A report of two bronchioloalveolar carcinoma cases which were rapidly improved by treatment with the epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 ("Iressa")

Bronchioloalveolar carcinoma (BAC), a form of pulmonary adenocarcinoma, presents unique clinical features, such as endobronchial spread and bronchorrhea in advanced stages. The prognosis for BAC patients in advanced stages is poor, as is the case for patients with other non-small-cell lung cancer (NSCLC) types, because of low susceptibility to conventional chemotherapy. Recently, an orally active, selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (EGFR-TKI), ZD1839 ("Iressa"), has been investigated in phase II clinical studies (IDEAL 1 and IDEAL 2) as monotherapy against chemotherapy-refractory NSCLC, and provided clinically significant antitumor activity. In this study, we examined the therapeutic efficiency of ZD1839 in chemotherapy-refractory BAC patients with bronchorrhea. Two female BAC patients with bronchorrhea were treated once daily with ZD1839 (250 mg/day). In both cases, serous sputum production was dramatically reduced within 3 days of starting the treatment, and hypoxia and radiographic signs of bilateral lung consolidation were visibly improved within 7 days. Following more than 8 months of treatment, no evidence of recurrence or severe adverse events has been observed. These results suggest that this selective EGFR-TKI, ZD1839, may be a powerful agent for treatment of chemotherapy-refractory BAC patients with bronchorrhea.     

The tyrosine kinase inhibitor ZD1839 ("Iressa") inhibits HER2-driven signaling and suppresses the growth of HER2-overexpressing tumor cells

The epidermal growth factor receptor (EGFR) is commonly overexpressed in many human tumors and provides a new target for anticancer drug development. ZD1839 ("Iressa"), a quinazoline tyrosine kinase inhibitor selective for the EGFR, has shown good activity in preclinical studies and in the early phase of clinical trials. However, because it remains unclear which tumor types are the best targets for treatment with this agent, the molecular characteristics that correlate with tumor sensitivity to ZD1839 have been studied. In a panel of human breast cancer and other epithelial tumor cell lines, HER2-overexpressing tumors were particularly sensitive to ZD1839. Growth inhibition of these tumor cell lines was associated with the dephosphorylation of EGFR, HER2, and HER3, accompanied by the loss of association of HER3 with phosphatidylinositol 3-kinase, and down-regulation of Akt activity. These studies suggest that HER2-overexpressing tumors are particularly susceptible to the inhibition of HER family tyrosine kinase signaling and suggest novel strategies to treat these particularly aggressive tumors.     

Enhancement of radiosensitivity by dual inhibition of the HER family with ZD1839 ("Iressa") and trastuzumab ("Herceptin")

PURPOSE: The aims of this study were twofold: (1) to examine the effects of dual inhibition of 2 members of the HER family, the epidermoid growth factor receptor (EGFR) and HER2/neu, by gefitinib (ZD1839) and trastuzumab on radiosensitivity; and (2) to explore the molecular mechanism of radiosensitization especially focusing on the survival signal transduction pathways by using A431 human vulvar squamous carcinoma cells expressing EGFR and HER2/neu. METHODS AND MATERIALS: The effects of inhibitors on the radiation-induced activation of EGFR and/or HER2/neu, and the intracellular proteins that are involved in their downstream signaling, were quantified by the Western blot. Radiosensitizing effects by the blockage of EGFR and/or HER2/neu were determined by a clonogenic assay. RESULTS: Radiation-induced activation of the EGFR and HER2/neu was inhibited with ZD1839 and/or trastuzumab. ZD1839 also inhibited the radiation-induced phosphorylation of HER2/neu. Radiation in combination with the HER family inhibitors inhibited the activation of Akt and MEK1/2, the downstream survival signaling of the HER family. ZD1839 enhanced radiosensitivity with a dose-modifying factor (DMF) (SF3) of 1.45 and trastuzumab did so with a DMF (SF3) of 1.11. Simultaneous blockade of EGFR and HER2/neu induced a synergistic radiosensitizing effect with a DMF (SF3) of 2.29. CONCLUSIONS: The present data suggest that a dual EGFR and HER2/neu targeting may have potential for radiosensitization in tumors in which both of these pathways are active.     

