypoxia. And EF-24 can inhibit the proliferation and invasive ability mediated by GLUT1 in ovarian cancer cell.In view of this, we adopted the GLUT1 as the starting point, used Western-blotting and RT-PCR to detect the effect of EF-24 on GLUT1 and gene in NSCLC cells; observed the effect of different concentration of EF-24 on cell proliferation in non-small cell lung cancer; observed the impact of EF-24 on invasion and metastasis of NCLC cells in vitro and vivo. Through the observation of EF-24 impacting the proliferation and metastasis activity and glycolysis of NSCLC cell at the cellular and organismic level, we could identify the relation between EF-24 and NSCLC cell proliferation and metastasis in order to provide a new drug target for the prevention and treatment of lung cancer.Objective:l.To identify the expression of GLUT1 in transcription and translation level from tumor and surrounding normal tissue and normal lung tissue of NSCLC patients.2.Taking GLUT1 as the breakthrough point and using different concentrations of EF-24 intervene A549 cell in order to identify the impact of EF-24 to cell proliferation activity of A549 by MMT.3.Exploring the relation between EF-24 and invasion and metastasis of NSCLC, and then GLUT1 may be a new drug target in lung cancer therapy.Methods:1. Specimens such as tumor and surrounding normal tissue from patients with newly diagnosed NSCLCs who underwent surgery were collected. This study was approved by the Ethics Committee of the Second Hospital of Shandong University.2.The expression of GLUT1 was evaluated by western blot assay and immunohistochemistry,meanwhile, the relationship between the expression of GLUT1 and clinical manifestation and pathological grading was also analysed by these results.3.Different concentrations of EF-24 were used to intervent cell line from NSCLC, and expression of GLUT1 in transcription and translation level was evaluated by RT-PCR, Western blot, immunofluorescence and so on. Cell cycle analysis, cell migration assay, and tumor formation in vivo, and MTT were combined in order to evaluate this phenomenon furthermore.4.The impact of GLUT1 to growth and proliferation capability of A549 cell line could be realized by the way of cell transfection, cell count and cell proliferation assay in vivo and vitro.5.Statistical analysis:Experimental data was performed using SPSS 16.0 software and presented as means ± SEM.A value of P<0.05 was considered statistically significant. The difference between two groups was identified by t-test, variance analysis and Tukey test.Results:1.In the lung tissue specimens from NSCLC patients, by RT-PCR and Western blotting, we found that comparing to normal lung tissue and paracancerous tissues, the levels of transcription and translation of GULT1 in lung tumor tissues were up-regulated obviously, and it had a positive correlation to clinical pathological grading.2.In the present study, A549 cell lines were transfected by GLUT1 siRNA and shRNA in order to cause gene silencing, and it confirmed that GLUT1 gene silencing can reduce growth and proliferation of A549 cells. In addition, A549 cells which were transfected with GLUT1 shRNA were injected into nude mice. Comparing to the control group, the tumor volume was significantly reduced, which further confirmed that GLUT1 gene silencing can significantly inhibit the growth of lung cancer cells. Therefore, we speculated that the growth and ability of tumorigenicity of A549 were inhibited by silence GLUT1 and vice versa.3.Different concentrations of EF-24 were used to intervene A549 cells, and EF-24 from A549 cells could significantly reduce the expression of GLUT1. This decline was gradient -dependent, which was in proportion to the concentration of EF-24.4.The A549 cells were treated by different concentrations of EF-24, absorption of glucose and the rate of glycolysis in A549 cells were tested, migration and invasion of Transwell cells were observed and cell proliferation was measured, we found that EF-24 could inhibit glycolytic pathway, invasion and migration of A549 cell.Conclusion:1.In this experiment, the expression of GLUT 1 in NSCLC tissue was significantly higher than that in surrounding and normal tissues.2.The research proved that EF-24 could inhibit the expression of GLUT1 in A549 cell line, and then it may inhibit the growth of NSCLC by impacting glycolysis, migration and invasion.3.As one kind of curcumin analogues, EF-24, may be a new choice of medication in lung cancer. |
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