Elotuzumab Enhances CD16-Independent NK Cell-Mediated Cytotoxicity against Myeloma Cells by Upregulating Several NK Cell-Enhancing Genes.

Multiple myeloma (MM) is an intractable hematological malignancy caused by abnormalities in plasma cells. Combination therapy using antibodies and natural killer (NK) effectors, which are innate immune cells with safe and potent antitumor activity, is a promising approach for cancer immunotherapy and can enhance antitumor effects. Elotuzumab (Elo) is an immune-stimulatory antibody that targets the signaling lymphocytic activation molecule family 7 (SLAMF7) expressed on the surface of MM and NK cells. We confirmed that Elo strongly promoted NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) against SLAMF7-positive MM cells in a CD16-dependent NK cell line, and also activated expanded NK cells derived from peripheral blood mononuclear cells of healthy donors and patients with MM in the present study. However, the antitumor effects and genes involved in the direct promotion of NK cell-mediated activation using Elo in CD16-independent NK cells are not clearly known. In this study, we demonstrated that Elo pretreatment significantly enhanced CD16-independent NK cell-mediated cytotoxicity in both SLAMF7-positive MM.1S and SLAMF7-negative K562, U266, and RPMI 8226 tumor cells. Upon direct simulation of CD16-independent NK cells with Elo, increased levels of CD107a degranulation and IFN-γ secretion were observed along with the upregulation of granzyme B, TNF-α, and IL-1α gene expression. The enhanced NK cell function could also be attributed to the increased expression of the transcription factors T-BET and EOMES. Furthermore, the augmentation of the antitumor effects of CD16-independent NK cells upon pretreatment with Elo enhanced the expression of CRTAM, TNFRSF9, EAT-2, and FOXP3 genes and reduced the expression of HSPA6. Our results suggest that Elo directly promotes the cytotoxic function of CD16-independent NK cells against target cells, which is associated with the upregulation of the expression of several NK cell-enhancing genes.

A nerve conduit filled with Wnt5a-loaded fibrin hydrogels promotes peripheral nerve regeneration.

AIMS: Peripheral nerve injury is a significant clinical problem with a substantial impact on quality of life, for which no optimal treatment has been found. This study aimed to analyze the effect and mechanism of Wnt5a-loaded fibrin hydrogel on a 10-mm rat sciatic nerve defect. METHODS: The Wnt5a-loaded fibrin hydrogel was synthesized by mixing a Wnt5a solution with thrombin and fibrinogen solutions. The loading capacity and release profile of Wnt5a-loaded fibrin hydrogel and the effect of Wnt5a on Schwann cells were evaluated in vitro. We also assessed the in vivo repair status via histological analysis of the regenerative nerve and gastrocnemius muscle, electrophysiological examination, gait analysis, and muscle wet weight. RESULTS: We developed a nerve conduit filled with Wnt5a-loaded fibrin hydrogel (Fn) as a sustained-release system to repair a 10-mm rat sciatic nerve defect. In vitro, Wnt5a could promote SC proliferation and the gene expression of vascular endothelial growth factor (VEGF), nerve growth factor (NGF), and cholinergic neurotrophic factor (CNTF), as well as the protein secretion of VEGF and NGF. In vivo, the Wnt5a/Fn group was superior to the hollow, fibrin hydrogel, and Wnt5a groups in terms of axonal growth, myelination, electrophysiological recovery, target organ innervation, and motor function recovery 12 weeks after the operation. CONCLUSION: The Wnt5a/Fn nerve conduit can promote peripheral nerve defect regeneration, with potential clinical applications. The mechanism for this may be the facilitation of multiple neurotrophin secretion, combining vascularization and neurotrophic growth cues.

Graphene Oxide Nanoparticle-Loaded Ginsenoside Rg3 Improves Photodynamic Therapy in Inhibiting Malignant Progression and Stemness of Osteosarcoma.

