Overexpression of Bcl2 abrogates chemo- and radiotherapy-induced sensitisation of NCI-H460 non-small-cell lung cancer cells to adenovirus-mediated expression of full-length TRAIL

TNF-related apoptosis-inducing ligand (TRAIL, also known as Apo-2L) is a promising novel anticancer agent that selectively induces apoptosis in tumour cells and the activity of which can be enhanced by combined treatment with chemo- or radiotherapy. For therapeutic purposes, the use of full-length TRAIL may be favourable to recombinant TRAIL based on its increased tumour cell killing potential, and the delivery of TRAIL at the tumour site by adenovirus vectors may provide an approach to overcome the short half-life of recombinant TRAIL and hepatocyte toxicity in vivo. Here, we constructed an adenoviral vector expressing full-length TRAIL (AdTRAIL) and studied the potential of chemo- and radiotherapy in enhancing AdTRAIL-induced apoptosis in non-small cell lung cancer (NSCLC) H460 cells and normal cells and, in addition, investigated the mechanism of AdTRAIL-induced apoptosis. AdTRAIL effectively killed H460 cells, which we previously showed to have a deficiency in mitochondria-dependent apoptosis by downstream activation of caspase-8 rather than caspase-9. Further analyses revealed that AdTRAIL induces death receptor- and mitochondria-dependent apoptosis that could be partially suppressed by Bcl2 overexpression. Combined treatment with doxorubicin (DOX), cisplatin (CDDP), paclitaxel (PTX) and radiation strongly enhanced AdTRAIL-induced cytotoxicity in a synergistic way. Synergy was accompanied by the cleavage of Bid and an increase in caspase-8 processing that was abolished by Bcl2 overexpression, indicating that the Bid-mitochondrial amplification loop is functional in H460 cells. Moreover, combination treatment did not alter the tumour selectivity of AdTRAIL since normal human fibroblasts (NHFs) remained resistant under these conditions. These findings further indicate that the combined use of chemo/radiotherapy and adenovirus-produced full-length TRAIL may provide a valuable treatment option for NSCLC.     

Tumor growth inhibition with cetuximab and chemotherapy in non-small cell lung cancer xenografts expressing wild-type and mutated epidermal growth factor receptor.

PURPOSE: Targeting the epidermal growth factor receptor (EGFR) is a validated approach to treat cancer. In non-small cell lung cancer (NSCLC), EGFR contains somatic mutations in 10% of patients, which correlates with increased response rates to small molecule inhibitors of EGFR. We analyzed the effects of the monoclonal IgG1 antibody Erbitux (cetuximab) in NSCLC xenografts with wild-type (wt) or mutated EGFR. EXPERIMENTAL DESIGN: NSCLC cell lines were grown s.c. in nude mice. Dose-dependent efficacy was established for cetuximab. To determine whether combination therapy produces tumor regressions, cetuximab was dosed at half-maximal efficacy with chemotherapy used at maximum tolerated dose. RESULTS: Cetuximab showed antitumor activity in wt (A549, NCI-H358, NCI-H292) and mutated [HCC-827 (delE746-A750), NCI-H1975 (L858R, T790M)] EGFR-expressing xenografts. In the H292 model, cetuximab and docetaxel combination therapy was more potent to inhibit tumor growth than cetuximab or docetaxel alone. Cisplatin augmented efficacy of cetuximab to produce 6 of 10 regressions, whereas 1 of 10 regressions was found with cetuximab and no regression was found with cisplatin. Using H1975 xenografts, gemcitabine increased efficacy of cetuximab resulting in 12 of 12 regressions. Docetaxel with cetuximab was more efficacious with seven of nine regressions compared with single treatments. Cetuximab inhibited autophosphorylation of EGFR in both H292 and H1975 tumor lysates. Exploring the underlying mechanism for combination effects in the H1975 xenograft model, docetaxel in combination with cetuximab added to the antiproliferative effects of cetuximab but was the main component in this drug combination to induce apoptosis. CONCLUSIONS: Cetuximab showed antitumor activity in NSCLC models expressing wt and mutated EGFR. Combination treatments increased the efficacy of cetuximab, which may be important for the management of patients with chemorefractory NSCLC.     

