Molecular Mechanism Of Inhibitory Effect Research Articles

Regulation of Metabolic Homeostasis By TRAF6 Contributes to the Leukemia Progression

TNF receptor associated factor 6 (TRAF6) is an E3 ubiquitin ligase that mediates innate immune signaling that converges on key mediators of Toll-like receptor and interleukin-1 signaling. Dysregulated TRAF6 expression and innate immune pathway activation is implicated in the pathogenesis of various myeloid malignancies. For instance, increased TRAF6-mediated signaling in hematopoietic stem and progenitor cells (HSPCs) results in myelodysplastic syndrome-like cellular features, such as the functional impairment of HSPCs. On the other hand, our previous study indicated that loss of TRAF6 induces the leukemic transformation of pre-leukemic cells ( Muto et al. Cell Stem Cell). These paradoxical observations suggests that TRAF6 acts as both an oncogene and tumor suppressor gene in hematopoietic cells in a disease type- and context-dependent manner. However, the direct consequence of TRAF6 loss in AML cells, rather than in pre-leukemic cells, has not yet been explored. Therefore, the role of TRAF6 in the pathogenesis of AML remains obscure. In this study, we found elevated level of TRAF6 expression in AML cells, and determined the effect of TRAF6 loss on the cellular and molecular features of AML cells. We proposed a novel role of TRAF6 as a regulator of metabolic homeostasis, which is important for the progression of AML. To determine the requirement of TRAF6 in AML, we first evaluated cell the effect of TRAF6 loss in AML cell lines on proliferative capacities. Multiple types of human AML cell lines showed significant growth impairment upon TRAF6-knockdown. Furthermore, transplantation of TRAF6-deficient HSPCs expressing the leukemic oncogene MLL-AF9 to the recipient mice resulted in suppression of overt leukemia. These findings confirm that loss of TRAF6 suppresses leukemic cell function in vitro and in vivo. To assess the molecular mechanism of inhibitory effect of TRAF6 loss in AML, we next performed gene expression analysis using human AML database and TRAF6-knockdown AML cell lines. Interestingly, we found that reduced levels of TRAF6 in AML is associated with changes in the expression of genes involved in mitochondria-related processes. Our extensive in vitro validation experiments including extracellular flux assay and metabolome analysis confirmed that TRAF6 loss in AML cells induces dynamic changes in metabolites and the impairment of mitochondrial function. To identify the mechanism of metabolic alterations by TRAF6 loss in leukemia, we compared the upregulated genes in TRAF6-knockdown AML cells with the 130 previously identified essential genes for AML cell survival in vitro and in vivo ( Yamauchi et al. Cancer Cell). We focused on O-GlcNAc transferase ( OGT) as a gene associated in mitochondrial function and AML cell survival. Importantly, TRAF6 gene expression in human AML patient samples was positively correlated with the expression of OGT. To confirm the importance of OGT in the regulation of mitochondrial function mediated by TRAF6 in leukemia, we utilized the inhibitor of O-GlcNAcase (OGA), a remover of O-GlcNAc from its substrates. The treatment with MK8719 (OGA inhibitor) mildly, but significantly, restored the number of TRAF6 knockdown AML cells, which was correlated with the changes in mitochondrial function, indicating that O-GlcNAc modification regulated by TRAF6 is important for AML progression. In summary, We provide evidence for the oncogenic function of TRAF6 in leukemia, and shed light on the novel TRAF6/OGT/O-GlcNAc axis that regulates the metabolic reprogramming required for leukemogenesis.

Molecular mechanism of inhibitory effects of melatonin on prostate cancer cell proliferation, migration and invasion.

The increasing incidence of prostate cancer (PCa) indicates an urgent need for the development of new effective drug therapy. There are limited options to treat the PCa, this study tried to determine a new therapy option for this acute cancer. Androgen-independent PCa cell lines PC3 and DU145 were treated with different melatonin concentrations (0.1~3.5 mM) for 1~3 days and assessed cell migration, cell invasion, cycle arrest in G0/G1 phase as well as apoptosis. We utilized RNA-seq technology to analyze the transcriptional misregulation pathways in DU145 prostate cancer cell line with melatonin (0.5 mM) treatment. Data revealed 20031 genes were up and down-regulated, there were 271 genes that differentially expressed: 97 up-regulated (P<0.05) and 174 down-regulated (P<0.05) genes. Furthermore, RNA-seq results manifested that the melatonin treatment led to a significant increase in the expression levels of HPGD, IL2Rβ, NGFR, however, IGFBP3 and IL6 (P <0.05) had decreased expression levels. The immunoblot assay revealed the expression of IL2Rβ and NGFR genes was up-regulated, qPCR confirmed the gene expression of HPGD and IL2RB were also up-regulated in Du145 cells. Consequently, we probed mechanisms that generate kinetic patterns of NF-κB-dependent gene expression in PCa cells responding to a NF-κB-activation, the significant results were indicated by the inhibition of the NF-kB pathway via IL2Rβ actions. Based on our investigation, it could be concluded that melatonin is a chemotherapeutic molecule against PCa and provides a new idea for clinical therapy of PCa.

