Katsunobu Oyama

Angiotensin II enhances epithelial-to-mesenchymal transition through the interaction between activated hepatic stellate cells and the stromal cell-derived factor-1/CXCR4 axis in intrahepatic cholangiocarcinoma

We previously reported that hepatic stellate cells (HSCs) activated by angiotensin II (AngII) facilitate stromal fibrosis and tumor progression in intrahepatic cholangiocarcinoma (ICC). AngII has been known as a growth factor which can promote epithelial-to-mesenchymal transition (EMT) in renal epithelial cells, alveolar epithelial cells and peritoneal mesothelial cells. However, in the past, the relationship between AngII and stromal cell-derived factor-1 (SDF-1) in the microenvironment around cancer and the role of AngII on EMT of cancer cells has not been reported in detail. SDF-1 and its specific receptor, CXCR4, are now receiving attention as a mechanism of cell progression and metastasis. In this study, we examined whether activated HSCs promote tumor fibrogenesis, tumor progression and distant metastasis by mediating EMT via the AngII/AngII type 1 receptor (AT-1) and the SDF-1/CXCR4 axis. Two human ICC cell lines and a human HSC line, LI-90, express CXCR4. Significantly higher concentration of SDF-1α was released into the supernatant of LI-90 cells to which AngII had been added. SDF-1α increased the proliferative activity of HSCs and enhanced the activation of HSCs as a growth factor. Furthermore, addition of SDF-1α and AngII enhanced the increase of the migratory capability and vimentin expression, reduced E-cadherin expression, and translocated the expression of β-catenin into the nucleus and cytoplasm in ICC cells. Co-culture with HSCs also enhanced the migratory capability of ICC cells. These findings suggest that SDF-1α, released from activated HSCs and AngII, play important roles in cancer progression, tumor fibrogenesis, and migration in autocrine and paracrine fashion by mediating EMT. Our mechanistic findings may provide pivotal insights into the molecular mechanism of the AngII and SDF-1α-initiated signaling pathway that regulates fibrogenesis in cancerous stroma, tumor progression and meta-stasis of tumor cells expressing AT-1 and CXCR4.

The role of human peritoneal mesothelial cells in the fibrosis and progression of gastric cancer

Peritoneal dissemination is the most frequent metastatic pattern of scirrhous gastric cancer. However, despite extensive research effort, disease outcomes have not improved sufficiently. Tumor progression and metastasis result from interactions between cancer and various cells in the stroma, including endothelial cells, immune cells and fibroblasts. Fibroblasts have been particularly well studied; they are known to change into carcinoma-associated fibroblasts (CAFs) and produce transforming growth factor β (TGF-β), which mediates cancer-stroma interactions. Here, we investigated whether TGF-β derived from cancer cells in the peritoneal microenvironment activates human peritoneal mesothelial cells (HPMCs), leading to the progression and fibrosis of gastric cancer. We found that activated HPMCs (a-HPMCs) took on a spindle shape formation, decreased the expression of E-cadherin and increased that of α-SMA. Furthermore, a-HPMCs became more invasive and upregulated proliferation of human gastric cancer-derived MKN45 cells following direct cell-cell contact. Notably, MKN45 cells co-cultured with a-HPMCs also acquired anchorage-independent cell growth and decreased expression of E-cadherin in vitro. To measure the effects of the co-culture in vivo, we developed a mouse xenograft model into which different culture products were subcutaneously injected. The largest tumors were observed in mice that had been given MKN45 cells co-cultured with a-HPMCs. Furthermore, these tumors contained HPMC-derived fibrous tissue. Thus, the epithelial-mesenchymal transition (EMT) of HPMCs appears to drive peritoneal dissemination and tumor fibrosis.

The Angiotensin II type 1 receptor blocker candesartan suppresses proliferation and fibrosis in gastric cancer

Gastric cancer with peritoneal dissemination has poor clinical prognosis because of the presence of rich stromal fibrosis and acquired drug resistance. Recently, Angiotensin II type I receptor blockers such as candesartan have attracted attention for their potential anti-fibrotic activity. We examined whether candesartan could attenuate tumor proliferation and fibrosis through the interaction between gastric cancer cell line (MKN45) cells and human peritoneal mesothelial cells. Candesartan significantly reduced TGF-β1 expression and epithelial-to-mesenchymal transition-like change, while tumor proliferation and stromal fibrosis were impaired. Targeting the Angiotensin II signaling pathway may therefore be an efficient strategy for treatment of tumor proliferation and fibrosis.

