ZD1839 (Gefitinib, 'Iressa'), an epidermal growth factor receptor-tyrosine kinase inhibitor, enhances the anti-cancer effects of TRAIL in human esophageal squamous cell carcinoma

The EGF (epidermal growth factor) receptor-tyrosine kinase inhibitor ZD1839 (Gefitinib, 'Iressa') blocks the cell signaling pathways involved in cell proliferation, survival, and angiogenesis in various cancer cells. TNF-related death apoptosis inducing ligand (TRAIL) acts as an anticancer agent. We investigated the antitumor effects of ZD1839 alone or in combination with TRAIL against human esophageal squamous cell cancer (ESCC) lines. Although all ESCC cells expressed EGF receptor at a protein level, the effect of ZD1839 on cell growth did not correlate with the level of EGFR expression and phosphorylation of EGF receptor protein in ESCC lines. ZD1839 caused a dose-dependent growth arrest at G0-G1 phase associated with increased p27 expression. As TE8 cells are resistant to TRAIL, we tested whether ZD1839 combined with TRAIL induced apoptosis of TE8 cells via the inhibition of EGF receptor signaling by ZD1839. ZD1839 inhibited the phosphorylation of Akt, and enhanced TRAIL-induced apoptosis via activation of caspase-3 and caspase-9, and inactivation of Bcl-xL. Our results indicated that ZD1839 has anti-cancer properties against human esophageal cancer cells. ZD1839 also augmented the anti-cancer activity of TRAIL, even in TRAIL-resistant tumors. These results suggest that treatment with ZD1839 and TRAIL may have potential in the treatment of ESCC patients.     

Critical role of both p27KIP1 and p21CIP1/WAF1 in the antiproliferative effect of ZD1839 ('Iressa'), an epidermal growth factor receptor tyrosine kinase inhibitor,in head and neck squamous carcinoma cells

High expression of the epidermal growth factor receptor (EGFR) has been implicated in the development of squamous-cell carcinomas of head and neck (SCCHN). ZD1839 ('Iressa') is an orally active, selective EGFR-TKI (EGFR-tyrosine kinase inhibitor) that blocks signal transduction pathways implicated in proliferation and survival of cancer cells, and other host-dependent processes promoting cancer growth. We have demonstrated that ZD1839 induces growth arrest in SCCHN cell lines by inhibiting EGFR-mediated signaling. Cell cycle kinetic analysis demonstrated that ZD1839 induces a delay in cell cycle progression and a G1 arrest together with a partial G2/M block; this was associated with increased expression of both p27(KIP1) and p21(CIP1/WAF1) cyclin-dependent kinase (CDK) inhibitors. The activity of CDK2, the main target of CIP/KIP CDK inhibitors, was reduced in a dose-dependent fashion after 24 h of ZD1839 treatment and this effect correlated to the increased amount of p27(KIP1) and p21(CIP1/WAF1) proteins associated with CDK2-cyclin-E and CDK2-cyclin-A complexes. In addition, ZD1839-induced growth inhibition was significantly reduced in cell transfectants expressing p27(KIP1) or p21(CIP1/WAF1) antisense constructs. Overall, these results as well as the timing of the effect of ZD1839 on G1 arrest and p27(KIP1) and p21(CIP1/WAF1) upregulation, suggest a mechanistic connection between these events.     

Inhibition of proliferation and induction of apoptosis in breast cancer cells by the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor ZD1839 ('Iressa') is independent of EGFR expression level

