Yukiko Nishiguchi

Pro-chemotherapeutic effects of antibody against extracellular domain of claudin-4 in bladder cancer

Bladder cancer displays an aggressive phenotype in the muscle-invasive phase, and is associated with a high mortality rate. Therefore, novel molecular therapeutic targets are needed to improve patient survival. A monoclonal antibody against the extracellular domain of the claudin-4 (CLDN4) tight junction protein was established by immunizing rats with a plasmid vector encoding human CLDN4. A hybridoma clone, producing a rat monoclonal antibody recognizing CLDN4 (clone 4D3), was obtained. Immunohistochemistry by using the 4D3 antibody showed that CLDN4 expression was associated with local invasion, nodal metastasis, distant metastasis, and advanced stage in 86 cases of bladder cancer. The 4D3 antibody inhibited growth, invasion, and survival, associated with abrogation of the intratumoral microenvironment; lowered concentrations of epidermal growth factor and vascular endothelial growth factor were found in three-dimensional cultures of T24 and RT4 cells. In combination with cisplatin therapy, 4D3 enhanced cisplatin cytotoxicity by increasing cellular permeability, leading to increased intracellular cisplatin concentrations. In mouse models of subcutaneous tumors and lung metastasis, 4D3 enhanced tumor growth inhibition, alone and with concurrent cisplatin treatment. The anti-tumor activity of the newly established 4D3 antibody suggests that it may be a powerful tool in CLDN4-targeting therapy, and in combination with chemotherapy.

Expression of MAS1 in breast cancer

MAS1 is a receptor for angiotensin 1‐7 (A1‐7), which is derived from angiotensin II (A‐II) by the action of angiotensin converting enzyme (ACE) 2. MAS1 induces anti‐A‐II phenotypes, such as vessel dilation and depression of blood pressure. Using immunohistochemistry, we examined the role of MAS1 in 132 cases of invasive ductal carcinoma (IDC) of the breast. While benign mammary tissues expressed MAS1 at high levels, MAS1 expression was attenuated in all IDC, especially in scirrhous IDC. The decrease in MAS1 expression was associated with tumor growth, lymph node metastasis, and grade. MAS1 expression was inversely associated with the proliferation index and epidermal growth factor receptor and human epidermal growth factor receptor‐2 expression. Of the 132 cases, 12 (9.1%) were triple‐negative breast cancer (TNBC) cases. All TNBC cases (the 12 cases and the additional 36 cases using a tissue array) expressed MAS1. Using the TNBC cell lines 4T1 and MDA‐MB‐468, which expresses MAS1, we found that cell growth, anti‐apoptotic survival and invasion were suppressed by MAS1 activation with A1‐7 treatment and enhanced by MAS1 knockdown. In contrast, synergic effect was found between tamoxifen and A1‐7 in a luminal A breast cancer cell line, MCF‐7. Combination treatment with cisplatin, an ACE2 activator, and an A‐II type 1 receptor blocker showed synergic effects on tumor growth inhibition of 4T1 tumors in a syngeneic mouse model. These findings suggest that MAS1 might act as an inhibitory regulator of breast cancer and may be a possible molecular target for this malignancy.

Storkhead box 2 and melanoma inhibitory activity promote oral squamous cell carcinoma progression

Background Storkhead box protein 2 (STOX2) is a transcriptional factor associated with pre-eclampsia with fetal growth restriction. We recently reported that melanoma inhibitory activity (MIA) promotes oral squamous cell carcinoma (OSCC) progression. However, the relationship between STOX2 and MIA remains unknown in malignancies. Methods We used immunohistochemistry and PCR to investigate MIA and STOX2 expression in OSCC. We also performed functional analysis in human OSCC cells. Results MIA and STOX2 mRNA levels were higher in OSCCs than in normal oral epithelial cells, and upregulation of STOX2 was significantly correlated with overexpression of MIA. Immunostaining for STOX2 was associated with nodal metastasis (P = 0.0002) and MIA expression (P < 0.0001). Furthermore, MIA expression (P = 0.0035) and STOX2 expression (P = 0.0061) were associated with poor outcome in OSCCs. In vitro analysis using OSCC cells revealed that MIA increased expression of STOX2 by paracrine manner. Moreover, STOX2 accelerated OSCC cell growth, invasion, suppressed apoptosis, and enhanced resistance to paclitaxel, cisplatin, and 5-FU. Conclusions Our results suggest that MIA-STOX2 signaling may be a useful diagnostic and therapeutic target in OSCCs.

