Newly synthesized dihydropyridine derivatives as modulators of P-glycoprotein-mediated multidrug resistance.

Newly synthesized 1,4-dihydropyridine derivatives possessing alkyl chains at the 4-position screened whether they could overcome P-glycoprotein-mediated multidrug resistance in cultured cancer cells and also leukemia-bearing animals. Of these derivatives, some could overcome drug resistance to doxorubicin and vincristine in multidrug resistant human cancer cell lines. Combined administration of vincristine and some of the derivatives significantly increased the life span of P-glycoprotein overexpressing multidrug-resistant P388 leukemia-bearing mice.

Expression of multidrug resistance protein gene in patients with glioma after chemotherapy.

Two different ATP-binding membrane glycoproteins, the 170 kDa P-glycoprotein (P-gp) and the 190 kDa multidrug resistance protein (MRP), are involved in the acquisition of multidrug resistance phenotypes in cancer cells. Overexpression of P-gp is often observed in various human tumors when treated with anticancer agents. In this study, we asked whether MRP was overexpressed in human gliomas after cancer chemotherapy.

Hypomethylation status of CpG sites at the promoter region and overexpression of the human MDR1 gene in acute myeloid leukemias.

Selection of human cells for resistance to vincristine or doxorubicin often induces overexpression of the multidrug resistance 1 gene (MDR1), which encodes the cell surface P-glycoprotein, as a result of gene amplification or transcriptional activation. Moreover, overexpression of the MDR1 gene has been shown to be associated closely with clinical outcome in various hematological malignancies, including acute myeloid leukemia (AML). However, the precise mechanism underlying overexpression of the MDR1 gene during acquisition of drug resistance remains unclear.

Enhanced expression of thrombospondin-1 and hypovascularity in human cholangiocarcinoma.

Cholangiocarcinoma (CCC) is relatively hypovascular, in contrast to hepatocellular carcinoma (HCC), which is often highly vascular. We investigated if the diminished vascularity of CCC is related to altered expression of thrombospondin-1 (TSP-1), an antiangiogenic factor, and/or vascular endothelial growth factor (VEGF), a potent angiogenic factor, comparing the relationships with those of high- and low-vascular HCC. We also investigated the relationship between the mutation of the p53 gene and TSP-1 expression or VEGF expression.

Isolation of a novel human canalicular multispecific organic anion transporter, cMOAT2/MRP3, and its expression in cisplatin-resistant cancer cells with decreased ATP-dependent drug transport.

The human multidrug resistance protein (MRP) gene encodes a membrane protein involved in the ATP-dependent transport of hydrophobic compounds. We previously isolated a canalicular multispecific organic anion transporter, cMOAT1/MRP2, that belongs to the ATP binding cassette (ABC) superfamily, which is specifically expressed in liver, and cMOAT1/MRP2 is responsible for the defects in hyperbilirubinemia II/Dubin-Johnson syndrome. In this study, we isolated a new cDNA of the ABC superfamily designated cMOAT2/MRP3 that is homologous to human MRP1 and cMOAT1/MRP2: cMOAT2/MRP3 is 56% identical to MRP1 and 45% identical to cMOAT1/MRP2, respectively.

Novel biological functions of interleukin-4: formation of tube-like structures by vascular endothelial cells in vitro and angiogenesis in vivo.

The effect of interleukin-4 (IL-4) on angiogenesis was studied in vitro and in vivo. Human recombinant IL-4 significantly stimulated the formation of tube-like structures in collagen gels by bovine aortic endothelial cells as well as by human microvascular endothelial cells. Human recombinant IL-4 at 50-500 U/ml stimulated by about two- to threefold the formation of tubes by bovine aortic endothelial cells; the rate was comparable to that of basic fibroblast growth factor.

Nuclear expression of YB-1 protein correlates with P-glycoprotein expression in human osteosarcoma.

The Y-box-binding protein, YB-1, is a member of the DNA-binding protein family. It binds to the Y-box, an inverted CCAAT box, in the promoter region of the human multidrug resistance 1 gene, which encodes P-glycoprotein (P-gp). Nuclear localization of YB-1 protein has been reported to be associated with the intrinsic expression of P-gp in human breast cancer.

Clinical significance of the increased multidrug resistance-associated protein (MRP) gene expression in patients with primary breast cancer.

The efficacy of cancer treatment is limited by either intrinsic or acquired resistance to various chemotherapeutic agents. To evaluate the clinically important factors related to prognosis in primary breast cancer retrospectively, we investigated the expression of the following genes involving acquirement of drug resistance: multidrug resistance 1 (MDRl), multidrug resistance-associated protein (MRP), and topoisomerase (Topo) I, II alpha, and II beta. Using an RT-PCR method, we semiquantified the gene expression level in untreated stage II breast cancer tissue (n = 27) and noncancerous breast tissue (n = 10).

