[Analysis of gene expression profiles in malignant brain tumors with respect to sensitivity to therapies].
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Antineoplastic Combined Chemotherapy Protocols↗
Biomedical subjects
Kazuto Nishio
Publications and source records attributed to Kazuto Nishio.
Small in-frame deletion in the epidermal growth factor receptor as a target for ZD6474.
ZD6474 is an inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2/KDR) tyrosine kinase, with additional activity against epidermal growth factor receptor (EGFR) tyrosine kinase. ZD6474 inhibits angiogenesis and growth of a wide range of tumor models in vivo. Gefitinib ("Iressa") is a selective EGFR tyrosine kinase inhibitor that blocks signal transduction pathways implicated in cancer cell proliferation. Here, the ability of gefitinib and ZD6474 to inhibit tumor cell proliferation was examined directly in eight cancer cell lines in vitro, and a strong correlation was noted between the IC(50) values of gefitinib and ZD6474 (r = 0.79). No correlation was observed between the sensitivity to ZD6474 and the level of EGFR or VEGFR expression. The NSCLC cell line PC-9 was seen to be hypersensitive to gefitinib and ZD6474, and a small (15-bp) in-frame deletion of an ATP-binding site (exon 19) in the EGFR was detected (delE746-A750-type deletion). To clarify the involvement of the deletional mutation of EGFR in the cellular sensitivity to ZD6474, we examined the effect of this agent on HEK293 stable transfectants expressing deletional EGFR that designed as the same deletion site observed in PC-9 cells (293-pDelta15). These cells exhibited a 60-fold higher sensitivity to ZD6474 compared with transfectants expressing wild-type EGFR. ZD6474 inhibited the phosphorylation of the mutant EGFR by 10-fold compared with cells with wild-type EGFR. In conclusion, the findings suggested that a small in-frame deletion in the EGFR increased the cellular sensitivity to ZD6474.
Base Sequence↗
hnRNP L enhances sensitivity of the cells to KW-2189.
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are involved in several RNA-related biological processes. We demonstrated hnRNP L as a candidate protein of DARP (duocarmycin-DNA adduct recognizing protein) by gel shift assay and amino acid sequencing. Stable transfectants of hnRNP L showed high sensitivity of the cells to the growth inhibitory effect of KW-2189, a duocarmycin derivative in vitro. Immunostaining of hnRNP L demonstrated differential intracellular localization of hnRNP L among human lung cancer cell lines. A transfection study using a series of deletion mutants of hnRNP L fused to indicated that the N-terminal portions of RRM(RNA recognition motif)1, RRM3 and RRM2 are involved in localization of hnRNP L. We identified sequences in these portions that have high homology with the sequences of known NLS (nuclear localization signal) and NES (nuclear export signal). hnRNP L is a factor that determines the sensitivities of cancer cells to the minor groove binder, and overexpression and differential intracellular localization of hnRNP L are involved in its function in lung cancer.
Amino Acid Sequence↗
Synergistic interaction between the EGFR tyrosine kinase inhibitor gefitinib ("Iressa") and the DNA topoisomerase I inhibitor CPT-11 (irinotecan) in human colorectal cancer cells.
