Kazushige Kawai

Chloroquine potentiates the anti-cancer effect of 5-fluorouracil on colon cancer cells

BackgroundChloroquine (CQ), the worldwide used anti-malarial drug, has recently being focused as a potential anti-cancer agent as well as a chemosensitizer when used in combination with anti-cancer drugs. It has been shown to inhibit cell growth and/or to induce cell death in various types of cancer. 5-Fluorouracil (5-FU) is the chemotherapeutic agent of first choice in colorectal cancer, but in most cases, resistance to 5-FU develops through various mechanisms. Here, we focused on the combination of CQ as a mechanism to potentiate the inhibitory effect of 5-FU on human colon cancer cells.MethodsHT-29 cells were treated with CQ and/or 5-FU, and their proliferative ability, apoptosis and autophagy induction effects, and the affection of the cell cycle were evaluated. The proliferative ability of HT-29 was analyzed by the MTS assay. Apoptosis was quantified by flow-cytometry after double-staining of the cells with AnnexinV/PI. The cell cycle was evaluated by flow-cytometry after staining of cells with PI. Autophagy was quantified by flow-cytometry and Western blot analysis. Finally, to evaluate the fate of the cells treated with CQ and/or 5-FU, the colony formation assay was performed.Results5-FU inhibited the proliferative activity of HT-29 cells, which was mostly dependent on the arrest of the cells to the G0/G1-phase but also partially on apoptosis induction, and the effect was potentiated by CQ pre-treatment. The potentiation of the inhibitory effect of 5-FU by CQ was dependent on the increase of p21Cip1 and p27Kip1 and the decrease of CDK2. Since CQ is reported to inhibit autophagy, the catabolic process necessary for cell survival under conditions of cell starvation or stress, which is induced by cancer cells as a protective mechanism against chemotherapeutic agents, we also analyzed the induction of autophagy in HT-29. HT-29 induced autophagy in response to 5-FU, and CQ inhibited this induction, a possible mechanism of the potentiation of the anti-cancer effect of 5-FU.ConclusionOur findings suggest that the combination therapy with CQ should be a novel therapeutic modality to improve efficacy of 5-FU-based chemotherapy, possibly by inhibiting autophagy-dependent resistance to chemotherapy.

The diagnosis of submucosal tumors of the stomach by endoscopic ultrasonography

The clinical value of endoscopic ultrasonography (EUS) in the diagnosis of submucosal tumors (SMTs) of the stomach was examined. We used echo endoscopes with a 7.5 or 10.0 MHz radial-scan transducer made by Olympus Co. Ltd. EUS was carried out on 80 patients with SMTs of the stomach including 54 cases confirmed histologically (24 cases of leiomyoma, 3 of leiomyosarcoma, 12 of cysts, 7 of aberrant pancreas, 4 of lipoma, 2 of carcinoid, and 3 of other diseases). Fifty-nine patients with extraluminal compression were detected by endoscopy and/or x-ray examination. We examined the effectiveness of EUS based on our analysis of the gastrointestinal tract wall seen in the EUS image as a five-layered structure corresponding with that of the histological layers. As a result, SMTs and extragastric compression were easily distinguishable in the EUS images of the lesions. The size, location, and origin of the SMTs could be detected. From the location of the SMT in the five-layered structure seen in the EUS image we could predict its histological nature. Thus, EUS was a most valuable method not only in the diagnosis of intramural and extramural SMTs but also in the detection of extragastric compressive lesions and organs.

Epigallocatechin gallate, the main component of tea polyphenol, binds to CD4 and interferes with gp120 binding

BACKGROUND Epigallocatechin gallate (EGCG), the major component of tea polyphenol, has been reported to have various physiologic modulatory activities. Several reports also have shown that catechin has a protective effect against HIV infection, part of which is mediated by inhibiting virions to bind to the target cell surface. OBJECTIVE We investigated the effect of EGCG on the expression of CD4 molecules and on its ability to bind gp120, an envelope protein of HIV-1. METHODS Peripheral blood CD4+ T cells were incubated in the presence of EGCG, and the expression of CD4 was evaluated by means of flow cytometry. The effect of EGCG on the antibody binding to CD4 was investigated by using a sandwich ELISA, and the effect on the gp120 binding to CD4 was analyzed by means of flow cytometry. RESULTS EGCG efficiently inhibited binding of anti-CD4 antibody to its corresponding antigen. This effect was mediated by the direct binding of EGCG to the CD4 molecule, with consequent inhibition of antibody binding, as well as gp120 binding. CONCLUSION The present results suggest a potential preventive effect of EGCG on HIV-1 infection by modulating binding to CD4.

