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Isocitrate dehydrogenase 1 and 2 (IDH1/2) mutations and their key effector 2-hydroxyglutarate (2-HG) have been reported to promote oncogenesis in various human cancers. To elucidate molecular mechanism(s) associated with IDH1/2 mutations, we established mouse embryonic fibroblasts (MEF) cells and human colorectal cancer cells stably expressing cancer-associated IDH1R132C or IDH2R172S, and analyzed the change in metabolic characteristics of the these cells. We found that IDH1/2 mutants induced intracellular 2-HG accumulation and inhibited cell proliferation. Expression profile analysis by RNA-seq unveiled that glucose transporter 1 (Glut1) was induced by the IDH1/2 mutants or treatment with 2-HG in the MEF cells. Consistently, glucose uptake and lactate production were increased by the mutants, suggesting the deregulation of glucose metabolism. Furthermore, PI3K/Akt/mTOR pathway and Hif1α expression were involved in the up-regulation of Glut1. Together, these results suggest that Glut1 is a potential target regulated by cancer-associated IDH1/2 mutations.

Background and Aim: The clinical outcome of Helicobacter pylori infection is strongly affected by the bacterial genotype. The aim of this study was to assess the relationship between the status of H. pylori sialic acid-binding adhesin (sabA) and the severity of gastric inflammation and diseases. Methods: Clinical isolates from 108 patients with duodenal ulcer, gastric ulcer, gastric cancer, or chronic gastritis were analyzed by sequencing and PCR assay. The degree of neutrophil infiltration in the antral region was evaluated by histopathological examination. The extent of atrophic change was evaluated using serum pepsinogen levels and the pepsinogen I/II ratio. Results: Functional sabA was present in 88 (81%) of the 108 patients. Although there was no correlation between the sabA status and gastric diseases, severe neutrophil infiltration and atrophy were associated with functional sabA. Conclusions: The results show a high prevalence of functional sabA in Japanese clinical isolates. The status of sabA may be an important virulence factor in inducing chronic gastric inflammation.

We developed a new method capable of detecting point mutations in the 23S rRNA gene of Helicobacter pylori using a LightCycler. Our method can detect a mutation in this gene in less than 1 h and can process many samples at once, thereby contributing to the selection of patients suitable for clarithromycin-based therapy.

Backgrounds/Aims: Pit cells are located in the hepatic sinusoids and are organ-associated natural killer cells that contribute to immune surveillance in the liver. In the present study, the interleukin-2 gene was introduced into hepatocytes using an adenovirus vector to induce interleukin-2 production in an attempt to enhance the natural killer activity of pit cells, leading to inhibition of metastasis of colon carcinoma. Methods: The recombinant adenovirus vector "Adex1CAmIL2" was constructed by inserting an expression unit which was composed of the CAG promotor (cytomegalovirus enhancer plus chicken beta-actin promotor), murine interleukin-2 cDNA, and a rabbit beta-globin polyadenylation signal. After administration of Adex1CAmIL2 to mice (4×10 7 pfu per animal), the expression of murine interleukin-2 in hepatocytes was examined by immunostaining and in situ hybridization, and the natural killer activity of hepatic mononuclear cells was measured. Inhibition of hepatic metastasis of colon carcinoma was examined after infusion of colon 38 tumor cells into the superior mesenteric vein. Results: After administration of Adex1CAmIL2, interleukin-2 mRNA expression was demonstrated in hepatocytes until day 7, and the serum interleukin-2 level was increased. The natural killer activity of hepatic mononuclear cells was markedly enhanced for 7–10 days. Hepatic metastasis was inhibited by administration of Adex1CAmIL2 until day 7 after tumor cell inoculation. Conclusion: These results suggest that gene therapy using Adex1CAmIL2 could be potentially useful for inhibiting hepatic micrometastasis by enhancing the natural killer activity of pit cells.

