精氨酸缺乏对硼替佐米治疗多发性骨髓瘤的作用研究

摘要 目的：探讨精氨酸缺乏对硼替佐米(Bortezomib,BTZ) 治疗多发性骨髓瘤细胞的影响。方法：通过CCK8筛选BTZ对骨髓瘤细胞株(H929和RPMI 8226)治疗的最适药物浓度,比较在缺乏和富含精氨酸的两种培养基中的细胞增殖情况;通过使用PI染料标记细胞检测不同试验组细胞周期的分布,以及使用Annexin V/7AAD凋亡试剂盒检测BTZ对不同试验组细胞凋亡的影响。结果：BTZ降低了骨髓瘤细胞的存活率,并通过将细胞周期阻滞于G2/M、S期,抑制骨髓瘤细胞的增殖。缺乏精氨酸使细胞周期阻滞于S期,也抑制了骨髓瘤细胞的增殖。BTZ作用于缺乏精氨酸组的骨髓瘤细胞后,细胞凋亡百分比明显低于富含精氨酸组（H929细胞由约40%降至13.6%,RPMI8226凋亡百分比分别7.13%和19.27%）。结论：缺乏精氨酸和给予BTZ均阻滞细胞周期,抑制骨髓瘤细胞增殖;同时缺乏精氨酸降低了BTZ诱导骨髓瘤细胞的凋亡作用。     

Fluvoxamine and sigma-1 receptor agonists dehydroepiandrosterone (DHEA)-sulfate induces the Ser473-phosphorylation of Akt-1 in PC12 cells

AimsThe expression of brain-derived neurotrophic factor (BDNF) may be a downstream target of a variety of antidepressant treatments, and selective serotonin reuptake inhibitors (SSRIs) are used clinically for the treatment of depression. BDNF binds to and activates tyrosine kinases receptor (TrkB) to exert its effects. TrkB, after activation by ligands, stimulates phosphoinositide 3-kinase (PI3K). The downstream target of PI3K is Akt-1, a serine-threonine kinase. BDNF has signaling through the PLC-?IP₃/Ca²⁺ pathway. Furthermore, the PLC-?γ/IP₃/Ca²⁺ pathway is regulated by the sigma-1 receptors. Here, we examined whether fluvoxamine (FLV) activated Akt-1 and increased phosphorylation of Akt-1 via sigma-1 receptor in PC12 cells.Main methodsWe examined the effect of the SSRI, FLV and BDNF on the phosphorylation levels of serine-threonine kinase Akt-1 in PC12 cells using immunoblotting techniques.Key findingsTreatment with 10 μM and 100 μM FLV of PC12 cells stimulated a 2.4- and 3.8-fold maximal increase in Ser⁴⁷³-phosphorylated Akt-1 levels at 40 min, respectively. Treatment with 50 ng/ml BDNF also stimulated Ser⁴⁷³ -phosphorylated Akt-1 by 2.6-fold with a maximal increase at 5 min. In addition, the phosphorylation induced by FLV and BDNF was blocked by LY294002, a selective inhibitor of PI3K. The sigma-1 receptor agonists dehydroepiandrosterone (DHEA)-sulfate also stimulated a 2.1-fold increase in the level of Ser473-phosphorylated Akt-1.SignificanceThis study demonstrates that fluvoxamine treatment rapidly increased phosphorylation of Akt-1. And BDNF activated Akt-1 phosphorylation by the TrkB/PI3K/Akt-1 pathway. We conclude that the phosphorylation of Akt-1, downstream of PI3K, was the key to their antidepressant effects.     