Epidermal growth factor receptor [EGFR (HER1, erbB1)] is a receptor with associated tyrosine kinase activity, and is expressed in colorectal cancers and many other solid tumors. We examined the effect of the selective EGFR tyrosine kinase inhibitor (EGFR-TKI) gefitinib ("Iressa") in combination with the DNA topoisomerase I inhibitor CPT-11 (irinotecan) on human colorectal cancer cells. EGFR mRNA and protein expression were detected by RT-PCR and immunoblotting in all 7 colorectal cancer cell lines studied. Gefitinib inhibited the cell growth of the cancer cell lines in vitro with an IC(50) range of 1.2-160 microM by 3,(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Lovo cells exhibited the highest level of protein and autophosphorylation of EGFR and were the most sensitive to gefitinib. The combination of gefitinib and CPT-11 induced supra-additive inhibitory effects in COLO320DM, WiDR and Lovo cells, assessed by an in vitro MTT assay. Administration of gefitinib and CPT-11 had a supra-additive inhibitory effect on WiDR cells and tumor shrinkage was observed in Lovo cell xenografts established in nude mice, whereas no additive effect of combination therapy was observed in COLO320DM cells. To elucidate the mechanisms of synergistic effects, the effect of CPT-11-exposure on phosphorylation of EGFR was examined by immunoprecipitation. CPT-11 increased phosphorylation of EGFR in Lovo and WiDR cells in time- and dose-dependent manners. This EGFR activation was completely inhibited by 5 microM gefitinib and gefitinib-induced apoptosis was enhanced by combination with CPT-11, measured by PARP activation although no PARP activation was induced by 5 microM CPT-11 alone. These results suggested that these modification of EGFR by CPT-11, in Lovo cells, is a possible mechanism for the synergistic effect of CPT-11 and gefitinib. These findings imply that the EGFR-TKI gefitinib and CPT-11 will be effective against colorectal tumor cells that express high levels of EGFR, and support clinical evaluation of gefitinib in combination with CPT-11, in the treatment of colorectal cancers.
Animals↗
Synergistic interaction between gefitinib (Iressa, ZD1839) and paclitaxel against human gastric carcinoma cells.
We have evaluated the antitumor effects of gefitinib (Iressa, ZD1839) in SNU-1 human gastric cancer cells (hMLH1-deficient and epidermal growth factor receptor-overexpressed) when given alone or as a doublet with oxaliplatin (LOHP), 5-fluorouracil (5-FU) or paclitaxel (PTX). The four drugs showed IC50s ranging from 1.81 nM to 13.2 microM. LOHP and PTX induced G2/M arrest, 5-FU increased S phase, and gefitinib increased G1 in a concentration-dependent manner. The analysis using the previously developed cytostatic TPi model showed that 64 and 80% of the overall growth inhibition was attributed to cell cycle arrest in cells exposed to 7.55 microM of LOHP or 10 nM of PTX for 72 h, respectively. PTX + gefitinib showed greatest synergism as determined by combination index analysis and apoptosis induced by PTX was potentiated by the co-administration of gefitinib. LOHP + gefitinib showed a similar, although to a lesser degree, synergistic effect. This study demonstrates the antitumor activity and the significant cell cycle arrest induced by gefitinib in SNU-1 human gastric carcinoma cells, and its synergistic interaction with LOHP and PTX.
Adaptor Proteins, Signal Transducing↗
Anticancer effects of ZD6474, a VEGF receptor tyrosine kinase inhibitor, in gefitinib ("Iressa")-sensitive and resistant xenograft models.
ZD6474 is a novel, orally available inhibitor of vascular endothelial growth factor (VEGF) receptor-2 (KDR) tyrosine kinase, with additional activity against epidermal growth factor receptor (EGFR) tyrosine kinase. ZD6474 has been shown to inhibit angiogenesis and tumor growth in a range of tumor models. Gefitinib ("Iressa") is an selective EGFR tyrosine kinase inhibitor (TKI) that blocks signal transduction pathways. We examined the antitumor activity of ZD6474 in the gefitinib-sensitive lung adenocarcinoma cell line, PC-9, and a gefitinib-resistant variant (PC-9/ZD). PC-9/ZD cells showed cross-resistance to ZD6474 in an in vitro dye formation assay. In addition, ZD6474 showed dose-dependent inhibition of EGFR phosphorylation in PC-9 cells, but inhibition was only partial in PC-9/ZD cells. ZD6474-mediated inhibition of tyrosine residue phosphorylation (Tyr992 and Tyr1045) on EGFR was greater in PC-9 cells than in PC-9/ZD cells. These findings suggest that the inhibition of EGFR phosphorylation by ZD6474 can contribute a significant, direct growth-inhibitory effect in tumor cell lines dependent on EGFR signaling for growth and/or survival. The effect of ZD6474 (12.5-50 mg/kg/day p.o. for 21 days) on the growth of PC-9 and PC-9/ZD tumor xenografts in athymic mice was also investigated. The greatest effect was seen in gefitinib-sensitive PC-9 tumors, where ZD6474 treatment (>12.5 mg/kg/day) resulted in tumor regression. Dose-dependent growth inhibition, but not tumor regression, was seen in ZD6474-treated PC-9/ZD tumors. These studies demonstrate that the additional EGFR TKI activity may contribute significantly to the antitumor efficacy of ZD6474, in particular in those tumors that are dependent on continued EGFR-signaling for proliferation or survival. In addition, these results provide a preclinical rationale for further investigation of ZD6474 as a potential treatment option for both EGFR-TKI-sensitive and EGFR-TKI-resistant tumors.