Heat shock factor 1 (HSF1), a major transactivator of stress responses, has been implicated in carcinogenesis in various organs. However, little is known about the biological functions of HSF1 in the development of hepatocellular carcinoma (HCC). To clarify the functional role of HSF1 in HCC, we established HSF1-knockdown (HSF1 KD) KYN2 HCC cells by stably expressing either small hairpin RNA (shRNA) against HSF1 (i.e. HSF1 KD) or control shRNA (HSF1 control). Tumorigenicity was significantly reduced in orthotopic mice with HSF1 KD cells compared with those with HSF1 control cells. Reduced tumorigenesis in HSF1 KD cells appeared attributable to increased apoptosis and decreased proliferation. Tumor necrosis factor-α-induced apoptosis was increased in HSF1 KD cells and HSF1(-/-) mouse hepatocytes compared with controls. Decreased expression of IκB kinase γ, a positive regulator of nuclear factor-κB, was also observed in HSF1 KD cells and HSF1(-/-) mouse hepatocytes. Furthermore, expression of bcl-2-associated athanogene domain 3 (BAG3) was dramatically reduced in HSF1 KD cells and HSF1(-/-) mouse hepatocytes. We also found that epidermal growth factor-stimulated mitogen-activated protein kinase signaling was impaired in HSF1 KD cells. Clinicopathological analysis demonstrated frequent overexpression of HSF1 in human HCCs. Significant correlations between HSF1 and BAG3 protein levels and prognosis were also observed. In summary, these results identify a mechanistic link between HSF1 and liver tumorigenesis and may provide as a potential molecular target for the development of anti-HCC therapies.

Alpha-feto protein (AFP) mRNA levels increase in hepatocellular carcinoma (HCC) cells as compared with non-neoplastic tissue. Therefore, detection of AFP mRNA in blood nuclear cells is useful for the evaluation of treatment efficacy and prognosis of HCC. In this study, simple and reproducible methods were developed to quantify AFP mRNA using the real-time RT-PCR assay (Taq Man assay). By using in vitro synthesized AFP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA, the sensitivity and dynamic range of the RT-PCR assay were established. AFP mRNA in both HCC and non-neoplastic tissue, as well as in cell lines, were measured using this assay system. The expression of the AFP mRNA level was normalized using the GAPDH house keeping gene product as an endogenous reference. AFP and GAPDH mRNA can be quantified in the range of 10–10 8 copies when using this quantitative assay. Among HCC cell lines, Huh 7 and HepG2 cells, respectively, represented 1.5×10 6 and 6.0×10 5 AFP mRNA/10 6 GAPDH mRNA, in contrast to 6, 23 and 230 AFP mRNA/10 6 GAPDH mRNA for HLE, HLF and PLC/PRF/5 cells, respectively. Other cell lines derived from stomach, pancreas, and colon cancers have 10 AFP mRNA copies/10 6 GAPDH mRNA. In liver tissue from patients with chronic hepatitis, and the non-neoplastic portion of the liver from HCC patients, AFP mRNA distributes from 2.5×10 3 to 5.8×10 4 /10 6 GAPDH transcripts. In contrast, AFP mRNA in tumor cells were more than 100-fold higher than that found in corresponding non-neoplastic portions in two patients who had a high level of AFP in serum. The establishment of the TaqMan quantifying system for AFP mRNA may have important clinical implications.

The immune defense system in the liver is known to consist of Kupffer cells, pit cells, lymphocytes, neutrophils, and other infiltrating immune responsive cells. In the liver, Kupffer cells as well as pit cells play an important role in tumor defense and the immune surveillance system. Activation of these cells could contribute to cytotoxicity against tumor cells. In an experimental model of hepatic metastasis of colon carcinoma, biological response modifiers potentially activate these immune responsive cells and reduce hepatic metastasis of tumor cells. Since the activation of pit cells may be induced by interleukin-2 (IL-2), the local tumor surveillance system of the liver can be activated by gene transduction of IL-2 cDNA in hepatocytes using adenovirus vector. Local production of IL-2 may enhance the natural killer (NK) activity of pit cells, leading to a reduction of hepatic metastasis of colon carcinoma. The modulation of sinusoidal cells by gene transduction (gene therapy) may provide a tool to change the pathophysiology of the liver, leading to a clinical treatment for liver diseases.