Anticancer effects of ZD6474, a VEGF receptor tyrosine kinase inhibitor, in gefitinib ("Iressa")-sensitive and resistant xenograft models

ZD6474 is a novel, orally available inhibitor of vascular endothelial growth factor (VEGF) receptor-2 (KDR) tyrosine kinase, with additional activity against epidermal growth factor receptor (EGFR) tyrosine kinase. ZD6474 has been shown to inhibit angiogenesis and tumor growth in a range of tumor models. Gefitinib ("Iressa") is an selective EGFR tyrosine kinase inhibitor (TKI) that blocks signal transduction pathways. We examined the antitumor activity of ZD6474 in the gefitinib-sensitive lung adenocarcinoma cell line, PC-9, and a gefitinib-resistant variant (PC-9/ZD). PC-9/ZD cells showed cross-resistance to ZD6474 in an in vitro dye formation assay. In addition, ZD6474 showed dose-dependent inhibition of EGFR phosphorylation in PC-9 cells, but inhibition was only partial in PC-9/ZD cells. ZD6474-mediated inhibition of tyrosine residue phosphorylation (Tyr992 and Tyr1045) on EGFR was greater in PC-9 cells than in PC-9/ZD cells. These findings suggest that the inhibition of EGFR phosphorylation by ZD6474 can contribute a significant, direct growth-inhibitory effect in tumor cell lines dependent on EGFR signaling for growth and/or survival. The effect of ZD6474 (12.5-50 mg/kg/day p.o. for 21 days) on the growth of PC-9 and PC-9/ZD tumor xenografts in athymic mice was also investigated. The greatest effect was seen in gefitinib-sensitive PC-9 tumors, where ZD6474 treatment (>12.5 mg/kg/day) resulted in tumor regression. Dose-dependent growth inhibition, but not tumor regression, was seen in ZD6474-treated PC-9/ZD tumors. These studies demonstrate that the additional EGFR TKI activity may contribute significantly to the antitumor efficacy of ZD6474, in particular in those tumors that are dependent on continued EGFR-signaling for proliferation or survival. In addition, these results provide a preclinical rationale for further investigation of ZD6474 as a potential treatment option for both EGFR-TKI-sensitive and EGFR-TKI-resistant tumors.     

Gefitinib ("Iressa", ZD1839), an epidermal growth factor receptor tyrosine kinase inhibitor, reverses breast cancer resistance protein/ABCG2-mediated drug resistance

Gefitinib ("Iressa", ZD1839) is an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor, and the single agent is clinically effective in non-small cell lung cancer. Although gefitinib combined with various cytotoxic agents has been reported to enhance cytotoxicity in vitro and in mouse models, the mechanism remains undetermined. Here, to explore the mechanism with topoisomerase I inhibitors, we focused on the efflux pump of the breast cancer resistance protein (BCRP/ABCG2), and then examined whether gefitinib restored drug sensitivity in multidrug-resistant cancer cells overexpressing BCRP. We used PC-6 human small cell lung cancer cells and multidrug-resistant PC-6/SN2-5H cells selected with SN-38 of the active metabolite of irinotecan, and BCRP-overexpressing MCF-7/MX cells selected with mitoxantrone and BCRP cDNA transfectant MCF-7/clone 8 cells. Drug sensitivity against anticancer drugs was determined by tetrazolium dye assay, and intracellular topotecan accumulation by FACScan. The topotecan transport study was done using the plasma membrane vesicles of PC-6/SN2-5H cells. The resistant PC-6/SN2-5H cells overexpressed BCRP but not epidermal growth factor receptor mRNA. Ten micromoles of gefitinib reversed topotecan, SN-38, and mitoxantrone resistance, and increased the intracellular topotecan accumulation in the resistant cells but not in the parental cells. Furthermore, gefitinib inhibited the topotecan transport into the vesicles, and the K(i) value was 1.01 +/- 0.09 micromol/L in the Dixon plot analysis, indicating direct inhibition of BCRP by gefitinib. However, gefitinib was not transported into the vesicles with the high-performance liquid chromatography method. These results indicate that gefitinib reverses BCRP-mediated drug resistance by direct inhibition other than competitive inhibition as a BCRP substrate. Combination of gefitinib and topoisomerase I inhibitors could be clinically effective in cancers expressing BCRP.     

