Cytotoxic effects toward human hematopoietic progenitor cells and tumor cell lines of paclitaxel, docetaxel, and newly developed analogues IDN5109, IDN5111, and IDN5127

The growth inhibitory effect of paclitaxel, docetaxel, and newly developed taxanes IDN5109, IDN5111, and IDN5127 was assessed on peripheral blood (PB) CD34+ maintained in liquid culture and on three human cancer cell lines (MDA-MB231, MCF-7 ADRr, CEM VBLr). Concomitantly, DNA analysis was also performed. For unfractionated peripheral blood progenitor cells (PBPC) toxicity was also assessed by clonogenic assay. The cytotoxic effects induced by taxanes toward PBPC as measured by clonogenic assay were correlated with those found for multidrug resistance (MDR)-positive cell lines (IDN5109 > IDN5111 > IDN5127 > docetaxel > paclitaxel). We established a therapeutic index (TI) between the antitumor activity in MDR-positive cells and the toxicity toward PBPC. Paclitaxel and IDN5109, as determined by TI, showed the best value in MDR-negative and MDR-positive cells, respectively. The ranking of the cytotoxic effects observed in PB CD34+ was not correlated with that obtained in clonogenic assay and in cancer cells (IDN5127 > IDN5109 > docetaxel > IDN5111). Remarkably, in DNA analysis docetaxel induced the maximal cell cycle blocking activity. Newly developed taxanes IDN5109 and IDN5111 are endowed of a profile of anticancer activity in MDR-bearing cells and toxicity toward hematopoietic progenitors better than that of docetaxel. However, mechanism(s) underlying toxicity toward hematopoietic progenitors could be, at least in part, different from that of docetaxel and likely dependent on the interaction with P-glycoprotein function in PB CD34+ cells.     

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.     

Antitumor effects of ZD6474, a small molecule vascular endothelial growth factor receptor tyrosine kinase inhibitor, with additional activity against epidermal growth factor receptor tyrosine kinase.

PURPOSE: Vascular endothelial growth factor (VEGF) is a major mitogen for endothelial cells and enhances vascular permeability. Enhanced VEGF secretion is found in human cancers and correlates with increased tumor neovascularization. ZD6474 is a p.o. bioavailable, VEGF flk-1/KDR receptor (VEGFR-2) tyrosine kinase inhibitor with antitumor activity in many human cancer xenografts and is currently in Phase I clinical development. EXPERIMENTAL DESIGN: We tested the effects of ZD6474 on EGFR phosphorylation in cell expressing functional epidermal growth factor receptor (EGFR) and the antiproliferative and the proapoptotic activity of ZD6474 alone or in combination taxanes in human cancer cell lines with functional EGFR but lacking VEGFR-2. The antitumor activity of this drug was also tested in nude mice bearing established GEO colon cancer xenografts. RESULTS: ZD6474 causes a dose-dependent inhibition of EGFR phosphorylation in mouse NIH-EGFR fibroblasts and human MCF-10A ras breast cancer cells, two cell lines that overexpress the human EGFR. ZD6474 treatment resulted in a dose-dependent inhibition of soft agar growth in seven human cell lines (breast, colon, gastric, and ovarian) with functional EGFR but lacking VEGFR-2. A dose-dependent supra-additive effect in growth inhibition and in apoptosis in vitro was observed by the combined treatment with ZD6474 and paclitaxel or docetaxel. ZD6474 treatment of nude mice bearing palpable GEO colon cancer xenografts (which are sensitive to inhibition of EGFR signaling) induced dose-dependent tumor growth inhibition. Immunohistochemical analysis revealed a significant dose-dependent reduction of neoangiogenesis. The antitumor activity of ZD6474 in GEO tumor xenografts was also found to be enhanced when combined with paclitaxel. Tumor regression was observed in all mice after treatment with ZD6474 plus paclitaxel, and it was accompanied by a significant potentiation in inhibition of angiogenesis. Six of 20 mice had no histological evidence of tumors after treatment with ZD6474 plus paclitaxel. CONCLUSIONS: This study suggests that in addition to inhibiting endothelial cell proliferation by blocking VEGF-induced signaling, ZD6474 may also be able to inhibit cancer cell growth by blocking EGFR autocrine signaling. These results provide also a rationale for the clinical evaluation of ZD6474 combined with taxanes in cancer patients.     