Brain derived neurotrophic factor is involved in the regulation of glycogen synthase kinase 3β (GSK3β) signalling

Glycogen synthase kinase 3β (GSK3β) is involved in several biochemical processes in neurons regulating cellular survival, gene expression, cell fate determination, metabolism and proliferation. GSK3β activity is inhibited through the phosphorylation of its Ser-9 residue. In this study we sought to investigate the role of BDNF/TrkB signalling in the modulation of GSK3β activity. BDNF/TrkB signalling regulates the GSK3β activity both in vivo in the retinal tissue as well as in the neuronal cells under culture conditions. We report here for the first time that BDNF can also regulate GSK3β activity independent of its effects through the TrkB receptor signalling. Knockdown of BDNF lead to a decline in GSK3β phosphorylation without having a detectable effect on the TrkB activity or its downstream effectors Akt and Erk1/2. Treatment with TrkB receptor agonist had a stimulating effect on the GSK3β phosphorylation, but the effect was significantly less pronounced in the cells in which BDNF was knocked down. The use of TrkB receptor antagonist similarly, manifested itself in the form of downregulation of GSK3β phosphorylation, but a combined TrkB inhibition and BDNF knockdown exhibited a much stronger negative effect. In vivo, we observed reduced levels of GSK3β phosphorylation in the retinal tissues of the BDNF^+/− animals implicating critical role of BDNF in the regulation of the GSK3β activity. Concluding, BDNF/TrkB axis strongly regulates the GSK3β activity and BDNF also exhibits GSK3β regulatory effect independent of its actions through the TrkB receptor signalling.     

Paracrine and autocrine functions of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in brain-derived endothelial cells

Brain-derived neurotrophic factor (BDNF) is expressed by endothelial cells. We investigated the characteristics of BDNF expression by brain-derived endothelial cells and tested the hypothesis that BDNF serves paracrine and autocrine functions affecting the vasculature of the central nervous system. In addition to expressing TrkB and p75NTR and BDNF under normoxic conditions, these cells increased their expression of BDNF under hypoxia. While the expression of TrkB is unaffected by hypoxia, TrkB exhibits a base-line phosphorylation under normoxic conditions and an increased phosphorylation when BDNF is added. TrkB phosphorylation is decreased when endogenous BDNF is sequestered by soluble TrkB. Exogenous BDNF elicits robust angiogenesis and survival in three-dimensional cultures of these endothelial cells, while sequestration of endogenous BDNF caused significant apoptosis. The effects of BDNF engagement of TrkB appears to be mediated via the phosphatidylinositol (PI) 3-kinase-Akt pathway. Modulation of BDNF levels directly correlate with Akt phosphorylation and inhibitors of PI 3-kinase abrogate the BDNF responses. BDNF-mediated effects on endothelial cell survival/apoptosis correlated directly with activation of caspase 3. These endothelial cells also express p75NTR and respond to its preferred ligand, pro-nerve growth factor (pro-NGF), by undergoing apoptosis. These data support a role for neurotrophins signaling in the dynamic maintenance/differentiation of central nervous system endothelia.     

The Trkb Receptor Tyrosine Kinase Regulates Cellular Proliferation Via Signal Transduction Pathways Involving Shc,Plcγ, and Cbl

Abstract

The TrkB protein tyrosine kinase is a high affinity receptor for brain derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). TrkB autophosphorylation occurs on five cytoplasmic tyrosines: Y484, Y670, Y674, Y675, and Y785. Using site directed mutagenesis, we have assessed the importance of TrkB tyrosines 484 and 785 in affecting TrkB-mediated signaling events leading to NIH 3T3 cell mitogenesis and survival. Mutation of TrkB tyrosine 484, while having no affect on BDNF-inducible PLCγ and Cbl tyrosine phosphorylation, is essential for the phosphorylation of Shc, the complete activation of extracellular regulated kinase 1/2 (ERK1/2) and the induction of c-fos protein synthesis. In contrast, mutation of Y785 does not significantly affect BDNF-inducible Shc phosphorylation, ERK1/2 activation, or c-fos protein synthesis, but completely inhibits the tyrosine phosphorylation of PLCγ and Cbl. These data indicate that both ERK-dependent and ERK-independent signaling pathways lead to BDNF-inducible mitogenesis and survival.     