Feasibility and efficacy of preoperative chemotherapy with docetaxel, cisplatin and S-1 in gastric cancer patients with para-aortic lymph node metastases.

We performed preoperative chemotherapy with combined docetaxel, cisplatin and S-1 (DCS therapy) for treatment of advanced gastric cancer with para-aortic lymph node metastases. The aim of this study was to determine the maximum tolerated dose (MTD) and the dose-limiting toxicities. Furthermore, we evaluated the feasibility of DCS therapy in a preoperative setting, and also examined the pathological response. Fifteen patients received intravenous docetaxel and cisplatin (30, 35 or 40 mg/m2, each dose escalation was reciprocal) on days 1 and 15 and oral S-1 (40 mg/m2 twice daily) on days 1–14 every 4 weeks. After one cycle of chemotherapy, toxicities were evaluated and after two cycles of chemotherapy, patients who were judged to be candidates for curative resection underwent gastrectomy with D2 lymphadenectomy plus para-aortic lymph node dissection. The MTD of this combination was presumed to be at dose level 3 (docetaxel 40 mg/m2 and cisplatin 35 mg/m2). The dose-limiting toxicities were grade 4 neutropenia in one patient, grade 3 febrile neutropenia in two patients and grade 3 diarrhoea in two patients. Thirteen of the 15 patients received complete resection and there was no operation-related death. Good pathological responses were observed in 12 cases with lesions in the lymph nodes (complete response, n = 4; partial response, n = 8) and 11 patients with primary stomach lesions (complete response, n = 2; partial response, n = 9). This preoperative DCS therapy was considered feasible and provided a high pathological response rate in gastric cancer patients with para-aortic lymph node metastases.

Valproic acid, a histone deacetylase inhibitor, enhances radiosensitivity in esophageal squamous cell carcinoma

Histone deacetylase (HDAC) inhibitors have been shown to enhance radiation response in various cancer cell lines. Valproic acid (VPA) has been used in clinical practice for the treatment of epilepsy and other seizure disorders and is also one of the most represented HDAC inhibitors. The aim of this study was to evaluate the radiosensitizing ability of VPA and its mechanisms in four esophageal squamous cell carcinoma (ESCC) cell lines (TE9, TE10, TE11 and TE14). VPA inhibited the viability of all ESCC cells in a dose-dependent manner. The 50% inhibitory concentration (IC50) value of VPA in each cell line was between 1.02-2.15 mM, which is higher than clinically used safe concentrations. VPA induced the hyperacetylation of histones H3 and H4, as well as apoptosis and had a radiosensitizing effect on all four ESCC cell lines at a concentration of 0.5 mM which is equivalent to the therapeutic plasma concentration of anti-epilepsy therapy in humans. The radiosensitization was accompanied by an increase in γH2AX levels, indicating the presence of double-strand breaks (DSBs), and decrease in Rad51 expression, a DSB repair protein. These results suggest that a clinically safe dose of VPA can enhance radiation-induced cytotoxicity in human ESCC cells by chromatin decondensation with histone hyperacetylation and downregulation of Rad51. In conclusion, VPA appears to be a safe and promising radiosensitizer for esophageal cancer radiotherapy.

Antitumor and anti-metastatic effects of cyclooxygenase-2 inhibition by celecoxib on human colorectal carcinoma xenografts in nude mouse rectum.