Decreased response to paclitaxel versus docetaxel in HER-2/neu transfected human breast cancer cells

Taxanes are effective in the treatment of metastatic breast cancer. Docetaxel has been shown to be more potent than paclitaxel in inducing bcl-2 phosphorylation and apoptosis and is clinically active in some paclitaxel-resistant breast tumors. HER-2/neu overexpression has been shown to correlate with resistance to hormonal therapy as well as chemotherapy. Using a HER-2/neu transfected MCF-7 human breast cancer cell line, we investigated the role of HER-2/neu overexpression on resistance to paclitaxel and docetaxel treatment. A control vector transfected MCF-7 human breast cancer cell line (MCF/neo) and a HER-2/neu transfected MCF-7 line (MCF/18) were treated with various concentrations of docetaxel or paclitaxel. Cell number was assessed using the MTT tetrazolium dye assay. In the control vector transfected MCF/neo cell line, paclitaxel and docetaxel gave similar dose-dependent growth inhibition ( p = 0.175). In HER-2/neu transfected MCF/18 cells, docetaxel treatment resulted in a dose-dependent inhibition similar to that seen in MCF/neo cells. Paclitaxel, however, gave significantly less growth inhibition than docetaxel in the HER-2/neu overexpressing MCF/18 cells (p = 0.0003). These data suggest that HER-2/neu overexpression may contribute to paclitaxel resistance. In contrast, the cytotoxic effects of docetaxel in these breast carcinoma cells are not affected by HER-2/neu expression. Therefore, docetaxel may be the preferred taxane therapy in HER-2/neu overexpressing breast tumors.     

Oral administration of a novel taxane, an antisense oligonucleotide targeting protein kinase A, and the epidermal growth factor receptor inhibitor Iressa causes cooperative antitumor and antiangiogenic activity.

PURPOSE: Protein kinase A type I (PKAI) and the epidermal growth factor receptor (EGFR) play a role in neoplastic transformation and interact with each other in transducing mitogenic signals. We developed different PKAI and EGFR inhibitors, demonstrating their cooperation with cytotoxic drugs and the therapeutic potential of the combined blockade of PKAI and EGFR. In this study, we investigated the effect of orally active PKAI and EGFR inhibitors in combination with a novel taxane. EXPERIMENTAL DESIGN: We combined a hybrid PKAI antisense oligonucleotide sequence (AS-PKAI), the EGFR inhibitor ZD1839 (Iressa), and the taxane IDN5109, studying their effect on human cancer growth, apoptosis, and angiogenesis and measuring vascular endothelial growth factor (VEGF) expression and vessel formation in vitro and after oral administration in nude mice. RESULTS: We demonstrated cooperative growth inhibitory and proapoptotic effects and inhibition of VEGF expression with any combination of two drugs and a marked synergistic effect when all three agents were combined. Oral administration of AS-PKAI, ZD1839, and IDN5109 in combination to nude mice caused a remarkable antitumor effect with no histological evidence of tumors in 50% of mice 5 weeks after treatment withdrawal, accompanied by complete suppression of vessel formation and VEGF expression. CONCLUSION: This is the first demonstration of the cooperative antitumor and antiangiogenic activity of three novel agents that block multiple signaling pathways after oral administration. Because all agents are under clinical evaluation in cancer patients, our results provide a rationale to translate this feasible therapeutic strategy in a clinical setting.     

Enhancement of antitumor activity of ionizing radiation by combined treatment with the selective epidermal growth factor receptor-tyrosine kinase inhibitor ZD1839 (Iressa).

