lncRNA HAND2-AS1 overexpression inhibits cancer cell proliferation in hepatocellular carcinoma by downregulating RUNX2 expression.

BACKGROUND: The long non-coding RNA HAND2 antisense RNA 1 (HAND2-AS1) acts as a tumor suppressor in several malignancies, but its role in hepatocellular carcinoma (HCC) remains unknown. In this study, we aimed to investigate the function of HAND2-AS1 in HCC. METHODS: The expression levels of HAND2-AS1 and runt-related transcription factor 2 (RUNX2) were determined in patients with HCC and HCC cell lines using quantitative real-time polymerase chain reaction and western blot analyses. Cell proliferation was determined using Cell Counting Kit-8 assay, and the correlation between HAND2-AS1 and RUNX2 expression was also investigated. RESULTS: The plasma level of HAND2-AS1 was downregulated and that of RUNX2 was upregulated in patients with early-stage HCC compared with those in healthy controls. No significant differences in the plasma levels of HAND2-AS1 and RUNX2 were found among hepatitis B virus (HBV)-positive, hepatitis C virus (HCV)-positive, and HBV- and HCV-negative patients with HCC. The plasma levels of HAND2-AS1 and RUNX2 were inversely correlated in the patient groups but not in the control group. HAND2-AS1 overexpression led to the downregulation of RUNX2 expression in human HCC cells, whereas RUNX2 failed to significantly affect HAND2-AS1 expression. HAND2-AS1 overexpression inhibited and RUNX2 overexpression promoted the proliferation of HCC cells. RUNX2 overexpression attenuated the inhibitory effects of HAND2-AS1 overexpression on cancer cell proliferation. CONCLUSION: HAND2-AS1 overexpression inhibits cancer cell proliferation in HCC by downregulating RUNX2 expression.

THBS1/CD47 Modulates the Interaction of γ-Catenin With E-Cadherin and Participates in Epithelial-Mesenchymal Transformation in Lipid Nephrotoxicity.

Hyperlipidemia, an important risk factor for cardiovascular and end-stage renal diseases, often aggravates renal injury and compromises kidney function. Here, histological analysis of human kidney samples revealed that high lipid levels induced the development of renal fibrosis. To elucidate the mechanism underlying lipid nephrotoxicity, we used two types of mouse models (Apoe-/- and C57BL/6 mice fed a 45 and 60% high-fat diet, respectively). Histological analysis of kidney tissues revealed high-lipid-induced renal fibrosis and inflammation; this was confirmed by examining fibrotic and inflammatory marker expression using Western blotting and real-time polymerase chain reaction. Oxidized low-density lipoprotein (OX-LDL) significantly induced the fibrotic response in HK-2 tubular epithelial cells. RNA-sequencing and Gene Ontology analysis of differentially expressed mRNAs in OX-LDL-treated HK-2 tubular epithelial cells and real-time PCR validation in Apoe-/- mice showed that the expression of thrombospondin-1 (THBS1) in the high-fat group was significantly higher than that of the other top known genes, along with significant overexpression of its receptor CD47. THBS1 knockdown cells verified its relation to OX-LDL-induced fibrosis and inflammation. Liquid chromatography tandem mass spectrometry and STRING functional protein association network analyses predicted that THBS1/CD47 modulated the interaction between γ-catenin and E-cadherin and was involved in epithelial-mesenchymal transition, which was supported by immunoprecipitation and immunohistochemistry. CD47 downregulation following transfection with small-hairpin RNA in OX-LDL-treated tubular epithelial cells and treatment with anti-CD47 antibody restored the expression of E-cadherin and attenuated renal injury, fibrosis, and inflammatory response in OX-LDL-treated cells and in type 2 diabetes mellitus. These findings indicate that CD47 may serve as a potential therapeutic target in long-term lipid-induced kidney injury.

Rosiglitazone Reduces Apoptosis and Inflammation in Lipopolysaccharide-Induced Human Umbilical Vein Endothelial Cells.