重组腺病毒TRAIL基因联合抗EGFR靶向药物对肺癌细胞增殖的影响

目的:观察重组腺病毒TRAIL基因制剂联合抗EGFR靶向药物对H460肺癌细胞株及裸鼠移植瘤增殖的影响。方法:重组腺病毒TRAIL基因治疗制剂分别与EGFR信号通路靶向治疗药物Iressa、Tarceva、以及C225联合应用于H460肺腺癌细胞株,采用MTT法以及流式细胞仪检测不同用药方案的抗肿瘤作用;在裸鼠H460肺癌模型中验证重组腺病毒TRAIL制剂与C225的协同抗肿瘤作用。结果:在体外实验中发现Iressa和Tarceva可以增加重组腺病毒TRAIL基因制剂对H460肺癌细胞的抗肿瘤作用(P<0.05);重组腺病毒TRAIL基因制剂在体外实验中与C225并不存在协同效应(P>0.05),但在裸鼠H460肺癌模型中重组腺病毒TRAIL基因制剂与C225有明显的协同抗肿瘤作用(P<0.05)。结论:本研究初步探讨了基因治疗与靶向治疗的联合抗肿瘤作用,实验发现EGFR信号通路上的靶向治疗药物包括小分子酪氨酸激酶抑制剂以及EGFR单克隆抗体均可以增加重组腺病毒TRAIL基因制剂抗肺癌作用。     

Aberrant epidermal growth factor receptor signaling and enhanced sensitivity to EGFR inhibitors in lung cancer

Epidermal growth factor receptor (EGFR) is occasionally amplified and/or mutated in non-small cell lung cancer (NSCLC) and can be coexpressed with other members of the HER receptor family to form functional heterodimers. We therefore investigated lung cancer cell lines for alterations in EGFR gene copy number, enhanced expression of EGFR and other HER family members, and EGFR coding sequence mutations and correlated these findings with response to treatment with the EGFR inhibitors and the kinetics of ligand-induced signaling. We show here that somatic deletions in the tyrosine kinase domain of EGFR were associated with increased EGFR gene copy number in NSCLC. Treatment with the specific EGFR tyrosine kinase inhibitors (TKI) gefitinib or erlotinib or the EGFR inhibitory antibody cetuximab induced apoptosis of HCC827, a NSCLC cell line with EGFR gene amplification and an exon 19 deletion. H1819, a NSCLC cell line that expresses high levels of EGFR, ErbB2, and ErbB3 but has wild-type EGFR, showed intermediate sensitivity to TKIs. In both cell lines, ligand-induced receptor tyrosine phosphorylation was delayed and prolonged and AKT was constitutively phosphorylated (but remained inhibitable by EGFR TKI). Thus, in addition to EGFR mutations, other factors in NSCLC cells, such as high expression of ErbB family members, may constitutively activate AKT and sensitize cells to EGFR inhibitors.     

Enhancement of the antitumor activity of ionising radiation by nimotuzumab, a humanised monoclonal antibody to the epidermal growth factor receptor, in non-small cell lung cancer cell lines of differing epidermal growth factor receptor status

