Teruya Nagahara

Potential of adenovirus-mediated REIC/Dkk-3 gene therapy for use in the treatment of pancreatic cancer

The reduced expression in immortalized cells REIC/the dickkopf 3 (Dkk‐3) gene, tumor suppressor gene, is downregulated in various malignant tumors. In a prostate cancer study, an adenovirus vector carrying the REIC/Dkk‐3 gene (Ad‐REIC) induces apoptosis. In the current study, we examined the effects of REIC/Dkk‐3 gene therapy in pancreatic cancer.

Loss of runt-related transcription factor 3 induces gemcitabine resistance in pancreatic cancer

Runt‐related transcription factor 3 (RUNX3) is a tumor suppressor gene that is expressed in gastric and other cancers including pancreatic cancer. However, the precise function of RUNX3 in pancreatic cancer has not been fully elucidated. In this study, we aimed to determine the effect of decreased RUNX3 expression in pancreatic cancer.

Novel REIC/Dkk-3-encoding adenoviral vector as a promising therapeutic agent for pancreatic cancer

Reduced expression in immortalized cells (REIC)/dickkopf-3 (Dkk-3), a tumor suppressor gene, is downregulated in various cancers. We previously reported the tumor-inhibitory effects of the REIC/Dkk-3 gene, delivered by a conventional adenoviral vector (Ad-CAG-REIC) in pancreatic cancer. Here, we developed an Ad-REIC vector with a novel gene expression system, termed the super gene expression (SGE) system, and assessed its therapeutic effects relative to those of Ad-CAG-REIC in pancreatic cancer cells. Human pancreatic cancer cell lines ASPC1 and MIAPaCa2 were used. REIC/Dkk-3 expression was assessed by western blot analysis. Relative cell viability and apoptotic effects were examined in vitro. The anti-tumor effects of Ad-REIC treatment were assessed in the mouse xenograft model. Compared with Ad-CAG-REIC, Ad-SGE-REIC elicited a significant increase in REIC protein expression in the cells studied. Relative to Ad-CAG-REIC, Ad-SGE-REIC reduced cell viability and induced apoptosis in the ASPC1 and MIAPaCa2 cell lines in vitro, and achieved superior tumor growth inhibition in the mouse xenograft model. Compared with conventional Ad-REIC agents, Ad-SGE-REIC provided enhanced inhibitory effects against tumor growth. Our results indicate that Ad-SGE-REIC is an innovative therapeutic tool for pancreatic cancer.

Synergistic anti‐pancreatic cancer immunological effects by treatment with REIC/DKK3 protein and peripheral blood mononuclear cells

Reduced expression in immortalized cells/dickkopf‐3 (REIC/DKK3) is a reported tumor suppressor gene and has potential to become an innovative therapy for various cancers. We examined the antitumor immunological effects of human REIC/DKK3 protein against pancreatic cancer.

Synergistic anti‐pancreatic cancer immunological effects by treatment with reduced expression in immortalized cells/dickkopf‐3 protein and peripheral blood mononuclear cells

Reduced expression in immortalized cells/dickkopf‐3 (REIC/DKK3) is a reported tumor suppressor gene and has potential to become an innovative therapy for various cancers. We examined the antitumor immunological effects of human REIC/DKK3 protein against pancreatic cancer.

Hepatic stellate cells promote upregulation of epithelial cell adhesion molecule and epithelial-mesenchymal transition in hepatic cancer cells

Microenvironment plays an important role in epithelial-mesenchymal transition (EMT) and stemness of cells in hepatocellular carcinoma (HCC). Epithelial cell adhesion molecule (EpCAM) is known as a tumor stemness marker of HCC. To investigate the relationship between microenvi-ronment and stemness, we performed an in vitro co-culture assay. Four HCC cell lines (HepG2, Hep3B, HuH-7 and PLC/PRF/5) were co-cultured with the TWNT-1 immortalized hepatic stellate cells (HSCs), which create a microenvironment with HCC. Cell proliferation ability was analyzed by flow cytometry (FCM) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide (MTT) assay, while migration ability was assessed by a wound healing assay. Expression of EpCAM was analyzed by immunoblotting and FCM. HCC cell lines were co-cultured with TWNT-1 treated with small interfering RNA (siRNA) for TGF-β and HB-EGF; we then analyzed proliferation, migration ability and protein expression using the methods described above. Proliferation ability was unchanged in HCC cell lines co-cultured with TWNT-1. Migration ability was increased in HCC cell lines (HepG2, Hep3B, HuH-7 and PLC/PRF/5) directly (216.2±67.0, 61.0±22.0, 124.0±66.2 and 51.5±40.3%) and indirectly (102.5±22.0, 84.6±30.9, 86.1±25.7 and 73.9±29.7%) co-cultured with TWNT-1 compared with the HCC uni-culture. Immunoblot analysis revealed increased EpCAM expression in the HCC cell lines co-cultured with TWNT-1. Flow cytometry revealed that the population of E-cadherin−/N-cadherin+ and EpCAM-positive cells increased and accordingly, EMT and stemness in the HCC cell line were activated. These results were similar in the directly and indirectly co-cultured samples, indicating that humoral factors were at play. Conversely, HCC cell lines co-cultured with siRNA-treated TWNT-1 showed decreased migration ability, a decreased population of EpCAM-positive and E-cadherin−/N-cadherin+ cells. Taken together, humoral factors secreted from TWNT-1 promote upregulation of EpCAM and EMT in hepatic cancer cells.

