MRNA and enzyme activity of hepatic 11beta-hydroxysteroid dehydrogenase type 1 are elevated in C57BL/KsJ-db/db mice.

To evaluate the importance of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in insulin resistant diabetic C57BL/KsJ-db/db mice, we measured the activity and mRNA level of 11beta-HSD1 in the liver of db/db mice and their heterozygote litter mates, db/+m mice. The blood glucose, plasma insulin, and corticosterone levels of db/db mice were significantly higher than those of db/+m mice. Despite hyperinsulinemia, the activity level of this enzyme was significantly higher in db/db mice, and the mRNA level of hepatic 11beta-HSD1 was also significantly higher in db/db mice. Since hepatic 11beta-HSD1 in vivo mainly functions as 11-keto-reductase and does not work as 11beta-oxidase, these results suggest that the rate of hepatic conversion of 11-dehydrocorticosterone to corticosterone is increased in db/db mice, resulting in higher glucocorticoid activity in the liver. The increased hepatic corticosterone concentration due to the elevation of 11beta-HSD1 and high plasma corticosterone concentration may antagonize the action of insulin and cause insulin resistance. These findings have a potentially important implication for relationships between increased hepatic 11beta-HSD1 and insulin resistance in db/db mice. The present paper is the first to demonstrate the increased activities and mRNA level of hepatic 11beta-HSD1 in db/db mice.

Expression of WNT14 and WNT14B mRNAs in human cancer, up-regulation of WNT14 by IFNgamma and up-regulation of WNT14B by beta-estradiol.

WNT proteins play key roles in carcinogenesis. We have previously cloned and characterized WNT14 and WNT14B/WNT15. WNT14 and WNT3A genes are clustered on human chromosome 1q42, while WNT14B and WNT3 genes are clustered on human chromosome 17q21. Here, we investigated expression of WNT14 and WNT14B mRNAs in human cancer. WNT14 was significantly up-regulated in 1 out of 9 cases of primary breast cancer. WNT14B was not expressed in primary breast, gastric and colorectal cancers. Among 3 human breast cancer cell lines, WNT14 mRNA was expressed in T-47D cells, and weakly expressed in MCF-7 cells. WNT14 mRNA was also detected in 7 out of 7 pancreatic cancer cell lines, 12 out of 12 esophageal cancer cell lines, 4 out of 4 cervical cancer cell lines, and 5 out of 7 brain tumor cell lines by using cDNA-PCR. These results indicate that WNT14 rather than WNT14B is preferentially expressed in various types of human cancer, such as breast cancer, gastric cancer, and pancreatic cancer. WNT14 mRNA was up-regulated by interferon gamma (IFNgamma), but not by tumor necrosis factor alpha (TNFalpha), in MKN45 cells derived from gastric cancer, while expression of WNT14B mRNA was not affected by IFNgamma and TNFalpha in MKN45 cells. Although expression of WNT14 mRNA was not affected by beta-estradiol in MCF-7 cells, WNT14B mRNA was transiently up-regulated by beta-estradiol in MCF-7 cells. These results indicate that WNT14 is a target gene of IFNgamma in MKN45 cells, and that WNT14B is a target gene of estrogen in MCF-7 cells.

Molecular cloning and characterization of human WNT11.

WNT signaling pathway is implicated in carcinogenesis. Here, we cloned and characterized human WNT11, which showed three amino-acid substitutions (Ala121Thr, Gly156Arg, and Ser271Trp) compared with human WNT11 cDNA previously isolated by another group. WNT11 encoded a 354 amino-acid polypeptide with five N-glycosylation sites. Gly156 of human WNT11 was conserved in other members of the human WNT family, such as WNT2B1, WNT2B2, WNT3, WNT3A, WNT5B, WNT6, WNT7B, WNT8A, WNT10A, and WNT14. The Ala121-Gly156-Ser271 WNT11 allele isolated in this study was also identified in human genome draft sequence AC069055. Expression profile of WNT11 was next investigated. The 4.3-kb WNT11 mRNA was expressed in fetal lung, kidney, adult heart, liver, skeletal muscle, and pancreas. WNT11 mRNA was significantly up-regulated in a gastric cancer cell line MKN45 and a cervical cancer cell line SKG-IIIa. Among various types of human primary tumors, WNT11 mRNA was up-regulated in four cases of colorectal adenocarcinoma, and a case of renal cell carcinoma. Up-regulation of WNT11 mRNA might play an important role in human carcinogenesis through activation of the WNT signaling pathway.

