Polymorphisms of the RET gene in hirschsprung disease, anorectal malformation and intestinal pseudo-obstruction in Taiwan.

BACKGROUND/PURPOSE: Mutations in the receptor tyrosine kinase RET gene are associated with Hirschsprung disease (HD), which is also known as congenital intestinal aganglionosis. We found an association with specific alleles in five single nucleotide polymorphism (SNP) sites of the RET gene in our HD patients. METHODS: We compared the association of specific RET SNP alleles in patients with severe GI disorders such as anorectal malformation (ARM) or pediatric intestinal pseudo-obstruction (IPO) to that in HD patients. Sixty-four HD, 23 ARM and 35 IPO patients were included. Genomic DNA extracted from blood samples was analyzed by polymerase chain reaction and DNA sequencing analysis. RESULTS: The allele distributions of all five RET SNPs in the HD patients deviated from those in the normal population (p < 0.05), whereas those of the ARM patients did not. The allele distributions of these RET SNPs in the IPO patients were all significantly different from those in the HD patients. Allele distributions of exon 2 and 13 in the IPO patients were also significantly different from those of the normal population. The frequencies of all the HD-predominant alleles were lower in the HD patients than the normal population, and were even lower in the IPO patients. CONCLUSION: This study strengthens the association of specific RET SNP alleles with typical HD in Taiwan.

Suppressive effects of extracts from the aerial part of Coriandrum sativum L. on LPS-induced inflammatory responses in murine RAW 264.7 macrophages.

BACKGROUND: Coriandrum sativum is used not only as a spice to aid flavour and taste values in food, but also as a folk medicine in many countries. Since little is known about the anti-inflammatory ability of the aerial parts (stem and leaf) of C. sativum, the present study investigated the effect of aerial parts of C. sativum on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We further explored the molecular mechanism underlying these pharmacological properties of C. sativum. RESULTS: Ethanolic extracts from both stem and leaf of C. sativum (CSEE) significantly decreased LPS-induced nitric oxide and prostaglandin E(2) production as well as inducible nitric oxide synthase, cyclooxygenase-2, and pro-interleukin-1beta expression. Moreover, LPS-induced IkappaB-alpha phosphorylation and nuclear p65 protein expression as well as nuclear factor-kappaB (NF-kappaB) nuclear protein-DNA binding affinity and reporter gene activity were dramatically inhibited by aerial parts of CSEE. Exogenous addition of CSEE stem and leaf significantly reduced LPS-induced expression of phosphorylated mitogen-activated protein kinases (MAPKs). CONCLUSION: Our data demonstrated that aerial parts of CSEE have a strong anti-inflammatory property which inhibits pro-inflammatory mediator expression by suppressing NF-kappaB activation and MAPK signal transduction pathway in LPS-induced macrophages.

p38 mitogen-activated protein kinase pathway is involved in protein kinase Calpha-regulated invasion in human hepatocellular carcinoma cells.

Protein kinase Calpha (PKCalpha) has been suggested to play an important role in tumorigenesis, invasion, and metastasis. In this study, we investigated the signal pathways selectively activated by PKCalpha in human hepatocellular carcinoma (HCC) cells to determine the role of mitogen-activated protein kinases (MAPK) in PKCalpha-mediated HCC migration and invasion. A stable SK-Hep-1 cell clone (siPKCalpha-SK) expressing DNA-based small interfering RNA (siRNA) PKCalpha was established and was then characterized by cell growth, migration, and invasion. The expression of PKCalpha was decreased in siPKCalpha-SK, and cell growth, migration, and invasion were reduced. These changes were associated with the decrease in p38 MAPK phosphorylation level, but not in c-jun-NH(2)-kinase-1/2 (JNK-1/2) and extracellular signal-regulated kinase-1/2 (ERK-1/2). This phenomenon was confirmed in the SK-Hep-1 cells treated with antisense PKCalpha olignucleotide. The p38 MAPK inhibitor SB203580 or dominant negative p38 mutant plasmid (DN-p38) was used to evaluate the dependency of p38 MAPK in PKCalpha-regulated migration and invasion. Attenuation of cell migration and invasion was revealed in the SK-Hep-1 cells treated with the SB203580 or DN-p38, but not with ERK-1/2 inhibitor PD98059 or JNK-1/2 inhibitor SP600125. Overexpression of constitutively active MKK6 or PKCalpha may restore the inactivation of p38 and the attenuation of cell migration and invasion in siPKCalpha-SK. Similar findings were observed in the stable HA22T/VGH cell clone expressing siRNA PKCalpha. This study provides new insight into the role of p38 MAPK in PKCalpha-mediated malignant phenotypes, especially in PKCalpha-mediated cancer cell invasion, which may have valuable implications for developing new therapies for some PKCalpha-overexpressing cancers.

