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Background: Indirubin, a constituent of the Chinese herbal medicine "Qing-Dai," has anti-cancer and anti-inflammatory activities. We aimed to evaluate the efficacy of indirubin for ameliorating colonic inflammation in a mouse model of inflammatory bowel disease., Methods: Mice with dextran sulfate sodium (DSS)-induced acute and chronic colitis were treated with indirubin in their diet. Clinical and histologic changes were evaluated. In addition, colon levels of interleukin-6, a critical pro-inflammatory mediator, was detected by enzyme-linked immunosorbent assay., Results: In the model of acute colitis, indirubin treatment improved the loss of body weight. Histology of colonic tissue revealed that indirubin treatment improved the histology grading of colitis ( P = 0.02), the extent of submucosal fibrosis ( P = 0.018), the number of mucosal toluidine blue-positive cells ( P = 0.004) and colon length ( P = 0.01). In the model of chronic colitis, indirubin treatment had no significant effect on pathologic findings except for colon length ( P = 0.003). However, indirubin administration significantly reduced colon levels of interleukin-6 in the chronic-colitis model ( P = 0.001)., Conclusion: Our study clearly showed that oral intake of indirubin can improve murine DSS-induced colitis (which mimics human inflammatory bowel disease).

The human ncRNA gene RGM249 regulates the extent of differentiation of cancer cells and the conversion of 293FT cells to hiPSCs. To identify the factors underlying this process, we investigated the effects of lentivirally inducing miR-520d expression in 293FT and HLF cells in vitro. Subsequently, we evaluated tumor formation in a xenograft model. Transformed HLF cells were Oct4 and Nanog positive within 24 h, showed p53 upregulation and hTERT downregulation, and mostly lost their migration abilities. After lentiviral infection, the cells were intraperitoneally injected into mice, resulting in benign teratomas (6%), the absence of tumors (87%) or differentiation into benign liver tissues (7%) at the injection site after 1 month. We are the first to demonstrate the loss of malignant properties in cancer cells in vivo through the expression of a single microRNA (miRNA). This miRNA successfully converted 293FT and hepatoma cells to hiPSC-like cells. The regulation of malignancy by miR-520d appears to be through the conversion of cancer cells to normal stem cells, maintaining p53 upregulation.

The incidence of advanced hepatocellular carcinoma (HCC) is increasing worldwide, and its prognosis is extremely poor. Interferon-alpha (IFN-α)/5-fluorouracil (5-FU) therapy is reportedly effective in some HCC patients. In the present study, to improve HCC prognosis, we identified the genes that are sensitizing to these agents. The screening strategy was dependent on the concentration of ribozymes that rendered HepG2 cells resistant to 5-FU by the repeated transfection of ribozymes into the cells. After 10 cycles of transfection, which was initiated by 5,902,875 sequences of a ribozyme library, three genes including protein kinase, adenosine monophosphate (AMP)-activated, gamma 2 non-catalytic subunit (PRKAG2); transforming growth factor-beta receptor II (TGFBR2); and exostosin 1 (EXT1) were identified as 5-FU-sensitizing genes. Adenovirus-mediated transfer of TGFBR2 and EXT1 enhanced IFN-α/5-FU-induced cytotoxicity as well as 5-FU, although the overexpression of these genes in the absence of IFN-α/5-FU did not induce cell death. This effect was also observed in a tumor xenograft model. The mechanisms of TGFBR2 and EXT1 include activation of the TGF-β signal and induction of endoplasmic reticulum stress, resulting in apoptosis. In HCC patients treated with IFN-α/5-FU therapy, the PRKAG2 mRNA level in HCC tissues was positively correlated with survival period, suggesting that PRKAG2 enhances the effect of IFN-α/5-FU and serves as a prognostic marker for IFN-α/5-FU therapy. In conclusion, we identified three genes that chemosensitize the effects of 5-FU and IFN-α/5-FU on HCC cells and demonstrated that PRKAG2 mRNA can serve as a prognostic marker for IFN-α/5-FU therapy.

Extrarenal rhabdoid tumors (ERRTs) are very rare neoplasms and have been reported in a range of organs, including sixteen cases in the stomach. We describe a woman aged 86 years who had an advanced gastric tumor with lymph node metastasis. The tumor mostly showed a diffuse arrangement with a small glandular region. The tumor cells were non-cohesive and had polygonal morphology with eccentric vesicular nuclei, prominent nucleoli, and abundant eosinophilic cytoplasm, i.e. they showed rhabdoid features. Immunohistochemically, the rhabdoid tumor cells were strongly positive for cytokeratins and vimentin. However, a candidate tumor suppressor gene of rhabdoid tumors, the INI1 gene, showed no mutations or loss of expression in the tumor cells. Although ERRTs typically have an aggressive clinical course, the patient was still alive without any evidence of recurrence or metastasis at 26 months after surgery. The rhabdoid features of the present case seemed to be a variant of gastric adenocarcinoma.

Background and Purpose: Nicorandil, an ATP-sensitive potassium (K(ATP) ) channel opener and nitric oxide donor, is used in the treatment of angina and acute heart failure. Here we investigated the effects of two K(ATP) channel openers, nicorandil and cromakalim on ischaemia reperfusion (I-R) injury in the kidney., Experimental Approach: Right nephrectomy was performed in 8-week-old male Sprague-Dawley rats and they were then divided into six groups: control group; I-R, including 30 min of left renal ischaemia followed by 24 h of reperfusion; I-R groups plus nicorandil 3 or 10 mg·kg⁻¹ i.p.; and I-R groups plus cromakalim 100 or 300 µg·kg⁻¹ i.p. After reperfusion, renal function was estimated by serum creatinine (SCr), urinary albumin:creatinine ratio (ACR) and urinary β2-microglobulin (β2-MG). Levels of K(ATP) channel subtypes were investigated by Western blot. Kidney sections were stained for 4-hydroxy-2-nonenal and 8-hydroxy-2'-deoxyguanosine., Key Results: Renal I-R induced significant increases in SCr, ACR and β2-MG levels compared with the control animals. Treatment with K(ATP) channel openers reduced urinary β2-MG levels, raised by I-R. Both K(IR) 6.1 and K(IR) 6.2 channels were expressed. Expression of K(IR) 6.2 channels in the I-R group was lower than in the control group, which was restored to normal by treatment with K(ATP) channel openers. Histologically, severe acute tubular damage was observed in the I-R kidney and this damage was ameliorated by K(ATP) channel openers, dose-dependently., Conclusions and Implications: ATP-sensitive potassium channel openers protected against proximal tubule damage after I-R injury. Nicorandil could represent a powerful additional component in the treatment of patients undergoing partial nephrectomy or renal transplantation., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

We previously reported that impaired retinoid signaling causes hepatocellular carcinoma (HCC) through oxidative stress. However, the interaction between oxidative stress and retinoid signaling has not been fully understood. To address this issue, the effects of hydrogen peroxide on the transcriptional activity of RAR/RXR heterodimers, RARα and RXRα proteins and intracellular signaling pathways were examined. The transcriptional activity of RAR/RXR examined by the DR5-tk-Luc reporter assay was significantly suppressed. The RARα protein level began to decrease at 6 h after treatment and declined thereafter. However, RARα mRNA were not changed. Activation of extracellular regulated kinases (ERK), p38, c-Jun N-terminal kinase (JNK) and Akt was observed after treatment of hydrogen peroxide. SP600125, an inhibitor of JNK, reversed the RARα protein level reduced by hydrogen peroxide. Anisomycin, an activator of JNK, reduced RARα protein. Transfection of wild-type JNK-constitutive actively expressing plasmid, but not kinase-negative JNK-expressing plasmid caused reduction of RARα protein. Proteasomal degradation of RARα was observed after anisomycin treatment; however, the mutant RARα, of which phosphorylation sites are replaced with alanines, was not degradated. In hepatitis C virus (HCV)-related human liver tissues, phospho-JNK and RARα reciprocally expressed with the progression of liver disease. Finally, the staining of 8-OHdG and thioredoxin was increased with the disease progression. These data indicate that JNK activation by oxidative stress suppresses retinoid signaling through proteasomal degradation of RARα, suggesting that a vicious cycle between aberrant retinoid signaling and oxidative stress accelerates hepatocarcinogenesis., (© 2011 Japanese Cancer Association.)

Objective: Under the current World Health Organization (WHO) classification, 'lung adenocarcinoma with mixed subtypes' is the most frequent type, even in small lung adenocarcinoma, with a diameter of 3 cm or less. For this type of lung adenocarcinoma, it has been reported that the high ratio of the peripheral bronchioloalveolar carcinoma (BAC)/lepidic growth (LG) component was a favorable prognostic factor. On the other hand, the central solid components of lung adenocarcinoma with mixed subtypes have not been focused on in the past. In this study, we took note of the histological features in central solid components of lung adenocarcinoma with mixed subtypes and evaluated whether the morphological diversity of these tumors is associated with local invasiveness and prognostic implication., Methods: A total of 103 surgically resected peripheral lung adenocarcinomas were reviewed. All the tumors were 3 cm or less in diameter and histologically diagnosed as lung adenocarcinoma with mixed subtypes, containing a BAC/LG component at the peripheral lesion of the tumor. The tumors were classified into two groups, according to the number of histological subtypes in the tumor, using the modified WHO classification (including the micropapillary subtype); group A (n = 76) has two or three histological subtypes, and group B (n = 27) has four or five subtypes in the tumor, respectively. Then, we evaluated the differences in clinicopathological factors and prognosis between these two groups., Results: Group B was significantly associated with positive lymphatic and vascular invasion, lymph node metastasis, and advanced pathological stage, compared with group A. The 5-year survival rates of all patients were 91.4% for group A and 43.3% for group B, respectively, with a significant difference (p < 0.01). Multivariate analysis showed that the group classification by the number of histological subtypes was an independent prognostic factor in stage IA patients (p < 0.01)., Conclusions: The morphological diversity of small lung adenocarcinoma with mixed subtypes is an independent prognostic factor and is associated with tumors' local invasiveness and patients' prognosis., (Copyright © 2010 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved.)

Basal cell adenoma (BCA) is an uncommon benign salivary gland neoplasm that includes isomorphic basaloid cells. We report on a female patient with BCA that developed in the right parotid gland in her 50s. The present patient demonstrated a few tumor nests in the fibrous capsule, and her tumor was larger than usual. These facts made us suspect of malignancy. Histopathologically, the tumor was characterized by multiple duct-like structures and tubular-trabecular masses composed of small isomorphic cells with hyperchromatic, round nuclei and an eosinophilic cytoplasm. It was difficult to determine whether the ductal structures noted in the tumor capsule were invasive. By immunohistochemistry, tumor cells of the tubular nests were positive for cytokeratin 7 and that the outer cells of tubular nests were positive for alpha smooth muscle actin (αSMA) and calponin. Tumor cells were immuno-negative for S-100 protein and glial fibrillary acidic protein. The Ki-67 labeling scores of the cells were extremely low (< 1%). We could achieve an accurate diagnosis of BCA by immunohistochemistry with MIB-1 and other markers.

Background: Geminin negatively regulates Cdt1 and induces the formation of prereplicative complexes by loading mini-chromosome maintenance proteins (Mcm) onto chromatin and limiting DNA replication to once per cell cycle. Recent studies have suggested that geminin expression is a marker of the S/G2/M phase of the cell cycle and is associated with a poor prognosis in various human malignancies. This study aimed to clarify the pathobiological role of geminin in intestinal-type gastric carcinoma, and its relationships with minichromosome maintenance 2 (Mcm2) and Ki67 expression., Methods: We performed western blot analysis of seven human gastric cancer cell lines, and immunohistochemical analysis of 72 gastric mucosal lesions and 128 surgically removed advanced intestinal-type gastric carcinomas. Double-labeling immuno-fluorescence was performed to identify the coexpression of geminin and Ki67., Results: Geminin was detected in all cell lines. Geminin labeling indices (LIs) in hyperplastic polyps, low-grade adenomas, high-grade adenomas, and intestinal-type adenocarcinomas were 3.9%, 10.5%, 18.6%, and 27.2%, respectively. The equivalent LIs for Ki67 and Mcm2 were 17.7%, 42.2%, 52.6%, and 59.7%; and 26.7%, 70.0%, 67.8%, and 77.8%, respectively. Double-labeling immunofluorescence revealed coexpression of geminin and Ki67 in both normal and tumor cells. The LI for geminin was significantly correlated with N stage, International Union Against Cancer (UICC) stage, Mcm2 LI, and Ki67 LI. Patients in stages I-IV and stage III with higher LIs for geminin (>25%) had significantly worse prognoses (P < 0.05 and P < 0.04, respectively). Univariate Cox regression analysis indicated that the overall survival of stage I-IV tumors was significantly correlated with high geminin LIs (relative risk [RR] = 1.94; P = 0.04)., Conclusions: Geminin expression might reflect the biological nature of gastric intramucosal neoplasms and could be a possible prognostic marker in advanced intestinal-type gastric carcinomas.

Minichromosome maintenance complex (MCM2-7) and Geminin are important in the prevention of DNA re-replication in the cell cycle, and are also prognostic markers for numerous human malignancies. The present study examined Minichromosome maintenance protein 7 (MCM7) and Geminin expression in human soft tissue sarcomas (STSs) to clarify their correlation to the clinicopathological factors. Immunohistochemistry was performed to detect the expression of MCM7, Geminin and Ki-67 on paraffin-embedded sections of 109 STSs. Labeling indices (LIs) of the molecules were evaluated in the tumors. Higher LIs of MCM7, Geminin and Ki-67 were significantly correlated with distant metastasis (P<0.01), histological grade (P<0.01) and poor prognosis (P<0.01), respectively. LIs of MCM7 and Geminin were significantly correlated with Ki-67 LIs, (MCM7/Ki-67: rs=0.745, P<0.01 and Geminin/Ki-67: rs=0.604, P<0.01). Multivariate analyses showed that the higher LIs of Geminin, but not MCM7 and Ki-67, were shown to be an independent factor of poorer prognosis (relative risk 2.72, P=0.013). The immunohistochemical expression of MCM7 and Geminin may be novel and useful markers for evaluating the prognosis in patients with human STS.

Backgrounds: We previously reported a highly sensitive method for serum human telomerase reverse transcriptase (hTERT) mRNA for hepatocellular carcinoma (HCC). alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) are good markers for HCC. In this study, we verified the significance of hTERTmRNA in a large scale multi-centered trial, collating quantified values with clinical course., Methods: In 638 subjects including 303 patients with HCC, 89 with chronic hepatitis (CH), 45 with liver cirrhosis (LC) and 201 healthy individuals, we quantified serum hTERTmRNA using the real-time RT-PCR. We examined its sensitivity and specificity in HCC diagnosis, clinical significance, ROC curve analysis in comparison with other tumor markers, and its correlations with the clinical parameters using Pearson relative test and multivariate analyses. Furthermore, we performed a prospective and comparative study to observe the change of biomarkers, including hTERTmRNA in HCC patients receiving anti-cancer therapies., Results: hTERTmRNA was demonstrated to be independently correlated with clinical parameters; tumor size and tumor differentiation (P < 0.001, each). The sensitivity/specificity of hTERTmRNA in HCC diagnosis showed 90.2%/85.4% for hTERT. hTERTmRNA proved to be superior to AFP, AFP-L3, and DCP in the diagnosis and underwent an indisputable change in response to therapy. The detection rate of small HCC by hTERTmRNA was superior to the other markers., Conclusions: hTERTmRNA is superior to conventional tumor markers in the diagnosis and recurrence of HCC at an early stage.

Polaprezinc (PZ), a chelate compound consisting of zinc and l-carnosine (Car), is an anti-ulcer drug developed in Japan. In the present study, we investigated whether PZ suppresses mortality, pulmonary inflammation, and plasma nitric oxide (NO) and tumor necrosis factor (TNF)-alpha levels in endotoxin shock mice after peritoneal injection of lipopolysaccharide (LPS), and how PZ protects against LPS-induced endotoxin shock. PZ pretreatment inhibited the decrease in the survival rate of mice after LPS injection. PZ inhibited the increases in plasma NO as well as TNF-alpha after LPS. Compatibly, PZ suppressed LPS-induced inducible NO synthase mRNA transcription in the mouse lungs. PZ also improved LPS-induced lung injury. However, PZ did not enhance the induction of heat shock protein (HSP) 70 in the mouse lungs after LPS. Pretreatment of RAW264 cells with PZ suppressed the production of NO and TNF-alpha after LPS addition. This inhibition likely resulted from the inhibitory effect of PZ on LPS-mediated nuclear factor-kappaB (NF-kappaB) activation. Zinc sulfate, but not Car, suppressed NO production after LPS. These results indicate that PZ, in particular its zinc subcomponent, inhibits LPS-induced endotoxin shock via the inhibition of NF-kappaB activation and subsequent induction of proinflammatory products such as NO and TNF-alpha, but not HSP induction.

Background: The origin licensing factor minichromosome maintenance 2 (MCM2) has recently been identified as a critical regulator of proliferation in both normal and neoplastic cells. This study examined whether MCM2 expression was of prognostic relevance in patients with stage III gastric carcinoma and whether the expression of this marker showed any correlation with clinicopathological characteristics. In addition, we evaluated whether the expression of this proliferation marker was correlated with that of another marker, Ki-67, in gastric carcinoma., Methods: We examined the immunohistochemical expression of MCM2, Ki-67, and p53 in 103 surgically removed stage III gastric carcinomas, which consisted of 60 intestinal-type and 43 diffuse-type carcinomas. The labeling indices (LIs) of MCM2 and Ki-67 in cancer cells were compared with clinicopathological characteristics, p53 expression, and overall survival rates., Results: The mean MCM2 and Ki-67 LIs were 69.1 +/- 11.8% and 48.2 +/- 14.5%, respectively, in the intestinal carcinomas, and 43.7 +/- 9.9% and 24.9 +/- 11.0%, respectively, in the diffuse carcinomas. The LIs of these proteins revealed no significant association with clinicopathological characteristics or with p53 expression in the carcinomas. Kaplan-Meier survival curves showed that, in the patients with diffuse carcinoma, those with higher MCM2 LIs had a poorer prognosis (P < 0.05), but the MCM2 LI was not correlated with prognosis for those with intestinal carcinoma (P = 0.25). Ki-67 expression had no significant correlation with prognosis in either intestinal-type or diffuse-type carcinomas. Multivariate Cox regression analysis confirmed that MCM2 was an independent prognostic factor in patients with diffuse carcinoma., Conclusion: Our data suggest that MCM2 is a useful prognostic marker in patients stage III diffuse-type gastric carcinoma.

