Takashi Kamigaki

Suppression of human pancreatic carcinoma cell growth and invasion by epigallocatechin-3-gallate.

Introduction The consumption of green tea is associated with a lower risk of several types of human carcinomas. A number of studies have focused on the possible mechanisms of cancer prevention by tea extracts, especially polyphenols such as epigallocatechin-3-gallate (EGCG). Aims and Methodology Green tea–derived EGCG was tested in human pancreatic carcinoma cells. The cells (PANC-1, MIA PaCa-2, and BxPC-3) were treated with different doses of EGCG (0, 25, 50, 100, and 200 &mgr;mol/L) for 48 hours in culture medium. Proliferation of pancreatic carcinoma cells was measured by means of the WST-1 colorimetric assay. For the study of cell invasion, the cells were incubated with 100 &mgr;mol/L EGCG for 2 hours. Then, the cells were added into the cell insert, coated with Matrigel basement membrane matrix. After incubation at 37°C for 24 hours, the cells that had invaded through the Matrigel were counted visually under the microscope. Results The growth of all three pancreatic carcinoma cells was significantly suppressed by EGCG treatment in a dose-dependent manner. EGCG treatment caused significant suppression of the invasive ability of pancreatic carcinoma cells PANC-1, MIA PaCa-2, and BxPC-3 but did not affect the cell cycle protein cyclin D1. Conclusion EGCG may be a potent biologic inhibitor of human pancreatic carcinomas, reducing their proliferative and invasive activities.

Zoledronate-activated Vγ9γδ T cell-based immunotherapy is feasible and restores the impairment of γδ T cells in patients with solid tumors

Gamma/delta (γδ) T cells play a role in innate immunity and exhibit cytotoxicity toward a large range of tumor types. Recent studies have shown that aminobisphosphonates may be applied to a culture in which a large number of γδ T cells are proliferated ex vivo. We carried out a clinical study of 25 patients with various solid tumors to determine further the safety, immunologic effect and feasibility of zoledronate-activated Vγ9γδ T cell-based immunotherapy. No severe toxicity was observed. In the cells used for the first treatment, the total cell number, frequency and number of CD3(+) Vγ9(+) γδ T cells were 409 ± 284 × 10(7) cells, 56 ± 33% and 255 ± 242 × 10(7) cells, respectively. Aminobisphosphonate therapy or chemotherapy resulted in the suppression of CD3(+) Vγ9(+) γδ T-cell proliferation. The numbers of CD3(+) T cells, CD3(+) Vγ9(+) γδ T cells and CD27(-) CD45RA(-) Vγ9(+) subsets in peripheral blood were significantly lower in patients than in healthy subjects (P < 0.05). From such an impaired immunologic condition, the numbers and frequencies of CD3(+) Vγ9(+) γδ T cells and CD27(-) CD45RA(-) subsets significantly increased in patients treated with this immunotherapy. Zoledronate-activated Vγ9γδ T cell-based immunotherapy that restores the number of Vγ9γδ T cells in cancer patients may provide another mode of adoptive immunotherapy.

Randomized clinical trial of preoperative intranasal mupirocin to reduce surgical-site infection after digestive surgery.

Compromised patients subjected to major digestive surgery frequently develop infective complications caused by methicillin‐resistant Staphylococcus aureus (MRSA), which may have dangerous consequences. This was a prospective randomized study to determine whether intranasal mupirocin could reduce postoperative infective complications in patients having digestive surgery.

Rab27a Negatively Regulates Phagocytosis by Prolongation of the Actin-coating Stage around Phagosomes

Rab27a, a Rab family small GTPase, is involved in the exocytosis of secretory granules in melanocytes and cytotoxic T-cells. Rab27a mutations cause type 2 Griscelli syndrome, which is characterized by immunodeficiency, including uncontrolled macrophage activation known as hemophagocytic syndrome. However, the role of Rab27a in phagocytosis remains elusive. Here, using macrophage-like differentiated HL-60 cells and C3bi-opsonized zymosan as a pathogen-phagocyte model, we show that Rab27a negatively regulates complement-mediated phagocytic activity in association with F-actin remodeling. We found that transfection of Rab27a shRNA into HL-60 cells enhances complement-mediated phagocytosis. To clarify the mechanisms underlying the elevated phagocytosis in Rab27a knockdown cells, we analyzed the process of phagosome formation focusing on F-actin dynamics: F-actin assembly, followed by F-actin extension around the particles and the subsequent degradation of F-actin, leading to internalization of the particles enclosed in phagosomes. Microscopic analysis revealed that these actin-related processes, including F-actin coating and F-actin degradation, proceed more rapidly in Rab27a knockdown cells than in control HL-60 cells. Both elevated phagocytosis and accelerated F-actin remodeling were restored by expression of rescue-Rab27a and Rab27a-Q78L (GTP-bound form), but not by Rab27a-T23N (GDP-bound form). Furthermore, an increased accumulation of Coronin 1A surrounding F-actin coats was observed in Rab27a knockdown cells, suggesting that the function of Coronin 1A is related to the regulation of the F-actin coating. Our findings demonstrate that Rab27a plays a direct regulatory role in the nascent process of phagocytosis by prolongation of the stage of actin coating via suppression of Coronin 1A. This study may contribute to an explanation of the underlying mechanisms of excessive phagocytosis observed in Griscelli syndrome.