Immunochemical localization of protein kinase Calpha in the biopsies of human hepatocellular carcinoma.

The purpose of this study was to elucidate the protein kinase C (PKC) alpha distribution in human hepatocellular carcinoma (HCC). The histoimmunopathologic technique was used to determine the localization and expression of PKCalpha in HCC biopsies. The HCC tissues were classified as cytosolic type (PKCalpha deposited in the cytoplasm in > 50% of cells) and membranous type for the remaining ones. There was a significant association of the membranous type with non-hepatitis C virus (HCV) infected patients. Moreover, the expression of PKCalpha in this type was significantly higher in HCC cells than that in the adjacent non-tumor liver cells. The result indicated that PKCalpha may play an important role in carcinogenesis of HCC patients with HBV infection and/or non-HCV infection.

Overexpression of protein kinase C alpha mRNA in human hepatocellular carcinoma: a potential marker of disease prognosis.

BACKGROUND: Members of the protein kinase C (PKC) isoenzyme family play a central role in the tumorigenesis of several tissues. However, little is known about subtype specific intracellular expression of PKC in human hepatocellular carcinomas. METHODS: We investigated PKC isoforms mRNA expression in 42 HCC specimens using reverse transcription polymerase chain reaction analysis, and the correlation between PKC isoforms expression and clinicopathologic parameters. RESULTS: We found that PKCalpha, PKCdelta and PKCiota mRNA were significantly increased in HCCs as compared to the corresponding non-cancerous liver tissues. PKCalpha expression also significantly correlated with tumor size (P<0.05) and TNM stage (P<0.05), but PKCdelta and PKCiota did not. The log-rank analysis revealed that patients with higher PKCalpha mRNA expression in the HCC tissues had significantly shorter survival rate than patients with lower PKCalpha mRNA expression (P<0.01). CONCLUSIONS: Our results suggested that the PKCalpha may be a prognostic factor for the survival of patients with HCC.

Suppression of tumorigenicity of human hepatocellular carcinoma cells by antisense oligonucleotide MZF-1.

Our previous studies have found that reducing expression of myeloid zinc finger-1 (MZF-1) inhibited protein kinase C alpha (PKCalpha) expression and decreased cell migration and invasion in human hepatocellular carcinoma (HCC). In this study, we further investigated the role of MZF-1 in tumorigenesis. The SK-Hep-1 HCC cells were transfected with the antisense oligonucleotide (ODN) of MZF-1. The pretreated cells was then detected the mRNA and protein levels by RT-PCR and western blotting, and the cell growth was assayed by MTT. Meanwhile, the pretreated-SK-Hep-1 HCC cells were implanted subcutaneously into nude mice to observe the tumor growth and calculated tumor inhibitory rate. The results showed that, at the dosage of 5 microM, the antisense ODN MZF-1 suppressed both MZF-1 and PKCalpha productions by SK-Hep-1 HCC cells after cationic liposome-mediated transfection, to 15% and 30% in control cultures of 0 microM dosage, respectively. The growth of SK-Hep-1 HCC cells was inhibited by the 2-5 microM doses of the antisense ODN MZF-1, whereas the control reagent, the sense ODN MZF-1, showed no effects. In BALB/nude mice, SK-Hep-1 HCC cells pretreated with the 5 microM antisense ODN MZF-1 formed tumors much smaller than the cells with sense ODN. The mean of inhibitory rate of tumor growth was 71.2 +/- 8.6%, and the tumor formation time was prolonged from 14 days to 26 days. These findings suggested the usefulness of antisense ODN MZF-1 as a new reagent for cancer therapy.