High expression of the epidermal growth factor receptor (EGFR) in breast carcinoma confers a growth advantage to the tumor cells. The EGFR tyrosine kinase inhibitor (EGFR-TKI) ZD1839 ('Iressa') has clinical activity in a wide range of tumor types, although the mechanism(s) by which it exerts its antitumor activity effects remain unclear. We analyzed the ability of ZD1839 to induce apoptosis and/or inhibition of proliferation in breast carcinoma cell lines, as well any association between this ability and the downregulation activity of MAPK and Akt, two recently proposed markers of ZD1839 activity. Proliferation, survival, and activation of Akt and MAPK were evaluated in six human breast cancer cell lines expressing various levels of EGFR and HER2 and exposed to ZD1839. EGFR and HER2 expression levels were determined using specific monoclonal antibodies and FACS analysis. The effects of ZD1839 were independent of EGFR expression levels, but were influenced by high HER2 expression. ZD1839 significantly reduced the rate of [3H]-thymidine incorporation in the four sensitive cell lines, while apoptosis was also induced in two of these cell lines. No correlation was found between the cytostatic or cytotoxic effects of ZD1839 and its ability to downregulate MAPK and Akt activity in the tumor cell lines. Our data suggest that the antitumor activity of ZD1839 is due to a cytostatic effect, and involves apoptosis induction in a subset of sensitive cells only, and that neither MAPK nor Akt is a reliable marker of ZD1839 activity.     

Activation of BAD by therapeutic inhibition of epidermal growth factor receptor and transactivation by insulin-like growth factor receptor

Novel cancer chemotherapeutics are required to induce apoptosis by activating pro-apoptotic proteins. Both epidermal growth factor (EGF) and insulin-like growth factor (IGF) provide potent survival stimuli in many epithelia, and activation of their receptors is commonly observed in solid human tumors. Here we demonstrate that blockade of the EGF receptor by a new drug in phase III clinical trails for cancer, ZD1839, potently induces apoptosis in mammary epithelial cell lines and primary cultures, as well as in a primary pleural effusion from a breast cancer patient. We identified the mechanism of apoptosis induction by ZD1839. We showed that it prevents cell survival by activating the pro-apoptotic protein BAD. Moreover, we demonstrate that IGF transactivates the EGF receptor and that ZD1839 blocks IGF-mediated phosphorylation of MAPK and BAD. Many cancer therapies kill tumor cells by inducing apoptosis as a consequence of targeting DNA; however, the threshold at which apoptosis can be triggered through DNA damage is often different from that in normal cells. Our results indicate that by targeting a growth factor-mediated survival signaling pathway, BAD phosphorylation can be manipulated therapeutically to induce apoptosis.     

Targeted inhibition of the epidermal growth factor receptor-tyrosine kinase by ZD1839 ('Iressa') induces cell-cycle arrest and inhibits proliferation in prostate cancer cells

The epidermal growth factor (EGF) plays a role in the development of prostate cancer, which becomes essential after androgen resistance has emerged. The EGF receptor (EGFR) is therefore a potential target for anticancer therapy. We evaluated the effects of ZD1839 ('Iressa'), an orally active EGFR-tyrosine kinase inhibitor, on prostate cancer cell lines. The effects of ZD1839 were evaluated on the anchorage dependent and independent growth of androgen-responsive (LNCaP) and androgen-independent (DU145 and PC3) cells by a cell proliferation assay, cell counting, and soft agar analysis. Flow cytometric analysis and Western blotting were used to assess the effects on the cell-cycle and on protein expression levels, respectively. ZD1839 caused a dose- and time-dependent growth inhibition in all three cell lines. A dose-dependent supra-additive increase in growth inhibition was observed when ZD1839 was combined with the antiandrogen flutamide or ionizing radiation (IR). The antiproliferative effect of ZD1839 was mainly cytostatic and associated with a block in the G(0)/G(1) phase of the cell-cycle, evident after about 12 h of treatment. In the DU145 cells this block was associated with an increase in expression of the CDK inhibitor p27(Kip1), both in the cytoplasmic and nuclear fractions. The increase in p27(Kip1) was not evident in the LNCaP and PC3 cells. No changes were observed in the expression of cyclin D1 protein. These results demonstrate the antiproliferative effects of ZD1839 on the growth of prostate cancer cells and suggest that inhibition of EGFR-associated signal transduction pathway might represent a promising novel therapeutic strategy for the treatment of prostate cancer.     

