Anti-tumoral Effects of miR-3189-3p in Glioblastoma

Glioblastoma is one of the most aggressive brain tumors. We have previously found up-regulation of growth differentiation factor 15 (GDF15) in glioblastoma cells treated with the anticancer agent fenofibrate. Sequence analysis of GDF15 revealed the presence of a microRNA, miR-3189, in the single intron. We then asked whether miR-3189 was expressed in clinical samples and whether it was functional in glioblastoma cells. We found that expression of miR-3189-3p was down-regulated in astrocytoma and glioblastoma clinical samples compared with control brain tissue. In vitro, the functionality of miR-3189-3p was tested by RNA-binding protein immunoprecipitation, and miR-3189-3p coimmunoprecipitated with Argonaute 2 together with two of its major predicted gene targets, the SF3B2 splicing factor and the guanine nucleotide exchange factor p63RhoGEF. Overexpression of miR-3189-3p resulted in a significant inhibition of cell proliferation and migration through direct targeting of SF3B2 and p63RhoGEF, respectively. Interestingly, miR-3189-3p levels were increased by treatment of glioblastoma cells with fenofibrate, a lipid-lowering drug with multiple anticancer activities. The attenuated expression of miR-3189-3p in clinical samples paralleled the elevated expression of SF3B2, which could contribute to the activation of SF3B2 growth-promoting pathways in these tumors. Finally, miR-3189-3p-mediated inhibition of tumor growth in vivo further supported the function of this microRNA as a tumor suppressor.     

稳恒磁场抑制肿瘤增殖的实验研究与理论探讨

作为模型处理最简单的稳恒磁场,其与肿瘤作用的研究是最具理论和实际意义的。分别介绍了稳恒磁场作用于微循环系统、免疫系统对肿瘤的间接抑制和杀伤作用,磁场影响自由基代谢和细胞膜及细胞内结构对肿瘤的抑制作用;回顾了磁场干扰细胞周期、诱导细胞凋亡对肿瘤的影响,并介绍了稳恒磁场联合抗癌药物在治癌中的应用现状;最后结合实验的进展情况,分析探讨了稳恒磁场抑制肿瘤细胞增殖的物理机理,对肿瘤磁疗的发展前景进行了展望。     

The tumor suppressor DiRas3 forms a complex with H-Ras and C-RAF proteins and regulates localization, dimerization, and kinase activity of C-RAF

The maternally imprinted Ras-related tumor suppressor gene DiRas3 is lost or down-regulated in more than 60% of ovarian and breast cancers. The anti-tumorigenic effect of DiRas3 is achieved through several mechanisms, including inhibition of cell proliferation, motility, and invasion, as well as induction of apoptosis and autophagy. Re-expression of DiRas3 in cancer cells interferes with the signaling through Ras/MAPK and PI3K. Despite intensive research, the mode of interference of DiRas3 with the Ras/RAF/MEK/ERK signal transduction is still a matter of speculation. In this study, we show that DiRas3 associates with the H-Ras oncogene and that activation of H-Ras enforces this interaction. Furthermore, while associated with DiRas3, H-Ras is able to bind to its effector protein C-RAF. The resulting multimeric complex consisting of DiRas3, C-RAF, and active H-Ras is more stable than the two protein complexes H-Ras·C-RAF or H-Ras·DiRas3, respectively. The consequence of this complex formation is a DiRas3-mediated recruitment and anchorage of C-RAF to components of the membrane skeleton, suppression of C-RAF/B-RAF heterodimerization, and inhibition of C-RAF kinase activity.     

NDV CN株对几种人肿瘤细胞的杀伤作用及其机理的分析

本文研究了NDV-CN株对5株不同的人肿瘤细胞的体外杀伤作用。结果表明5株肿瘤细胞对NDV-CN敏感,于感染早期出现细胞收缩变圆,失去贴壁性,感染第5天时细胞存活率低于10％。其中尤以HEP3B细胞最敏感。但NDV-CN株对人二倍体细胞2BS有弱杀伤性。病毒感染早期可检测到感染细胞中有病毒核酸的复制、感染细胞表面有病毒蛋白的表达,脑浆内有病毒粒子存在。NDV-CN株主要诱导HEP3B及T24细胞产生凋亡,主要诱导Hep2、Hela及A549细胞产生坏死。     

Carrier Cells for Delivery of Oncolytic Measles Virus into Tumors: Determinants of Efficient Loading