The expression and activity of the epidermal growth factor receptor (EGFR) are determinants of radiosensitivity in several tumour types, including non-small cell lung cancer (NSCLC). However, little is known of whether genetic alterations of EGFR in NSCLC cells affect the therapeutic response to monoclonal antibodies (mAbs) to EGFR in combination with radiation. We examined the effects of nimotuzumab, a humanised mAb to EGFR, in combination with ionising radiation on human NSCLC cell lines of differing EGFR status. Flow cytometry revealed that H292 and Ma-1 cells expressed high and moderate levels of EGFR on the cell surface, respectively, whereas H460, H1299, and H1975 cells showed a low level of surface EGFR expression. Immunoblot analysis revealed that EGFR phosphorylation was inhibited by nimotuzumab in H292 and Ma-1 cells but not in H460, H1299, or H1975 cells. Nimotuzumab augmented the cytotoxic effect of radiation in H292 and Ma-1 cells in a clonogenic assay in vitro, with a dose enhancement factor of 1.5 and 1.3, respectively. It also enhanced the antitumor effect of radiation on H292 and Ma-1 cell xenografts in nude mice, with an enhancement factor of 1.3 and 4.0, respectively. Nimotuzumab did not affect the radioresponse of H460 cells in vitro or in vivo. Nimotuzumab enhanced the antitumor efficacy of radiation in certain human NSCLC cell lines in vitro and in vivo. This effect may be related to the level of EGFR expression on the cell surface rather than to EGFR mutation.     

Matuzumab and cetuximab activate the epidermal growth factor receptor but fail to trigger downstream signaling by Akt or Erk

Molecular inhibition of the epidermal growth factor receptor (EGFR) is a promising anticancer strategy, and monoclonal antibodies (mAbs) to EGFR are undergoing extensive evaluation in preclinical and clinical trials. However, the effects of anti-EGFR mAbs on EGFR signaling have remained unclear. We have now examined the effects of 2 anti-EGFR mAbs, matuzumab (EMD72000) and cetuximab (Erbitux), both of which are currently under assessment for treatment of various cancers, on EGFR signal transduction and cell survival in nonsmall cell lung cancer cell lines. Similar to EGF, matuzumab and cetuximab each induced phosphorylation of EGFR at several tyrosine phosphorylation sites as a result of receptor dimerization and activation of the receptor tyrosine kinase. In contrast to the effects of EGF, however, EGFR activation induced by these antibodies was not accompanied by receptor turnover or by activation of downstream signaling pathways that are mediated by Akt and Erk and are important for regulation of cell proliferation and survival. In addition, clonogenic survival assays revealed that matuzumab and cetuximab reduced the survival rate of H292 cells, in which they also inhibited the EGF-induced activation of Akt and Erk. Although we have examined only a few cell lines, our results indicate that the antitumor effects of matuzumab and cetuximab depend on inhibition of EGFR downstream signaling mediated by Akt or Erk rather than on inhibition of EGFR itself.     

Epidermal growth factor competes with EGF receptor inhibitors to induce cell death in EGFR-overexpressing tumor cells

Epidermal growth factor receptor (EGFR) signaling plays an important role in cell growth and differentiation. Mutations in the EGFR gene and EGFR gene amplifications have been associated with increased responsiveness to selective EGFR tyrosine kinase inhibitors (EGFR-TKIs). By contrast, EGF may also stimulate apoptosis in tumor cells, depending on EGFR and Her2 (erbB-2) expression levels. In the present study, we investigated cellular responses after EGFR activation by EGF, or inhibition by cetuximab and gefitinib. EGF treatment induced a near-immediate increase in p38 MAPK phosphorylation together with inactivation of ERK1/2. In contrast, gefitinib- and cetuximab-induced phosphorylation of p38 MAPK was much delayed, and gefitinib also induced a delayed activation of ERK1/2. EGF induced progressive cell death of A431 cells with prolonged treatment, whereas cetuximab- or gefitinib-treated cells showed temporary growth arrest and subsequent re-growth. Moreover, in combination treatment experiments, cetuximab or gefitinib competitively inhibited EGF-induced cell death. Normal WI38-VA13 cells did not display any noticeable changes in cell proliferation in response to EGF, gefitinib or cetuximab. EGF-induced death signaling is apparently irreversible: EGF induced significant EGFR phosphorylation/internalization and activated caspase-3, -8 and -9, effects that were not observed in cetuximab- or gefitinib-treated cells. Collectively, these results indicate that EGF may be a more potent cytotoxic agent than EGFR blockers in EGFR-overexpressing cancer cells.     

Implication of the insulin-like growth factor-IR pathway in the resistance of non-small cell lung cancer cells to treatment with gefitinib.

