MicroRNA-451 regulates stemness of side population cells via PI3K/Akt/mTOR signaling pathway in multiple myeloma

Side population (SP) cells are an enriched source of cancer-initiating cells with stemness characteristics, generated by increased ABC transporter activity, which has served as a unique hallmark for multiple myeloma (MM) stem cell studies. Here we isolated and identified MM SP cells via Hoechst 33342 staining. Furthermore, we demonstrate that SP cells possess abnormal cell cycle, clonogenicity, and high drug efflux characteristics-all of which are features commonly seen in stem cells. Interestingly, we found that bortezomib, As₂O₃, and melphalan all affected apoptosis and clonogenicity in SP cells. We followed by characterizing the miRNA signature of MM SP cells and validated the specific miR-451 target tuberous sclerosis 1 (TSC1) gene to reveal that it activates the PI3K/Akt/mTOR signaling in MM SP cells. Inhibition of miR-451 enhanced anti-myeloma novel agents'' effectiveness, through increasing cells apoptosis, decreasing clonogenicity, and reducing MDR1 mRNA expression. Moreover, the novel specific PI3K/Akt/mTOR signaling inhibitor {"type":"entrez-protein","attrs":{"text":"S14161","term_id":"98844","term_text":"pir||S14161"}}S14161 displayed its prowess as a potential therapeutic agent by targeting MM SP cells. Our findings offer insights into the mechanisms regulating MM SP cells and provide a novel strategy to overcome resistance to existing therapies against myeloma.     

Activation of the anti-apoptotic Akt/protein kinase B pathway in human malignant gliomas in vivo

In the present study we investigated two important signal transduction pathways in glioma biopsies. By Western analysis we found an overexpression of the epidermal growth factor receptor in 10 out of 27 (37%) glioblastoma multiforme (GBM), but not in astrocytomas WHO II/III which demonstrated only weak or absent expression. Only two GBM (8%) but none of the astrocytomas WHO II/III exhibited loss of PTEN expression. Activation of Akt/protein kinase B showed a close correlation with EGF receptor overexpression in human malignant gliomas since 6 out of 7 GBMs with high degrees of protein kinase B activation exhibited overexpression of the EGF receptor. In contrast, no significant differences in MAP kinase activation could be detected between individual GBMs. Our data show that EGF receptor overexpression seems to be responsible for activation of the protein kinase B whereas PTEN deletion seems to play a minor role in the dysregulation of this important pathway in human GBM in vivo.     

Inactivation of NF-kappaB by genistein is mediated via Akt signaling pathway in breast cancer cells

Genistein, a natural isoflavonoid found in soybean products, has been proposed to be associated with a lower rate of breast cancer in Asian women. Studies from our laboratory and others have shown that genistein can induce apoptosis by regulating the expression of apoptosis-related genes in breast cancer cells. However, the precise molecular mechanism(s) by which genistein induces apoptotic cell death is not clear. In order to investigate such mechanism, we tested the role of Akt and NF-kappaB in genistein-treated MDA-MB-231 breast cancer cells. We found that inhibition of cell growth and induction of apoptosis by genistein are partly mediated through the downregulation of Akt and NF-kappaB pathways. Gel shift assay showed that NF-kappaB DNA-binding activity in MDA-MB-231 cells transfected with Akt cDNA was induced, suggesting that there is a cross-talk between NF-kappaB and Akt signaling pathway. Moreover, we found that genistein could abrogate EGF and Akt induced NF-kappaB activation. From these results, we conclude that the inactivation of NF-kappaB by genistein in MDA-MB-231 breast cancer cells is partly mediated via Akt pathway, which could be useful for rational design of strategies for the prevention and/or treatment of breast cancer.     