Inhibition of apoptosis by amphiregulin via an insulin-like growth factor-1 receptor-dependent pathway in non-small cell lung cancer cell lines

Several abnormalities in the insulin-like growth factor-1 (IGF1) and erbB receptors pathways stimulate the growth and survival of lung cancer cells, but their mechanisms of action and cooperation are poorly understood. In this report, we have identified a new mechanism of apoptosis inhibition by amphiregulin through an IGF1-dependent survival pathway in non-small cell lung cancer (NSCLC) cells: amphiregulin activates the IGF1 receptor that in turn induces the secretion of amphiregulin and IGF1. In the absence of serum, the NSCLC cell line H358 resists apoptosis and secretes factors protecting the NSCLC cell line H322 from serum deprivation apoptosis. IGF1 receptor inhibitor AG1024 as well as epidermal growth factor receptor inhibitors AG556 and ZD1839 restore apoptosis in H322 cells cultured in H358-conditioned medium. Accordingly, the anti-apoptotic activity of H358-conditioned medium is completely abolished after incubation with anti-amphiregulin neutralizing antibody and only partially with anti-IGF1 neutralizing antibody. H358-conditioned medium and amphiregulin induce IGF1 receptor phosphorylation in H322 cells, which is prevented by anti-amphiregulin neutralizing antibody but not by AG556 or ZD1839. H358 cells secrete a high level of amphiregulin that, in combination with IGF1, prevents serum deprivation apoptosis. Finally, IGF1 receptor inhibitor blocks amphiregulin and IGF1 release by H358 cells.     

The mitogenic action of insulin-like growth factor I in normal human mammary epithelial cells requires the epidermal growth factor receptor tyrosine kinase

The signals used by insulin-like growth factor I (IGF-I) to stimulate proliferation in human mammary epithelial cells have been investigated. IGF-I caused the activation of both ERKs and Akt. Activation of ERKs was slower and more transient than that of Akt. ZD1839, a specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, prevented activation of ERKs but not Akt by IGF-I. Inhibition of the EGFR with function-blocking monoclonal antibodies also specifically blocked IGF-I-induced ERK activation. These effects occurred in primary mammary epithelial cells and in two cell lines derived from normal mammary epithelium but not in mammary fibroblasts or IGF-I-responsive breast carcinoma cell lines. Although IGF-I stimulated the proliferation of both normal and carcinoma cell lines, ZD1839 blocked this only in the normal line. ZD1839 had no effect on IGF-I receptor (IGF-IR) autophosphorylation in intact cells. IGF-I-induced ERK activation was insensitive to a broad spectrum matrix-metalloproteinase inhibitor and to CRM-197, an inhibitor of the EGFR ligand heparin-bound epidermal growth factor. EGFR was detectable within IGF-IR immunoprecipitates from normal mammary epithelial cells. Treatment of cells with IGF-I led to an increase in the amount of tyrosine-phosphorylated EGFR within these complexes. ZD1839 had no effect on complex formation but completely abolished their associated EGFR tyrosine phosphorylation. These findings indicate that IGF-I utilizes a novel EGFR-dependent signaling pathway involving the formation of a complex between the IGF-IR and the EGFR to activate the ERK pathway and to stimulate proliferation in normal human mammary epithelial cells. This form of regulation may be lost during malignant progression.     

a controlled trial of intratumoral ONYX-015, a selectively-replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer

ONYX-015 is an adenovirus with the E1B 55-kDa gene deleted, engineered to selectively replicate in and lyse p53-deficient cancer cells while sparing normal cells. Although ONYX-015 and chemotherapy have demonstrated anti-tumoral activity in patients with recurrent head and neck cancer, disease recurs rapidly with either therapy alone. We undertook a phase II trial of a combination of intratumoral ONYX-015 injection with cisplatin and 5-fluorouracil in patients with recurrent squamous cell cancer of the head and neck. There were substantial objective responses, including a high proportion of complete responses. By 6 months, none of the responding tumors had progressed, whereas all non-injected tumors treated with chemotherapy alone had progressed. The toxic effects that occurred were acceptable. Tumor biopsies obtained after treatment showed tumor-selective viral replication and necrosis induction.     

Studies leading to the identification of ZD1839 (IRESSA): an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor targeted to the treatment of cancer.

