Identification of three elevenin receptors and roles of elevenin disulfide bond and residues in receptor activation in Aplysia californica.

Neuropeptides are ubiquitous intercellular signaling molecules in the CNS and play diverse roles in modulating physiological functions by acting on specific G-protein coupled receptors (GPCRs). Among them, the elevenin signaling system is now believed to be present primarily in protostomes. Although elevenin was first identified from the L11 neuron of the abdominal ganglion in mollusc Aplysia californica, no receptors have been described in Aplysia, nor in any other molluscs. Here, using two elevenin receptors in annelid Platynereis dumerilii, we found three putative elevenin GPCRs in Aplysia. We cloned the three receptors and tentatively named them apElevR1, apElevR2, and apElevR3. Using an inositol monophosphate (IP1) accumulation assay, we demonstrated that Aplysia elevenin with the disulfide bond activated the three putative receptors with low EC50 values (ranging from 1.2 to 25 nM), supporting that they are true receptors for elevenin. In contrast, elevenin without the disulfide bond could not activate the receptors, indicating that the disulfide bond is required for receptor activity. Using alanine substitution of individual conserved residues other than the two cysteines, we showed that these residues appear to be critical to receptor activity, and the three different receptors had different sensitivities to the single residue substitution. Finally, we examined the roles of those residues outside the disulfide bond ring by removing these residues and found that they also appeared to be important to receptor activity. Thus, our study provides an important basis for further study of the functions of elevenin and its receptors in Aplysia and other molluscs.

IP6 reduces colorectal cancer metastasis by mediating the interaction of gut microbiota with host genes.

Inositol hexaphosphate (IP6) is a phytochemical widely found in grains and legumes that plays an anti-cancer role. However, the mechanism underlying the inhibition of colorectal cancer metastasis by IP6 through host genes, gut microbiota, and their interactions remain elusive. In this study, 16S rRNA sequencing was used to study the effect of IP6 on gut microbiota in an orthotopic transplantation model of colorectal cancer mice. The transcriptome was used to study the changes of host genes in metastasis and the relationship with gut microbiota. The results showed that the gut microbiota composition of model mice was significantly different from that of normal mice. The beta diversity partly tended to return to the normal level after IP6 intervention. Especially, Lactobacillus helveticus and Lactococcus lactis were recovered after IP6-treated. Enrichment analysis showed that the enrichment score of the Cytokine-Cytokine receptor interaction signal pathway decreased after IP6 treatment compared to the model group. Further analysis of differentially expressed genes (DEGs) in this pathway showed that IP6 reduced the expression of the Tnfrsf1b gene related to the area of liver metastasis, and the Tnfrsf1b gene was negatively correlated with the relative abundance of Lactobacillus helveticus. Our results presented that host gene, microbiome and their interaction may serve as promising targets for the mechanism of IP6 intervention in colorectal cancer metastasis.

Increased expression of Myosin X contributes to the metastasis in patients with laryngeal squamous cell carcinoma.

Laryngeal Squamous Cell Carcinoma (LSCC) is one of the most common malignancy in Head and neck cancer for which the mechanism underlying its metastasis is poorly understood. Myosin X, a molecular motor in cells has been demonstrated to play an important role in cell migration. However, whether Myosin X is involved in the metastasis of LSCC remains unclear. To investigate the expression of Myosin X and its implication in the metastasis of LSCC, we recruited 30 patients with LSCC and 6 patients with vocal cord polyp range from October 2016 to October 2018. Tissue samples were obtained during surgery and the expression of Myosin X, Cortactin, MMP2, MMP9, E-cadherin, and ß-catenin in tissue samples were evaluated by RT-PCR, Western blot, immunohistochemistry or ELISA. Patients with LSCC were further followed-up 2 year after surgery for metastasis analysis. We found that the level of Myosin X, Cortactin, MMP2, and MMP9 was much higher in poorly differentiated LSCC compared to that in moderately and highly LSCC, as well as the control tissues. In contrast, the expression of epithelial-mesenchymal transition related marker, E-cadherin, and ß-catenin, were much lower in poorly differentiated LSCC tissues compared to that in moderately and highly differentiated LSCC tissues, as well as the control tissues. Moreover, the expression of Myosin X was positively correlated with Cortactin, MMP2, and MMP9 levels. Increased expression of Myosin X in LSCC tissues was related to higher risk of metastasis. In conclusion, our findings showed that. Myosin X augments the expression of Cortactin, MMP2 and MMP9, which could upregulate the cell migration and the matrix degradation, and consequently reduce the expression of E-cadherin and ß-catenin, thereby activating epithelial-mesenchymal transformation and promoting the metastasis of LSCC. Targeting Myosin X may have potential therapeutic effect in the metastasis of LSCC.