PURPOSE: The epidermal growth factor receptor (EGFR) is expressed in the majority of human epithelial cancers and has been implicated in the development of cancer cell resistance to cyotoxic drugs and to ionizing radiation. Experimental Design: We used ZD1839, a selective small molecule EGFR tyrosine kinase inhibitor currently in clinical development. We tested the antiproliferative and the proapoptotic activity of ZD1839 in combination with ionizing radiation in human colon (GEO), ovarian (OVCAR-3), non-small cell lung (A549 and Calu-6), and breast (MCF-7 ADR) cancer cell lines. The antitumor activity of this combination was also tested in nude mice bearing established GEO colon cancer xenografts. RESULTS: With ionizing radiation or ZD1839, a dose-dependent growth inhibition was observed in all of the cancer cell lines growing in soft agar. A cooperative antiproliferative and proapoptotic effect was obtained when cancer cells were treated with ionizing radiation followed by ZD1839. This effect was accompanied by inhibition in the expression of the antiapoptotic proteins bcl-xL and bcl-2, and by a suppression of the activated (phosphorylated) form of akt protein. Treatment of mice bearing established human GEO colon cancer xenografts with radiotherapy (RT) resulted in a dose-dependent tumor growth inhibition that was reversible upon treatment cessation. Long term GEO tumor growth regressions were obtained after RT in combination with ZD1839. This resulted in a significant improvement in survival of these mice as compared with the control group (P < 0.001), the RT-treated group (P < 0.001), or the ZD1839-treated group (P < 0.001). The only mice alive 10 weeks after tumor cell injection were in the RT-plus-ZD1839 group. Furthermore, 10% of mice in this group were alive and tumor-free after 26 weeks. Similar results were obtained in mice bearing established human A549 lung adenocarcinoma xenografts. Finally, the combined treatment with RT plus ZD1839 was accompanied by a significant potentiation in the inhibition of transforming growth factor alpha, vascular epidermal growth factor, and basic fibroblast growth factor expression in cancer cells, which resulted in significant antiangiogenic effects as determined by immunohistochemical count of neovessels within the GEO tumors. CONCLUSION: This study provides a rationale for evaluating in cancer patients the combination of ionizing radiation and selective EGFR tyrosine kinase inhibitors such as ZD1839.     

ZD1839, a specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, induces the formation of inactive EGFR/HER2 and EGFR/HER3 heterodimers and prevents heregulin signaling in HER2-overexpressing breast cancer cells.

PURPOSE: ZD1839 is a tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR) that has shown clinical activity against EGFR-expressing tumors. Our aim was to explore the effects of ZD1839 in breast cancer cell lines expressing different levels of EGFR and the closely related HER2 receptor. EXPERIMENTAL DESIGN: We studied the growth-inhibitory effects of ZD1839 in a series of breast carcinoma cell lines. In HER2-overexpressing BT-474 breast cancer cells, we studied the effects of ZD1839 on cell growth and heterodimerization of receptors under basal and ligand-stimulated conditions. RESULTS: ZD1839 was an equally potent inhibitor of growth in breast cancer cells expressing high levels of EGFR and HER2. In BT-474 breast cancer cells, ZD1839 abolished EGF- and heregulin-induced activation of ErbB receptors and downstream signaling molecules. Because ZD1839 does not inhibit the HER2 tyrosine kinase in vitro, and because heregulin is a ligand that activates HER2 by binding to HER3 and HER4 but does not bind to the EGFR, our findings suggested that ZD1839 interfered with HER2 function in intact cells. Searching for mechanisms, we report that ZD1839 induces the formation of inactive unphosphorylated EGFR/HER2 and EGFR/HER3 heterodimers. Furthermore, ZD1839 completely abolishes basal and heregulin-induced formation of active phosphorylated HER2/HER3 heterodimers. CONCLUSIONS: ZD1839 inhibits the growth of HER2-overexpressing breast cancer cells, possibly by sequestration of HER2 and HER3 receptors in an inactive heterodimer configuration with the EGFR. Our findings suggest that there is a strong rationale to conduct clinical trials of ZD1839 in patients with HER2-overexpressing breast tumors.     

Efficacy of cytotoxic agents against human tumor xenografts is markedly enhanced by coadministration of ZD1839 (Iressa), an inhibitor of EGFR tyrosine kinase.