Osteosarcoma serves as a prevalent bone cancer with a high metastasis and common drug resistance, resulting in poor prognosis and high mortality. Photodynamic therapy (PDT) is a patient-specific and non-invasive tumor therapy. Nanoparticles, like graphene oxide have been widely used in drug delivery and PDT. Ginsenoside Rg3 is a principal ginseng component and has presented significant anti-cancer activities. Here, we constructed the nanoparticles using GO linked with photosensitizer (PS) indocyanine green (ICG), folic acid, and polyethylene glycol (PEG), and loaded with Rg3 (PEG-GO-FA/ICG-Rg3). We aimed to explore the effect of PEG-GO-FA/ICG-Rg3 combined with PDT for the treatment of osteosarcoma. Significantly, we found that Rg3 repressed proliferation, invasion, and migration, and enhanced apoptosis and autophagy of osteosarcoma cells, while the PEG-GO-FA/ICG-Rg3 presented a higher activity, in which NIR laser co-treatment could remarkably increase the effect of PEG-GO-FA/ICG-Rg3. Meanwhile, stemness of osteosarcoma cell-derived cancer stem cells was inhibited by Rg3 and PEG-GO-FA/ICG-Rg3, and the combination of PEG-GO-FA/ICG-Rg3 with NIR laser further significantly attenuated this phenotype in the system. Moreover, NIR laser notably improved the inhibitor effect of PEG-GO-FA/ICG-Rg3 on the tumor growth of osteosarcoma cells in vivo. Consequently, we concluded that PEG-GO-FA/ICG-Rg3 improved PDT in inhibiting malignant progression and stemness of osteosarcoma cell. Our finding provides a promising and practical therapeutic strategy for the combined treatment of osteosarcoma.

High PRAME expression is associated with poor survival and early disease progression in myelodysplastic syndromes with a low bone marrow blast percentage.

We investigated the clinical implications of preferentially expressed antigen in melanoma (PRAME) expression in bone marrow cells of 116 patients with myelodysplastic syndromes (MDS). Quantitative RT-PCR was carried out to examine the PRAME expression level. High PRAME expression was observed in MDS patients classified into higher revised International Prognostic Scoring System (IPSS-R) risk categories (Very high and High) with a high bone marrow blast percentage (5% or higher). Kaplan-Meier analysis demonstrated that high PRAME expression is significantly associated with a poorer overall survival (OS) in MDS patients with a low bone marrow blast percentage (less than 5%) (log-rank test p = .0014) and those classified into lower IPSS-R risk categories (Very Low, Low, and Intermediate) (log-rank test, p = .0035). In contrast, there was no significant association between PRAME expression and OS in MDS patients with a high bone marrow blast percentage or those classified into higher IPSS-R risk categories. In addition, high PRAME expression was associated with early disease progression in MDS patients with a low bone marrow blast percentage. This study suggested PRAME expression to be a prognostic factor in MDS.

The clinical significance, prognostic value and biological role of lncRNA LINC01793 in oral squamous cell carcinoma.

OBJECTIVE: The present study aimed to investigate the clinical significance and prognostic value of LINC01793 in OSCC patients, and to explore its role in the modulation of OSCC development. METHODS: LINC01793 expression was analyzed in 80 cases of OSCC patients and SCC9, SCC25, Cal27, and HN6 cell lines by qRT-PCR. The association of LINC01793 expression with clinicopathological features and prognosis in OSCC patients was analyzed. The effects of LINC01793 on cell proliferation, cell cycle, migration, and invasion of SCC9 and Cal27 cells were detected by MTT, flow cytometry, and Transwell assays in vitro, respectively. RESULTS: LINC01793 level was upregulated in cancer tissues and cell lines of OSCC, and its expression was increased in cancer tissues from patients with lymph node metastasis. ROC curve for LINC01793 expression and lymph node metastasis revealed a significant AUC of 0.84 (95 % CI: 0.75-0.93), with 76.51 % sensitivity and 83.69 % specificity. Moreover, high LINC01793 level was positively correlated with T category, TNM stage, lymph node metastasis, and local recurrence. OSCC patients with high level of LINC01793 was followed by low overall survival rate, and LINC01793 expression was an independent prognostic indicator for overall survival in patients with OSCC. Functionally, cell proliferation, invasion and migration of SCC9 and Cal27 cells were decreased after knockdown of LINC01793. Consistently, silence of LINC01793 induced G0/G1 cell cycle arrest in OSCC cells. CONCLUSION: High LINC01793 level is correlated with adverse clinicopathological features and poor prognosis of patients with OSCC. LINC01793 act as an oncogenic role in the development of OSCC.

Integrated Omics of Metastatic Colorectal Cancer.