Butyrate ameliorates alcoholic fatty liver disease via reducing endotoxemia and inhibiting liver gasdermin D-mediated pyroptosis.

BACKGROUND: Alcoholic fatty liver disease (AFLD) is characterized by hepatic steatosis and carries an elevated risk of cirrhosis and hepatocellular carcinoma. However, the mechanism of AFLD has not been elucidated thoroughly, and there are still no efficient therapies in clinic. Notably, butyrate, one kind of short-chain fatty acids produced by gut microbiota, has been shown to improve methionine-choline-deficient diet-induced non-alcoholic steatohepatitis. And our previous study found that butyrate ameliorated endotoxemia in db/db mice. In this study, we aimed to explore the role of butyrate in the development of AFLD. METHODS: C57BL/6 mice were treated with saline (normal control), alcohol with or without butyrate by gavage for 6 months. AFLD was evaluated by the levels of serum alcohol, aspartate aminotransferase (AST), alanine transaminase (ALT), triglyceride (TG) and intrahepatic TG. And the histology and inflammation in liver and colon were analyzed using hematoxylin-eosin (H&E) staining, immunohistochemistry and western blot. In addition, gut microbiota composition was analyzed using the V3-V4 regions of the bacterial 16S ribosomal RNA gene by sequence. Furthermore, we performed in vitro experiment to verify the role of butyrate in hepatocyte by western blot and transmission electron microscopy. RESULTS: We found that butyrate ameliorated alcohol-induced hepatic steatosis and inflammation. Furthermore, chronic alcohol feeding induced dysbiosis and dysfunction of the gut microbiota, disrupted the intestinal barrier, and increased serum endotoxin levels. Meanwhile, butyrate improved the intestinal barrier disruption and endotoxemia induced by alcohol, but did not significantly alleviate the microbiome dysfunction. Mechanistically, butyrate ameliorated AFLD by inhibiting gasdermin D (GSDMD)-mediated pyroptosis. CONCLUSIONS: In summary, we found butyrate ameliorated alcoholic fatty liver by down-regulating GSDMD-mediated pyroptosis. We speculate that butyrate improves AFLD mainly by maintaining intestinal barrier function and alleviating gut leakage. These findings suggest that butyrate may have the potential to serve as a novel treatment for AFLD.

Dysoxylactam A: A Macrocyclolipopeptide Reverses P-Glycoprotein-Mediated Multidrug Resistance in Cancer Cells.

A 17-membered macrocyclolipopeptide, named dysoxylactam A (1) comprising an unprecedented branched C19 fatty acid and an l-valine, was isolated from the plants of Dysoxylum hongkongense. The challenging relative configuration of 1 was established by means of residual dipolar coupling-based NMR analysis. The absolute configuration of 1 was determined by single-crystal X-ray diffraction on its p-bromobenzoate derivative (2). Compound 1 dramatically reversed multidrug resistance in cancer cells with the fold-reversals ranging from 28.4 to 1039.7 at the noncytotoxic concentration of 10 µM. The mode-of-action study of 1 revealed that it inhibited the function of P-glycoprotein (P-gp), a key mediator in multidrug resistance.

From Hyper- to Hypoinsulinemia and Diabetes: Effect of KCNH6 on Insulin Secretion.

