A novel intraperitoneal therapy for gastric cancer with DFP-10825, a unique RNAi therapeutic targeting thymidylate synthase, in a peritoneally disseminated xenograft model.

PURPOSE: In advanced gastric cancer, peritoneal dissemination is a life-threatening mode of metastasis. Since the treatment options with conventional chemotherapy remain limited, any novel therapeutic strategy that could control such metastasis would improve the outcome of treatment. We recently developed a unique RNA interference therapeutic regimen (DFP-10825) consisting of short hairpin RNA against thymidylate synthase (TS shRNA) and cationic liposomes. The treatment with DFP-10825 has shown remarkable antitumor activity in peritoneally disseminated human ovarian cancer-bearing mice via intraperitoneal administration. In this study, we expanded DFP-10825 to the treatment of peritoneally disseminated gastric cancer. METHODS: DFP-10825 was administered intraperitoneally into mice with intraperitoneally implanted human gastric cancer cells (MKN45 or NCI-N87). Antitumor activity and host survival benefits were monitored. Intraperitoneal distribution of fluorescence-labeled DFP-10825 was monitored in this MKN45 peritoneally disseminated mouse model. RESULTS: Intraperitoneal injection of DFP-10825 suppressed tumor growth in two peritoneally disseminated cancer models (MKN45 and NCI-N87) and increased the survival time of the MKN45 model without severe side effects. Throughout the treatment regimen, no significant body weight loss was associated with the administration of DFP-10825. Interestingly, after intraperitoneal injection, fluorescence-labeled DFP-10825 retained for more than 72 hours in the peritoneal cavity and selectively accumulated in disseminated tumors. CONCLUSIONS: Intraperitoneal injection of DFP-10825 demonstrated effective antitumor activity without systemic severe adverse effects via the selective delivery of RNAi molecules into disseminated tumors in the peritoneal cavity. Our current study indicates that DFP-10825 could become an alternative option to improve the outcomes of patients with peritoneally disseminated gastric cancer.

A simplified method for manufacturing RNAi therapeutics for local administration.

RNA interference (RNAi) is one of the most promising strategies for cancer therapeutics. The successful translation of RNAi therapeutics to a clinic setting requires a delivery system that is efficient and simple to upscale. In this study, we devised a simple industrial method to manufacture lipoplex, which includes short hairpin RNA against the expression of thymidylate synthase (TS shRNA) - a key molecule for DNA biosynthesis. An aqueous solution of TS shRNA was gently mixed with either a precursor of cationic liposome (Presome DF-1) or a cationic lipid mixture in an o/w emulsion. This solution was subsequently lyophilized under optimal conditions to obtain either FD-lipoplex-1 or FD-lipoplex-2, respectively. With this method, a lipoplex in activated form was obtained via a simple "one-step" hydration with saline. Both forms of FD-lipoplex showed physicochemical properties comparable to those of conventional lipoplex. FD-lipoplexes stably retained TS shRNA within their formulations in the presence of tumor ascites fluid. Intraperitoneal treatment with either FD-lipoplex-1 or FD-lipoplex-2 provided a therapeutic level of efficacy comparable to that of conventional lipoplex in the treatment of a peritoneal disseminated gastric cancer mouse model. Collectively, established freeze-drying-based methods for RNAi-therapeutic preparation could realistically be used in a clinical setting for the treatment of patients with peritoneal disseminated cancer.

Anticancer activity of the intraperitoneal-delivered DFP-10825, the cationic liposome-conjugated RNAi molecule targeting thymidylate synthase, on peritoneal disseminated ovarian cancer xenograft model.