Adenocarcinoma↗
[Resistance to target-based therapy and its circumvention].
Intrinsic and acquired resistance to molecular target therapy critically limits the outcome of cancer treatments. Target levels including quantitative and gene alteration should be determinants for the resistance. Downstream of the target molecules, drug metabolism, and drug transport influences the tumor sensitivity to molecular target therapy. The mechanisms of resistance to antibody therapy have not been fully clarified. Correlative clinical studies using these biomarkers of resistance are extremely important for circumvention of clinical resistance to target based therapy.
Antibodies, Monoclonal↗
Novel cisplatin-incorporated polymeric micelles can eradicate solid tumors in mice.
Polymeric micelles incorporating cisplatin (CDDP) were prepared through the polymer-metal complex formation between CDDP and poly(ethylene glycol)-poly(glutamic acid) block copolymers, and their utility as a tumor-targeted drug delivery system was investigated. CDDP-incorporated micelles (CDDP/m) had a size of 28 nm with remarkably narrow distribution. CDDP/m were very stable in distilled water even in long-time storage, but exhibited a sustained drug release accompanied with the decay of the carrier itself in physiological saline. These micelles showed remarkably prolonged blood circulation and effectively accumulated in solid tumors (Lewis lung carcinoma cells) according to the passive targeting manner (20-fold higher than free CDDP). Reduced accumulation of the micelles in normal organs provided high selectivity to the tumor. In vivo antitumor activity assay demonstrated that both free CDDP and the CDDP/m had significant antitumor activity in C 26-bearing mice compared with nontreatment (P < 0.05 for free CDDP; P < 0.01 for CDDP/m), but complete tumor regression was observed only for the treatment with CDDP/m. Four of 10 mice treated with CDDP/m (4 mg/kg; five times administration at 2-day intervals) showed complete tumor regression with no significant body weight loss, whereas free CDDP treatment at the same drug dose and regime resulted in tumor survivals and approximately 20% of body weight loss. These data suggest that CDDP/m could be a promising formulation of CDDP for the targeted therapy of solid tumors.
Adenocarcinoma↗
Osteopontin overproduced by tumor cells acts as a potent angiogenic factor contributing to tumor growth.
Angiogenesis, which is essential for tumor growth, is regulated by various angiogenic factors. Osteopontin (OPN) is expressed in various human tumors and is postulated to be involved in tumor progression. We have recently reported that culture medium with murine neuroblastoma C1300 cells transfected with OPN gene significantly stimulates human umbilical vein endothelial cell migration and induces neovascularization in mice by dorsal air sac assay. However, the effect of OPN on tumorigenesis as an angiogenic factor remains to be clarified. In this study, we injected the OPN-transfected C1300 cells and control cells into the nude mice subcutaneously. OPN-overexpressing C1300 cells significantly formed rapidly growing tumor as compared to the control cells in mice, although in vitro and in vivo cell growth rates were similar. In vivo tumorigenecity of these cells correlated with the amount of secreted OPN protein. In addition, neovascularization of OPN-transfected tumor was significantly increased in comparison with those of control cells by immunohistochemistry for CD31. In vitro chemoinvasiveness and gene expression of proteases including uPA, MMP2, 9, MT1-MMP, and cathepsin B, D, L, were not different between OPN-transfected and control cells determined with matrigel invasion assay and cDNA expression macroarray, respectively. Conclusively, these results strongly imply that OPN plays an important role in tumor growth through the enhancement of angiogenesis in vivo.
Angiogenesis Inducing Agents↗
Strategy for the development of novel anticancer drugs.
