Toshiki Hirakawa

Hypoxia Stimulates the EMT of Gastric Cancer Cells through Autocrine TGFβ Signaling

Epithelial mesenchymal transition (EMT) is considered to be correlated with malignancy of cancer cells and responsible for cancer invasion and metastasis. We previously reported that distant metastasis was associated with hypoxia in gastric cancer. We therefore investigated the effect of hypoxic condition on EMT of gastric cancer cells. Gastric cancer cells were cultured in normoxia (21% O2) or hypoxia (1% O2) for 24 h. EMT was evaluated as the percentage of spindle-shaped cells in total cells. Effect of transforming growth factor β1 (TGFβ1) or tyrosine kinase inhibitors on the EMT was evaluated. The expression level of TGFβ1 and TGFβR was evaluated by real time RT-PCR. The TGFβ1 production from cancer cells was measured by ELISA. Hypoxia stimulated EMT of OCUM-2MD3 and OCUM-12 cells, but not that of OCUM-2M cells. The expression level of TGFβ1 mRNA under hypoxia was significantly higher than that under normoxia in all of three cell lines. The expression level of TGFβR mRNA was significantly increased by hypoxia in OCUM-2MD3 cells, but not in OCUM-2M cells. TGFβR inhibitor, SB431542 or Ki26894, significantly suppressed EMT of OCUM-2MD3 and OCUM-12. TGFβ1 production from OCUM-2MD3 and OCUM-12 cells was significantly increased under hypoxia in comparison with that under normoxia. These findings might suggest that hypoxia stimulates the EMT of gastric cancer cells via autocrine TGFβ/TGFβR signaling.

IGF-1 receptor and IGF binding protein-3 might predict prognosis of patients with resectable pancreatic cancer

BackgroundThe present study aimed to elucidate the clinicopathologic role of insulin-like growth factor-1 receptor (IGF1R) and IGF binding protein-3 (IGFBP3) in patients with pancreatic cancer. The function of IGFBP3 is controversial, because both inhibition and facilitation of the action of IGF as well as IGF-independent effects have been reported. In this study, IGF1R and IGFBP3 expression was examined, and their potential roles as prognostic markers in patients with pancreatic cancer were evaluated.MethodsClinicopathological features of 122 patients with curatively resected pancreatic cancer were retrospectively reviewed, and expression of IGF1R and IGFBP3 was immunohistochemically analyzed.ResultsExpression of IGF1R and IGFBP3 was observed in 50 (41.0%) and 37 (30.3%) patients, respectively. IGF1R expression was significantly associated with histological grade (p = 0.037). IGFBP3 expression had a significant association with tumor location (p = 0.023), and a significant inverse association with venous invasion (p = 0.037). Tumors with IGF1R-positive and IGFBP3-negative expression (n = 32) were significantly frequently Stage II and III (p = 0.011). The prognosis for IGF1R positive patients was significantly poorer than that for IGF1R negative patients (p = 0.0181). IGFBP3 protein expression did not correlate significantly with patient survival. The subset of patients with both positive IGF1R and negative IGFBP3 had worse overall survival (8.8 months versus 12.6 months, respectively, p < 0.001).ConclusionIGF1R signaling might be associated with tumor aggressiveness, and IGFBP3 might show antiproliferative effects in pancreatic cancer. Both high IGF1R expression and low IGFBP3 expression represent useful prognostic markers for patients with curatively resected pancreatic cancer.

HER3 Overexpression as an Independent Indicator of Poor Prognosis for Patients with Curatively Resected Pancreatic Cancer

Objective: The association between human epidermal growth factor receptor 3 (HER3) overexpression and survival in patients with curatively resected pancreatic cancer was investigated. Methods: Tissue samples from 126 pancreatic cancers without hematogenous or peritoneal metastases recovered from macroscopically curative resection were fixed with formalin, embedded in paraffin and subjected to immunohistochemical staining. Semiquantitative scores of zero (no staining or staining in less than 10% of cancer cells), 1+, 2+, or 3+ were assigned to each sample based on the intensity of staining. Scores of 2+ or 3+ were defined as HER3-positive staining, i.e., HER3 overexpression. Results: HER3 overexpression was observed in 52 of the 126 tissue samples (41.3%). There were no associations between HER3 overexpression and clinicopathological factors, including tumor location, tumor size, tumor differentiation, T/N categories according to the International Union against Cancer, and serum carbohydrate antibody 19-9 level (CA19-9). Univariate analysis demonstrated the median survival time of patients with HER3 overexpression was 37.2 months, while that of patients with HER3-negative samples was 58.6 months (p = 0.008). HER3 overexpression, lymph node metastasis, and elevated serum CA19-9 level were independent predictors of poor prognosis based on multivariate survival analysis. Conclusion: A new prognostic predictor, HER3 overexpression, was identified for resected pancreatic cancer.