Hedyotis Diffusa Willd. Extracts Inhibit the Proliferation and Migration of Ovarian Cancer Cells by Modulating the AKT/ERK Signaling Pathway

Hedyotis diffusa Willd. (HDW) is a traditional Chinese medicine used for the treatment of colorectal cancer. The aim of this study was to investigate the molecular mechanism of inhibitory effects of HDW on the growth of ovarian cancer cells. Treatment of ovarian cancer cells with increasing doses of HDW for 48 or 72 h resulted in a significant reduction in the viability and colony formation of cancer cells. In HDW-treated cancer cells, the cell cycle was blocked at the G0/G1 phase and cell apoptosis was 4- to 5-fold higher than that of control group. HDW treatment also markedly inhibited the migration and invasion of ovarian cancer cells. Mechanistically, the phosphorylation levels of AKT and ERK were down-regulated in HDW-treated ovarian cancer cells. Therefore, HDW is likely to become a promising therapeutic agent for ovarian cancer treatment.

Molecular mechanism of inhibitory effects of bovine lactoferrin on the growth of oral squamous cell carcinoma.

BackgroundLactoferrin (LF), a member of the transferrin family, recently has been demonstrated to have anticancer effects on various cancers including oral squamous cell carcinoma (OSCC). However, little is known about the underlying mechanisms of its effects on OSCC. Therefore, we aimed to investigate the mechanism of the suppressive effects of bovine LF (bLF) on the growth of OSCC cells.MethodsIn the current study, HSC2, HSC3, HSC4 and normal human oral keratinocytes (RT7) cell lines were tested with bLF 1, 10, and 100 μg/ml. The effects and detail mechanisms of bLF on proliferation and apoptosis of cells were investigated using flow cytometry and western blotting.ResultsWe found that bLF (1, 10, and 100 μg/ml) induced activation of p53, a tumor suppressor gene, is associated with the induction of cell cycle arrest in G1/S phase and apoptosis in OSCC. Moreover, bLF downregulated the phosphorylation of Akt and activated suppressor of cytokine signaling 3 (SOCS3), thereby attenuating multiple signaling pathways including mTOR/S6K and JAK/STAT3. Interestingly, we revealed that bLF exerted its effect selectively against HSC3 but not on RT7 via different effects on the phosphorylation status of NF-κB and Akt.ConclusionThis is the first report showing that bLF selectively suppresses proliferation through mTOR/S6K and JAK/STAT3 pathways and induction of apoptosis in OSCC. This study provides important new findings, which might be useful in the prevention and treatment of OSCC.

Molecular mechanism of inhibitory effects of C-phycocyanin combined with all-trans-retinoic acid on the growth of HeLa cells in vitro

We studied the effects of all-trans-retinoic acid (ATRA), C-phycocyanin (C-PC), or ATRA+C-PC on the growth of cervical cells (HeLa cells), cell cycle distribution, and apoptosis. The anticancer mechanism of the drug combination was revealed. MTT assay was adopted to determine the effects of C-PC and ATRA on the growth of HeLa cells. The expression quantities of cyclin-dependent kinase (CDK) 4, cyclin D1, Bcl-2, caspase-3, and CD59 were determined by in situ hybridization, immunofluorescence, immunohistochemistry staining, Western blot, and RT-PCR. TUNEL assay was adopted to determine the cellular apoptosis levels. Both C-PC and ATRA could inhibit the growth of HeLa cells, and the combination of ATRA+C-PC functioned cooperatively to induce apoptosis in HeLa cells. The dosage of ATRA was reduced when it cooperated with C-PC to reduce the toxicity. ATRA treated with C-PC could induce more cell cycle arrests than the single drug used by decrease in cyclin D1 and CDK4 expression. The combination of the two drugs could upregulate caspase-3 and downregulate the Bcl-2 gene and induce cell apoptosis. Moreover, the combination therapy has an important immunological significance in decreased expression of the CD59 protein. Singly, C-PC or ATRA could inhibit the growth of HeLa cells, and the effects of treatment were further enhanced in the combination group. In combination with C-PC, the dosage of ATRA was effectively reduced. The C-PC + ATRA combination might take effect by inhibiting the progress of the cell cycle, inducing cell apoptosis and promoting complement-mediated cytolysis.