Oncolytic measles virus (OMV) is a promising antitumor agent. However, the presence of anti-measles neutralizing antibodies (NAbs) against the hemagglutinin (H) protein of OMV is a major barrier to the therapeutic application of OMV in clinical practice. In order to overcome this challenge, specific types of cells have been used as carriers for OMV. Differential loading strategies appear to result in different therapeutic outcomes; despite this, only few studies have reported practical ex vivo loading strategies required for effective treatment. To this end, we systematically evaluated the antitumor efficacy of OMV using different loading strategies; this involved varying the in vitro loading duration and loading dose of OMV. We found that improved oncolysis of carrier cells was achieved by a prolonged loading duration in the absence of NAbs. However, the enhanced oncolytic effect was abrogated in the presence of NAbs. Further, we found that the expression of H protein on the surface of carrier cells was predominantly determined by the loading duration rather than the loading dose. Finally, we showed that NAbs blocked viral transfer by targeting H protein prior to the occurrence of cell-to-cell interactions. Our results provide comprehensive information on the determinants of an effective loading strategy for carrier cell-based virotherapy; these results may be useful for guiding the application of OMV as an antitumor agent in clinical practice.     

p53-induced gene 3 mediates cell death induced by glutathione peroxidase 3

Expression of glutathione peroxidase 3 (GPx3) is down-regulated in a variety of human malignancies. Both methylation and deletion of GPx3 gene underlie the alterations of GPx3 expression in prostate cancer. A strong correlation between the down-regulation of GPx3 expression and progression of prostate cancer and the suppression of prostate cancer xenografts in SCID mice by forced expression of GPx3 suggests a tumor suppression role of GPx3 in prostate cancer. However, the mechanism of GPx3-mediated tumor suppression remains unclear. In this report, GPx3 was found to interact directly with p53-induced gene 3 (PIG3). Forced overexpression of GPx3 in prostate cancer cell lines DU145 and PC3 as well as immortalized prostate epithelial cells RWPE-1 increased apoptotic cell death. Expression of GPx3(x73c), a peroxidase-negative OPAL codon mutant, in DU145 and PC3 cells also increased cell death. The induced expression of GPx3 in DU145 and PC3 cells resulted in an increase in reactive oxygen species and caspase-3 activity. These activities were abrogated by either knocking down PIG3 or mutating the PIG3 binding motif in GPx3 or binding interference from a peptide corresponding to PIG3 binding motif in GPx3. In addition, UV-treated RWPE-1 cells underwent apoptotic death, which was partially prevented by knocking down GPx3 or PIG3, suggesting that GPx3-PIG3 signaling is critical for UV-induced apoptosis. Taken together, these results reveal a novel signaling pathway of GPx3-PIG3 in the regulation of cell death in prostate cancer.     

sTRAIL levels and TRAIL gene polymorphisms in Chinese patients with fatty liver disease

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily, and has been identified as a novel mediator of fatty liver disease (FLD). The aim of our study was to further investigate the relationship between TRAIL and FLD. We found that soluble TRAIL (sTRAIL) concentrations in non-alcoholic FLD (NAFLD) patients were significantly higher than those of controls, and that sTRAIL levels positively correlated with triglyceride concentrations in NAFLD patients. Our results also indicated that the AA/TT genotypes of TRAIL at 1525/1595 engendered a lower risk of FLD attack and a less severe form of steatosis for NAFLD patients in Chinese population. This study provides a means to test for susceptibility to FLD and may assist in the diagnosis of FLD. In addition, we found that 1525G/A and 1595C/T sites were in complete linkage disequilibrium in Chinese population. This might indicate a haplotype with high genetic frequency of TRAIL.     

NAMPT 真核表达载体构建及其对肝癌细胞增殖的影响

目的:为探索烟酰胺磷酸核糖基转移酶(NAMPT)对肝癌细胞增殖的影响,构建pc DNA3.1(+)-NAMPT真核表达载体。方法:以正常肝细胞的c DNA为模板,通过PCR扩增得到NAMPT全长序列,经酶切、连接、转化等步骤构建真核表达载体。重组质粒经酶切鉴定及测序验证后,用脂质体转染法转染肝癌细胞MHCC-97H和正常肝细胞HL-7702,免疫印迹检测NAMPT蛋白表达情况;WST实验检测过表达NAMPT后对MHCC-97H和HL-7702增殖的影响。结果:真核表达载体pc DNA3.1(+)-NAMPT构建成功,转染HL-7702和MHCC-97H细胞后,NAMPT蛋白表达水平较空白组分别升高了83%和180%;过表达NAMPT可促进细胞增殖,而抑制NAMPT活性后,细胞增殖被抑制(P0.001)。结论:成功构建了真核表达载体pc DNA3.1(+)-NAMPT,并发现NAMPT表达水平与细胞增殖密切相关,为进一步探索NAMPT表达在肝癌细胞增殖的分子作用机制和筛选新的肿瘤药物靶点奠定了基础。     