Insulin-like growth factor-1 induces adhesion and migration in human multiple myeloma cells via activation of beta1-integrin and phosphatidylinositol 3'-kinase/AKT signaling

Insulin-like growth factor-1 (IGF-I) is a growth and survival factor in human multiple myeloma (MM) cells. Here we examine the effect of IGF-I on MM cell adhesion and migration, and define the role of beta1 integrin in these processes. IGF-I increases adhesion of MM.1S and OPM6 MM cells to fibronectin (FN) in a time- and dose-dependent manner, as a consequence of IGF-IR activation. Conversely, blocking anti-beta1 integrin monoclonal antibody, RGD peptide, and cytochalasin D inhibit IGF-I-induced cell adhesion to FN. IGF-I rapidly and transiently induces association of IGF-IR and beta1 integrin, with phosphorylation of IGF-IR, IRS-1, and p85(PI3-K). IGF-I also triggers phosphorylation of AKT and ERK significantly. Both IGF-IR and beta1 integrin colocalize to lipid rafts on the plasma membrane after IGF-I stimulation. In addition, IGF-I triggers polymerization of F-actin, induces phosphorylation of p125(FAK) and paxillin, and enhances beta1 integrin interaction with these focal adhesion proteins. Importantly, using pharmacological inhibitors of phosphatidylinositol 3'-kinase (PI3-K) (LY294002 and wortmannin) and extracellular signal-regulated kinase (PD98059), we demonstrate that IGF-I-induced MM cell adhesion to FN is achieved only when PI3-K/AKT is activated. IGF-I induces a 1.7-2.2 (MM.1S) and 2-2.5-fold (OPM6) increase in migration, whereas blocking anti-IGF-I and anti-beta1 integrin monoclonal antibodies, PI3-K inhibitors, as well as cytochalasin D abrogate IGF-I-induced MM cell transmigration. Finally, IGF-I induces adhesion of CD138+ patient MM cells. Therefore, these studies suggest a role for IGF-I in trafficking and localization of MM cells in the bone marrow microenvironment. Moreover, they define the functional association of IGF-IR and beta1 integrin in mediating MM cell homing, providing the preclinical rationale for novel treatment strategies targeting IGF-I/IGF-IR in MM.     

Mutations in the NF-kappaB signaling pathway: implications for human disease

The nuclear factor-kappa B (NF-kappaB) signaling pathway is a multi-component pathway that regulates the expression of hundreds of genes that are involved in diverse and key cellular and organismal processes, including cell proliferation, cell survival, the cellular stress response, innate immunity and inflammation. Not surprisingly, mis-regulation of the NF-kappaB pathway, either by mutation or epigenetic mechanisms, is involved in many human and animal diseases, especially ones associated with chronic inflammation, immunodeficiency or cancer. This review describes human diseases in which mutations in the components of the core NF-kappaB signaling pathway have been implicated and discusses the molecular mechanisms by which these alterations in NF-kappaB signaling are likely to contribute to the disease pathology. These mutations can be germline or somatic and include gene amplification (e.g., REL), point mutations and deletions (REL, NFKB2, IKBA, CYLD, NEMO) and chromosomal translocations (BCL-3). In addition, human genetic diseases are briefly described wherein mutations affect protein modifiers or transducers of NF-kappaB signaling or disrupt NF-kappaB-binding sites in promoters/enhancers.     

Bone marrow stromal cells prevent apoptosis of lymphoma cells by upregulation of anti-apoptotic proteins associated with activation of NF-kappaB (RelB/p52) in non-Hodgkin's lymphoma cells.

Stromal cells are an essential component of the bone marrow microenvironment that regulate or supports tumor survival. In this study we therefore studied the role of stromal cells in lymphoma cell survival. We demonstrated that adhesion of the B-cell lymphoma cell lines SUDH-4 and 10 to bone marrow stroma inhibited mitoxantrone-induced apoptosis. This adhesion-dependent inhibition of mitoxantrone-induced apoptosis correlated with decreased activation of caspases-8 and 9, and cleavage of caspase 3 and PARP. Electrophoretic mobility shift assays (EMSA) analysis demonstrated significantly increased NF-kappaB binding activity in lymphoma cells adhered to stroma cells compared to lymphoma cells in suspension. This DNA binding activity could be attributed to cell adhesion-mediated proteolysis of the NF-kappaB precursor, p100 (NF-kappaB2). This resulted in the generation of active p52, which translocated to the nucleus in complex with p65 and RelB. Coculture with stromal cells also induced expression of the NF-kappaB-regulated anti-apoptotic molecules, XIAP, cIAP(1) and cIAP(2). Inhibition of NF-kappaB significantly suppressed HS-5-induced protection against apoptosis in lymphoma cell lines as well as in primary lymphoma cells. Thus, bone marrow stroma protects B-cell lymphoma cells against apoptosis, at least in part through activation of NF-kappaB dependent mechanism involving up-regulation of NF-kappaB regulated antiapoptotic proteins. Consequently, this study suggests a new approach to decrease the resistance of lymphoma to chemotherapy.     