Elaidic Acid, a Trans-Fatty Acid, Enhances the Metastasis of Colorectal Cancer Cells.

The effects of trans-fatty acids (TFAs) on cardiovascular disorders have been extensively studied, and the effect of TFAs on cancers has recently been recognized. This study examined the effects of elaidic acid (EA), a TFA, on colorectal cancer (CRC) progression. We demonstrated that EA enhanced the growth, survival, and invasion of the CRC cell lines, CT26, and HT29. Tumor growth and metastasis in the lung, liver, and peritoneum were significantly more enhanced in CRC cells treated with EA than those treated with the cis form of EA, oleic acid (OA), or vehicle. Spheres of CRC cells were formed at more pronounced numbers in EA-treated cells than in OA-treated cells. Compared to OA, EA treatment also induced expression of the stemness factors, nucleostemin, CD133, and Oct4. Moreover, spheres of EA-treated CRC cells were larger and more proliferative than spheres of OA-treated cells. Oral intake of EA also enhanced liver metastasis and CD133 expression of CRC cells in a dose-dependent manner. EA intake also increased resistance to 5-fluorouracil. Inhibition of Wnt and ERK1/2 abrogated EA-induced enhancement of metastasis. Our findings demonstrate that EA might provide prominent metastatic potential to CRC cells, which shows important implications for the treatment of CRC.

Remodeling of energy metabolism by a ketone body and medium-chain fatty acid suppressed the proliferation of CT26 mouse colon cancer cells

Normal and cancerous cells are suggested to have differential utilization of fatty acids and ketone bodies, which could be exploited in cancer therapy. The present study examined the effect of 3-hydroxybutyric acid (3-HBA), which is a ketone body generating acetyl-CoA, and lauric acid (LAA, C12:0), which is a medium-chain saturated fatty acid translocated to mitochondria in a carnitine-independent manner to produce acetyl-CoA, on the energy metabolism of mouse CT26 colon cancer cells. In CT26 cells expressing 3-HBA and LAA transporters, 3-HBA and LAA reduced cell proliferation, mitochondrial volume and lactate production, and increased oxidative stress, particularly in low-glucose conditions. Concurrent treatment with 3-HBA and LAA under glucose starvation had a synergistic effect on cell growth inhibition. In addition, LAA and LAA + 3-HBA promoted an imbalance in the expression of enzymes in the electron transport chain. These findings suggested that treatment with 3-HBA and/or LAA during glucose starvation may reprogram energy metabolism and decrease the proliferation of cancer cells.

A comprehensive expression analysis of the MIA gene family in malignancies: MIA gene family members are novel, useful markers of esophageal, lung, and cervical squamous cell carcinoma

Melanoma inhibitory activity (MIA) gene family members include MIA, MIA2, and Transport and Golgi organization protein 1 (TANGO). Although MIA gene family members have several tumor-related functions, their detailed roles in malignancies remain poorly elucidated. In this study, 477 tumor specimens were subjected to immunohistochemical screening to evaluate MIA gene family expression. For a validation analysis, we also examined the association between MIA gene family expression and clinicopathological factors in 66 cases of esophageal cancer, 145 cases of lung cancer, and 126 cases of cervical cancer. The frequency of MIA gene family expression was higher among squamous cell carcinomas than among other tumor types subjected to screening. In the validation analysis, MIA gene family staining was observed frequently in esophageal and lung cancers associated with nodal and/or distant metastasis. In cervical cancers, MIA and TANGO immunostaining also correlated with tumor progression and metastasis. Furthermore, MIA2 expression levels in invasive cervical cancer were upregulated relative to those in cervical intraepithelial neoplasia 3. A disease-free survival analysis revealed that MIA-, MIA2, or TANGO-positive patients had a significantly shorter disease-free survival than did those patients who were negative. Our results suggest that MIA, MIA2, and TANGO may be useful diagnostic and therapeutic molecular targets in human malignancies.

Pancreatic adenocarcinoma up‐regulated factor has oncogenic functions in oral squamous cell carcinoma

Pancreatic adenocarcinoma up‐regulated factor (PAUF) is a novel secretory protein which promotes tumour progression, metastasis and poor prognosis in pancreatic, cervical and colorectal carcinoma. It is also associated with gemcitabine resistance in pancreatic cancer cells. However, the expression and function of PAUF in oral squamous cell carcinoma (OSCC) remain unknown.