Clinical and pathological features of five-year survivors after pancreatectomy for pancreatic adenocarcinoma

BackgroundClinical factors determining short-term survival after pancreatectomy have been well studied, but factors predicting long-term survival with curative resection are poorly understood in pancreatic carcinoma. Our objective was to identify clinical and pathological features of five-year disease-free survivors after surgical resection of pancreatic adenocarcinoma.MethodsThe clinical and pathological data from 147 patients who underwent a potentially curative resection for pancreatic adenocarcinoma at our institution between 1988 and 2012 were retrospectively analyzed.ResultsOf 147 patients, 18 survived for more than five years after surgery without disease recurrence. A univariate analyses demonstrated that: two or fewer lymph node metastases (P = 0.014), a preoperative serum carbohydrate antigen 19-9 (CA19-9) level of 40 U/mL or less (P = 0.0018), an absence of intrapancreatic nerve invasion (P = 0.028), and undergoing an R0 resection (P = 0.011) were significantly associated with five-year survival. A logistic regression model identified the following independent cancer-related predictors of five-year survivors: having two or fewer lymph node metastases (odds ratio (OR): 6.02; 95% confidence interval (CI): 1.08 to 112.98; P = 0.0385), a preoperative serum CA19-9 level of 40 U/mL or less (OR: 5.02; 95% CI: 1.68 to 16.48; P = 0.0036), and undergoing an R0 resection (OR: 3.63; 95% CI: 1.12 to 14.28; P = 0.0316).ConclusionsWe conclude that number of lymph node metastases being two or less, a preoperative serum CA19-9 level of 40 U/mL or less, and undergoing an R0 resection may be independent predictive factors to identify actual five-year survivors after pancreatectomy for pancreatic adenocarcinoma.

Abstract 3337: TGF-β from niche fibroblasts increases the stemness cancer stem cell-like side population cells in gastric cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Cancer Stem Cells (CSCs) are thought to possess tumor initiation and self-renewal and are associated with tumor progression. The niche cells of microenvironment, such as fibroblast might play an important role for the progression of cancer cells. The aim of this study is to examine the effect of cancer-associated fibroblasts on the stemness of CSCs. Material and Methods: A gastric cancer cell line, OCUM-12, and cancer-associated gastric fibroblast, CaF-37, were used. SP cells, known as CSCs rich population, were isolated from OCUM-12 cells by flowcytometry using Hoechest33342, and were named as OCUM-12/SP. The percentage of SP cells was evaluated by flowcytometry, after it was cultured with condition medium from CaF-37 for three days. SP fraction was evaluated by flowcytometry, in the presence or absence of TGF-β receptor inhibitor, FGF receptor inhibitor, and c-Met Receptor inhibitor. In vivo, tumorigenicity of OCUM-12/SP cells were evaluated by subcutaneous inoculation in the presence or abcence of CaF-37. Results: The inoculation of 1.5x105, 5x104, and 1.5x104 OCUM-12/SP cells with 5x105 CaF-37 fibroblasts resulted in tumor formation in all of mice, respectively. However, the inoculation of 1.5x105, 5x104, and 1.5x104 OCUM-12/SP cells alone results in poor tumor formation at 3(60%) of 5 mice, 0(0%) of 5 mice and 0(0%) of 5 mice, respectively. The percentage of SP fraction of OCUM-12/SP was significantly increased in the presence of condition medium from CaF-37. The increase of SP fraction of OCUM-12/SP cells by condition medium from CaF-37 was inhibited by TGF-β receptor inhibitor, but not FGF receptor inhibitor and c-Met inhibitor. Conclusion: TGF-β from the niche, fibroblasts, might sustain the stemness of CSCs and might be associated with the progression of Cancer Cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3337. doi:1538-7445.AM2012-3337