INTRODUCTION: Peritoneal disseminated ovarian cancer is one of the most difficult cancers to treat with conventional anti-cancer drugs and the treatment options are very limited, although an intraperitoneal (ip) paclitaxel has shown some clinical benefit. Therefore, treatment of peritoneal disseminated ovarian cancer is a highly unmet medical need and it is urgent to develop a new ip delivered drug regulating the fast DNA synthesis. METHODS: We developed a unique RNAi molecule consisting of shRNA against the thymidylate synthase (TS) and a cationic liposome (DFP-10825) and tested its antitumor activity and PK profile in peritoneally disseminated human ovarian cancer ascites models by the luciferase gene-transfected SCID mice. DFP-10825 alone, paclitaxel alone or combination with DFP-10825 and paclitaxel were administered in an ip route to the tumor-bearing mice. The TS expression level was measured by conventional RT-PCR. The anti-tumor activity and host survival benefit by DFP-10825 treatment on tumor-bearing mice were observed as resulting from the specific TS mRNA knock-down in tumors. RESULTS: DFP-10825 alone significantly suppressed the growth of SKOV3-luc tumore ascites cells and further extended the survival time of these tumor-bearing mice. Combination with the ip paclitaxel augmented the antitumor efficacy of DFP-10825 and significantly prolonged the survival time in the tumor-bearing mice. Short-hairpin RNA for TS (TS shRNA) levels derived from DFP-10825 in the ascetic fluid were maintained at a nM range across 24 hours but not detected in the plasma, suggesting that TS shRNA is relatively stable in the peritoneal cavity, to be able to exert its anti-tumor activity, but not in blood stream, indicating little or no systemic effect. CONCLUSION: Collectively, the ip delivery of DFP-10825, TS shRNA conjugated with cationic liposome, shows a favorable antitumor activity without systemic adverse events via the stable localization of TS shRNA for a sufficient time and concentration in the peritoneal cavity of the peritoneally disseminated human ovarian cancer-bearing mice.

Analysis of the prolonged infusion of DFP-10917, a deoxycytidine analog, as a therapeutic strategy for the treatment of human tumor xenografts in vivo.

2'-C-cyano-2'-deoxy-1-ß-D-arabino-pentofranocyl-cytosine (DFP-10917, CNDAC) is a 2'-deoxycytidine analog with antitumor activity against various tumor cells. However, a clinically available therapeutic regimen for this compound needs to be established and its functional mechanisms in relation to the dosing schedule need to be clarified. In this study, we evaluated the antitumor activity and toxicity of DFP-10917 by varying the dose and administration schedule in human solid tumor and leukemia xenografts in vivo. Compared to a 1-day infusion with a high-dose of DFP-10917 (30 mg/kg/day), a prolonged 14-day infusion with a low-dose (4.5 mg/kg/day) exerted superior tumor growth inhibitory effects without decreasing the body weights of mice in our human tumor xenograft model. In addition, we found that a 14-day infusion of low-dose DFP-10917 markedly prolonged the lifespan of nude mice bearing both acute leukemia and ovarian cancer cell-derived tumors. On the other hand, gemcitabine (GEM) and cytosine arabinoside (Ara-C), which are similar deoxycytidine analogs and are widely used clinically as standard regimens, exerted less potent antitumor effects than DFP-10917 on these tumors. To elucidate the possible functional mechanisms of the prolonged infusion of DFP-10197 compared with that of GEM or Ara-C, the rate of DNA damage in CCRF-CEM and HeLa cells treated with DFP-10917, Ara-C and GEM was detected using a comet assay. DFP-10917, at a range of 0.05 to 1 µM, induced a clear tailed-DNA pattern in both the CCRF-CEM and HeLa cells; Ara-C and GEM did not have any effect. It was thus suggested that a low concentration and long-term exposure to DFP-10917 aggressively introduced the fragmentation of DNA molecules, namely the so-called double-strand breaks in tumor cells, leading to potent cytotoxicity. Moreover, treatment with DFP-10917 at a low-dose with a long-term exposure specifically increased the population of cells in the G2/M phase, while GEM reduced this cell population, suggesting a unique function (G2/M arrest) of DFP-10917. On the whole, our findings indicate that the prolonged infusion of low-dose DFP-10917 mainly displays a novel functional mechanism as a DNA-damaging drug and may thus prove to be useful in the treatment of cancer patients who are resistant to other cytosine nucleosides, or in patients in which these other nucleosides have been shown to be ineffective.

Development of new promising antimetabolite, DFP-11207 with self-controlled toxicity in rodents.