Molecular cloning and characterization of WNT14B, a novel member of the WNT gene family.

WNT14B was cloned and characterized in this study. WNT14B encoded 357-amino acid WNT family protein with the signal peptide and an N-linked glycosylation site. WNT14B was most homologous to WNT14 (61.4% total amino acid identity). WNT15 cDNA fragment previously isolated by another group corresponds to a part of ORF of the WNT14B cDNA (codon 216-335). Exon-intron boundaries were conserved between WNT14B and WNT14 genes. WNT14B and WNT3 genes were clustered in the human chromosome 17q21 region in head to head manner. Intergenic region between WNT14B and WNT3 genes was about 33 kb in size. The 6.6-kb WNT14B mRNA was moderately expressed in fetal kidney and adult kidney. Although WNT14B mRNA was not detected in fetal brain and adult brain by northern blot analyses, WNT14B mRNA was detected in brain, especially in occipital lobe, by RNA dot blot analysis. Among 48 human cancer cell lines derived from various tissues, WNT14B was expressed in a teratocarcinoma cell line NT2 with the potential to differentiate into neuronal cells. WNT14B mRNA was significantly up-regulated by all-trans retinoic acid in NT2 cells. These results strongly suggest that WNT14B might be implicated in the early process of neuronal differentiation of NT2 cells induced by retinoic acid.

Expression of WNT10A in human cancer.

WNT signaling pathway is implicated in carcinogenesis and embryogenesis. We have previously cloned and characterized WNT10A, and demonstrated up-regulation of WNT10A in gastric cancer. Here, we investigated expression of WNT10A mRNA in various types of human cancer. WNT10A mRNA was detected in 10 out of 12 esophageal cancer cell lines by cDNA-PCR, and was significantly up-regulated in esophageal cancer cell lines TE2, TE3, TE4, and a brain tumor cell line A-172. WNT10A mRNA was not up-regulated by retinoic acid in a teratocarcinoma cell line NT2. TFF1/pS2 mRNA, but not WNT10A mRNA, was up-regulated by beta-estradiol in a breast cancer cell line MCF-7. Expression of WNT10A mRNA in various types of primary cancers was next investigated by using Matched tumor/normal expression array filter. WNT10A mRNA was significantly up-regulated in 2 out of 8 cases of primary gastric cancer, and in 1 out of 7 cases of primary rectal cancer. Expression of WNT10A mRNA in esophageal cancer was not investigated, because such samples were not blotted on the expression array filter. Up-regulation of WNT10A mRNA might play key roles in some cases of esophageal, gastric, and colorectal cancer.

A long-term survival case of pancreatic poorly differentiated adenocarcinoma with transcatheter arterial embolization and combination chemotherapy.

A 66-year-old woman complained of supraclavicular lymph node swelling during her initial visit to an outpatient clinic. Computed tomography revealed a hypervascular tumor in the uncus of the pancreas (2.0 x 2.0 cm), therefore a needle biopsy of the pancreas was performed. A poorly differentiated adenocarcinoma was identified. Transcatheter arterial embolization using adriamycin and gelatin sheet was performed. To alleviate her symptoms (movement disorder of neck, etc.), initial chemotherapy; FP (5-fluorouracil and cisplatin intravenously) was continued for 6 cycles with her consent, and subsequently MF (methotrexate and 5-fluorouracil intravenously) for 14 cycles. This patient survived with transcatheter arterial embolization, FP and MF combination chemotherapies for 24 months after presenting with the symptoms. To our knowledge, this is the longest surviving case of pancreatic poorly differentiated adenocarcinoma (Stage IV). In conclusion, this present case suggests that transcatheter arterial embolization, FP and MF combination therapy may have an effect at prolonging survival in poorly differentiated pancreatic adenocarcinoma.