The blockade of epidermal growth factor receptor (EGFR) function with monoclonal antibodies has major antiproliferative effects against human tumors in vivo. Similar antiproliferative effects against some of these same tumors have also been observed with specific inhibitors of the EGFR-associated tyrosine kinase. One such inhibitor, the p.o. active ZD1839 (Iressa), has pronounced antiproliferative activity against human tumor xenografts. We now show that coadministration of ZD1839, as with anti-EGFR, will enhance the efficacy of cytotoxic agents against human vulvar (A431), lung (A549 and SK-LC-16 NSCL and LX-1), and prostate (PC-3 and TSU-PR1) tumors. Oral ZD1839 (five times daily x 2) and cytotoxic agents (i.p. every 3-4 days x 4) were given for a period of 2 weeks to mice with well-established tumors. On this schedule, the maximum tolerated dose (150 mg/kg) of ZD1839 induced partial regression of A431, a tumor that expresses high levels of EGFR, 70-80% inhibition among tumors with low but highly variable levels of EGFR expression (A549, SKLC-16, TSU-PR1, and PC-3), and 50-55% inhibition against the LX-1 tumor, which expresses very low levels of EGFR. ZD1839 was very effective in potentiating most cytotoxic agents in combination treatment against all of these tumors, irrespective of EGFR status, but dose reduction of ZD1839 below its single-agent maximum tolerated dose was required for optimum tolerance. The pronounced growth inhibitory action of the platinums, cisplatin and carboplatinum, as single agents against A431 vulvar, A549 and LX-1 lung, and TSU-PR1 and PC-3 prostate tumors was increased several-fold when ZD1839 was added, with some regression of A431 and PC-3 tumors. Although the taxanes, paclitaxel or docetaxel, as single agents markedly inhibited the growth of A431, LX-1, SK-LC-16, TSU-PR1, and PC-3, when combined with ZD1839, partial or complete regression was usually seen. Against A549, the growth inhibition of doxorubicin was increased 10-fold (>99%) with ZD1839. The folate analogue, edatrexate, was highly growth inhibitory against A549, LX-1, and TSU-PR1, whereas edatrexate combined with ZD1839 resulted in partial or complete regression in these tumors. Against the A431 tumor, paclitaxel alone either was highly growth inhibitory or induced some regression, but when combined with ZD1839, pronounced regression was obtained. Combination with gemcitabine neither added nor detracted from baseline cytotoxic efficacy, whereas ZD1839 combined with vinorelbine was poorly tolerated. Overall, these results suggest that potentiation of cytotoxic treatment with ZD1839 does not require high levels of EGFR expression in the target tumors. They also suggest significant clinical benefit from ZD1839 in combination with a variety of widely used cytotoxic agents.     

Combined targeted inhibition of bcl-2, bcl-XL, epidermal growth factor receptor, and protein kinase A type I causes potent antitumor, apoptotic, and antiangiogenic activity.

PURPOSE: This study investigated whether the functional and structural interactions between epidermal growth factor receptor (EGFR), protein kinase AI (PKAI), and bcl-2/bcl-xL could be exploited to obtain cooperative antitumor effects against models of human colon and breast cancer. EXPERIMENTAL DESIGN: Antisense bcl-2/bcl-xL (4625), antisense PKAI (AS-PKAI), and ZD1839 ("Iressa"), a selective EGFR tyrosine kinase inhibitor, were administered as single agents and in combination against GEO colon and ZR-75-1 breast cancer cell lines in vitro and to mice bearing s.c. GEO human tumor xenografts in vivo. Effects on growth inhibition, vascular endothelial growth factor secretion, and induction of apoptosis were assessed. RESULTS: Antisense bcl-2/bcl-xL inhibited the growth of GEO and ZR-75-1 cells in vitro, reducing bcl-2 and bcl-xL expression and vascular endothelial growth factor secretion. Supra-additive growth inhibition and apoptosis induction were observed when 4625 was combined with ZD1839 or AS-PKAI. Combining all three agents resulted in a complete growth inhibitory effect in vitro. Antisense bcl-2/bcl-xL, AS-PKAI, and ZD1839 administered in vivo as single agents caused growth inhibition of GEO xenografts. Combining all three agents caused a marked and sustained effect, with 50% growth inhibition and 50% of mice tumor free 5 weeks after treatment withdrawal. The combination was well tolerated. CONCLUSIONS: The combination of 4625, AS-PKAI, and ZD1839 resulted in a strong antiproliferative, proapoptotic, and antiangiogenic response, suggestive of a functional interaction between EGFR, PKAI, and bcl-2/bcl-xL and providing a rationale for the selection of specific molecular treatments for the development of therapeutic strategies. Iressa is a trademark of the AstraZeneca group of companies.     

Anti-tumour activity of a panel of taxanes toward a cellular model of human cervical cancer