To reduce 5-fluorouracil (5-FU)-induced serious toxicities without loss of antitumor activity, we have developed DFP-11207, a novel fluoropyrimidine, which consists of 1-ethoxymethyl-5-fluorouracil (EM-FU; a precursor form of 5-FU), 5-chloro-2,4-dihydroxypyridine (CDHP; an inhibitor of 5-FU degradation), and citrazinic acid (CTA; an inhibitor of 5-FU phosphorylation). In vitro studies of DFP-11207 indicated that it strongly inhibited the degradation of 5-FU by dihydropyrimidine dehydrogenase (DPD) in homogenates of the rat liver, and also inhibited the phosphorylation of 5-FU by orotate phosphoribosyltransferase (OPRT) in tumor tissues in a similar magnitude of potency by CDHP and CTA, respectively. Especially, DFP-11207 inhibited the intracellular phosphorylation of 5-FU in tumor cells in a dose-dependent manner whereas CTA alone did not protect intracellular 5-FU phosphorylation. These results postulate that DFP-11207 rapidly entered into the cell and the free CTA produced from DFP-11207 inhibited the phosphorylation of 5-FU in the cell. Furthermore, following oral administration of DFP-11207, CTA was found to be highly retained in the gastrointestinal (GI) tract compared to other tissues in rats. Interestingly, EM-FU, the prodrug of 5-FU was found to specifically produce 5-FU by various species of liver microsomes. When DFP-11207 was administered to rats, the plasma level of 5-FU was persisted for a long-time with lower Cmax and longer half-life than that from other 5-FU prodrugs. The antitumor activity of DFP-11207 was evaluated in human tumor xenografts in nude rats and found that DFP-11207 showed an antitumor activity in a dose-dependent fashion and its efficacy is equivalent to reference 5-FU drugs. In striking contrast, DFP-11207 manifested no or less 5-FU-related toxicities, such as a decrease in body weights, GI injury, and myelosuppression, especially thrombocytopenia. Taken together, the preclinical evaluation of DFP-11207 strongly indicates that DFP-11207 be a potential new version of the oral fluoropyrimidine prodrug for further clinical development.

Co-administration of liposomal l-OHP and PEGylated TS shRNA-lipoplex: A novel approach to enhance anti-tumor efficacy and reduce the immunogenic response to RNAi molecules.

Many therapeutic strategies have been applied in efforts to conquer the development and/or progression of cancer. The combination of chemotherapy and an RNAi-based approach has proven to be an efficient anticancer therapy. However, the feasibility of such a therapeutic strategy has been substantially restricted either by the failure to achieve the efficient delivery of RNAi molecules to tumor tissue or by the immunostimulatory response triggered by RNAi molecules. In this study, therefore, we intended to investigate the efficacy of using liposomal oxaliplatin (liposomal l-OHP) to guarantee the efficient delivery of RNAi molecules, namely shRNA against thymidylate synthase (TS shRNA) complexed with cationic liposome (TS shRNA-lipoplex), to solid tumors, and to suppress the immunostimulatory effect of RNAi molecules, TS shRNA, following intravenous administration. Herein, we describe how liposomal l-OHP enhanced the intra-tumor accumulation of TS shRNA-lipoplex and significantly reduced the immunostimulatory response triggered by TS shRNA. Consequently, such enhanced accumulation of TS shRNA-lipoplex along with the cytotoxic effect of liposomal l-OHP led to a remarkable tumor growth suppression (compared to mono-therapy) following systemic administration. Our results, therefore, may have important implications for the provision of a safer and more applicable combination therapy of RNAi molecules and anti-cancer agents that can produce a more reliable anti-tumor effect.

Role of the uridine/cytidine kinase 2 mutation in cellular sensitiveness toward 3'-ethynylcytidine treatment of human cancer cells.

A nucleosidic medicine, 1-(3-C-ethynyl-ß-D-ribo-pentofuranosyl)cytosine [3'-ethynylcytidine (ECyd)], is a potent inhibitor of RNA polymerase I and shows anticancer activity to various human solid tumors in vitro and in vivo. ECyd is phosphorylated to 3'-ethyntlcytidine 5'-monophosphate by uridine/cytidine kinase 2 (UCK2) and subsequently further to diphosphate and triphosphate (3'-ethyntlcytidine 5'-diphosphate, 3'-ethyntlcytidine 5'-triphosphate). 3'-Ethyntlcytidine 5'-triphosphate is an active metabolite that can inhibit RNA polymerase I competitively, causing cancer cell death. Here, to identify the UCK2 mutation for detecting responder or nonresponder to ECyd, we investigated the relationship between point mutation of the UCK2 gene and response to ECyd in various human solid tumors. We identified several functional point mutations including the splice-site mutation of the UCK2 gene IVS5+5 G>A. In addition, we found that the IVS5+5 G>A variant generates an aberrant mRNA transcript, namely, truncated mRNA was produced and normal mRNA levels were markedly decreased in the ECyd-resistant cancer cell line HT1080. We concluded that these findings strongly suggest that the IVS5+5 G>A variant would affect the expression level of the UCK2 transcript, resulting in decreased sensitivity to ECyd.

