Drug-Drug Interaction Study of Benznidazole and E1224 in Healthy Male Volunteers.

E1224 is a prodrug of ravuconazole (RVZ), an antifungal drug with promising anti-Trypanosoma cruzi activity, the causative organism of Chagas disease (CD). This study was designed to assess the pharmacokinetics (PK) and safety interactions of benznidazole (BNZ), the drug of choice for treatment of CD, and E1224 in healthy volunteers. This open-label, single-center, sequential, single- and multiple-oral-dose study enrolled 28 healthy male subjects. These subjects received BNZ (2.5 mg/kg) once daily on days 1 and 9 and twice daily from day 12 to day 15 and E1224 once daily from day 4 to day 15 (loading dose of 400 mg for 3 days and maintenance dose of 100 mg for 9 days). The maximum concentration (Cmax) and area under the concentration curve from zero to infinity for BNZ were comparable, whether BNZ was given alone or with E1224 at steady state, with ratios of geometric means for BNZ-RVZ to BNZ of 0.96 and 0.83 and corresponding 90% confidence intervals (CIs) of 0.91 to 1.10 and 0.80 to 0.87, respectively. However, RVZ Cmax and area under the concentration curve from zero to 24 h increased by about 35% when concomitantly administered with BNZ at steady state (ratio of geometric means for RVZ-BNZ/RVZ of 1.31 and 1.36 and corresponding 90% CIs of 1.23 to 1.39 and 1.31 to 1.41, respectively). Both compounds were well tolerated. There were no clinically relevant safety interactions between E1224 and BZN. Given these results, coadministration of RVZ and BNZ should not require any adaptation of E1224 dosing.

[Development and Distribution of Drugs for NTDs: Efforts of One Pharmaceutical Company].

The Pharmaceutical Industry is expected to play a proactive global role in combatting neglected tropical diseases (NTDs) and other tropical diseases affecting low-income countries. Such a role would include novel medicine R&D, manufacturing and distribution. In order to succeed in this role, several challenges need to be overcome: a) the economic challenge or cost benefit balance for the development of these medicines, and b) sparse in-house experience with these diseases within the Industry. During the last decade, the Product Development Partnership (PDP) model has become an effective strategy to address such challenges. Organizations such as the Medicines for Malaria Venture (MMV), Drugs for Neglected Diseases initiative (DNDi), TB alliance, PATH (formerly the Program for Appropriate Technology in Health), and others have linked pharmaceutical companies, funding organizations, academic researchers and others, and have thus been able to successfully populate treatment pipelines directed at NTDs, Malaria, tuberculosis (TB), and human immunodeficiency virus (HIV)/AIDS. In this paper, our experience working with one of these organizations, DNDi, is described. We have been collaborating with DNDi in evaluating the actions of Eisai's antifungal compound, E1224, in a clinical study for treating Chagas Disease. In addition, other Eisai initiatives directed at NTDs and improving patients' access to medicines are introduced.

Lenvatinib, an angiogenesis inhibitor targeting VEGFR/FGFR, shows broad antitumor activity in human tumor xenograft models associated with microvessel density and pericyte coverage.

BACKGROUND: Lenvatinib is an oral inhibitor of multiple receptor tyrosine kinases (RTKs) targeting vascular endothelial growth factor receptor (VEGFR1-3), fibroblast growth factor receptor (FGFR1-4), platelet growth factor receptor α (PDGFR α), RET and KIT. Antiangiogenesis activity of lenvatinib in VEGF- and FGF-driven angiogenesis models in both in vitro and in vivo was determined. Roles of tumor vasculature (microvessel density (MVD) and pericyte coverage) as biomarkers for lenvatinib were also examined in this study. METHOD: We evaluated antiangiogenesis activity of lenvatinib against VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. Effects of lenvatinib on in vivo angiogenesis, which was enhanced by overexpressed VEGF or FGF in human pancreatic cancer KP-1 cells, were examined in the mouse dorsal air sac assay. We determined antitumor activity of lenvatinib in a broad panel of human tumor xenograft models to test if vascular score, which consisted of high MVD and low pericyte coverage, was associated with sensitivity to lenvatinib treatment. Vascular score was also analyzed using human tumor specimens with 18 different types of human primary tumors. RESULT: Lenvatinib inhibited VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. In vivo angiogenesis induced by overexpressed VEGF (KP-1/VEGF transfectants) or FGF (KP-1/FGF transfectants) was significantly suppressed with oral treatments of lenvatinib. Lenvatinib showed significant antitumor activity in KP-1/VEGF and five 5 of 7 different types of human tumor xenograft models at between 1 to 100 mg/kg. We divided 19 human tumor xenograft models into lenvatinib-sensitive (tumor-shrinkage) and relatively resistant (slow-growth) subgroups based on sensitivity to lenvatinib treatments at 100 mg/kg. IHC analysis showed that vascular score was significantly higher in sensitive subgroup than relatively resistant subgroup (p < 0.0004). Among 18 types of human primary tumors, kidney cancer had the highest MVD, while liver cancer had the lowest pericyte coverage, and cancers in Kidney and Stomach had highest vascular score. CONCLUSION: These results indicated that Lenvatinib inhibited VEGF- and FGF-driven angiogenesis and showed a broad spectrum of antitumor activity with a wide therapeutic window. MVD and pericyte-coverage of tumor vasculature might be biomarkers and suggest cases that would respond for lenvatinib therapy.