Overexpression of anion exchanger 2 in human hepatocellular carcinoma.

To compare gene expression patterns between the poorly-differentiated (HA22T/VGH) and well-differentiated (HepG2) hepatocellular carcinoma cells, messenger RNA was isolated form both kinds of cells and subjected to differential displays reversing transcriptase-polymerase chain reaction (DDRT-PCR) technology. Gene fragments showing difference in the expression were recovered, reamplified, cloned and sequenced. Anion exchanger 2 (AE2), an isoform of band 3 protein, was identified and chosen for further characterization. AE2 was strongly expressed in HA22T/VGH cells, while it was little expressed in the well-differentiated hepatocellular carcinoma cells (PLC/PRF5 and HepG2) or little in the normal liver cell (Chang liver). In the 28 pairs of HCC and adjacent non-tumor tissues, levels in the cancer tissue (32.7 +/- 5.0) were significantly higher than that in the adjacent non-tumor tissue (9.1 +/- 2.4) (P < 0.01). Moreover, 19 cases (68%) showed over-expression of AE2 in HCC tissues, 3 cases were similar in both tissues, and 6 cases exhibited little or undetectable signals. Twenty cases (71%) of adjacent normal tissue showed little or undetectable signals. The results indicated that overexpression of AE2 may be involved in the development of human HCC.

Congenital mesoblastic nephroma presenting with massive hematuria and hemorrhagic shock: report of one case.

Congenital mesoblastic nephroma (CMN) is a rare benign tumor that occurs during the neonatal period and early infancy. The vast majority of these tumors present as asymptomatic palpable abdominal masses. We describe an unusual presentation of a CMN in a 10-month-old male infant who presented with massive hematuria and the development of hemorrhagic shock. Abdominal ultrasound showed a heterogeneous solid complex mass measuring 4.8 x 3.5 cm arising from the upper pole of the left kidney. The patient was resuscitated using intravenous fluids and blood transfusions because persistent massive bloody urine leading to progressive shock occurred the night of the admission day. Preoperative diagnosis was possible Wilms tumor of the left kidney. The histopathological findings were consistent with the character of a cellular variant of CMN. The patient was free of recurrence and metastasis at the 2-year follow-up examination. Our case report suggests that CMN is a rare benign renal tumor during infancy and may present with unusual massive hematuria and shock.

Expression of protein kinase C alpha in biopsies and surgical specimens of human hepatocellular carcinoma.

Variations in the activity of protein kinase C (PKC) have been observed in different types of tumors. Although these inconsistent findings may be attributed to the alterations of individual PKC isoforms, the effects of general anesthetic may not be neglected. In this study, biopsies and surgical specimens were obtained from patients with HCC, and the levels of PKCalpha were analyzed by immunohistochemistry. PKCalpha expression in biopsies was mainly revealed on the cell membrane of hepatocytes whereas that in the surgical specimens was in the cytosol and on the membrane. In both types of specimens, the PKCalpha level in HCC was significantly higher than that in the adjacent non-tumorous tissue. Moreover, the level of PKCalpha in biopsies was significantly higher than that in surgical specimens of the corresponding tissue type. These findings suggested that general anesthetics may significantly affect the expression of PKCalpha, and the effects of anesthetics should not be neglected in observations which were made only based on surgical specimens.

Augmented release of matrix metalloproteinase-9 by PKC activation in organotypic cultures of human breast cancer and adjacent normal breast tissue and fibroadenoma.