Vaccination with autologous endothelium inhibits angiogenesis and metastasis of colon cancer through autoimmunity

Overcoming immune tolerance of tumor angiogenesis should be useful for adjuvant therapy of cancer. We hypothesized that vaccination with autologous endothelium would induce an autoimmune response targeting tumor angiogenesis. To test this concept, we immunized BALB/c mice with a vaccine of glutaraldehyde‐fixed murine hepatic sinusoidal endothelial cells (HSEs) in a lung metastasis model of Colon‐26 cancer. Vaccination with autologous HSEs induced both preventive and therapeutic anti‐tumor immunity that significantly inhibited the development of metastases. ELISA revealed an immunoglobulin response involving IgM and IgG subclasses. These antibodies had a strong affinity for antigens of both murine and human endothelium, and lyzed endothelial cells in the CDC assay. Flow‐cytometry and chromium‐release cytotoxicity assay revealed a specific CTL response against endothelial cells, which were lyzed in an effector: target ratio‐dependent manner. Neither antibodies nor CTLs reacted with Colon26. The effect of autologous HSEs was more pronounced than that of xenogeneic human umbilical vein endothelial cells (HU‐VECs), which were tested in the same experimental setting. Our results suggest that vaccination with autologous endothelium can overcome peripheral tolerance of self‐angiogenic antigens and therefore should be useful for adjuvant immunotherapy of cancer. (Cancer Sci 2004; 95: 85–90)

Circulating lymphocyte number has a positive association with tumor response in neoadjuvant chemoradiotherapy for advanced rectal cancer

Although neoadjuvant chemoradiotherapy (CRT) is the standard treatment for advanced rectal cancer (RC), markers to predict the treatment response have not been fully established. In 73 patients with advanced RC who underwent CRT in a neoadjuvant setting, we retrospectively examined the associations between the clinical effects of CRT and blood cell counts before and after CRT. Clinical or pathological complete response (CR) was observed in 10 (14%) cases. The CR rate correlated significantly with the size and the circumferential extent of the tumor. Hemoglobin level, white blood cell (WBC) count and platelet count before CRT did not show a significant difference between CR and non-CR cases. Interestingly, however, lymphocyte ratio in WBC was significantly higher (p = 0.020), while neutrophil ratio tended to be lower (p = 0.099), in CR cases, which was shown to be an independent association by multivariate analysis. When all the blood data obtained in the entire treatment period were evaluated, circulating lymphocyte count was most markedly decreased in the CRT period and gradually recovered by the time of surgery, while the numbers of neutrophils and monocytes were comparatively stable. Moreover, the lymphocyte percentage in samples obtained from CR patients was maintained at a relatively higher level than that from non-CR patients. Since tumor shrinkage is known to be dependent not only on the characteristics of tumor cells but also on various host conditions, our data raise the possibility that a lymphocyte-mediated immune reaction may have a positive role in achieving complete eradication of tumor cells. Maintenance of circulating lymphocyte number may improve the response to CRT in rectal cancer.

Circulating lymphocyte is an important determinant of the effectiveness of preoperative radiotherapy in advanced rectal cancer

BackgroundAlthough preoperative radiotherapy (RT) is widely used as the initial treatment for locally advanced rectal cancer (RC) in the neoadjuvant setting, factors determining clinical response have not been adequately defined. In order to find other factors possibly related with radiosensitivity, we evaluated the relationships between circulating blood cell counts and RT effects.MethodsIn 179 cases with advanced RC, we retrospectively examined hemoglobin (Hb) levels and counts of white blood cells (WBC), platelets and WBC subsets before and after RT and investigated their associations with the complete response (CR) rate together with other clinicopathological factors.ResultsThe ratio of lymphocytes in WBC taken before RT was significantly greater in 15 CR cases as compared with those in non-CR cases. Patients with high lymphocyte percentages (25.7%) showed better outcome than the counterparts. Conversely, the ratio of neutrophiles was reduced in CR cases. The lymphocyte ratio showed an independent association with CR with multivariate analysis, and tended to be maintained at relatively high levels in CR cases.ConclusionsIn RC patients, peripheral blood lymphocytes have a significant impact on the CR rate in response to RT. Lymphocyte-mediated immune reactions are supposed to have positive roles on clinical response in radiotherapy for RC.

Targeting Id1 and Id3 inhibits peritoneal metastasis of gastric cancer

Inhibitor of DNA binding (Id) proteins are essential for cell differentiation, proliferation, migration, invasion and angiogenesis. Recently, they have been shown to correlate with less differentiated phenotypes, high malignant potential and poor clinical outcome in various kinds of tumors. In an attempt to develop new strategies for the treatment of peritoneal metastasis of gastric cancer, we prepared an Id1, 3 double‐knockdown gastric cancer cell line, MKN45, by RNA interference and investigated its effects on the development of metastatic nodules in the peritoneal cavity. Both cell proliferation and migration capabilities were decreased in Id1, 3 double‐knockdown cells, as was their ability to bind to laminin, which could be explained by the decreased expression of integrin α6. These are important steps in the metastatic process. In a mouse model, the number of peritoneal metastatic nodules formed by Id1, 3 double‐knockdown cells was reduced compared to mock‐transfected control cells, as was the size of individual tumors. In this study, we clearly demonstrated that Id1, 3 double‐knockdown significantly impaired the ability of gastric cancer cells to form peritoneal metastasis. Id should be considered an ideal target for the treatment and prevention of gastric cancer, and RNA interference is an attractive and promising strategy to achieve it. (Cancer Sci 2005; 96: 784–790)