Enhanced anti-angiogenic effect of E7820 in combination with erlotinib in epidermal growth factor receptor-tyrosine kinase inhibitor-resistant non-small-cell lung cancer xenograft models.

Most non-small-cell lung cancers (NSCLCs) harboring activating mutations in the epidermal growth factor receptor (EGFR) are initially responsive to EGFR tyrosine kinase inhibitors (EGFR-TKIs); however, they invariably develop resistance to these drugs. E7820 is an angiogenesis inhibitor that decreases integrin-α2 expression and is currently undergoing clinical trials. We investigated whether E7820 in combination with erlotinib, an EGFR-TKI, could overcome EGFR-TKI-resistance in the NSCLC cell lines A549 (KRAS; G12S), H1975 (EGFR; L858R/T790M), and H1650 (PTEN; loss, EGFR; exon 19 deletion), which are resistant to erlotinib. Immunohistochemical analysis was carried out in xenografted tumors to investigate anti-angiogenesis activity and endothelial cell apoptosis levels by endothelial cell marker CD31 and TUNEL staining, respectively. Treatment with E7820 (50 mg/kg) with erlotinib (60 mg/kg) showed a synergistic antitumor effect in three xenograft models. Immunohistochemical analysis indicated that combined treatment with E7820 and erlotinib significantly decreased microvessel density and increased apoptosis of tumor-associated endothelial cells compared with use of only one of the agents. This combination increased apoptosis in HUVECs through activation of both intrinsic and extrinsic apoptosis pathways in vitro. The combination of E7820 with erlotinib is an alternative strategy to overcome erlotinib resistance in NSCLC by enhancement of the anti-angiogenic activity of E7820.

Therapeutic potential and molecular mechanism of a novel sulfonamide anticancer drug, indisulam (E7070) in combination with CPT-11 for cancer treatment.

PURPOSE: Indisulam (N-(-3-chloro-7-indolyl)-1,4-benzenedisulfonamide; E7070) is an experimental anticancer agent. Microarray analysis indicates that indisulam downregulates several genes involved in drug resistance, and this finding led us to test the effect of combining indisulam with other anticancer drugs. We investigated the antitumor effect and mechanism of synergism when indisulam was administered in combination with CPT-11. METHODS: In vitro cytotoxic activity was examined using a cell counter kit, and the combination effect was determined by isobologram analysis. The level of topoisomerase IIα was measured by Western blotting. The in vivo antitumor effect was assessed in mice inoculated with human colorectal cancer SW620 cells. RESULTS: Isobologram analysis indicated that a 24-h exposure to indisulam and SN-38, an active metabolite of CPT-11, had a synergistic effect in HCT116 and SW620 cells and an additive effect in HCT15 and WiDr cells. Prolongation of exposure to 48 h resulted in a synergistic effect in HCT15 and WiDr cells. Treatment with SN-38 alone increased the amount of intracellular topoisomerase IIα in all cell lines tested. Co-treatment with indisulam suppressed the SN-38-induced upregulation of topoisomerase IIα after 24 h of exposure in HCT116 and SW620 cells and after 48 h of exposure in HCT15 and WiDr cells. This apparent association between a synergistic effect and suppression of SN-38-mediated upregulation of topoisomerase IIα suggests that indisulam enhances SN-38 cytotoxicity by suppressing topoisomerase IIα upregulation to compensate for topoisomerase I inhibition by SN-38. Synergy was also observed in xenografted tumors and was accompanied by complete suppression of topoisomerase IIα upregulation induced by CPT-11 treatment. CONCLUSION: These observations prompted the clinical evaluation of indisulam and CPT-11 combination therapy.