ZD1839 (Iressa) induces antiangiogenic effects through inhibition of epidermal growth factor receptor tyrosine kinase

Epidermal growth factor receptor (EGFR) tyrosine kinase is a potential target for anticancer therapy. ZD1839 (Iressa) is a selective inhibitor of EGFR tyrosine kinase. In this study, we investigated the question as to whether the antitumor effect of ZD1839 is partly attributable to antiangiogenic activity and the potential mechanisms involved. Both ZD1839 and SU5416 [a vascular endothelial growth factor (VEGF)-receptor tyrosine kinase inhibitor] inhibited the migration of human umbilical vein endothelial cell cocultivated with EGF-stimulated cancer cells. ZD1839 also inhibited EGF-induced migration and the formation of tube-like structures by human microvascular endothelial cells. Moreover, ZD1839 almost completely blocked EGF-induced neovascularization of mice cornea, and SU5416 partially blocked neovascularization. In contrast, ZD1839 did not inhibit VEGF-induced angiogenesis. However, EGF-induced up-regulation of the angiogenic factors, VEGF and IL-8, was almost completely blocked by ZD1839. The antitumor effects of ZD1839 could, therefore, be mediated in part by the inhibition of tumor angiogenesis through direct effects on microvascular endothelial cells that express EGFR and also through reduced production of proangiogenic factors by tumor cells.     

Differential radiosensitisation by ZD1839 (Iressa), a highly selective epidermal growth factor receptor tyrosine kinase inhibitor in two related bladder cancer cell lines

The epidermal growth factor receptor (EGFR) is expressed in a wide variety of epithelial tumours including carcinoma of the bladder. Stimulation of the EGFR pathway is blocked by ZD1839 (Iressa), a highly selective EGFR tyrosine kinase inhibitor. Radical radiotherapy is an established organ sparing treatment option for muscle invasive bladder cancer and this study has explored the possibility for the use of ZD1839 as a radiosensitiser in this scenario. The effect of combination treatment with ZD1839 (0.01 microM) and ionising radiation in the established bladder cancer cell lines MGH-U1 and its radiosensitive mutant clone S40b was measured by clonogenic assays. A highly significant radiosensitising effect was seen in both cell lines (P < 0.001 for MGH-U1 and S40b cell lines). This effect was independent of the concentration of the drug and the duration of exposure prior to treatment with ionising radiation. Cell cycle kinetics of both cell lines was not significantly altered with ZD1839 (0.01 microM) as a single agent. A modest induction of apoptosis was observed with ZD1839 (0.01 microM) as a single agent, but a marked induction was observed with the combination treatment of ZD1839 and ionising radiation. These results suggest a potentially important role for ZD1839 in combination with radiotherapy in the treatment of muscle invasive bladder cancer.     

The epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 (Iressa) suppresses c-Src and Pak1 pathways and invasiveness of human cancer cells.

PURPOSE: Abnormalities in the expression and signaling pathways downstream of the epidermal growth factor receptor (EGFR) contribute to the progression, invasion, and maintenance of the malignant phenotype in human cancers, including those of the head and neck and breast. Accordingly, agents such as the EGFR tyrosine kinase inhibitor (EGFR-TKI) ZD1839 (Iressa) are promising, biologically based treatments that are in various stages of preclinical and clinical development. The process of tumor progression requires, among other steps, increased transformation, directional migration, and enhanced cell survival; this study explored the effect of ZD1839 on the stimulation of c-Src and p21-activated kinase 1 (Pak1), which are vital for transformation, directional motility, and cell survival of cancer cells. EXPERIMENTAL DESIGN: We examined the effect of ZD1839 on biochemical and functional assays indicative of directional motility and cell survival, using human head and neck squamous cancer cells and breast cancer cells. RESULTS: ZD1839 effectively inhibited c-Src activation and Pak1 activity in exponentially growing cancer cells. In addition, ZD1839 suppressed EGF-induced stimulation of EGFR autophosphorylation on Y1086 and Grb2-binding Y1068 sites, c-Src phosphorylation on Y215, and Pak1 activity. ZD1839 also blocked EGF-induced cytoskeleton remodeling, redistribution of activated EGFR, and in vitro invasiveness of cancer cells. CONCLUSIONS: These studies suggest that the EGFR-TKI ZD1839 may cause potent inhibition of the Pak1 and c-Src pathways and, therefore, have potential to affect the invasiveness of human cancer cells deregulated in these growth factor receptor pathways.     