We examined the effects of the preferential cyclooxygenase-2 (COX-2) inhibitor celecoxib on tumorigenesis, angiogenesis, apoptosis, vascular endothelial growth factor (VEGF) protein expression and metastasis in HT-29 human colorectal carcinoma cell xenografts in nude mouse rectum. COX-2 mRNA expression was examined in the xenograft and metastatic sites. The antitumor effect of celecoxib in the xenografts was evaluated by measuring the weight of the peri-ano-rectal tumor. The anti-metastatic effect of celecoxib was assessed by quantification of lymph node and lung metastases by amplification of a cancer-related human DNA by TaqMan PCR. The effects of celecoxib on angiogenesis, apoptosis, prostaglandin E2 (PGE2) production and VEGF protein expression in the xenografts were evaluated by means of microvessel density (MVD) counting, terminal deoxynucleotidyl transferase-mediated nick end labeling assay, quantitative enzyme-linked immunosorbent assay and western blotting, respectively. The rectal xenograft model showed lymph node and lung metastases with enhanced expression of COX-2 mRNA in each organ. Celecoxib inhibited rectal xenograft growth in a dose-dependent manner as follows: 150 ppm, 33.0% (p=0.000220); 750 ppm, 46.4% (p=0.0000292); 1500 ppm, 63.4% (p=0.0000109). Celecoxib inhibited lymph node metastasis in a dose-dependent manner as follows: 150 ppm, 86.7% (p=0.0263); 750 ppm, 90.3% (p=0.00638); 1500 ppm, 96.0% (p=0.000894). Celecoxib also inhibited lung metastasis as follows: 750 ppm, 53.3% (p=0.0107); 1500 ppm, 78.3% (p=0.00022). Celecoxib (1500 ppm) significantly inhibited PGE2 production by 68.4% (p=0.000157) and MVD counting by 48.2% (p=1.3×10−12) and induced apoptosis 2.5-fold (p=3.0×10−14) in the rectal xenograft. Celecoxib suppressed VEGF protein expression in the rectal xenograft. These studies demonstrate that celecoxib reduces the growth and metastatic potential of colorectal carcinoma in mice through COX-2 inhibition, anti-angiogenesis and apoptosis induction. The studies using HT-29 human colorectal carcinoma cell xenografts in nude mouse rectum also provide important information that supports that the COX-2 inhibitor celecoxib has a high potential for use as a clinical agent for inhibition of hematological and lymphatic metastases of colorectal cancer.

Effects of valproic acid on the cell cycle and apoptosis through acetylation of histone and tubulin in a scirrhous gastric cancer cell line

BackgroundManagement of peritoneal dissemination is the most critical problem in gastric cancer. This study was performed to investigate the inhibitory effects of valproic acid (VPA) on a highly peritoneal-seeding cell line of human scirrhous gastric cancer, OCUM-2MD3, and to explore the mechanism and the potential of VPA.MethodsThe effects of VPA on the growth of OCUM-2MD3 cells were assessed by MTT assay. In addition, paclitaxel (PTX) was combined with VPA to evaluate their synergistic effects. HDAC1 and HDAC2 expression were evaluated by western blotting in OCUM-2MD3 cells and other gastric cancer cell lines (TMK-1, MKN-28). The acetylation status of histone H3 and α-tubulin after exposure to VPA were analyzed by western blotting. The activities of cell cycle regulatory proteins and apoptosis-modulating proteins were also examined by western blotting. The effects of VPA in vivo were evaluated in a xenograft model, and apoptotic activity was assessed by TUNEL assay.ResultsOCUM-2MD3 cells showed high levels of HDAC1 and HDAC2 expression compared with TMK-1 and MKN-28. The concentration of VPA required for significant inhibition of cell viability (P < 0.05) was 5 mM at 24 h and 0.5 mM at 48 h and 72 h. The inhibition of VPA with PTX showed dose-dependent and combinatorial effects. VPA increased acetyl-histone H3, acetyl-α-tubulin, and p21WAF1 levels accompanied by upregulation of p27, caspase 3, and caspase 9, and downregulation of bcl-2, cyclin D1, and survivin. In the xenograft model experiment, the mean tumor volume of the VPA-treated group was significantly reduced by 36.4%, compared with that of the control group at 4 weeks after treatment (P < 0.01). The apoptotic index was significantly higher in the VPA-treated group (42.3% ± 3.5%) than in the control group (7.7% ± 2.5%) (P < 0.001).ConclusionsVPA induced dynamic modulation of histone H3 and α-tubulin acetylation in relation with the anticancer effect and the enhancement of PTX in the OCUM-2MD3 cell line. Therefore, VPA in combination with PTX is expected to be a promising therapy for peritoneal dissemination of scirrhous gastric cancer.

Prospective randomized controlled study on the effects of perioperative administration of a neutrophil elastase inhibitor to patients undergoing video-assisted thoracoscopic surgery for thoracic esophageal cancer.