Glucose-stimulated insulin secretion from islet ß cells is mediated by KATP channels. However, the role of non-KATP K+ channels in insulin secretion is largely unknown. Here, we show that a non-KATP K+ channel, KCNH6, plays a key role in insulin secretion and glucose hemostasis in humans and mice. KCNH6 p.P235L heterozygous mutation co-separated with diabetes in a four-generation pedigree. Kcnh6 knockout (KO) or Kcnh6 p.P235L knockin (KI) mice had a phenotype characterized by changing from hypoglycemia with hyperinsulinemia to hyperglycemia with insulin deficiency. Islets from the young KO mice had increased intracellular calcium concentration and increased insulin secretion. However, islets from the adult KO mice not only had increased intracellular calcium levels but also had remarkable ER stress and apoptosis, associated with loss of ß cell mass and decreased insulin secretion. Therefore, dysfunction of KCNH6 causes overstimulation of insulin secretion in the short term and ß cell failure in the long term.

The truncated mutant HBsAg expression increases the tumorigenesis of hepatitis B virus by regulating TGF-ß/Smad signaling pathway.

BACKGROUND: It has been reported that the emergence of HBV rtA181T/sW172* mutant could result in a dominant secretion defect of HBsAg and increase the risk of HCC development. This study was designed to reveal the role and possible pathogenic mechanism of truncated mutant HBsAg in tumorigenesis of HBV rtA181T/sW172* mutant. RESULTS: As compared to wide type or substituted mutant HBsAg, the ratio of cell clones was significant higher in L02 cells stable expressing truncated mutant HBsAg. Injection of L02 cells stable expressing truncated mutant HBsAg into the dorsal skin fold of nude mice resulted in increased primary tumor growth compared to L02 cells stable expressing wide-type and substituted mutant HBsAg. In HBV replication L02 cell lines, the key molecular involved in TGF-ß/Smad pathway was also investigated. We found that the mRNA and protein levels of Smad3/2, CREB and CyclinD1 were significantly higher and TGFBI level was significantly lower in cells stably expressing truncated mutant HBsAg as compared to cells stably expressing wide-type and substituted mutant HBsAg. Additionally, after administration of TGF-ß1 (increasing TGFBI level), the volume of tumor is obviously reduced in nude mice with injection of L02 cells stable expressing truncated HBsAg. CONCLUSIONS: The emergence of sW172* mutant may increase the tumorigenesis of HBV, and its mechanism may be associated with down-regulated expression of TGFBI in TGF-ß/Smad signaling pathway.

Preliminary research on DNA methylation changes during murine palatogenesis induced by TCDD.

2,3,7,8-Tetrachlrodibenzo-p-dioxin (TCDD) has been shown to induce cleft palate through growth factor and receptor expression changes during palatogenesis. DNA methylation is an important epigenetic modification that can regulate gene expressions and may be involved in TCDD-induced cleft palate. In this study, we investigated the effects of TCDD on the global and CpG DNA methylation status and the expression levels of DNA methyltransferases (Dnmts) in palate tissue of fetal mice. Pregnant C57BL/6J mice were administered with corn oil or TCDD 28 µg/kg at gestation day 10.5(GD10.5), and sacrificed at GD13.5, 14.5, 15.5. Fetal palates were collected for molecular analysis. Global DNA methylation status was detected by Methylamp™ Global DNA Methylation Quantification Ultra Kit. The expression of DNA methyltransferases were examined by quantitative real-time PCR(q-PCR). Methylation Specific PCR (MSP) was performed to analyze CpG methylation status of Dnmts. We found that the global DNA methylation level and the expression of Dnmt3a were higher at GD13.5 in the TCDD group. The methylation level of CpG site 2 in the promoter region of Dnmt3a in the control group was higher than that of the TCDD group at GD13.5. The low CpG methylation level of Dnmt3a at GD13.5 which causes the up-expression of Dnmt3a may induce global hypermethylation in fetal palate tissue. The aberrant global methylation status at GD13.5 may be the cause of palate malformation in fetal mice induced by TCDD.