Xanthohumol exhibits anti-myeloma activity in vitro through inhibition of cell proliferation,induction of apoptosis via the ERK and JNK-dependent mechanism,and suppression of sIL-6R and VEGF production

BackgroundXanthohumol (XN, a hop-derived prenylflavonoid) was found to exert anticancer effects on various cancer types. However, the mechanisms by which XN affects the survival of multiple myeloma cells (MM) are little known. Therefore, our study was undertaken to address this issue.MethodsAnti-proliferative activity of XN towards two phenotypically distinct MM cell lines U266 and RPMI8226 was evaluated with the MTT and BrdU assays. Cytotoxicity was determined with the LDH method, whereas apoptosis was assessed by flow cytometry and fluorescence staining. The expression of cell cycle- and apoptosis-related proteins and the activation status of signaling pathways were estimated by immunoblotting and ELISA assays.ResultsXN reduced the viability of RPMI8226 cells more potently than in U266 cells. It blocked cell cycle progression through downregulation of cyclin D1 and increased p21 expression. The marked apoptosis induction in the XN-treated RPMI8226 cells was related to initiation of mitochondrial and extrinsic pathways, as indicated by the altered p53, Bax, and Bcl-2 protein expression, cleavage of procaspase 8 and 9, and elevated caspase-3 activity. The apoptotic process was probably mediated via ROS overproduction and MAPK (ERK and JNK) activation as N-acetylcysteine, or specific inhibitors of these kinases prevented the XN-induced caspase-3 activity and, hence, apoptosis. Moreover, XN decreased sIL-6R and VEGF production in the studied cells.ConclusionsERK and JNK signaling pathways are involved in XN-induced cytotoxicity against MM cells.General Significance: The advanced understanding of the molecular mechanisms of XN action can be useful in developing therapeutic strategies to treat multiple myeloma.     

Co-activation of the phosphatidylinositol-3-kinase/Akt signaling pathway by N-methyl-D-aspartate and TrkB receptors in cerebellar granule cell neurons

Summary.  Neuroprotective concentrations of N-methyl-D-aspartate (NMDA) promote survival of cerebellar granule cell neurons against glutamate excitotoxicity through a TrkB receptor-mediated brain-derived neurotrophic factor (BDNF) autocrine loop. However, the intracellular signaling pathway(s) are not clear. Our results show that PI-3 kinase/Akt is activated by either NMDA or BDNF displaying differential kinetics. BDNF and NMDA increased Akt phosphorylation within 5 minutes but maximal activation by NMDA was observed at 3 hours. Akt phosphorylation was completely blocked by the PI-3 kinase inhibitor LY294002. NMDA-mediated activation of Akt was completely blocked by MK-801 and partially blocked by the TrkB receptor inhibitor, K252a, indicating the requirement of TrkB receptors for maximal activation by NMDA. In contrast, BDNF-induced Akt phosphorylation was abolished by K252a, but not by the addition of MK-801. Therefore, the PI-3 kinase/Akt pathway is co-activated by NMDA and TrkB receptors. The kinetics of BDNF and NMDA-mediated activation of PI-3 kinase/Akt suggests that they have different roles in intraneuronal time-related events. Received June 29, 2001 Accepted August 6, 2001 Published online June 3, 2002     

The release of glutamate from cortical neurons regulated by BDNF via the TrkB/Src/PLC‐γ1 pathway