Identification of Mixed Lineage Leukemia 1(MLL1) Protein as a Coactivator of Heat Shock Factor 1(HSF1) Protein in Response to Heat Shock Protein 90 (HSP90) Inhibition

Heat shock protein 90 (HSP90) inhibition inhibits cancer cell proliferation through depleting client oncoproteins and shutting down multiple oncogenic pathways. Therefore, it is an attractive strategy for targeting human cancers. Several HSP90 inhibitors, including AUY922 and STA9090, show promising effects in clinical trials. However, the efficacy of HSP90 inhibitors may be limited by heat shock factor 1 (HSF1)-mediated feedback mechanisms. Here, we identify, through an siRNA screen, that the histone H3 lysine 4 methyltransferase MLL1 functions as a coactivator of HSF1 in response to HSP90 inhibition. MLL1 is recruited to the promoters of HSF1 target genes and regulates their expression in response to HSP90 inhibition. In addition, a striking combination effect is observed when MLL1 depletion is combined with HSP90 inhibition in various human cancer cell lines and tumor models. Thus, targeting MLL1 may block a HSF1-mediated feedback mechanism induced by HSP90 inhibition and provide a new avenue to enhance HSP90 inhibitor activity in human cancers.     

Caspase-1 protein induces apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-mediated necrosis independently of its catalytic activity

The adaptor protein, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), connects pathogen/danger sensors such as NLRP3 and NLRC4 with caspases and is involved in inflammation and cell death. We have found that ASC activation induced caspase-8-dependent apoptosis or CA-074Me (cathepsin B inhibitor)-inhibitable necrosis depending on the cell type. Unlike necroptosis, another necrotic cell death, ASC-mediated necrosis, was neither RIP3-dependent nor necrostatin-1-inhibitable. Although acetyl-YVAD-chloromethylketone (Ac-YVAD-CMK) (caspase-1 inhibitor) did not inhibit ASC-mediated necrosis, comprehensive gene expression analyses indicated that caspase-1 expression coincided with the necrosis type. Furthermore, caspase-1 knockdown converted necrosis-type cells to apoptosis-type cells, whereas exogenous expression of either wild-type or catalytically inactive caspase-1 did the opposite. Knockdown of caspase-1, but not Ac-YVAD-CMK, suppressed the monocyte necrosis induced by Staphylococcus and Pseudomonas infection. Thus, the catalytic activity of caspase-1 is dispensable for necrosis induction. Intriguingly, a short period of caspase-1 knockdown inhibited IL-1β production but not necrosis, although longer knockdown suppressed both responses. Possible explanations of this phenomenon are discussed.     

Triggering Receptor Expressed on Myeloid Cells 1 (TREM-1)-mediated Bcl-2 Induction Prolongs Macrophage Survival

Triggering receptor expressed on myeloid cells 1 (TREM-1) is a superimmunoglobulin receptor expressed on myeloid cells that plays an important role in the amplification of inflammation. Recent studies suggest a role for TREM-1 in tumor-associated macrophages with relationship to tumor growth and progression. Whether the effects of TREM-1 on inflammation and tumor growth are mediated by an alteration in cell survival signaling is not known. In these studies, we show that TREM-1 knock-out macrophages exhibit an increase in apoptosis of cells in response to lipopolysaccharide (LPS) suggesting a role for TREM-1 in macrophage survival. Specific ligation of TREM-1 with monoclonal TREM-1 (mTREM-1) or overexpression of TREM-1 with adeno-TREM-1 induced B-cell lymphoma-2 (Bcl-2) with depletion of the key executioner caspase-3 prevents the cleavage of poly(ADP-ribose) polymerase. TREM-1 knock-out cells showed lack of induction of Bcl2 with an increase in caspase-3 activation in response to lipopolysaccharide. In addition overexpression of TREM-1 with adeno-TREM-1 led to an increase in mitofusins (MFN1 and MFN2) and knockdown of TREM-1 decreased the expression of mitofusins suggesting that TREM-1 contributes to the maintenance of mitochondrial integrity favoring cell survival. Investigations into potential mechanisms by which TREM-1 alters cell survival showed that TREM-1-induced Bcl-2 in an Egr2-dependent manner. Furthermore, our data shows that expression of Egr2 in response to specific ligation of TREM-1 is ERK mediated. These data for the first time provide novel mechanistic insights into the role of TREM-1 as an anti-apoptotic protein that prolongs macrophage survival.     