Biological characteristics comparison of HBV rtA181T mutants with truncated or substituted HBsAg expression in vitro and in vivo model systems.

The hepatitis B virus(HBV) polymerase rtA181T mutation is selected during long-term antiviral therapy. As the polymerase gene completely overlaps with the envelope (S) gene, HBV rtA181T mutation also carries sW172 mutations. In this study, we investigated whether there were biological differences between rtA181T/sW172* (coding truncated HBsAg) and rtA181T/sW172L (coding substituted HBsAg) mutants. In cell experiments, a slight decline of viral replication was observed in both two mutants as compared to wild-type strains, but the levels of supernatant HBsAg and HBV DNA in rtA181T/sW172* were significantly lower than those in rtA181T/sW172L transfected cells. In animal experiments, we were amazed to find that viral replication in rtA181T/sW172* mutant increased and maintained significantly longer than that in rtA181T/sW172L mutant, while no significant difference was observed between rtA181T/sW172L and wild-type strains. Compared with wild-type strains, there were intracellular accumulations of HBsAg and HBcAg in rtA181/sW172* but none in rtA181/sW172L mutant strains. Importantly, we also found that truncated HBsAg could increase the activity of HBV core promoter, but substituted HBsAg could not. In summary, the characteristics of above two rtA181T mutants mentioned above were significantly different, and it is necessary and important for us to distinguish sW172* truncated mutation from sW172L substituted mutation.

Ciliatonoids A and B, Two Limonoids from Toona ciliata.

Three new ring B-seco limonoids, ciliatonoids A-C (1-3), were isolated from Toona ciliate and structurally characterized by spectroscopic data, X-ray crystallography, and electronic circular dichroism analysis. Ciliatonoids A and B feature an unprecedented limonoid architecture, while ciliatonoid C belongs to a rare class of limonoids. Biological evaluation showed that compound 3 exhibited modest activities against the tested tumor cell lines.

Enhancement of CD4(+) T cell response and survival via coexpressed OX40/OX40L in Graves' disease.

OX40/OX40L pathway plays a very important role in the antigen priming T cells and effector T cells. In the present study, we aimed to examine the involvement of OX40/OX40L pathway in the activation of autoreactive T cells in patients with Grave's disease (GD). We found that OX40 and OX40L were constitutively coexpressed on peripheral CD4(+) T cells from GD patients using flow cytometry analysis. The levels of OX40 and OX40L coexpression on CD4(+) T cells were shown to be correlated with TRAbs. Cell proliferation assay showed that blocking OX40/OX40L signal inhibited T cell proliferation and survival, which suggested that OX40/OX40L could enhance CD4(+) T cell proliferation and maintain their long-term survival in GD by self-enhancing loop of T cell activation independent of APCs. Confocal microscopy and coimmunoprecipitation analysis further revealed that OX40 and OX40L formed a functional complex, which may facilitate signal transduction from OX40L to OX40 and contribute to the pathogenesis of GD.

Diagnostic Value of Common Inflammatory Markers on Fever of Unknown Origin.

This study was designed to evaluate the diagnostic value of common inflammatory markers with regard to fever of unknown origin (FUO). We investigated 383 patients who were hospitalized with FUO at the Henan Province People's hospital between January 2009 and June 2015. Of all the cases, infectious diseases accounted for 33.9%, neoplasms for 21.1%, collagen vascular diseases for 25.1%, miscellaneous diseases for 4.7%, and no diagnosis for 15.1%. Patients in the neoplasm group were older than those in the infectious disease, collagen vascular disease, and miscellaneous disease groups (p = 0.006, p < 0.0001, and p = 0.001, respectively). The duration of fever before admission of patients in the neoplasm and collagen vascular disease group was longer than that of patients in the infectious disease group (p = 0.002 and p = 0.007, respectively). The diagnostic time after admission of patients from the neoplasm and collagen vascular disease groups was longer than that for patients from the infectious disease group (both p < 0.0001). Serum ferritin levels of patients in the infectious disease group were lower than those of patients in the neoplasm and collagen vascular disease groups (p = 0.029 and p = 0.032, respectively), while serum procalcitonin (PCT) levels in the infectious disease group was higher than that in the neoplasm and collagen vascular disease groups (p = 0.016 and p = 0.007, respectively). Therefore, FUO remains a clinical problem in China and serum ferritin and PCT may be useful in discriminating infectious from non-infectious causes (neoplasms and collagen vascular diseases) in patients with FUO.