自分泌IGF-1/IGF-1R环路促进NK/T细胞淋巴瘤细胞株迁移和侵袭

目的 探讨IGF-1/IGF-1R信号通路对NK/T细胞淋巴瘤(NK/TCL)细胞株迁移和侵袭的作用及其调控机制.方法 采用反转录聚合酶链反应(RT-PCR)及免疫荧光技术检测IGF-1和IGF-1R的表达,Transwell技术观察NK/TCL细胞迁移和侵袭.酶联免疫吸附实验(ELISA)检测MMP-2、MMP-9及TIMP1水平.结果 NK/TCL细胞株SNK-1和SNK-6均表达IGF-1与IGF-1R,而健康人NK细胞不表达IGF-1R.IGF-1R抑制剂显著抑制SNK-1和SNK-6的迁移和侵袭.外源性IGF-1则促进细胞迁移及侵袭,而IGF-1R抑制剂能阻断该效应.IGF-1R下游相关激酶p38、PI3K及JNK的抑制剂均可降低细胞迁移能力.外源性IGF-1可上调MMP-2、MMP-9的分泌水平,IGF-1R抑制剂则抑制MMP-2、MMP-9的分泌.结论 NK/TCL细胞株中存在IGF-1/IGF-1R自分泌环路,并通过IGF-1/IGF-1R通路促进MMP-2、MMP-9分泌及IGF-1R下游p38、PI3K及JNK等激酶促进肿瘤细胞的迁移和侵袭.     

硼替佐米对人类多发性骨髓瘤细胞系U266细胞抗凋亡蛋白HSP-90的影响

 目的　探讨硼替佐米对人类多发性骨髓瘤（MM）细胞系U266细胞抗凋亡蛋白HSP-90的影响。方法　采用RT-PCR技术研究经不同浓度的硼替佐米作用于U266细胞4 h后其内HSP-90 mRNA表达情况的变化。结果　随着硼替佐米浓度的增加,其MM细胞中HSP-90α的mRNA表达量逐渐增加。由低浓度组到高浓度组（0、50、100、150、200 nmol／L）所对应的HSP-90α的灰度值分别为0.343±0.017、0.505±0.039、0.640±0.029、0.760±0.059、0.963±0.054;两两组间比较差异有统计学意义（P＜0.05 ）。浓度由低到高组对应HSP-90β的灰度值分别为0.610±0.022、0.765±0.050、0.645±0.052、0.770±0.059、0.790±0.027,而HSP-90β的灰度值 0 nmol／L组与50、150、200 nmol／L组之间差异有统计学意义（P＜0.05）。50、100 nmol／L浓度组对应的HSP-90β的灰度值不同（P＜0.05）。100 nmol／L组与150、200 nmol／L组对应的HSP-90β的灰度值差异有统计学意义（P＜0.05）。HSP-90β的mRNA表达量增加趋势不很明显,但总体差异仍具统计学意义（P＜0.05）。结论　硼替佐米可以上调HSP-90的分子水平的表达,并且以上调HSP-90α的表达为主。     

