[Corrigendum] Ouabain suppresses the growth and migration abilities of glioma U­87MG cells through inhibiting the Akt/mTOR signaling pathway and downregulating the expression of HIF­1α.

Subsequently to the publication of the above paper, an interested reader drew to the authors' attention that several pairings of panels in Fig. 5, as shown on p. 5599, were strikingly similar. After having examined their original data, the authors realized that they uploaded some images incorrectly during the process of compiling this figure, and that there were duplicated data panels in this figure. However, the authors were able to consult their original data, and had access to the correct images. The revised version of Fig. 5, showing the correct data for the Akt/Control, p­Akt/Control, mTOR/0.05 µM Ouabain, HIF­1α/0.05 µM Ouabain and Akt/0.5 µM Ouabain experiments, is shown opposite. Note that the replacement of the erroneous data does not affect either the results or the conclusions reported in this paper, and all the authors agree to this Corrigendum. The authors are grateful to the Editor of Molecular Medicine Reports for granting them this opportunity to publish a Corrigendum, and apologize to the readership for any inconvenience caused. [the original article was published in Molecular Medicine Reports 17: 5595­5600, 2018; DOI: 10.3892/mmr.2018.8587].

Ouabain suppresses the growth and migration abilities of glioma U­87MG cells through inhibiting the Akt/mTOR signaling pathway and downregulating the expression of HIF­1α.

Glioma is one of the most malignant forms of brain tumor, and has been of persistent concern due to its high recurrence and mortality rates, and limited therapeutic options. As a cardiac glycoside, ouabain has widespread applications in congestive heart diseases due to its positive cardiac inotropic effect by inhibiting Na+/K+­ATPase. Previous studies have demonstrated that ouabain has antitumor activity in several types of human tumor, including glioma. However, the exact underlying mechanism remains to be elucidated. The purpose of present study was to elucidate the effect of ouabain on human glioma cell apoptosis and investigate the exact mechanism. U­87MG cells were treated with various concentrations of ouabain for 24 h, following which cell viability and survival rate were assessed using a 3­(4,5-dimethylthiazol-2­yl)­2,5­diphenyltetrazolium bromide assay. The dynamic changes and cell motility were observed using digital holographic microscopy. Additionally, western blot analysis and high­content screening assays were used to detect the protein expression levels of phosphorylated (p­)Akt, mammalian target of rapamycin (mTOR), p­mTOR and hypoxia­inducible factor (HIF)­1α, respectively. Compared with the control group, ouabain suppressed U­87MG cell survival, and attenuated cell motility in a dose­dependent manner (P<0.01). The downregulation of p­Akt, mTOR, p­mTOR and HIF­1α were observed following treatment with 2.5 and 25 µmol/l of ouabain. These results suggested that ouabain exerted suppressive effects on tumor cell growth and motility, leading to cell death via regulating the intracellular Akt/mTOR signaling pathway and inhibiting the expression of HIF­1α in glioma cells. The present study examined the mechanism underlying the antitumor property of ouabain, providing a novel potential therapeutic agent for glioma treatment.

The role of ZFP580, a novel zinc finger protein, in TGF-mediated cytoprotection against chemical hypoxia­induced apoptosis in H9c2 cardiac myocytes.

Zing finger protein 580 (ZFP580) is a novel Cys2-His2 zinc-finger transcription factor that has an anti-apoptotic role in myocardial cells. It is involved in the endothelial transforming growth factor­ß1 (TGF­ß1) signal transduction pathway as a mothers against decapentaplegic homolog (Smad)2 binding partner. The aim of the present study was to determine the involvement of ZFP580 in TGF­ß1­mediated cytoprotection against chemical hypoxia­induced apoptosis, using H9c2 cardiac myocytes. Hypoxia was chemically induced in H9c2 myocardial cells by exposure to cobalt chloride (CoCl2). In response to hypoxia, cell viability was decreased, whereas the expression levels of hypoxia inducible factor-1α and ZFP580 were increased. Pretreatment with TGF­ß1 attenuated CoCl2­induced cell apoptosis and upregulated ZFP580 protein expression; however, these effects could be suppressed by SB431542, an inhibitor of TGF­ß type I receptor and Smad2/3 phosphorylation. Furthermore, suppression of ZFP580 expression by RNA interference reduced the anti­apoptotic effects of TGF­ß1 and thus increased CoCl2­induced apoptosis. B­cell lymphoma (Bcl)­2­associated X protein/Bcl­2 ratio, reactive oxygen species generation and caspase­3 activation were also increased following ZFP580 inactivation. In conclusion, these results indicate that ZFP580 is a component of the TGF-ß1/Smad signaling pathway, and is involved in the protective effects of TGF­ß1 against chemical hypoxia­induced cell apoptosis, through inhibition of the mitochondrial apoptotic pathway.