Nonresectable hepatocellular carcinoma: improved percutaneous ethanol injection therapy guided by CO(2)-enhanced sonography.

OBJECTIVE: The purpose of our study was to evaluate the usefulness of percutaneous ethanol installation using CO(2)-enhanced sonography for patients with nonresectable hepatocellular carcinoma (HCC). SUBJECTS AND METHODS: Forty-six patients with 65 HCC lesions were examined with contrast-enhanced sonography with direct injection of CO(2) into the proper hepatic artery during arteriography. We performed percutaneous ethanol injection guided by CO(2)-enhanced sonography for the treatment of hypervascular HCC lesions that could not be treated with conventional percutaneous ethanol injection or with transcatheter arterial embolization. RESULTS: CO(2)-enhanced sonography detected five additional small HCC lesions before treatment (p<0.05) and 14 new lesions during follow-up (p<0.01), than conventional sonography detected. CO(2)-enhanced sonography showed positive enhancement of residual lesions after initial treatment (n = 3) and incomplete local treatment (n = 5) that were not detected on conventional sonography. These 27 lesions were successfully treated with percutaneous ethanol injection using a mixture of iodized oil and ethanol and guided by CO(2)-enhanced sonography. CONCLUSION: CO(2)-enhanced sonography is a sensitive method for detecting residual viable lesions and small new HCC lesions that cannot be detected with conventional sonography. Percutaneous ethanol injection guided by CO(2)-enhanced sonography can treat hypervascular HCC lesions that cannot be treated with conventional percutaneous ethanol injection or transcatheter arterial embolization.

Expression profiles of 10 members of Frizzled gene family in human gastric cancer.

Frizzled (FZD) genes encode seven-transmembrane type WNT receptors, which are implicated in carcinogenesis and embryogenesis. We have previously cloned and characterized FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, and FZD10. Here, we investigated expression profiles of all members of the FZD gene family in human gastric cancer. FZD mRNAs were detected by one-step cDNA-PCR. Specificity of cDNA-PCR was confirmed by nucleotide sequence analyses of cDNA-PCR products. Among seven gastric cancer cell lines, FZD7 was up-regulated in MKN7, which was consistent with a previous report. FZD5 was up-regulated in MKN45. FZD9 and FZD10 were up-regulated together in TMK1 and MKN74. FZD2 was up-regulated in TMK1, MKN7, MKN28, MKN45, MKN74 and KATO-III. Among 10 cases of primary gastric cancer, FZD9 was up-regulated in 2 cases, FZD2 and FZD8 were up-regulated in 4 cases. Effects of Helicobacter pylori (H. pylori) on expression of FZDs were further investigated, and it was revealed that FZDs were not up-regulated by H. pylori in MKN45 cells. These results indicate that FZD2, FZD8, and FZD9 might play key roles in human gastric cancer.

Molecular cloning and characterization of human WNT7B.

WNT signaling molecules are implicated in carcinogenesis and embryogenesis. Only partial coding sequence of human WNT7B is reported so far, and human genome draft sequence corresponding to the WNT7B gene in human chromosome 22q13 region is not available at present. Here, we have cloned human WNT7B cDNAs, spanning the complete coding sequence, by using rapid amplification of cDNA ends (RACE) and cDNA-PCR. WNT7B encoded a 349-amino-acid polypeptide with three N-linked glycosylation sites and consensus amino-acid residues conserved among members of the WNT family. WNT7B showed 77.1% total-amino-acid identity with WNT7A. The 4.0-kb WNT7B was moderately expressed in fetal brain, weakly expressed in fetal lung and kidney, and faintly expressed in adult brain, lung and prostate. Expression levels of WNT7B mRNA in a lung cancer cell line A549, esophageal cancer cell lines TE2, TE3, TE4, TE5, TE6, TE7, TE10, TE12, a gastric cancer cell line TMK1, and pancreatic cancer cell lines BxPC-3, AsPC-1 and Hs766T were significantly higher than that in fetal kidney. In addition, WNT7B was up-regulated in 5 out of 10 cases of primary gastric cancer. These results strongly suggest that WNT7B might play important roles in various types of human cancer.

