Fumito Tamura

Targeting anticancer drug delivery to pancreatic cancer cells using a fucose-bound nanoparticle approach.

Owing to its aggressiveness and the lack of effective therapies, pancreatic ductal adenocarcinoma has a dismal prognosis. New strategies to improve treatment and survival are therefore urgently required. Numerous fucosylated antigens in sera serve as tumor markers for cancer detection and evaluation of treatment efficacy. Increased expression of fucosyltransferases has also been reported for pancreatic cancer. These enzymes accelerate malignant transformation through fucosylation of sialylated precursors, suggesting a crucial requirement for fucose by pancreatic cancer cells. With this in mind, we developed fucose-bound nanoparticles as vehicles for delivery of anticancer drugs specifically to cancer cells. L-fucose-bound liposomes containing Cy5.5 or Cisplatin were effectively delivered into CA19-9 expressing pancreatic cancer cells. Excess L-fucose decreased the efficiency of Cy5.5 introduction by L-fucose-bound liposomes, suggesting L-fucose-receptor-mediated delivery. Intravenously injected L-fucose-bound liposomes carrying Cisplatin were successfully delivered to pancreatic cancer cells, mediating efficient tumor growth inhibition as well as prolonging survival in mouse xenograft models. This modality represents a new strategy for pancreatic cancer cell-targeting therapy.

Fucosylated TGF-β receptors transduces a signal for epithelial-mesenchymal transition in colorectal cancer cells.

Background:Transforming growth factor-β (TGF-β) is a major inducer of epithelial–mesenchymal transition (EMT) in different cell types. TGF-β-mediated EMT is thought to contribute to tumour cell spread and metastasis. Sialyl Lewis antigens synthesised by fucosyltransferase (FUT) 3 and FUT6 are highly expressed in patients with metastatic colorectal cancer (CRC) and are utilised as tumour markers for cancer detection and evaluation of treatment efficacy. However, the role of FUT3 and FUT6 in augmenting the malignant potential of CRC induced by TGF-β is unclear.Methods:Colorectal cancer cell lines were transfected with siRNAs for FUT3/6 and were examined by cell proliferation, invasion and migration assays. The expression and phosphorylation status of TGF-β downstream molecules were analysed by western blot. Fucosylation of TGF-β receptor (TβR) was examined by lectin blot analysis.Results:Inhibition of FUT3/6 expression by siRNAs suppressed the fucosylation of type I TβR and phosphorylation of the downstream molecules, thereby inhibiting the invasion and migration of CRC cells by EMT.Conclusion:Fucosyltransferase 3/6 has an essential role in cancer cell adhesion to endothelial cells by upregulation of sialyl Lewis antigens and also by enhancement of cancer cell migration through TGF-β-mediated EMT.

RNAi-mediated gene silencing of ST6GalNAc I suppresses the metastatic potential in gastric cancer cells

BackgroundST6GalNAc I is a sialyltransferase controlling the expression of sialyl-Tn antigen (STn), which is overexpressed in several epithelial cancers, including gastric cancer, and is highly correlated with cancer metastasis. However, the functional contribution of ST6GalNAc I to development or progression of gastric cancer remains unclear. In this study, we investigated the effects of suppression of ST6GalNAc I on gastric cancer in vitro and in vivo.MethodsGastric cancer cell lines were transfected with ST6GalNAc I siRNA and were examined by cell proliferation, migration, and invasion assays. We also evaluated the effect of ST6GalNAc I siRNA treatment in a peritoneal dissemination mouse model. The differences in mRNA levels of selected signaling molecules were analyzed by polymerase chain reaction (PCR) arrays associated with tumor metastasis in MKN45 cells. The signal transducer and activator of transcription 5b (STAT5b) signaling pathways that reportedly regulate the insulin-like growth factor-1 (IGF-1) were analyzed by Western blot.ResultsST6GalNAc I siRNA inhibited gastric cancer cell growth, migration, and invasion in vitro. Furthermore, intraperitoneal administration of ST6GalNAc I siRNA- liposome significantly inhibited peritoneal dissemination and prolonged the survival of xenograft model mice with peritoneal dissemination of gastric cancer. PCR array confirmed that suppression of ST6GalNAc I caused a significant reduction in expression of IGF-1 mRNA. Decreased IGF-1 expression in MKN45 cells treated with ST6GalNAc I siRNA was accompanied by reduced phosphorylation of STAT5b.ConclusionST6GalNAc I may regulate the gene expression of IGF-1 through STAT5b activation in gastric cancer cells and may be a potential target for treatment of metastasizing gastric cancer.