We integrate the genomics, proteomics, and phosphoproteomics of 480 clinical tissues from 146 patients in a Chinese colorectal cancer (CRC) cohort, among which 70 had metastatic CRC (mCRC). Proteomic profiling differentiates three CRC subtypes characterized by distinct clinical prognosis and molecular signatures. Proteomic and phosphoproteomic profiling of primary tumors alone successfully distinguishes cases with metastasis. Metastatic tissues exhibit high similarities with primary tumors at the genetic but not the proteomic level, and kinase network analysis reveals significant heterogeneity between primary colorectal tumors and their liver metastases. In vivo xenograft-based drug tests using 31 primary and metastatic tumors show personalized responses, which could also be predicted by kinase-substrate network analysis no matter whether tumors carry mutations in the drug-targeted genes. Our study provides a valuable resource for better understanding of mCRC and has potential for clinical application.

MiR-375 promotes human periodontal ligament stem cells proliferation and osteogenic differentiation by targeting transducer of ERBB2, 2.

BACKGROUND: MiRNAs have been demonstrated to be important regulators during osteogenic differentiation in multiple types of stem cells. In the study, the interaction between miR-375 and TOB2 was analyzed to identify their functions on the proliferation and osteogenic differentiation of hPDLSCs. METHODS: hPDLSCs were isolated from human first premolars, and hPDLSCs stably expressing and silenced miR-375 were constructed using miR-375-ago and miR-375-antago, respectively. miR-375 and RUNX2 mRNA expression levels in hPDLSCs during osteogenic differentiation were investigated using qRT-PCR. The impact of miR-375 expression on hPDLSCs proliferation and osteogenic differentiation was determined using MTT assay, ALP assay, and alizarin red S staining. The protein expression levels of COL1A1, RUNX2 and OCN were detected using Western blot. The targeting of TOB2 by miR-375 was validated using dual luciferase reporter assay. RESULTS: The expression levels of miR-375 were increased in hPDLSCs during osteogenic differentiation in a time-dependant manner, and was positively correlated with RUNX2 mRNA expression. miR-375 facilitated the proliferation and osteogenic differentiation of hPDLSCs, and promoted the protein expression levels of COL1A1, RUNX2 and OCN. Moreover, TOB2 protein expression was reduced in hPDLSCs during osteogenic differentiation in a time-dependant manner, and miR-375 directly targeted TOB2 expression. In addition, targeting TOB2 expression in hPDLSCs could rescue the suppression of cell proliferation and osteogenic differentiation by miR-375-antago. CONCLUSION: In summary, miR-375 promotes proliferation and osteogenic differentiation of hPDLSCs by targeting TOB2, which reveals a new regulatory mechanism underlying osteogenic differentiation of hPDLSCs by miR-375/TOB2 axis.

Reduced PLCG1 expression is associated with inferior survival for myelodysplastic syndromes.

The PLCG1 gene, which encodes the phospholipase C γ1 isoform, is located within the commonly deleted region of the long arm of chromosome 20 (del(20q)) observed in myelodysplastic syndromes (MDS). Phospholipase C is involved in diverse physiological and pathological cellular processes through inositide signaling. We hypothesized that reduced PLCG1 expression because of haploinsufficiency by del(20q) plays a role in the molecular pathogenesis of MDS. Therefore, we analyzed PLCG1 expression in bone marrow mononuclear cells at diagnosis in 116 MDS patients with or without del(20q) by quantitative RT-PCR to evaluate its clinical significance. The expression level of PLCG1 was significantly lower not only in MDS patients with del(20q) but also in those without del(20q) compared to that of the controls, which suggests that reduced PLCG1 expression is a common molecular event in MDS. Patients in the lowest quartile (Q4) group for PLCG1 expression had lower overall survival (OS) compared to that of other patients (Q1-Q3) (log-rank test, P = .0004) with estimated median OS times of 22 in the Q4 group and 106 months in the Q1-3 group. Univariate and multivariate analysis indicated reduced PLCG1 expression (Q4) was associated with lower OS (hazard ratio 2.58, 95% CI 1.35-4.84, P = .0049), which suggests that reduced PLCG1 expression is an independent prognostic factor for OS. In addition, patients were well-stratified for OS by combining PLCG1 expression level (Q4 vs Q1-3) and bone marrow blast percentage (5% or more vs less than 5%). Thus, the level of PLCG1 expression at time of diagnosis is a prognostic biomarker for MDS.

Expression analysis of genes located within the common deleted region of del(20q) in patients with myelodysplastic syndromes.

