Na+,K+-ATPase inhibitor induces cell cycle arrest in liver cancer HepG2 cells by regulating expression of DNA damage Mre11/Rad50/Nbs1 complex / 中国药理学通报

Aim To explore the relationship between Mre11/Rad50/Nbs1 ( MRN ) complex focus formation and DNA double-strand breaks( DSBs) caused by cinob-ufagin in human hepatocellular carcinoma HepG2 cells. Methods The Na+,K+-ATPaseα1 subunit expression level in liver cancer tissues was detected by immunohis-tochemistry. After HepG2 cells were treated with 5μmol·L-1 cinobufagin for 6, 12 and 24 h, the drug-in-duced DSBs were assessed by single cell gel electro-phroesis ( SCGE ) , the gene transcription and protein levels of Mrel1, Nbs1, Rad50 and p53 were evaluated by Real time-PCR and Western blot. The cell cycle in parallel was analyzed by flow cytometry. Results The Na+, K+-ATPase α1 subunit expression level in liver cancer tissues was significantly increased compared with the tissue adjacent to carcinoma ( P <0. 05 ) . The 5μmol · L-1 cinobufagin could induce the DSBs in a time-dependent manner (P <0. 05), and it could up-regulate the gene expression levels of Mre11, Nbs1, Rad50 and p53 in HepG2 cells ( P<0. 05 ) . The pro-portions of HepG2 cells in S phase were ( 21. 32 ± 4. 21) % in the control group, and (33. 25 ± 5. 72) %, (56. 72 ± 6. 29) % and (67. 32 ± 9. 42) % in HepG2 cells treated with 5 μmol · L-1 cinobufagin for 6, 12 and 24 h, respectively. The proportions of cells in S phase in cinobufagin groups were significantly increased compared with the control group ( P<0. 05 ) . Conclu-sion Cinobufagin could induce the cell cycle arrest in liver cancer HepG2 cells by activation of Mre11/Rad50/Nbs1 Complex.

ZFP580, a novel transcription factor, is involved in cardioprotection of hypoxic preconditioning against hypoxia-reoxygenation injury in myocar-dial cells / 中国病理生理杂志

AIM:To elucidate whether ZFP580 is involved in the cardioprotective effects of hypoxic precondi-tioning (HPC) against hypoxia-reoxygenation (H/R) injury in H9c2 myocardial cells.METHODS: Rat heart-derived H9c2 cells were cultured in DMEM .H/R was induced by incubation under ischemic hypoxia for 3 h and reoxygenation for 2 h.HPC was induced by exposing the H 9c2 cells to 10 min of hypoxia and 20 min of reoxygenation for 3 cycles before H/R treatment.MTT staining and LDH leakage detection were used to evaluate the effects of HPC .Western blotting was used to detect the protein levels of ZFP580, phosphorylated ERK1/2 and cleaved caspased-3.The effects of ZFP580 overexpre-ssion or knockdown on H/R induced apoptosis were determined .RESULTS:The results of MTT staining and LDH leakage detection showed evidence of HPC cytoprotection against H /R-induced cell death in H9c2 cells.ZFP580 protein level and ERK1/2 phosphorylation were significantly increased in the HPC group compared with control group and H /R group. PD98059, an inhibitor of ERK1/2 phosphorylation , significantly suppressed the HPC-induced up-regulation of ZFP580 pro-tein expression.ZFP580 overexpression significantly inhibited apoptosis and caspase-3 activation in H9c2 cells.CON-CLUSION:HPC exhibits cytoprotection against H/R and leads to high level of ZFP 580 protein in H9c2 cells.ZFP580 is regulated by ERK1/2 activation and mediates the anti-apoptotic effect of the ERK1/2 signaling pathway in HPC cytoprotec-tion.

The linkage between cell cycle S phase arrest and apoptosis on human hepatocellular carcinoma HepG2 induced by Na~+,K~+-ATPase inhibitors via regulating proteins associated with cell cycle / 中国药理学通报

Aim To investigate the effect of ouabain and cinobufogenin on cell proliferation,apoptosis and cell cycle on HepG2,and explore their molecular mechanism.Methods The anti-proliferative effect on HepG2 cells was determined by MTT assay.The HepG2 cells were stained with Hoechst 33342,and its morphological changes were observed under fluorescence microscope;The cell cycle was measured by flow cytometry.The Cyclin A1,CDK 2,PCNA and p21~(CIP1) expression levels of HepG2 cells treated with ouabain and cinobufogenin were dectected in mRNA and protein by Real time PCR and Western blot.Results Ouabain and cinobufogenin could inhibit cell proliferation on HepG2 cells,and the inhibitory effects were in time and dose dependent manners.The HepG2 cells treated with ouabain and cinobufogenin showed the typical morphological features of apoptosis.Cell cycle analysis showed that the S phase of HepG2 cells treated with ouabain and cinobufogenin increased significantly compared with the control group.Real-time quantitative PCR and Western blot results showed that ouabain and cinobufogenin could down-regulate Cyclin A1,CDK 2,and PCNA expressions(P<0.05)and up-regulate p21~(CIP1) expression(P<0.05).Conclusion Nα~+,K~+-ATPase inhibitor has the anti-proliferative effect on HepG2 cells and induce apoptosis and S phase arrest.These effects might be related with proteins associated with cell cycle closely.