A PTCH1 Homolog Transcriptionally Activated by p53 Suppresses Hedgehog Signaling

The p53-mediated responses to DNA damage and the Hedgehog (Hh) signaling pathway are each recurrently dysregulated in many types of human cancer. Here we describe PTCH53, a p53 target gene that is homologous to the tumor suppressor gene PTCH1 and can function as a repressor of Hh pathway activation. PTCH53 (previously designated PTCHD4) was highly responsive to p53 in vitro and was among a small number of genes that were consistently expressed at reduced levels in diverse TP53 mutant cell lines and human tumors. Increased expression of PTCH53 inhibited canonical Hh signaling by the G protein-coupled receptor SMO. PTCH53 thus delineates a novel, inducible pathway by which p53 can repress tumorigenic Hh signals.     

Intermediate domain of receptor-interacting protein kinase 1 (RIPK1) determines switch between necroptosis and RIPK1 kinase-dependent apoptosis

Receptor-interacting protein kinase 1 (RIPK1) is an important component of the tumor necrosis factor receptor 1 (TNFR1) signaling pathway. Depending on the cell type and conditions, RIPK1 mediates MAPK and NF-κB activation as well as cell death. Using a mutant form of RIPK1 (RIPK1ΔID) lacking the intermediate domain (ID), we confirm the requirement of this domain for activation of these signaling events. Moreover, expression of RIPK1ΔID resulted in enhanced recruitment of caspase-8 to the TNFR1 complex II component Fas-associated death domain (FADD), which allowed a shift from TNF-induced necroptosis to apoptosis in L929 cells. Addition of the RIPK1 kinase inhibitor necrostatin-1 strongly reduced recruitment of RIPK1 and caspase-8 to FADD and subsequent apoptosis, indicating a role for RIPK1 kinase activity in apoptotic complex formation. Our study shows that RIPK1 has an anti-apoptotic function residing in its ID and demonstrates a cellular system as an elegant genetic model for RIPK1 kinase-dependent apoptosis that, in contrast to the Smac mimetic model, does not rely on depletion of cellular inhibitor of apoptosis protein 1 and 2 (cIAP1/2).     

Protein interactions of phosphatase and tensin homologue (PTEN) and its cancer-associated G20E mutant compared by using stable isotope labeling by amino acids in cell culture-based parallel affinity purification

The tumor suppressor PTEN (phosphatase and tensin homologue) negatively regulates the PI3K pathway through its lipid phosphatase activity and is one of the most commonly lost tumor suppressors in human cancers. Though the tumor suppressive function involves the lipid phosphatase-dependent and -independent activities of PTEN, the mechanism leading to the phosphatase-independent function of PTEN is understood poorly. Some PTEN mutants have lipid phosphatase activity but fail to suppress cell growth. Here, we use a cancer-associated mutant, G20E, to gain insight into the phosphatase-independent function of PTEN by investigating protein-protein interactions using MS-based stable isotope labeling by amino acids in cell culture (SILAC). A strategy named parallel affinity purification (PAP) and SILAC has been developed to prioritize interactors and to compare the interactions between wild-type and G20E PTEN. Clustering of the prioritized interactors acquired by the PAP-SILAC approach shows three distinct clusters: 1) wild-type-specific interactors, 2) interactors unique to the G20E mutant, and 3) proteins common to wild-type and mutant. These interactors are involved mainly in cell migration and apoptosis pathways. We further demonstrate that the wild-type-specific interactor, NUDTL16L1, is required for the regulatory function of wild-type PTEN in cell migration. These findings contribute to a better understanding of the mechanisms of the phosphatase-dependent and -independent functions of PTEN.     

