Erlotinib Induces Mitochondrial-Mediated Apoptosis in Human H3255 Non-Small-Cell Lung Cancer Cells with Epidermal Growth Factor ReceptorL858R Mutation through Mitochondrial Oxidative Phosphorylation-Dependent Activation of BAX and BAK

Division of Medical Oncology, Albert Einstein College of Medicine, Bronx, New York, USA.
Molecular pharmacology (Impact Factor: 4.13). 09/2008; 74(3):793-806. DOI: 10.1124/mol.107.044396
Source: PubMed


Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib shows potent antitumor activity in some non-small-cell lung cancer (NSCLC) cell lines and is approved by the Food and Drug Administration as second and third line treatment for NSCLC. However, the molecular mechanisms by which erlotinib induces apoptosis remain to be elucidated. Here, we investigated the effect of erlotinib on apoptotic signal pathways in H3255 cells with the EGFR(L858R) mutation. Erlotinib induces apoptosis associated with the activation of caspases in a dose- and time-dependent manner. Erlotinib did not alter the expression of apoptotic receptors FAS and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), although it induced caspase-8 activation and BID cleavage. In addition, cell death caused by erlotinib was not prevented by coincubation with FAS and TRAIL antagonists, ZB-4 monoclonal antibody and TRAIL/Fc recombinant, suggesting that erlotinib-induced apoptosis is not associated with receptor-mediated pathways. Erlotinib induces loss of mitochondrial membrane potential and release of cytochrome c and second mitochondria-derived activator of caspases/direct IAP binding protein with low pI from mitochondria. Furthermore, erlotinib causes BAX translocation to mitochondria, BAX and BAK conformational changes, and oligomerization. Erlotinib did not induce reactive oxygen species generation, and cotreatment with antioxidants did not alter erlotinib-induced activation of BAX and BAK and apoptosis. However, cotreatment with inhibitors of mitochondrial oxidative phosphorylation significantly blocked erlotinib-induced activation of BAX and BAK and cell death. Benzyloxycarbiny-VAD-fluoromethyl ketone had no effect on erlotinib-induced BAX and BAK activation but effectively prevented apoptosis. Overexpression of BCL-2 caused a significant attenuation of erlotinib-induced cell death, but no effect on BAX and BAK activation. Down-regulation of BAX and BAK gene expression with small interfering RNA led to an effective reduction of erlotinib-induced apoptosis. Our data indicate that activation of BAX and BAK plays a critical role in the initiation of erlotinib-induced apoptotic cascades.