ZD1839 (Iressa) in non-small-cell lung cancer

The epidermal growth factor receptor (EGFR) signaling pathway plays an important role in a number of processes that are key to tumor progression, including cell proliferation, angiogenesis, metastatic spread, and inhibition of apoptosis. EGFR is expressed or overexpressed in non-small-cell lung cancer (NSCLC), and EGFR-mediated growth has been associated with advanced disease and poor prognosis in NSCLC patients. ZD1839 (Iressa) is an orally active, selective EGFR-tyrosine kinase inhibitor that blocks EGFR signal transduction. In preclinical studies using NSCLC cell lines, ZD1839 has been shown to inhibit tumor cell growth. In addition, ZD1839, as monotherapy and in combination with commonly used cytotoxic agents, has produced growth delay in NSCLC human xenografts. Preliminary results from phase I trials in patients with advanced disease have shown that ZD1839 has excellent bioavailability, an acceptable tolerability profile, and promising clinical activity in patients with a variety of tumor types, particularly in NSCLC. ZD1839 is currently in phase III clinical development for the treatment of advanced NSCLC.     

Inhibition of growth factor production and angiogenesis in human cancer cells by ZD1839 (Iressa), a selective epidermal growth factor receptor tyrosine kinase inhibitor.

The transforming growth factor-alpha/epidermal growth factor receptor (TGF-alpha-EGFR) autocrine pathway, which is involved in the development and the progression of human epithelial cancers, controls, in part, the production of angiogenic factors. These angiogenic factors, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), are secreted by cancer cells to stimulate normal endothelial cell growth through paracrine mechanisms. ZD1839 (Iressa) is a p.o.-active, selective EGFR-tyrosine kinase inhibitor (TKI) in clinical trials in cancer patients. In this study, we evaluated the antiangiogenic and antitumor activity of ZD1839 in human colon (GEO, SW480, and CaCo2), breast (ZR-75-1 and MCF-7 ADR), ovarian (OVCAR-3), and gastric (KATO III and N87) cancer cells that coexpress TGF-alpha and EGFR. ZD1839 treatment determined a dose- and time-dependent growth inhibition accompanied by the decrease of VEGF, bFGF and TGF-alpha production in vitro. Treatment of immunodeficient mice bearing well-established, palpable GEO xenografts with ZD1839 determined a cytostatic dose-dependent tumor growth inhibition. Immunohistochemical analysis of GEO tumor xenografts after ZD1839 treatment revealed a significant dose-dependent reduction of TGF-alpha, bFGF, and VEGF expression in cancer cells and of neoangiogenesis, as determined by microvessel count. Furthermore, the antitumor activity of ZD1839 was potentiated in combination with the cytotoxic drug paclitaxel in GEO tumor xenografts. Tumor regression was observed in all mice after treatment with ZD1839 plus paclitaxel, and it was accompanied by a significant potentiation in inhibition of TGF-alpha, VEGF, and bFGF expression with a few or no microvessels. Furthermore, 6 of 16 mice bearing well-established, palpable GEO xenografts had no histological evidence of GEO tumors at the end of treatment with ZD1839 plus paclitaxel. These results demonstrate that the antitumor effect of ZD1839 is accompanied by inhibition in the production of autocrine and paracrine growth factors that sustain autonomous local growth and facilitate angiogenesis, and that this effect can be potentiated by the combined treatment with certain cytotoxic drugs, such as paclitaxel.     

