The Biological Activity Of Egf To Stimulate Lung Cells To Secrete Il-8 And Affects Lung Cancer Cells And Its Regulation Mechanism

ObjectiveThis study aimed to address epidermal growth factor (EGF) induced secretion of IL-8 in non-small cell lung cancer (NSCLC) cell line SPC-A1 and its potential molecular mechanism.Materials and MethodsSPC-A1 cells were cultured in 24 well plate and then treated with EGF at concentrations of 0.01,0.1 and 1μg/ml respectively for 24h. Concentrations of interleukin 8(IL-8) in supernatant were measured by the enzyme-linked immunosorbent assay (ELISA). After then, SPC-A1 cells were treated with EGF (0.1μg/ml) alone or in combination with several inhibitors including EGFR-Tki (Erotinibat 1 and 10μM), phosphoinositide-3-kinas (PI3K) inhibitor (GDC-0941 at 0.1 and 1μM) and ERK1/2 inhibitor (PD98059 at 1 and 10μM) for 24h. Finally, SPC-A1 cells were treated by EGF (0.1μg/ml) and in combination with new PI3K inhibitor SHBM1009 (10,6,3,1μM) for 48 hours, then the concentrations of IL-8 were measured over 12h,24h and 48h. All the experiments included an untreated control group.ResultsEGF at all concentrations stimulated a significant increase of IL-8 production from SPC-A1 cells in a concentration-associated pattern, as compared with cells without EGF stimulation over 24 hours. The concentration of IL-8 in the control group was 43.07±12.26pg/ml, and the concentrations of IL-8 in EGF (0.01μg/ml,0.1μg/ml and 1μg/ml) timulated groups were 103.68±1/36,216.91±8.07 and 232.21±42.44pg/ml respectively (p values were p<0.001, p＜0.001 and p<0.01, respectively). EGFR-Tki (10μM和1μM), GDC-0941, BEZ-235 (1μM) and PD98059 (10μM) inhibited the secretion of IL-8 (p value, p＜0.01or p<0.05, compared to EGF stimulated group) after SPC-A1 cells were treated by EGF (100ng/ml) alone and in combination with inhibitors for 24 hours. The EGF stimulated secretion of IL-8 was inhibited by d ifferent concentrations of SHBM1009 (10,6,3,1μM). ConclusionThis study demonstrated that lung cancer cells could secrete chemokine IL-8. EGF could induce the secretion of IL-8 by SPC-A1 cell lines, which was possibly related to the pathway of EGF/EGFR/PI3K/ERK1/2. ObjectiveThis study aimed to observe the changes of molecular biologic behavior of SPC-A1 cell linces stimulated by EGF alone and in combination with several inhibitors such as EGFR-Tki, GDC-0941, BEZ-235, SHBM1009 and PD98059.Materials and MethodsMTT experiment:SPC-A1 cells were treated by EGF (0.1μg/ml) alone and in combination with five inhibitors including EGFR-Tki, three PI3K inhibitors GDC-0941, BEZ-235, SHBM 1009 and ERK1/2 inhibitor PD98059 (all inhibitors at four concentrations:0.01μM,0.1μM,1μM and 10μM) for 24 hours. The survival cell s were calculated by MTT method.Cell-IQ system:SPC-A1 cells were treated by EGF(0.1μg/ml) alone and in combination with inhibitors for72 hours. Cell bio-behaviors including total cell numbers, cell differentiation and cell movement were observed by real-time cell monitoring system.Western-blot:SPC-A1 cells were treated by EGF (0.1μg/ml) alone and in combination with inhibitors, then the levels of intracellular proteins pAkt, Akt, pERK and ERKwere measured by western-blot.ResultsMTT results showed that During cell culture with EGF at 0.1μg/ml, PD98059 and GDC-0941 only had about 10% inhibitory effects at 10μM (p<0.05), while EGFR-Tki (Erlotinib) about 25% at 10μM (p<0.05, vs Cells without inhibitors). BEZ235 showed about 30% and 40% inhibitory effects at 0.01-1.0 and 10μM, respectively, while SHBM 1009's inhibitory effect was strongest and in a dose-dependent manner, at 0.1-1.0 and 10μM had about 25-35% to 60% inhibitory effects on cell proliferation, significantly vehicle (p<0.01, respectively).In the Cell-IQ real-time monitoring system, EGF changed the cell-bio behaviors of SPC-A1 cells such as promoting cell proliferation, increasing the cell numbers in dividing stage, and accelerating cell movement. The effects of EGF on cell-bio behaviors of SPC-A1 cells inhibited by five inhibitors as described abve. The levels of intracellular proteins pAkt and Perk 1/2 were increased after the SPC-A1 cells were stimulated by EGF, while EGFR-Tki inhibited this effect entirely. PI3K inhibitor could inhibit the increase of pAkt stimulated by EGF, ERK1/2 inhibitor could the increase of pERK1/2 protein induced by EGF.ConclusionEGF could change the cell-bio behaviors of non-small cell lung cancer cells such as promoting cell proliferation, accelerating cell dividing and cell movement. These effects EGF were associated with the pathway of EGFR/PI3K/Akt/ERK1/2.