| Objective: Lung cancer is the highest incidence of malignant tumors in the world, and the traditional Chemoradiotherapy can not overcome the tumor better. In recent years, the epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs) as the representative of more efficient and low toxic targeted therapy,HAve been widely amplicated in lung cancer patients and been obtained more satisfactory results. EGFR is a kind of glycoprotein receptor, having tyrosine kinase activity. Its overexpression can promote the proliferation, angiogenesis, invasion and metastasis of tumor. Erlotinib, known targets located on the tumor cells, is a well-known EGFR-TKI. It is showing strong antitumor activity against, which inhibits downstream signaling pathways, and induces apoptosis so that the tumor growth was inhibited. Clinical findings EGFR-TKIs also can inhibit metastasis trend. So we guess that it could also potentially pathway through inhibition of tumor invasions and metastasis behavior. EGFR-TKIs also can inhibit metastasis trend in clinical treatment. It could also through potentially pathway to inhibit invasions and metastasis behavior in tumor cells.Integrin and CD44 are the important members of the adhesion molecules family in tumor microenvironment. They play an important role in cell and intracellular adhesion. And the high level of the cell adhesion molecules has a very important role in promoting tumor survival, invasions and metastasis process. Current domestic and foreign study confirmed: EGFR with Integrin, CD44 exist cross relationships in head and neck cancer, lung cancer and other tumors. Whether existing relationship of such lung cancer. EGFR-TKIs whether or not through this pathway inhibiting tumor invasions and metastasis is still unknowned.Methods:1 Cell cultureHCC827 cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin 100 U/m L, streptomycin 100 ug/m L at 37℃ in an incubator, containing 5% CO2.2 Cell viability measured by MTT assay in vitroThe exponential growth phase of HCC827, stimulated by HA and FN, were planted into 96-well multiplates and after adhesion the cells were treated with series concentrations of elotinib. The cells were cultured for 24 h. The 50% inhibitory concentration(IC50) was calculated, and growth curve line chart was made.3 The expression levels of CD44+, Integrinβ3+ were detected with flow cytometry after the cells was or was not sitimulated by HA, FN, and were detected t HAt was sitimulated then was added with elotinib.4 The metastatic levels detected with transwell assay after the cells was or was not sitimulated by HA, FN, and were detected that was sitimulated then was added with elotinib.5 The expression levels of EGFR, CD44, Integrinβ3, FAK m RNA were detected with q RT-PCR assays after the cells were or were not sitimulated by HA, FN, and were detected t HAt was sitimulated then were added with elotinib.6 The expression levels of FAK, p FAK, p EGFR, EGFR protein were detected with Western-blot assay after the cells were or were not sitimulated by HA, FN, and were detected that were sitimulated then were added with elotinib.7 Statistical analysesThe results were expressed as Mean ± Standard or Median±Interquartile Range deviation and evaluated with SPSS 21.0 solftware by analysis of variance(One-Way ANOVA) and Kruskal-Wallis H test. P<0.05 was considered statistically significant.Results:1 The cells were treated with different concentrations of elotinib for 24 h, and MTT assay showed elotinib significantly suppressed HCC827 cells proliferation and viability which were : 82.36±6.53%, 68.06±3.76%, 42.30±3.33%, 34.28±4.91%, 29.32±4.64%, and cells proliferation and viability were in a concentration-dependent manner. IC50 values were 0.127 μM for 24 h respectively.2 Compared with the expression levels of CD44+, Integrinβ3+ in cell surface by flow cytometryThe expression levels of CD44+ in cell surface of Group A, Group B, Group C were: 24.48±2.35%, 40.77±3.66%, 20.01±1.43%, Group B compared with Group A and Group C compared with Group B were both significant in HCC827 cells(P<0.05);the expression levels of Integrinβ3+ in in cell surface of Group A, Group B, Group C were : 0.44±0.25%, 0.42±0.17%, 0.26±0.07%, Group B compared with Group A and Group C compared with Group B were neither significant in HCC827 cells(P>0.05).3 Compared with the metastatic level by transwell assayThe metastatic cell numbers of Group A, Group B, Group C were: 7.40±1.50,18.27±1.87,1.87±1.19. Group B compared with Group A and Group C compared with Group B were both significant in HCC827 cells(P<0.05).4 Compared with the expression levels of EGFR, CD44, Integrinβ3, FAK m RNA by q RT-PCR assays.The expression levels of EGFR m RNA of Group A, Group B, Group C were: 1.24±2.2, 8.74±496.06, 0.64±2.4; the expression levels of CD44 m RNA of Group A, Group B, Group C were : 176.71±192.59, 18079.52±12618.91, 312.09±478.78; the expression levels of Integrinβ3 m RNA of Group A, Group B, Group C were : 0.11±8.15, 80.24±336.14, 0.01±0.03;the expression levels of FAK m RNA of Group A, Group B, Group C were : 17.40±55.40, 1094.06±1035.37, 12.67±146.22. Group B compared with Group A and Group C compared with Group B were both significant in HCC827 cells(P<0.05).5 Compared with the expression levels of EGFR, p EGFR,p FAK, FAK m RNA by Western-blot.The expression levels of p FAK protein of Group A, Group B, Group C were: 1.08±0.26, 1.22±0.13, 1.05±0.11; the expression levels of FAK protein of Group A, Group B, Group C were: 0.99±0.11, 0.95±0.42, 0.92±0.07; the expression levels of p EGFR protein of Group A, Group B, Group C were: 0.96±0.08, 1.36±0.11, 0.37±0.05; the expression levels of EGFR protein of Group A, Group B, Group C were : 0.93±0.06, 0.98±0.05, 0.94±0.07; Group B compared with Group A and Group C compared with Group B were both significant in HCC827 cells(P<0.05).Conclusion:1 Elotinib significantly suppressed HCC827 cells proliferation and viability and which was in a concentration-dependent manner.2 HA and FN could stimulate the expression level of CD44+, Integrinβ3+, futher promote the metastatic level of HCC827 cells. Elotinib could suppress the CAMs, reduced the metastatic level.3 HA and FN could stimulate the expression of CD44+, Integrinβ3+ in m RNA and protein, simultaneously, they could activate FAK, thus activate EGFR from the reverse. Elotinib could suppress the expression level of EGFR, CD44, Integrinβ3, FAK, futhur decrease the metastatic level. And FAK is a key factor in cross-signal transduction with EGFR, CD44, Integrinβ3. |
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