The organotypic culture technique and quantitative gelatin zymography were used to determine the expression of matrix metalloproteinase (MMP)-9 and MMP-2 in human breast cancer and adjacent normal breast tissue and fibroadenoma. MMP-9 and MMP2 were constitutively expressed in all cultures. The release of these two enzymes in breast cancer was higher than that in adjacent normal breast tissue and fibroadenoma. Administration of 12-o-tetradecanoyl- phorbol-13-acetate (TPA) increased the release of MMP-9 but not of MMP-2. This response was inhibited by protein kinase C (PKC) inhibitor (H7), transcription inhibitor (actinomycin D) and translation inhibitor (cycloheximide). Moreover, the increased level of MMP-9 by TPA in breast cancer was also higher than that in adjacent normal breast tissue and fibroadenoma. These phenomena were also observed in the DAG-treated culture. These findings suggested that the MMP-9 expression in the breast cancer tissue may be more sensitive for the PKC activation.

Herlyn-Werner-Wunderlich syndrome consisting of uterine didelphys, obstructed hemivagina and ipsilateral renal agenesis in a newborn.

Herlyn-Werner-Wunderlich (HWW) syndrome is a rare variant of Müllerian duct anomalies consisting of uterine didelphys, obstructed hemivagina, and ipsilateral renal agenesis. Patients with HWW syndrome are usually asymptomatic until menarche, when they present with acute lower abdominal pain. Here we report a case of a female newborn with right renal agenesis diagnosed during the pregnancy. The patient presented with a protruding mass over the vaginal introitus that was associated with an obstructed hemivagina and uterine didelphys.

The predominant protein arginine methyltransferase PRMT1 is critical for zebrafish convergence and extension during gastrulation.

Protein arginine methyltransferase (PRMT)1 is the predominant type I methyltransferase in mammals. In the present study, we used zebrafish (Danio rerio) as the model system to elucidate PRMT1 expression and function during embryogenesis. Zebrafish prmt1 transcripts were detected from the zygote period to the early larva stage. Knockdown of prmt1 by antisense morpholino oligo (AMO) resulted in delayed growth, shortened body-length, curled tails and cardiac edema. PRMT1 protein level, type I protein arginine methyltransferase activity, specific asymmetric protein arginine methylation and histone H4 R3 methylation all decreased in the AMO-injected morphants. The morphants showed defective convergence and extension and the abnormalities were more severe at the posterior than the anterior parts. Cell migration defects suggested by the phenotypes were not only observed in the morphant embryos, but also in a cellular prmt1 small-interfering RNA knockdown model. Rescue of the phenotypes by co-injection of wild-type but not catalytic defective prmt1 mRNA confirmed the specificity of the AMO and the requirement of methyltransferase activity in early development. The results obtained in the present study demonstrate a direct link of early development with protein arginine methylation catalyzed by PRMT1.

Reduction of PKC alpha decreases cell proliferation, migration, and invasion of human malignant hepatocellular carcinoma.

Protein kinase C (PKC) superfamily play key regulatory roles on the development of cancer. However, the exact role of these enzymes in human hepatocellular carcinoma (HCC) has not been well established. Using the RT-PCR and Western blotting to analyze the levels of PKC isoforms mRNA and protein in the five different differentiated hepatoma cell lines, we found that PKC alpha was highly expressed in the poor-differentiated HCC cell lines (SK-Hep-1 and HA22T/VGH) as compared with that in the well-differentiated HCC cell lines (PLC/PRF/5, Hep3B, and HepG2). When treated with PKC alpha antisense oligonucleotides (ODN), both HA22T/VGH and SK-Hep-1 cells lines showed the reduction of PKC alpha expression, as well as a deceleration in the growth rate and in the level of cyclin D1, but the increase in the levels of p53 and p21(WAF1/CIP1). Moreover, the reduction of PKC alpha expression also inhibited the migratory and invasive potential of both HA22T/VGH and SK-Hep-1 cells lines, and revealed a down-regulation of several migration/invasion-related genes (MMP-1, u-PA, u-PAR, and FAK). These phenomenon were also confirmed by DNA-based small interfering RNA (siRNA) PKC alpha and PKC alpha/beta specific inhibitor Go6976. Thus, the results indicated that PKC alpha may be associated with regulation of cell proliferation/migration/invasion in human poorly differentiated HCC cells, suggesting a role for the PKC alpha in the malignant progression of human HCC.