BACKGROUND Although the peroxisome proliferator-activated receptor-g (PPARg) agonist rosiglitazone has significant anti-inflammatory properties, no scientific studies have provided new insights in its pharmacological properties with respect to acute respiratory distress syndrome (ARDS). The present investigation aimed to evaluate whether rosiglitazone can reduce apoptosis and inflammation in a lipopolysaccharide (LPS)-induced acute respiratory distress syndrome in vitro model. MATERIAL AND METHODS Human umbilical vein endothelial cells (HUVECs) were treated with 1 µg/ml LPS in the absence or presence of 10 µM rosiglitazone for 24 h. Cell viability was measured by MTT assay. Flow cytometry was used to examine the cell apoptosis and ROS production in HUVECs response to LPS and rosiglitazone. The levels of pro-inflammatory cytokine factors, including TNF-α, IL-6, CXCL12, and CXCR4, were measured by ELISA, real-time PCR, and Western blot assay, respectively. The expression of PPARg, Bcl-2, and Bax and the activity of JAK2 and STAT3 were also investigated by Western blot assay. RESULTS We found that rosiglitazone significantly inhibited LPS-induced cell apoptosis, ROS production, and inflammation in HUVECs. Furthermore, we found a significant reduction of JAK2/STAT3 activation and the Bax/Bcl-2 ratio in LPS-induced HUVECs response to rosiglitazone treatment. CONCLUSIONS Treatment with rosiglitazone can reduce apoptosis and inflammation in HUVECs induced by LPS.

[Downregulation of MCL-1 by Diallyl Disulfide Induces G2/M Arrest in Human Leukemia K562 Cells and Its Mechanism].

OBJECTIVE: To investigate the inducing effect of down-regulation of MCL-1 by diallyl disulfide(DADS) on the G2/M arrest of human leukemia K562 cells and its mechanisms. METHODS: CCK-8 was used to detect the effect of DADS on proliferation of K562 cells, flow cytometry was employed to observe the effect of cycle arrest by DADS and RNAi silencing MCL-1 gene in K562 cells. The expressions of MCL-1, PCNA and CDK1 in K562 cells treated with DADS were detected by Western blot. The amphigamy of MCL-1 with PCNA and CDK1 was detected by Coimmunoprecipitation. RESULTS: CCK-8 detection showed that the inhibition rates of K562 cells treated with 15, 30, 60, 120, 240 µmol/L DADS were 32.48%, 59.34%, 66.42%, 77.06%, 81.05% respectively (P<0.05). Flow cytometry analysis revealed that the perecentages of G2/M cells were increased to 18.6% and 34.4%, 17.5% and 28.5%, respectively at 24 and 48 h after treating K562 cells with 60 and 120 µmol/L DADS (P<0.05). And the perecentage of G2/M cells of silencing MCL-1 was significantly increased (P<0.05). Silencing effects of MCL-1+DADS on the cells were enhanced more significantly as compared with DADS or MCL-1 alone (P<0.05). Western blot exhibited that DADS could markedly downregulate the expression of MCL-1, PCNA and CDK1(P<0.05). Coimmunoprecipitation revealed that MCL-1 bound with PCNA and CDK1, then forming heterodimers, which were downregulated respectively more significantly than that in the control group after treating K562 cells with DADS for 8 h (P<0.05). CONCLUSION: DADS can inhibit the K562 cell proliferation and induce them arrest G2/M through downregulation of MCL-1, then decreasing the expression of PCNA and CDK1 in hunan leukemia K562 cells. Moreover, silencing MCL-1 can enhance the effect of DADS.

Involvement of Mcl1 in diallyl disulfide-induced G2/M cell cycle arrest in HL-60 cells.

Diallyl disulfide (DADS) has shown potential as a therapeutic agent in various cancers. Previously, we found that myeloid cell leukemia sequence 1 (Mcl1) was downregulated in DADS-induced cell cycle arrest in HL-60 human leukemia cells. Here, we investigated the role of this protein in DADS-induced G2/M cell cycle arrest in HL-60 cells. We demonstrated that DADS treatment significantly increased the proportion of G2/M phase HL-60 cells (P<0.05) and caused a time-dependent significant downregulation of Mcl1 and the cell cycle-related proteins PCNA and CDK1 (P<0.05). Small interfering RNA-mediated knockdown of Mcl1 expression in HL-60 cells arrested the cell cycle in G2/M phase. By co-immunoprecipitation, we demonstrated that Mcl1 associated with PCNA and CDK1 in G2/M cell cycle arrest in DADS-treated HL-60 cells. DADS decreased the interaction of Mcl1 with PCNA and CDK1, leading to G2/M cell cycle arrest in HL-60 cells. Mcl1 plays an important role in DADS-induced G2/M cell cycle arrest in HL-60 human leukemia cells.

Diallyl disulfide induces apoptosis in human leukemia HL-60 cells through activation of JNK mediated by reactive oxygen.