Clinical utility of capsule endoscopy with flexible spectral imaging color enhancement for diagnosis of small bowel lesions

Background and study aims: The clinical utility of computed virtual chromoendoscopy with flexible spectral imaging color enhancement (FICE) in capsule endoscopy (CE) remains controversial. To clarify the clinical utility of FICE-enhanced CE in evaluating small bowel lesions, we quantitatively assessed white light (WL), FICE, and blue mode (BM) images and examined the sensitivity of these 3 imaging modes of small-bowel lesions from patients who underwent CE. Methods: The CIELAB color difference (∆E) and visual analogue scales (VAS) were measured in 261 CE images (3 different lesion categories) using WL and FICE set 1, 2, and 3, and BM images, respectively. Three endoscopists reviewed CE videos with WL, 3 FICE mode settings, and BM, and compared the sensitivity and detectability for small intestinal diseases from 50 patients who underwent CE. Results: In the assessment of visibility in the 152 vascular lesion images, the ∆E and VAS of FICE set 1, 2, and BM images were significantly higher than that of WL images. In 88 erosion/ulceration images, the ∆E and VAS of FICE set 1 and 2 images were significantly higher than that of WL images. In 21 tumor images, there were no significant differences in ∆E among these modalities. When analyzed on a per-patient basis, FICE settings 1 and 2 had the highest sensitivity (100 %) and specificity (97.3 – 100 %) for vascular lesions. As for erosive/ulcerative lesions, FICE setting 2 had the highest sensitivity (100 %) and specificity (97.2 %). For tumors or polyps, WL had the highest sensitivity (90.9 %) and specificity (87.1 %). In per-lesion analysis, FICE settings 1 and 2 showed significantly superior detection ability over WL for vascular lesions. In the detection of erosive/ulcerative lesions, FICE setting 2 was significantly superior to WL. In tumor images, there was no significant improvement with any of the settings relative to WL images. Conclusions: FICE is most useful for improving CE image quality and detection in cases of angioectasia and erosion/ulceration of the small intestine.

Relationship Between Increased Fucosylation and Metastatic Potential in Colorectal Cancer

BACKGROUND Fucose is utilized for the modification of different molecules involved in blood group determination, immunological reactions, and signal transduction pathways. We have recently reported that enhanced activity of the fucosyltransferase 3 and/or 6 promoted TGF-ß-mediated epithelial mesenchymal transition and was associated with increased metastatic potential of colorectal cancer (CRC), suggesting that fucose is required by CRC cells. With this in mind, we examined requirement of L-fucose in CRC cells and developed fucose-bound nanoparticles as vehicles for delivery of anticancer drugs specific to CRC. METHODS In this study, we first examined the expression of fucosylated proteins in 50 cases of CRC by immunochistochemical staining with biotinylated Aleuria aurantia lectin (AAL). Then we carried out an L-fucose uptake assay using three CRC cell lines. Finally, we developed fucose-bound nanoparticles as vehicles for the delivery of an anticancer drug, SN38, and examined tumor growth inhibition in mouse xenograft model (n = 6 mice per group). All statistical tests were two-sided. RESULTS We found a statistically significant relationship between vascular invasion, clinical stage, and intensity score of AAL staining (P≤ .02). L-fucose uptake assay revealed that L-fucose incorporation, as well as fucosylated protein release, was high in cells rich in fucosylated proteins. L-fucose-bound liposomes effectively delivered Cy5.5 into CRC cells. The excess of L-fucose decreased the efficiency of Cy5.5 uptake through L-fucose-bound liposomes, suggesting an L-fucose receptor dependency. Intravenously injected, L-fucose-bound liposomes carrying SN38 were successfully delivered to CRC cells, mediating efficient tumor growth inhibition (relative tumor growth ratio: no treatment group [NT], 8.29 ± 3.09; SN38-treated group [SN38], 3.53 ± 1.47; liposome-carrying, SN38-treated group [F0], 3.1 ± 1.39; L-fucose-bound, liposome-carrying, SN38-treated group [F50], 0.94 ± 0.89; F50 vs NT,P= .003; F50 vs SN38,P= .02, F50 vs F0,P= .04), as well as prolonging survival of mouse xenograft models (log-rank test,P< .001). CONCLUSIONS Thus, fucose-bound liposomes carrying anticancer drugs provide a new strategy for the treatment of CRC patients.