This paper describes the development of the epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 from a lead series of 4-anilinoquinazoline compounds. ZD1839 has suitable properties for use as a clinically effective drug and shows activity against human tumours. In particular, the use of pharmacokinetic data in the development of ZD1839 is discussed.     

Epidermal growth factor receptor signaling modulates apoptosis via p38alpha MAPK-dependent activation of Bax in intestinal epithelial cells

Previous studies have demonstrated that the proapoptotic protein Bax plays an important role in the elevated enterocyte apoptosis that occurs during the intestinal adaptation response to massive small bowel resection (SBR). Additionally, epidermal growth factor receptor (EGFR) activation prevents SBR-induced enterocyte apoptosis. The present study aims to delineate the relationship between EGFR activity and intestinal epithelial cell apoptosis. Treatment of model intestinal epithelial cells (RIEC-18) with both a selective EGFR inhibitor (ZD1839) and EGFR small interfering RNA knockdown resulted in a dramatic increase in apoptosis, accompanied by rapid phosphorylation of p38alpha. Concurrently, Bax underwent conformational changes consistent with activation and translocated to mitochondria. In contrast, EGF stimulation enhanced cell survival by attenuating p38alpha phosphorylation, Bax conformational change, mitochondrial trafficking, and apoptosis. These results demonstrate that that diminished EGFR activity initiates the intrinsic pathway of apoptosis through p38alpha-dependent Bax activation in intestinal epithelial cells. These finding provide mechanistic insight into the role that EGFR signaling plays in the regulation of enterocyte apoptosis following massive intestinal loss.     

Porcine epidermal stem cells as a biomedical model for wound healing and normal/malignant epithelial cell propagation

This article summarizes research using cells derived from epidermis of the miniature pigs for use as a cell therapy for skin repair and as a model for squamous carcinoma of the head and neck. Stem cells are an important "tool" for biomedical research. Adult stem cells are defined functionally, as cells that have the capacity to self-renew as well as the ability to generate differentiated cells. They are present in defined tissue microenvironments called niches. Asymmetric mitosis allows them to produce one daughter cell with the properties of stem cells (self-renewal) and a second cell with characteristics of progenitor cells, or transit amplifying cells, which proliferate quickly but with a limited number of mitotic divisions. Porcine epidermal stem cells, located in the bulge region of the outer root sheath of hair follicles, migrate in vitro from hair sheaths and because they are resistant to anoikis (detachment induced apoptosis), survive in non-adhesive conditions to form spheroids. These cells express keratins, galectin-1 and their nuclei are rich in DeltaNp63alpha. Interestingly, the multiple phenotype analysis of the human tumor cells in squamous carcinoma of head and neck revealed similarities with epidermal stem cells. These cancer stem cells are usually located on the periphery of the tumor where the invasive front of the tumor responsible for its aggressive behavior is located. In contrast, extensive expression of markers of terminal differentiation such as expression of glycoligands reactive for the endogenous lectin, galectin-3, indicates better tumor prognosis.     

ZD6474 enhances paclitaxel antiproliferative and apoptotic effects in breast carcinoma cells

Chemotherapy employing paclitaxel and docetaxel is widely used for treating early‐stage breast cancer and metastasis, which is frequently associated with overexpression of epidermal growth factor receptor (EGFR) and resistance to apoptosis. ZD6474, a dual tyrosine kinase inhibitor of EGFR and VEGFR, inhibits cell proliferation of solid tumors, including breast. Phase III clinical trials using ZD6474 in non‐small cell lung carcinoma when combined with standard chemotherapy appear promising. In order to improve the antineoplastic activity of paclitaxel, we presently investigated the effects of ZD6474 in combination with paclitaxel in EGFR and VEGFR expressing human breast cancer cell lines MCF‐7 and MDA‐MB‐231. ZD6474 synergistically decreased cell viability when used in combination with paclitaxel. ZD6474 inhibited cyclin D1 and cyclin E expression and induced p53 expression when combined with paclitaxel. The combination of ZD6474 with paclitaxel versus either agent alone also more potently down‐regulated the antiapoptotic bcl‐2 protein, up‐regulated pro‐apoptotic signaling events involving expression of bax, activation of caspase‐3 and caspase‐7 proteins, and induced poly(ADP‐ribose) polymerase resulting in apoptosis. ZD6474 combined with paclitaxel inhibited anchorage‐independent colony formation and invasion of breast cancer cells in vitro as compared to either single agent, indicating a potential involvement of altered expression and reorganization of cytoskeletal proteins in combinatorial treated breast cancer cells. Collectively, our studies indicate that incorporating an anti‐EGFR plus VEGFR strategy (ZD6474) with chemotherapy (paclitaxel), where clinical studies of dose‐intensive paclitaxel therapy are currently in progress, may be more effective in treating patients with locally advanced or metastatic breast cancer than either approach alone. J. Cell. Physiol. 226: 375–384, 2011. © 2010 Wiley‐Liss, Inc.     