PURPOSE: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors have been found to be effective against lung cancer in vitro, but clinical resistance to these agents has developed as their usage has increased. In this study, we determined whether the insulin-like growth factor I (IGF-I) signaling pathway induces resistance of non-small cell lung cancer (NSCLC) cells to the EGFR tyrosine kinase inhibitor gefitinib. EXPERIMENTAL DESIGN: The effects of gefitinib and cetuximab on NSCLC cells, alone or with an IGF-I receptor (IGF-IR) inhibitor, were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the flow cytometry-based terminal nucleotidyl transferase-mediated nick end labeling assay, coimmunoprecipitation, and Western blot analysis. EGFR and IGFR expression in NSCLC tissues were examined by Western blot analysis. RESULTS: Gefitinib inhibited NSCLC cell proliferation by inducing apoptosis when IGF-IR signaling was suppressed. Treatment with gefitinib, but not cetuximab, induced EGFR:IGF-IR heterodimerization and activation of IGF-IR and its downstream signaling mediators, resulting in increased survivin expression in NSCLC cell lines with high levels of IGF-IR expression. Inhibition of IGF-IR activation and knockdown of survivin expression led to increased apoptosis. In contrast, overexpression of survivin protected cells with low IGF-IR expression from gefitinib-induced apoptosis. Most NSCLC tissues with EGFR overexpression had associated high levels of IGF-IR expression. CONCLUSIONS: IGF-IR expression may be useful as a predictive marker for gefitinib treatment of NSCLC. Suppression of IGF-IR signaling pathways may prevent or delay development of gefitinib resistance in patients with NSCLC.     

Dual-agent molecular targeting of the epidermal growth factor receptor (EGFR): combining anti-EGFR antibody with tyrosine kinase inhibitor

Molecular inhibition of epidermal growth factor receptor (EGFR/HER1) signaling is under active investigation as a promising cancer treatment strategy. We examined the potency of EGFR inhibition achieved by combining anti-EGFR monoclonal antibody and tyrosine kinase inhibitor, which target extracellular and intracellular domains of the receptor, respectively. We specifically studied the combination of cetuximab (Erbitux, C225; ImClone Systems, New York, NY) with either gefitinib (Iressa, ZD1839; AstraZeneca, Macclesfield, UK) or erlotinib (Tarceva, OSI-774; Genentech, South San Francisco, CA) across a variety of human cancer cells. The combination of cetuximab plus gefitinib or erlotinib enhanced growth inhibition over that observed with either agent alone. As measured by immunostaining, inhibition of EGFR phosphorylation with the combination of cetuximab plus gefitinib or erlotinib was augmented over that obtained with single-agent therapy in head and neck (H&N) cancer cell lines. Phosphorylation inhibition of downstream effector molecules [mitogen-activated protein kinase (MAPK) and AKT] also was enhanced in tumor cells treated with the combination of cetuximab plus gefitinib or erlotinib. Flow cytometry and immunoblot analysis demonstrated that treatment of H&N tumor cells with cetuximab in combination with either gefitinib or erlotinib amplified the induction of apoptosis. Following establishment of cetuximab-resistant cell lines, we observed that gefitinib or erlotinib retained the capacity to inhibit growth of lung and H&N tumor cells that were highly resistant to cetuximab. Treatment with gefitinib or erlotinib, but not cetuximab, also could further inhibit the activation of downstream effectors of EGFR signaling in cetuximab-resistant cells, including MAPK and AKT. These data suggest that tyrosine kinase inhibitors may further modulate intracellular signaling that is not fully blocked by extracellular anti-EGFR antibody treatment. Finally, animal studies confirmed that single EGFR inhibitor treatment resulted in partial and transient tumor regression in human lung cancer xenografts. In contrast, more profound tumor regression and regrowth delay were observed in mice treated with the combination of cetuximab and gefitinib or erlotinib. Immunohistochemical staining, which demonstrated significant reduction of the proliferative marker proliferating cell nuclear antigen in mice treated with dual EGFR inhibitors, further supported this in vivo observation. Together, these data suggest that combined treatment with distinct EGFR inhibitory agents can augment the potency of EGFR signaling inhibition. This approach suggests potential new strategies to maximize effective target inhibition, which may improve the therapeutic ratio for anti-EGFR-targeted therapies in developing clinical trials.     