In vitro activity of E1210, a novel antifungal, against clinically important yeasts and molds.

E1210 is a new antifungal compound with a novel mechanism of action and broad spectrum of antifungal activity. We investigated the in vitro antifungal activities of E1210 compared to those of fluconazole, itraconazole, voriconazole, amphotericin B, and micafungin against clinical fungal isolates. E1210 showed potent activities against most Candida spp. (MIC(90) of ≤0.008 to 0.06 µg/ml), except for Candida krusei (MICs of 2 to >32 µg/ml). E1210 showed equally potent activities against fluconazole-resistant and fluconazole-susceptible Candida strains. E1210 also had potent activities against various filamentous fungi, including Aspergillus fumigatus (MIC(90) of 0.13 µg/ml). E1210 was also active against Fusarium solani and some black molds. Of note, E1210 showed the greatest activities against Pseudallescheria boydii (MICs of 0.03 to 0.13 µg/ml), Scedosporium prolificans (MIC of 0.03 µg/ml), and Paecilomyces lilacinus (MICs of 0.06 µg/ml) among the compounds tested. The antifungal action of E1210 was fungistatic, but E1210 showed no trailing growth of Candida albicans, which has often been observed with fluconazole. In a cytotoxicity assay using human HK-2 cells, E1210 showed toxicity as low as that of fluconazole. Based on these results, E1210 is likely to be a promising antifungal agent for the treatment of invasive fungal infections.

Efficacy of oral E1210, a new broad-spectrum antifungal with a novel mechanism of action, in murine models of candidiasis, aspergillosis, and fusariosis.

E1210 is a first-in-class, broad-spectrum antifungal with a novel mechanism of action-inhibition of fungal glycosylphosphatidylinositol biosynthesis. In this study, the efficacies of E1210 and reference antifungals were evaluated in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. Oral E1210 demonstrated dose-dependent efficacy in infections caused by Candida species, Aspergillus spp., and Fusarium solani. In the treatment of oropharyngeal candidiasis, E1210 and fluconazole each caused a significantly greater reduction in the number of oral CFU than the control treatment (P < 0.05). In the disseminated candidiasis model, mice treated with E1210, fluconazole, caspofungin, or liposomal amphotericin B showed significantly higher survival rates than the control mice (P < 0.05). E1210 was also highly effective in treating disseminated candidiasis caused by azole-resistant Candida albicans or Candida tropicalis. A 24-h delay in treatment onset minimally affected the efficacy outcome of E1210 in the treatment of disseminated candidiasis. In the Aspergillus flavus pulmonary aspergillosis model, mice treated with E1210, voriconazole, or caspofungin showed significantly higher survival rates than the control mice (P < 0.05). E1210 was also effective in the treatment of Aspergillus fumigatus pulmonary aspergillosis. In contrast to many antifungals, E1210 was also effective against disseminated fusariosis caused by F. solani. In conclusion, E1210 demonstrated consistent efficacy in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. These data suggest that further studies to determine E1210's potential for the treatment of disseminated fungal infections are indicated.

Synthesis and evaluation of novel antifungal agents-quinoline and pyridine amide derivatives.

Quinoline amide, azaindole amide and pyridine amides were synthesized and tested for in vitro antifungal activity against fungi. These synthesized amides have potent antifungal activity against Candida albicans and Aspergillus fumigatus. Our results suggest that hetero ring amides may be potent antifungal agents that operate by inhibiting the function of Gwt1 protein in the GPI biosynthetic pathway.

E7050: a dual c-Met and VEGFR-2 tyrosine kinase inhibitor promotes tumor regression and prolongs survival in mouse xenograft models.

