title: Effects of PI3K inhibitor NVP-BKM120 on acquired resistance to gefitinib of human lung adenocarcinoma H1975 cells.
abstract: The effects of class I PI3K inhibitor NVP-BKM120 on cell proliferation, cell cycle distribution, cellular apoptosis, phosphorylation of several proteins of the PI3K/AKT signaling pathway and the mRNA expression levels of HIF1-α, VEGF and MMP9 in the acquired gefitinib resistant cell line H1975 were investigated, and whether NVP-BKM120 can overcome the acquired resistance caused by the EGFR T790M mutation and the underlying mechanism were explored. MTT assay was performed to detect the effect of gefitinib, NVP-BKM120, NVP-BKM120 plus 1 μmol/L gefitinib on growth of H1975 cells. The distribution of cell cycle and apoptosis rate of H1975 cells were examined by using flow cytometry. The mRNA expression levels of tumor-related genes such as HIF1-α, VEGF and MMP9 were detected by using real-time quantitative PCR. Western blotting was used to detect the expression level of phosphorylated proteins in the PI3K/AKT signaling pathway, such as Ser473-p-AKT, Ser235/236-p-S6 and Thr70-p-4E-BP1, as well as total AKT, S6 and 4E-BP1. The results showed that the NVP-BKM120 could inhibit the growth of H1975 cells in a concentration-dependent manner, and H1975 cells were more sensitive to NVP-BKM120 than gefitinib (IC50:1.385 vs. 15.09 μmol/L respectively), whereas combination of NVP-BKM120 and gefitinib (1 μmol/L) did not show more obvious effect than NVP-BKM120 used alone on inhibition of cell growth (P>0.05). NVP-BKM120 (1 μmol/L) increased the proportion of H1975 cells in G0-G1 phase and the effect was concentration-dependent, and 2 μmol/L NVP-BKM120 promoted apoptosis of H1975 cells. There was no significant difference in the proportion of H1975 cells in G0-G1 phase and apoptosis rate between NVP-BKM120-treated alone group and NVP-BKM120 plus genfitinib (1 μmol/L)-treated group or between DMSO-treated control group and gefitinib (1 μmol/L)-treated alone group (P>0.05 for all). It was also found that the mRNA expression levels of these genes were down-regulated by NVP-BKM120 (1 μmol/L), and NVP-BKM120 (1 μmol/L) or NVP-BKM120 (1 μmol/L) plus gefitinib (1 μmol/L) obviously inhibited the activation of Akt, S6 and 4E-BP1 as compared with control group, but single use of gefitinib (1 μmol/L) exerted no significant effect. These data suggested that NVP-BKM120 can overcome gefitinib resistance in H1975 cells, and the combination of NVP-BKM120 and gefitinib did not have additive or synergistic effects. It was also concluded that NVP-BKM120 could overcome the acquired resistance to gefitinib by down-regulating the phosphorylated protein in PI3K/AKT signal pathways in H1975 cells, but it could not enhance the sensitivity of H1975 cells to gefitinib.
authors: Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors: Yi-Chen
authors: YC
authors: Liang
authors: Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors: Hong-Ge
authors: HG
authors: Wu
authors: Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors: Hong-Jian
authors: HJ
authors: Xue
authors: Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors: Qing
authors: Q
authors: Liu
authors: Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors: Liang-Liang
authors: LL
authors: Shi
authors: Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors: Tao
authors: T
authors: Liu
authors: Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China. xhzlwg@163.com.
authors: Gang
authors: G
authors: Wu
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism; D000631:Aminopyridines / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D000077156:Gefitinib; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D008175:Lung Neoplasms / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011799:Quinazolines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
chemical_list: D000631:Aminopyridines; D000970:Antineoplastic Agents; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009025:Morpholines; C571178:NVP-BKM120; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011799:Quinazolines; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; C579270:MMP9 protein, human; D020780:Matrix Metalloproteinase 9; D000077156:Gefitinib