Progress in molecular pharmacology has demonstrated each anticancer drug to have a unique molecular target. Recent drug development has focused on compounds that specifically inhibit and/or modify tumor-specific molecular biological changes (target-based drug development). These compounds are generally classified as either small molecules or macromolecules. With the exception of antibodies, the majority of recently developed target-based drugs are small molecules. Assessing the effects of these compounds on their targets would probably help researchers to predict the antitumor effects of these anticancer drugs; however, actually assessing this hypothesis, even in preclinical models, is difficult. Most preclinical experiments attempt to show tumor growth inhibition or shrinkage, leading to a longer survival period or higher cure rate. Few experiments have examined the correlation between antitumor activity and the effect of a compound on its target. In phase I clinical trials of target-based drugs, the determination of maximum tolerated dose is not enough; the effect of the drug on its target should also be evaluated. Recently, dose-escalation strategies based on the effects of drugs on their targets have been proposed, even though an appropriate target effect is necessary but not sufficient to demonstrate clinical efficacy. Compounds that are not specifically directed against molecular targets on or within tumor cells, but against blood vessels, matrix, etc., usually do not cause tumor shrinkage. These compounds include angiogenesis inhibitors and matrix metalloproteinase inhibitors and are usually used in combination with other treatments at the start of clinical trials. Whether the methodology of clinical trials is sensitive enough to detect the subtle effects of these compounds remains uncertain. The effects of experimental drugs on their targets are rarely examined in clinical trials. Few data from translational studies are available and data obtained using surrogate tissues do not necessarily reflect the effects of the drugs on in situ tumors. Parameters such as time to progression, changes in tumor markers, and growth rates often vary significantly and are regarded as soft endpoints. Phase III trials evaluating survival benefit require extensive resources, including a large number of patients, a sophisticated data center, and well-trained study groups. The problems and future prospects of novel anticancer drug development are discussed.
Antineoplastic Agents↗
Translational and clinical studies of target-based cancer therapy.
The increasing number of negative trials in cancer treatment has prompted a systematic re-evaluation of target-based drugs. It is our impression that the sensitivity of current clinical trial methodology is inadequate to demonstrate the effectiveness of target-based drugs, in terms of tumor shrinkage, delay in progression, or improved survival. Enrichment of sensitive populations, based on the data from translational studies, is essential for a better understanding of the role of novel therapies in cancer. Translational and clinical studies are key to the identification and maximization of benefit of target-based therapy
Antineoplastic Agents↗
Antitumor activity of TZT-1027 (Soblidotin) against vascular endothelial growth factor-secreting human lung cancer in vivo.
TZT-1027 (Soblidotin), an antimicrotubule agent, has been demonstrated to show potent antitumor effects, though the relationships among antitumor effect, cytotoxicity and anti-vascular effect of TZT-1027 have not been studied. We established in vivo human lung vascular-rich tumor models using a vascular endothelial growth factor-secreting tumor (SBC-3/VEGF). SBC-3/VEGF tumors exhibited a high degree of angiogenesis in comparison with the mock transfectant (SBC-3/Neo) tumors in a dorsal skinfold chamber model and grew much faster and larger than SBC-3/Neo tumors in the tumor growth study. The antitumor activity of antimicrotubule agents, including TZT-1027, was evaluated in both early- and advanced-stage SBC-3/Neo and SBC-3/VEGF tumor models to elucidate the relationship between the antitumor activity and anti-vascular effect of these agents. TZT-1027 exhibited potent antitumor activity against both early- and advanced-stage SBC-3/Neo and SBC-3/VEGF tumors, whereas combretastatin A4 phosphate did not. Vincristine and docetaxel exhibited potent antitumor activity against early-stage SBC-3/Neo and SBC-3/VEGF tumors, and advanced-stage SBC-3/Neo tumors, but did not exhibit activity against advanced-stage SBC-3/VEGF tumors. The difference in antitumor activity between these agents could be ascribed to differences in direct cytotoxicity and anti-vascular effect. Furthermore, a prominent accumulation of erythrocytes in the tumor vasculature, followed by leakage and scattering of these erythrocytes from the tumor vasculature, was observed after TZT-1027 administration to mice bearing advanced-stage SBC-3/VEGF tumors. These findings strongly suggest that TZT-1027 has a potent anti-vascular effect, in addition to direct cytotoxicity.