Using a model of human cervical cancer (ME-180 cells), the anti-tumour activity of paclitaxel was compared to that of docetaxel and IDN5109, a newly developed taxane. The growth inhibition effect of taxanes was assessed after 3 days of exposure. DNA analysis, the taxane-dependent modulation of the expression of the α and β subunits of tubulin and DNA fragmentation were assessed by flow cytometry. The presence of apoptosis was confirmed by morphological analysis using a laser scan cytometer. For the evaluation of “in vivo” anti-tumour activity, taxanes were administered to nude mice intravenously once daily, according to a q3/4d × 4 schedule. Docetaxel, IDN5109 and paclitaxel obtained “in vitro” IC₅₀ values of 0.86, 1.4 and 2.4 nM, respectively. DNA analysis demonstrated a transient block at the G₂/M phase of the cell cycle only after 12 h of culture in the presence of taxanes and an increase of nuclear fragmentation suggestive for apoptosis after additional 12 and 60 h of exposure. Morphological analysis confirmed the presence of apoptosis. Taxanes induced a down-modulation of the α subunit of tubulin in the G_0/1 phase of the cell cycle, and an overexpression of the β subunit in the G₂/M phase. A strong anti-tumour activity was obtained “in vivo” for nude mice xenografted using ME-180 cells (T/C=0% for all drugs). These data indicate that the three taxanes are strongly active both “in vitro” and “in vivo” toward ME-180 cells. Clinical studies are now needed to ascertain if the higher anti-tumour activity observed “in vitro” using docetaxel and IDN5109 yields a better clinical response in advanced cervical carcinoma with respect to paclitaxel. Received: 4 May 1999 / Accepted: 9 July 1999     

Epidermal growth factor receptor (HER1) tyrosine kinase inhibitor ZD1839 (Iressa) inhibits HER2/neu (erbB2)-overexpressing breast cancer cells in vitro and in vivo.

Aberrrant signaling by the epidermal growth factor receptor [EGFR (HER1, erbB1)] and/or HER2/neu tyrosine kinases is present in a cohort of breast carcinomas. Because HER2 is constitutively phosphorylated in some breast tumors, we speculated that, in these cancers, transmodulation of HER2 may occur via EGFR signaling. To test this possibility, we examined the effect of EGFR-specific kinase inhibitors against the HER2-overexpressing human breast tumor lines BT-474, SKBR-3, MDA-361, and MDA-453. ZD1839 (Iressa) is an ATP-mimetic that inhibits the purified EGFR and HER2 kinases in vitro with an IC(50) of 0.033 and >3.7 microM, respectively. The specificity of ZD1839 against EGFR was confirmed in Rat1 fibroblasts transfected with EGFR or HER2 chimeric receptors activated by synthetic ligands without the interference of endogenous receptors. Treatment of all breast cancer cell lines (except MDA-453) with 1 microM ZD1839 almost completely eliminated HER2 phosphorylation. In contrast, the incorporation of [gamma-(32)P]ATP in vitro onto HER2 receptors isolated from BT-474 cells was unaffected by 1 microM ZD1839. EGFR is expressed by BT-474, SKBR-3, and MDA-361 but not by MDA-453 cells, suggesting that ZD1839-mediated inhibition of the EGFR kinase explained the inhibition of HER2 phosphorylation in vivo. In SKBR-3 cells, ZD1839 exhibited a greater growth-inhibitory effect than Herceptin, a monoclonal antibody against the HER2 ectodomain. In both SKBR-3 and BT-474 cells, treatment with ZD1839 plus Herceptin induced a greater apoptotic effect than either inhibitor alone. Finally, ZD1839 completely prevented growth of BT-474 xenografts established in nude mice and enhanced the antitumor effect of Herceptin. These data imply that EGFR tyrosine kinase inhibitors will be effective against HER2-overexpressing breast tumor cells that also express EGFR and support their use in combination with HER2 antibodies, such as Herceptin, against mammary carcinomas with high levels of the HER2 proto-oncogene.     

Antitumor effect and potentiation of cytotoxic drugs activity in human cancer cells by ZD-1839 (Iressa), an epidermal growth factor receptor-selective tyrosine kinase inhibitor.