SILAC-based proteomic analysis reveals that salidroside antagonizes cobalt chloride-induced hypoxic effects by restoring the tricarboxylic acid cycle in cardiomyocytes.

Hypoxic status alters the energy metabolism and induces cell injury in cardiomyocytes, and it further triggers the occurrence and development of cardiovascular diseases. Our previous studies have shown that salidroside (SAL) exhibits anti-hypoxic activity. However, the mechanisms remain obscure. In the present study, we successfully screened 92 different expression proteins in CoCl2-induced hypoxic conditions, 106 different expression proteins in the SAL-mediated anti-hypoxic group were compared with the hypoxic group using quantitative proteomics strategy, respectively. We confirmed that SAL showed a positive protective function involving the acetyl-CoA metabolic, tricarboxylic acid (TCA) cycle using bioinformatics analysis. We also demonstrated that SAL plays a critical role in restoring the TCA cycle and in protecting cardiomyocytes from oxidative injury via up-regulation expressions of PDHE1-B, ACO2, SUCLG1, SUCLG2 and down-regulation of MDH2. SAL also inhibited H9c2 cell apoptosis by inhibiting the activation of pro-apoptotic molecules caspase 3 and caspase 9 as well as activation of the anti-apoptotic molecular Bcl-2. Additionally, SAL also improved mitochondrial membrane potential (ΔΨm), reduced reactive oxygen species (ROS) and intercellular Ca(2+) concentration ([Ca(2+)]i) accumulation and inhibited the excessive consumption of ATP in H9c2 cells.

ZFP580, a novel zinc-finger transcription factor, is involved in cardioprotection of intermittent high-altitude hypoxia against myocardial ischemia-reperfusion injury.

BACKGROUND: ZFP580 is a novel C2H2 type zinc-finger transcription factor recently identified by our laboratory. We previously showed that ZFP580 may be involved in cell survival and growth. The aim of this study was to elucidate whether ZFP580 is involved in the cardioprotective effects of intermittent high-altitude (IHA) hypoxia against myocardial ischemia-reperfusion (I/R) injury. METHODS AND RESULTS: After rats were subjected to myocardial ischemia for 30 min followed by reperfusion, ZFP580 expression in the left ventricle was measured. ZFP580 protein expression was found to be up-regulated within 1 h and decreased at 2 h after reperfusion. Comparing normoxic and IHA hypoxia-adapted rats (5000 m, 6 h day-1, 6 weeks) following I/R injury (30 min ischemia and 2 h reperfusion), we found that adaptation to IHA hypoxia attenuated infarct size and plasma leakage of lactate dehydrogenase and creatine kinase-MB. In addition, ZFP580 expression in the myocardium was up-regulated by IHA hypoxia. Consistent with this result, ZFP580 expression was found to be significantly increased in cultured H9c2 myocardial cells in the hypoxic preconditioning group compared with those in the control group following simulated I/R injury (3 h simulated ischemic hypoxia and 2 h reoxygenation). To determine the role of ZFP580 in apoptosis, lentivirus-mediated gene transfection was performed in H9c2 cells 72 h prior to simulated I/R exposure. The results showed that ZFP580 overexpression significantly inhibited I/R-induced apoptosis and caspase-3 activation. H9c2 cells were pretreated with or without PD98059, an inhibitor of ERK1/2 phosphorylation, and Western blot results showed that PD98059 (10 µM) markedly suppressed I/R-induced up-regulation of ZFP580 expression. CONCLUSIONS: Our findings demonstrate that the cardioprotective effect of IHA hypoxia against I/R injury is mediated via ZFP580, a downstream target of ERK1/2 signaling with anti-apoptotic roles in myocardial cells.

[The effects of intermittent hypobaric hypoxia on myocardial ischemia/reperfusion injury and ZFP580 expression].

OBJECTIVE: To elucidate whether ZFP580 is involved in the cardioprotective effects of intermittent hypobaric hypoxia (IHH) against myocardial ischemia/reperfusion (I/R) injury. METHODS: Thirty two male Wistar rats were randomly divided into 2 groups (n = 16): normoxia control group and IHH preconditioning group. Rats in IHH group were exposed in a hypobaric chamber (equivalent to an altitude of 5 000 m) for a 6 h period each day for 42 d. Plasma was collected and lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) were measured after 2 h of myocardial I/R injury. ZFP580 protein expression in myocardial tissue was assayed by Western blot. Other 8 rats in each group were used to evaluate I/R-induced cardiac infarction by TTC staining. Lentivirus-mediated gene transfection was performed in H9c2 cells 72 h prior to simulated ischemia/reperfusion (SI/R) exposure. The degree of cell apoptosis was determined by annexin V/7-AAD staining and flow cytometry analysis. RESULTS: Compared with normoxia control group, adaptation to IHH attenuated infarct size and plasma leakage of LDH and CK-MB. In addition, ZFP580 expression in the myocardium was up-regulated by IHH. The results of gene transfection showed that ZFP580 overexpression significantly inhibited cells apoptosis induced by SI/R. CONCLUSION: Our findings demonstrate that the cardioprotective effect of IHH against I/R injury is mediated via ZFP580, a novel transcription factor, with anti-apoptotic roles in myocardial cells.