ik3-2, a relative to ik3-1/Cables, is involved in both p53-mediated and p53-independent apoptotic pathways

ik3-2 is a close relative to ik3-1/Cables, an associator with cdk3 and cdk5. ik3-1/Cables has been identified to be a candidate tumor suppressor for colon and head/neck cancers. In agreement, it has been pointed out that ik3-1/Cables is a regulator for both p53- and p73-induced apoptosis [J. Biol. Chem. 277 (2002) 2951] although ectopic expression of ik3-1/Cables does not induce apoptosis. Here we show that adenovirus-mediated overexpression of ik3-2 results in apoptosis of p53-intact U2OS cells. ik3-2 binds to p53 in vivo and ectopic coexpression of ik3-2 enhances apoptosis induced by adenovirus-mediated expression of p53. Furthermore, ectopic expression of ik3-2 results in apoptosis of primary p53/Mdm2- and p53/ARF-null mouse embryo fibroblasts, indicating that ik3-2-induced apoptosis is partially p53-independent. Both the highly conserved C-terminal cyclin box-homologous domain (ik3-2-C) and the N-terminal region consisting of 70 amino acids (ik3-2-N) are responsible for ik3-2-mediated enhancement of p53-induced apoptosis. In contrast, ik3-2-induced p53-independent apoptosis is mediated through ik3-2-N. We thus identified ik3-2 as a proapoptotic factor involved in both p53-mediated and p53-independent apoptotic pathways.     

Enhanced gene transfer and cell death following p53 gene transfer using photochemical internalisation of glucosylated PEI-DNA complexes

BACKGROUND: p53 is frequently mutated in many cancers including human head and neck squamous cell carcinoma and pancreatic cancer. In tumor models, wild-type (wt) p53 gene transfer induces apoptosis and tumor regression in vivo, justifying the extensive clinical investigation of p53 gene therapy. METHODS: p53 nonviral-mediated gene transfer was achieved using glucosylated polyethylenimine (PEI) in conjunction with photochemical internalisation (PCI). Experimental conditions were optimised using the green fluorescent protein (GFP) as a reporter. p53 gene transfer was then evaluated using semi-quantitative RT-PCR in p53-deleted PANC3 and p53-mutated FaDu cell lines. Following gene transfer, induction of apoptosis was investigated using phosphatidylserine externalisation and nuclear fragmentation assays. Induction of long-term cell death was analysed using colony-forming assays. RESULTS: PCI was found to enhance GFP gene transfer after 48 h in both cell lines. Whether using glucosylated-PEI alone or associated with PCI, p53 gene transfer was achieved with subsequent recovery of p53 mRNA expression in PANC3 cells and a significant 4-fold increase in p53 mRNA expression in FaDu cells. PCI was found to further enhance p53 mRNA expression by 2.3-fold in PANC3 cells. Spontaneous induction of apoptosis following wt-p53 gene transfer was achieved in both cell lines. PCI was found to enhance apoptosis up to levels similar to those achieved with chemotherapy. As a consequence, long-term cell death was significantly enhanced after wt-p53 gene transfer when PCI was used in both cell lines, yielding up to 60% cell death. CONCLUSIONS: PCI increases glucosylated-PEI-mediated p53 gene transfer, apoptosis as well as cell death in mutant p53 human cancer cells.