Kazuhito Sasaki

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.

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.

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.

Id1/Id3 Knockdown Inhibits Metastatic Potential of Pancreatic Cancer

BACKGROUND The Id (inhibitor of DNA binding/differentiation) proteins belong to the helix-loop-helix transcriptional regulatory factors, and play important roles in tumor development. Previously, we and others have shown that targeting Id in tumor cells could have important clinical implications. In the present study, we aimed to evaluate the effects of Id inhibition in human pancreatic cancer cells. MATERIALS AND METHODS Id1 and Id3 were stably double-knockdown in human pancreatic cancer cell line MIA-Paca2 by means of RNA interference. Expression of Id and integrins were analyzed by flow-cytometry. Cell proliferation was evaluated by MTS assay. Migration was measured by wound closure assay. Adhesion assay was performed to evaluate binding capacity for different extracellular matrix proteins. Finally, in vivo properties of tumor cells were observed in a mouse model of peritoneal metastasis. RESULTS Id1/Id3 double-knockdown resulted in decreased ability of pancreatic cancer cells to proliferate and migrate. In addition, Id1/Id3 double-knockdown caused decreased expression of integrins alpha3, alpha6, and beta1, and consequently reduced adhesion of tumor cells to laminin. Finally, peritoneal metastases of Id1/Id3 double-knockdown tumor cells were significantly reduced. CONCLUSIONS We concluded that the Id proteins play a pivotal role in the development of peritoneal metastasis of pancreatic cancer, and consequently, their targeting would be a novel strategy for the prevention and treatment of pancreatic cancer.

Hypoxia enhances colon cancer migration and invasion through promotion of epithelial-mesenchymal transition.

BACKGROUND A hypoxic environment exists in most solid tumors because in rapidly growing tumors, the development of angiogenic vasculature is heterogenous, usually not enough to overcome the necessary oxygen supply. In an ischemic condition, cancer cells develop escape mechanisms to survive and leave the unfavorable environment. That result in the acquisition of increased potential for local invasion and evasion to distant organs. However, the escape mechanisms of cancer cells from hypoxic stress have not been fully characterized. MATERIALS AND METHODS The human colon cancer cell line LoVo was cultured in hypoxia, and the adhesive and migratory properties were analyzed. The expression of cell surface and cytoplasmic molecules was also investigated. RESULTS Under hypoxic conditions, cells developed epithelial-mesenchymal transition. The expression levels of α2, α5, and β1 integrins were significantly upregulated and, as a consequence, the ability to adhere to and migrate on collagen and fibronectin was increased. On the other hand, the expression of 67-kDa laminin receptor and the abilities to adhere to and migrate on laminin were decreased. Additionally, the expression of CXCR4 was significantly increased on cells cultured in hypoxia, and the chemotactic activity to stromal cell-derived factor 1α was remarkably increased. CONCLUSIONS Hypoxic stress induced active epithelial-mesenchymal transition in colon cancer cells, with the typical morphologic and functional changes. These morphologic and functional changes of β1 integrins, the 67-kDa laminin receptor, and CXCR4 may be essential for the acquisition of the invasive and metastatic features in colorectal cancer.

One-stage segmental colectomy and primary anastomosis after intraoperative colonic irrigation and total colonoscopy for patients with obstruction due to left-sided colorectal cancer.

BACKGROUND: Intraoperative colonic irrigation and intraoperative on-table colonoscopy may be useful for a more accurate diagnosis of colorectal cancer before colectomy in patients with obstructive left-sided colorectal cancer, but the clinical benefit of this technique has not been investigated in large-scale studies. OBJECTIVE: The aim of this study was to evaluate the usefulness of intraoperative colonic irrigation with a Y-shaped irrigation device and intraoperative colonoscopy in the management of obstructive colorectal cancer in patients undergoing elective surgery. DESIGN AND SETTING: This was a retrospective cohort study of patients undergoing surgical treatment at a single tertiary care institution in Japan. PATIENTS AND INTERVENTION: Among 715 consecutive patients with left-sided colorectal cancer, 101 patients (14.1%) with obstructing tumor received intraoperative colonic irrigation and intraoperative colonoscopy before colectomy and primary anastomosis, and 614 patients with nonobstructive colorectal cancer underwent preoperative colonoscopy with mechanical bowel preparation. MAIN OUTCOME MEASURES: Detection rates of proximal synchronous lesions, occurrence of postoperative complications, and changes in the surgical procedure prompted by the results of the intraoperative colonoscopy were evaluated. RESULTS: Intraoperative colonoscopy detected synchronous adenomatous polyps in 27 patients (26.8%), carcinoma in 4 patients (4%), and obstructive colitis in 2 patients (2%). Findings of the intraoperative colonoscopy prompted changes in surgical procedure in 9 patients (8.9%). The overall morbidity in the intraoperative group was 17%, with anastomotic leakages in 3 patients, wound infection in 5, and postoperative ileus in 3 patients. The risk of these complications was not increased in patients with intraoperative colonoscopy with intraoperative colonic irrigation compared with those receiving preoperative colonoscopy with mechanical bowel preparation. The operation time was 28 minutes longer in the intraoperative than in the preoperative group, but neither the time to start of oral intake nor the length of postoperative hospital stay was significantly different between the 2 groups. LIMITATIONS: The study is limited by its retrospective nature. CONCLUSIONS: In patients with obstructive colorectal cancer, intraoperative colonic irrigation with intraoperative colonoscopy is a useful strategy for detecting synchronous lesions located proximally to the obstructing tumor, without increasing patient morbidity.