Effect of retinoic acid on morphological changes of human pancreatic cancer cells on collagen gels: a possible association with the metastatic potentials.

Pancreatic carcinoma is an invasive and metastasizing type of malignancy. We established six pancreatic cancer cell lines from human pancreatic carcinomas, three highly metastatic lines (KP-1NL, KP-4, and SUIT-2) and three minimally metastatic lines (KP-2, KP-3, and BxPC-3). The three highly metastatic cell lines grew in a fibroblastoid pattern on collagen gels, whereas the three minimally metastatic cell lines grew in an epithelioid pattern under similar conditions.

Glutathione homeostasis in human hepatic cells: overexpression of gamma-glutamylcysteine synthetase gene in cell lines resistant to buthionine sulfoximine, an inhibitor of glutathione synthesis.

The synthesis of glutathione (GSH) and its conjugation to xenobiotics are essential for detoxification in liver cells. To understand how cellular levels of GSH are balanced in response to environmental stress, we cloned two cell lines, HLE/BSO1-1 and HLE/BSO1-2, from human hepatic HLE/WT cells resistant to buthionine sulfoximine (BSO), an irreversible inhibitor of gamma-glutamylcysteine synthetase (GCS). HLE/BSO1-1 and HLE/BSO1-2 showed 35- and 40-fold higher resistance respectively, than the wild type to BSO.

The role of an inverted CCAAT element in transcriptional activation of the human DNA topoisomerase IIalpha gene by heat shock.

Expression of the DNA topoisomerase IIalpha (topoIIalpha) gene is highly sensitive to various environmental stimuli including heat shock. The amount of topoIIalpha mRNA was increased 1.5-3-fold 6-24 h after exposure of T24 human urinary bladder cancer cells to heat shock stress at 43 degreesC for 1 h.

Involvement of vascular endothelial growth factor and urokinase-type plasminogen activator receptor in microvessel invasion in human colorectal cancers.

To evaluate the association among known angiogenic growth factors or factors related to the plasminogen activation system and clinicopathological factors in patients with colorectal cancer, we examined the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor-alpha (TGF-alpha), urokinase-type plasminogen activator (u-PA), u-PA receptor (u-PA-R) and plasminogen activator inhibitor-1 (PAI-1) in clinical specimens of colorectal cancers by Northern blot analysis. In comparison with the expression of these angiogenesis-related genes in 7 paired samples of colorectal cancers and the adjacent normal mucosa, VEGF mRNA level was significantly higher in the cancer tissues than in the adjacent normal mucosa (p < 0.05).

Localization of 67 exons on a YAC contig spanning 1.5 Mb around the multidrug resistance gene region of human chromosome 7q21.1.

A contig of 21 nonchimeric yeast artificial chromosomes (YACs) was previously assembled across 1.5 Mb of the multidrug resistance (MDR) gene (PGY1 and PGY3) region of human chromosome 7q21.1.

Direct involvement of the Y-box binding protein YB-1 in genotoxic stress-induced activation of the human multidrug resistance 1 gene.

The human multidrug resistance 1 (MDR1) gene encoding P-glycoprotein is often overexpressed in various human tumors after chemotherapy. During treatment with various chemotherapeutic agents, the MDR1 gene is activated at the transcriptional level and/or amplified, resulting in overexpression. Our previous studies demonstrated that an inverted CCAAT box (Y-box) might be a critical cis-regulatory element regulating UV or drug-induced MDR1 gene expression.

Mutations in the canilicular multispecific organic anion transporter (cMOAT) gene, a novel ABC transporter, in patients with hyperbilirubinemia II/Dubin-Johnson syndrome.

Members of the ATP-binding cassette (ABC) transporter superfamily are mutated to cause diseases that include cystic fibrosis, hyperinsulinemia, adrenoleukodystrophy, Stargardt disease and multidrug resistance. We recently isolated a novel human member of ABC transporter superfamily as the candidate transporter for the glucuronide and glutathione-conjugated antitumor agents, and found it highly homologous to the rat cmoat gene. consistent with recent findings of defects in the homologous cmoat gene in two rat models of hyperbilirubinemia (TR- and Eisai), we report two deletions and a missense mutation in the active transport family signature region in the gene in patients with hyperbilirubinemia II/Dubin-Johnson syndrome (DJS; MIM 237500), respectively.