Loss of Runt-related transcription factor 3 induces resistance to 5-fluorouracil and cisplatin in hepatocellular carcinoma.

Runt-related transcription factor 3 (RUNX3) is known to function as a tumor suppressor in gastric cancer and other types of cancers, including hepatocellular carcinoma (HCC). However, its role has not been fully elucidated. In the present study, we aimed to evaluate the role of RUNX3 in HCC. We used the human HCC cell lines Hep3B, Huh7 and HLF; RUNX3 cDNA was introduced into Hep3B and Huh7 cells, which were negative for endogenous RUNX3 expression, and RUNX3 siRNA was transfected into HLF cells, which were positive for endogenous RUNX3. We analyzed the expression of RUNX3 and multidrug resistance-associated protein (MRP) by immunoblotting. MTT assays were used to determine the effects of RUNX3 expression on 5-fluorouracil (5-FU) and cisplatin (CDDP) sensitivity. Finally, 23 HCC specimens resected from patients with HCC at Okayama University Hospital were analyzed, and correlations among immunohistochemical expression of RUNX3 protein and MRP protein were evaluated in these specimens. Exogenous RUNX3 expression reduced the expression of MRP1, MRP2, MRP3 and MRP5 in the RUNX3-negative cells, whereas knockdown of RUNX3 in the HLF cells stimulated the expression of these MRPs. An inverse correlation between RUNX3 and MRP expression was observed in the HCC tissues. Importantly, loss of RUNX3 expression contributed to 5-FU and CDDP resistance by inducing MRP expression. These data have important implications in the study of chemotherapy resistance in HCC.

Dipeptide γ‑secretase inhibitor treatment enhances the anti‑tumor effects of cisplatin against gastric cancer by suppressing cancer stem cell properties

The γ-secretase inhibitor blocks Notch activity by preventing its cleavage at the cell surface. In the present study, the effect of the γ-secretase inhibitor on the viability of gastric cancer cells when administered in combination with cisplatin was investigated, with particular focus on CD44highLgr-5high cancer cells. The four gastric cancer cell lines, MKN45, MKN74, SC-6-JCK and SH-10-TC, were used for the experiments. In the MTT assay, treatment with 25 µM dipeptide γ-secretase inhibitor (DAPT) alone did not affect cell proliferation in any of the four cell lines. Gastric cancer cells subjected to combination treatment with DAPT and cisplatin exhibited decreased viability when compared with those treated with cisplatin alone. Flow cytometry was performed to evaluate the expression of cluster of differentiation (CD)-44 and leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr-5), two cancer stem cell markers in gastric cancers. Treatment with cisplatin alone significantly increased the proportion of CD44highLgr-5high cells. However, the addition of DAPT to cisplatin reduced the CD44highLgr-5high fraction, suggesting that DAPT reduced the number of gastric cancer cells. In conclusion, the present study demonstrated the synergistic effects of DAPT in combination with cisplatin by decreasing the survival of gastric cancer cells. In addition, combination treatment with DAPT reduced the number of CD44highLgr-5high cells, which are thought to exhibit cancer stem cell properties. These results highlight the therapeutic potential of DAPT in gastric cancer treatment.

Promising therapeutic efficacy of a novel reduced expression in immortalized cells/dickkopf-3 expressing adenoviral vector for hepatocellular carcinoma

Reduced expression in immortalized cells (REIC)/dickkopf‐3 (Dkk‐3) is a tumor suppressor gene that is downregulated in various cancers. In our previous study of prostate cancer, the REIC/Dkk‐3‐expressing adenoviral vector (Ad‐REIC) was found to induce cancer‐selective apoptosis. This study recently developed a novel super gene expression (SGE) system and used this system to re‐construct an Ad‐REIC vector, termed the Ad‐SGE‐REIC, to achieve more effective therapeutic outcomes. In this study, the therapeutic effects of Ad‐SGE‐REIC on hepatocellular carcinoma (HCC) was assessed.

Corrigendum to “Loss of runt-related transcription factor 3 induces gemcitabine resistance in pancreatic cancer” [Mol. Oncol. 7 (2013) 840–849]

Shigeru Horiguchi, Hidenori Shiraha*, Teruya Nagahara, Jyunnro Kataoka, Masaya Iwamuro, Minoru Matsubara, Shinichi Nishina, Hironari Kato, Akinobu Takaki, Kazuhiro Nouso, Takehiro Tanaka, Koichi Ichimura, Takahito Yagi, Kazuhide Yamamoto Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and, Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan Department of Pathology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical, Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan Department of Gastroenterological Surgery, Transplant, and Surgical Oncology, Okayama University Graduate, School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan