Toshio Kokuryo

Nek2 as a novel molecular target for the treatment of breast carcinoma

We investigated the role of Nek2, a member of the serine‐threonine kinase family, in the tumorigenic growth of breast carcinoma. Increased expression of Nek2 was observed in all breast carcinoma cell lines examined (BT20, BT474, Hs578T, MCF7, MDA‐MB‐231, T47D, and ZR‐75‐1) by immunoblotting. By treatment with Nek2 short interfering RNA (siRNA), expression of Nek2 was clearly decreased in both estrogen receptor (ER)‐positive (MCF7) and ER‐negative (MDA‐MB‐231) breast carcinoma cell lines. Cell growth, colony formation in soft agar, and in vitro invasiveness of these cell lines were substantially suppressed by Nek2 siRNA treatment. In a xenograft nude mouse model with subcutaneous implantation of MCF7 or MDA‐MB‐231, subcutaneous injection of Nek2 siRNA around the tumor nodules resulted in a reduction of tumor size compared with those of control siRNA injection. Taken together, Nek2 appears to play a pivotal role in tumorigenic growth of breast carcinoma cells, and could be a useful therapeutic molecular target for the treatment of breast carcinoma both in ER‐positive and ER‐negative cases. (Cancer Sci 2009; 100: 111–116)

Nek2 as an Effective Target for Inhibition of Tumorigenic Growth and Peritoneal Dissemination of Cholangiocarcinoma

We investigated the role of Nek2, a member of the serine/threonine kinase family, Nek, in the tumorigenic growth of cholangiocarcinoma cells. Expression of Nek2 is elevated in cholangiocarcinoma in a tumor-specific manner as compared with that of normal fibroblast cells. Expression of exogenous Nek2 did not perturb the growth of cholangiocarcinoma cells, whereas suppression of the Nek2 expression with siRNA resulted in the inhibition of cell proliferation and induced cell death. In xenograft-nude mouse model, s.c. injection of Nek2 siRNA around the tumor nodules resulted in reduction of tumor size as compared with those of control siRNA injection. In peritoneal dissemination model, Nek2 siRNA-treated mice showed statistically longer survival periods in comparison with those of the control siRNA-treated mice. Taken together, our data indicate a pivotal role of Nek2 in tumorigenic growth of cholangiocarcinoma.

Novel combination treatment for colorectal cancer using Nek2 siRNA and cisplatin

Nek2 (NIMA‐related kinase 2) is involved in cell division and mitotic regulation by centrosome splitting. We previously reported that Nek2 depletion causes growth suppression and cell death in cholangiocarcinoma and breast cancer cells. In this report, we examine the effect of a combination treatment using Nek2 siRNA with the cytotoxic chemotherapeutic agent cisplatin (CDDP) on colorectal cancer. Nek2 was overexpressed in all colorectal cancer cell lines examined (HCT‐15, DLD‐1, Colo205, and Colo320). Nek2 short‐interfering RNA (siRNA) resulted in the inhibition of cell proliferation and the induction of apoptosis in vitro. Nek2 siRNA suppressed tumor growth compared to control siRNA in a xenograft mouse model. To investigate the potential utility of Nek2 siRNA for clinical cancer therapy, we examine the effect of a combination treatment using Nek2 siRNA with CDDP on colorectal cancer. The combined administration of both Nek2 siRNA and CDDP inhibited cell proliferation and induced apoptotic cell death in vitro. Furthermore, the combined administration of both Nek2 siRNA and CDDP suppressed tumor growth compared to either the single administration of Nek2 siRNA or the combined administration of control siRNA and CDDP. Our results suggest that combination treatment using Nek2 siRNA and chemotherapeutic agents may be an effective therapeutic option for colorectal cancer.

Adipose tissue-derived mesenchymal stem cell transplantation promotes hepatic regeneration after hepatic ischemia-reperfusion and subsequent hepatectomy in rats.

BACKGROUND Adipose tissue-derived mesenchymal stem cells (ADSCs) are an attractive source for regenerative medicine because they are easily accessible through minimally invasive methods. We investigated the efficacy of ADSC transplantation on outcome after hepatic ischemia-reperfusion and subsequent hepatectomy in rats. METHODS ADSCs were isolated from subcutaneous adipose tissue of rats. After clamping the hepatoduodenal ligament for 15 min, the rats were subjected to a 70% partial hepatectomy. After releasing the clamp, 2 × 10(6) ADSCs per rat were injected through the penile vein. Phosphate buffered saline was injected as a control. The parameters of hepatic regeneration, such as hepatic regeneration rate, mitotic index, and anti-proliferating cell nuclear antigen levels, were examined. Furthermore, the expression of hepatic regeneration-associated proteins and genes in the regenerating liver was determined. RESULTS The hepatic regeneration rate 2 d after hepatectomy was significantly greater in the ADSC transplanted group compared with the sham group. Mitotic index, anti-proliferating cell nuclear antigen levels, and other regeneration-associated proteins in the liver were significantly higher in the ADSC transplanted group than the sham group on 1 d after hepatectomy. A number of hepatic regeneration-associated genes also were significantly upregulated in the ADSC transplanted group. CONCLUSIONS These results indicate that ADSC transplantation may provide beneficial effects in the process of liver regeneration after hepatic ischemia-reperfusion and subsequent hepatectomy.

SATB2 suppresses the progression of colorectal cancer cells via inactivation of MEK5/ERK5 signaling

Special AT‐rich sequence binding protein 2 (SATB2) is an evolutionarily conserved transcription factor that has multiple roles in neuronal development, osteoblast differentiation, and craniofacial patterning. SATB2 binds to the nuclear matrix attachment region, and regulates the expression of diverse sets of genes by altering chromatin structure. Recent studies have reported that high expression of SATB2 is associated with favorable prognosis in colorectal and laryngeal cancer; however, it remains uncertain whether SATB2 has tumor‐suppressive functions in cancer cells. In this study, we examined the effects of SATB2 expression on the malignant characteristics of colorectal cancer cells. Expression of SATB2 repressed the proliferation of cancer cells in vitro and in vivo, and also suppressed their migration and invasion. Extracellular signal‐regulated kinase 5 (ERK5) is a mitogen‐activated protein kinase that is associated with an aggressive phenotype in various types of cancer. SATB2 expression reduced the activity of ERK5, and constitutive activation of ERK5 restored the proliferation, anchorage‐independent growth, migration and invasion of SATB2‐expressing cells. Our results demonstrate the existence of a novel regulatory mechanism of SATB2‐mediated tumor suppression via ERK5 inactivation.

Recent advances in cancer stem cell research for cholangiocarcinoma

Cancer stem cells have been identified as cells with the capacity to self-renew and differentiate into multiple lineages of human malignancies. Cholangiocarcinoma is one of the most difficult intra-abdominal malignancies that can be treated using a surgical approach. Chemotherapy in addition to surgery is necessary to improve patient survival. However, its clinical benefit is limited, and, to date, no other effective anticancer drug is available for this disease. Several reports have shown the existence of cholangiocarcinoma stem cells. Cell surface antigens such as CD133, CD24, EpCAM, CD44, and others have been used to isolate cholangiocarcinoma stem cells. In general, enhanced expression of these markers in resected specimens of cholangiocarcinoma was associated with malignant potential. Distinct and specific pathways are expected to be present in cancer stem cells compared to other cancer cells that have no stem cell properties. To date, reports showing possible signaling pathways in cholangiocarcinoma stem cells are limited. More research is anticipated. Targeting therapies for surface molecular markers or specific signaling pathways of cholangiocarcinoma stem cells may be important in order to change the clinical outcome of patients with this disease. However, no clinical trial has been performed so far. This review will focus on the markers and signaling pathways used to define cholangiocarcinoma stem cells. A novel therapeutic approach of targeting cholangiocarcinoma stem cells will also be discussed.

Antitumor effects of α‐bisabolol against pancreatic cancer

In the present study, we investigated whether α‐bisabolol, a sesquiterpene alcohol present in essential oils derived from a variety of plants, has antitumor effects against pancreatic cancer. α‐Bisabolol induced a decrease in cell proliferation and viability in pancreatic cancer cell lines (KLM1, KP4, Panc1, MIA Paca2), but not in pancreatic epithelial cells (ACBRI515). α‐Bisabolol treatment induced apoptosis and suppressed Akt activation in pancreatic cancer cell lines. Furthermore, α‐bisabolol treatment induced the overexpression of early growth response‐1 (EGR1), whereas EGR1 siRNA decreased the α‐bisabolol‐induced cell death of KLM1 cells. Tumor growth in both subcutaneous and peritoneal xenograft nude mouse models was significantly inhibited by intragastric administration of 1000 mg/kg of α‐bisabolol, once a week for three weeks. The results indicate that α‐bisabolol could be a novel therapeutic option for the treatment of pancreatic cancer. (Cancer Sci 2011; 102: 2199–2205)

Inchinkoto, an herbal medicine, exerts beneficial effects in the rat liver under stress with hepatic ischemia-reperfusion and subsequent hepatectomy.

Objective:To investigate the beneficial effects of inchinkoto (ICKT) in the liver after 70% hepatectomy following ischemia reperfusion. Methods:Wistar rats were divided into 3 groups: simple laparotomy and 70% hepatectomy (Hx), 70% hepatectomy following ischemia reperfusion (IR) with vehicle (IRHxV), 70% hepatectomy following IR with ICKT (1 or 2 g/kg of body weight; IRHxK). Vehicle or ICKT was administered for 3 days preoperatively. The hepatoduodenal ligament was clamped for 15 minutes before hepatectomy in the IRHx groups. Rats were killed 1 hours after hepatectomy. In other experiments, the hepatoduodenal ligament was clamped for 30 minutes, with or without ICKT treatment, to evaluate the effect of ICKT on IR injury-induced mortality. Serum transaminase levels and the gene expression of inflammatory cytokines and inducible nitric oxide synthase in the remnant liver were determined. Furthermore, the expression of antioxidant genes was evaluated by PCR array. Results:The elevation of serum transaminase levels, the upregulation of genes for inflammatory cytokines and inducible nitric oxide synthase, and the increased formation of nitrotyrosine observed in the remnant livers of the IRHxV group were all significantly attenuated by preoperative administration of ICKT in the IRHxK group. The expression of antioxidant genes was also higher in the IRHxK group compared with that of the IRHxV group. Moreover, administration of ICKT significantly reduced the mortality induced by IRHx after 30-minute ischemia. Conclusions:Preoperative administration of ICKT provides beneficial effects through attenuating inflammatory responses and oxidative stress in the liver following IR and subsequent hepatectomy.

MEK inhibitor enhances the inhibitory effect of imatinib on pancreatic cancer cell growth

Imatinib mesylate (imatinib) inhibits the c-Kit-dependent tyrosine kinase activities and highly effective in the treatment of CML and GIST patients. Although pancreatic cancer is reported to express c-Kit, imatinib does not effectively inhibit pancreatic cancer cell growth at physiological concentrations. Therefore, we investigated the mechanism of resistance of pancreatic cancer to imatinib treatment. Imatinib inhibited growth of pancreatic cancer cell lines in concentration and time-dependent fashion regardless of c-Kit expression. However, 5 microM imatinib, which is almost a mean maximal plasma concentration in clinical setting, failed to suppress pancreatic cancer cell growth. Western blot analysis demonstrated that 5 microM imatinib treatment for 1h activated the MEK-MAPK pathway and the activation was independent of Ras activation. Administration of 5 microM imatinib and 1 microM U0126 (MEK inhibitor) significantly suppressed pancreatic cell growth. Our results indicate that a combination therapy of imatinib and MEK inhibitor can be a new therapeutic strategy to suppress the progression of pancreatic cancer.

Inflammation and Tumor Progression: A Lesson from TNF-α-Dependent FAK Signaling in Cholangiocarcinoma

Focal Adhesion Kinase (FAK) is implicated in a wide array of cellular processes and also involved in the production of matrix metalloproteinases (MMPs) which degrade extracellular matrix (ECM). We have shown that FAK plays a critical role in MMP-9 production and subsequent invasion of the cholangiocarcinoma activated by an inflammatory cytokine, TNF-alpha. By nature, cholangiocarcinoma is frequently associated with hepatolithiasis that causes recurrent inflammation. As degradation of the ECM is a prerequisite step for the invasion and metastasis of cancer cells, we used an assay of gelatin-degrading MMPs by Zymography to clarify the characteristic feature of the matrix degrading systems of the cancer cells. Immunoprecipitation and western blot analysis together with site specific phosphorylated FAK antibodies showed aberrant FAK activity in inflammation-mediated tumor cells. Confocal immunofluorescence staining could confirm not only localization but also phosphotyrosine contents of phosphorylated FAK by TNF-alpha stimulation. Destruction or penetration of the basement membrane is thought to be an essential step in successful metastasis by tumor cells, we used a matrix of basement membrane which was reconstituted on to a filter in the Boyden Chamber and assayed the ability of cancer cells to penetrate through matrigel-coated filter. We demonstrated the effectiveness of FAK siRNA treatment to prevent tumor invasion. Our observations suggested the importance of FAK as a therapeutic target for malignant neoplasm.