Targeting Notch-1 positive acute leukemia cells by novel fucose-bound liposomes carrying daunorubicin.

Complete remission by induction therapy in acute myelogenous leukemia (AML) can be achieved due to improvements in supportive and optimized therapy. However, more than 20% of patients will still need to undergo salvage therapy, and most will have a poor prognosis. Determining the specificity of drugs to leukemia cells is important since this will maximize the dose of chemotherapeutic agents that can be administered to AML patients. In turn, this would be expected to lead to reduced drug toxicity and its increased efficacy. We targeted Notch-1 positive AML cells utilizing fucose-bound liposomes, since activation of Notch-1 is required for O-fucosylation. Herein, we report that intravenously injected, L-fucose-bound liposomes containing daunorubicin can be successfully delivered to AML cells that express fucosylated antigens. This resulted in efficient tumor growth inhibition in tumor-bearing mice and decreased proliferation of AML patient-derived leukemia cells. Thus, biological targeting by fucose-bound liposomes that takes advantage of the intrinsic characteristics of AML cells could be a promising new strategy for Notch-1 positive-AML treatment.

Abstract 4463: Targeting SN38 delivery to gastrointestinal cancer cells using a fucose-bound nanoparticle approach

Background & Aims: Prognosis of patients with metastatic colorectal cancer (CRC) has been improving by introduction of molecular targeting therapy combined with chemotherapeutic agents. However, metastatic CRC still needs new strategies to improve treatment efficacy and survival rate. Increased expression of fucosyltransferases (FUTs) is common in CRC cells. FUTs are key enzymes accelerating malignant transformation through the fucosylation of different sialylated precursors, suggesting a crucial requirement for fucose by metastatic CRC cells. With this in mind, we developed fucose-bound nanoparticles as vehicles for delivery of anticancer drugs specifically to CRC cells. Methods: L-fucose-bound liposomes were produced by the modified cholate dialysis method as vehicles for delivery of Cy5.5, FAM and SN38. The production of CA19-9 was measured by enzyme-linked immunosorbent assay. The CRC cell lines were exposed to SN38 or L-fucose-bound liposomes carrying SN38, and cytotoxicity was monitored with BrdU assay. Introduction of Cy5.5 or FAM by L-fucose-bound liposomes was analyzed by flow cytometry or fluorescence microscopy. The effect of L-fucose-bound liposome carrying SN38 in vivo was analyzed by intravenous injection in mice engrafted with CRC cell lines. All statistical tests were two-sided. Results: L-fucose-bound liposomes containing Cy5.5 or SN38 were effectively delivered into CA19-9 expressing CRC cancer cells. Excess L-fucose decreased the efficiency of Cy5.5 introduction by L-fucose-bound liposomes, suggesting L-fucose-receptor dependency. Intravenously injected L-fucose-bound liposomes carrying SN38 were successfully delivered to CRC cells, mediating efficient tumor growth inhibition as well as prolonging survival in mouse xenograft models. Conclusions: Fucose-bound liposomes carrying anti-cancer drugs should provide a new strategy for patients with colorectal cancer. Citation Format: Junji Kato, Rishu Takimoto, Takahiro Osuga, Michihiro Ono, Masahiro Hirakawa, Makoto Yoshida, Yasushi Sato, Fumito Tamura. Targeting SN38 delivery to gastrointestinal cancer cells using a fucose-bound nanoparticle approach. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4463. doi:10.1158/1538-7445.AM2014-4463

The prognostic factor and the optimal timing of conversion surgery in unresectable stage IV gastric cancer: A retrospective analysis.