Deletion of the long arm of chromosome 20 (del(20q)) is observed in 5-10% of patients with myelodysplastic syndromes (MDS). We examined the expression of 28 genes within the common deleted region (CDR) of del(20q), which we previously determined by a CGH array using clinical samples, in 48 MDS patients with (n = 28) or without (n = 20) chromosome 20 abnormalities and control subjects (n = 10). The expression level of 8 of 28 genes was significantly reduced in MDS patients with chromosome 20 abnormalities compared to that of control subjects. In addition, the expression of BCAS4, ADA, and YWHAB genes was significantly reduced in MDS patients without chromosome 20 abnormalities, which suggests that these three genes were commonly involved in the molecular pathogenesis of MDS. To evaluate the clinical significance, we analyzed the impact of the expression level of each gene on overall survival (OS). According to the Cox proportional hazard model, multivariate analysis indicated that reduced BCAS4 expression was associated with inferior OS, but the difference was not significant (HR, 3.77; 95% CI, 0.995-17.17; P = 0.0509). Functional analyses are needed to understand the biological significance of reduced expression of these genes in the pathogenesis of MDS.

[Effect of modified Buyang Huanwu Decoction on hemorrhagic transformation after rt-PA intravenous thrombolysis in patients with super early cerebral infarction].

To study the effect of modified Buyang Huanwu Decoction on the hemorrhagic transformation after intravenous thrombolysis of recombinant tissue type plasminogen activator(rt-PA) in patients with super early(onset time<4. 5 h) cerebral infarction. From March 2016 to July 2018,at the brain disease zone of the First Affiliated Hospital of Henan University of Traditional Chinese Medicine,212 cases of super early cerebral infarction were selected and divided into two group according to the randomized complete blocks designs: control group(106 cases) and traditional Chinese medicine group(106 cases). The control group was treated with rt-PA intravenous thrombolysis,while the traditional Chinese medicine group was treated with modified Buyang Huanwu Decoction in addition to the therapy of the control group. Both groups were treated for 14 days. Neurological deficit score,serum matrix metalloproteinase-9(MMP-9),neuron specific enolase(NSE),vascular endothelial growth factor(VEGF) and plasma cellular fibronectin(c-FN) levels,the incidence of hemorrhagic transformation,clinical efficacy and adverse drug reactions before and after treatment were compared between the two groups. According to the findings,at the 14 thday after treatment,the rank sum test of the grade data showed that the clinical efficacy of the traditional Chinese medicine group was better than that of the control group(Z =-2. 033,P = 0. 042); on the basis of χ2 test,the total efficiency of the traditional Chinese medicine group was higher than that of the control group(χ2= 4. 895,P =0. 027); the hemorrhagic transformation rate of the traditional Chinese medicine group was lower than that of the control group within14 days of treatment(χ2= 3. 962,P = 0. 047). MMP-9 levels in the traditional Chinese medicine group were lower than those in the control group at the 3 rd,5 th,7 th,10 th,14 thd after treatment(t =-2. 474,-3. 022,-5. 163,-6. 998,-9. 821; P = 0. 014,0. 003,0,0,0). The improvement of c-FN,NSE,VEGF and NIHSS scores in the traditional Chinese medicine group was superior to that of the control group after 14 days of treatment(t =-2. 343,-3. 187,-2. 129,-3. 105; P = 0. 020,0. 002,0. 034,0. 002). No obvious adverse reactions of modified Buyang Huanwu Decoction were observed during 14 days of treatment. Modified Buyang Huanwu Decoction could reduce the expressions of MMP-9,c-FN,NSE and VEGF after rt-PA intravenous thrombolysis in patients with super early cerebral infarction,and decrease the hemorrhagic transformation rate after thrombolysis,with high safety.

Long noncoding RNA SBF2-AS1 act as a ceRNA to modulate cell proliferation via binding with miR-188-5p in acute myeloid leukemia.

LncRNA SBF2-AS1 has been reported to be implicated in the deterioration of multiple human cancers. However, the roles and underlying mechanisms of SBF2-AS1 in acute myeloid leukemia (AML) are still unclear. In the present study, the online GEPIA database showed that SBF2-AS1 expression was significantly increased in AML samples. QRT-PCR results showed that SBF2-AS1 expression was upregulated in AML cells. CCK-8 assay revealed that SBF2-AS1 inhibition decreased AML cells proliferation ability in vitro. Flow cytometry assays showed that SBF2-AS1 inhibition induced AML cells apoptosis and arrested AML cells in G0/G1 phase. Mechanistically, miR-188-5p was identified as a direct target of SBF2-AS1. SBF2-AS1 upregulated the expression level of ZFP91 by sponging miR-188-5p. And the effects of SBF2-AS1 suppression on AML cells progression could be abolished by miR-188-5p inhibitors. Moreover, we found that SBF2-AS1 inhibition reduced tumor growth in vivo. Taken together, our findings elucidated that SBF2-AS1 could act as a miRNA sponge in AML progression, and provided a potential therapeutic strategy for AML treatment.