Interleukin-6 signaling regulates anchorage-independent growth, proliferation, adhesion and invasion in human ovarian cancer cells.

It has been widely reported that Interleukin-6 (IL-6) is overexpressed in the serum and ascites of ovarian cancer (OVCA) patients, and elevated IL-6 level correlates with poor prognosis and survival. However, the exact role that IL-6 plays in this malignancy or whether IL-6 can regulate tumorigenic properties has not been established. Here we demonstrate that overexpression of IL-6 in non-IL-6-expressing A2780 cells (by transfecting with plasmid encoding for sense IL-6) increases anchorage-independent growth, proliferation, adhesion and invasion, while depletion of endogenous IL-6 expression in IL-6-overexpressing SKOV-3 cells (by transfecting with plasmid encoding for antisense IL-6) decreases. Further investigation indicates that IL-6 promotes OVCA cell proliferation by altering cell cycle distribution rather than inhibiting apoptosis and that IL-6-enhanced OVCA cell invasive may be associated with increased matrix metalloproteinase (MMP)-9 but not MMP-2 proteolytic activity. In addition, overexpressing or deleting of IL-6 in OVCA cells enhances or reduces its receptor (IL-6Rα and gp130) expression and basal phosphorylation levels of both ERK and Akt, and additional treatment with specific inhibitor of the ERK or Akt signaling pathway significantly inhibits the proliferation of IL-6-overexpressing A2780 cells. Our data suggest that the autocrine production of IL-6 by OVCA cells regulates tumorigenic properties of these cells by inducing IL-6 signaling pathways. Thus, regulation of IL-6 expression or its related signaling pathway may be a promising strategy for controlling the progression of OVCA.

Interleukin-8 secretion by ovarian cancer cells increases anchorage-independent growth, proliferation, angiogenic potential, adhesion and invasion.

It has been shown that IL-8 is elevated in ovarian cyst fluid, ascites, serum, and tumor tissue from ovarian cancer (OVCA) patients, and increased IL-8 expression correlates with poor prognosis and survival. However, the exact role that IL-8 plays in this malignancy or whether IL-8 can regulate malignant behavior has not been established. Here we demonstrate that overexpression of IL-8 in non-IL-8-expressing A2780 cells (by transfecting with plasmid encoding for sense IL-8) increases anchorage-independent growth, proliferation, angiogenic potential, adhesion and invasion while depletion of endogenous IL-8 expression in IL-8-overexpressing SKOV-3 cells (by transfecting with plasmid encoding for antisense IL-8) decreases the above effects. Further investigation indicates that IL-8-stimulated cell proliferation correlates with alteration of cell cycle distribution by increasing levels of cell cycle-regulated Cyclin D1 and Cyclin B1 proteins as well as activation of PI3K/Akt and Raf/MEK/ERK, whereas IL-8-enhanced OVCA cell invasive correlates with increased MMP-2 and MMP-9 activity and expression. Our data suggest that IL-8 secreted by OVCA cells promotes malignant behavior of these cells via inducing intracellular molecular signaling. Therefore, modulation of IL-8 expression or its related signaling pathway may be a promising strategy for controlling the progression and metastasis of OVCA.

[Cycle arrest of prostate carcinoma DU-145 cells induced by pseudolaric acid B].

OBJECTIVE: To study the effect of pseudolaric acid B (PLAB) on cell proliferation and cycle of human prostate carcinoma DU-145 cells. method: Its inhibitory effect on the cell growth was measured by MTT method. Characteristics of cell death were determined by Hoechest 33342 staining. The cell cycle was detected by flow cytometry. The expressions of cyclin B1, cyclin D1 and CDK1 were detected by Real time-PCR and Western blot, respectively. RESULT: PLAB notably inhibited DU-145 cell growth in a dose- and time dependent manner (P < 0.05). Its IC50 values of PLAB for DU-145 cells for 24, 48 and 72 h were 4.53, 2.39 and 2.08 micromol x L(-1), respectively. Having been treated with 5 micromol x L(-1) PLAB for 24 h, the cells showed such apoptosis characteristics as nuclei chromatin condensation and apoptotic body. With the increase in PLAB concentration, the proportion of G2/M phase cells strikingly increased in a dose- and time dependent manner (P < 0.05), meanwhile cyclin B1 and CDK1 showed over-expressions (P < 0.05), and the cyclin D1 showed under-expression (P < 0.05). CONCLUSION: PLAB can inhibit the growth of DU-145 cells and induce the cell cycle G2/M arrest, accompanied with the over-expression of cyclin B1 and CDK1, which may be related with its regulation cycle-associated protein degradation.