DF3/MUC1 signaling in multiple myeloma cells is regulated by interleukin-7

The human DF3/MUC1 transmembrane protein is aberrantly expressed in multiple myeloma cells and other B cell malignancies. The regulation of MUC1 in B cells and its potential function as a signaling molecule are unknown. The present results demonstrate that interleukin-7 (IL-7) stimulates MUC1 expression in multiple myeloma cells. The results also demonstrate the IL-7 induces binding of MUC1 to the Lyn tyrosine kinase. The MUC1 C-terminal subunit binds directly to Lyn through interactions with the Lyn SH3 and SH2 domains. Activation of Lyn in response to IL-7 stimulation results in increased tyrosine phosphorylation of the MUC1 C-terminal subunit. In vitro and in vivo studies show that Lyn phosphorylates MUC1, at least in large part, on a YEKV site in the MUC1 cytoplasmic tail. The functional significance of the MUC1-Lyn interaction is supported by the demonstration that Lyn-mediated phosphorylation of MUC1 on YEKV induces binding of MUC1 and the beta-catenin signaling protein. In concert with these results, IL-7 treatment is associated with binding of MUC1 to beta-catenin and targeting of the MUC1-beta-catenin complex to the nucleus. These findings indicate that IL-7 regulates MUC1 expression and function in multiple myeloma cells.     

Activation of T cells via tumor antigen specific chimeric receptors: the role of the intracellular signaling domain

T cells engineered to express hybrid receptors with antibody defined specificity can successfully be targeted to tumor cells. In order to select intracellular domains of chimeric receptors capable of efficiently activate T cells in vitro and in vivo, we compared the function of receptors, which share the same extracellular antigen-binding part, joined to different intra-cellular signal transduction units. The antigen binding domain of the receptors was a single-chain fragment of a monoclonal antibody, which recognize a High Molecular Weight Melanoma-Associated Antigen with high affinity. The intracellular tails were derived from the T-cell receptor zeta chain (TCR-zeta), from the B-cell receptor Ig-alpha molecule and from a mutated Ig-alpha molecule able of stronger signal transduction. We compared the activity of the different chimeric receptors at a single-cell level by using a T-cell line that expressed an activation-dependent EGFP-reporter gene. Upon cross-linking with immobilized antibodies, all receptors were able to induce EGFP expression in the majority of the T cells. In contrast, EGFP expression was induced by contact to melanoma cells in vitro only in T cells that expressed the chimeric receptor that contained the TCR-zeta intracellular tail. In these T cells, the co-expression of chimeric receptors that contain a mutated Ig-alpha tail lowers the threshold of T-cell activation and facilitates tumor recognition in vitro and in vivo. Given their specificity and efficiency, T cells grafted with these type of receptors may represent potential candidates for cancer passive immunotherapy.     

Akt/c-Myc通路对胃癌细胞化疗效果的影响

背景与目的：Akt信号通路是调节原癌基因c-myc蛋白表达水平的主要机制之一,Akt信号通路激活后可降低胃癌细胞的化疗敏感性。然而,目前对Akt/c-Myc通路在胃癌化疗过程中的作用尚不明确。因此,本课题通过观察Akt/c-Myc信号通路对胃癌细胞化疗效果的影响并探讨其作用机制。方法：依托泊苷作用于胃癌SGC7901和BGC823细胞,MTT法检测细胞的生存率;分别采用流式细胞仪和Hoechst33258荧光染色法检测细胞周期分布和凋亡;Akt活性的检测采用非放射性蛋白激酶活性分析法;Western印迹法检测c-Myc和p-AktSer473蛋白的表达水平;RT-PCR法检测c-MycmRNA的表达;同时观察PI3-K/Akt通路抑制剂Wortmannin对依托泊苷诱导的上述变化的影响。结果：依托泊苷呈时间-剂量依赖性抑制SGC7901和BGC823细胞的生长,明显诱导细胞的凋亡;同时上调SGC7901和BGC823细胞中Akt活性及p-AktSer473蛋白表达水平,并增强c-Myc蛋白和mRNA的表达。Wortmannin可明显增强依托泊苷的细胞生长抑制作用并提高细胞的凋亡水平;抑制依托泊苷诱导的c-Myc蛋白的表达,但对c-MycmRNA表达没有明显的影响。结论：依托泊苷激活SGC7901和BGC823细胞中的Akt信号通路,并上调c-Myc蛋白的表达水平而影响胃癌的化疗效果。Akt信号通路可能主要是通过转录后调节来调控c-Myc蛋白的水平。     

Differential expression of multiple anti-apoptotic proteins in epidermis of IGF-1 transgenic mice as revealed by 2-dimensional gel electrophoresis/mass spectrometry analysis