Elevated Neutrophil to Lymphocyte Ratio Predicts Poor Prognosis in Advanced Colorectal Cancer Patients Receiving Oxaliplatin-Based Chemotherapy

Background: The aim of this study was to assess whether the neutrophil to lymphocyte ratio (NLR) and other laboratory markers may predict the prognosis of advanced colorectal cancer (CRC) patients receiving palliative chemotherapy. Methods: The study population included 50 patients with far advanced or recurrent unresectable CRC who received oxaliplatin-based combination chemotherapy as first-line treatment in our hospital between June 2005 and November 2010. Seven clinical variables and 7 laboratory indices before chemotherapy were evaluated retrospectively as the possible prognostic factors of overall and progression-free survival. Results: During the study period, 27 patients (54%) died of CRC. Elevated NLR (≥4.0) was observed in 15 patients (30%). By univariate analysis, elevated NLR, performance status and hypoalbuminemia were significantly associated with both poor overall and progression-free survivals. Multivariate analysis showed that elevated NLR (hazard ratio 4.39, 95% confidence interval 1.82–10.7; p = 0.0013) and thrombocytosis (hazard ratio 5.02, 95% confidence interval 1.69–13.4; p = 0.0066) were independently associated with overall survival. Conclusion: Elevated NLR is a powerful predictor of poor response to oxaliplatin-based chemotherapy in patients with unresectable CRC. The ratio is a simply accessible and inexpensive but useful biomarker in CRC patients receiving chemotherapy.

Expression of platelet-derived endothelial cell growth factor in inflammatory bowel disease

Background: Platelet-derived endothelial cell growth factor (PD-ECGF) is reported to be highly expressed in tumors and inflammatory tissues, but its expression and role in inflammatory bowel disease (IBD) are still unclear. In this study we examined the location and tissue density of cells immunoreactive for PD-ECGF in the colonic mucosa of IBD. Methods: Paraffin-embedded sections of colonic tissue from patients with ulcerative colitis (UC) or Crohns disease (CD) were immunostained for PD-ECGF. As controls, noninflamed mucosa of IBD, as well as normal colonic mucosa from patients with colorectal cancer, were used. Also, cancer tissues were evaluated. In addition, changes in the expression of PD-ECGF in human umbilical vein endothelial cells (HUVEC) after treatment with inflammatory cytokines and angiogenic factors, as well as after coculture with colon cancer cell lines, were evaluated by flow cytometry. Results: In normal colonic mucosa and noninflamed mucosa of IBD, PD-ECGF expression was negligible. In inflamed colonic mucosa, strong expression was observed, predominantly in macrophages and fibroblasts. Vascular endothelial cells of the inflamed colonic mucosa, but not of normal colonic mucosa or of neoplastic tissues, stained for PD-ECGF, and the microvessel density was significantly increased in the severely inflamed mucosa. Flow cytometry demonstrated that PD-ECGF was constitutively expressed in HUVEC. Inflammatory cytokines and vascular endothelial growth factor (VEGF) increased its expression, whereas basic fibroblast growth factor (bFGF) decreased it. Coculture with colon cancer cell lines in direct contact, but not in those without contact, also resulted in an important decrease in the expression of PD-ECGF in HUVEC. Conclusions: Autocrine production of PD-ECGF by endothelial cells may be a mechanism of inflammatory angiogenesis, but not tumor angiogenesis, and may be particularly important for the maintenance of damaged vasculature in IBD.

Resistance of colon cancer to 5-fluorouracil may be overcome by combination with chloroquine, an in vivo study.

Autophagy is a complex of adaptive cellular response that enhances cancer cell survival in the face of cellular stresses such as chemotherapy. Recently, chloroquine diphosphate (CQ), a widely used antimalarial drug, has been studied as a potential inhibitor of autophagy. Here, we aimed to investigate the role of CQ in potentiating the effect of 5-fluorouracil (5-FU), the chemotherapeutic agent of first choice for the treatment of colorectal cancer, in an animal model of colon cancer. The mouse colon cancer cell line colon26 was used. For the in-vivo study, colon26 cells were injected subcutaneously into BALB/c mice, which were treated with saline as a control, CQ (50 mg/kg/day), 5-FU (30 mg/kg/day), or the combination therapy (CQ plus 5-FU). The tumor volume ratio and body weight were monitored. After the sacrifice, tumor tissue protein extracts and tumor sections were prepared and subjected to immunoblotting for the analysis of autophagy-related and apoptosis-related proteins, and the terminal transferase uridyl end labeling assay. The combination of CQ resulted in the inhibition of 5-FU-induced autophagy and a significant enhancement in the 5-FU-induced inhibition of tumor growth. Furthermore, the combination treatment of CQ and 5-FU resulted in a significant increase in the ratio of apoptotic cells compared with other treatments. The expression levels of the proapoptotic proteins, namely Bad and Bax, were increased by the CQ treatment in the protein extracts from tumors. Our findings suggest that the combination therapy of CQ and 5-FU should be considered as an effective strategy for the treatment of colorectal cancer.