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    • "On receiving a death signal, the mitochondrial membrane is disrupted, and cytochrome c is released from mitochondria into the cytosol (30). Bcl-2 family proteins have been shown to play an important role in the regulation of mitochondria-mediated apoptosis, and are the upstream regulators of the ΔΨm (32). The pro-apoptotic member Bax translocates to the mitochondrion and integrates into the OMM, where Bax promotes the excretion of cytochrome c into the cytosol and the disruption of ΔΨm, whereas anti-apoptotic protein Bcl-2 prevents this process by preserving mitochondrial integrity (33). "
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    ABSTRACT: Emerging evidence indicates that the redistribution of phosphatidylethanolamine (PE) across the bilayer of the plasma membrane is an important molecular marker for apoptosis. However, the effect of PE on apoptosis and the underlying mechanism of PE remain unclear. In the current study, MTT and flow cytometric assays were used to examine the effects of PE on apoptosis in SMMC‑7721 cells. The level of mitochondrial membrane potential (ΔΨm) and the expression of Bax, Bcl‑2, caspase‑3, phospho‑Erk and phospho‑Stat1/2 in SMMC‑7721 cells that were exposed to PE were also investigated. The results showed that PE inhibited proliferation, caused G0/G1 phase cell cycle arrest and induced apoptosis in SMMC‑7721 cells in a dose‑dependent manner. Rhodamine 123 staining showed that the treatment of SMMC‑7721 cells with different concentrations of PE for 24 h significantly decreased the level of ΔΨm and exerted dose‑dependent effects. Using immunofluorescence and western blotting, we found that the expression of Bax was upregulated, whereas that of Bcl‑2 was downregulated in PE‑induced apoptotic cells. In addition, these events were accompanied by an increase in caspase‑3 expression in a dose‑dependent manner following PE treatment. PE‑induced apoptosis was accompanied by a decrease in Erk phospho-rylation and by the activation of Stat1/2 phosphorylation in SMMC‑7721 cells. In conclusion, the results suggested that PE‑induced apoptosis is involved in upregulating the Bax/Bcl‑2 protein ratio and decreasing the ΔΨm. Moreover, the results showed that the Erk and Stat1/2 signalling pathways may be involved in the process of PE‑induced apoptosis.
    International Journal of Molecular Medicine 05/2014; 34(1). DOI:10.3892/ijmm.2014.1777 · 2.09 Impact Factor
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    • "Erlotinib has been shown to inhibit proliferation of cancer cells, including HCC cells, and block cellcycle progression at the G1 phase [15] [16] [17]. Erlotinib has also been shown to induce mitochondrial-mediated apoptosis through activation of BAX and BAK in human H3255 NSCLC cells (which harbor EGFR L858R mutation) [18]. Clinically, erlotinib is currently an approved drug for treatment for NSCLC and pancreatic cancer [15]. "
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    ABSTRACT: Erlotinib is a small-molecular inhibitor of epidermal growth factor receptor (EGFR). Here, we identify that cancerous inhibitor of protein phosphatase 2A (CIP2A) is a major determinant mediating erlotinib-induced apoptosis in hepatocellular carcinoma (HCC). Erlotinib showed differential effects on apoptosis in 4 human HCC cell lines. Erlotinib induced significant apoptosis in Hep3B and PLC5 cell lines; however, Huh-7 and HA59T cell lines showed resistance to erlotinib-induced apoptosis at all tested doses. Down-regulation of CIP2A, a cellular inhibitor of protein phosphatase 2A (PP2A), mediated the apoptotic effect of erlotinib in HCC. Erlotinib inhibited CIP2A in a dose- and time-dependent manner in all sensitive HCC cells whereas no alterations in CIP2A were found in resistant cells. Overexpression of CIP2A upregulated phospho-Akt and protected Hep3B cells from erlotinib-induced apoptosis. In addition, silencing CIP2A by siRNA restored the effects of erlotinib in Huh-7 cells. Moreover, adding okadaic acid, a PP2A inhibitor, abolished the effects of erlotinib on apoptosis in Hep3B cells; and forskolin, a PP2A agonist enhanced the effect of erlotinib in resistant HA59T cells. Combining Akt inhibitor MK-2206 with erlotinib restored the sensitivity of HA59T cells to erlotinib. Furthermore, in vivo xenograft data showed that erlotinib inhibited the growth of PLC5 tumor but had no effect on Huh-7 tumor. Erlotinib downregulated CIP2A and upregulated PP2A activity in PLC5 tumors, but not in Huh-7 tumors. In conclusion, inhibition of CIP2A determines the effects of erlotinib on apoptosis in HCC. CIP2A may be useful as a therapeutic biomarker for predicting clinical response to erlotinib in HCC treatment.
    Biochemical pharmacology 11/2012; 85(3). DOI:10.1016/j.bcp.2012.11.009 · 5.01 Impact Factor
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    • "Topoisomerase I inhibitors Camptothecin Lung cancer Lymphoma Ovarian cancer Traganos et al. (1996), Sanchez-Alcazar et al. (2003) Irinotecan Colorectal cancer Xu and Villalona-Calero (2002), Li et al. (2009) Topotecan Cervical cancer Ovarian cancer SCLC Caserini et al. (1997), Nakashio et al. (2000) Topoisomerase II inhibitors Etoposide Ewing's sarcoma Glioblastoma multiforme Lung cancer Lymphoma Non-lymphocytic leukemia Testicular cancer Karpinich et al. (2002), Cosse et al. (2007) Mitoxantrone AML Breast cancer (metastatic) NHL Bhalla et al. (1993), Cao et al. (2009) Tyrosine kinase inhibitors Dasatinib (Sprycel ® ) ALL CML Prostate cancer Talpaz et al. (2006), Guerrouahen et al. (2010) Erlotinib (Tarceva ® ) NSCLC Pancreatic cancer Ling et al. (2008), Felip et al. (2008) "
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    ABSTRACT: For a long time, it was commonly believed that efficient anticancer regimens would either trigger the apoptotic demise of tumor cells or induce a permanent arrest in the G(1) phase of the cell cycle, i.e., senescence. The recent discovery that necrosis can occur in a regulated fashion and the increasingly more precise characterization of the underlying molecular mechanisms have raised great interest, as non-apoptotic pathways might be instrumental to circumvent the resistance of cancer cells to conventional, pro-apoptotic therapeutic regimens. Moreover, it has been shown that some anticancer regimens engage lethal signaling cascades that can ignite multiple oncosuppressive mechanisms, including apoptosis, necrosis, and senescence. Among these signaling pathways is mitotic catastrophe, whose role as a bona fide cell death mechanism has recently been reconsidered. Thus, anticancer regimens get ever more sophisticated, and often distinct strategies are combined to maximize efficacy and minimize side effects. In this review, we will discuss the importance of apoptosis, necrosis, and mitotic catastrophe in the response of tumor cells to the most common clinically employed and experimental anticancer agents.
    Frontiers in Oncology 05/2011; 1:5. DOI:10.3389/fonc.2011.00005
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