Overexpression of insulin-like growth factor-1 (IGF-1) has been associated with a number of human tumors, including breast, colon, lung, and prostate cancers. In previous studies, we found that mice overexpressing human IGF-1 in the basal layer of the epidermis (BK5.IGF-1 mice) developed skin tumors following treatment with the skin tumor initiator, 7,12-dimethylbenz[a]anthracene, indicating that IGF-1 can act as a skin tumor promoter. In the present study, we employed a proteomics approach of two-dimensional (2-D) gel electrophoresis and mass spectrometry to profile differentially expressed proteins in skin epidermis between BK5.IGF-1 transgenic and nontransgenic littermates. Two-D gels from each of three transgenic and three age/sex matched wild-type littermates were compared at two different pH ranges. Differentially expressed protein spots were identified by Bio-Rad's PDQuest image analysis, in-gel digested, and analyzed on a MALDI-TOF MS system. A total of 23 proteins were identified as differentially expressed, 17 of them overexpressed in transgenic mice. These proteins included 14-3-3 sigma, galectin-7, an apoptosis-related protein, three heat shock proteins, four calcium binding proteins, three proteases or protease inhibitors, one actin regulatory capping protein, and translation initiation factor 5A. The differential expression of GRP78, alpha enolase, and galectin-7 was verified by 1-D western blot analysis. Two-D western blot analyses of alpha enolase and galectin-7 further revealed that alpha enolase had more than one protein spot dependent on charge. The current data suggest that some of the differentially expressed proteins may play a role in the tumor promoting action of IGF-1 in mouse skin.     

Activation of reactive oxygen species/AMP activated protein kinase signaling mediates fisetin-induced apoptosis in multiple myeloma U266 cells

We investigated the molecular mechanisms responsible for fisetin-induced apoptosis in U266 cells. Fisetin elicited the cytotoxicity in U266 cells, manifested as an increased fraction of the cells with sub-G1 content or stained positively with TUNEL labeling. Fisetin enhanced caspase-3 activation, downregulation of Bcl-2 and Mcl-1(L), and upregulation of Bax, Bim and Bad. Fisetin activated AMPK as well as its substrate acetyl-CoA carboxylase (ACC), along with a decreased phosphorylation of AKT and mTOR. Fisetin also stimulated generation of ROS in U266 cells. Conversely, compound C or N-acetyl-L-cystein blocked fisetin-induced apoptosis. Our data suggest that fisetin-induced apoptosis in U266 cells is through ROS and AMPK pathways.     

Triptolide inhibits transcription factor NF-kappaB and induces apoptosis of multiple myeloma cells

Triptolide has been reported to be effective in the treatment of auto-immune diseases. This study investigates the cytotoxic function of triptolide on multiple myeloma (MM) cells. We found that triptolide inhibited the proliferation of both RPMI8226 and U266 cells in a dose-dependent manner (10-80 ng/mL). Triptolide induced apoptosis in MM cells through activation of the cystein protease caspase 8, 9 and 3, and subsequent cleavage of the DNA repair enzyme poly (ADP-ribose) polymerase. Apoptosis was confirmed with cell-cycle analysis and annexin V staining. Moreover, triptolide down-regulated nuclear factor (NF)-kappaB activity in MM cell lines. In addition, triptolide also induced chemosensitivity to doxorubicin and suppressed cell proliferation of fresh MM cells. Therefore, triptolide appears to be a potent inducer of apoptosis in myeloma cells, and might have some benefit in the treatment of myeloma patients.     

Ovarian cancer-associated mesothelial cells induce acquired platinum-resistance in peritoneal metastasis via the FN1/Akt signaling pathway