Diallyl disulfide (DADS) is a chemopreventive agent that can induce apoptosis in many tumor cells. Reactive oxygen species (ROS) are important mediators in apoptosis induced by various stimuli, including chemopreventive agents. The phosphotransferase c-JUN N-terminal kinase (JNK) has been shown to regulate apoptosis. In this study, we found that DADS-induced apoptosis in human leukemia HL-60 cells is mediated by ROS-activated JNK. The DADS-treated HL-60 cells showed a dose- and time-dependent decrease in cell viability and proliferation. Agarose gel electrophoresis of cells treated with 10.0 or 20.0 mg/L DADS for 24 h showed a characteristic ladder pattern in their DNA. Levels of DADS-induced ROS, as measured by 2',7'-dichlorofluorescein diacetate (DCFH-DA) fluorescence, also showed dose- and time-dependent increases in HL-60 cells. Activity of JNK was induced by DADS in a dose-dependent manner; HL-60 cells exposed to 10.0 mg/L DADS for 8 h showed maximum levels of phosphorylated JNK, which decreased when exposed for additional 4h. In contrast, Sp600125, a specific inhibitor of JNK, blocked apoptosis of HL-60 cells exposed to DADS. N-Acetylcysteine (NAC), a known antioxidant, also decreased ROS generation, effectively blocked apoptosis, and decreased DADS-induced phosphorylated JNK levels. These results suggest that JNK is involved in DADS-induced ROS-mediated apoptosis in HL-60 cells.

Role of Ras-related C3 botulinum toxin substrate 2 (Rac2), NADPH oxidase and reactive oxygen species in diallyl disulphide-induced apoptosis of human leukaemia HL-60 cells.

1. Diallyl disulphide (DADS) has potential as a chemopreventive and therapeutic agent. Previous studies have reported that Ras-related C3 botulinum toxin substrate 2 (Rac2), a regulatory subunit of the NADPH oxidase complex, is upregulated in DADS-induced apoptosis in human leukaemia HL-60 cells. The aim of the present study was to investigate the role of Rac2, NADPH oxidase and reactive oxygen species (ROS) in DADS-induced apoptosis. 2. Expression of the Rac2 gene along with that of five other genes of NADPH oxidase subunits were in HL-60 cells measured by Sybergreen quantitative real-time polymerase chain reaction. RNA interference was used to test the effect of Rac2. Protein expression was evaluated using western blot analysis and ROS levels were measured by 2',7'-dichlorofluorescein diacetate (DCFH-DA) fluorescence. DNA fragmentation and flow cytometry analysis were used to detect apoptotic cells. 3. Levels of Rac2 gene and protein were significantly upregulated and NADPH oxidase was activated in DADS-induced apoptosis. Pretreatment of HL-60 cells with small interfering (si) RNAs to inhibit Rac2 blocked DADS-induced apoptosis. Diallyl disulphide-induced intracellular ROS production was increased in phorbol myristate acetate-stimulated cells, but decreased in Rac2 siRNA-treated cells. In Rac2 siRNA-treated cells, activator protein-1 and caspase 3 levels decreased, c-myc protein levels were increased and p38 protein levels were unchanged compared with Rac2-competent, DADS-treated cells. 4. These results demonstrate that NADPH oxidase is the main source of DADS-induced ROS. In addition, Rac2 selectively activates the c-Jun N-terminal kinase pathway, but not the p38 pathway, in DADS-induced apoptosis. So, Rac2, NADPH oxidase and ROS have a critical role in DADS-induced apoptosis in human leukaemia HL-60 cells.

Proteomics analysis of apoptosis initiation induced by diallyl disulfide in human leukemia HL-60 cells.

BACKGROUND AND OBJECTIVE: Diallyl disulfide (DADS), an antitumor reagent, has increasingly gained attention. This study was to explore the related proteins in DADS-induced apoptosis initiation in human leukemia HL-60 cells. METHODS: Total protein of HL-60 cells with or without 2-day treatment of 3.6 mg/L DADS was extracted, separated by two-dimensional polyacrylamide gel electrophoresis, and analyzed by PDQuest 2-DE software. Differentially expressed proteins were separated and identified by peptide mass fingerprinting analysis and bioinformatics, and verified by western blot and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: As compared with those in untreated HL-60 cells, 29 proteins were differentially expressed in DADS-treated HL-60 cells: 22 were upregulated and seven were downregulated. Among nine proteins which were randomly selected for peptide mass fingerprinting analysis and bioinformatics, seven were meaningful. These proteins were associated with cellular responses, gene transcription and regulation, cytoskeleton, metabolism, and so on. western blot showed that Ras-related C3 botulinum toxin substrate 2 (Rac2) was upregulated in DADS-treated HL-60 cells; this result was accordant with RT-PCR results. CONCLUSION: Some proteins, including Rac2, may be involved in DADS-induced apoptosis in human leukemia HL-60 cells, but the exact mechanism needs to be explored.