New Molecular Targeted Therapies Integrated With Radiation Therapy in Lung Cancer

Non–small-cell lung cancer (NSCLC) accounts for approximately 80%-85% of all cases of lung cancer; for patients with stage III disease, it accounts for approximately 40% of all cases. The treatment for unresectable stage III NSCLC is the combination of platinum-based chemotherapy and thoracic radiation. In this article, new targeted agents under investigation for possible integration into the combined therapy are reviewed. One of the most promising strategies is the inhibition of the epidermal growth factor receptor (EGFR) pathway. Radiation activates EGFR signaling, leading to radio-resistance by inducing cell proliferation and enhanced DNA repair. Several preclinical models have shown synergistic activity when cetuximab was combined with radiation therapy. Some phase II trials have evaluated the safety and efficacy of synchronous cetuximab and radiation therapy with promising results. Gefitinib has a radiosensitizing effect on cell lines and has been investigated in combination with radiation therapy for unresectable stage III NSCLC. However, disappointing results were observed in the maintenance treatment with gefitinib after chemoradiation therapy. Erlotinib has been tested in a phase I trial with chemoradiation therapy. Radiation induces tumor death by damaging cell membranes, DNA, and microvascular endothelial cells, which in response increase proangiogenic growth factors. Antiangiogenic agents reduce vascular density but improve tumor oxygenation. Use of vascular endothelial growth factor receptor (VEGFR) inhibitors enhances the therapeutic efficacy of irradiation in human NSCLC by hindering the repair of sublethal radiation damage. Trials combining erlotinib and bevacizumab with thoracic radiation are ongoing. New strategies must be developed for the integration of this triple-combination treatment. As radiation therapy enhances HSP90 chaperone function, causing radio-resistant lung cancer cells, therapeutic agents that block this path are likely candidates for decreasing radio-resistance by suppressing HIF-1α and VEGF expression and thus inhibiting the survival and angiogenic potential of lung cancer cells. Aurora kinase inhibitors with radiation therapy seem to have an additive effect in preclinical models in NSCLC and mesothelioma.     

Primary resistance to cetuximab in a panel of patient-derived tumour xenograft models: activation of MET as one mechanism for drug resistance

Cetuximab (Erbitux®) targets the epidermal growth factor receptor (EGFR) and is approved for treatment of colorectal and head and neck cancer. Despite wide expression of EGFR, only a subgroup of cancer patients responds to cetuximab therapy. In the present study we assessed the cetuximab response in vivo of 79 human patient-derived xenografts originating from five tumour histotypes. We analysed basic tumour characteristics including EGFR expression and activation, mutational status of KRAS, BRAF and NRAS, the expression of EGFR ligands and the activation of HER3 (ErbB3) and the hepatocyte growth factor receptor MET. Based on these results, a cetuximab response score including positive and negative factors affecting therapeutic response is proposed. Positive factors are high expression and activation of EGFR and its ligands epiregulin or amphiregulin, negative factors are markers for downstream pathway activation independent of EGFR. In cetuximab resistant NSCL adenocarcinoma LXFA 526 and LXFA 1647, overexpression due to gene amplification and strong activation of MET was identified. Knock-down of MET by siRNA in the corresponding cell lines showed that anchorage-independent growth and migration are dependent on MET. MET knock down sensitized LXFA 526L and LXFA 1647L to EGF. Combined treatments of a MET inhibitor and cetuximab were additive. Therefore, combination therapy of cetuximab and a MET inhibitor in selected lung cancer patients could be of high clinical significance.     