The brain‐derived neurotrophic factor (BDNF) participates in the regulation of cortical neurons by influencing the release of glutamate. However, the specific mechanisms are unclear. Hence, we isolated and cultured the cortical neurons of Sprague Dawley rats. Specific inhibitors of TrkB, Src, PLC‐γ1, Akt, and MEK1/2 (i.e., K252a, PP2, U73122, LY294002, and PD98059, respectively) were used to treat cortical neurons and to detect the glutamate release from cortical neurons stimulated with BDNF. BDNF significantly increased glutamate release, and simultaneously enhanced phosphorylation levels of TrkB, Src, PLC‐γ, Akt, and Erk1/2. For BDNF‐stimulated cortical neurons, K252a inhibited glutamate release and inhibited the phosphorylation levels of TrkB, Src, PLC‐γ, Erk1/2, and Akt (P < 0.05). PP2 reduced the glutamate release from BDNF‐stimulated cortical neurons (P < 0.05) and inhibited the phosphorylation levels of TrkB and PLC‐γ1 (P < 0.05). However, PP2 had no effect on the phosphorylation levels of Erk1/2 or Akt (P > 0.05). U73122 inhibited the glutamate release from BDNF‐stimulated cortical neurons, but had no influence on the phosphorylation levels of TrkB, Src, Erk1/2, or Akt (P > 0.05). LY294002 and PD98059 did not affect the BDNF‐stimulated glutamate release and did not inhibit the phosphorylation levels of TrkB, Src, or PLC‐γ1. In summary, BDNF stimulated the glutamate release from cortical neurons via the TrkB/Src/PLC‐γ1 signaling pathway. J. Cell. Biochem. 114: 144–151, 2012. © 2012 Wiley Periodicals, Inc.     

Neosetophomone B induces apoptosis in multiple myeloma cells via targeting of AKT/SKP2 signaling pathway

Multiple myeloma (MM) is a hematologic malignancy associated with malignant plasma cell proliferation in the bone marrow. Despite the available treatments, drug resistance and adverse side effects pose significant challenges, underscoring the need for alternative therapeutic strategies. Natural products, like the fungal metabolite neosetophomone B (NSP-B), have emerged as potential therapeutic agents due to their bioactive properties. Our study investigated NSP-B's antitumor effects on MM cell lines (U266 and RPMI8226) and the involved molecular mechanisms. NSP-B demonstrated significant growth inhibition and apoptotic induction, triggered by reduced AKT activation and downregulation of the inhibitors of apoptotic proteins and S-phase kinase protein. This was accompanied by an upregulation of p21Kip1 and p27Cip1 and an elevated Bax/BCL2 ratio, culminating in caspase-dependent apoptosis. Interestingly, NSP-B also enhanced the cytotoxicity of bortezomib (BTZ), an existing MM treatment. Overall, our findings demonstrated that NSP-B induces caspase-dependent apoptosis, increases cell damage, and suppresses MM cell proliferation while improving the cytotoxic impact of BTZ. These findings suggest that NSP-B can be used alone or in combination with other medicines to treat MM, highlighting its importance as a promising phytoconstituent in cancer therapy.     

Activation of STAT3/HIF-1α/Hes-1 axis promotes trastuzumab resistance in HER2-overexpressing breast cancer cells via down-regulation of PTEN

BackgroundResistance to the HER2-targeted antibody trastuzumab remains to be a major clinical challenge in the treatment of HER2-positive breast cancer. Hyper-activation of STAT3 is proposed to be a predictive biomarker of trastuzumab resistance. However, the precise mechanism(s) remains poorly defined. Evidence is emerging that HIF-1α, a central downstream element of STAT3 pathway, serves a pivotal role in the complex signaling network with subsequent diverse cellular events.Material and methodsWe have established trastuzumab resistant SKBR3 cells (SKBR3-TR). The cell viability, apoptosis as well as western blot, siRNA transfection and co-immunoprecipitation assays were performed to evaluate the involvement of STAT3/HIF-1α in modulation of trastuzumab resistance.ResultsWe found that in SKBR3-TR cells and conditioned medium-treated parental cells, constitutive phosphorylated STAT3 coincided with prominent up-regulation of HIF-1α which was accompanied with PTEN attenuation. Moreover, the inhibition of STAT3 activation by Stattic and/or genetically STAT3 knocking down decreased HIF-1α level in SKBR3-TR cells. Additionally, treatment with Stattic and/or STAT3 siRNA engendered the up-regulation of PTEN protein in STAT3-inhibited resistant cells. Restoration of PTEN was also observed following siRNA-mediated silencing of HIF-1α expression. Moreover, down-regulation of HIF-1α caused a reduction in the HES-1 content. Further study with HES-1 specific siRNA revealed the elevation of PTEN expression in HES-1 knock-down trastuzumab resistant cells.ConclusionThe impairment of STAT3-HIF-1α-HES-1 pathway restored trastuzumab sensitivity through up-regulation of PTEN protein.General significanceThese findings highlighted the signal integrator role of HIF-1α in STAT3-mediated trastuzumab resistance induction which would be valuable in designing more efficient chemosensitization strategies.     