Metronomic S-1 dosing and thymidylate synthase silencing have synergistic antitumor efficacy in a colorectal cancer xenograft model.

Metronomic chemotherapy is currently considered an emerging therapeutic option in clinical oncology. S-1, an oral formulation of Tegafur (TF), a prodrug of 5-fluorouracil (5-FU), is designed to improve the antitumor activity of 5-FU in tandem with reducing its toxicity. Clinically, metronomic S-1 dosing has been approved for the standard first- and second-line treatment of metastatic or advanced stage of colorectal (CRC). However, expression of intratumor thymidylate synthase (TS), a significant gene in cellular proliferation, is associated with poor outcome to 5-FU-based chemotherapeutic regimens. In this study, therefore, we examined the effect of a combination of TS silencing by an RNA interfering molecule, chemically synthesized short hairpin RNA against TS (shTS), and 5-FU on the growth of human colorectal cancer cell (DLD-1) both in vitro and in vivo. The combined treatment of both shTS with 5-FU substantially inhibited cell proliferation in vitro. For in vivo treatments, the combined treatment of metronomic S-1 dosing with intravenously injected polyethylene glycol (PEG)-coated shTS-lipoplex significantly suppressed tumor growth, compared to a single treatment of either S-1 or PEG-coated shTS-lipoplex. In addition, the combined treatment increased the proportion of apoptotic cells in the DLD-1 tumor tissue. Our results suggest that metronomic S-1 dosing combined with TS silencing might represent an emerging therapeutic strategy for the treatment of patients with advanced CRC.

Systemically Administered RNAi Molecule Sensitizes Malignant Pleural Mesotheliomal Cells to Pemetrexed Therapy.

Pemetrexed (PMX) is a key drug for the management of malignant pleural mesothelioma (MPM). However, its therapeutic efficacy is cruelly restricted in many clinical settings by the overexpression of thymidylate synthase (TS) gene. Recently, we emphasized the efficacy of locally administered shRNA designed against TS gene in enhancing the cytotoxic effect of PMX against orthotopically implanted MPM cells in tumor xenograft tumor model. Herein, we explored the efficiency of systemic, rather than local, delivery of TS RNAi molecule in sensitizing MPM cells to the cytotoxic effect of PMX. We here designed a PEG-coated TS shRNA-lipoplex (PEG-coated TS shRNA-lipoplex) for systemic injection. PEG modification efficiently delivered TS shRNA in the lipoplex to tumor tissue following intravenous administration as indicated by a significant suppression of TS expression level in tumor tissue. In addition, the combined treatment of PMX with systemic injection of PEG-coated TS shRNA-lipoplex exerted a potent antitumor activity in a s.c. xenograft tumor model, compared to a single treatment with either PMX or PEG-coated TS shRNA-lipoplex. Metastasis, or the spread, of mesothelioma substantially dedicates the effectiveness of treatment options. The systemic, in addition to local, delivery of tumor targeted anti-TS RNAi system we propose in this study might be an effective option to extend the clinical utility of PMX in treating malignant mesothelioma.

Downregulation of thymidylate synthase by RNAi molecules enhances the antitumor effect of pemetrexed in an orthotopic malignant mesothelioma xenograft mouse model.

Malignant pleural mesothelioma (MPM) is an incurable cancer with an increasing incidence. Currently, pemetrexed (PMX)-based chemotherapy is the mainstay of chemotherapy for MPM, however, the outcome of PMX-based chemotherapy in patients with MPM is dismal. RNA interference (RNAi) technology has been considered as an effective tool to substantially enhance the therapeutic efficacy of chemotherapeutic agents in many preclinical and clinical settings. In this study, therefore, we investigated whether non-viral anti-thymidylate synthase RNAi embedded liposome (TS shRNA lipoplex) would effectively guide the downregulation of TS in human malignant mesothelioma MSTO-211H cells. Consequently, it enhanced the antitumor effect of PMX both in vitro and in vivo. TS shRNA effectively enhanced the in vitro cell growth inhibition upon treatment with PMX via downregulating TS expression in the MSTO-211H cell line. In in vivo orthotopic tumor model, the combined treatment of PMX and TS shRNA lipoplex efficiently combated the progression of orthotopic thoracic tumors and as a result prolonged mouse survival, compared to each single treatment. Our findings emphasize the pivotal relevance of RNAi as an effective tool for increasing the therapeutic efficacy of PMX, a cornerstone in the treatment regimens of MPM, and thereby, raising the possibility for the development of a novel therapeutic strategy, combination therapy of TS-shRNA and PMX, that can surpass many of the currently applied, but less effective, therapeutic regimens against lethal MPM.

