A Novel Epidermal Growth Factor Receptor Inhibitor Promotes Apoptosis in Non–Small Cell Lung Cancer Cells Resistant to Erlotinib

Abstract

Non-small cell lung cancer (NSCLC) with activating mutations in the epidermal growth factor receptor (EGFR) responds to EGFR tyrosine kinase inhibitors such as erlotinib. However, secondary somatic EGFR mutations (e.g., T790M) confer resistance to erlotinib. BMS-690514, a novel panHER/vascular endothelial growth factor receptor (VEGFR) inhibitor described here, exerted antiproliferative and proapoptotic effects on NSCLC cell lines, with prominent efficacy on H1975 cells expressing the T790M mutation. In this model, BMS-690514 induced a G 1 cell cycle arrest, as well as ultrastructural hallmarks of apoptosis, mitochondrial release of cytochrome c, and activation of caspases involved in the intrinsic (e.g., caspase-2, caspase-3, caspase-7, and caspase-9), but not in the extrinsic (e.g., caspase-8), pathway. Caspase inhibition conferred partial protection against BMS-690514 cytotoxicity, pointing to the involvement of both caspasedependent and caspase-independent effector mechanisms. Transcriptome analyses revealed the up-regulation of proapoptotic (e.g., Bim, Puma) and cell cycle inhibitory (e.g., p27Kip1 , p57 Kip2 ) factors, as well as the down-regulation of antiapoptotic (e.g., Mcl1), heat shock (e.g., HSP40, HSP70, HSP90), and cell cycle promoting [e.g., cyclins B1, D1, and D3; cyclin-dependent kinase 1 (CDK1); MCM family proteins; proliferating cell nuclear antigen (PCNA)] proteins. BMS-690514-induced death of H1975 cells was modified in a unique fashion by a panel of small interfering RNAs targeting apoptosis modulators. Down-regulation of components of the nuclear factor-KB survival pathway (e.g., p65, Nemo/IKK;, TAB2) sensitized cells to BMS-690514, whereas knockdown of proapoptotic factors (e.g., Puma, Bax, Bak, caspase-2, etc.) and DNA damage-related proteins (e.g., ERCC1, hTERT) exerted cytoprotective effects. BMS-690514 is a new pan-HER/VEGFR inhibitor that may become an alternative to erlotinib for the treatment of NSCLC. [Cancer Res 2007;67(13):6253-62]

Preferential killing of tetraploid tumor cells by targeting the mitotic kinesin Eg5

“…36 at 30-40% confluence by means of the HiPerFect transfection reagent (Qiagen, Hilden, Germany) as previously described. 40 72 h later, the transfection efficiency was determined by immunoblot. The compound dimethylenastron (Eg5 Inhibitor III, DIMEN) was purchased from Calbiochem-Merck (Darmstadt, Germany).…”

Molecular mechanisms of ATP secretion during immunogenic cell death

“…siRNAs specific for murine Atg5 (sense 5 0 -CAUCAACCGGAAACUCAUdTdT-3 0 ), murine Atg7 (sense 5 0 -AGUUUC CAGUCCGUUGAAdTdT-3 0 ), human ATG5 (sense 5 0 -CCUUUGGCCUAAGAAGA AAdTdT-3 0 ), human ATG7 (sense 5 0 -CGACUUGUUCCUUACGGAAdTdT-3 0 ) human BCN1 (sense 5 0 -CAGUGGAUCUAAAUCUCAAdTdT-3 0 ), human LAMP1 (siLAMP1a sense 5 0 -CGAGAAAUGCAACACGUUAdTdT-3 0 ; siLAMP1b sense, 5 0 -GGAAUCCAGUUGAAUACAAdTdT-3 0 ), human ROCK1 (siROCK1a sense 5 0 -GCCGCCGGGACCCAACUAUdTdT-3 0 ; siROCK1b sense 5 0 -CAGCCAUCACU AUCAAGAUdTdT-3 0 ; siROCK1c sense 5 0 -GACUAUACAAAACUAUUUUdTdT-3 0 ) and human VAMP1 (sense 5 0 -GGACAUCAUGCGUGUGAAUdTdT-3 0 ), as well as a siRNA unrelated to the murine and human genome (siUNR, sense 5 0 -GCC GGUAUGCCGGUUAAGUdTdT-3 0 ) 57 were purchased from Sigma-Proligo (Woodlands, TX, USA). In addition, pre-designed commercial siRNAs specific for murine Bcn1 (sc-29798) and human SLC17A9 (best known as VNUT; sc-72740) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).…”

Preferential killing of p53-deficient cancer cells by reversine

“…The knockdown of proteins reported in Table S1 was performed with validated specific small interfering RNAs (siRNA) purchased from Sigma-Proligo. 54,55 Alternatively, siRNA duplexes for the downregulation of AURKA (SIHK0142) were purchased from Sigma-Aldrich and siRNAs for the downregulation of BAK1 and BAX (Hs_BAK1_5 and Hs_BAX_10 HP Validated siRNAs, respectively) from Qiagen. Cells pre-seeded in…”