Characterization of TrkB Receptor-Mediated Signaling Pathways in Rat Cerebellar Granule Neurons: Involvement of Protein Kinase C in Neuronal Survival

Abstract: TrkB belongs to the Trk family of tyrosine kinase receptors and mediates the response to brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5). Here, we report that both truncated and full-length forms of TrkB receptors are expressed in developing cerebellar granule neurons. BDNF and NT-4/5 increased the survival of cultured cerebellar granule neurons. BDNF and NT-4/5 also induced an autophosphorylation of TrkB receptors and subsequently resulted in a phosphorylation and binding of phospholipase C-γ (PLC-γ) and SH2-containing sequence to the autophosphorylated TrkB receptors. Both contain src homology 2 (SH2) regions. In keeping with a signaling function of PLC-γ, BDNF increased the phosphatidylinositol (PI) turnover and elevated intracellular calcium levels. To investigate the involvement of protein kinase C (PKC) in the survival of granular neurons, we show here activation of PKC after BDNF or TPA treatment and blocking of the observed survival-promoting effects of BDNF and TPA with calphostin C, a specific PKC inhibitor. In addition, BDNF activated c- ras in a concentration-dependent manner. These results suggest that two different pathways, the c- ras and the PLC-γ pathway, are activated by TrkB receptors in primary neurons and that PKC activation is involved in the survival promoting effect of BDNF.     

Cell Signaling and Differential Protein Expression in Neuronal Differentiation of Bone Marrow Mesenchymal Stem Cells with Hypermethylated Salvador/Warts/Hippo (SWH) Pathway Genes

Human mesenchymal stem cells (MSCs) modified by targeting DNA hypermethylation of genes in the Salvador/Warts/Hippo pathway were induced to differentiate into neuronal cells in vitro. The differentiated cells secreted a significant level of brain-derived neurotrophy factor (BDNF) and the expression of BDNF receptor tyrosine receptor kinase B (TrkB) correlated well with the secretion of BDNF. In the differentiating cells, CREB was active after the binding of growth factors to induce phosphorylation of ERK in the MAPK/ERK pathway. Downstream of phosphorylated CREB led to the functional maturation of differentiated cells and secretion of BDNF, which contributed to the sustained expression of pERK and pCREB. In summary, both PI3K/Akt and MAPK/ERK signaling pathways play important roles in the neuronal differentiation of MSCs. The main function of the PI3K/Akt pathway is to maintain cell survival during neural differentiation; whereas the role of the MAPK/ERK pathway is probably to promote the maturation of differentiated MSCs. Further, cellular levels of protein kinase C epsilon type (PKC-ε) and kinesin heavy chain (KIF5B) increased with time of induction, whereas the level of NME/NM23 nucleoside diphosphate kinase 1 (Nm23-H1) decreased during the time course of differentiation. The correlation between PKC-ε and TrkB suggested that there is cross-talk between PKC-ε and the PI3K/Akt signaling pathway.     