A pharmacodynamic study of the epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 in metastatic colorectal cancer patients.

PURPOSE: Epidermal growth factor receptor (EGFR) appears to play an important role in the pathogenesis of colorectal cancer. We have performed a Phase I/II study of the EGFR tyrosine kinase inhibitor ZD1839 in metastatic colorectal cancer patients in which serial biopsies were taken pre- and posttreatment to assess biological activity. Experimental Design: Paired biopsies were obtained from colorectal cancer patients before and after treatment. Proliferation and apoptosis were assessed using Ki67 immunohistochemistry and terminal deoxynucleotidyl transferase-mediated nick end labeling assays, respectively. Immunohistochemistry for EGFR, activated EGFR, phosphorylated Akt, phosphorylated ERK, p27(Kip1), and beta-catenin was also performed. RESULTS: Posttreatment samples showed a statistically significant reduction in the cancer cell proliferation index (mean proliferation index pretreatment 31%; posttreatment 21%; P = 0.047). The mean cancer cell apoptosis index also increased from 6 to 12% in posttreatment samples, although this difference did not achieve statistical significance. All pretreatment samples showed strong staining for EGFR. Loss of immunohistochemical staining for activated EGFR, phosphorylated Akt, and phosphorylated ERK in cancer cells was observed in some patients after treatment. p27(Kip1) was absent in the cancer cells of most pretreatment biopsies; two patients showed a marked increase in staining for nuclear p27(Kip1) after treatment with ZD1839. These two patients also showed large increases in apoptotic index. CONCLUSIONS: ZD1839 inhibits EGFR signaling and proliferation in the cancer cells of patients with metastatic colorectal cancer. ZD1839 may also induce cancer cell apoptosis in a subset of colorectal cancer patients via up-regulation of p27(Kip1).     

Cell growth after withdrawal of gefitinib ("Iressa", ZD1839) in human lung cancer cells

Gefitinib ("Iressa", ZD1839) is a selective epidermal growth factor receptor-tyrosine kinase inhibitor, which blocks signal transduction pathways implicated in proliferation and survival of cancer cells. However, in vitro and in vivo studies concerning cell growth after withdrawal of gefitinib are limited. To determine whether cancer cells would resume proliferation upon removal of gefitinib, an in vitro study was undertaken using 4 human non-small cell lung cancer cell lines. With immunocytochemical staining and Western blot analysis, we confirmed positive expression of EGFR in these cell lines. We next evaluated growth inhibition before and after withdrawal of gefitinib. After incubation with 0-100 microM gefitinib for 24 h, medium containing gefitinib was removed, followed by addition of fresh growth medium to cell cultures. MTT assays were performed daily over a 6-day time course. Even at very low levels of gefitinib (<1 microM), these 4 TKB cells had 1-10% growth inhibition, respectively. With these levels of gefitinib, continued inhibitory effects after withdrawal of getitinib were observed in 3 of the 4 cell lines. Furthermore, none of the 4 cell lines, including the cell line which had abolished growth inhibition, showed accelerated re-growth rate after the withdrawal of gefitinib even in these exposure conditions. Based on our in vitro experiments, additional in vivo studies, which can compare the pre- and post-treatment growth rate, will be necessary to help better understand the mechanisms behind cell growth recovery after temporary gefitinib treatment.     