Transforming growth factor alpha (TGF-alpha) is an autocrine growth factor for human cancer. Overexpression of TGF-alpha and its specific receptor, the epidermal growth factor receptor (EGFR), is associated with aggressive disease and poor prognosis. The EGFR has been proposed as a target for anticancer therapy. Compounds that block ligand-induced EGFR activation have been developed. ZD-1839 (Iressa) is a p.o.-active, quinazoline derivative that selectively inhibits the EGFR tyrosine kinase and is under clinical development in cancer patients. The antiproliferative activity of ZD-1839 alone or in combination with cytotoxic drugs differing in mechanism(s) of action, such as cisplatin, carboplatin, oxaliplatin, paclitaxel, docetaxel, doxorubicin, etoposide, topotecan, and raltitrexed, was evaluated in human ovarian (OVCAR-3), breast (ZR-75-1, MCF-10A ras), and colon cancer (GEO) cells that coexpress EGFR and TGF-alpha. ZD-1839 inhibited colony formation in soft agar in a dose-dependent manner in all cancer cell lines. The antiproliferative effect was mainly cytostatic. However, treatment with higher doses resulted in a 2-4-fold increase in apoptosis. A dose-dependent supra-additive increase in growth inhibition was observed when cancer cells were treated with each cytotoxic drug and ZD-1839. The combined treatment markedly enhanced apoptotic cell death induced by single-agent treatment. ZD-1839 treatment of nude mice bearing established human GEO colon cancer xenografts revealed a reversible dose-dependent inhibition of tumor growth because GEO tumors resumed the growth rate of controls at the end of the treatment. In contrast, the combined treatment with a cytotoxic agent, such as topotecan, raltitrexed, or paclitaxel, and ZD-1839 produced tumor growth arrest in all mice. Tumors grew slowly for approximately 4-8 weeks after the end of treatment, when they finally resumed a growth rate similar to controls. GEO tumors reached a size not compatible with normal life in all control mice within 4-6 weeks and in all single agent-treated mice within 6-8 weeks after GEO cell injection. In contrast, 50% of mice treated with ZD-1839 plus topotecan, raltitrexed, or paclitaxel were still alive 10, 12, and 15 weeks after cancer cell injection, respectively. These results demonstrate the antitumor effect of this EGFR-selective tyrosine kinase inhibitor and provide a rationale for its clinical evaluation in combination with cytotoxic drugs.     

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.     

A novel taxane with improved tolerability and therapeutic activity in a panel of human tumor xenografts

Clinically available taxanes represent one of the most promising class of antitumor agents, despite several problems with their solubility and toxicity. In an attempt to improve the pharmacological profile of taxanes, a new series of analogues was synthesized from 14beta-hydroxy-10-deacetylbaccatin III and tested in a panel of human tumor cell lines. On the basis of the pattern of cytotoxicity and lack of cross-resistance in tumor cell lines expressing the typical multidrug-resistant phenotype, a compound (IDN5109) was selected for preclinical development. A comparative efficacy study of IDN5109 and paclitaxel was performed using a large panel of human tumor xenografts, characterized by intrinsic (seven tumors) or acquired (four tumors) resistance to cisplatin or doxorubicin, including four ovarian, one breast, one cervical, three lung, one colon, and one prostatic carcinoma. Drugs were delivered i.v. according to the same schedule (four times every 4th day). IDN5109 achieved a very high level of activity (percentage tumor weight inhibition >70%; log10 cell kill >1) in all but one of the tested tumors. Compared to paclitaxel, IDN5109 exhibited a significantly superior activity in six tumors (including the four tumors that were resistant to paclitaxel) and a comparable activity against the other five paclitaxel-responsive tumors. Additional advantages of IDN5109 over paclitaxel were also suggested by its toxicity profile. IDN5109 was not only less toxic (maximal tolerated doses were 90 and 54 mg/kg for IDN5109 and paclitaxel, respectively), but it also appeared to be endowed with a reduced neurotoxic potential and an improved profile of tolerability compared to the parent drug. Furthermore, the best antitumor efficacy was often already reached with doses lower than the maximal tolerated dose, suggesting an improved therapeutic index for the new drug. In conclusion, the results support the preclinical interest of IDN5109 in terms of the toxicity profile and of the efficacy with particular reference to the ability to overcome multiple mechanisms of drug resistance.     

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.     

In vitro comparative evaluation of trastuzumab (Herceptin) combined with paclitaxel (Taxol) or docetaxel (Taxotere) in HER2-expressing human breast cancer cell lines.

