1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)-3-(2-(dimethylamino)ethyl)imidazolidin-2-one (ZX-42), a novel ALK inhibitor, induces apoptosis and protective autophagy in H2228 cells.

OBJECTIVES: To examine the antiproliferative effects of 1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)-3-(2-(dimethylamino)ethyl)imidazolidin-2-one (ZX-42) on the echinoderm microtubule-associated protein-4/anaplastic lymphoma kinase fusion gene (EML4-ALK) positive lung cancer cell line H2228 and its underlying mechanism. METHODS: The MTT assay was used to study the effect of ZX-42 on H2228 cell growth. Propidium iodide (PI) staining and Western blotting were used to investigate the cell cycle changes. ZX-42-induced cell apoptosis was determined using the Annexin V-FITC/PI (AV/PI) apoptotic assay kit, acridine orange/ethidium bromide (AO/EB) and Hoechst 33258 staining, Rhodamine 123 (Rh 123) fluorescence assay and Western blotting. ZX-42-induced reactive oxygen species (ROS) production was examined by ROS assay kit. Transmission electron microscope, monodansylcadaverine (MDC) staining and the AV/PI apoptotic assay kit were used to demonstrate the relationship between autophagy and apoptosis. KEY FINDINGS: ZX-42 had good cell viability inhibitory effect on H2228 cells. ZX-42 dramatically inhibited ALK and its downstream pathways. ZX-42 also blocked H2228 cell cycle at G1 phase and then induced apoptosis by activating the mitochondrial pathway. Next, ZX-42 induced the production of ROS, and antioxidant N-acetylcysteine (NAC) reduced ROS production and also decreased apoptotic rates. We also found that ZX-42 induced protective autophagy in H2228 cells. CONCLUSIONS: In summary, ZX-42 is a novel ALK inhibitor that significantly inhibits the cell viability of H2228 cells and ultimately induces apoptosis through the mitochondrial pathway, in which autophagy plays a protective role. Therefore, inhibition of autophagy might enhance the anti-cancer effect of ZX-42.

Role of HGF/c-Met signaling pathway in crizotinib-induced apoptosis of different lung carcinoma cell lines / 中国病理生理杂志

AIM: To investigate the role of HGF/c-Met signaling pathway in crizotinib-induced apoptosis of different lung carcinoma cell lines and to analyze its potential regulatory mechanisms .METHODS: EML4-ALK positive cell line H2228, c-Met proliferation cell line H1993 and control cell line A549 were treated with crizotinib at different doses for different time periods .The viability of the cell lines was measured by MTT assay .The apoptosis was analyzed by flow cytometry with PI staining.The protein levels of MET and phosphorylated MET (p-MET) of HGF/c-Met signaling pathway as well as its down-stream key proteins AKT , ERK, p-AKT and p-ERK in the cell lines before and after crizotinib treatment were examined by Western blot .RESULTS:The growth of H1993, H2228 and A549 cell lines was inhibited after crizoti-nib treatment for 72 h in a dose-dependent manner .Apoptotic rates of H1993 cells and H2228 cells were increased with the crizotinib concentration and exposure time .Down-regulation of p-MET, p-AKT and p-ERK at protein levels in H1993 cells and H2228 cells after exposure to crizotinib for 72 h was confirmed by Western blot .No obvious change of the related-pro-teins of HGF/c-Met signaling pathway was found in A 549 cell line.CONCLUSION: HGF/c-Met signaling pathway may contribute to crizotinib-induced apoptosis of H1993 cells and H2228 cells, which provides the experimental basis for MET-targeting treatment of lung cancer .

Role of mTOR signaling pathway in crizotinib-induced apoptosis of EML4-ALK fusion gene-positive lung adenocarcinoma cell line H2228 / 中国病理生理杂志

AIM:To investigate the mammalian target of rapamycin ( mTOR) signaling pathway as the center playing a role in the crizotinib-induced apoptosis of non-small cell lung cancer (NSCLC) cell line H2228, which represents positive echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene. METHODS:H2228 cells were processed according to different purposes .Fluorescence quantitative PCR is used to ob-serve the gene states .MTT assay is used to detect the cell inhibition rates .The cell apoptosis and cell cycle were analyzed by flow cytometry .The expression and activation levels of the key proteins in the mTOR signaling pathway were determined by Western blotting .RESULTS:Crizotinib promoted the apoptosis of H 2228 cells in a time-and dose-dependent manner . Crizotinib blocked the H2228 cells staying at the G1 phase.In apoptotic H2228 cells processed with crizotinib, the activa-tion level of mTOR was decreased , and the activation levels of the key proteins in upstream and downstream of mTOR path -way were both declined .The expression level of the fusion protein EML 4-ALK variant 3 was not affected , but its active form of p-ALK was significantly suppressed .CONCLUSION:mTOR signaling pathway has a certain relationship with the crizotinib-induced apoptosis of lung cancer cell H 2228, which represents positive EML4-ALK fusion gene.