Diterpenoids from Croton laui and their cytotoxic and antimicrobial activities.

Fourteen new diterpenoids including clerodane (1-12), labdane (13), and norlabdane (14) types, as well as nine known analogues were isolated from the aerial parts of Croton laui. Their structures were established on the basis of spectroscopic analysis, and that of crotonolide H (11) was confirmed by single-crystal X-ray crystallography. Crotonolide A (1) exhibited moderate cytotoxicity against two tumor cell lines, HL-60 (human premyelocytic leukemia, IC50 9.42 µM) and P-388 (murine leukemia, IC50 7.45 µM), and crotonolide G (10) displayed significant antibacterial activity against a panel of Gram-positive bacteria.

Activated effects of parathyroid hormone-related protein on human hepatic stellate cells.

BACKGROUND & AIMS: After years of experiments and clinical studies, parathyroid hormone-related protein(PTHrP) has been shown to be a bone formation promoter that elicits rapid effects with limited adverse reaction. Recently, PTHrP was reported to promote fibrosis in rat kidney in conjunction with transforming growth factor-beta1 (TGF-ß1), which is also a fibrosis promoter in liver. However, the effect of PTHrP in liver has not been determined. In this study, the promoting actions of PTHrP were first investigated in human normal hepatic stellate cells (HSC) and LX-2 cell lines. METHODS: TGF-ß1, alpha-smooth muscle actin (α-SMA), matrix metalloproteinase 2 (MMP-2), and collagen I mRNA were quantified by real-time polymerase chain reaction (PCR) after HSCs or LX-2 cells were treated with PTHrP(1-36) or TGF-ß1. Protein levels were also assessed by western-blot analysis. Alpha-SMA were also detected by immunofluorescence, and TGF-ß1 secretion was measured with enzyme-linked immunosorbent assay (ELISA) of HSC cell culture media. RESULTS: In cultured human HSCs, mRNA and protein levels of α-SMA, collagen I, MMP-2, and TGF-ß1 were increased by PTHrP treatment. A similar increasing pattern was also observed in LX-2 cells. Moreover, PTHrP significantly increased TGF-ß1 secretion in cultured media from HSCs. CONCLUSIONS: PTHrP activated HSCs and promoted the fibrosis process in LX-2 cells. These procedures were probably mediated via TGF-ß1, highlighting the potential effects of PTHrP in the liver.

[The antagonistic effect of folic acid and resveratrol on cleft palate in mice induced by TCDD].