An in vitro chemoresponse assay defines a subset of colorectal and lung carcinomas responsive to cetuximab

Cetuximab is a chimeric monoclonal antibody for the epidermal growth factor receptor (EGFR) that may provide benefit to select cancer patients; however, identification of the characteristics of those patients who may benefit from its use is not complete. The ChemoFx? drug response marker (DRM) is an in vitro assay that can provide drug response data on tumor specimens before any patient treatment is initiated. We determined the feasibility of using the ChemoFx DRM to test tumor samples for sensitivity to cetuximab. We exposed four non-small cell lung carcinoma (NSCLC) cell lines (H358, H520, HCC827, and H1666) to cetuximab and determined their sensitivity using the ChemoFx DRM and, in parallel, EGFR status using immunocytochemistry, Western blotting, and In-Cell Western (TM) analysis. We used the ChemoFx DRM to determine cetuximab sensitivity of primary NSCLC and colorectal tumor samples. The ChemoFx DRM distinguished between cetuximab-sensitive and -resistant cell lines. Cetuximab sensitivity was not dependent on EGFR mutational status; H358 cells were non-responsive to cetuximab yet contain wild-type EGFR, whereas H1666 cells were intermediately responsive to cetuximab and contain wild-type EGFR. HCC827 (EGFR-mutant) cells were intermediately responsive and, as expected, H520 cells (EGFR-null) were non-responsive to cetuximab. ChemoFx-determined cetuximab sensitivity of primary NSCLC and colorectal tumor samples was 9.0% and 7.5%, respectively. Use of the ChemoFx DRM is feasible for determining cetuximab sensitivity. The ChemoFx-determined cetuximab responses of primary NSCLC and colorectal tumor specimens were similar to published response rates of patients to treatment with cetuximab monotherapy.     

Autocrine growth stimulation by transforming growth factor-alpha in human non-small cell lung cancer.

We studied the biological response to and production of transforming growth factor-alpha (TGF-alpha) by the non-small cell lung carcinoma (NSCLC) clonal cell lines H226b, H322a, H460a, H596b. Each of these cell lines expressed epidermal growth factor receptor (EGFR) as determined by [125I]EGF competitive binding and Scatchard analysis and by phosphorylation. The receptors were functionally active as determined in immune complex kinase assays. H322a, H226b, H460a, and H596b cells showed stimulated [3H]thymidine (Thd) uptake in response to TGF-alpha. Exogenously added TGF-alpha increased colony formation in soft agar for three of the cell lines in media containing serum. All cell lines expressed TGF-alpha detected by immunohistochemistry and TGF-alpha mRNA, although to differing degrees. Cell lysates and spent media competed for EGFR binding with EGF, thus demonstrating production of TGF-alpha-like activity. The anti-TGF-alpha monoclonal antibody AB-3 inhibited the uptake of [3H]Thd by proliferating H322a and H226b cells but not H460a and H596b cells. No inhibition occurred with MOPC21 antibody and inhibition was completely reversed by addition of TGF-alpha to the culture. Suramin inhibited cell proliferation and [3H]Thd uptake by all cell lines. Inhibition of H460a and H596b cells was reversed with exogenous TGF-alpha but not PDGF. Our data suggests that TGF-alpha is a mediator of autocrine growth stimulation for NSCLC cells, and that for some NSCLC cells cytoplasmic binding of receptor and ligand is the primary mechanism for autocrine growth stimulation.     

Antitumor activity of ZD6474, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, in human cancer cells with acquired resistance to antiepidermal growth factor receptor therapy.