Association between mRNA expression of chemotherapy-related genes and clinicopathological features in colorectal cancer: A large-scale population analysis.

To establish the individualized treatment of patients with colorectal cancer, factors associated with chemotherapeutic effects should be identified. However, to the best of our knowledge, few studies are available on this topic, although it is known that the prognosis of patients and sensitivity to chemotherapy depend on the location of the tumor and that the tumor location is important for individualized treatment. In this study, primary tumors obtained from 1,129 patients with colorectal cancer were used to measure the mRNA expression levels of the following genes associated with the effects of standard chemotherapy for colorectal cancer: 5-fluorouracil (5-FU)-related thymidylate synthase (TYMS), dihydropyrimidine dehydrogenase (DPYD) and thymidine phosphorylase (TYMP); folate-related dihydrofolate reductase (DHFR), folylpolyglutamate synthase (FPGS) and gamma-glutamyl hydrolase (GGH); irinotecan-related topoisomerase I (TOP1); oxaliplatin-related excision repair cross-complementing 1 (ERCC1); biologic agent-related vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR). Large-scale population analysis was performed to determine the association of gene expression with the clinicopathological features, in particular, the location of the colorectal cancer. From the results of our analysis of the mRNA expression of these 10 genes, we noted the strongest correlation between DPYD and TYMP, followed by TYMS and DHFR. The location of the colorectal cancer was classified into 4 regions (the right­ and left-sided colon, rectosigmoid and rectum) and was compared with gene expression. A significant difference in all genes, apart from VEGF, was noted. Of the remaining 9 genes, the highest expression of TYMS and DPYD was observed in the right­sided colon; the highest expression of GGH and EGFR was noted in the left-sided colon; the highest expression of DHFR, FPGS, TOP1 and ERCC1 was noted in the rectosigmoid, whereas TYMP expression was approximately equivalent in the right-sided colon and rectum, and higher than that in other locations. The data generated from this study may prove to be useful for the development of individualized chemotherapeutic treatments for patients with colorectal cancer, and will mean that the tumor location is taken into account.

Dynamic modulation of thymidylate synthase gene expression and fluorouracil sensitivity in human colorectal cancer cells.

Biomarkers have revolutionized cancer chemotherapy. However, many biomarker candidates are still in debate. In addition to clinical studies, a priori experimental approaches are needed. Thymidylate synthase (TS) expression is a long-standing candidate as a biomarker for 5-fluorouracil (5-FU) treatment of cancer patients. Using the Tet-OFF system and a human colorectal cancer cell line, DLD-1, we first constructed an in vitro system in which TS expression is dynamically controllable. Quantitative assays have elucidated that TS expression in the transformant was widely modulated, and that the dynamic range covered 15-fold of the basal level. 5-FU sensitivity of the transformant cells significantly increased in response to downregulated TS expression, although being not examined in the full dynamic range because of the doxycycline toxicity. Intriguingly, our in vitro data suggest that there is a linear relationship between TS expression and the 5-FU sensitivity in cells. Data obtained in a mouse model using transformant xenografts were highly parallel to those obtained in vitro. Thus, our in vitro and in vivo observations suggest that TS expression is a determinant of 5-FU sensitivity in cells, at least in this specific genetic background, and, therefore, support the possibility of TS expression as a biomarker for 5-FU-based cancer chemotherapy.

The novel thymidylate synthase inhibitor trifluorothymidine (TFT) and TRAIL synergistically eradicate non-small cell lung cancer cells.