OBJECTIVE: To evaluate whether or not administration of folic acid and resveratrol have preventive effects on cleft palate formation as well as the comparison of the two drugs' s effects. METHODS: Pregnant mice were randomly divided into 9 groups, with 8 mice in each group. The TCDD group mice were dosed with TCDD 28 microg/kg body weight on gestation day 10 (GD 10) animals in folic acid group were respectively dosed with folic acid 15, 10, 5 mg/kg and TCDD 28 microg/kg; resveratrol treated mice were divided into 3 groups: resveratrol 50 mg/kg were orally administered for 6 consecutive days, from gestational day GD 8 to GD13 in resveratrol (GD8-13 ) group; resveratrol 50 mg/kg were orally administered for 6 consecutive days, from gestational day GD 8 to GD13, followed hy an oral administered with TCDD on GD10 in resveratrol (GD8-13) + TCDD group; resveratrol 50mg/kg and TCDD 28 microg/kg were used by gavage administration at GD10 in resveratrol (GD10) + TCDD group. Control mice were treated with the same volume of water for 6 consecutive days from GD8 to GD13 and were given a single dose of corn oil on GD10. The pregnant mice weight and embryos, the number of live, cleft palate, dead and resorption fetal mice were recorded on GD 17.5. The coronal sections of the fetal mice heads were prepared at GD 17.5 and observed by microscopy. RESULTS: Total frequency of clefts was 92.86% in TCDD group, 84.00% (15 mg), 73.08% (10 mg), 84.00% (5 mg) in folic acid + TCDD groups, 0% in resveratrol (GD10) group, 74.51% (GD10), 57.78% (GD8-13) in resveratrol + TCDD groups. The frequency of cleft was 0% in the control group. Compared with the control and the TCDD groups, there were significant differences in the number of live, dead and resorption fetal mice in TCCD + resveratrol (GD8-13) group (P < 0.05). No significant differences in embryonic weight, live fetuses weight, the number of live, dead and resorption fetal mice were found in the other groups (P > 0.05). CONCLUSION: Test dose of folic acid and resveratrol both had certain antagonistic effect on cleft palate in mice induced by TCDD, with folic acid 10 mg/kg, resveratrol 50 mg/kg GD8-13 doses having stronger antagonistic action. Effects of both the two drugs have no significant difference, but resveratrol (50 mg/kg, GD8-13) significantly affects the fetal mice's growth and development under TCDD exposure in utero.

[Expression of B7-H3 costimulatory molecule in peripheral blood of myasthenia gravis patients].

AIM: To reveal the clinical significance of B7-H3 costimulatory molecule in myasthenia gravis (MG) patients by analyzing membranous B7-H3 (mB7-H3) and soluble B7-H3 (sB7-H3) expressions. METHODS: We collected peripheral blood samples of 35 MG patients and 44 health controls (HC) and detected the expression of mB7-H3 on peripheral blood mononuclear cells (PBMCs) using flow cytometry. ELISA was performed to analyze the levels of sB7-H3 in plasma samples from MG patients and HC. RESULTS: There was no significant difference in the expressions of mB7-H3 on T lymphocytes, monocytes or B cells between MG patients and HC. However, the level of sB7-H3 from MG patients was (2.166±0.958) ng/mL, significantly lower than that from HC (3.379±0.768) ng/mL. The level of sB7-H3 in general MG (GMG) patients (1.664±0.699) ng/mL was lower than that in ocular MG (OMG) patients (2.396±0.985) ng/mL. In MG patients complicated with abnormal thymus, the level of sB7-H3 was (1.593±0.441) ng/mL, also lower than that in MG patients with normal thymus (2.364±1.014) ng/mL. In addition, a significant negative correlation was found between the levels of sB7-H3 and QMGS in MG patients (r=-0.4189, P=0.012), but sB7-H3 was not associated with mB7-H3 in MG patients. CONCLUSION: In MG patients, down-regulation of sB7-H3 is finely correlated to the severity of the disease. Its different expression levels in various types of MG patients indicate that this costimulatory molecule may be involved in the immunopathogenesis of MG.

[Allergen analysis on patients with allergic rhinitis in Lanzhou].

OBJECTIVE: To investigate the effective clinical diagnosis and treatment for allergic rhinitis (AR) by examining the allergens and dietary habits of the patients. METHODS: A total of 347 patients from Lanzhou area were sampled between April 2010 and March 2012. Free point skin prick test were performed to analyze the allergens. SPSS 17.0 software was used to analyze the data. RESULTS: Among 347 patients, 266 (76.7%) were found to be positive in the skin test. Major aeroallergens were Artemisia pollen (38.0%, 132 cases), house dust mite (35.4%, 123 cases), and Dermatophagoides farinae (30.3%, 105 cases). CONCLUSIONS: The major aeroallergens in Lanzhou with AR were artemisia pollen and mite.

Association analysis of the RET proto-oncogene with Hirschsprung disease in the Han Chinese population of southeastern China.