BACKGROUND: Trastuzumab (Herceptin) has clinical indication in association with paclitaxel (Taxol) for the treatment of human epidermal growth factor receptor 2 (HER2)-expressing breast cancer. Synergistic interactions have been reported with taxane derivatives in HER2-expressing breast cancer cells. However, no direct comparison of the potential interest in combining trastuzumab with either paclitaxel or docetaxel (Taxotere) has been reported. MATERIALS AND METHODS: The present study was designed to evaluate in a comparative way the interaction of trastuzumab with paclitaxel or docetaxel in HER2-overexpressing human breast cancer cell lines. HER2 expression was documented in MCF-7, MDA-MB453 and SK-BR3 cell lines using immunocytochemistry with purified mouse anti-human monoclonal antibody. Cytotoxicity assays were performed using the sulforhodamine B assay and in vitro interactions between trastuzumab and taxanes were analyzed using the median-effect principle. RESULTS: Trastuzumab cytotoxicity was confirmed to be directly related to HER2 expression level. At the IC(50), the combination of trastuzumab with either paclitaxel or docetaxel led to synergism in all cell lines. However, considering mean values calculated in the IC(30)-IC(70) range of concentrations, trastuzumab interacted additively with docetaxel in SK-BR3 and MDA-MB453 cell lines while additive and synergistic interactions were achieved with paclitaxel in SK-BR3 and MDA-MB453, respectively. On the same basis, trastuzumab yielded synergistic interaction with both taxanes in the MCF-7 cell line. CONCLUSIONS: The present study shows that at least additive interactions are observed when trastuzumab is combined with either paclitaxel or docetaxel in weak to moderate or more than moderate HER2-expressing cells. Some interesting results were achieved in cells displaying weak HER2 expression which could suggest some further potential interest in trastuzumab.     

Anti-proliferative activity of a new class of taxanes (14β-hydroxy-10-deacetylbaccatin III derivatives) on multidrug-resistance-positive human cancer cells

Paclitaxel, docetaxel and a series of new analogs synthesized from 14β-hydroxy-10-deacetylbaccatin III (14-OH-DAB), a natural diterpene closely related to the core synthon of the 2 above prototypes, were tested in vitro for their growth-inhibitory activity on different human cancer cell lines, including some expressing the classic multidrug-resistant (MDR) phenotype (MCF-7 ADRr and CEM VBLr). The 14-OH-DAB derivatives showed enhanced anti-proliferative activity as compared to the parent compounds on the MDR-positive cancer cell lines. Particularly, IDN 5109 showed a 25- to 30-fold higher activity than paclitaxel. The fold change in activity between paclitaxel and analogs (IC₅₀ paclitaxel/IC₅₀ analogs) on the MDR-positive cell lines was calculated and a significant correlation observed. As far as the MDR-negative MDA-MB 231 cells are concerned, docetaxel and IDN 5109 exhibited a more potent activity than paclitaxel. On the basis of the data obtained on cell growth inhibition, we selected the most active compounds to study their effect on the cell cycle. Cell cycle analysis showed that all of the compounds tested were able to induce cell cycle block at G₂/M in a concentration-dependent manner. The amount of cell block, measured as a G₁/G₂ ratio, was correlated significantly (p < 0.001) with apoptosis, as evaluated in the sub-G₁ region (% of DNA fragmentation), thereby suggesting that the G₂/M-blocked cells underwent apoptosis. To confirm the occurrence of apoptosis in this system, DNA gel agarose electrophoresis was performed and showed the typical ladder pattern. Int. J. Cancer 72:844–850, 1997. © 1997 Wiley-Liss, Inc.     

The role of EGFR-directed therapy in the treatment of breast cancer

The epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase group of growth factor receptors. Following the binding of ligands such as the epidermal growth factor (EGF) with EGFR, activation of signal transduction pathways can lead to changes in cellular proliferation and differentiation. EGFR is highly expressed by many tumors, and is associated with disease with poor prognostic features. EGFR and its downstream signaling pathways are, therefore, promising antitumor targets for drug development. Using ZD1839 ('Iressa') as an example of a small molecule inhibitor of EGFR, the utility of targeting EGFR in the treatment of breast cancer will be discussed. ZD1839 is an example of an orally active, selective EGFR tyrosine kinase inhibitor, which has shown extensive preclinical activity and evidence of being active combined with a favorable tolerability profile. Breast cancers have been shown to express high levels of EGFR and hormone-resistant disease is associated with an increased expression of both EGFR and EGFR ligands. ZD 1839 has shown in vitro activity as both monotherapy and in combination with other agents such as paclitaxel or doxorubicin, inhibiting the growth of breast cancer cells that are resistant to endocrine agents such as tamoxifen. Against hormone-responsive cells, the combination of ZD1839 with endocrine therapy produces synergistic activity. ZD1839 has also shown growth inhibitory activity against xenografts initiated from ductal carcinoma in situ tissues, indicating that EGFR inhibition may have a role in the treatment of early-stage breast cancer. EGFR is, therefore, a rational target for the development of novel therapies, and promising preclinical studies indicate that EGFR-targeted therapy may have a role in the treatment of breast cancer. The results of clinical trials with ZD 1839 in breast cancer patients are awaited with interest.     

