Retrospective analysis of efficacy and safety of amrubicin in refractory and relapsed small-cell lung cancer.

BACKGROUND: Amrubicin, a totally synthetic 9-aminoanthracycline, was evaluated retrospectively for the treatment of refractory and relapsed small-cell lung cancer (SCLC). METHODS: Retrospective analysis was performed in 32 patients. Amrubicin was infused over 5 min on days 1-3, with courses repeated at 3- or 4-week intervals. Amrubicin was given at a dose of 45 mg/m(2) per day, 40 mg/m(2) per day, 35 mg/m(2) per day, 30 mg/m(2) per day, or 25 mg/m(2) per day depending on medical conditions (patients' age and performance status [PS]), and the dose was modulated according to myelosuppression. RESULTS: The median number of treatment cycles was 3 (range, 1-6). Seventeen patients (53.1%) had a partial response. Median progression-free survival time for all patients was 96 days, and median survival time was 166 days. Grade 3 or 4 hematologic toxicities comprised neutropenia (78.1%), anemia (65.6%), and thrombocytopenia (50.0%). Febrile neutropenia was observed in 8 patients (25.0%). Nonhematologic toxicities were mild. Treatment-related death was observed in 1 patient. CONCLUSION: Treatment with amrubicin appeared effective in SCLC patients previously treated with chemotherapy, although it was not necessarily safe, because of myelosuppression. Further research is warranted to investigate amrubicin treatment for patients with SCLC.

The laminin alpha-1 chain derived peptide, AG73, increases fibronectin levels in breast and melanoma cancer cells.

Laminin-111 promotes the malignant phenotype, and a 12-mer synthetic peptide (AG73, RKRLQVQLSIRT) from the carboxyl terminus of the alpha1 chain increases B16F10 melanoma metastasis to the lung and liver. Using an antibody array, fibronectin was identified as an up-regulated protein in B16F10 cells after incubation with this peptide. The increased fibronectin is cell-associated with no increase in soluble fibronectin. The AG73 peptide increased the number and size of bone metastases with both B16F10 melanoma and MDA-231 breast carcinoma cells in an intracardiac injection model. Using siRNA transfection, we found that a reduction in fibronectin expression did not reduce bone metastasis in the presence of the metastasis-promoting peptide AG73. We conclude that the laminin peptide AG73 increases metastasis independently of fibronectin expression.

Laminin alpha5 chain metastasis- and angiogenesis-inhibiting peptide blocks fibroblast growth factor 2 activity by binding to the heparan sulfate chains of CD44.

Recently, we reported that the laminin alpha5 synthetic peptide A5G27 (RLVSYNGIIFFLK, residues 2,892-2,904) binds to the CD44 receptor of B16-F10 melanoma cells via the glycosaminoglycans on CD44 and inhibits tumor cell migration, invasion, and angiogenesis in a dominant-negative manner. Here, we have identified the potential mechanism of A5G27 activity using WiDr human colorectal carcinoma cells. WiDr cells bound to the laminin A5G27 peptide via the heparin-like and chondroitin sulfate B glycosaminoglycan side chains of CD44. Cell binding to fibroblast growth factor (FGF2) was blocked by laminin peptide A5G27 but not by either a scrambled version of this peptide or by another laminin peptide known to bind cell surface proteoglycans. FGF2 signaling involving tyrosine phosphorylation was also blocked by laminin peptide A5G27 but was not affected by peptide controls. Finally, we have shown that peptide A5G27 directly blocks FGF2 binding to heparin. Peptide A5G27 has sequence homology to a region on FGF2 that binds heparin and the FGF receptor and is important in FGF2 central cavity formation. We conclude that peptide A5G27 inhibits metastasis and angiogenesis by blocking FGF2 binding to the heparan sulfate side chains of CD44 variant 3, thus decreasing FGF2 bioactivity.

Identification of an active site on the laminin alpha5 chain globular domain that binds to CD44 and inhibits malignancy.

