Kumiko Hongo

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.

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.

Immunohistochemical detection of high-mobility group box 1 correlates with resistance of preoperative chemoradiotherapy for lower rectal cancer: a retrospective study.

BackgroundHigh-mobility group box 1 (HMGB1) is a nucleoprotein that is related to inflammation. It has been implicated in a variety of biologically important processes, including transcription, DNA repair, differentiation, development, and extracellular signaling. Recently, its important role in the process of tumor invasion, metastasis, and resistance to anti-cancer therapies has been demonstrated. In this study, we aimed to investigate the correlation of HMGB1 expression and resistance of rectal cancer patients to chemoradiotherapy (CRT) prior to curative operation.MethodsWe retrospectively reviewed the data of 75 lower rectal cancer patients without complete pathological response who had received preoperative CRT and had undergone curative resection at the University of Tokyo Hospital between May 2003 and June 2010. HMGB1 expression in surgically resected specimens was evaluated using immunohistochemical detection and specimens were classified into high or low HMGB1 expression groups. Clinicopathologic features, degree of tumor reduction, regression of tumor grade, and patient survival were compared between the groups using non-paired Student’s t-tests and Kaplan-Meier analysis.ResultsA total of 52 (69.3%) patients had high HMGB1 expression, and 23 (30.7%) had low expression. HMGB1 expression was significantly correlated with histologic type (P = 0.02), lymphatic invasion (P = 0.02), and venous invasion (P = 0.05). Compared to patients with low HMGB1 expression, those with high expression had a poorer response to CRT, in terms of tumor reduction ratio (42.2 versus 28.9%, respectively; P <0.01) and post-CRT histological tumor regression grade (56.5 versus 30.8% grade 2; respectively; P = 0.03). However, no significant correlation was found between HMGB1 expression and recurrence-free and overall survival rates.ConclusionsHMGB1 expression may be one of the key factors regulating the response of rectal cancer to preoperative CRT in terms of tumor invasiveness and resistance to therapy.

The expression of phospholipase A2 group X is inversely associated with metastasis in colorectal cancer

Among the secretory phospholipase A2s (sPLA2), sPLA2 group X (PLA2GX) has the most potent hydrolyzing activity toward phosphatidylcholine, and has recently been shown to be implicated in chronic inflammatory diseases. The aim of the present study was to investigate PLA2GX expression in colorectal cancer (CRC) and its correlation with patient clinicopathological features. The present study comprises a series of 158 patients who underwent surgical resection for primary CRC. PLA2GX expression in CRC tissues was examined by immunohistochemistry and compared with patient clinicopathological findings and survival. A total of 64% of the tumors expressed PLA2GX at high levels. Statistical analysis revealed that PLA2GX expression was inversely correlated with hematogenous metastasis (P=0.005). In the subgroup analysis, left-sided tumors with high PLA2GX expression showed an inverse correlation with lymph node metastasis (P=0.018) and hematogenous metastasis (P=0.017). Patients with high PLA2GX expression tended to have a longer disease-specific survival compared with those with low PLA2GX expression in left-sided, but not right-sided, CRC (P=0.08). In light of the present results, we suggest that PLA2GX has an inhibitory effect on the progression of CRC.

Six Cases of Primary Colorectal Cancer After Living-donor Liver Transplantation: A Single-institution Experience in Japan

OBJECTIVE Liver-transplant patients have an increased risk of developing primary malignancies, possibly due to prolonged immunosuppression. However, no information on the incidence and biological characteristics of colorectal cancer after living-donor liver transplantation is available. METHODS The medical records of 392 consecutive adult patients who had undergone living-donor liver transplantation were retrospectively analyzed. RESULTS Colorectal cancer developed in 6 (1.5%) patients; 3 of 204 (1.5%) presented with hepatic cirrhosis, 2 of 77 (2.5%) with primary biliary cirrhosis and 1 (2.6%) of 39 with subacute fulminant hepatitis, but none of 13 patients with primary sclerosing cholangitis. Four patients were successfully treated with curative surgery and one with endoscopic resection, while another patient died 3 months after palliative surgery because of the progression of peritoneal metastasis. A pathological study revealed vessel invasion in all the five cases of surgically removed colorectal cancer and nodal metastasis in four (80%) cases. CONCLUSIONS Colorectal cancer develops at a relatively high frequency after living-donor liver transplantation, even in non-primary sclerosing cholangitis cases, and might have high malignant potential. The screening program for colorectal cancer should be more intensified after living-donor liver transplantation compared with that in the general population.

Abstract 85: Chloroquine potentiates the 5-fluorouracil-induced cell cycle arrest on HT-29 colon cancer cells

Introduction Autophagy is a self-degradation process that is essential for cell survival, development, and homeostasis. During autophagy, cytoplasmic organelles and proteins damaged by the cellular stress, such as starvation, hypoxia and chemotherapy, are recycled and converted into more essential proteins. Thus, induction of autophagy in cancer cells may result in cell adaptation against anticancer agents, consequently leading to resistance to therapy. Chloroquine (CQ), a worldwide used anti-malarial drug, is a lysosomotropic drug, also known to inhibit autophagy by impairing the autophagic protein degradation. On the other hand, 5-Fluorouracil (5-FU) is the most common chemotherapeutic agent for colorectal cancer, which inhibits DNA synthesis and induces apoptosis and/or cell cycle arrest in colon cancer cells. In the present study, we aimed to investigate the induction of autophagy as a mechanism of resistance of colon cancer cells to 5-FU-based chemotherapy, and the role of autophagy inhibition, by CQ, in potentiating the effect of 5-FU, especially focusing on cell cycle arrest. Procedure The human colon cancer cell line HT-29 was used. Cells were treated with CQ and/or 5-FU at various concentrations, and the changes in the proliferative activity were investigated, especially focusing on the induction of autophagy, the changes of cell cycle, and the development of apoptosis. The proliferative ability of HT-29 was analyzed by the MTS assay. The cell cycle was evaluated by flow-cytometry after staining of cells with PI, and autophagy was quantified by the analysis of the formation of acidic vesicular organelles (AVOs) by flow-cytometry after staining of cells with acridine orange (AO), and additionally, by the analysis of the expression of LC3-II by Western blot. Apoptosis was quantified by flow-cytometry after double-staining of the cells with AnnexinV/PI. Finally, to evaluate the long-term viability of the cells, the colony formation assay was performed. Results Treatment of HT-29 with 5-FU resulted in significant inhibition of the proliferative activity, compared with untreated cells. Treatment of the cells with CQ prior to exposure to 5-FU, potentiated the inhibitory effect. And it was dependent on the increase of the percentage of cells in G0/G1-phase, and the simultaneous decrease of cells in G2/M-phase. Also a partial increase of apoptotic cells was observed. These effects were dependent on the decreased expression of cyclin-dependent kinase-2 and the increased expression of p21Cip1, p27Kip1, important regulators of the cell cycle progression from G1 to S phase. Therefore, pre-treatment of HT-29 with the autophagy inhibitor, CQ, resulted in potentiation of the G0/G1 phase cell cycle arrest induced by 5-FU. Conclusion: The combination therapy using inhibitor of autophagy, such as CQ, should be a novel therapeutic modality to improve efficacy of 5-FU-based chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 85.