Peritoneal dissemination of ovarian cancer (OvCa) arises from the surface of the peritoneum, covered by monolayer of mesothelial cells (MCs). Given that both OvCa cells and MCs are present in the same peritoneal metastatic microenvironment, they may establish cell-to-cell crosstalk or phenotypic alterations including the acquisition of platinum-resistance in OvCa cells. Herein, we report how OvCa-associated mesothelial cells (OCAMs) induce platinum-resistance in OvCa cells through direct cell-to-cell crosstalk. We evaluated mutual associations between OvCa cells and human primary MCs with in vitro coculturing experimental models and in silico omics data analysis. The role of OCAMs was also investigated using clinical samples and in vivo mice models. Results of in vitro experiments show that mesenchymal transition is induced in OCAMs primarily by TGF-β1 stimulation. Furthermore, OCAMs influence the behavior of OvCa cells as a component of the tumor microenvironment of peritoneal metastasis. Mechanistically, OCAMs can induce decreased platinum-sensitivity in OvCa cells via induction of the FN1/Akt signaling pathway via cell-to-cell interactions. Histological analysis of OvCa peritoneal metastasis also illustrated FN1 expression in stromal cells that are supposed to originate from MCs. Further, we also confirmed the activation of Akt signaling in OvCa cells in contact with TGF-β1 stimulated peritoneum, using an in vivo mice model. Our results suggest that the tumor microenvironment, enhanced by direct cell-to-cell crosstalk between OvCa cells and OCAMs, induces acquisition of platinum-resistance in OvCa cells, which may serve as a novel therapeutic target for prevention of OvCa peritoneal dissemination.     

Growth factor receptors signaling in glioblastoma cells: therapeutic implications

In this study, we investigated the protein expression of platelet-derived growth factor receptor (PDGFR), insulin like growth factor-1 receptor (IGF-1R), phosphatidylinositol 3-kinase (PI3-K) and extracellular signal-regulated kinase (ERK1/2) in five primary glioblastoma (GB), with a view to their possible use as therapeutic targets. Our results demonstrated that appreciable levels of these proteins could be detected in the analysed GB cell lines, except for a low level of PDGFR and ERK1/2 expression in one GB cell line. The small molecule inhibitors towards IGF-1R, PDGFR, PI3-K and ERK1/2 respectively, have only modest or no anti-tumour activity on GB cells and therefore their combination with other therapy modalities was analysed. The interaction between small inhibitors and radiation was mostly additive or sub-additive; synergistic interaction was found in five of forty analysed combinations. Our results showed that GB cells are in general resistant to treatment and illustrate the difficulties in predicting the treatment response in malignant gliomas.     

Decursin chemosensitizes human multiple myeloma cells through inhibition of STAT3 signaling pathway

Early detection of lung cancer provides the highest potential for saving lives. To date, no routine screening method enabling early detection is available, which is a key factor in the disease’s high mortality rate. Copy number changes and DNA methylation alterations are good indicators of carcinogenesis and cancer prognosis. In this study, we attempted to combine profiles of DNA copy number and methylation patterns in 20 paired cancerous and noncancerous tissue samples from non-small cell lung cancer (NSCLC) patients, and we detected several clinically important genes with genetic and epigenetic relationships. Using array comparative genomic hybridization (aCGH), statistically significant differences were observed across the histological subtypes for gains at 1p31.1, 3q26.1, and 3q26.31-3q29 as well as for losses at 1p21.1, 2q33.3, 2q37.3, 3p12.3, 4q35.2, and 13q34 in squamous cell carcinoma (SQ) patients, and losses at 12q24.33 were measured in adenocarcinoma (AD) patients (p < 0.05). In an analysis of DNA methylation at 1505 autosomal CpG loci that are associated with 807 cancer-related genes, we identified six and nine loci with higher and lower DNA methylation levels, respectively, in tumor tissue compared to non-tumor lung tissues from AD patients. In addition, three loci with higher and seven loci with lower DNA methylation levels were identified in tumor tissue from SQ patients compared to non-tumor lung tissue. Subsequently, we searched for regions exhibiting concomitant hypermethylation and genomic loss in both ADs and SQs. One clone representing 7p15.2 (which includes candidate genes such as HOXA9 and HOXA11) and one target ID representing HOXA9_E252_R were detected. Quantitative real-time PCR identified the potential candidate gene HOXA9 as being down-regulated in the majority of NSCLC patients. Moreover, following HOXA9 over-expression, the invasion of representative cell lines, A549 and HCC95, were significantly inhibited. Taken together, our results show that the combined profiling analysis technique is a useful tool for identifying biomarkers in lung cancer and that HOXA9 might be a potential candidate gene for the pathogenesis and diagnosis of NSCLC patients.