Pancreatic Fibroblasts Stimulate the Motility of Pancreatic Cancer Cells through IGF1/IGF1R Signaling under Hypoxia

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its hypovascularity, with an extremely poor prognosis because of its highly invasive nature. PDAC proliferates with abundant stromal cells, suggesting that its invasive activity might be controlled by intercellular interactions between cancer cells and fibroblasts. Using four PDAC cell lines and two pancreas cancer-associated fibroblasts (CAFs), the expression of insulin-like growth factor-1 (IGF1) and IGF1 receptor (IGF1R) was evaluated by RT-PCR, FACScan, western blot, or ELISA. Correlation between IGF1R and the hypoxia marker carbonic anhydrase 9 (CA9) was examined by immunohistochemical staining of 120 pancreatic specimens. The effects of CAFs, IGF1, and IGF1R inhibitors on the motility of cancer cells were examined by wound-healing assay or invasion assay under normoxia (20% O2) and hypoxia (1% O2). IGF1R expression was significantly higher in RWP-1, MiaPaCa-2, and OCUP-AT cells than in Panc-1 cells. Hypoxia increased the expression level of IGF1R in RWP-1, MiaPaCa-2, and OCUP-AT cells. CA9 expression was correlated with IGF1R expression in pancreatic specimens. CAFs produced IGF1 under hypoxia, but PDAC cells did not. A conditioned medium from CAFs, which expressed αSMA, stimulated the migration and invasion ability of MiaPaCa-2, RWP-1, and OCUP-AT cells. The motility of all PDAC cells was greater under hypoxia than under normoxia. The motility-stimulating ability of CAFs was decreased by IGF1R inhibitors. These findings might suggest that pancreas CAFs stimulate the invasion activity of PDAC cells through paracrine IGF1/IGF1R signaling, especially under hypoxia. Therefore the targeting of IGF1R signaling might represent a promising therapeutic approach in IGF1R-dependent PDAC.

Plasma pharmacokinetics after combined therapy of gemcitabine and oral S-1 for unresectable pancreatic cancer

BackgroundThe combination of gemcitabine (GEM) and S-1, an oral 5-fluorouracil (5-FU) derivative, has been shown to be a promising regimen for patients with unresectable pancreatic cancer.MethodsSix patients with advanced pancreatic cancer were enrolled in this pharmacokinetics (PK) study. These patients were treated by oral administration of S-1 30 mg/m2 twice daily for 28 consecutive days, followed by a 14-day rest period and intravenous administration of GEM 800 mg/m2 on days 1, 15 and 29 of each course. The PK parameters of GEM and/or 5-FU after GEM single-administration, S-1 single-administration, and co-administration of GEM with pre-administration of S-1 at 2-h intervals were analyzed.ResultsThe maximum concentration (Cmax), the area under the curve from the drug administration to the infinite time (AUCinf), and the elimination half-life (T1/2) of GEM were not significantly different between GEM administration with and without S-1. The Cmax, AUCinf, T1/2, and the time required to reach Cmax (Tmax) were not significantly different between S-1 administration with and without GEM.ConclusionThere were no interactions between GEM and S-1 regarding plasma PK of GEM and 5-FU.

Prostaglandin D synthase is a potential novel therapeutic agent for the treatment of gastric carcinomas expressing PPARγ.

The antitumor activity of prostaglandin (PG) D2 has been demonstrated against some types of cancer, including gastric cancer. However, exogenous PGD2 is not useful from a clinical point of view because it is rapidly metabolized in vivo. The aim of this study was to clarify the antitumor efficacy of an alternative, PGD synthase (PGDS), on gastric cancer cells. The effects of PGD2 and PGDS on the proliferation of gastric cancer cells were examined in vivo and in vitro. The expression levels of PGD2 receptors and peroxisome proliferator‐activated receptor γ (PPARγ) were evaluated by RT‐PCR. The effects of a PPARγ antagonist or siPPARγ on the proliferation of cancer cells and the c‐myc and cyclin D1 expression were examined in the presence or absence of PGD2 or PGDS. PPARγ was expressed in gastric cancer cell lines, but PGD2 receptors were not. PGD2 and PGDS significantly decreased the proliferation of gastric cancer cells that highly expressed PPARγ. PGDS increased the PGD2 production of gastric cancer cells. A PPARγ antagonist and siPPARγ transfection significantly suppressed the growth‐inhibitory effects of PGD2 and PGDS. Expression of c‐myc and cyclin D1 was significantly decreased by PGD2; this inhibitory effect was suppressed by PPARγ antagonist. Both PGD2 and PGDS significantly decreased subcutaneous tumor growth in vivo. Tumor volume after PGDS treatment was significantly less than PGD2 treatment. These findings suggest that PGDS and PGD2 decrease the proliferation of gastric cancer cells through PPARγ signaling. PGDS is a potentially promising therapeutic agent for gastric cancers that express PPARγ.