Family history of cancer in Japanese gastric cancer patients

The aim of this study was to evaluate the family history of cancer in Japanese gastric cancer patients and to investigate the clinicopathological features of gastric cancer patients with and without a family history of cancer. Four hundred and forty gastric cancer patients were enrolled in this study. The family history (first- and second-degree relatives) was investigated. The 440 patients were divided into three groups: (1) patients with a family history of gastric cancer; (2) patients with a family history of other cancers; and (3) patients without a family history of cancer. Two hundred and four patients (46.4%) reported a family history of cancer. Gastric cancer was the most frequent, with 98 patients having a total of 123 reports of gastric cancer in the family; colorectal cancer was the second most frequent and lung cancer was the third most frequent. The average ages of the group with a family history of gastric cancer and the group with a family history of other cancers were significantly lower than that of the patients without a family history of cancer. Other clinicopathological factors examined showed no significant difference between the groups. Japanese gastric cancer showed aggregation within second-degree relatives. The average age of the patients in the group with a family history of gastric cancer was the only significant factor that differed between gastric cancer patients with and without a family history of cancer.

Experimental evaluation of the mechanical strength of stapling techniques.

BackgroundThe single stapling technique (SST) and the double stapling technique (DST) are common anastomoses for rectal cancer. Although many mechanical devices have been developed, the best choice remains unclear. In this study we examined the strength of anastomoses by determining their bursting pressures using an animal model.MethodsThe intestines of pigs were used. In experiment 1, we compared the bursting pressures for Endo GIA™ 60 blue, Endo GIA™ 60 green, and GIA™ 60 blue. In experiment 2, the bursting pressures of a buttressed cutting site and a nonbuttressed cutting site were measured. In experiment 3, the SST, DST, and DST with buttress using PCEEA™ were performed and the bursting pressures and points of these anastomoses were examined.ResultsThe bursting pressure of Endo GIA 60 blue (80.3 ± 10.5 mmHg) was significantly higher than that of Endo GIA 60 green (37.3 ± 4.2 mmHg) and GIA 60 blue (31.7 ± 5.8 mmHg) (p < 0.01). When a cut end was buttressed, the bursting pressure (149.6 ± 37.6 mmHg) was significantly higher than that of the nonbuttressed end (75.3 ± 25.1 mmHg) (p < 0.01). The bursting pressure among SST, DST, and DST with buttress was not significantly different. Only one bursting point was the crossing point of the PCEEA and Endo GIA and the bursting pressure of this point was much lower than that of the others.ConclusionEndo GIA was most suitable for DST. The SST, DST, and DST with buttress had almost the same strength. The crossing point of PCEEA and Endo GIA may be a dangerous point for DST.

Chemosensitivity Assessed by Collagen Gel Droplet Embedded Culture Drug Sensitivity Test, and MDR1, MRP1, and MRP2 mRNA Expression in Human Colorectal Adenocarcinomas

AbstractPurpose. To evaluate chemosensitivity and its correlation with expression levels of the multidrug resistant transporter (MDR1) and the multidrug resistance-associated proteins 1 and 2 (MRP1, MRP2) mRNA in human colorectal adenocarcinomas. Methods. Colorectal adenocarcinomas were obtained as surgical samples from 25 patients. The chemosensitivity of 12 anticancer drugs was assessed by the collagen gel droplet embedded culture drug sensitivity test (CD-DST). The expression levels of MDR1, MRP1, and MRP2 mRNA in colorectal adenocarcinomas were also evaluated by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Results. The chemosensitivity was successfully evaluated for 16 of 25 patients, and the anticancer drugs were effective against the samples showing a relatively high growth rate. Gemcitabine hydrochloride was found to be more promising than those often prescribed for the treatment of colorectal adenocarcinoma. There was no correlation of the mRNA expression levels of MDR1 and MRP1 with the chemosensitivity of any anticancer drugs tested, but mitomycin C was found to be more effective for the colorectal adenocarcinoma with relatively high expression of MRP2 mRNA.