The laminin alpha5 chain is a component of laminin-10 (alpha5beta1gamma1) and -11 (alpha5beta2gamma1). In this study, we have screened 113 overlapping synthetic peptides from the laminin alpha5 globular domain (G-domain) for cell attachment activity with B16-F10 cells using peptide-coated dishes. Eleven attachment-active peptides were identified. In vivo experimental B16-F10 pulmonary metastasis and primary tumor growth assays found that 4 of the 11 peptides inhibited tumor metastasis and growth and increased apoptosis. These four peptides also blocked tumor cell migration, invasion, and angiogenesis. Two of the peptides were highly homologous and showed significant similarity to sequences in collagens. We sought to identify the B16-F10 cell surface receptors for each of the four active peptides using peptide affinity chromatography. Only one peptide recognized a cell surface protein. Peptide A5G27 (RLVSYNGIIFFLK, residues 2892-2904) bound a diffuse M(r) approximately 120,000-180,000 band that eluted with 2 m NaCl. Glycosidase digestion of the 2 m eluate yielded protein bands of M(r) 90,000 and 60,000 that reacted in Western blot analysis with antibodies to CD44. Immunoprecipitation of the A5G27-bound membrane proteins with various cell surface proteoglycan antibodies confirmed CD44 as the surface receptor for A5G27. Finally, attachment assays to A5G27 in the presence of soluble glycosaminoglycans (GAGs) identified the GAGs of CD44 as the binding sites for A5G27. Our results suggest that A5G27 binds to the CD44 receptor of B16-F10 melanoma cells via the GAGs on CD44 and, thus, inhibits tumor cell migration, invasion, and angiogenesis in a dominant-negative manner.

The difference of angiogenesis in human lung adenocarcinoma cell lines with different metastatic potency.

We investigated the ability of angiogenesis in PC9/F9 cells (from a highly metastatic human lung adenocarcinoma cell line) as compared with PC9 cells (from a low metastatic human lung adenocarcinoma cell line). In vivo tumor growth assay using BALB/c nude mice (7 mice/group), showed that the tumor volume of PC9/F9 cells on day 35 (230.7+/-31.3 mm(3)) was significantly larger than that of PC9 cells (90.9+/-24.7 mm(3)) (p<0.001). However, there was no significant difference between PC9/F9 cells and PC9 cells in an in vitro growth assay. In a dorsal air sac assay (DAS assay) using ICR mice (3 mice/group), PC9/F9 cells (4.7+/-1.2 vessels) showed stronger neovascurizationin in compared with PC9 cells (0.3+/-0.4 vessels) (p<0.05). In an enzyme linked immunosorbent assay (ELISA) and Western blotting analysis there were no significant differences between PC9/F9 cells and PC9 cells in the protein expression of vascular endothelial growth factor (VEGF). There was no significant difference between the gene expression levels of PC9/F9 cells and PC9 cells on cDNA array analysis. Matrix metalloproteinase-2 (MMP-2) activity in PC9/F9 cells was remarkably stronger than that of PC9 cells in Gelatin Zymography. From these results, we considered that of the increased metastasis of PC9/F9 cells might be induced by augmented angiogenesis. Furthermore, we speculated that the augmented angiogenesis of the highly metastatic PC9/F9 cell line might be induced by increased MMP-2 activity.

Weekly administration of irinotecan (CPT-11) plus cisplatin for refractory or relapsed small cell lung cancer.

PURPOSE: Weekly administrations of CPT-11 plus cisplatin together with an anti-diarrheal program, the Oral Alkalization and Control of Defecation [Int J Cancer 1999;83:491; Int J Cancer 2001;92:269; Cancer Res 2002;62:179], were evaluated in this phase II study for patients with refractory or relapsed small cell lung cancer. METHODS: Patients were treated by weekly administrations of 60 mg/m(2) CPT-11 plus 30 mg/m(2) cisplatin on Days 1, 8 and 15 over 4 weeks. Coinciding with the infusions and for 4 days thereafter, the anti-diarrheal program was practiced using orally administered sodium bicarbonate, magnesium oxide and basic water. RESULTS: Twenty-five patients who had prior treatments of etoposide and platinum containing regimens (16 refractory patients and nine relapsed patients) were entered. The mean dose-intensities of CPT-11 and cisplatin were 154.8 and 77.4 mg/m(2) per course, respectively. Therefore, 86% of the planned dose was delivered. There were 20 partial responses and an overall response rate of 80% (95% confidence interval, 62-96%) was obtained. The median time to progression and the median survival after starting this regimen were 3.6 and 7.9 months, respectively. The major toxicity was myelosuppression. Grades 3 and 4 neutropenia occurred in 24 and 12% of patients, respectively. One patient with febrile neutropenia was experienced, and Grade 3 diarrhea was observed in 8%. But there was no treatment death. CONCLUSION: Weekly administrations of CPT-11 plus cisplatin together with Oral Alkalization and Control of Defecation provide a practical and well tolerated regimen that was active for refractory or relapsed small cell lung cancer.

Identification of a potent peptide antagonist to an active laminin-1 sequence that blocks angiogenesis and tumor growth.