Genomic organization of the human Y-box protein (YB-1) gene.

The human Y-box protein (YB-1) is a member of a family of DNA-binding proteins containing a highly conserved cold shock domain. The genomic organization of the human YB-1 gene was determined from five overlapping genomic clones that encompassed all exons of the gene. Sequence analysis of these clones revealed that human YB-1 spans approximately 19 kb of genomic DNA and contains eight exons.

Stimulation of cell-surface urokinase-type plasminogen activator activity and cell migration in vascular endothelial cells by a novel hexapeptide analogue of neurotensin.

To investigate if neurotensin (NT) could induce activation of urokinase-type plasminogen activator (uPA) in vascular endothelial cells, we utilized the acetyl-NT (8-13) analogue, TJN-950, in which the C-terminal leucine is reduced to leucinol. TJN-950 inhibited the binding of 125I-NT to membranes of newborn rat brains and of COS-7 cells transfected with rat NT receptor cDNA, but at 10(4) higher doses than NT (8-13). However, TJN-950 was as effective as NT in inducing the fibrinolytic activity in bovine vascular aortic and human umbilical vein endothelial cells, and enhanced the migration of vascular endothelial cells.

[Novel molecular targets for anticancer drugs].

Numbers of molecular targets for anticancer agents have been increased during the recent scientific progress in cancer biology. Of these targets, one should understand which target is clinically applicable. The forthcoming approach to evaluate such "molecular targets" is expected to provide novel diagnostic markers for adequate cancer therapy, and also novel targets for development of potent and useful therapeutic agents.

A canalicular multispecific organic anion transporter (cMOAT) antisense cDNA enhances drug sensitivity in human hepatic cancer cells.

The human cMOAT gene encodes a membrane protein involved in the ATP-dependent transport of hydrophobic compounds. To determine whether cMOAT is associated with drug sensitivity, we transfected an expression vector containing cMOAT antisense cDNA into the HepG2 human hepatic cancer cell line. We observed a reduction in cMOAT protein, as well as an enhanced level of glutathione, in the antisense transfectants.

All-trans-retinoic acid-dependent inhibition of E-cadherin-based cell adhesion with concomitant dephosphorylation of beta-catenin in metastatic human renal carcinoma cells.

We previously described an in vitro invasion assay model, using a monolayer of vascular endothelial cells grown on collagen gel, that mimics the metastatic abilities of the highly metastatic human renal carcinoma cell lines, MM-1,3 and 8 and their poorly metastatic counterparts, SN12C and Cl-8. MM-1, 3 and 8 cells were observed to penetrate the monolayer of vascular endothelial cells and grew in a spreading or scattering manner with loose cell-cell contact on collagen gel or on vascular endothelial cells. SN12C and Cl-8 cells failed to penetrate and grew in a clustering manner with tight cell-cell contact.

Nuclear translocation of the Y-box binding protein by ultraviolet irradiation.

The Y-box binding protein, YB-1, is a member of a DNA binding protein family with a structurally and functionally conserved cold shock domain. Using Western blotting and immunohistochemical methods, larger amounts of YB-1 were detected in the cytosol, particularly at the perinuclear region, than in the nucleus of human cancer cells. UV irradiation increased accumulation of YB-1 in the nucleus at 20 min and thereafter.

Upregulation of low density lipoprotein receptor by gemfibrozil, a hypolipidemic agent, in human hepatoma cells through stabilization of mRNA transcripts.

Gemfibrozil reduces the plasmal levels of cholesterol and triglyceride in patients with hyperlipidemia by a mechanism that is not well understood. The present study evaluated the effect of gemfibrozil on the LDL receptor in human hepatoma cells compared with that of pravastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Exposure to gemfibrozil, 40 mumol/L, for 3 days increased the binding of 125I-LDL to the surface of three lines of human hepatoma cell, HepG2, HuH7, and HLE by 1.

Human canalicular multispecific organic anion transporter (cMOAT) is expressed in human lung, gastric, and colorectal cancer cells.

Human canalicular multispecific organic anion transporter (cMOAT), a glutathione conjugate membrane transporter, has been isolated from cisplatin-resistant cancer cells and is distributed mainly in normal liver. We analyzed the expression of human cMOAT in 14 lung, 11 gastric, and 9 colorectal non-drug-selected human cancer cells, two multidrug-resistant cells, and one cisplatin-resistant cells, using quantitative RT-PCR and newly developed anti-human cMOAT antibody. All cell lines analyzed here expressed human cMOAT at the level of mRNA and protein, and some of them expressed higher levels of human cMOAT than the cisplatin-resistant cells.