Direct Injection of Recombinant AAV-Containing Solution into the Oviductal Lumen of Pregnant Mice Caused In Situ Infection of Both Preimplantation Embryos and Oviductal Epithelium.

Adeno-associated virus (AAV) vector is an efficient viral-based gene delivery tool used with many types of cells and tissues, including neuronal cells and muscles. AAV serotype 6 (AAV-6), one of numerous AAV serotypes, was recently found to efficiently transduce mouse preimplantation embryos. Furthermore, through coupling with a clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system-a modern genome editing technology-AAV-6 has been shown to effectively create a mutation at a target locus, which relies on isolation of zygotes, in vitro viral infection, and transplantation of the infected embryos to recipient females. Unfortunately, this procedure, termed "ex vivo handling of embryos", requires considerable investment of capital, time, and effort. Direct transduction of preimplantation embryos through the introduction of AAV-6 into the oviductal lumen of pregnant females would be an ideal approach. In this study, we injected various types of recombinant AAV vectors (namely, rAAV-CAG-EGFP-1, -2, -5, and -6, each carrying an enhanced green fluorescent protein [EGFP] cDNA whose expression is under the influence of a cytomegalovirus enhancer + chicken ß-actin promoter) into the ampulla region of oviducts in pregnant female mice at Day 0.7 of pregnancy (corresponding to the late 1-cell stage), and EGFP-derived green fluorescence was assessed in the respective morulae. The highest levels of fluorescence were observed in rAAV-CAG-EGFP-6. The oviductal epithelium was distinctly fluorescent. The fluorescence in embryos peaked at the morula stage. Our results indicate that intra-oviductal injection of AAV-6 vectors is the most effective method for transducing zona pellucida-enclosed preimplantation embryos in situ. AAV-6 vectors could be a useful tool in the genetic manipulation of early embryos, as well as oviductal epithelial cells.

E-cadherin loss in RMG-1 cells inhibits cell migration and its regulation by Rho GTPases.

E-cadherin is an adherens junction protein that forms intercellular contacts in epithelial cells. Downregulation of E-cadherin is frequently observed in epithelial tumors and it is a hallmark of epithelial-mesenchymal transition (EMT). However, recent findings suggest that E-cadherin plays a more complex role in certain types of cancers. Previous studies investigating the role of E-cadherin mainly used gene-knockdown systems; therefore, we used the CRISPR/Cas9n system to develop E-cadherin-knockout (EcadKO) ovarian cancer RMG-1 cell to clarify the role of E-cadherin in RMG-1 cells. EcadKO RMG-1 cells demonstrated a complete loss of the adherens junctions and failed to form cell clusters. Cell-extracellular matrix (ECM) interactions were increased in EcadKO RMG-1 cells. Upregulation of integrin beta1 and downregulation of collagen 4 were confirmed. EcadKO RMG-1 cells showed decreased ß-catenin levels and decreased expression of its transcriptional target cyclin D1. Surprisingly, a marked decrease in the migratory ability of EcadKO RMG-1 cells was observed and the cellular response to Rho GTPase inhibitors was diminished. Thus, we demonstrated that E-cadherin in RMG-1 cells is indispensable for ß-catenin expression and ß-catenin mediated transcription and Rho GTPase-regulated directionally persistent cell migration.

Thymidine catabolism promotes NADPH oxidase-derived reactive oxygen species (ROS) signalling in KB and yumoto cells.

Thymidine phosphorylase (TP) is a rate-limiting enzyme in the thymidine catabolic pathway. TP is identical to platelet-derived endothelial cell growth factor and contributes to tumour angiogenesis. TP induces the generation of reactive oxygen species (ROS) and enhances the expression of oxidative stress-responsive genes, such as interleukin (IL)-8. However, the mechanism underlying ROS induction by TP remains unclear. In the present study, we demonstrated that TP promotes NADPH oxidase-derived ROS signalling in cancer cells. NADPH oxidase inhibition using apocynin or small interfering RNAs (siRNAs) abrogated the induction of IL-8 and ROS in TP-expressing cancer cells. Meanwhile, thymidine catabolism induced by TP increased the levels of NADPH and intermediates of the pentose phosphate pathway (PPP). Both siRNA knockdown of glucose 6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme in PPP, and a G6PD inhibitor, dihydroepiandrosterone, reduced TP-induced ROS production. siRNA downregulation of 2-deoxy-D-ribose 5-phosphate (DR5P) aldolase, which is needed for DR5P to enter glycolysis, also suppressed the induction of NADPH and IL-8 in TP-expressing cells. These results suggested that TP-mediated thymidine catabolism increases the intracellular NADPH level via the PPP, which enhances the production of ROS by NADPH oxidase and activates its downstream signalling.