PURPOSE: The epidermal growth factor receptor (EGFR) autocrine signaling pathway is involved in cancer development and progression. EGFR inhibitors such as C225 (cetuximab), a chimeric human-mouse anti-EGFR monoclonal antibody, and ZD1839 (gefitinib), a small molecule EGFR-selective tyrosine kinase inhibitor, are in advanced clinical development. The potential emergence of cancer cell resistance in EGFR-expressing cancers treated with EGFR inhibitors could determine lack of activity of these drugs in some cancer patients. Vascular endothelial growth factor (VEGF) is secreted by cancer cells and plays a key role in the regulation of tumor-induced endothelial cell proliferation and permeability. ZD6474 is a small molecule VEGF flk-1/KDR (VEGFR-2) tyrosine kinase inhibitor that also demonstrates inhibitory activity against EGFR tyrosine kinase. EXPERIMENTAL DESIGN: The antitumor activity of ZD1839, C225, and ZD6474 was tested in athymic mice bearing human GEO colon cancer xenografts. GEO cell lines resistant to EGFR inhibitors were established from GEO xenografts growing in mice treated chronically with ZD1839 or C225. Expression of EGFR was evaluated by flow cytometry. Expression of various proteins involved in intracellular cell signaling was assessed by Western blotting. Tumor growth data were evaluated for statistical significance using the Student's t test. All Ps were two-sided. RESULTS: Although chronic administration of optimal doses of C225 or ZD1839 efficiently blocked GEO tumor growth in the majority of mice, tumors slowly started to grow within 80-90 days, despite continuous treatment. In contrast, continuous treatment of mice bearing established GEO xenografts with ZD6474 resulted in efficient tumor growth inhibition for the entire duration of dosing (up to 150 days). ZD6474 activity was also determined in mice pretreated with ZD1839 or C225. When GEO growth was apparent after 4 weeks of treatment with EGFR inhibitors, mice were either re-treated with EGFR inhibitors or treated with ZD6474. GEO tumor growth was blocked only in mice treated with ZD6474, whereas tumor progression was observed in mice re-treated with C225 or ZD1839. GEO tumors growing during treatment with C225 or with ZD1839 were established as cell lines (GEO-C225-RES and GEO-ZD1839-RES, respectively). Cell membrane-associated EGFR expression was only slightly reduced in these cell lines compared with parental GEO cells. Western blotting revealed no major change in the expression of the EGFR ligand transforming growth factor alpha of bcl-2, bcl-xL, p53, p27, MDM-2, akt, activated phospho-akt, or mitogen-activated protein kinase. However, both GEO-C225-RES and GEO-ZD1839-RES cells exhibited a 5-10-fold increase in activated phospho-mitogen-activated protein kinase and in the expression of cyclooxygenase-2 and of VEGF compared with GEO cells. GEO-C225-RES and GEO-ZD1839-RES growth as xenografts in nude mice was not significantly affected by treatment with either C225 or ZD1839 but was efficiently inhibited by ZD6474. CONCLUSIONS: Long-term treatment of GEO xenografts with selective EGFR inhibitors results in the development of EGFR inhibitor-resistant cancer cells. Growth of EGFR inhibitor-resistant tumors can be inhibited by ZD6474. These data indicate that inhibition of VEGF signaling has potential as an anticancer strategy, even in tumors that are resistant to EGF inhibitors.     

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines

Background

EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF.

Methods

Cell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A.

Results

Caco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment. In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases.

Conclusion

This is the first study to have systematically investigated the effect of cetuximab or gefitinib, alone and in combination with EGF, on human colon cancer cell lines. The EGFR inhibitors have a weaker effect in the presence of EGF that binds EGFR. Cetuximab treatment showed an expression pattern that inversely correlates with EGF treatment. We found interesting cyto-morphological features closely relating to gene expression profile. Both drugs have an effect on differentiation towards cellular death.     

The novel thymidylate synthase inhibitor trifluorothymidine (TFT) and TRAIL synergistically eradicate non-small cell lung cancer cells

Purpose

TRAIL, a tumor selective anticancer agent, may be used for the treatment of non-small cell lung cancer (NSCLC). However, TRAIL resistance is frequently encountered. Here, the combined use of TRAIL with trifluorothymidine (TFT), a thymidylate synthase inhibitor, was examined for sensitizing NSCLC cells to TRAIL.

Methods

Interactions between TRAIL and TFT were studied in NSCLC cells using growth inhibition and apoptosis assays. Western blotting and flow cytometry were used to investigate underlying mechanisms.