c-Met is the cellular receptor for hepatocyte growth factor (HGF) and is known to be dysregulated in various types of human cancers. Activation of the HGF/c-Met pathway causes tumor progression, invasion, and metastasis. Vascular endothelial growth factor (VEGF) is also known as a key molecule in tumor progression through the induction of tumor angiogenesis. Because of their key roles in tumor progression, these pathways provide attractive targets for therapeutic intervention. We have generated a novel, orally active, small molecule compound, E7050, which inhibits both c-Met and vascular endothelial growth factor receptor (VEGFR)-2. In vitro studies indicate that E7050 potently inhibits phosphorylation of both c-Met and VEGFR-2. E7050 also potently represses the growth of both c-met amplified tumor cells and endothelial cells stimulated with either HGF or VEGF. In vivo studies using E7050 showed inhibition of the phosphorylation of c-Met and VEGFR-2 in tumors, and strong inhibition of tumor growth and tumor angiogenesis in xenograft models. Treatment of some tumor lines containing c-met amplifications with high doses of E7050 (50-200 mg/kg) induced tumor regression and disappearance. In a peritoneal dissemination model, E7050 showed an antitumor effect against peritoneal tumors as well as a significant prolongation of lifespan in treated mice. Our results indicate that E7050 is a potent inhibitor of c-Met and VEGFR-2 and has therapeutic potential for the treatment of cancer.

Multi-kinase inhibitor E7080 suppresses lymph node and lung metastases of human mammary breast tumor MDA-MB-231 via inhibition of vascular endothelial growth factor-receptor (VEGF-R) 2 and VEGF-R3 kinase.

PURPOSE: Vascular endothelial growth factor (VEGF)-C/VEGF-receptor 3 (VEGF-R3) signal plays a significant role in lymphangiogenesis and tumor metastasis based on its effects on lymphatic vessels. However, little is known about the effect of inhibiting VEGF-R3 on lymphangiogenesis and lymph node metastases using a small-molecule kinase inhibitor. EXPERIMENTAL DESIGN: We evaluated the effect of E7080, a potent inhibitor of both VEGF-R2 and VEGF-R3 kinase, and bevacizumab on lymphangiogenesis and angiogenesis in a mammary fat pad xenograft model of human breast cancer using MDA-MB-231 cells that express excessive amounts of VEGF-C. Lymphangiogenesis was determined by lymphatic vessel density (LVD) and angiogenesis by microvessel density (MVD). RESULTS: In contrast to MDA-MB-435 cells, which expressed a similar amount of VEGF to MDA-MB-231 cells with an undetectable amount of VEGF-C, only MDA-MB-231 exhibited lymphangiogenesis in the primary tumor. E7080 but not bevacizumab significantly decreased LVD within the MDA-MB-231 tumor. E7080 and bevacizumab decreased MVD in both the MDA-MB-231 and MDA-MB-435 models. E7080 significantly suppressed regional lymph nodes and distant lung metastases of MDA-MB-231, whereas bevacizumab significantly inhibited only lung metastases. E7080 also decreased both MVD and LVD within the metastatic nodules at lymph nodes after resection of the primary tumor. CONCLUSIONS: Inhibition of VEGF-R3 kinase with E7080 effectively decreased LVD within MDA-MB-231 tumors, which express VEGF-C. Simultaneous inhibition of both VEGF-R2 and VEGF-R3 kinases by E7080 may be a promising new strategy to control regional lymph node and distant lung metastases.

E7080, a novel inhibitor that targets multiple kinases, has potent antitumor activities against stem cell factor producing human small cell lung cancer H146, based on angiogenesis inhibition.

E7080 is an orally active inhibitor of multiple receptor tyrosine kinases including VEGF, FGF and SCF receptors. In this study, we show the inhibitory activity of E7080 against SCF-induced angiogenesis in vitro and tumor growth of SCF-producing human small cell lung carcinoma H146 cells in vivo. E7080 inhibits SCF-driven tube formation of HUVEC, which express SCF receptor, KIT at the IC(50) value of 5.2 nM and it was almost identical for VEGF-driven one (IC(50) = 5.1 nM). To assess the role of SCF/KIT signaling in tumor angiogenesis, we evaluated the effect of imatinib, a selective KIT kinase inhibitor, on tumor growth of H146 cells in nude mice. Imatinib did not show the potent antitumor activity in vitro (IC(50) = 2,200 nM), because H146 cells did not express KIT. However, oral administration of imatinib at 160 mg/kg clearly slowed tumor growth of H146 cells in nude mice, accompanied by decreased microvessel density. Oral administration of E7080 inhibited tumor growth of H146 cells at doses of 30 and 100 mg/kg in a dose-dependent manner and caused tumor regression at 100 mg/kg. While anti-VEGF antibody also slowed tumor growth, it did not cause tumor regression. These results indicate that KIT signaling has a role in tumor angiogenesis of SCF-producing H146 cells, and E7080 causes regression of H146 tumors as a result of antiangiogenic activity mediated by inhibition of both KIT and VEGF receptor signaling. E7080 may provide therapeutic benefits in the treatment of SCF-producing tumors.