NIPA-like domain containing 1 is a novel tumor-promoting factor in oral squamous cell carcinoma

PurposeIn our previous global gene expression analysis, we identified NIPA-like domain containing 1 (NIPAL1), which encodes a magnesium transporter, as one of the most overexpressed genes in recurrent oral squamous cell carcinoma (OSCC). Although has been NIPAL1 linked with gout pathogenesis, little is known about its expression and function in human malignancies.MethodsIn this study, we examined NIPAL1 expression in 192 cases of OSCC by immunohistochemistry and performed a functional analysis of human OSCC cells.ResultsNIPAL1 immunostaining was observed in 39 of 192 OSCC patients (20.3%). NIPAL1 expression correlated significantly with cancer cell intravsation (P = 0.0062), as well as with poorer disease-free survival in a Kaplan–Meier analysis (P < 0.0001). Moreover, a multivariate Cox proportional hazards model analysis revealed that NIPAL1 expression was an independent predictor of disease-free survival in OSCC (P < 0.0001). In a functional analysis, NIPAL1 regulated the growth and adhesion of OSCC tumor cells and endothelial cells.ConclusionsOur findings suggest that NIPAL1 might be a novel factor promoting OSCC tumorigenesis, as well as a useful molecular marker of OSCC.

Proton pump inhibitor induced collagen expression in colonocytes is associated with collagenous colitis

AIM To elucidate the role of proton pump inhibitors (PPIs) in collagenous disease, direct effect of PPI on colonocytes was examined. METHODS Collagenous colitis is a common cause of non-bloody, watery diarrhea. Recently, there has been increasing focus on the use of proton PPIs as a risk factor for developing collagenous colitis. Mouse CT26 colonic cells were treated with PPI and/or PPI-induced alkaline media. Expression of fibrosis-associated genes was examined by RT-PCR. In human materials, collagen expression was examined by immunohistochemistry. RESULTS CT26 cells expressed a Na+-H+ exchanger gene (solute carrier family 9, member A2). Treatment with PPI and/or PPI-induced alkaline media caused growth inhibition and oxidative stress in CT26 cells. The treatment increased expression of fibrosis inducing factors, transforming growth factor β and fibroblast growth factor 2. The treatment also decreased expression of a negative regulator of collagen production, replication factor C1, resulting in increased expression of collagen types III and IV in association with lipid peroxide. In biopsy specimens from patients with collagenous colitis, type III and IV collagen were increased. Increase of type III collagen was more pronounced in PPI-associated collagenous colitis than in non-PPI-associated disease. CONCLUSION From these findings, the reaction of colonocytes to PPI might participate in pathogenesis of collagenous colitis.

Fatty acids inhibit anticancer effects of 5-fluorouracil in mouse cancer cell lines

The present study investigated the effects of two major dietary fatty acid components, linoleic acid (LA) and elaidic acid (EA), on the antitumor effects of 5-fluorouracil (5-FU) in the LL2, CT26 and CMT93 mouse cancer cell lines. Concurrent treatment with LA and 5-FU elicited a decreased cell viability compared with treatment with 5-FU alone. In addition, increased inhibition of growth was observed following concurrent treatment with EA and 5-FU. Sequential treatment of LA followed by 5-FU abrogated the anticancer effects of 5-FU, and treatment with EA followed by 5-FU increased cancer cell growth in addition to abrogating the anticancer effects of 5-FU. The expression of the stem cell markers CD133 and nucleostemin (NS) increased in all three cell lines treated concurrently with 5-FU and either LA or EA when compared with cells treated with 5-FU alone. Aldehyde dehydrogenase activity in the cancer stem cells (CSCs), in response to concurrent treatment with 5-FU and either LA or EA, was increased compared with 5-FU treatment alone. 5-FU inhibited the growth of CT26 tumors, but co-treatment with either LA or EA abrogated this effect. NS-positive CSCs were more abundant in CT26 tumors treated with 5-FU and either LA or EA compared with those treated with 5-FU alone. The results of the present study suggested that, rather than altering the sensitivity of cancer cells to 5-FU, LA and EA may promote the survival of CSCs. The results indicated that dietary composition during chemotherapy is an important issue.