Animals↗
Restoration of p53 gene function in 12-O-tetradecanoylphorbor 13-acetate-resistant human leukemia K562/TPA cells.
The human leukemia K562 cell line does not express wild-type p53 protein. Due to the loss of one p53 allele and an insertion mutation in exon 5 of the other allele resulting in a frameshift mutation, K562 cells express a truncated p53 protein of 148 amino acids. A human leukemia phorbol ester-resistant subline, K562/TPA, is cross-resistant to some anticancer agents. A remarkable difference in cell cycle progression at G1/S phase was observed in the synchronised K562/TPA cells as compared with K562 cells. Southern blot and DNA sequence analysis revealed no mutation in exon 5 of the p53 gene in K562/TPA cells. p21Cip1 expression was also restored in K562/TPA cells confirming that the reversal of this p53 gene mutation restored wild-type p53 function in these cells. This is a unique report describing reversal of p53 gene mutation by drugs. This was associated with the expression of wild-type p53 mRNA and protein in K562/TPA cells. The K562/TPA cell line may provide a very useful tool for the investigation of the relationship between p53 status and chemosensitization.
Blotting, Southern↗
Differential gene expression profiles and identification of the genes relevant to clinicopathologic factors in colorectal cancer selected by cDNA array method in combination with principal component analysis.
The clinical outcome of patients with colorectal cancer frequently varies even if they are at the same clinicopathologic stage. Alternative superior tumor markers of colorectal cancer are needed for prediction of clinical outcome. To clarify the regulatory factors in colorectal cancers, we examined differential expression profiles using cDNA macroarray technique with surgically resected specimens obtained from the patients with colorectal cancer. The gene profiles by an average-linkage hierarchical clustering analysis were found to be almost separable into two groups: tumor group and normal mucosa group. The relationship between several clinicopathologic factors and cancer related genes were investigated by using statistical analyses including principal component analysis (PCA). c-myc-binding protein MM-1, and c-jun proto-oncogene were identified as possible markers of tumor histology and clinical prognosis and early growth response protein 1 (EGR1) was selected to play an important role in progression of clinical stage. We conclude that, with PCA method, we successfully selected the genes relevant to clinicopathologic factors using limited population of clinical samples.
Aged↗
Selection of surrogate marker genes in primary central nervous system lymphomas for radio-chemotherapy by DNA array analysis of gene expression profiles.
Primary central nervous system lymphomas (PCNSLs) are extra nodal B-cell non-Hodgkin's lymphomas with primary manifestation in the brain, and their incidence has been increasing among both immunocompetent and immunocompromised populations. Samples of oligodendroglioma (n=5), glioblastoma (n=7), PCNSL (n=6), and normal brain (n=3) were studied (total of 21 samples) using cDNA array technology. The hierarchical clustering algorithm was used to obtain a phylogenetic tree, and it revealed a striking feature: PCNSL was clearly separated. The genes encoding laminin receptor 2, thioredoxin peroxidase, and elongation factor-1 were selected as specific genes in PCNSL by principal component analysis (PCA). When Mann-Whitney tests were performed to identify genes responsible for the differences between responders and non-responders to the treatment schedule for PCNSL, 76 known genes were found to show significantly different expression patterns between the two groups at the P<0.01 level. The two groups were clearly separated by the re-clustering method using the selected genes related to response to chemo-radiotherapy. This is the first report describing the gene expression profiles of PCNSL. In conclusion, accumulation of data with respect to the expression profiles of PCNSL specimens, clinicopathological data, susceptibility to treatment, and outcome will provide information for identifying optimal therapeutic modalities for individual patients and novel therapeutic targets.
Adult↗
Osteopontin induces angiogenesis of murine neuroblastoma cells in mice.