Up-regulation of WNT10A by tumor necrosis factor alpha and Helicobacter pylori in gastric cancer.

WNT signaling pathway is implicated in carcinogenesis and embryogenesis. We have previously cloned and characterized WNT10A and WNT6, which are clustered in human chromosome 2q35 region. In this study, we investigated expression of WNT10A and WNT6 in gastric cancer. The 3.0- and 2.4-kb WNT10A mRNAs were expressed in gastric cancer cell lines MKN7, MKN45 and MKN74. The 2.0-kb WNT6 mRNA was expressed in gastric cancer cell lines MKN28 and MKN74. WNT10A was up-regulated in 3 out of 6 cases of primary gastric cancer, while WNT6 was not up-regulated in primary gastric cancer. Effects of inflammatory cytokines and Helicobacter pylori (H. pylori) on expression of WNT10A and WNT6 were next investigated. Interferon gamma (IFNgamma) failed to induce up-regulation of WNT10A and WNT6. Tumor necrosis factor alpha (TNFalpha) induced up-regulation of WNT10A in MKN45 cells. Up-regulation of WNT10A reached maximum at 6 h after TNFalpha treatment. H. pylori also induced up-regulation of WNT10A in MKN45 cells. These results strongly suggest that up-regulation of WNT10A induced by TNFalpha and H. pylori might play key roles in human gastric cancer through activation of WNT--beta-catenin--TCF signaling pathway.

Insulin-independent and wortmannin-resistant targeting of IRS-3 to the plasma membrane via its pleckstrin homology domain mediates a different interaction with the insulin receptor from that of IRS-1.

AIMS/HYPOTHESIS: In primary adipocytes, although IRS-1 and IRS-3 are expressed in comparable amounts, these proteins manifest distinct distribution and significance in insulin signalling. We investigated the molecular basis of the difference between these two proteins. METHODS: In Cos-1 cells transiently expressing rat IRS-1, IRS-3, or chimeric proteins of these two proteins we examined the tyrosine phosphorylation via the wild-type or mutant insulin receptors and evaluated their targeting to the plasma membrane by immunostaining the membrane ghost. RESULTS: In contrast to IRS-1, IRS-3 was tyrosine-phosphorylated by the insulin receptor altering Tyr960 to Phe (Y960F), which disrupts the binding site of the PTB domain of IRSs, to an extent comparable to the wild-type receptor. The tyrosine phosphorylation of IRS-3 with the PH domain replacement via the Y960F insulin receptor markedly decreased, whereas that of IRS-3 with the PTB domain alteration was mildly impaired. Insulin-stimulated translocation of IRS-1 to the plasma membrane, as well as that of IRS-3 with the PH domain replacement, was wortmannin-sensitive, although that of IRS-3 was insulin-independent and wortmannin-resistant. CONCLUSIONS/INTERPRETATION: The affinity of the PH domain for the phospholipids in the plasma membrane seems to influence the receptor-substrate interaction required for IRS tyrosine phosphorylation, indicating that the PH domain and the PTB domain of IRSs cooperatively function in insulin-stimulated tyrosine phosphorylation of these proteins.

Up-regulation of Frizzled-7 (FZD7) in human gastric cancer.