Anion exchanger inhibitor DIDS induces human poorly-differentiated malignant hepatocellular carcinoma HA22T cell apoptosis.

Anion exchangers (AEs) of the Cl(-)/HCO3(-) exchanger family contribute to the regulation of intracellular acid-base balance. Recently, we found that anion exchanger 2 (AE2) was significantly expressed in human hepatocellular carcinoma (HCC) and in poorly-differentiated human HCC HA22T/VGH cells. In the present study, we further explored the pharmacological function of AE in four human HCC cell lines (SK-Hep-1, HA22T/VGH, HepG2, and Hep3B) following the treatment of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an AEs specific inhibitor. After administrations with 400-1000 microM of DIDS, cell proliferation was greatly inhibited in a dose-dependent manner from 47.5 to 65.0% in higher malignant HA22T/VGH cells, but not in other cell lines. The results of 4,6-diamidino-2-phenylindole (DAPI) staining, DNA fragmentation and flow cytometric analysis further revealed that cell apoptosis induced by DIDS was also observed in HA22T/VGH cells. Therefore, these findings suggested that AE may be involved, in part, in the proliferation and survival of HA22T cells and could be a new potential therapeutic target against specific human HCC.

PKCalpha expression regulated by Elk-1 and MZF-1 in human HCC cells.

Our previous study found that PKCalpha was highly expressed in the poor-differentiated human HCC cells and associated with cell migration and invasion. In this study, we further investigated the gene regulation of this enzyme. We showed that PKCalpha expression enhancement in the poor-differentiated human HCC cells was found neither by DNA amplification nor by increasing mRNA stability using differential PCR and mRNA decay assays. After screening seven transcription factors in the putative cis-acting regulatory elements of human PKCalpha promoters, only Elk-1 and MZF-1 antisense oligonucleotide showed a significant reduction in the PKCalpha mRNA level. They also reduced cell proliferation, cell migratory and invasive capabilities, and DNA binding activities in the PKCalpha promoter region. Over-expression assay confirmed that the PKCalpha expression may be modulated by these two factors at the transcriptional level. Therefore, these results may provide a novel mechanism for PKCalpha expression regulation in human HCC cells.

Low RET mutation frequency and polymorphism analysis of the RET and EDNRB genes in patients with Hirschsprung disease in Taiwan.

Hirschsprung disease (HSCR), or congenital intestinal aganglionosis, is a relatively common disorder characterized by the absence of ganglion cells in the nerve plexuses of the lower digestive tract, resulting in intestinal obstruction in neonates. Mutations in genes of the RET receptor tyrosine kinase and endothelin receptor B (EDNRB) signaling pathways have been shown to be associated in HSCR patients. In this study, we collected genomic DNA samples from 55 HSCR patients in central Taiwan and analyzed the coding regions of the RET and EDNRB genes by PCR amplification and DNA sequencing. In the 55 patients, an A to G transition was detected in two (identical twin brothers). The mutation was at the end of RET exon 19 at codon 1062 (Y1062C), a reported critical site for the signaling pathways. Single nucleotide polymorphisms (SNP) in exons 2, 7, 11, 13, and 15 of RET and exon 4 of EDNRB in the HSCR patients or controls were detected. The differences between patients and controls in allele distribution of the five RET polymorphic sites were statistically significant. The most frequent genotype encompassing exons 2 and 13 SNPs (the polymorphic sites with the highest percentage of heterozygotes) was AA/GG in patients, which was different from the AG/GT in the normal controls. Transmission disequilibrium was observed in exons 2, 7, and 13, indicating nonrandom association of the susceptibility alleles with the disease in the patients. This study represents the first comprehensive genetic analysis of HSCR disease in Taiwan.