Impaired NHEJ function in multiple myeloma

Multiple myeloma (MM) is characterized by multiple chromosomal aberrations. To assess the contribution of DNA repair to this phenotype, ionizing radiation was used to induce DNA double strand breaks in three MM cell lines. Clonogenic survival assays showed U266 (SF4 = 15.3 + 6.4%) and RPMI 8226 (SF4 = 12.6.0 + 1.7%) were radiation sensitive while OPM2 was resistant (SF4 = 78.9 + 4.1%). Addition of the DNA-PK inhibitor NU7026 showed the expected suppression in radiation survival in OPM2 but increased survival in both radiation sensitive cell lines. To examine non-homologous end joining (NHEJ) repair in these lines, the ability of protein extracts to support in vitro DNA repair was measured. Among the three MM cell lines analyzed, RPMI 8226 demonstrated impaired blunt ended DNA ligation using a ligation-mediated PCR technique. In a bacterial based functional assay to rejoin a DNA break within the β-galactosidase gene, RPMI 8226 demonstrated a 4-fold reduction in rejoining fidelity compared to U266, with OPM2 showing an intermediate capacity. Ionizing radiation induced a robust γ-H2AX response in OPM2 but only a modest increase in each radiation sensitive cell line perhaps related to the high level of γ-H2AX in freshly plated cells. Examination of γ-H2AX foci in RPMI 8226 cells confirmed data from Western blots where a significant number of foci were present in freshly plated untreated cells which diminished over 24 h of culture. Based on the clonogenic survival and functional repair assays, all three cell lines exhibited corrupt NHEJ repair. We conclude that suppression of aberrant NHEJ function using the DNA-PK inhibitor NU7026 may facilitate access of DNA ends to an intact homologous recombination repair pathway, paradoxically increasing survival after irradiation. These data provide insight into the deregulation of DNA repair at the site of DNA breaks in MM that may underpin the characteristic genomic instability of this disease.     

Brain-derived neurotrophic factor induces phosphorylation of fibroblast growth factor receptor substrate 2

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival. Gaining an understanding of how BDNF, via the tropomyosin-related kinase B (TRKB) receptor, elicits specific cellular responses is of contemporary interest. Expression of mutant TrkB in fibroblasts, where tyrosine 484 was changed to phenylalanine, abrogated Shc association with TrkB, but only attenuated and did not block BDNF-induced phosphorylation of mitogen-activated protein kinase (MAPK). This suggests there is another BDNF-induced signaling mechanism for activating MAPK, which compelled a search for other TrkB substrates. BDNF induces phosphorylation of fibroblast growth factor receptor substrate 2 (FRS2) in both fibroblasts engineered to express TrkB and human neuroblastoma (NB) cells that naturally express TrkB. Additionally, BDNF induces phosphorylation of FRS2 in primary cultures of cortical neurons, thus showing that FRS2 is a physiologically relevant substrate of TrkB. Data are presented demonstrating that BDNF induces association of FRS2 with growth factor receptor-binding protein 2 (GRB2) in cortical neurons, fibroblasts, and NB cells, which in turn could activate the RAS/MAPK pathway. This is not dependent on Shc, since BDNF does not induce association of Shc and FRS2. Finally, the experiments suggest that FRS2 and suc-associated neurotrophic factor-induced tyrosine-phosphorylated target are the same protein.     

Brain-derived neurotrophic factor promotes proliferation and progesterone synthesis in bovine granulosa cells