表皮生长因子受体酪氨酸激酶抑制剂ZD1839与伊利替康联合效应的实验研究

目的 探讨ZD1839与伊利替康的活性代谢产物7-乙基-10羟基-喜树碱（SN38）联合的最佳方案及其机制。方法 以药物联合效应测定方法,评价ZD1839和SN38不同给药顺序对人结肠癌细胞HT-29和LoVo的抑制作用;以Western blot和免疫共沉淀方法,分析ZD1839与化疗不同联合方案对各自靶蛋白及其下游分子表达的影响;以流式细胞仪测定不同联合方案对细胞周期的影响;以组蛋白相关的DNA碎片分析,比较不同方案对细胞凋亡指数的影响。结果 先SN38后ZD1839的序贯给药方案表现出明显的协同作用;反之,则表现为拈抗作用。SN38明显抑制细胞拓扑异构酶-1（Topo-1）活性;ZDl839不影响表皮生长因子受体（EGFR）的表达,但能抑制EGFR的磷酸化。与SN38单药相比,SN38联合ZDl839对Topo-1的抑制无增强;与单药ZD1839相比,联合方案对EGFR、MAPK磷酸化的抑制作用无增强,但ZD1839、SN38同时给药,和先SN38后ZD1839序贯给药对AKT的抑制有所增强。同时,联合方案对细胞周期分布的改变影响明显。ZD1839可明显维持化疗诱导的DNA损伤和细胞凋亡。结论 先SN38后ZD1839序贯给药可能是治疗结肠癌的最佳联合模式。     

Small cell lung cancer cells express EGFR and tyrosine phosphorylation of EGFR is inhibited by gefitinib ("Iressa", ZD1839)

The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, gefitinib ("Iressa", ZD1839) has demonstrated anti-tumor activity in non-small cell lung cancer (NSCLC) and has been approved in over 20 countries. NSCLC has been reported to express high levels of EGFR. However, gefitinib appears to be more effective against adenocarcinoma than squamous cell carcinoma, the latter expressing more EGFR. In the present study, we evaluated the effect of gefitinib against the small cell lung cancer (SCLC) cell lines NCI-H82, NCI-H209, NCI-H510, NCI-H526 and NCI-H660. SCLC has been reported to express a low to undetectable level of EGFR. We compared the effects of gefitinib between cell lines with detectable and undetectable EGFR expression. First, we evaluated expression levels of EGFR and HER2/neu by Western blotting and immunoprecipitation respectively; EGFR protein was detected in two of the five SCLC cell lines, whereas HER2/neu was not detected in any. Next, we analyzed expression levels of phosphorylated ERK1/2 and compared these results with EGFR (HER-1/ErbB1) and HER2/neu (ErbB2) expression levels, as EGFR conducts signals through Ras-Raf-MAPK pathway; gefitinib inhibited phosphorylation of ERK1/2 by EGF addition in cell lines with detectable and undetectable EGFR expression. These data suggest that gefitinib is potentially effective against cancers with low EGFR expression such as SCLC.     

ZD1839 (Iressa) in non-small cell lung cancer

Despite the advent of cisplatin-based combination chemotherapy for advanced non-small cell lung cancer (NSCLC), the prognosis for this patient population remains poor. Novel biologically targeted agents currently in development have the potential for greater efficacy against NSCLC, and possibly less toxicity than is associated with conventional cytotoxic chemotherapy. The epidermal growth factor receptor (EGFR) is recognized as a potentially useful target, and the small molecule, orally active EGFR-tyrosine kinase inhibitor ZD1839 (Iressa) is currently the furthest along in clinical development of the anti-EGFR agents. This review summarizes the currently available clinical data on the use of ZD1839 in the treatment of NSCLC.     

Proteomic signature corresponding to the response to gefitinib (Iressa, ZD1839), an epidermal growth factor receptor tyrosine kinase inhibitor in lung adenocarcinoma.

PURPOSE: We aimed to identify candidate proteins for tumor markers to predict the response to gefitinib treatment. EXPERIMENTAL DESIGN: We did two-dimensional difference gel electrophoresis to create the protein expression profile of lung adenocarcinoma tissues from patients who showed a different response to gefitinib treatment. We used a support vector machine algorithm to select the proteins that best distinguished 31 responders from 16 nonresponders. The prediction performance of the selected spots was validated by an external sample set, including six responders and eight nonresponders. The results were validated using specific antibodies. RESULTS: We selected nine proteins that distinguish responders from nonresponders. The predictive performance of the nine proteins was validated examining an additional six responders and eight nonresponders, resulting in positive and negative predictive values of 100% (six of six) and 87.5% (seven of eight), respectively. The differential expression of one of the nine proteins, heart-type fatty acid-binding protein, was successfully validated by ELISA. We also identified 12 proteins as a signature to distinguish tumors based on their epidermal growth factor receptor gene mutation status. CONCLUSIONS: Study of these proteins may contribute to the development of personalized therapy for lung cancer patients.     