A role for loss of p53 function in sensitivity of ovarian carcinoma cells to taxanes

Loss of p53 function has been linked to increased responsiveness to taxane treatment of ovarian carcinoma in clinical studies. We recently reported that the acquisition of cisplatin resistance in an ovarian carcinoma cell line (IGROV-1) was associated with mutation of p53 and collateral sensitivity to paclitaxel. The increased sensitivity to paclitaxel of the cisplatin-resistant subline appeared to be pharmacologically relevant since it was reflected in an in vivo sensitization to taxanes. To investigate the cellular and molecular basis of this phenomenon, we performed a comparative study of cellular response to taxanes (paclitaxel and the novel analog IDN 5109) in the parental cell line, containing wild-type p53 and its cisplatin-resistant p53 mutant subline (IGROV-1/Pt1). IDN 5109 was included in this study because of its higher potency and efficacy compared with paclitaxel on both tumor systems. The pattern of cellular response of the two ovarian cell lines was different. In IGROV-1 cells, apoptosis was an early event consequent to a transient mitotic arrest. The cell death of IGROV-1/Pt1 cells was a somewhat slow and delayed event, following mitotic arrest and appearance of hyperploid cells. The increased cytotoxic effect of IDN 5109, compared with paclitaxel, was associated with more marked p34(cdc2) dephosphorylation in IGROV-1 cells and higher Bcl-2 phosphorylation in IGROV-1/Pt1 cells after 24 hr of treatment. In each cell line, these biochemical events were not correlated with parallel levels of mitotic cells. Attempts to reintroduce wild-type p53 in IGROV-1/Pt1 were unsuccessful. However, in other p53-deficient cells (osteosarcoma SAOS), taxane treatment was associated with hyperploid progression and the introduction of wild-type p53 resulted in a reduced sensivity. Although our approach does not allow definitive conclusions, these results suggest that loss of p53-dependent post-mitotic checkpoint results in a different time-course of taxane-induced cell death following DNA reduplication. These events, more evident after exposure to the potent analog IDN 5109, support the notion that the enhanced sensitivity of p53 mutant cells is closely related to the different mode of cell death.     

Studies with CWR22 xenografts in nude mice suggest that ZD1839 may have a role in the treatment of both androgen-dependent and androgen-independent human prostate cancer.

PURPOSE: These studies examined the effect of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor ZD1839 ("Iressa")(3) on CWR22 prostate tumors in nude mice. The effect of ZD1839 was also examined in combination with either bicalutamide ("Casodex") or cytotoxic agents against a hormone-dependent or -independent variant of CWR22, respectively. EXPERIMENTAL DESIGN: The xenografts were grown for 4-7 days, then tumor measurements were made and therapy initiated. ZD1839 and bicalutamide were given p.o. on a once-daily, 5-day schedule for 2 successive weeks. Carboplatin and paclitaxel were given every 3-4 days for a total of four doses. Measurements of tumor volume were made twice weekly during treatment and for 2 weeks after treatment. The effect of ZD1839 on EGFR function was assessed by Western blotting of EGFR and its phosphorylated form in CWR22 and variant tumors before and after treatment with this agent. RESULTS: ZD1839 at its maximum tolerated dose (150 mg/kg) inhibited the growth of androgen-dependent CWR22 by 54%, and the growth of two variants with different degrees of androgen independence and androgen receptor gene expression (CWR22LD1 and CWR22RV1) by 76%. The effects of ZD1839 were similar to those recorded for phosphorylation of EGFR as determined by Western blotting. Coadministration of ZD1839 at its maximum tolerated dose markedly increased the antiproliferative action of the antiandrogen bicalutamide against CWR22LD1. In fact, combining ZD1839 with a suboptimal dose of bicalutamide was more effective than a higher dose of bicalutamide alone. Coadministration of ZD1839, which required a 2-3-fold attenuation of dose to avoid toxicity, also markedly increased the therapeutic activity of carboplatin and paclitaxel against CWR22RV1, bringing about regression to a degree not seen with either agent alone. Tumor-free mice were seen only with the combination of ZD1839 and paclitaxel. CONCLUSIONS: The results obtained in these related and highly relevant models of human prostate cancer suggest that ZD1839 may have a role in enhancing existing treatments of androgen-dependent and -independent forms of this disease in patients.