Sivelestat sodium hydrate (Ono Pharmaceutical Co., Osaka, Japan) is a selective inhibitor of neutrophil elastase (NE) and is effective in reducing acute lung injury associated with systemic inflammatory response syndrome (SIRS). We conducted a prospective randomized controlled study to investigate the efficacy of perioperative administration of sivelestat sodium hydrate to prevent postoperative acute lung injury in patients undergoing thoracoscopic esophagectomy and radical lymphadenectomy. Twenty-two patients with thoracic esophageal cancer underwent video-assisted thoracoscopic esophagectomy with extended lymph node dissection in our institution between April 2007 and November 2008. Using a double-blinded method, these patients were randomly assigned to one of two groups preoperatively. The active treatment group received sivelestat sodium hydrate intravenously for 72 hours starting at the beginning of surgery (sivelestat-treated group; n= 11), while the other group received saline (control group; n= 11). All patients were given methylprednisolone immediately before surgery. Postoperative clinical course was compared between the two groups. Two patients (one in each group) were discontinued from the study during the postoperative period because of surgery-related complications. Of the remaining 20 patients, 2 patients who developed pneumonia within a week after surgery were excluded from some laboratory analyses, so data from 18 patients (9 patients in each group) were analyzed based on the arterial oxygen pressure/fraction of inspired oxygen ratio, white blood cell count, serum C-reactive protein level, plasma cytokine levels, plasma NE level, and markers of alveolar type II epithelial cells. In the current study, the incidence of postoperative morbidity did not differ between the two groups. The median duration of SIRS in the sivelestat-treated group was significantly shorter than that in the control group: 17 (range 9-36) hours versus 49 (15-60) hours, respectively (P= 0.009). Concerning the parameters used for the diagnosis of SIRS, the median heart rates on postoperative day (POD) 2 were significantly lower in the sivelestat-treated group than in the control group (P= 0.007). The median arterial oxygen pressure/fraction of inspired oxygen ratio of the sivelestat-treated group were significantly higher than those of the control group on POD 1 and POD 7 (POD 1: 372.0 [range 284.0-475.0] vs 322.5 [243.5-380.0], respectively, P= 0.040; POD 7: 377.2 [339.5-430.0] vs 357.6 [240.0-392.8], P= 0.031). Postoperative white blood cell counts, serum C-reactive protein levels, plasma interleukin-1beta, tumor necrosis factor-alpha levels, and plasma NE levels did not differ significantly between the two groups at any point during the postoperative course, nor did serum Krebs von den Lungen 6, surfactant protein-A, or surfactant protein-D levels, which were used as markers of alveolar type II epithelial cells to evaluate the severity of lung injury. Plasma interleukin-8 levels were significantly lower in the sivelestat-treated group than in the control group on POD 3 (P= 0.040). In conclusion, perioperative administration of sivelestat sodium hydrate (starting at the beginning of surgery) mitigated postoperative hypoxia, partially suppressed postoperative hypercytokinemia, shortened the duration of SIRS, and stabilized postoperative circulatory status after thoracoscopic esophagectomy.

Efficacy of pre-operative chemotherapy with docetaxel, cisplatin, and S-1 (DCS therapy) and curative resection for gastric cancer with pathologically positive para-aortic lymph nodes

The prognosis of gastric cancer with para‐aortic lymph node (PAN) metastasis is poor. We applied triple combination chemotherapy with docetaxel, cisplatin, and S‐1 (DCS therapy) as pre‐operative chemotherapy and investigated the outcome of the combination of this therapy and gastrectomy with para‐aortic lymph node dissection (PAND).

Cyclooxygenase-2 (COX-2) in Carcinogenesis and Selective COX-2 Inhibitors for Chemoprevention in Gastrointestinal Cancers

IntroductionNonsteroidal anti-inflammatory drugs (NSAIDs) have been reported to have a property to inhibit tumor development in some cancers while it shows various side effects such as gastrointestinal bleeding and renal disorder. Selective cyclooxygenase (COX)-2 inhibitors (coxibs) were originally developed as one of anti-inflammatory drugs to avoid side effect of NSAIDs. Fortunately, the coxibs was also proved to have an inhibiting effect on tumorigenesis by many experimental studies using cell lines and animal models like NSAIDs.DiscussionSince a randomized study for polyp chemoprevention by celecoxib in familial adenomatous polyposis (FAP) patients demonstrated a significant reduction in the number of colorectal polyps, the clinical use of celecoxib was approved for FAP patients. Three large trials using celecoxib (the Adenoma Prevention with Celebrex and the Prevention of Spontaneous Adenomatopus Polyps) or refecoxib (the Adenomatous Polyp Prevention on Vioxx) for the recurrence of colorectal polyps in patients with a history of colorectal adenoma polypectomized confirmed chemopreventive effects on colorectal adenoma but two of three trails alerted us a hazard of cardiovascular (CV) events. Thereafter, some coxibs were withdrawn from the market because they showed to increase risk of serious CV events including heart attacks and strokes. But recent reports concluded that a merit of the reduction in gastrointestinal events by coxibs exceeded a demerit of the increase in serious CV events. In this review, a role of COX-2 in carcinogenesis of gastrointestinal tract and a future of coxibs for chemoprevention are discussed