Human Frizzled-7 (FZD7) and human FzE3, showing 98.8% nucleotide identity, encode almost identical WNT receptors with nine amino-acid substitutions. FzE3 is claimed to be expressed specifically in esophageal cancer. We determined the structure of the FZD7 gene and the FZD7 cDNA expressed in esophageal cancer. The FZD7 gene without intron and the FZD7 cDNAs isolated from esophageal cancer cell lines TE4 and TE5 were found to encode WNT receptor identical to FZD7, but not to FzE3. Nucleotide sequence of FzE3 was not identified on the human genome draft sequence. Thus, we could not obtain any data suggesting the existence of FzE3. Expression profile of FZD7 was also investigated. FZD7 was expressed throughout normal gastrointestinal tract, from esophagus to rectum. Among human esophageal and gastric cancer cell lines, expression level of FZD7 was relatively lower in esophageal cancer cell lines, and was highest in the gastric cancer cell line MKN7. FZD7 was up-regulated in one out of six cases of human primary gastric cancer. As over-expression of Frizzled-7 leads to activation of the WNT-beta-catenin-TCF pathway, up-regulation of FZD7 in human gastric cancer might play key roles in carcinogenesis through activation of the WNT-beta-catenin-TCF pathway.

Subcellular localization of insulin receptor substrate family proteins associated with phosphatidylinositol 3-kinase activity and alterations in lipolysis in primary mouse adipocytes from IRS-1 null mice.

To clarify the roles of insulin receptor substrate (IRS) family proteins in phosphatidylinositol (PI) 3-kinase activation and insulin actions in adipocytes, we investigated the intracellular localization of IRS family proteins and PI 3-kinase activation in response to insulin by fractionation of mouse adipocytes from wild-type and IRS-1 null mice. In adipocytes from wild-type mice, tyrosine-phosphorylated IRS-1 and IRS-2, which were found to associate with PI 3-kinase in response to insulin, were detected in the plasma membrane (PM) and low-density microsome (LDM) fractions. By contrast, tyrosine-phosphorylated IRS-3 (pp60), which was found to associate with PI 3-kinase, was predominantly localized in the PM fraction. In adipocytes from IRS-1-null mice, insulin-stimulated PI 3-kinase activity in anti-phosphotyrosine (alphaPY) immunoprecipitates in the LDM fraction was almost exclusively mediated via IRS-2 and was reduced to 25%; however, insulin-stimulated PI 3-kinase activity in the PM fraction was primarily mediated via IRS-3 and was reduced to 60%. To determine the potential functional impact of the distinct subcellular localization of IRSs and associating PI 3-kinase activity on adipocyte-specific metabolic actions, we examined lipolysis in IRS-1 null mice. The level of isoproterenol-induced lipolysis was increased 5.1-fold in adipocytes from IRS-1 null mice as compared with wild-type mice. Moreover, hormone-sensitive lipase (HSL) protein was increased 4.3-fold in adipocytes from IRS-1-null mice compared with wild-type mice, and HSL mRNA expression was also increased. The antilipolytic effect of insulin in IRS-1 null adipocytes, however, was comparable to that in wild-type mice. Thus, discordance between these two insulin actions as well as the transcriptional and translational effect (HSL mRNA and protein regulation) and the PM effect (antilipolysis) of insulin may be explained by distinct roles of both PI 3-kinase activity associated with IRS-1/IRS-2 and PI 3-kinase activity associated with IRS-3 in insulin actions related to their subcellular localization.

Combination therapy of percutaneous mitoxantrone injection, percutaneous ethanol injection, and transcatheter arterial embolization for intrahepatic hepatocellular carcinoma and adrenal metastasis.

We treated a 63-year-old man who had recurrent large hepatocellular carcinomas (> 5 cm in diameter) and left adrenal metastasis with the combination approach of percutaneous intratumoral chemotherapy with mitoxantrone, percutaneous ethanol injection, and transcatheter arterial embolization. He received repeated transcatheter arterial embolization and percutaneous ethanol injection combination therapy for intrahepatic hepatocellular carcinomas, which controlled his disease for 6 months from the first treatment. After that, left adrenal metastasis was detected by biopsy specimen. Therefore, we repeated more transcatheter arterial embolization and percutaneous ethanol injection to the liver and left adrenal gland, but this combination therapy could not control the hepatocellular carcinomas in these organs. With the patient's consent, he was treated with the combination approach of percutaneous intratumoral chemotherapy with mitoxantrone, percutaneous ethanol injection, and transcatheter arterial embolization for hepatocellular carcinomas of the liver and left adrenal gland. After this combination therapy, we followed-up the viable lesions by color Doppler ultrasonography and computed tomography examination. However, we could not detect these viable lesions of hepatocellular carcinomas in his body until one month before he died. When the degree of hepatic failure worsened due to the natural course of cirrhosis, this combination therapy was stopped 7 months before he died. He died of pulmonary tumor emboli from metastasis of inferior vena cava 24 months after the combination therapy started. However, on autopsy there was almost no remaining hepatocellular carcinoma found in the main lesions of liver and left adrenal gland. We suggest that a combination approach of percutaneous intratumoral chemotherapy with mitoxantrone, percutaneous ethanol injection, and transcatheter arterial embolization may be indicated in elderly cases of intrahepatic large hepatocellular carcinoma and adrenal metastasis, which are not under control only by transcatheter arterial embolization and percutaneous ethanol injection.

Risk factors for recurrence of large HCC in patients treated by combined TAE and PEI.

BACKGROUND/AIMS: In this report, risk factors of intrahepatic recurrence of a large solitary hepatocellular carcinoma after combination therapy with transcatheter arterial embolization followed by percutaneous ethanol injection were studied. METHODOLOGY: The series included 61 patients with an unresectable large solitary hepatocellular carcinoma, the largest size of which was greater than 3 cm in diameter. All patients completely responded to combination therapy and recurrence rates were determined. The following parameters; age, sex, hepatitis B virus surface antigen, hepatitis C virus antibodies, Child's classification, alcohol abuse, alanine aminotransferase, aspartate aminotransferase, alpha-fetoprotein, indocyanine green retention rate, hepatocellular carcinoma size, hepatocellular carcinoma capsule, total amount of injected ethanol and the alpha-fetoprotein 1 month after treatment were evaluated. RESULTS: The 1-, 3-, and 5-year cancer-free survival rates of all patients were calculated to be 61%, 23%, and 13%, respectively. Among pretreatment parameters, the log-rank test and subsequent Cox's proportional hazards model showed that a tumor size of more than 5 cm in diameter was independently associated with recurrence. The posttreatment parameters of total amount of injected ethanol was also shown to be significantly related to recurrence by the log-rank test. CONCLUSIONS: Lesions more than 5 cm in diameter and insufficient injected ethanol were associated with intrahepatic recurrence after this combination therapy.

WNT10A and WNT6, clustered in human chromosome 2q35 region with head-to-tail manner, are strongly coexpressed in SW480 cells.

Human WNT10A and WNT6 were cloned and characterized. WNT10A encoded a 417-amino-acid polypeptide with WNT core domain, and WNT6 encoded a 365-amino-acid polypeptide with N-terminal signal peptide, WNT core domain, and RGD motif. WNT10A and WNT6 genes were clustered in the head-to-tail manner with an interval less than 7.0 kb in human chromosome 2q35 region. Among human WNT family, WNT10A was most homologous to WNT10B (59.2% amino-acid identity), and WNT6 was most homologous to WNT1 (47.4% amino-acid identity). WNT10B and WNT1 genes were also clustered in human chromosome 12q13 region. Two WNT gene clusters in human chromosome 2q35 and 12q13 regions might be generated due to duplication of ancestral gene cluster. The 3.0- and 2.4-kb WNT10A mRNAs were expressed in fetal kidney, placenta, adult spleen and kidney. The 2.0-kb WNT6 mRNA was coexpressed with WNT10A in placenta and adult spleen. WNT10A and WNT6 were strongly coexpressed in SW480 (colorectal cancer). In addition to SW480, WNT10A was strongly expressed in HL-60 (promyelocytic leukemia) and Raji (Burkitt's lymphoma), and WNT6 in HeLa S3 (cervical cancer). Overexpression WNT10A and WNT6 might play key roles in human carcinogenesis through activation of WNT-beta-catenin-TCF signaling pathway, just like Wnt10b and Wnt1.