Human umbilical cord mesenchymal stem cells inhibit C6 glioma growth via secretion of dickkopf-1 (DKK1)

Mesenchymal stem cells (MSCs) represent a potential therapeutic target for glioma. We determined the molecular mechanism of inhibitory effect of human umbilical cord-derived MSCs (hUC-MSCs) on the growth of C6 glioma cells. We demonstrated that hUC-MSCs inhibited C6 cell growth and modulated the cell cycle to G0/G1 phase. The expression of β-catenin and c-Myc was downregulated in C6 cells by conditioned media from hUC-MSCs, and the levels of secreted DKK1 were positively correlated with concentrations of hUCMSCs-CM. The inhibitory effect of hUC-MSCs on C6 cell proliferation was enhanced as the concentration of DKK1 in hUCMSCs-CM increased. When DKK1 was neutralized by anti-DKK1 antibody, the inhibitory effect of hUC-MSCs on C6 cells was attenuated. Furthermore, we found that conditioned media from hUC-MSCs transfection with siRNA targeting DKK1 mRNA or pEGFPN1-DKK1 plasmid lost or enhanced the abilities to regulate the Wnt signaling in C6 cells. Therefore, hUC-MSCs inhibited C6 glioma cell growth via secreting DKK1, an inhibitor of Wnt pathway, may represent a novel therapeutic strategy for malignant glioma.

Molecular mechanism of inhibitory effects of CD59 gene on atherosclerosis in ApoE (−/−) mice

BackgroundHow to find an effective gene locus resistant to atherosclerosis has become a hotspot of today's medicine. Membrane attack complex (MAC) has proved to be related with the occurrence and development of atherosclerosis. Complement regulatory protein CD59 is a key regulator of complement MAC assembly. So this study aimed at discussing the effects of CD59 gene on occurrence and development of atherosclerosis and relative mechanism. MethodsApolipoprotein E knockout (ApoE (−/−)) mice were randomly divided into four groups: control group, empty plasmid-treated group, 0.5ml CD59-treated group and 1.0ml CD59-treated group. At the end of the 12th week, CD59 mRNA levels in whole blood were determined by RT-PCR and CD59 protein expressions were detected by western blot. The biochemical indexes in blood serum were detected. The paraffin sections of aortic root of mice were made and the degrees of atherosclerotic plaques formation were observed by hematoxylin/eosin (HE) staining. The expressions of cell apoptosis-related proteins (Bcl-2 and Fas) and plaque stability related protein (MMP-2) were detected by immunohistochemistry. Then the cell apoptosis levels were detected by TUNEL, the expression of Cyclin D1 and the mRNA level of cyclin dependent protein kinase 4 (CDK4) were detected by immunofluorescence and in situ hybridization, respectively. ResultsAtherosclerotic mouse model was successfully established. CD59 gene was overexpressed in blood cells and tissue cells after liposome transfection. CD59 could reduce blood lipid levels, promote the expression of anti-apoptotic Bcl-2 protein and inhibit pro-apoptotic Fas proteins, so finally lead to degradation of apoptosis levels of endothelial cells. In addition, Cyclin D1 protein and CDK4 mRNA levels were restrained by CD59 so as to inhibit the proliferation of smooth muscle cells. CD59 could inhibit the formation of atherosclerotic vulnerable plaque by suppressing the MMP-2 expression, which was further confirmed by HE staining. The anti-atherosclerotic effects were enhanced with the increase of CD59 gene dose. ConclusionsCD59 could lower blood lipid levels, positively regulate cell cycle, maintain the stability of cell proliferation and apoptosis of aorta cells, slow down the development of atherosclerotic vulnerable plaque, and finally inhibit the progress of atherosclerosis. So CD59 gene might be a new genetic locus for the therapy of atherosclerosis.