Results

The combined treatment of TFT and TRAIL showed synergistic cytotoxicity in A549, H292, H322 and H460 cells. For synergistic activity, the sequence of administration was important; TFT treatment followed by TRAIL exposure did not show sensitization. Combined TFT and TRAIL treatment for 24 h followed by 48 h of TFT alone was synergistic in all cell lines, with combination index values below 0.9. The treatments affected cell cycle progression, with TRAIL inducing a G1 arrest and TFT, a G2/M arrest. TFT activated Chk2 and reduced Cdc25c levels known to cause G2/M arrest. TRAIL-induced caspase-dependent apoptosis was enhanced by TFT, whereas TFT alone mainly induced caspase-independent death. TFT increased the expression of p53 and p21/WAF1, and p53 was involved in the increase of TRAIL-R2 surface expression. TFT also caused downregulation of cFLIP and XIAP and increased Bax expression.

Conclusions

TFT enhances TRAIL-induced apoptosis in NSCLC cells by sensitizing the apoptotic machinery at different levels in the TRAIL pathway. Our findings suggest a possible therapeutic benefit of the combined use of TFT and TRAIL in NSCLC.     

EGF activates intracellular and intercellular calcium signaling by distinct pathways in tumor cells

Epidermal growth factor (EGF)-mediated Ca2+ signaling in multiple cell lines derived from human gliomas and in the A431 epidermoid carcinoma cell line was observed using fluorescence videomicroscopy. Bath application of EGF evoked an oscillatory increase in [Ca2+]i in 4 different human glioma cell lines as well as the A431 cell line. This effect was blocked by the EGF receptor tyrosine kinase inhibitors gefitinib and erlotinib, as well as by the EGFR antibody cetuximab. In addition to this acute Ca2+ signaling response, transient exposure to EGF also potentiated subsequent Ca2+ signaling responses to other stimuli. Tumor cells transiently exposed to EGF (5 minutes), showed a sustained increase in propagation of intercellular Ca2+ waves, which have been previously shown to involve release of ATP and activation of purinergic receptors. Cells transiently exposed to EGF also showed a sustained potentiation of the Ca2+ signaling response to ATP. In contrast to the acute Ca2+ signaling response to EGF, this sustained potentiation of purinergic intercellular signaling was not blocked by gefitinib or erlotinib, while it was blocked by cetuximab. These results indicate that while the acute Ca2+ signaling response requires tyrosine kinase activation, the sustained potentiation of intercellular signaling occurs via a distinct pathway. Distinct intra- and intercellular Ca2+ signaling pathways may be mechanisms by which EGF modulates the growth and migration of tumor cells.     

Lack of AKT activation in lung cancer cells with EGFR mutation is a novel marker of cetuximab sensitivity

Epidermal growth factor receptor (EGFR) mutation is the best marker of sensitivity to the EGFR tyrosine kinase inhibitor gefitinib, but a marker for the anti-EGFR antibody cetuximab has not been identified in lung cancer. The present study investigated markers for sensitivity to cetuximab. Sensitivity to cetuximab and gefitinib was compared with EGFR expression, EGFR and KRAS mutation, and EGFR gene copy numbers in lung cancer cell lines. We also studied the effect of these agents on the activation of EGFR, ERK, AKT, and STAT3 in cetuximab-sensitive and -resistant cell lines. We found one cetuximab-sensitive cell line with EGFR mutation among 19 lung cancer cell lines. Analysis of molecules downstream from EGFR revealed that AKT phosphorylation was suppressed in this cell line. Augmentation of AKT phosphorylation by transfection of a plasmid induced resistance to cetuximab. Acquisition of cetuximab resistance was associated with AKT activation in this cell line, while pharmacological inhibition of AKT markedly enhanced the growth inhibitory effect of cetuximab. Dephosphorylation of AKT in association with EGFR mutation is a candidate marker for sensitivity to cetuximab, and combined use of an AKT pathway inhibitor with cetuximab could be a novel therapeutic strategy for lung cancer.