Misako Haraguchi

Targeting of Antigen to Dendritic Cells with Poly(γ-Glutamic Acid) Nanoparticles Induces Antigen-Specific Humoral and Cellular Immunity

Nanoparticles are considered to be efficient tools for inducing potent immune responses by an Ag carrier. In this study, we examined the effect of Ag-carrying biodegradable poly(γ-glutamic acid) (γ-PGA) nanoparticles (NPs) on the induction of immune responses in mice. The NPs were efficiently taken up by dendritic cells (DCs) and subsequently localized in the lysosomal compartments. γ-PGA NPs strongly induced cytokine production, up-regulation of costimulatory molecules, and the enhancement of T cell stimulatory capacity in DCs. These maturational changes of DCs involved the MyD88-mediated NF-κB signaling pathway. In vivo, γ-PGA NPs were preferentially internalized by APCs (DCs and macrophages) and induced the production of IL-12p40 and IL-6. The immunization of mice with OVA-carrying NPs induced Ag-specific CTL activity and Ag-specific production of IFN-γ in splenocytes as well as potent production of Ag-specific IgG1 and IgG2a Abs in serum. Furthermore, immunization with NPs carrying a CD8+ T cell epitope peptide of Listeria monocytogenes significantly protected the infected mice from death. These results suggest that Ag-carrying γ-PGA NPs are capable of inducing strong cellular and humoral immune responses and might be potentially useful as effective vaccine adjuvants for the therapy of infectious diseases.

Snail Regulates Cell-Matrix Adhesion by Regulation of the Expression of Integrins and Basement Membrane Proteins

Snail, a transcriptional repressor of E-cadherin expression, plays a role in the process of epithelial-mesenchymal transition. However, the molecular basis of the role of snail in epithelial-mesenchymal transition has not been fully clarified. Here we show that the expression of snail in epithelial Madin-Darby canine kidney (MDCK) and A431 cells enhances both cell detachment and attachment. Snail did not confer resistance to anoikis induced by loss of contact but instead enhanced cell attachment to extracellular matrices such as fibronectin. This attachment was inhibited by Arg-Gly-Asp (RGD) peptides. Up-regulation of the promoter activity of integrin αV was observed in snail-expressing MDCK (MDCK/snail) cells. Snail also enhanced MDCK cell migration toward osteopontin that is a ligand for integrin αVβ3. We confirmed the reduction of basement membrane proteins such as laminin (LN) α3, β3, and γ2 (laminin-5/LN-5) and of receptors for LN-5 such as integrins α3, α6, or β4 in MDCK/snail or in snail-expressing A431 (A431/snail) cells. Nevertheless, suppression of LN-α3 chain by transient transfection of small interference RNAs resulted in no enhancement of cell detachment. We also found an induction of matrix metalloproteinase-3 in MDCK/snail and A431/snail cells. However, the inhibition of matrix metalloproteinase-3 showed no significant effect on the detachment of MDCK/snail cells. These results suggest that snail enhances cell detachment by multiple mechanism and leads to cell migration and reattachment at a second site, at least in part, by changing the expression of integrins in the cells.

The role of thymidine phosphorylase, an angiogenic enzyme, in tumor progression

Thymidine phosphorylase (TP), an enzyme involved in pyrimidine metabolism, is identical with an angiogenic factor, platelet‐derived endothelial cell growth factor (PD‐ECGF). TP is overex‐pressed in various tumors and plays an important role in angiogenesis, tumor growth, invasion and metastasis. The enzymatic activity of TP is required for the angiogenic effect of TP. A novel, specific TP inhibitor, TPI, inhibits angiogenesis induced by overexpression of TP in KB/TP cells (human KB epidermoid carcinoma cells transfected with TP cDNA), as well as the growth and metastasis of KB/TP cells in vivo. 2‐Deoxy‐D‐ribose, the degradation product of thymidine generated by TP activity, has both angiogenic and chemotactic activity. Both 2‐deoxy‐D‐ribose and TP inhibit a hypoxia‐induced apoptotic pathway. These findings suggest that 2‐deoxy‐D‐ribose is a downstream mediator of TP function. 2‐Deoxy‐L‐ribose, a stereoisomer of 2‐deoxy‐D‐ribose, inhibits the promotion of angiogenesis, tumor growth and metastasis by TP. Although the mechanism of the action of 2‐deoxy‐D‐ribose is still unknown, 2‐deoxy‐L‐ribose may inhibit the physiological activities of 2‐deoxy‐D‐ribose, and consequently those of TP. Inhibition of TP activity and function appears to be a promising approach for the chemotherapy of various tumors.

Reversal of the resistance to STI571 in human chronic myelogenous leukemia K562 cells.