Tyr-301 Phosphorylation Inhibits Pyruvate Dehydrogenase by Blocking Substrate Binding and Promotes the Warburg Effect

The mitochondrial pyruvate dehydrogenase complex (PDC) plays a crucial role in regulation of glucose homoeostasis in mammalian cells. PDC flux depends on catalytic activity of the most important enzyme component pyruvate dehydrogenase (PDH). PDH kinase inactivates PDC by phosphorylating PDH at specific serine residues, including Ser-293, whereas dephosphorylation of PDH by PDH phosphatase restores PDC activity. The current understanding suggests that Ser-293 phosphorylation of PDH impedes active site accessibility to its substrate pyruvate. Here, we report that phosphorylation of a tyrosine residue Tyr-301 also inhibits PDH α 1 (PDHA1) by blocking pyruvate binding through a novel mechanism in addition to Ser-293 phosphorylation. In addition, we found that multiple oncogenic tyrosine kinases directly phosphorylate PDHA1 at Tyr-301, and Tyr-301 phosphorylation of PDHA1 is common in EGF-stimulated cells as well as diverse human cancer cells and primary leukemia cells from human patients. Moreover, expression of a phosphorylation-deficient PDHA1 Y301F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at distinct serine and tyrosine residues inhibits PDHA1 through distinct mechanisms to impact active site accessibility, which act in concert to regulate PDC activity and promote the Warburg effect.     

Simvastatin Potently Induces Calcium-dependent Apoptosis of Human Leiomyoma Cells

Statins are drugs commonly used for the treatment of high plasma cholesterol levels. Beyond these well known lipid-lowering properties, they possess broad-reaching effects in vivo, including antitumor effects. Statins inhibit the growth of multiple tumors. However, the mechanisms remain incompletely understood. Here we show that simvastatin inhibits the proliferation of human leiomyoma cells. This was associated with decreased mitogen-activated protein kinase signaling and multiple changes in cell cycle progression. Simvastatin potently stimulated leiomyoma cell apoptosis in a manner mechanistically dependent upon apoptotic calcium release from voltage-gated calcium channels. Therefore, simvastatin possesses antitumor effects that are dependent upon the apoptotic calcium release machinery.     

Microglial Toll-like receptor 2 contributes to kainic acid-induced glial activation and hippocampal neuronal cell death

Recent studies indicate that Toll-like receptors (TLRs), originally identified as infectious agent receptors, also mediate sterile inflammatory responses during tissue damage. In this study, we investigated the role of TLR2 in excitotoxic hippocampal cell death using TLR2 knock-out (KO) mice. TLR2 expression was up-regulated in microglia in the ipsilateral hippocampus of kainic acid (KA)-injected mice. KA-mediated hippocampal cell death was significantly reduced in TLR2 KO mice compared with wild-type (WT) mice. Similarly, KA-induced glial activation and proinflammatory gene expression in the hippocampus were compromised in TLR2 KO mice. In addition, neurons in organotypic hippocampal slice cultures (OHSCs) from TLR2 KO mouse brains were less susceptible to KA excitotoxicity than WT OHSCs. This protection is partly attributed to decreased expression of proinflammatory genes, such as TNF-α and IL-1β in TLR2 KO mice OHSCs. These data demonstrate conclusively that TLR2 signaling in microglia contributes to KA-mediated innate immune responses and hippocampal excitotoxicity.     

Tumor-suppressive Function of Protein-tyrosine Phosphatase Non-receptor Type 23 in Testicular Germ Cell Tumors Is Lost upon Overexpression of miR142–3p microRNA

Protein-tyrosine phosphatase non-receptor type 23 (PTPN23) is a candidate tumor suppressor involved in the tumorigenesis of various organs. However, its physiological role(s) and detailed expression profile(s) have not yet been elucidated. We investigated the function and regulation of PTPN23 in the formation of testicular germ cell tumors (TGCTs). Expression of PTPN23 in human TGCT cell lines was significantly lower than that in spermatogonial stem cells in mice. Overexpression of PTPN23 in NEC8, a human TGCT cell line, suppressed soft agar colony formation in vitro and tumor formation in nude mice in vivo. These data indicate that PTPN23 functions as a tumor suppressor in TGCTs. Multiple computational algorithms predicted that the 3′ UTR of human PTPN23 is a target for miR-142-3p. A luciferase reporter assay confirmed that miR-142-3p bound directly to the 3′ UTR of PTPN23. Introduction of pre-miR-142 in the PTPN23 transfectant of NEC8 led to suppressed expression of PTPN23 and increased soft agar colony formation. Quantitative RT-PCR data revealed a significantly higher expression of miR-142-3p in human seminomas compared with normal testes. No difference in mRNA expression between seminoma and non-seminoma samples was detected by in situ hybridization. Both quantitative RT-PCR and immunohistochemical analyses revealed that PTPN23 expression was significantly lower in TGCTs than in normal testicular tissues. Finally, a lack of PTPN23 protein expression in human TGCTs correlated with a relatively higher miR-142-3p expression. These data suggest that PTPN23 is a tumor suppressor and that repression of PTPN23 expression by miR-142-3p plays an important role in the pathogenesis of TGCTs.