HIF-1α Inhibits Wnt Signaling Pathway by Activating Sost Expression in Osteoblasts

The nature of the cellular and molecular mechanisms for the transition of avascular cartilage replacement with bone during endochondral ossification remains poorly understood. One of the driving forces is hypoxia. As a master regulator of hypoxia, hypoxia-inducible factor-1α (HIF-1α) has been reported to couple angiogenesis to osteogenesis. Our recent study has demonstrated that osteoblast growth is inhibited under hypoxia and that HIF-1α cooperates with Osterix (Osx) to inhibit Wnt pathway. However, molecular mechanisms for inhibitory effects of HIF-1α on Wnt pathway are not well understood. In this study, our quantitative RT-PCR results revealed that the expression of a Wnt antagonist Sclerostin (Sost) was upregulated in osteoblasts during hypoxia while HIF-1α was upregulated. Treatment of desferrioxamine (DFO), a HIF-1α activator, led to further increase of Sost expression, suggesting that HIF-1α may activate Sost expression. The regulation of Sost gene expression by HIF-1α was then investigated. We performed loss-of-function experiments to examine Sost expression by using siRNA approach against HIF-1α, and found that the inhibition of HIF-1α by siRNA in osteoblasts led to the decrease of Sost expression. To address transcriptional regulation of Sost gene by HIF-1α, transient transfection assay was performed and showed that HIF-1α activated Sost-1 kb promoter reporter activity in a dose-dependent manner. To narrow down the minimal region of Sost promoter activated by HIF-1α, we generated a series of deletion mutants of Sost constructs. It was demonstrated that Sost-260 was the minimal region of Sost promoter for HIF-1α activation and that Sost-106 construct, which lack hypoxia response element, abolished HIF-1α-mediated Sost reporter activation. Gel shift assay showed that HIF-1 bound to the promoter sequence of Sost directly. These findings support our hypothesis that HIF-1α activates Sost expression. This study provides a novel molecular mechanism through which HIF-1α inhibits Wnt signaling in osteoblasts.

The cellular and molecular mechanism of inhibitory effect of asiaticoside on capsular contracture following breast augmentation

To explore the cellular and molecular mechanism of the inhibitory effect of asiaticoside on capsular contracture following breast augmentation. Contracture capsule derived fibroblasts were cultured in medium with different concentration of asiaticoside. The cell proliferation, collage synthesis and alpha-SMA expression were detected by means of 3H-thymidine incorporation, 3H-proline incorporation, and Western-blot. The results were analyzed by SPSS 11.0 with t test. DNA and collagen synthesis of fibroblasts were dramatically inhibited when the asiaticoside reached the concentration of 50 mg/L. The inhibitory rate was 34.7% and 30.1% respectively, showing a significant difference from that in control group (P < 0.05). The inhibitory effect increased with the rise of the asiaticoside concentration in a dose-dependent manner. When the concentration of asiaticoside reached 25 mg/L, the expression of alpha-SMA was down-regulated with an activation index of 1.673, showing a significant difference when compared with that in control group (P < 0.05). asiaticoside can effectively inhibit the DNA and collagen synthesis of capsule-derived fibroblasts. The trans-differentiation of fibroblast to myo-fibroblasts is also prevented by it.

Abstract 1639: The novel c-MET inhibitor, ARQ 197, shows additive growrh-inhibitory effect with erlotinib through enhanced degradation of c-MET protein via ubiquitin/proteasome pathway

Abstract Background c-MET, a receptor of hepatocyte growth factor (HGF), is known to be overexpressed in a variety of human cancers. Recently several c-MET inhibitors are being developed as cancer chemotherapy against this compelling molecular target. ARQ 197 is a new small molecule c-MET inhibitor. Since ARQ 197 shows efficient anti-cancer activity without many severe adverse events in Phase I clinical trials, it is expected to be a useful anticancer agent for clinical treatment. This drug is known to bind to c-MET at neighbor area of its ATP binding site and it does not work as an ATP mimic. In this study, we investigated the molecular mechanism of inhibitory effect on c-MET by ARQ 197 in a non-small cell lung cancer cell (NSCLC) line. Materials and Method We established a c-MET-overexpressed NSCLC line, PC-9/MET, by long term exposure of PC-9 cells to EGFR-tyrosine kinase inhibitor. The cytotoxicity of ARQ 197 was measured by MTT assay. The expression of c-MET protein and mRNA were detected by Western blotting analysis and real time RT-PCR method, respectively. In a c-MET degradation analysis, bortezomib was used for inhibition of proteasome. Results ARQ 197 is equally cytotoxic to parental PC-9 and EGFRTKI-resistant PC-9/MET cell line with GI50 values around 200 nM (?). ARQ 197 showed an additive growth- inhibitory effect with erlotinib in PC-9/MET cells as assessed by an Isobologram analysis. In ARQ 197-treated PC-9/MET cells, a clear decrease of phospho c-MET protein as well as phospho AKT and ERK proteins was observed, however, ARQ 197 also induced a down-regulation of c-MET protein in the cells without any effect on c-MET mRNA level. This ARQ 197 induced degradation was reversed by a concomitant treatment with a proteasome inhibitor, bortezomib in the cells. However, bortezomib did not affect cytotoxicity of ARQ 197 in the same cells. Conclusion These data suggest that ARQ 197 may inhibit c-MET through downregulation of this protein in addition to inhibition of its kinase activity. Furthermore, it may enhance c-MET degradation by the ubiquitin/proteasome pathway. These c-MET inhibitory activities by ARQ 197 should be important for the additive combined effect with erlotinib in PC-9/MET cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1639.