The extracellular matrix plays an important role in many physiological processes. We have identified >20 angiogenic sites in the extracellular matrix protein laminin-1. The most potent sites are A13 (RQVFQVAYIIIKA) and C16 (KAFDITYVRLKF), which are present in homologous NH(2)-terminal domains of the alpha 1 and gamma 1 chains, respectively. We reported recently that a scrambled C16 sequence, C16S (DFKLFAVTIKYR), acts as an antagonist to both peptides. Here, we have identified a stronger antiangiogenic peptide, C16Y (C16S with a T to Y substitution), with potent activity in several biological assays including tumor growth. C16Y is more potent in promoting endothelial cell attachment and inhibiting attachment to laminin-1 than either C16 or C16Y. Disruption of tube formation by C16Y is also observed at concentrations at least five times lower than C16S. The minimal active sequence was found to be DFKLFAVY. C16Y is more potent in blocking C16-induced chick chorioallantoic membrane angiogenesis than C16S. Tumor growth studies on the chick chorioallantoic membrane showed that C16Y reduces breast cancer cell growth without affecting cell proliferation. This result suggests that angiogenesis is being inhibited by the peptide. In vivo animal studies demonstrated that C16Y treatment significantly reduced tumor growth and decreased tumor vessel number, as compared with controls, additionally suggesting that angiogenesis was affected. These results indicate that we have identified a more potent antiangiogenesis inhibitor peptide that may be used as a therapeutic to treat cancer.

Increased expression of the LGALS3 (galectin 3) gene in human non-small-cell lung cancer.

Patients with lung cancer have a poor prognosis because of the high metastatic potential of the neoplasm. Therefore, identifying new molecular targets for anti-metastatic therapy is very important. To identify novel key factors of tumor metastasis in lung cancer, we established the gene expression profiles of two adenocarcinoma cell line variants, PC9/f9 and PC9/f14, by use of genome-wide human cDNA microarray analysis and comparing these profiles with that of the parental cell line, PC9. The PC9/f9 and PC9/f14 cell lines were selected for analysis because of their high metastatic potential. We identified five genes in the highly metastatic cell lines that showed a significantly enhanced or reduced expression and that had not been reported to be involved in metastasis of lung cancer. One of the overexpressed genes that was identified encoded the beta-galactoside-binding protein LGALS3 (Galectin 3). LGALS3 has been reported to be overexpressed in a variety of human cancers, but not in lung cancer, and to be involved in tumor metastasis. We examined the expression of LGALS3 by use of real-time quantitative reverse transcription-polymerase chain reaction in 38 lung cancer cell lines and in tumor tissue obtained by thoracoscopic biopsy. A population (10/30) of the non-small-cell lung cancers examined was found to overexpress the LGALS3 gene at levels three times higher than those of normal epithelial cells. In contrast, all small-cell lung cancers either failed to express the gene or expressed it at a very low level. The mean of the relative expression of the LGALS3 gene in non-small-cell lung cancer (3.065 +/- 3.976) was significantly higher than those of small-cell lung cancer (0.02 +/- 0.03) (P < 0.025). This is the first report of alterations of LGALS3 gene expression in lung cancer. These results, together with the previous reports on Galectin 3 function, suggest that Galectin 3 may play a role in the process of metastasis in non-small-cell lung cancer that overexpresses Galectin 3, but not in small-cell cancer. Accordingly, LGALS3 may be a phenotypic marker that excludes small-cell lung cancer and may represent a novel target molecule in non-small-cell lung cancer therapy.

A novel matrix metalloproteinase inhibitor, FYK-1388 suppresses tumor growth, metastasis and angiogenesis by human fibrosarcoma cell line.

The matrix metalloproteinases (MMPs) are likely to contribute to tumor cell invasion, metastasis and angiogenesis. Several MMP inhibitors have been developed, recently and their anti-tumor efficacy is being evaluated in clinical trials. FYK-1388 is a novel broad MMP inhibitor which blocks the activity of MMP-1, -2, -3, -7, -9, -13 and -14 (MT-MMP-1). It is especially effective against MMP-2 and -9 more so than other MMP inhibitors such as Marimastat, Ro 32-3555 and D-2163. Here, we investigated the anti-tumor efficacy of FYK-1388 using the human fibrosarcoma cell line HT-1080. These cells produced MMP-2 and -9, which FYK-1388 inhibited at a dose of 10(-8) M. FYK-1388 at 0.2 mg/mouse/day significantly suppressed tumor growth when given by s.c. injection for 22 days, experimental lung metastasis after 5 days s.c. injection and also suppressed tumor-induced angiogenesis in the dorsal air sac assay after 7 days s.c. injection. In the MTT assay, FYK-1388 had no effect on the in vitro growth of HT-1080 cells. These results suggest that FYK-1388 possesses anti-tumor efficacy as a result of inhibiting angiogenesis through the suppression of MMP-2 and -9 activity.