STI571, an Abl‐specific tyrosine kinase inhibitor, selectively kills Bcr‐Abl‐containing cells in vitro and in vivo. However, some chronic myelogenous leukemia (CML) cell lines are resistant to STI571. We evaluated whether STI571 interacts with P‐glycopro‐tein (P‐gp) and multidrug resistance protein 1 (MRP1), and examined the effect of agents that reverse multidrug resistance (MDR) on the resistance to SI571 in MDR cells. STI571 inhibited the [125l]azidoagosterol A‐photolabeling of P‐gp, but not that of MRP1. K562/MDR cells that overexpress P‐gp were 3.67 times more resistant to STI571 than the parental Philadelphia‐chromosome‐positive (Ph+) CML K562 cells, and this resistance was most effectively reversed by cepharanthine among the tested reversing agents. The concentration of STI571 required to completely inhibit tyrosine phosphorylation in K562/MDR cells was about 3 times higher than that in K562 cells, and cepharanthine abolished the difference. In KB‐G2 cells that overexpress P‐gp, but not Bcr‐Abl, 2.5 μM STI571 partly reversed the resistance to vincristine (VCR), paclitaxel, etoposide (VP‐16) and actinomycin D (ACD) but not to Adriamycin (ADM) or colchicine. STI571 increased the accumulation of VCR, but not that of ADM in KB‐G2 cells. STI571 did not reverse resistance to any agent in KB/MRP cells that overexpress MRP1. These findings suggest that STI571 is a substrate for P‐gp, but is less efficiently transported by P‐gp than VCR, and STI571 is not a substrate for MRP1. Among the tested reversing agents that interact with P‐gp, cepharanthine was the most effective agent for the reversal of the resistance to STI571 in K562/ MDR cells. Furthermore, STI571 itself was a potent reversing agent for MDR in P‐gp‐expressing KB‐G2 cells.

Inhibition of Metastasis of Tumor Cells Overexpressing Thymidine Phosphorylase by 2-Deoxy-l-Ribose

Thymidine phosphorylase (TP) catalyzes the reversible conversion of thymidine to thymine, thereby generating 2-deoxy-d-ribose-1-phosphate, which upon dephosphorylation forms 2-deoxy-d-ribose (d-dRib), a degradation product of thymidine. We have previously shown that d-dRib promotes angiogenesis and chemotaxis of endothelial cells and also confers resistance to hypoxia-induced apoptosis in some cancer cell lines. 2-Deoxy-l-ribose (l-dRib), a stereoisomer of d-dRib, can inhibit d-dRib anti-apoptotic effects and suppressed the growth of KB cells overexpressing TP (KB/TP cells) transplanted into nude mice. In this study, we examined the ability of l-dRib to suppress metastasis of KB/TP cells using two different models of metastasis. The antimetastatic effect of l-dRib was first investigated in a liver-metastasis model in nude mice inoculated with KB/TP cells. Oral administration of l-dRib for 28 days at a dose of 20 mg/kg/day significantly reduced the number of metastatic nodules in the liver and suppressed angiogenesis and enhanced apoptosis in KB/TP metastatic nodules. Next, we compared the ability of l-dRib and tegafur alone or in combination to decrease the number of metastatic nodules in organs in the abdominal cavity in nude mice receiving s.c. of KB/TP cells into their backs. l-dRib (20 mg/kg/day) was significantly (P < 0.05) more efficient than tegafur (100 mg/kg/day) in decreasing the number of metastatic nodules in organs in the abdominal cavity. By in vitro invasion assay, l-dRib also reduced the number of invading KB/TP cells. l-dRib anti-invasive activity may be mediated by its ability to suppress the enhancing effect of TP and d-dRib on both mRNA and protein expression of vascular endothelial growth factor and interleukin-8 in cultured KB cells. These findings suggest that l-dRib may be useful in a clinical setting for the suppression of metastasis of tumor cells expressing TP.

Targeted Deletion of Both Thymidine Phosphorylase and Uridine Phosphorylase and Consequent Disorders in Mice

ABSTRACT Thymidine phosphorylase (TP) regulates intracellular and plasma thymidine levels. TP deficiency is hypothesized to (i) increase levels of thymidine in plasma, (ii) lead to mitochondrial DNA alterations, and (iii) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). In order to elucidate the physiological roles of TP, we generated mice deficient in the TP gene. Although TP activity in the liver was inhibited in these mice, it was fully maintained in the small intestine. Murine uridine phosphorylase (UP), unlike human UP, cleaves thymidine, as well as uridine. We therefore generated TP-UP double-knockout (TP−/− UP−/−) mice. TP activities were inhibited in TP−/− UP−/− mice, and the level of thymidine in the plasma of TP−/− UP−/− mice was higher than for TP−/− mice. Unexpectedly, we could not observe alterations of mitochondrial DNA or pathological changes in the muscles of the TP−/− UP−/− mice, even when these mice were fed thymidine for 7 months. However, we did find hyperintense lesions on magnetic resonance T2 maps in the brain and axonal edema by electron microscopic study of the brain in TP−/− UP−/− mice. These findings suggested that the inhibition of TP activity caused the elevation of pyrimidine levels in plasma and consequent axonal swelling in the brains of mice. Since lesions in the brain do not appear to be due to mitochondrial alterations and pathological changes in the muscle were not found, this model will provide further insights into the causes of MNGIE.