78 Background: Conversion surgery could be an option for unresectable stage IV gastric cancer when distant metastasis (M1) is disappeared by chemotherapy. However, the indication and the optimal timing of conversion surgery in stage IV gastric cancer remain unclear, even if metastatic lesions disappear with chemotherapy. Guideline of National Comprehensive Cancer Network also shows no principle after down-staging. Methods: This retrospective study examined 34 gastric cancer patients who underwent curative conversion surgery at our institute between 2005 and 2014. Clinicopathologic characteristics and patient outcomes were analyzed, with particular focus on the potential to select patients who might benefit from surgical resection. Results: The number of M1 factors was one in 31 patients and two in 3, including metastases to non-regional lymph node in 21, peritoneum in 8, liver in 5, and lung in 3. The regimen of chemotherapy was Docetaxel/CDDP/S-1 in 23 patients, Docetaxel/CDDP/S-1+Trastuzmab in 6, S-1/CD...

Tu1967 Accumulation of L-Fucose Is a Functional Target for Colorectal Cancer Therapy

Background MK-2206, a novel Akt inhibitor, has shown anticancer effect in many types of cancers, but its effect in gastric cancer was unclear. We aimed to investigate the anticancer effect of MK-2206 in gastric cancer cells. Method Gastric cancer cell lines (SGC7901 and MKN45) were used in this study. The effect of MK-2206 on cell proliferation was determined by cell viability assays and colony formation assays. Apoptosis in SGC-7901 cells treated with MK-2206 was analyzed by flow cytometry. The interactions between MK2206 and other chemotherapeutics were determined by calculating the combination index (CI) based on the Talalay-Chou equation. The expression levels of Akt/pAkt, caspase-3/ caspase-7/caspase-9, PARP, cleaved caspase-3/caspase-7/caspase-9, and cleaved PARP were determined by western blot analysis in circumstances with treatment of chemotherapeutics alone or combined with MK-2206. Results Cell viability assays and colony formation assays showed that MK-2206 could effectively inhibit the proliferation of SGC-7901 and MKN45 cells. The cytotoxicity of MK-2206 increased when gastric cancer cells were treated with

Abstract 1123: Fucosylated TGF-ß receptors transduce a signal for epithelial-mesenchymal transition in colorectal cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Transforming growth factor-s (TGF-s) is a major inducer of epithelial-mesenchymal transition (EMT) in different cell types. TGF-s-mediated EMT is thought to contribute to tumor cell spread and metastasis. Sialyl Lewis antigens synthesized by fucosyltransferase (FUT) 3 and FUT6 are highly expressed in patients with metastatic colorectal cancer (CRC) and are utilized as tumor markers for cancer detection and evaluation of treatment efficacy. However, the role of FUT3 and FUT6 in augmenting the malignant potential of CRC induced by TGF-s is unclear. Methods: CRC cell lines were transfected with siRNAs for FUT3/6 and were examined by cell proliferation, invasion, and migration assays. The expression and phosphorylation status of TGF-s downstream molecules were analyzed by western blot. Fucosylation of TGF-s receptor was examined by lectin blot analysis. Results: Inhibition of FUT3/6 expression by siRNAs suppressed the fucosylation of type I TGF-s receptor and phosphorylation of the downstream molecules, thereby inhibiting the invasion and migration of CRC cells by EMT. Citation Format: Rishu Takimoto, Masahiro Hirakawa, Fumito Tamura, Makoto Yoshida, Michihiro Ono, Yasushi Sato, Takahiro Osuga, Junji Kato. Fucosylated TGF-s receptors transduce a signal for epithelial-mesenchymal transition in colorectal cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1123. doi:10.1158/1538-7445.AM2014-1123