We have recently isolated a gene, Ankyrin-repeated protein with a proline-rich region (ARPP), that is highly expressed in the skeletal and cardiac muscle. Our previous immunohistochemical analysis revealed that ARPP expression was augmented in rhabdomyosarcoma but scarcely detectable in leiomyosarcoma, showing that ARPP is a useful marker for rhabdomyosarcoma. In the present study, we generated the anti-ARPP monoclonal antibody, YAS11, immunoreactive with the N-terminal region (amino-acids residues 1-145) of the ARPP protein. Further, we immunohistochemically analyzed 100 renal tumors including 14 oncocytomas, and 86 renal cell carcinomas (RCCs). We found that ARPP was highly expressed in 12 of the 14 (85.7%) oncocytomas, but was detectable in only four of the 86 (4.7%) RCCs. Interestingly, ARPP was not detected in any of 11 chromophobe RCCs, suggesting that ARPP may be useful for differential diagnosis between oncocytoma and chromophobe RCC. Furthermore, we found that ARPP was selectively expressed in part of the distal renal tubule in normal kidney. Immunoelectron microscopy with anti-ARPP antibody revealed that ARPP was localized in mitochondria and nuclei in both the normal distal renal tubule and oncocytoma, suggesting that oncocytoma may be derived from the distal nephron, and probably from part of the distal renal tubule.

Using a newly developed assay of telomerase reverse transcriptase (hTERT) mRNA in serum by real-time RT-PCR, we previously reported this assay to be superior to other tumor markers for hepatoma. In this study, we aimed to clarify its clinical significance as a biomarker for lung cancer. In 112 patients with lung tumor and 80 individuals without cancer, we measured serum hTERT mRNA and epidermal growth factor receptor (EGFR) mRNA levels, using a quantitative one-step real-time RT-PCR assay. We examined its sensitivity and specificity in lung cancer diagnosis, its clinical significance in comparison with other tumor markers, and its correlation with the clinical parameters using multivariate analyses and correlation relative tests. The copy number of serum hTERT mRNA was independently correlated with tumor size, tumor number, presence of metastasis and recurrence, and smoking (all P < 0.05). EGFR mRNA correlated with tumor number and clinical stage (both P < 0.05). The sensitivity and specificity in lung cancer diagnosis were 89.0% and 72.7% for hTERT mRNA, and 71.3% and 80.0% for EGFR mRNA, respectively. hTERT mRNA was superior to other tumor markers in lung cancer diagnosis. For both mRNAs, serum levels were significantly correlated with levels in lung cancer tissues (both P < 0.05). The copy number of hTERT mRNA significantly decreased after the surgical treatment. The data suggest that hTERT mRNA, especially when combined with EGFR mRNA, is a novel and excellent biomarker for pulmonary malignancies to diagnose and assess the clinical stage.

This study examined the clinicopathological significance of minichromosome maintenance-2 (MCM2) expression in 38 human malignant fibrous histiocytomas (MFHs) and 36 benign fibrohistiocytic tumors (BFHTs) immunohistochemically, and in 9 human sarcoma or carcinoma cell lines, as well as 7 surgical specimens by Western blotting. MCM2 was detected in all the cell lines and surgical specimens as a single band at 120 kDa, while P53 expression was variable. Nuclear expression of MCM2 was noted in tumor but not mitotic cells of all the MFHs and 26 (72.2%) of the BFHTs, the labeling indices (LIs) being 62.0% in the 28 ordinary types, 38.5% in the 10 myxoid types, and 11.2% in the BFHTs with significant difference. Moreover, the LI was significantly higher for MCM2 than that for Ki-67 in the MFHs of both types (p<0.05). No correlation was noted between the MCM2-LI and P53 expression or apoptotic indices, which were significantly higher in the MFHs than BFHTs (p<0.01). These results indicate that MCM2 would correlate with cell proliferation rather than apoptosis in MFHs, and the expression is ubiquitous in proliferating cells, regardless of the expression of P53. Thus, MCM2 might be a reliable marker of proliferating cells in human MFH.