Reversal of P-glycoprotein mediated multidrug resistance by a newly synthesized 1,4-benzothiazipine derivative, JTV-519

A newly synthesized 1,4-benzothiazipine derivate, 4-[3-(4-benzylpiperidin-1-yl) propionyl]-7-methoxy-2,3,4,5-tetrahydro-1, 4-benzothiazepine monohydrochloride (JTV-519) was examined for its ability to reverse P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1) mediated multidrug resistance (MDR) in K562/MDR and KB/MRP cells, respectively. JTV-519 at 3 microM reversed the resistance of K562/MDR cells to vincristine (VCR), taxol, etoposide (VP16), adriamycin (ADM) and actinomycin D and at 0.5 or 1 microM reversed their resistance to STI571. JTV-519 at 10 microM enhanced the accumulation of ADM in K562/MDR cells to the level in parental K562 cells and inhibited the efflux of ADM from K562/MDR cells. Photoaffinity labeling of P-gp with 3H-azidopine was almost completely inhibited by 500 microM JTV-519. JTV-519 at 3 microM also partially reversed the resistance of KB/MRP cells to VCR and at 500 microM partially inhibited the photoaffinity labeling of MRP1 with (125)I-II-azidophenyl agosterol A (125I-azidoAG-A). These results suggest that JTV-519 reversed the resistance to the anti-cancer agents in P-gp and MRP1 overexpressing multidrug-resistant cells by directly binding to P-gp and MRP1, and competitively inhibiting transport of the anti-cancer agents.

Expression of the multidrug‐resistance‐associated protein (MRP) gene in human colorectal, gastric and non‐small‐cell lung carcinomas

MRP has been identified as another multidrug‐resistance (MDR) gene and may be involved in an alternative MDR mechanism in some solid tumors. We investigated the expression of MRP mRNA in multidrug‐resistant KB sublines (KB‐8‐5, KB‐C2, C‐A40 and C‐A120), human non‐small‐cell lung carcinomas (NSCLC), gastric and colorectal carcinomas, and compared it with that in drug‐sensitive human KB cells, MRP gene expression was elevated in 8 of 9 (89%) squamous‐cell carcinomas of the lung. Furthermore, MRP expression in 4 squamous‐cell carcinomas (L13, 18, 19 and 20) was more than 3.6 times higher than in KB‐3‐I cells, and the average MRP mRNA expression level of all squamous‐cell carcinomas was significantly higher than that of adenocarcinoma of the lung and of colorectal and gastric carcinomas. These results suggested that the MRP is responsible, at least in part, for drug resistance in some squamous‐cell carcinomas of the lung.

Thymidine phosphorylase inhibits apoptosis induced by cisplatin.

An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP, partially prevents hypoxia-induced apoptosis. TP is expressed at higher levels in tumor tissues compared to the adjacent non-neoplastic tissues in a variety of human carcinomas. High expression of TP is associated with an unfavorable prognosis. To investigate the effect of TP on cisplatin-induced apoptosis, human leukemia Jurkat cells were transfected with wild-type or mutant (L148R) TP cDNA. TP inhibited a number of steps in the cisplatin-induced apoptotic pathway, activation of caspases 3 and 9 and mitochondrial cytochrome c release. These findings suggest a mechanism by which TP confers resistance to apoptosis induced by cisplatin. Moreover, mutant TP that has no enzymatic activity also suppressed cisplatin-induced apoptosis. These findings indicate that TP has cytoprotective functions against cytotoxic agents which are independent of its enzymatic activity.

Down regulation of c-Myc and induction of an angiogenesis inhibitor, thrombospondin-1, by 5-FU in human colon cancer KM12C cells

5-FU is commonly used for treatment of various solid tumors including colon carcinoma. We have previously demonstrated that Egr-1 induced by 5-FU enhanced TSP-1 expression in human colon cancer KM12C cells. In this study, a Genechip analysis of KM12C cells treated with 5-FU revealed down-regulation of 924 genes and up-regulation of 460 genes. The decreased expression of c-Myc mRNA and phosphorylated c-Myc were detected and confirmed by RT-PCR and immunoblotting. Since 5-FU induced the expression of TSP-1, we examined the effect of c-Myc on the TSP-1 promoter. Deletion of the TSP-1 promoter region in which binding sites for c-Myc reside had no effect on the TSP-1 promoter activity induced by 5-FU. Meanwhile, 5-FU dose-dependently decreased the expression of miR-17-92 cluster. These findings suggest that 5-FU decreased the expression of c-Myc and consequently miR-17-92 cluster and increased the expression of TSP-1 mRNA.