Laparoscopic resection of ileal lipoma diagnosed by multidetector-row computed tomography.

Intussusception is rare in adults and it is difficult to diagnose on admission. We present the case of a 43-year-old woman with the chief complaint of nausea and upper abdominal pain. Abdominal multidetector-row computed tomography showed ileo-ileal small bowel intussusception with an intraluminal soft tissue mass with attenuation numbers suggestive of a lipoma. The patient was treated with a laparoscopic-assisted extracorporeal partial resection of the small bowel including ileal lipoma, followed by a functional end-to-end anastomosis. Histologic diagnosis of the resected tumor, 2.4×2.0×2.0 cm, was an intestinal lipoma. This case serves as the basis of a review of small bowel intussusception in adults secondary to lipomas. It focuses on the utility of multidetector-row computed tomography and the cosmetic, physical, and economic benefits of laparoscopic surgery as well as the rarity of the disease.

Abstract 3995: Antitumor effect of prostaglandin D2 by peroxisome proliferator-activated receptor gamma (PPARγ)-dependent pathway in gastric carcinoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: Prostaglandin D2 (PGD2) has been demonstrated to have not only physiological responses but also anti-tumor effects against some types of cancer cells such as a lung cancer. PGD2 act through two major receptors, DP1 and DP2 of chemoattractant receptor-like molecule on the Th2 cell. Recently, some papers reported that PGD2 may act through PPARγ, suggesting that the effects of PGD2 are involved in either PGD2 dependent or independent pathway. PGD2 metabolite, 15-deoxy-Δ12 14-PGJ2, activates peroxisome proliferator-activated receptor γ (PPARγ) which induces growth inhibition of various cells. PPARγ is expressed in various types of cancers including gastric cancer. Since the role of PGD2 on gastric cancer cells is still unknown, this study is aimed to investigate the effect of PGD2 signalings on the proliferation of gastric cancer cells. Materials and Methods: Three human gastric cancer cell lines, OCUM-2M, OCUM-12, and MKN-74, were used. Effect of PGD2 or PPARγ antagonist on the proliferation of cancer cells was examined by MTT assay. The expression level of PGD2 receptor, DP1 and DP2, and PPARγ of gastric cancer cells was examined by RT-PCR. The effect of PGD2 on cell cycle was examined with PI staining by flowcytometry. Results: PGD2 significantly decreased the proliferation of OCUM-2M and MKN-74 cells at a dose-dependent manner, but not that of OCUM-12. PGD2 receptors, DP1 and DP2, were not expressed in 3 gastric cancer cell lines while PPARγ was expressed in OCUM-2M and MKN-74. BADGE, a PPARγ antagonist, significantly suppressed the growth-inhibitory effects of PGD2 on OCUM-2M and MKN-74 cells. PGD2 increased G0/G1 phase of cancer and decereased G2/M phase. Conclusion: PGD2 decreased the proliferation of gastric cancer cells through PPARγ dependent pathway. PGD2 might be a promising therapeutic agent for gastric cancer with PPARγ expression. Citation Format: Tatsunari Fukuoka, Masakazu Yashiro, Hiroshi Takeda, Takayuki Maruyama, Haruhito Kinoshita, Tamammi Morisaki, Tsuyoshi Hasegawa, Toshiki Hirakawa, Naoki Aomatsu, Katsunobu Sakurai, Kenjiro Kimura, Hisashi Nagahara, Takahiro Toyokawa, Ryosuke Amano, Eiji Noda, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Hiroshi Otani, Kiyoshi Maeda, Masaichi Ohira, Kosei Hirakawa. Antitumor effect of prostaglandin D2 by peroxisome proliferator-activated receptor gamma (PPARγ)-dependent pathway in gastric carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3995. doi:10.1158/1538-7445.AM2013-3995