Thymidine Catabolism as a Metabolic Strategy for Cancer Survival.

Thymidine phosphorylase (TP), a rate-limiting enzyme in thymidine catabolism, plays a pivotal role in tumor progression; however, the mechanisms underlying this role are not fully understood. Here, we found that TP-mediated thymidine catabolism could supply the carbon source in the glycolytic pathway and thus contribute to cell survival under conditions of nutrient deprivation. In TP-expressing cells, thymidine was converted to metabolites, including glucose 6-phosphate, lactate, 5-phospho-α-D-ribose 1-diphosphate, and serine, via the glycolytic pathway both in vitro and in vivo. These thymidine-derived metabolites were required for the survival of cells under low-glucose conditions. Furthermore, activation of thymidine catabolism was observed in human gastric cancer. These findings demonstrate that thymidine can serve as a glycolytic pathway substrate in human cancer cells.

Snail1 expression in human colon cancer DLD-1 cells confers invasive properties without N-cadherin expression.

The epithelial-mesenchymal transition (EMT) is a fundamental characteristic of carcinoma cells. EMT is generally associated with a change in cellular morphology from cobblestone to spindle shape, reduced expression of epithelial markers such as E-cadherin, and enhanced expression of mesenchymal markers such as N-cadherin. This EMT-associated reciprocal expression of E-cadherin and N-cadherin has been called the "cadherin switch". Downregulation of E-cadherin enables cells to dissociate from colonies while upregulation of N-cadherin is associated with increased invasiveness. The transcription factor Snail1 induces these changes in various epithelial cell lines, including canine MDCK cells and human A431 cells. In the present study, we introduced a Snail1 expression vector into human DLD-1 cells and isolated stable transfectants. These cells showed changes in morphology, reduced expression of epithelial marker E-cadherin and occludin, and elevated invasion and migration. However, neither expression of N-cadherin protein nor its corresponding mRNA was detected. Therefore, elevated N-cadherin expression is not required for invasiveness of the cells.

CRISPR/Cas9n-Mediated Deletion of the Snail 1Gene (SNAI1) Reveals Its Role in Regulating Cell Morphology, Cell-Cell Interactions, and Gene Expression in Ovarian Cancer (RMG-1) Cells.

Snail1 is a transcription factor that induces the epithelial to mesenchymal transition (EMT). During EMT, epithelial cells lose their junctions, reorganize their cytoskeletons, and reprogram gene expression. Although Snail1 is a prominent repressor of E-cadherin transcription, its precise roles in each of the phenomena of EMT are not completely understood, particularly in cytoskeletal changes. Previous studies have employed gene knockdown systems to determine the functions of Snail1. However, incomplete protein knockdown is often associated with these systems, which may cause incorrect interpretation of the data. To more precisely evaluate the functions of Snail1, we generated a stable cell line with a targeted ablation of Snail1 (Snail1 KO) by using the CRISPR/Cas9n system. Snail1 KO cells show increased cell-cell adhesion, decreased cell-substrate adhesion and cell migration, changes to their cytoskeletal organization that include few stress fibers and abundant cortical actin, and upregulation of epithelial marker genes such as E-cadherin, occludin, and claudin-1. However, morphological changes were induced by treatment of Snail1 KO cells with TGF-beta. Other transcription factors that induce EMT were also induced by treatment with TGF-beta. The precise deletion of Snail1 by the CRISPR/Cas9n system provides clear evidence that loss of Snail1 causes changes in the actin cytoskeleton, decreases cell-substrate adhesion, and increases cell-cell adhesion. Treatment of RMG1 cells with TGF-beta suggests redundancy among the transcription factors that induce EMT.

The ectopic expression of Snail in MDBK cells does not induce epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell-cell junctions and cell polarity, as well as by the acquisition of migratory and invasive properties. However, the precise molecular events that initiate this complex EMT process are poorly understood. Snail expression induces EMT in Madin-Darby canine kidney (MDCK) cells and the human epidermoid carcinoma cell line, A431. Snail is a zinc finger transcription factor and triggers EMT by suppressing E-cadherin expression. In the present study, to broaden our knowledge of Snail­induced EMT, we generated stable Snail transfectants using Madin-Darby bovine kidney (MDBK) cells. Contrary to the MDCK or A431 cells examined in our previous studies, the MDBK cells transfected with the Snail construct maintained an epithelial morphology and showed no sign of reduced cell-cell adhesiveness compared to the control cells. Consistent with these observations, the downregulation of epithelial marker proteins, e.g. E-cadherin and desmoglein, and the upregulation of mesenchymal marker proteins, e.g., N-cadherin and fibronectin, were not detected. Furthermore, the E-cadherin promoter was not methylated. Therefore, in the MDBK cells, the ectopic expression of Snail failed to induce EMT. As previously demonstrated, in MDCK cells, Snail expression is accompanied by the increased expression of other EMT-inducing transcription factors, e.g., Slug and zinc finger E-box-binding homeobox 1 (ZEB1). However, the MDBK cells transfected with the Snail construct did not exhibit an increased expression of these factors. Thus, it is possible that the failure to upregulate other EMT-related transcription factors may explain the lack of Snail-mediated induction of EMT in MDBK cells.

Snail modulates cell metabolism in MDCK cells.

Snail, a repressor of E-cadherin gene transcription, induces epithelial-to-mesenchymal transition and is involved in tumor progression. Snail also mediates resistance to cell death induced by serum depletion. By contrast, we observed that snail-expressing MDCK (MDCK/snail) cells undergo cell death at a higher rate than control (MDCK/neo) cells in low-glucose medium. Therefore, we investigated whether snail expression influences cell metabolism in MDCK cells. Although gylcolysis was not affected in MDCK/snail cells, they did exhibit reduced pyruvate dehydrogenase (PDH) activity, which controls pyruvate entry into the tricarboxylic acid (TCA) cycle. Indeed, the activity of multiple enzymes involved in the TCA cycle was decreased in MDCK/snail cells, including that of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH2), succinate dehydrogenase (SDH), and electron transport Complex II and Complex IV. Consequently, lower ATP content, lower oxygen consumption and increased survival under hypoxic conditions was also observed in MDCK/snail cells compared to MDCK/neo cells. In addition, the expression and promoter activity of pyruvate dehydrogenase kinase 1 (PDK1), which phosphorylates and inhibits the activity of PDH, was increased in MDCK/snail cells, while expression levels of glutaminase 2 (GLS2) and ATP-citrate lyase (ACLY), which are involved in glutaminolysis and fatty acid synthesis, were decreased in MDCK/snail cells. These results suggest that snail modulates cell metabolism by altering the expression and activity of key enzymes. This results in enhanced glucose dependency and leads to cell death under low-glucose conditions. On the other hand, the reduced requirements for oxygen and nutrients from the surrounding environment, might confer the resistance to cell death induced by hypoxia and malnutrition.

The transcription factor Snail enhanced the degradation of E-cadherin and desmoglein 2 in oral squamous cell carcinoma cells.

Epithelial-mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell-cell junctions and cell polarity as well as the acquisition of migratory and invasive properties. However, the precise molecular events that initiate this complex EMT process are poorly understood. Snail is a regulator of EMT that represses E-cadherin transcription through its interaction with proximal E-boxes in the promoter region of target genes. To investigate the role of Snail in EMT, we generated stable Snail transfectants using the oral squamous cell carcinoma cell line HSC-4 (Snail/HSC-4). Snail/HSC-4 cells had a spindle-shaped mesenchymal morphology, and enhanced migration and invasiveness relative to control cells. Consistent with these EMT changes, the downregulation of epithelial marker proteins, E-cadherin and desmoglein 2, and the upregulation of mesenchymal marker proteins, vimentin and N-cadherin were detected. Despite these observations, the mRNA levels of E-cadherin and desmoglein 2 did not decrease significantly. Although E-cadherin and desmoglein 2 proteins were stable in parental HSC-4 cells, these proteins were rapidly degraded in Snail/HSC-4 cells. The degradation of E-cadherin, but not desmoglein 2, was inhibited by dynasore, an inhibitor of dynamin-dependent endocytosis. Therefore, in HSC-4 cells Snail regulates levels of these proteins both transcriptionally and post-translationally.

The transcription factor Snail expressed in cutaneous squamous cell carcinoma induces epithelial-mesenchymal transition and down-regulates COX-2.

Cutaneous spindle cell squamous cell carcinoma (SCC) is a rare, but highly malignant variant of SCC. The presence of spindle-shaped cells with a sarcomatous appearance, which are derived from squamous cells, suggests that these cells are produced as a result of epithelial-mesenchymal transition (EMT). EMT is a complex process in which epithelial cells lose their polarity and cell-cell contacts, while also acquiring increased motility and invasiveness. Snail regulates EMT by binding to proximal E-boxes in the promoter region of E-cadherin and repressing its transcription. When examining the expression of EMT markers and Snail in spindle cell SCCs, we found that cyclooxygenase-2 (COX-2) expression was down-regulated. Since it has been shown that COX-2 is constitutively overexpressed in a variety of malignancies, including colon, gastric, and lung carcinomas, the down-regulation of COX-2 expression was unexpected. The presence of E-box-like sequences in the promoter region of COX-2 prompted us to perform a more detailed analysis. We introduced a Snail expression vector into keratinocyte-derived cell lines (HaKaT, HSC5, and A431 cells), and isolated stable transfectants. We determined that COX-2 expression was down-regulated in cells expressing Snail. Consistent with these observations, reporter assays revealed that COX-2 promoter activity was repressed upon Snail overexpression. Thus Snail down-regulates COX-2 in these cells.

Thymidine phosphorylase enhances reactive oxygen species generation and interleukin-8 expression in human cancer cells.

Thymidine phosphorylase (TP) is an angiogenic factor that plays a pivotal role in tumor angiogenesis. Various kinds of solid tumors express TP and high TP activity is correlated with microvessel density. We have previously reported that TP enhances interleukin-8 (IL-8) expression in KB human epidermoid carcinoma cells. In this study, TP was shown to be involved in enhanced expression of IL-8 in EJ human bladder cancer cells and Yumoto human cervical cancer cells as well as KB human epidermoid carcinoma cells. The enzymatic activity of TP was required for the enhanced expression of IL-8. A degradation product of thymidine was implicated in the enhanced expression of IL-8. TP augmented reactive oxygen species (ROS) generation in KB and Yumoto cells, and the enzymatic activity of TP was again required for the generation of ROS. An antioxidant, N-acetylcysteine (NAC), attenuated the generation of ROS and IL-8 mRNA expression in KB and Yumoto cells, and H2O2 increased IL-8 mRNA expression in Yumoto cells, suggesting that ROS generated by TP caused the increased expression of IL-8 mRNA. Since TP also reduced cellular glutathione levels and transcription of γ-GCS in KB cells, the TP-induced augmentation of ROS may be partially attributed to the decreased glutathione. Our findings suggest that thymidine-derived sugars enhanced ROS generation and consequently increased IL-8 expression.

The role of the transcriptional regulator snail in cell detachment, reattachment and migration.

In order to metastasize, cancer cells must first detach from the primary tumor, migrate, invade through tissues and attach to a second site. The transcription factor snail is an important mediator of epithelial-mesenchymal transitions and is involved in tumor progression. Recent data have provided evidence for a requirement for snail expression in metastatic dissemination. Although very little is known about the molecular mechanisms governing metastatic dissemination, we review the possible roles of snail expression in this process. We also review the regulation of snail expression.

Molecular basis for the induction of an angiogenesis inhibitor, thrombospondin-1, by 5-fluorouracil.

5-Fluorouracil (5-FU) is one of the most commonly used anticancer drugs in chemotherapy against various solid tumors. 5-FU dose-dependently increased the expression levels of intrinsic antiangiogenic factor thrombospondin-1 (TSP-1) in human colon carcinoma KM12C cells and human breast cancer MCF7 cells. We investigated the molecular basis for the induction of TSP-1 by 5-FU in KM12C cells. Promoter assays showed that the region with the Egr-1 binding site is critical for the induction of TSP-1 promoter activity by 5-FU. The binding of Egr-1 to the TSP-1 promoter was increased in KM12C cells treated with 5-FU. Immunofluorescence staining revealed that 5-FU significantly increased the level of Egr-1 in the nuclei of KM12C cells. The suppression of Egr-1 expression by small interfering RNA decreased the expression level of TSP-1. Furthermore, 5-FU induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27). Blockade of the p38 MAPK pathway by SB203580 remarkably inhibited the phosphorylation of HSP27 induced by 5-FU and decreased the induction of Egr-1 and TSP-1 by 5-FU in KM12C cells. These findings suggest that the p38 MAPK pathway plays a crucial role in the induction of Egr-1 by 5-FU and that induced Egr-1 augments TSP-1 promoter activity, with the subsequent production of TSP-1 mRNA and protein.