A CASPR1-ATP1B3 protein interaction modulates plasma membrane localization of Na+/K+-ATPase in brain microvascular endothelial cells.

Contactin-associated protein 1 (CASPR1 or CNTNAP1) was recently reported to be expressed in brain microvascular endothelial cells (BMECs), the major component of the blood-brain barrier. To investigate CASPR1's physiological role in BMECs, here we used CASPR1 as a bait in a yeast two-hybrid screen to identify CASPR1-interacting proteins and identified the ß3 subunit of Na+/K+-ATPase (ATP1B3) as a CASPR1-binding protein. Using recombinant and purified CASPR1, RNAi, GST-pulldown, immunofluorescence, immunoprecipitation, and Na+/K+-ATPase activity assays, we found that ATP1B3's core proteins, but not its glycosylated forms, interact with CASPR1, which was primarily located in the endoplasmic reticulum of BMECs. CASPR1 knockdown reduced ATP1B3 glycosylation and prevented its plasma membrane localization, phenotypes that were reversed by expression of full-length CASPR1. We also found that the CASPR1 knockdown reduces the plasma membrane distribution of the α1 subunit of Na+/K+-ATPase, which is the major component assembled with ATP1B3 in the complete Na+/K+-ATPase complex. The binding of CASPR1 with ATP1B3, but not the α1 subunit, indicated that CASPR1 binds with ATP1B3 to facilitate the assembly of Na+/K+-ATPase. Furthermore, the activity of Na+/K+-ATPase was reduced in CASPR1-silenced BMECs. Interestingly, shRNA-mediated CASPR1 silencing reduced glutamate efflux through the BMECs. These results demonstrate that CASPR1 binds with ATP1B3 and thereby contributes to the regulation of Na+/K+-ATPase maturation and trafficking to the plasma membrane in BMECs. We conclude that CASPR1-mediated regulation of Na+/K+-ATPase activity is important for glutamate transport across the blood-brain barrier.

Establishment of trauma treatment teams within a regional severe trauma treatment system in China: study protocol for a national cluster-randomised trial.

INTRODUCTION: The implementation of first aid processes for patients with trauma in China faces significant challenges. These challenges include long response times of prehospital first aid services, lack of information exchange between prehospital first aid services and in-hospital emergency services, lack of a professional rescue team in the majority of hospitals, and lack of standardised training for prehospital and in-hospital emergency personnel. The purpose of the trial is to guide the establishment of an urban trauma treatment system in China, highlight the construction of a trauma treatment system tailored to the Chinese context and improve levels of medical treatment by selecting approximately 100 counties across China as pilots to establish a regional trauma treatment system. METHODS AND ANALYSIS: A cluster-randomised controlled trial will be performed in 98 county-level research institutes. Included research institutes will be randomised into an experimental group and a control group. Patients in both experimental and control groups will receive basic treatments. A trauma treatment team will be established in the experimental group. The primary outcome measure is in-hospital mortality rate of patients. The secondary outcome measures include mortality rate of patients within 30 days after trauma attack and within 30 days after discharge, the time between arrival in the institution and receiving consultation, and the time from admission to the start of surgery. The effects of establishment of trauma treatment teams on the treatment of severe trauma will be evaluated in all counties. ETHICS AND DISSEMINATION: The procedures have been approved by The Medical Ethics Committee of Peking University People's Hospital (No.2017PHB098-01) and conform to the Declaration of Helsinki. Data will be collected and analysed in accordance with participant privacy laws and regulations. Results will be disseminated through policy briefs, workshops, peer-reviewed publications and conferences. TRIAL REGISTRATION NUMBER: NCT03363880; Pre-results.

Homocysteine-induced proliferation of vascular smooth muscle cells occurs via PTEN hypermethylation and is mitigated by Resveratrol.