Protective effects of ouabain conjugated peptide from Ph. D.-7 Library on vascular endothelial cell / 细胞与分子免疫学杂志

AIM: To find one kind of peptide that will conjugate with ouabain and inhibit its biological function, and provide a new treatment strategy for primary hypertension. METHODS: In this study, ouabain was used as a target to screen ouabain conjugated peptide (OCP) from Ph. D. -7 phage display peptide library. After 3 rounds of bio-panning, the products were identified by ELISA and DNA electrophoresis analysis and sequencing. Peptide was synthesized and analyzed the activity by radioligand binding assay. The inhibitory ratio of cell proliferation was measured by MTT and the cell morphology changing was measured by Hoechst 33342/PI staining. The expression of Na~+-K~+-ATPase α1-subunit and β1-subunit were detected by RT-PCR and immunocytochemistry. The levels of the free intracellular Na~+ in EAhy926 cells were measured by laser confocal microscope. RESULTS: The ouabain conjugated peptide was found out, and it was occupied in 0.64(9/14). The analysis of protein showed that the obtained peptides had no homology with Na~+-K~+-ATPase. The amino acid sequence of synthesized peptide was Arg-Cys-Met-Thr-Ser-Arg-Ser. There was binding activity between OCP and ~3H-ouabain. The MTT assay showed that OCP could reverse the inhibition action of ouabain on vascular endothelial EAhy926 cells in a dose and time-dependent manner. The number of apoptotic cells had significantly decreased detected by Hoechst 33342/PI staining. The results of RT-PCR and immunocytochemistry showed that OCP could inhibit the up-regulated expression of Na~+-K~+-ATPase α1-subunit and down-regulated expression of Na~+-K~+-ATPase β1-subunit induced by ouabain in EAhy926 cells. CONCLUSION: The OCP could reverse the growth inhibition and death induction of ouabain in EAhy926 cells, which would provide the basis for studying the interaction between ouabain and Na~+-K~+-ATPase and explore novel anti-ouabain agents.

Up-expression of peroxisome proliferator-activated receptor-γand of retinoid X receptor-αenhances the inhibitory effect on leukemia cells / 白血病·淋巴瘤

Objective To investigate the relationship between the expression of peroxisome proliferators-activated recepter-γ (PPARγ) and retinoid X receptor-or (RXRα) and the inhibitory effect of PLAB, ligand of PPARγ and 9-cisRA, ligand of RXRα on growth of human leukemia cell lines (HL-60, K562and U937) in vitro. Methods The antiproliferative effect was evaluated by MTT assay. The mRNA expression of PPARγ and RXRα was semi-quantified by RT-PCR. Results PPARγ and RXRα mRNA was both expressed in HL-60, K562 and U937 cells, and the expression in HI,-60 was significantly higher than that in K562 and U937. The significant inhibitory effect on the growth of HL-60 cells was observed in K562 and U937 cells. The combination group showed more inhibitory effect in HL-60 cells than PLAB alone(P＜0.05).PLAB significantly up-regulates the expression of PPARγ in HL-60 cells, the expression of PPARγ and RXRα were higher in combination group than PLAB alone (P＜0.05). Conclusion The expression of PPARγand RXRα in HL-60, K562 and U937 cell lines predicts their response to PLAB and 9-cisRA treatment, and the inhibitory effect is different in these three kinds of cell lines, which may be related to their ligandsmediated signal pathway.

Up-expression of peroxisome proliferator-activated receptor-? and of retinoid X receptor-? enhances the inhibitory effect on tumor cells growth / 基础医学与临床

LOVO.PGZ significantly up-regulates the expression of PPAR? in MGC803 and LOVO cells,the expression of PPAR? was higher in combination group than PGZ alone(P

The ouabain signal transduction and its role in cell growth and death / 中国药理学通报

The ouabain is a member of the cardiotonic steroids (CT S) that specifically bind to Na+,K+-ATPase. Binding of ouabain to Na+,K +-ATPase causes rapid activation of Src family kinases, and forms a Na+,K +-ATPase-Src complex as a active binary receptor to phosphorylate and assem ble other proteins into different signaling modules.Oubain stimulates hypertroph ic growth in cardiac myocytes and proliferation in smooth muscle cells, it also induces apoptosis and a newly recognized "hybrid cell death" of concurrent apopt osis and necrosisin the same cells.