Brain-derived neurotrophic factor (BDNF) is involved in regulating the growth of ovarian follicles, maturation of the oocyte, and development of the early embryo through its receptor, tyrosine kinase receptor B (TrkB). However, it is still unclear as to how BDNF influences proliferation and steroidogenesis of bovine granulosa cells (GCs). In this paper, we confirmed that BDNF and TrkB were expressed in bovine GCs, and that proliferation and steroidogenesis by bovine GCs were reduced by knockdown of BDNF or inhibition of TrkB. With respect to GC proliferation, BDNF enhanced cellular viability and the percentage of cells in the S phase. BDNF also activated both protein kinase B (PKB, also known as AKT) and the extracellular signal-regulated protein kinase 1/2 (ERK1/2)-signaling pathway. Through the AKT-signaling pathway, BDNF increased the expression of proliferation-related genes, including cyclin A1 (CCNA1), cyclin E2 (CCNE2), cyclin D1 (CCND1), and cyclin-dependent kinase 1 (CDK1). However, through the ERK1/2 signaling pathway, BDNF only increased the expression of CCNA1 and CCNE2. Regarding steroidogenesis by bovine GCs, BDNF promoted progesterone (P ₄) synthesis, but had no effect on estradiol; it also activated the AKT-signaling pathway and increased the expression of steroidogenesis-related genes, including steroidogenic acute regulatory protein (STAR) and hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 1 (HSD3B1). In summary, our data are the first to show that BDNF promotes the proliferation of bovine GCs through TrkB–AKT and ERK1/2 signaling pathways and increases P₄ synthesis by bovine GCs through the TrkB–AKT signaling pathway.     

miR-451a targeting IL-6R activates JAK2/STAT3 pathway,thus regulates proliferation and apoptosis of multiple myeloma cells

Objectives:To investigate the effects of miR-451a targeting interleukin-6 (IL-6) on the proliferation and apoptosis of multiple myeloma (MM) cells and its potential mechanism via JAK2/STAT3 pathway.Methods:mRNA expression of miR-451a and IL-6R in the plasma of patients with MM and normal controls were determined by RT-qPCR. U266 cells were cultured, transfected with miR-451a mimics, the proliferative ability of U266 cells was determined by CCK-8. Potential targets of miR-451a were predicted with the biological software TargetScan, and the direct relationship between miR-451a and the target IL-6R was analyzed by a dual-luciferase reporter assay. U266 cells were stimulated with IL-6R (100 ng/ml), and the proliferative ability and apoptosis rate were determined by CCK-8 and flow cytometry after 48h.Results:In the plasma of patients with MM, miR-451a expression was low and IL-6R expression was high. miR-451a targeted and negatively regulated IL-6R. Overexpressing miR-451a inhibited the proliferation and promoted the apoptosis of U266 cells. IL-6R acting on U266 cells promoted the proliferation and inhibited the apoptosis of U266 cells. Overexpressing miR-451a inhibited the activation of JAK2/STAT3 pathway and down-regulating miR-451a promoted the activation of JAK2/STAT3 pathway.Conclusions:miR-451a targeting IL-6R activates JAK2/STAT3 pathway, thus regulates the proliferation and apoptosis in MM cells.     

Improved antitumoral properties of pure antiestrogen RU 58668-loaded liposomes in multiple myeloma

In most of multiple myeloma (MM) cells, the "pure" antiestrogen (AE) RU 58668 (RU) induced either a G1-arrest (LP-1, OPM-2, NCI-H929, U266 cells) or apoptosis (RPMI 8226 cells). In RPMI 8226 cells, RU activates a caspase-dependent cell death pathway leading to the release of cytochrome c, the decrease of the essential MM survival factor Mcl-1, the cleavage of Bid and the activation of caspases-3 and -8. Incorporation of RU in pegylated cholesterol-containing liposomes allowed a controlled RU release, improving its anti-proliferative and apoptotic effects in cells. In RPMI 8226 xenografts, i.v. injected RU-liposomes but not free RU, exhibited antitumor activity. In vivo, RU-liposomes triggered the mitochondrial death pathway, concomitantly with a down-regulation of Mcl-1 and Bid cleavage. The decrease of CD34 immunoreactivity indicated a reduction of angiogenesis. The decrease of VEGF secretion in vitro supported a direct effect of RU on angiogenesis. These pro-apoptotic and antiangiogenic effects were explained by a prolonged exposure to the drug and to the endocytosis capacity of liposomes which might increase RU uptake and bypass a membrane export of free RU. Thus, these combined enhanced activities of RU-liposomes support that such a delivery of an AE may constitute a strategy of benefit for MM treatment.     

