EGFR as targets for cancer therapy have been investigated for over 20 years and the field of targeted therapy is evolving rapidly as new insights in receptor biology and signal transduction [18]. Gefitinib (Iressa™, ZD1839), a small molecule tyrosine kinase inhibitor, has been demonstrated the promising antitumor activity [74]. It targets the catalytic domain of EGFR to compete with the ATP binding site [75]. The induction of apoptosis has been considered to be the major mechanism for these gefitinib mediated anticancer effects [76]. However, the precise downstream signaling molecules of EGFR-independent have not yet been elucidated. In this study, we investigated the effects of gefitinib on the EGFR-independent cell death signaling pathways in human cancer cells. Here, we found that gefitinib significantly induced cancer cell death in various human cancer cell lines. Interestingly, A375, MCF7 and RKO cells expressed very low protein level of the phosphorylated-EGFR and total EGFR; in contrast, BFTC905 and A549 cells expressed the high level. However, gefitinib more effectively induced cytotoxicity in A375 cells (low level EGFR) compared with BFTC905 cells (high level EGFR). In our data, treatment with 60 μM gefitinib for 24 h in cell cycle progression analysis increased apoptosis by about 26.6% and 12.3% in A375 and BFTC905 cells, respectively. And in annexin V and PI staining assay, gefitinib at 60 μM induced about 61.6% of apoptosis in A375 cells and 28.9% of apoptosis in BFTC905 cells. We suggest that gefitinib induces cancer cell death that may be through the EGFR-independent pathway (Fig. 15A). Although the underlined mechanism concerning the gefitinib-independent EGFR remains unknown, our analyses reveal a plausible hypothesis centered without EGFR, explaining how gefitinib elicits cell apoptosis (Fig. 15B).
Apoptosis plays an important role in development and maintenance of tissue homeostasis [77]. Caspases are central effectors of apoptosis [78]. Cells undergo apoptosis through two major pathways, the extrinsic pathway (death receptor pathway) or the intrinsic pathway (the mitochondrial pathway) [79]. PARP is one of the prime target proteins for caspase-3 [38]. Finally, the contents of dead cells are packaged into apoptotic bodies, which are recognized by neighboring cells or macrophages and cleared by phagocytosis [80]. In present study, we demonstrated that gefitinib induced
apoptosis mediated by the activation of caspase-3 induced and the protein cleavage of PARP.
Subsequently, it is the first time to prove that gefitinib inhibits the securin protein expression in human cancer cells. Treatment with anticancer agents including ultraviolet, doxorubicin, and bleomycin also decrease the securin expression in cancer cells [81; 82].
It had been demonstrated that oxaliplatin inhibits the securin protein expression via a p53-dependent pathway, and p53 and securin may modulate the oxaliplatin-induced cytotoxicity in human colorectal cancer cells [83]. And p38 MAPK may oppositely regulate securin protein expression and γ-H2AX formation in the oxaliplatin-induced apoptosis of human colorectal cancer cells [84]. As well as the blockage of survivin and securin expression increases the cytochalasin B-induced cell death and growth inhibition in human cancer cells [85]. Furthermore, we found that the securin null cells were more sensitive than the wild type cells on the induction of cytotoxicity and apoptosis following treatment with gefitinib. These findings indicate that the loss of securin in cancer cells may increase the cytotoxicity and apoptosis after gefitinib treatment. Vector-based overexpression experiments provide compelling evidence for a role of securin in gefitinib-induced tumor cell apoptosis. We provide the securin signal that plays an important role for gefitinib-induced apoptosis in human cancer cells (Fig.
16). Recently, securin has been shown to participate in the cellular DNA repair [52].
Moreover, securin prevents abnormal sister chromatid segregation during mitosis and maintains genomic stability, and its defects can result in chromosomal instability [51]. It also had been demonstrated that depletion of securin increases arsenite-induced chromosome instability and apoptosis via a p53-independent pathway [86]. In our study, gefitinib markedly decreased the fractions of S phase in cancer cells and increased the fraction of sub-G1 phase indicating apoptotic induction. Therefore, we propose that the loss of securin maybe decline cellular DNA repair ability and increases chromosome instability after treatment with gefitinib. Nevertheless, the precise mechanism and role of securin on the regulation of DNA repair in the gefitinib-exposed cells require further investigation.
ATF3 ia a transcription factor and has been shown to dimerise with ATF3 or other ATF/CREB proteins [87; 88; 89]. Depending on the promoter context, these homodimers or heterodimers can act as either repressors or activators of transcription [90]. Therefore, the role of ATF3 as a repressor or activator of transcription cannot be
generalised. ATF3 expression is maintained at low levels in quiescent cells [91]. In tumor, the role of ATF3 in the regulation of the cell cycle and apoptosis has important implications for understanding susceptibility to and progression of several cancers. In our study, we found that gefitinib increased ATF3 protein expression and it seemed to translocate to nuclei. According to literature reviews, the potential role of ATF3 is as an oncogene and a tumour suppressor gene. In tumorigenesis, ATF3 overexpression protected malignant MCF10CA1a human breast cancer cells from apoptosis and promoted their metastatic potential, associated with an upregulation of fibronectin-1, TWIST-1, and Slug transcripts, which are key regulators of cell-cell or cell- extracellular matrix interaction [69]. On the other hand, the potential role of ATF3 as a tumour suppressor is also supported by its defined role in transforming growth factor beta (TGFβ) signaling [92]. TGFβ is a potent tumour suppressor in epithelial cells, which signals via Smad3 activation to directly induce ATF3 [93]. In our data, if induction of ATF3 by treatment with gefitinib and translocate to nuclei that does indeed act as a tumour suppressor, it may be responsible in part for the action of anticancer.