Preparation and root growth-inhibitory activity of N-substituted 2-(2-chloroacetamido)-3-(furan-2-yl)propanamides.

A series of N-substituted 2-(2-chloroacetamido)-3-(furan-2-yl)propanamides (16--18) was prepared through the reaction of chloroacetyl chloride with N-substituted 2-amino-3-(furan-2-yl)propanamides (15), which were obtained via condensation of 2-(tert-butoxycarbonylamido)-3-(furan-2-yl)propanoic acid (Boc-furylalanine) (8) with amines (9, 11, 13), followed by hydrolysis of the resultant N-substituted Boc-furylalanine acid amides (10, 12, 14) in the presence of HCl/dioxane. The biological activity of the prepared 16, 17 and 18 as root growth inhibitors was examined by germination assay using rape seed. At the concentration of 5.0 x10 (-5) M, the most active compound, 2-(2-chloroacetamido)-N-(2,6-diethylphenyl)-3-(furan-2-yl)propanamide (16 n), showed potent root growth-inhibitory activity of 76% towards rape seedlings.

Improved preparation of racemic 2-amino-3-(heteroaryl)propanoic acids and related compounds containing a furan or thiophene nucleus.

Racemic 2-amino-3-(heteroaryl)propanoic acids (1), mostly with a furan or thiophene nucleus as a heteroaryl group, were synthesized in 48-94% yield by the reduction of 3-(heteroaryl)-2-(hydroxyimino)propanoic acids (5) with zinc dust and formic acid in the presence of a catalytic amount of iron dust at 60 degrees C for 2 h. Under these conditions, unfavorable hydrogenolysis of bromine on the thiophene nucleus does not occur. Traditional N-formylation of the prepared 3-(heteroaryl)alanine (1) with a mixture of formic acid and acetic anhydride afforded 2-(formylamino)-3-(heteroaryl)propanoic acids (6) in 51-95% yield.

Pladienolides, new substances from culture of Streptomyces platensis Mer-11107. III. In vitro and in vivo antitumor activities.

We have discovered seven novel 12-membered macrolides, pladienolides A to G, from Streptomyces platensis Mer-11107, with pladienolide B the most potently inhibiting hypoxia induced-VEGF expression and proliferation of the U251 cancer cell line. A growth inhibitory study using a 39-cell line drug-screening panel demonstrated that pladienolide B has strong antitumor activities in vitro. A COMPARE analysis reveals that it has a unique antitumor spectrum that sets it apart from anticancer drugs currently in clinical use. This result suggests that pladienolide B has a novel mechanism of action. A series of xenograft studies were conducted to evaluate the in vivo potency of pladienolides. Pladienolide B extensively inhibited tumor growth in xenograft models. In the most sensitive model, using BSY-1 xenografts, tumors were completely regressed by administration of pladienolide B. For the reason of their novel mechanism of action and excellent in vivo efficacy, pladienolides appear to have major potential for use in cancer treatment.

Stem cell factor/c-kit signaling promotes the survival, migration, and capillary tube formation of human umbilical vein endothelial cells.

c-kit receptor tyrosine kinase is a marker of progenitor cells, which differentiate into blood and/or vascular endothelial cells, and has an important role in the amplification/mobilization of progenitor cells. c-kit is expressed in mature endothelial cells, but its role there is unclear. Stem cell factor, a c-kit ligand, dose-dependently promoted survival, migration, and capillary tube formation of human umbilical vein endothelial cells. These effects mimicked those of vascular endothelial growth factor, except that stem cell factor did not sufficiently support proliferation of these cells. After exposing cells to this factor, Akt, Erk1/2, and c-kit were immediately (

An angiogenesis inhibitor E7820 shows broad-spectrum tumor growth inhibition in a xenograft model: possible value of integrin alpha2 on platelets as a biological marker.