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 alphaV was observed in snail-expressing MDCK (MDCK/snail) cells. Snail also enhanced MDCK cell migration toward osteopontin that is a ligand for integrin alphaVbeta3. We confirmed the reduction of basement membrane proteins such as laminin (LN) alpha3, beta3, and gamma2 (laminin-5/LN-5) and of receptors for LN-5 such as integrins alpha3, alpha6, or beta4 in MDCK/snail or in snail-expressing A431 (A431/snail) cells. Nevertheless, suppression of LN-alpha3 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.

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.

Isolation and characterization of arsenite-resistant human epidermoid carcinoma KB cells.

Arsenic trioxide (As2O3) has been used with success in the treatment of acute promyelocytic leukemia. However, resistance to arsenite agents reduces their efficacy. We have isolated arsenite-resistant human epidermoid carcinoma KB cells, termed KAS. KAS cells were resistant to sodium arsenite (22-fold) and showed a reduced accumulation of arsenite as a result of an active efflux mechanism. Further analysis indicated that resistance of KAS cells extended to other drugs including cisplatin (17-fold), antimony potassium tartrate (11-fold) and doxorubicin (27-fold). Although increased expression of multidrug resistance protein 1 (MRP1) in KAS cells was confirmed by quantitative RT-PCR and immunoblot analysis, specific inhibitors of MRP1 did not completely eliminate arsenite resistance. The level of glutathione (GSH) in KAS cells was 3-fold higher than that in KB-3-1 cells, and the inhibition of GSH synthesis by buthionine sulfoximine (BSO) considerably increased the cytotoxic effect of arsenite on KAS cells. A pyridine analog, 2-[4-(diphenylmethyl)-1-piperazinyl ethyl 5-(trans-4,6-dimethyl-1,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-3-pyridine-carboxylate P oxide (PAK-104P), partially reversed the arsenite resistance and increased the arsenite accumulation in KAS cells. We suggest that the increased level of GSH is involved in arsenite resistance and an as yet unidentified arsenite transporter is expressed in the arsenite-resistant KAS cells.

Targeting of antigen to dendritic cells with poly(gamma-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(gamma-glutamic acid) (gamma-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. gamma-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-kappaB signaling pathway. In vivo, gamma-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-gamma 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 gamma-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.

Molecular basis for the involvement of thymidine phosphorylase in cancer invasion.

Thymidine phosphorylase (TP), also known as platelet-derived endothelial cell growth factor (PD-ECGF), has been implicated in bladder cancer angiogenesis and invasion. However, the molecular basis of its role in invasion remains unclear. We investigated the expression of TP and 10 invasion-related genes in bladder cancers from 72 randomly selected patients by real-time two-step RT-PCR assay. We found that the expression levels of TP, MMP-9, uPA, and MMP-2 were significantly higher in invasive tumors than those in superficial tumors. Also, the expression level of TP significantly correlated with that of uPA, MMP-1, MMP-9, PAI-1 and VEGF. KK47/TP cells, bladder cancer cells that overexpress TP, had a higher expression of MMP-7 and MMP-9 than KK/CV cells that express lower level of TP in hypoxic condition. PC/TP cells, prostate cancer cells that overexpress TP, also had a higher expression of MMP-1 and MMP-7 than PC/CV cells that express no detectable TP. Taken together these data indicate that TP enhances the invasion of tumor cells through the induction of invasion-related genes.

2-Deoxy-L-ribose inhibits the invasion of thymidine phosphorylase-overexpressing tumors by suppressing matrix metalloproteinase-9.

Thymidine phosphorylase (TP), an enzyme involved in pyrimidine metabolism, is identical with an angiogenic factor, platelet-derived endothelial cell growth factor. 2-Deoxy-D-ribose (D-dRib), the degradation product of thymidine generated by TP activity, has been suggested to be a downstream mediator of TP function. 2-Deoxy-L-ribose (L-dRib), a stereoisomer of D-dRib, inhibited the promotion of angiogenesis, tumor growth and metastasis by TP. In our study, we have shown that nude mice inoculated with TP-overexpressing KB/TP cells had shorter survival times than those injected with control KB/CV cells. KB/TP tumors were also more highly invasive than KB/CV tumors in mice. The expression levels of matrix metalloproteinase (MMP)-9 in KB/TP tumors were significantly higher than those in KB/CV tumors. L-dRib and a TP inhibitior, TPI, extended the survival period of KB/TP tumor-bearing mice. L-dRib also reduced MMP-9 mRNA levels in KB/TP tumors. Furthermore, L-dRib suppressed the mRNA level of MMP-9 in cultured KB/TP cells, and the invasive activity of the cells. L-dRib may be useful for the suppression of invasion of TP-expressing tumor cells.