Angiogenesis is an essential process for tumor growth and is regulated by tumor-derived angiogenic cytokines. Osteopontin (OPN) is one of the cytokines produced by various tumor cells and is suggested to be involved in angiogenesis by upregulating endothelial cell migration in cooperation with vascular endothelial cell growth factor (VEGF). To provide evidence of OPN involvement in a causal role in tumor angiogenesis, we generated a stable transfectant from murine neuroblastoma C1300 cells to constitutively secrete high levels of murine OPN. The OPN mRNA expression and protein secretion were confirmed by RT-PCR and ELISA, respectively. The biological activity of secreted OPN was determined with migration assay by using human umbilical vein endothelial cells (HUVEC). Transfection with OPN gene did not increase VEGF production and did not affect gene expression of other angiogenic factors confirmed by complementary DNA macroarray system. To demonstrate the effect of OPN on tumor-induced angiogenesis in vivo, millipore chambers containing OPN-transfected or control cells were implanted to the dorsal air sac of mice. The OPN-transfected cells significantly induced neovascularization in comparison to the control cells in mice. Conclusively, these results provide direct evidence of OPN involvement in the role of tumor angiogenesis.
Animals↗
Antitumor activity of the selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) Iressa (ZD1839) in an EGFR-expressing multidrug-resistant cell line in vitro and in vivo.
Selective tyrosine kinase inhibitors are regarded as promising antitumor agents for cancer treatment. Iressa (ZD1839) is an orally active, selective EGFR-TKI (epidermal growth factor receptor-tyrosine kinase inhibitor) that blocks signal transduction pathways implicated in cancer cell proliferation, survival and other host-dependent processes promoting cancer growth. The cellular mechanisms of ZD1839 action against human malignant cells and drug-resistant cells were evaluated in vitro. Among the cell lines tested, ZD1839 showed a strong growth-inhibitory effect in vitro on human leukemic cells resistant to phorbol ester. This cell line, K562/TPA, shows a non-P-glycoprotein-mediated multidrug-resistant phenotype. The IC50 value of ZD1839 on K562/TPA was approximately 400-fold lower than that on the parental K562 cell (K562 = 12 +/- 2 microM; K562/TPA = 0.025 +/- 0.002 microM) in vitro as determined by a dye formation assay. The expression of EGFR and EGFR mRNA was clearly present in K562/TPA but not in parental K562 cells as determined by Western blotting and RT-PCR. EGFR was autophosphorylated in K562/TPA detected by the antiphosphotyrosine antibody. The in vivo antitumor effects of ZD1839 on K562 and K562/TPA cells were also investigated in BALB/c nude mice. K562/TPA cells transplanted subcutaneously into mice disappeared completely with ZD1839 treatment (20 mg/kg/day, days 3-9). This was not the case in K562 cells. These results suggest that ZD1839 is highly active against tumor cells with non-P-glycoprotein-mediated multidrug resistance that express EGFR. Iressa is a trademark of AstraZeneca (Cheshire, UK).
Animals↗
Mechanism of vinorelbine-induced radiosensitization of human small cell lung cancer cells.
Vinorelbine (Navelbine, KW-2307), a semisynthetic vinca alkaloid, is a potent inhibitor of mitotic microtubule polymerization. The aims of this study were to demonstrate vinorelbine-induced radiosensitization of human small cell lung cancer (SCLC) SBC-3 cells and to elucidate the mechanisms of radiosensitization. A clonogenic assay demonstrated that SBC-3 cells were sensitized to radiation by vinorelbine using different schedules combining exposure to both. The sensitizer enhancement ratios (SERs) at a cell survival level of 10% were 1.42+/-0.21 to 1.33+/-0.06, and 1.22+/-0.07 depending on schedule. Vinorelbine-induced radiosensitization did not depend on the schedule of the combined exposure. Flow cytometric analyses showed that the cells did not accumulate in the radiosensitive G(2)/M phase of the cell cycle after concurrent treatment with vinorelbine and radiation. The results of an alkaline filter elution assay demonstrated that in the presence of vinorelbine at 1 n M radiation-induced DNA strand breaks were not completely repaired at 24 h postradiation. We conclude that human SCLC SBC-3 cells are sensitized to radiation by vinorelbine and that a possible mechanisms of vinorelbine-induced radiosensitization may at least in part be associated with impairment of DNA repair following radiation-induced DNA damage.
Carcinoma, Small Cell↗