Danshen injection decrease interleukin-13 and eotaxin expression in the lung of asthmatic rats

Objective To investigate the molecular mechanism of inhibitory effect of Danshen injection on allergic airway inflamma-tion of asthma. Methods 30 SD rats were random divided into control group, asthma group and Danshen injection group. Bronchoalveolar lavage fluid (BALF) was collected, and cytology study was conducted. The lung tissue was obtained and pathologic analysis was done through HE staining, lnterleukin-13 (IL-13) and Eotaxin in lung tissue were measured by RT - PCR and immunohistochemistry SP method. Results Compared with the asthma group, less infiltration of inflammatory cells in lung tissues was observed in the Danshen injection group. Total cell number, the percentage of lymphocytes, neutrophils and eosinophils (Eos) in BALF of Danshen injection group were lower than that of asthma group (P<0.05, P<0.01). The expression of IL-13 and eotaxin in lung tissue of asthma group was higher than that of control group and Danshen injection group (P<0.05). The expression of IL-13 was negatively correlated with eotaxin (r=0.90, P< 0.05). Conclusion Danshen injection could suppress airway inflammation of asthmatic rats, which probably be through decreasing the ex-pression of IL-13 and eotaxin in the lung tissue of asthmatic rats. Key words: DANSHEN INJECTION/PD; Asthma; Interleukin-13; Chemotactic factors,eosinophil

Dkk-1 secreted by mesenchymal stem cells inhibits growth of breast cancer cells via depression of Wnt signalling

We determined the molecular mechanism of inhibitory effect of human mesenchymal stem cells (hMSCs) on the growth of human MCF-7 breast cancer cells. Our finding showed that β-catenin was down-regulated in MCF-7 cells by conditioned media from Z3 hMSCs, and the expression level of dickkopf-1 (Dkk-1) was higher in Z3 cells than that in MCF-7 cells. Neutralization of Dkk-1 and small interference RNA targeting Dkk-1 mRNA in Z3 cells attenuated the inhibitory effect of Z3 cells on MCF-7 cells. Overexpression of Dkk-1 in Z3 cells enhanced the inhibition. Therefore, Dkk-1 secreted by Z3 cells involves the inhibition via the Wnt pathway.

The molecular mechanisms of inhibitory effect of androstenone on hepatic skatole metabolism in relation to boar taint

Boar taint is off-odours in cooked pork from uncastrated male pigs. It is caused by an excessive accumulation of skatole and androstenone in backfat. Accumulation of skatole is due to a low expression and activity of hepatic enzyme CYP2E1. The mechanism of androstenone accumulation is not clear. It could be due to low activity and expression of 3ß-hydroxysteroid dehydrogenase (HSD), an enzyme metabolising androstenone in liver. On the basis of our previous in vivo experiments with castrated animals we suggest that accumulation of skatole is regulated by androstenone. Castrated pigs manifest lower levels of skatole and androstenone and higher CYP2E1 expression. We hypothesise that high levels of androstenone inhibits CYP2E1 expression and hence, reduces the rate of hepatic skatole metabolism. The aims of the present study were (i) to investigate the expression of androstenone-metabolising enzyme HSD in liver of pigs with high and low skatole and androstenone deposition; (ii) to investigate the effect of androstenone on expression of the skatole-metabolising enzyme CYP2E1 in vitro (in cell culture).