PURPOSE: TRAIL, a tumor selective anticancer agent, may be used for the treatment of non-small cell lung cancer (NSCLC). However, TRAIL resistance is frequently encountered. Here, the combined use of TRAIL with trifluorothymidine (TFT), a thymidylate synthase inhibitor, was examined for sensitizing NSCLC cells to TRAIL. METHODS: Interactions between TRAIL and TFT were studied in NSCLC cells using growth inhibition and apoptosis assays. Western blotting and flow cytometry were used to investigate underlying mechanisms. RESULTS: The combined treatment of TFT and TRAIL showed synergistic cytotoxicity in A549, H292, H322 and H460 cells. For synergistic activity, the sequence of administration was important; TFT treatment followed by TRAIL exposure did not show sensitization. Combined TFT and TRAIL treatment for 24 h followed by 48 h of TFT alone was synergistic in all cell lines, with combination index values below 0.9. The treatments affected cell cycle progression, with TRAIL inducing a G1 arrest and TFT, a G2/M arrest. TFT activated Chk2 and reduced Cdc25c levels known to cause G2/M arrest. TRAIL-induced caspase-dependent apoptosis was enhanced by TFT, whereas TFT alone mainly induced caspase-independent death. TFT increased the expression of p53 and p21/WAF1, and p53 was involved in the increase of TRAIL-R2 surface expression. TFT also caused downregulation of cFLIP and XIAP and increased Bax expression. CONCLUSIONS: TFT enhances TRAIL-induced apoptosis in NSCLC cells by sensitizing the apoptotic machinery at different levels in the TRAIL pathway. Our findings suggest a possible therapeutic benefit of the combined use of TFT and TRAIL in NSCLC.

Effects of depletion of dihydropyrimidine dehydrogenase on focus formation and RPA phosphorylation.

Gimeracil, an inhibitor of dihydropyrimidine dehydrogenase (DPYD), partially inhibits homologous recombination (HR) repair and has a radiosensitizing effect as well as enhanced sensitivity to Camptothecin (CPT). DPYD is the target protein for radiosensitization by Gimeracil. We investigated the mechanisms of sensitization of radiation and CPT by DPYD inhibition using DLD-1 cells treated with siRNA for DPYD. We investigated the focus formation of various kinds of proteins involved in HR and examined the phosphorylation of RPA by irradiation using Western blot analysis. DPYD depletion by siRNA significantly restrained the formation of radiation-induced foci of Rad51 and RPA, whereas it increased the number of foci of NBS1. The numbers of colocalization of NBS1 and RPA foci in DPYD-depleted cells after radiation were significantly smaller than in the control cells. These results suggest that DPYD depletion is attributable to decreased single-stranded DNA generated by the Mre11/Rad50/NBS1 complex-dependent resection of DNA double-strand break ends. The phosphorylation of RPA by irradiation was partially suppressed in DPYD-depleted cells, suggesting that DPYD depletion may partially inhibit DNA repair with HR by suppressing phosphorylation of RPA. DPYD depletion showed a radiosensitizing effect as well as enhanced sensitivity to CPT. The radiosensitizing effect of DPYD depletion plus CPT was the additive effect of DPYD depletion and CPT. DPYD depletion did not have a cell-killing effect, suggesting that DPYD depletion may not be so toxic. Considering these results, the combination of CPT and drugs that inhibit DPYD may prove useful for radiotherapy as a method of radiosensitization.

Role of miR-19b and its target mRNAs in 5-fluorouracil resistance in colon cancer cells.

BACKGROUND: Drug resistance in colorectal cancers is assumed to be mediated by changes in the expression of microRNAs, but the specific identities and roles of microRNAs are largely unclear. We examined the effect of 5-fluorouracil (5-FU) resistance on microRNA expression. METHODS: Two types of 5-FU-resistant colon cancer cells were derived from the DLD-1 and KM12C cell lines. The expressions of microRNAs were profiled with a microarray containing 723 microRNAs and validated by quantitative real-time polymerase chain reaction (qRT-PCR). To survey the downstream mediators of microRNA, we used a microRNA:mRNA immunoprecipitation (RIP)-Chip and pathway analysis tool to identify potential direct targets of microRNA. RESULTS: In response to 5-FU, miR-19b and miR-21 were over-expressed in 5-FU-resistant cells. Of note, miR-19b was up-regulated 3.47-fold in the DLD-1 resistant cells, which exhibited no alteration in cell cycle profiles despite exposure to 5-FU. After transfection of miR-19b, specific mRNAs were recruited to microRNA:mRNA complexes isolated with Ago2 antibody and subjected to whole-genome transcriptional analysis. In this analysis, 66 target mRNAs were enriched by at least 5.0-fold in the microRNA:mRNA complexes from DLD-1 resistant cells. Ingenuity pathway analysis of mRNA targets significantly (P < 0.05) indicated the category "Cell Cycle" as a probable area of the molecular and cellular function related with 5-FU resistance. Among candidate mRNA targets, SFPQ and MYBL2 have been linked to cell cycle functions. CONCLUSIONS: We revealed up-regulation of miR-19b in response to 5-FU and potential targets of miR-19b mediating the cell cycle under treatment with 5-FU. Our study provides an important insight into the mechanism of 5-FU resistance in colorectal cancers.