Antiangiogenic Effect of a Selective 5-HT4 Receptor Agonist

BACKGROUND Serotonin (5-hydroxytryptamine, 5-HT) is reported to regulate cell growth in a wide variety of cell types in different carcinomas. 5-HT exerts complex actions on blood vessels, dependent on its interactions with a multiplicity of 5-HT receptors. In the present study, we aimed to investigate the potential antiangiogenic effect of mosapride citrate, a selective 5-HT4 receptor agonist, known to have prokinetic properties on the gastrointestinal tract. For this purpose, cultured human umbilical vein endothelial cells (HUVECs) were used as an in vitro model. MATERIAL AND METHODS The effect of mosapride citrate on the proliferative activity of HUVECs was assessed by the MTS assay. Then, the apoptosis and the cell cycle detection assays were performed. The effect of mosapride citrate on the ability of HUVECs to adhere and migrate on extracellular matrix proteins (ECMs), as well as their ability to form vascular-like structures on Matrigel was investigated. RESULTS Mosapride citrate inhibited the proliferative activity of HUVECs, dependent on cell cycle arrest, and not on apoptosis. A dose-dependent increase in the percentage of cells in the G0/G1 phase of the cell cycle in mosapride-treated HUVECs was observed. Mosapride citrate also significantly inhibited the ability of HUVECs to migrate, but not to adhere on ECMs. Additionally, mosapride citrate dose-dependently inhibited the tube-like formation ability of HUVECs on matrigel, an important event in the process of angiogenesis. CONCLUSION The present results demonstrate the antiangiogenic activity of mosapride citrate in vitro and the possibility of its application as a new anti-cancer agent is suggested.

Retrocecal hernia successfully treated with laparoscopic surgery: A case report and literature review of 15 cases in Japan

Highlights • We report an interesting case of a retrocecal hernia.• On the 12th hospital day, laparoscopic surgery was performed.• Using laparoscopic exploration and suturing, we were able to perform a minimally invasive operation.

Small colorectal cancers resembling submucosal tumor with massive submucosal invasion and lymph node metastasis: A report of two cases and review of the literature

Colorectal cancer resembling submucosal tumor (SMT) is very rare. We herein report two cases of small colon carcinoma resembling SMT (80-year-old female and 67-year-old male), which massively invaded into the submucosal layer and accompanied marked lymphatic invasion and lymph node metastasis. We also reviewed the reported cases of colorectal carcinoma resembling SMT (SMT-like group, n=70) and analyzed the clinicopathological characteristics of this group compared with typical colorectal carcinoma cases operated at our institution (control group, n=1723). Tumors in the SMT-like group were significantly smaller in size compared with the control group; the median diameter measured 22mm vs. 37mm (P<0.01), respectively. Histologically, although the tumors in the SMT-like group were small in diameter, they almost all invaded into the submucosal (T1) or deeper layer (T2-4), and the rate of poorly differentiated adenocarcinoma or mucinous adenocarcinoma was significantly higher than that in the control group (48.6% vs. 7.7%; P<0.01). In the subgroup analysis of T1 tumors, the rate of lymphatic invasion in the SMT-like group was also significantly higher than that in the control group (43.8% vs. 15.4%; P<0.01). Carcinoma resembling SMT appears to be invasive and has a high risk of lymphatic invasion even if small in size. Therefore, surgical treatment with dissection of the regional lymph nodes might be necessary in cases with any signs of massive submucosal invasion.

A high preoperative carbohydrate antigen 19-9 level is a risk factor for recurrence in stage II colorectal cancer

Abstract Background: Many risk factors for recurrence in stage II colorectal cancer (CRC) have been proposed, and the efficacy of adjuvant chemotherapy is still controversial. This study aimed to identify risk factors for tumor recurrence and assess whether they are related to the benefits of adjuvant chemotherapy in stage II CRC. Material and methods: Patients with stage II CRC that was curatively operated on in a tertiary hospital between 2005 and 2014 were analyzed. Cox’s proportional hazards models were applied to identify risk factors for recurrence and overall mortality. Kaplan-Meier methods were used to evaluate whether adjuvant chemotherapy was beneficial in terms of recurrence-free survival (RFS). Results: A total of 384 patients were identified, among whom 38 (10%) received adjuvant chemotherapy. In a median follow-up of 48.6 months, 52 patients (14%) developed recurrence. Multivariate analyses identified two independent parameters that significantly decreased RFS; pathological T4 [hazard ratio (HR), 2.34; 95% confidence interval (CI), 1.31–4.15; p = .0045) and preoperative carbohydrate antigen (CA) 19-9 > 37 U/ml (HR 1.96; 95% CI 1.02–3.58; p = .045). These factors also inversely correlated with overall survival; T4: HR 2.10, p = .019) and CA 19-9 > 37 U/ml (HR 2.15, p = .025). The combination of T4 and CA 19-9 > 37 U/ml resulted in an increased HR (3.52) for recurrence. However, adjuvant chemotherapy did not improve RFS in patients with these features. Conclusion: The present study demonstrated elevated CA 19-9 levels as well as T4 independently predicted worse long-term outcomes in patients with stage II CRC. However, the characterization of patients who gain survival advantages by adjuvant chemotherapy requires further investigation.