Hirschsprung disease (HSCR) is a complex congenital disorder characterized by intestinal obstructions caused by the absence of the intestinal ganglion cells of the nerve plexuses in variable lengths of the digestive tract. This study investigated a possible role of the RET proto-oncogene in sporadic HSCR patients in the Han Chinese population. Our results indicated that rs1800858, rs1800860, rs1800863, and rs2075912, located in exons 2, 7, 15, and intron 19 of RET, are strongly associated with the disease (P < 0.01), with rs1800860 and rs1800863 playing a protective role in the pathogenesis of HSCR in the Chinese population. We also showed that the haplotype consisting of four SNPs is significantly associated with HSCR. We did not find a significant difference in the CA-repeat in intron 5 of RET between cases and controls. Our study provided further evidence that the RET gene is involved in the susceptibility to HSCR in the Han Chinese population.

Dexamethasone enhances trichosanthin-induced apoptosis in the HepG2 hepatoma cell line.

AIMS: Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) with antitumor activities for various cancers. In this paper, we aimed to investigate whether dexamethasone, an important synthetic member of the glucocorticoid steroids, in combination with TCS can be a potential therapy in treating hepatoma. MAIN METHODS: Cell viability was investigated using MTT assay, and apoptosis was evaluated with Hoechst 33258 staining. Western blot analysis was used to examine the changes in the expression levels of IkappaB-alpha, NF-kappaB p65 subunit and Cox-2. Additionally, we took advantage of dominant-negative IkappaB (IkappaB-DM) over-expression and chemical inhibitor PDTC to inhibit NF-kappaB activation. KEY FINDINGS: Our results demonstrated that dexamethasone could enhance TCS-induced apoptosis in the hepatoma cell line HepG2, decreasing IC50 values from in excess of 200microg/ml to 50microg/ml. In addition, our results demonstrated that TCS could induce rapid degradation of IkappaB-alpha, nuclear translocation of NF-kappaB and decrease of COX-2 expression in HepG2 cells. Inhibition of NF-kappaB by biological (IkappaB-DM) or chemical inhibitor (PDTC) increased HepG2 cells' sensitivity to TCS, resulting in cell viability rate decreasing and apoptotic rate increasing. Simultaneously, dexamethasone increased the level of IkappaB-alpha protein and effectively inhibited TCS-induced degradation of IkappaB-alpha. SIGNIFICANCE: These results suggest that dexamethasone could enhance trichosanthin-induced apoptosis in the HepG2, at least in part, by inhibiting the NF-kappaB signaling pathway and thus strengthening the antitumor effects of TCS, which highlights the possibility of combined drug application of TCS and dexamethasone in the clinical treatment of hepatoma.

[Methylation of Id4 gene and inhibitive effect of arsenic trioxide on it in Raji cells].

OBJECTIVE: To study methylation of Id4 gene and demethylation effect of arsenic trioxide (ATO) in Raji cells. METHODS: Human Burkitt's Raji lymphoma cells were cultared and treated with ATO at different concentrations and different time points. Methylated degree of Id4 gene was detected by methylation specificity polymerase chain reaction (MS-PCR), Id4 mRNA expression in Raji cell by reverse transcription polymerase chain reaction (RT-PCR), the growth of cell by MTT assay, and cell apoptosis and cycle distribution by Flow Cytometry (FCM). RESULTS: (1) The Id4 gene exhaustive methylation in control group, and hypermethylation in experimental group were reversed by ATO in a dose-dependent manner. (2) Id4 mRNA expression in Raji cells treated with ATO for 48 h increased gradually with ATO concentration increasing in experimental group. (3) Raji cell growth inhibited rates after different concentrations of ATO treatment for 24, 48, 72 h were 12.15% ∼ 92.17% in the experimental group (P < 0.05). (4) Apoptosis peak emerged after ATO treatment for 48 hours in experimental group, while a much lower apoptosis in control group. (5) After ATO treatment for 48 h in experiment group, the cells were arrested at G(0)/G(1) phase in a dose-dependent manner. CONCLUSION: Id4 gene presents exhaustive methylation in Raji cells. ATO can reverse the hypermethylation of Id4 gene and recover the expression of Id4 mRNA. Hypermethylation of Id4 gene is one of the reasons of Raji cells malignant proliferations.