We reported previously that an angiogenesis inhibitor, E7820, inhibits in vitro tube formation of human umbilical vein endothelial cell through the suppression of integrin alpha2 expression. Here we describe the antiangiogenic and antitumor effects of E7820 in mice and discuss the feasibility of using platelet integrin alpha2 expression on platelets as a biological marker of the efficacy of E7820. Oral administration of E7820 significantly inhibited basic fibroblast growth factor-induced angiogenesis in Matrigel implants and human colon WiDr tumor-induced angiogenesis in a dorsal air sac model. Twice-daily treatment with E7820 clearly inhibited the s.c. tumor growth of seven tumor cell lines derived from human colon, breast, pancreas, and kidney, and completely suppressed the growth of human pancreatic KP-1 and human colon LoVo cell lines. Moreover, E7820 significantly inhibited the growth of KP-1 and human colon tumor Colo320DM cells orthotopically implanted in the pancreas and cecum, respectively. The efficacy of E7820 was comparable in the s.c. and orthotopic transplantation models. Immunohistochemical analyses using anti-CD31 antibody showed that E7820 significantly reduced microvessel density in orthotopically implanted KP-1 tumor. E7820 reduced integrin alpha2 expression on a megakaryocytic cell line, Dami cells, induced by phorbol 12-myristate 13-acetate treatment. It also decreased the expression level of integrin alpha2 on platelets withdrawn from mice bearing s.c. KP-1 tumor at a dosage close to that affording antitumor activity. These data demonstrate that E7820 showed a broad-spectrum antitumor effect in mice through inhibition of angiogenesis and indicate that the decrease of integrin alpha2 on platelets might serve as a biological marker for the antitumor efficacy of E7820.

Alpha6beta1 integrin induces proteasome-mediated cleavage of erbB2 in breast cancer cells.

ErbB2 and alpha6 integrin have been implicated in malignancy of breast cancer cells. Here we have determined the influence of alpha6beta1 integrin on erbB2 signaling in anchorage-independent growth, using MDA-MB435 breast cancer cells. Firstly, we transfected the cells with erbB2 cDNA, and isolated cells with high or low levels of alpha6beta1 integrin by cell sorting (alpha6H-ErbB and alpha6L-ErbB). We found that an erbB ligand, heregulin beta1, enhanced growth activity of alpha6L-ErbB cells, but not alpha6H-ErbB cells. Secondly, we established cells expressing a beta4 integrin deletion mutant (beta4-deltacyt), which selectively inhibited alpha6beta1 integrin expression and adhesion to laminin-1. Again, heregulin beta1 enhanced the growth of erbB2 cDNA-transfected beta4-deltacyt cells, but not mock cells. Western blot analysis revealed that heregulin beta1 stimulated phosphorylation of Akt and its downstream molecules, GSK3beta and p70S6kinase, and that the extent of phosphorylation was greater in ErbB2/beta4-deltacyt cells than ErbB2/mock cells. Furthermore, we found that the erbB2 cytoplasmic domain was truncated in ErbB2/mock cells, which was independent of ligand stimulation and adhesion, and was suppressed by proteasome inhibitors. These results suggest that alpha6beta1 integrin inhibits erbB2 signals by inducing proteasome-dependent proteolytic cleavage of the erbB2 cytoplasmic domain, and may thereby contribute to the regulation of tumor growth.

Aberrant expression of integrin and erbB subunits in breast cancer cell lines.

Integrin and growth factor receptors play an important role in cell functions and their aberrant expressions are implicated in breast cancer malignancy. Recent studies have shown that integrins physically and functionally associate with growth factor receptors suggesting the cooperative regulation of these two signals. We studied the expression of integrin and erbB subunits by flow cytometer in human normal mammary epithelial (HME) cell, non-metastatic (MCF-7, ZR-75-1, MDA-MB453) and metastatic tumor cell lines (MDA-MB231, MDA-MB435). Compared with HME cells, all of non-metastatic and metastatic cell lines showed decreased expressions of alpha2 and beta4 integrin subunits. Two metastatic cell lines, but not three non-metastatic tumor cell lines, expressed alpha5 and alpha6 comparable to HME cells. There was no correlation of erbB2 expression with integrin expressions. We isolated MDA-MB435 subpopulations expressing lower amount of alpha6 integrin and found that alpha5, but not alpha2 and alphav integrins, was concomitantly decreased while erbB family was not affected. Then we transfected erbB2 gene into MDA-MB435 and found the induction of erbB3 expression but not erbB1 and erbB4. However, erbB2 transfection had no effect on the expression of alpha6 and beta4 integrin subunits. These data suggest that the expression of alpha5 and alpha6 integrins may contribute to metastasis, and that the regulation of erbB2 and alpha6 integrin expressions is independent in breast cancer cells.