Influence of epidermal growth factor receptor (EGFR), p53 and intrinsic MAP kinase pathway status of tumour cells on the antiproliferative effect of ZD1839 ("Iressa")

of ZD1839 ("Iressa") is an orally active, selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), which blocks signal transduction pathways implicated in proliferation and survival of cancer cells, and other host-dependent processes promoting cancer growth. Permanent downstream activation of the mitogen-activated protein kinase pathway can theoretically bypass the upstream block of epidermal growth factor receptor-dependent mitogen-activated protein kinase activation at the epidermal growth factor receptor level. We investigated the impact of epidermal growth factor receptor content, p53 status and mitogen-activated protein kinase signalling status on ZD1839 sensitivity in a panel of human tumour cell lines: seven head and neck cancer cell lines and two colon cancer cell lines (LoVo, HT29) with derivatives differing only by a specific modification in p53 status (LoVo p53 wt + p53 mut cells, HT29 p53 mut + p53 wt rescued cells). The antiproliferative activity of ZD1839 was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test. ZD1839 concentrations ranged from 0.2-200 microM (48 h exposure). Epidermal growth factor receptor expression, p53 status and p42/p44 (for testing a constitutively active mitogen-activated protein kinase pathway status) were determined by competition analysis (Scatchard plots), denaturing gradient cell electrophoresis and Western blot, respectively. Epidermal growth factor receptor levels ranged from 388 to 33794 fmol mg(-1) protein, a range that is similar to that found in head and neck tumours. The IC(50) values for cell sensitivity to ZD1839 ranged from 6 to 31 microM and a significant inverse correlation (P=0.022, r=0.82) between IC(50) values and epidermal growth factor receptor levels was observed. There was no influence of p53 status on the sensitivity to ZD1839. In two head and neck cancer cell lines with comparably elevated epidermal growth factor receptor expression, a two-fold higher ZD1839 IC(50) value was found for the one with a constitutively active mitogen-activated protein kinase. In conclusion, ZD1839 was active against cells with a range of epidermal growth factor receptor levels, although more so in cells with higher epidermal growth factor receptor expression. Activity was unaffected by p53 status, but was reduced in cells strongly dependent on epidermal growth factor receptor signalling in the presence of an intrinsically activated mitogen-activated protein kinase pathway.     

Synergistic interaction between the EGFR tyrosine kinase inhibitor gefitinib ("Iressa") and the DNA topoisomerase I inhibitor CPT-11 (irinotecan) in human colorectal cancer cells

Epidermal growth factor receptor [EGFR (HER1, erbB1)] is a receptor with associated tyrosine kinase activity, and is expressed in colorectal cancers and many other solid tumors. We examined the effect of the selective EGFR tyrosine kinase inhibitor (EGFR-TKI) gefitinib ("Iressa") in combination with the DNA topoisomerase I inhibitor CPT-11 (irinotecan) on human colorectal cancer cells. EGFR mRNA and protein expression were detected by RT-PCR and immunoblotting in all 7 colorectal cancer cell lines studied. Gefitinib inhibited the cell growth of the cancer cell lines in vitro with an IC(50) range of 1.2-160 microM by 3,(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Lovo cells exhibited the highest level of protein and autophosphorylation of EGFR and were the most sensitive to gefitinib. The combination of gefitinib and CPT-11 induced supra-additive inhibitory effects in COLO320DM, WiDR and Lovo cells, assessed by an in vitro MTT assay. Administration of gefitinib and CPT-11 had a supra-additive inhibitory effect on WiDR cells and tumor shrinkage was observed in Lovo cell xenografts established in nude mice, whereas no additive effect of combination therapy was observed in COLO320DM cells. To elucidate the mechanisms of synergistic effects, the effect of CPT-11-exposure on phosphorylation of EGFR was examined by immunoprecipitation. CPT-11 increased phosphorylation of EGFR in Lovo and WiDR cells in time- and dose-dependent manners. This EGFR activation was completely inhibited by 5 microM gefitinib and gefitinib-induced apoptosis was enhanced by combination with CPT-11, measured by PARP activation although no PARP activation was induced by 5 microM CPT-11 alone. These results suggested that these modification of EGFR by CPT-11, in Lovo cells, is a possible mechanism for the synergistic effect of CPT-11 and gefitinib. These findings imply that the EGFR-TKI gefitinib and CPT-11 will be effective against colorectal tumor cells that express high levels of EGFR, and support clinical evaluation of gefitinib in combination with CPT-11, in the treatment of colorectal cancers.     