Vascular smooth muscle cell (VSMC) proliferation is a primary pathological event in the development of atherosclerosis (AS), and the presence of homocysteine (Hcy) acts as an independent risk factor for AS. However, the underlying mechanisms remain to be elucidated. Phosphatase and tensin homologue on chromosome 10 (PTEN), is endogenously expressed in VSMCs and induces multiple signaling networks involved in cell proliferation, survival and inflammation, however, the specific role of PTEN is still unknown. The present study detected the proliferation ratio of VSMCs following treatment with Hcy and Resveratrol (RSV). In the 100 µM Hcy group, the proliferation ratio increased, and treatment with RSV decreased the proliferation ratio induced by Hcy. Reverse transcription­quantitative polymerase chain reaction and western blotting were used to analyze PTEN expression, RSV treatment was associated with decreased PTEN expression levels in VSMCs. PTEN levels were decreased in Hcy treated cells, and the proliferation ratio of VSMCs were increased following treated with Hcy. To study the mechanism of regulation of PTEN by Hcy, the present study detected PTEN methylation levels in VSMCs, and PTEN DNA methylation levels were demonstrated to be increased in the 100 µM Hcy group, whereas treatment with RSV decreased the methylation status. DNA methyltransferase 1 is important role in the regulation of PTEN methylation. Overall, Hcy impacts the methylation status of PTEN, which is involved in cell proliferation, and induces the proliferation of VSMCs. This effect is alleviated by treatment with RSV, which exhibits an antagonistic mechanism against Hcy.

Knockdown of the Wnt receptor Frizzled-1 (FZD1) reduces MDR1/P-glycoprotein expression in multidrug resistant leukemic cells and inhibits leukemic cell proliferation.

Multidrug resistance (MDR) is a major obstacle to leukemia treatment. The Frizzled-1 (FZD1) Wnt receptor is involved in MDR in some solid cancers, but has rarely been reported to act in acute myeloid leukemia (AML). We investigated whether the knockdown of FZD1 affects MDR1 expression and P-glycoprotein (P-gp) function in multidrug resistant leukemic cell lines, as well as FZD1 and MDR1/P-gp expression in leukemic cells taken from patients with AML (n = 112). FZD1 knockdown significantly reduced MDR1 expression through the Wnt/ß-catenin pathway, disrupted the P-gp efflux function, induced the recovery of sensitivity to chemotherapeutic agents, and hindered cell proliferation in cell lines. FZD1 expression in leukemic cells was significantly higher in patients experiencing relapse (n = 34) than in those with no relapse (n = 44, P = .003). Leukemic cells unable to achieve complete response (CR) showed an increased expression of MDR1 and P-gp, compared to patients who achieved CR. Obtaining CR in patients with higher FZD1 expression at diagnosis is difficult. Moreover, they tend to present instances of relapse, suggesting that AML cells with increased FZD1 expression are resistant to chemotherapy. We conclude that the activated FZD1 observed in leukemic cells likely confers acquired drug resistance, whereas FZD1 silencing may be more effective in reversing MDR.

Homocysteine inhibits endothelial progenitor cells proliferation via DNMT1-mediated hypomethylation of Cyclin A.

Endothelial progenitor cells (EPCs) contribute to neovasculogenesis and reendothelialization of damaged blood vessels to maintain the endothelium. Dysfunction of EPCs is implicated in the pathogenesis of vascular injury induced by homocysteine (Hcy). We aimed to investigate the role of Cyclin A in Hcy-induced EPCs dysfunction and explore its molecular mechanism. In this study, by treatment of EPCs with Hcy, we found that the expression of Cyclin A mRNA and protein were significantly downregulated in a dose-dependent manner. Knockdown of Cyclin A prominently reduced proliferation of EPCs, while over-expression of Cyclin A significantly promoted the cell proliferation, suggesting that Hcy inhibits EPCs proliferation through downregulation of Cyclin A expression. In addition, epigenetic study also demonstrated that Hcy induces DNA hypomethylation of the Cyclin A promoter in EPCs through downregulated expression of DNMT1. Moreover, we found that Hcy treatment of EPCs leads to increased SAM, SAH and MeCP2, while the ratio of SAM/SAH and MBD expression decrease. In summary, our results indicate that Hcy inhibits Cyclin A expression through hypomethylation of Cyclin A and thereby suppress EPCs proliferation. These findings demonstrate a novel mechanism of DNA methylation mediated by DNMT1 in prevention of Hcy associated cardiovascular disease.

Aberrant promoter methylation of multiple genes in VSMC proliferation induced by Hcy.