Platelet-derived endothelial cell growth factor/thymidine phosphorylase inhibitor augments radiotherapeutic efficacy in experimental colorectal cancer.

PURPOSES: A lot of radiosensitizers have been developed. However, there are few to be available in the clinical setting. Thymidine phosphorylase inhibitor (TPI) regulates the phosphorolysis of thymidine to thymine and 2-deoxy-d-ribose-1-phosphate which is essential for tumor angiogenesis. The aim of this study is to evaluate whether TPI augments the radiotherapy for colorectal cancer in vitro and in vivo studies. MATERIALS AND METHODS: The cytotoxicity of TPI with irradiation on HT29 and HCT116 cells was examined using MTT- and colony formation assay. At 10days post-inoculation, HT29 bearing orthotopic model mice (n=28) were divided into four groups and orally treated with TPI- (50mg/kg/day for 2weeks), radiation (RT, 2Gy×4: Total 8Gy), their combination or the vehicle. The mechanisms underlying the efficacy were assessed genomically and immunohistochemically. RESULTS: Compared to each single treatment, the combination of TPI and RT synergistically inhibited the cell viability in a time- and dose-dependent manner. In the HT-29 bearing mice, the combination of TPI and RT reduced the tumor growth compared with RT alone. Notably, the mRNA levels of VEGF, TGF-ß and, Rad51 and the protein expressions of VEGF and CD34 were significantly lower in the combination than the others. Furthermore, the combination markedly increased the TUNEL-positive cells, suggesting that TPI augments the cancer cell death through inhibition of angiogenesis and DNA repair system in the radiotherapy. CONCLUSIONS: Our study first demonstrated that the combination of TPI and irradiation was effective in colon cancer. TPI would provide a promising therapeutic strategy as a radiosensitizer.

A nucleoside anticancer drug, 1-(3-C-ethynyl-ß-D-ribo-pentofuranosyl)cytosine (TAS106), sensitizes cells to radiation by suppressing BRCA2 expression.

BACKGROUND: A novel anticancer drug 1-(3-C-ethynyl-ß-D-ribo-pentofuranosyl)cytosine (ECyd, TAS106) has been shown to radiosensitize tumor cells and to improve the therapeutic efficiency of X-irradiation. However, the effect of TAS106 on cellular DNA repair capacity has not been elucidated. Our aim in this study was to examine whether TAS106 modified the repair capacity of DNA double-strand breaks (DSBs) in tumor cells. METHODS: Various cultured cell lines treated with TAS106 were irradiated and then survival fraction was examined by the clonogenic survival assays. Repair of sublethal damage (SLD), which indicates DSBs repair capacity, was measured as an increase of surviving cells after split dose irradiation with an interval of incubation. To assess the effect of TAS106 on the DSBs repair activity, the time courses of γ-H2AX and 53BP1 foci formation were examined by using immunocytochemistry. The expression of DNA-repair-related proteins was also examined by Western blot analysis and semi-quantitative RT-PCR analysis. RESULTS: In clonogenic survival assays, pretreatment of TAS106 showed radiosensitizing effects in various cell lines. TAS106 inhibited SLD repair and delayed the disappearance of γ-H2AX and 53BP1 foci, suggesting that DSB repair occurred in A549 cells. Western blot analysis demonstrated that TAS106 down-regulated the expression of BRCA2 and Rad51, which are known as keys among DNA repair proteins in the homologous recombination (HR) pathway. Although a significant radiosensitizing effect of TAS106 was observed in the parental V79 cells, pretreatment with TAS106 did not induce any radiosensitizing effects in BRCA2-deficient V-C8 cells. CONCLUSIONS: Our results indicate that TAS106 induces the down-regulation of BRCA2 and the subsequent abrogation of the HR pathway, leading to a radiosensitizing effect. Therefore, this study suggests that inhibition of the HR pathway may be useful to improve the therapeutic efficiency of radiotherapy for solid tumors.

Gimeracil, an inhibitor of dihydropyrimidine dehydrogenase, inhibits the early step in homologous recombination.