表皮生长因子受体酪氨酸激酶抑制剂ZD1839与奥沙利铂联合对人肺癌A549细胞的作用

背景与目的:ZD1839是一种表皮生长因子受体酪氨酸激酶的小分子抑制剂,是目前肺癌分子靶向治疗中较为成熟的药物,因其单药临床有效率低,如何与化疗联合提高抗癌效应受到关注.本实验通过研究ZD1839联合奥沙利铂的不同给药方案对人肺腺癌细胞A549的杀伤作用,探讨两药联合的最佳模式.方法:以药物联合效应测定方法,评价ZD1839和奥沙利铂不同给药顺序对A549细胞的抑制作用,以流式细胞仪测定不同联合方案对A549细胞周期分布及凋亡率的影响.观察ZD1839、奥沙利铂不同给药方案作用时,裸鼠A549细胞移植瘤生长情况,测定肿瘤生长抑制率.结果:先奥沙利铂后ZD1839的序贯给药方案表现出明显的协同作用,药物联合指数为0.51±0.01;而先ZD1839后给予奥沙利铂时,药物联合指数为1.56±0.03,两药间为拮抗作用.先奥沙利铂后ZD1839组G2/M期细胞比例与其他组相比明显增加,达到37.9%(P＜0.05),细胞凋亡率达到22.3%.体内实验表明,先奥沙利铂后ZD1839组抑瘤率最高,达到58.9%;而先奥沙利铂 ZD1839 24 h后ZD1839 48 h组的抑瘤率为52.4%,ZD1839后奥沙利铂组的抑瘤率为30.6%.结论:先奥沙利铂后ZD1839序贯给药对A549细胞增殖的抑制作用更强.     

Additive antitumour effect of the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib (Iressa, ZD1839) and the antioestrogen fulvestrant (Faslodex, ICI 182,780) in breast cancer cells

A high expression level of epidermal growth factor receptor (EGFR)/HER1 has been suggested to lead to a shorter survival time and resistance to endocrine therapy in patients with breast cancer. To test the hypothesis that inhibition of the EGFR signalling pathway affects the antitumour effect of endocrine therapy, an EGFR tyrosine kinase inhibitor (EGFR-TKI), gefitinib, and an oestrogen receptor (ER) antagonist, fulvestrant, were administered to human breast cancer cells. A total of five human breast cancer cell lines were used. The effects of single or combined treatments with gefitinib and/or fulvestrant on cell growth, cell cycle progression and apoptosis were analysed. Changes in the expression levels of cyclin-dependent kinase inhibitors, p21 and p27, an antiapoptotic factor, Bcl-2, and a proapoptotic factor, Bax, were also investigated. All cell lines tested were sensitive to gefitinib (50% growth inhibitory concentration, 10-28.5 microM). Breast cancer cell lines with a high expression level of HER1 or HER2 were more sensitive to gefitinib than the others. Gefitinib induced a significant G1-S blockade in ER-positive KPL-3C cells. Gefitinib induced significant apoptosis in HER1-overexpressing MDA-MB-231 cells. Gefitinib additively increased the antitumour effect of fulvestrant in all three ER-positive cell lines in a medium supplemented with 17beta-oestradiol. The combined treatment promoted cell cycle retardation in KPL-3C cells, which is associated with an upregulation of p21 by fulvestrant and gefitinib, respectively. Apoptosis was associated with downregulation of Bcl-2 by gefitinib in MDA-MB-231 cells. These results suggest an additive interaction between the EGFR-TKI gefitinib and the antioestrogen fulvestrant in ER-positive breast cancer cells.