Vascular smooth muscle cell (VSMC) proliferation is a primary pathological event in atherosclerosis (AS), and homocysteine (Hcy) is an independent risk factor for AS. However, the underlying mechanisms are still lagging. Studies have used the combination of methylation of promoters of multiple genes to diagnose tumors, thus the aim of the current study was to investigate the role of methylation status of several genes in VSMCs treated with Hcy. CpG islands were identified in the promoters of platelet­derived growth factor (PDGF), p53, phosphatase and tensin homologue on chromosome 10 (PTEN) and mitofusin 2 (MFN2). Hypomethylation was observed to occur in the promoter region of PDGF, hypermethylation in p53, PTEN and MFN2, and hypomethylation in two global methylation indicators, aluminium (Alu) and long interspersed nucleotide element­1 (Line­1). This was accompanied by an increase in the expression of PDGF, and reductions of p53, PTEN and MFN2, both in mRNA and protein levels. An elevation of S­adenosylmethionine (SAM) and a reduction of S­adenosylhomocysteine (SAH) and the SAM/SAH ratio were also identified. In conclusion, Hcy impacted methylation the of AS­associated genes and global methylation status that mediate the cell proliferation, which may be a character of VSMCs treated with Hcy. The data provided evidence for mechanisms of VSMCs proliferation in AS induced by Hcy and may provide a new perspective for AS induced by Hcy.

FoxM1 promotes epithelial-mesenchymal transition of hepatocellular carcinoma by targeting Snai1.

Forkhead box protein M1 (FoxM1) is aberrantly expressed in several types of human malignancy, and serves an important role in tumor metastasis. Epithelial­mesenchymal transition (EMT) of cancer cells has been associated cancer metastasis; however, the implication of FoxM1 in EMT and its putative roles in the regulation of cancer metastasis remain to be elucidated. In the present study, the expression of FoxM1, Snai1 and E­cadherin in hepatocellular carcinoma (HCC) cell lines with various metastatic potentials, and in normal liver cells, was investigated using western blot analysis and reverse transcription­quantitative polymerase chain reaction. The effects of FoxM1 on the invasive and migratory capabilities of HCC cells were evaluated using wound healing and Transwell migration assays. The present results demonstrated that FoxM1 expression was significantly upregulated in HCC cells compared with in normal hepatocytes (P<0.05). In addition, FoxM1 expression was significantly increased in MHCC­LM3 cells, characterized by higher metastatic potential, compared with in SMMC­7721 cells, which have a lower metastatic potential. Furthermore, overexpression of FoxM1 was demonstrated to be negatively correlated with E­cadherin (P<0.05) and positively associated with Snai1 (P<0.05) expression. These observations suggested that FoxM1 may enhance the invasion and migration of cancer cells, and thus promotes their EMT, in a mechanism that may involve the regulation of Snai1. Therefore, it may be hypothesized that FoxM1 has potential as a novel diagnostic marker and therapeutic target for the treatment of patients with HCC.

Histone deacetylase inhibitor panobinostat induces calcineurin degradation in multiple myeloma.

Multiple myeloma (MM) is a relapsed and refractory disease, one that highlights the need for developing new molecular therapies for overcoming of drug resistance. Addition of panobinostat, a histone deacetylase (HDAC) inhibitor, to bortezomib and dexamethasone improved progression-free survival (PFS) in relapsed and refractory MM patients. Here, we demonstrate how calcineurin, when inhibited by immunosuppressive drugs like FK506, is involved in myeloma cell growth and targeted by panobinostat. mRNA expression of PPP3CA, a catalytic subunit of calcineurin, was high in advanced patients. Panobinostat degraded PPP3CA, a degradation that should have been induced by inhibition of the chaperone function of heat shock protein 90 (HSP90). Cotreatment with HDAC inhibitors and FK506 led to an enhanced antimyeloma effect with a greater PPP3CA reduction compared with HDAC inhibitors alone both in vitro and in vivo. In addition, this combination treatment efficiently blocked osteoclast formation, which results in osteolytic lesions. The poor response and short PFS duration observed in the bortezomib-containing therapies of patients with high PPP3CA suggested its relevance to bortezomib resistance. Moreover, bortezomib and HDAC inhibitors synergistically suppressed MM cell viability through PPP3CA inhibition. Our findings underscore the usefulness of calcineurin-targeted therapy in MM patients, including patients who are resistant to bortezomib.