Gimeracil (5-chloro-2, 4-dihydroxypyridine) is an inhibitor of dihydropyrimidine dehydrogenase (DPYD), which degrades pyrimidine including 5-fluorouracil in the blood. Gimeracil was originally added to an oral fluoropyrimidine derivative S-1 to yield prolonged 5-fluorouracil concentrations in serum and tumor tissues. We have already reported that gimeracil had radiosensitizing effects by partially inhibiting homologous recombination (HR) in the repair of DNA double strand breaks. We investigated the mechanisms of gimeracil radiosensitization. Comet assay and radiation-induced focus formation of various kinds of proteins involved in HR was carried out. siRNA for DPYD were transfected to HeLa cells to investigate the target protein for radiosensitization with gimeracil. SCneo assay was carried out to examine whether DPYD depletion by siRNA inhibited HR repair of DNA double strand breaks. Tail moments in neutral comet assay increased in gimeracil-treated cells. Gimeracil restrained the formation of foci of Rad51 and replication protein A (RPA), whereas it increased the number of foci of Nbs1, Mre11, Rad50, and FancD2. When HeLa cells were transfected with the DPYD siRNA before irradiation, the cells became more radiosensitive. The degree of radiosensitization by transfection of DPYD siRNA was similar to that of gimeracil. Gimeracil did not sensitize DPYD-depleted cells. Depletion of DPYD by siRNA significantly reduced the frequency of neopositive clones in SCneo assay. Gimeracil partially inhibits the early step in HR. It was found that DPYD is the target protein for radiosensitization by gimeracil. The inhibitors of DPYD, such as gimeracil, could enhance the efficacy of radiotherapy through partial suppression of HR-mediated DNA repair.

Mode of action of trifluorothymidine (TFT) against DNA replication and repair enzymes.

Trifluorothymidine (TFT) is well known to be converted to TFT-monophosphate by thymidine kinase and to inhibit thymidylate synthase. In addition, TFT-triphosphate (TFT-TP) is also incorporated into DNA, resulting in cytocidal effects. However, the precise mechanism of TFT-induced DNA damage is still unclear. Therefore, we investigated the modes of action of TFT against DNA replication and repair enzymes, as compared with those of 5FU and FdUrd. When HeLa cells were treated with TFT at a concentration of 1 µM (IC50 value), the concentration of TFT in the DNA was calculated as 62.2±0.9 pmol/1x106 cells for 4 h. On the other hand, following treatment of the cells with FdUrd (0.5 µM) and 5FU (10 µM) at their IC50 doses, the drug concentrations in the DNA were 7.53, and 0.17 pmol/1 x 106 cells for 4 h, respectively. These results show the markedly greater degree of incorporation of TFT into the DNA of the HeLa cells compared with that of 5FU (approximately more than 300-fold for 4 h) or FdUrd (approximately more than 8-fold for 4 h). The primer extension assay demonstrated that TFT-TP was also incorporated into the T-sites of the growing DNA strand, however, it competed only weakly with thymidine triphosphate. The DNA glycosylase assay was performed using commercially available DNA glycosylase and fractionated HeLa cell extracts obtained by gel filtration. There was no detectable excision of the TFT pairing to adenine by uracil DNA glycosylase (UDG), thymine DNA glycosylase (TDG), methyl-CpG binding domain 4 (MBD4) or the fractionated HeLa cell extracts, however, TDG and MBD4 were able to excise the TFT pairing to guanine. Additional data indicate that small-interfering RNA-mediated knockdown of TDG or MBD4 significantly increased the resistance to the cytotoxic effects of FdUrd, but not to that of TFT. These studies show the greater degree of incorporation of TFT into the DNA than that of 5FU or FdUrd, and that such a high degree of incorporation of TFT residues into the DNA might be related to exhibit potent cytotoxic activity to be refractory to cleavage by these DNA glycosylases; thus, the DNA-directed cytotoxic effect of the compound is quite different from that of 5FU.

Total synthesis of the bicyclic marine sesquiterpenoid drechslerine B.

The total synthesis of the recently isolated bicyclic sesquiterpenoid drechslerine B (2), isolated from the algicolous fungus Drechslera dematioidearare in the marine red alga Liagora viscida, has been achieved, starting from (S)-carvone (8), via an intramolecular aldol reaction and palladium-catalyzed carbon monoxide insertion as key reactions.