High Expression of Thymidylate Synthase Leads to Resistance to 5‐Fluorouracil in Biliary Tract Carcinoma in vitro

To evaluate the effect of chemotherapy of 5‐fluorouracil (5‐FU) in human biliary tract carcinoma, we studied 5‐FU sensitivity, thymidylate synthase (TS) content, and dihydropyrimidine dehydroge‐nase (DPD) activity in 4 human biliary tract carcinoma cell lines compared to 12 various digestive carcinoma cell lines of human organs in vitro. 5‐FU sensitivity in the cell lines was analyzed by MTT assay. TS content was analyzed by the [6‐3H]FdUMP binding assay method, and DPD activity was analyzed by thin‐layer chromatography (TLC). 5‐FU lC50 values of biliary tract carcinoma cell lines were significantly higher than those of the carcinoma cell lines of the other digestive organs: 97, 45, 119, and 194 tunes the concentration of the other digestive, pancreas, colon, and gastric carcinoma cell lines, respectively. TS content of biliary tract carcinoma cell lines was also significantly greater than that of the carcinoma cell lines of the other organs. No difference in DPD activity, however, was recognized between the carcinoma cell lines of each organ. TS content in the cell lines significantly correlated with 5‐FU sensitivity, but DPD activity did not. Therefore, in the present study, TS expression was concluded to influence the high resistance to 5‐FU of biliary tract carcinoma in comparison with the carcinomas of the other digestive organs.

Impaired and imbalanced cellular immunological status assessed in advanced cancer patients and restoration of the T cell immune status by adoptive T-cell immunotherapy.

Recent progress has been made in understanding the mechanisms of antitumor immune responses, which may further clarify the immune status of cancer patients. In this study, we performed a detailed evaluation of the immunological status of 47 patients with advanced solid cancer, who had received no immunosuppressive treatment, and compared the results with 32 healthy subjects. Flow-cytometry data for peripheral blood were obtained using 19 monoclonal antibodies against various cell surface and intracellular molecules. Absolute numbers of T cells, several T cell subsets, B cells, and NK cells were significantly decreased in patients compared with healthy subjects. The percentage of CD27(+)CD45RA(+) T cells was lower and that of CD27(-)CD45RA(-) T cells was higher in patients compared with controls. Regulatory and type 2 helper T cells were elevated in patients relative to healthy subjects. The percentage of perforin(+) NK cells was significantly lower in patients than in controls. These results suggest a dysfunctional anti-tumor immune response in cancer patients. Furthermore, peripheral blood from 26 of 47 cancer patients was analyzed after adoptive T cell immunotherapy (ATI). ATI increased the number of T cell subsets, but not B and NK cells. The number and percentage of regulatory T cells decreased significantly. These results suggest that ATI can restore impaired and imbalanced T cell immune status.

Antitumor Effect of Gemcitabine on Orthotopically Inoculated Human Gallbladder Cancer Cells in Nude Mice

BackgroundThe prognosis of gallbladder carcinoma is poor; therefore, investigating the efficacy of new chemotherapy agents is essential for the treatments for this tumor. Recently, several studies have reported clinical trials using gemcitabine as treatment for advanced gallbladder cancers. However, the antitumor effects of gemcitabine on gallbladder carcinoma have not been examined in in vitro and in vivo model systems.MethodsWe examined the cytotoxicity of gemcitabine in four biliary tract cancer cell lines using the WST-1 assay. In addition, we examined the effect of gemcitabine on gallbladder cancers resulting from orthotopic inoculation of NOZ gallbladder tumor cells into nude mice. One week after transplantation, the mice were randomized into two groups: In Group A, the mice were treated by an intra-peritoneal injection of 0.9% sodium chloride for three weeks after inoculation (control). In Group B, the mice were treated by an intra-peritoneal injection of gemcitabine (125 mg / kg) for three weeks. All mice were sacrificed one week after the end of treatment, and macroscopic and histological findings were evaluated. The expression levels of proliferating-cell nuclear antigen (PCNA) were examined to investigate cellular proliferation activity, and Tunnel assays were performed to determine apoptotic status. Survival duration of the mice after gemcitabine treatment was compared to that of untreated mice.ResultsThe gemcitabine sensitivity of the four biliary tract cancer cell lines was similar in a dose dependent manner. In the in vivo models, the Group A mice showed huge tumors of the gallbladder, with liver invasion and lymph node metastases. However, there were no abdominal tumors in the Group B mice, and microscopic gallbladder cancer could only be detected from histological findings. The mean percent of PCNA-positive tumor cells was significantly higher in tumors from mice in Group A (71.9%) compared to those of Group B (34.7%). The mean percent of Tunnel-positive tumor cells was significantly lower in mice from Group A (2.0%) than those from Group B (5.7%). Survival duration was prolonged significantly in the gemcitabine-treated mice relative to untreated mice.ConclusionsGemcitabine treatment may inhibit tumor progression and prolong survival in gallbladder cancer by inhibiting cell proliferation and inducing apoptosis.