Exosomal mRNAs and lncRNAs involved in multiple myeloma resistance to bortezomib

The bone marrow microenvironment plays an essential role in multiple myeloma (MM) progression. We aimed to explore the alterations of levels of long noncoding RNAs and messenger RNAs (mRNAs), derived from exosomes in peripheral blood, in resistance to bortezomib (Btz) of MM patients. Peripheral blood samples were collected from five Btz-resistant and five Btz-sensitive MM patients. Exosomes in patients' peripheral blood were enriched, and the profiles of long noncoding RNAs (lncRNAs) and mRNAs in exosomes were determined using deep sequencing. Bioinformatics analysis was performed to explore biological function. MTS was employed to determine the viability of Roswell Park Memorial Institute (RPMI) 8226 and LP-1 cells incubated with exosomes derived from Btz-resistant patients. Quantitative polymerase chain reaction (qPCR) was used to evaluate the levels of exosomal FFAR1, SP9, HIST1H2BG, and ITIH2. Incubation with Btz-resistant patient-derived exosomes significantly increased the viability of Btz-treated RPMI 8226 and LP-1 cells in a dose-dependent manner. We identified 482 lncRNAs and 2099 mRNAs deregulated in exosomes of the Btz-resistance group; and 78 mRNAs were enriched in DR-related pathways, including mammalian target of rapamycin, platinum drug resistance, and the cAMP and phosphoinositide 3-kinase–Akt signaling pathways. qPCR results verified the increases in FFAR1 and SP9 and decreases in HIST1H2BG and ITIH2 in Btz-resistant patient-derived exosomes. Moreover, exosomal FFAR1 and SP9 exhibited potential as independent prognostic indicators of survival of MM patients. Our study reveals significant dysregulation of exosomal RNA components in the Btz-resistant group of MM patients as well as several mRNAs that may be used as biomarkers of prognosis of MM patients that are resistant to Btz.     

A novel trimeric peptide, Neuropep-1-stimulating brain-derived neurotrophic factor expression in rat brain improves spatial learning and memory as measured by the Y-maze and Morris water maze

Abundant studies have shown possible links between low levels of brain-derived neurotrophic factor (BDNF) and neurological diseases such as Alzheimer's disease, Parkinson's disease, and depression, as well as stress and anxiety; therefore, BDNF could be a therapeutic target for neurological disorders. In the present study, a positional scanning-synthetic peptide combinatorial library was utilized to identify a peptide modulator of BDNF expression in the hippocampal neuronal cell line, H19-7. A novel tripeptide (Neuropep-1) induced a significant increase of BDNF mRNA and protein levels in H19-7 cells. Pre-treatment of TrkB inhibitor (K252a) did not block Neuropep-1-induced BDNF up-regulation. These results indicate that Neuropep-1 may up-regulate BDNF expression that might be independent of the TrkB receptor pathway. Tail vein injection of Neuropep-1 significantly up-regulated BDNF expression, TrkB phosphorylation, and its downstream signals including activation of Akt, ERK, and cAMP response element binding in the rat hippocampus. To evaluate improvement of spatial learning and memory (SLM) by Neuropep-1-induced BDNF up-regulation, the Y-maze and Morris water maze tests were performed. These results showed Neuropep-1 injection improved SLM performance with increase of BDNF and TrkB expression, activation of TrkB downstream signals in rat hippocampus compared with the control group. However, phosphorylation levels of TrkB were not changed when it was normalized to the level of TrkB expression. The difference on TrkB phosphorylation in Neuropep-1-injected rats may be affected by behavioral tests. These results suggest that Neuropep-1 may improve SLM via activation of the BDNF/TrkB signaling pathway in the rat hippocampus. Therefore, our findings represent that Neuropep-1 might be a potential candidate for treatment of learning and memory disorders as well as neurological diseases involving the abnormal expression of BDNF.