Cardiotonic steroids attenuate ERK phosphorylation and generate cell cycle arrest to block human hepatoma cell growth.

Recent studies revealed the potential of Na(+)/K(+)-ATPase as a target for anticancer therapy and showed additional modes of action of cardiotonic steroids (CSs), a diverse family of naturally derived compounds, as inhibitors of Na(+)/K(+)-ATPase. The results from epidemiological studies showed significantly lower mortality rates in cancer patients receiving CSs, which sparked interest in the anticancer properties of these drugs. The present study was designed to investigate the anticancer effect of CSs (ouabain or cinobufagin) and to elucidate the molecular mechanisms of CS activity in hepatoma cell lines (HepG2 and SMMC-7721). Ouabain and cinobufagin significantly inhibited cell proliferation by attenuating the phosphorylation of extracellular regulated kinase (ERK) and down-regulating the expression of C-myc. These CSs also induced cell apoptosis by increasing the concentration of intracellular free calcium ([Ca(2+)](i)) and induced S phase cell cycle arrest by down-regulating the expression of Cyclin A, cyclin dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA) as well as up-regulating the expression of cyclin dependent kinase inhibitor 1A (p21(CIP1)). Overexpression of ERK reversed the antiproliferation effect of ouabain or cinobufagin in HepG2 and SMMC-7721 cells. Currently, the first generation of CS-based anticancer drugs (UNBS1450 and Anvirzel) are in Phase I clinical trials. These data clearly support their potential use as cancer therapies.

[Effect of Na(+)/K(+)-ATPase alpha1 siRNA and ouabain upon cell cycle in human hepatoma HepG2 cell and its mechanism].

OBJECTIVE: To investigate the in vitro anti-cancer effects of ouabain combined Na(+)/K(+)-ATPase alpha1 siRNA upon HepG2 cells and its molecular mechanism. METHODS: The Na(+)/K(+)-ATPase alpha1 subunit expressions of human hepatoma tissue, normal liver tissue and human hepatoma cell lines (HepG2, SMC7721 and Bel7402) were determined. The cells received different treatments (0.03 micromol/L siRNA, 0.1 micromol/L ouabain and combination). The proliferation of HepG2 was observed by CCK-8 assay. The activity of Na(+)/K(+)-ATPase was measured. The HepG2 cell cycle distribution was detected by flow cytometry. The mRNA and protein levels of Na(+)/K(+)-ATPase alpha1 subunit, MAPK1, Cyclin A, CDK2, PCNA and P21WAF1 were detected by quantitative RT-PCR and Western blot. RESULTS: The gray value Na(+)/K(+)-ATPase alpha1 subunit in hepatoma tissue was 174.74 +/- 16.77 and 65.31 +/- 7.88 respectively in normal liver tissue. The Na(+)/K(+)-ATPase alpha1 subunit expression of hepatoma tissue was significantly higher than normal liver tissue(P < 0.05).The Na(+)/K(+)-ATPase alpha1 subunit expression level of HepG2 was higher than that in SMMC-7721 and Bel-7402 cells. The CCK-8 experiments demonstrated that siRNA, ouabain and combination could inhibit the HepG2 proliferation, and the combination group was different from the ouabain or siRNA group (P < 0.05). The 24, 48 and 72 h inhibitory rates of 0.03 micromol/L siRNA, 0.1 micromol/L ouabain and combination group were (17.4%, 20.3%, 24.3%), (37.5%, 44.3%, 51.2%) and (52.3%, 70.2%, 88.2%) respectively. The activity of Na(+)/K(+)-ATPase decreased in siRNA, ouabain and combination group. The S phase proportion of ouabain and combination group increased from 24.2% to 66.5% and 75.2% respectively. The 0.1 micromol/L ouabain and combination group could down-regulate the expression of Na(+)/K(+)-ATPsae alpha1, MAPK1, Cyclin A, CDK2, PCNA and up-regulate the expression of P21WAF1 in HepG2 cell. CONCLUSION: The Na(+)/K(+)-ATPase alpha1 siRNA combined ouabain inhibits the proliferation of HepG2 cells by decreasing the expression of MAPK1 and induces the cell cycle S arrest by decreasing the production of Cyclin A/CDK2/PCNA complex and increasing the expression of P21(WAF1).

Targeting the Na(+)/K(+)-ATPase alpha1 subunit of hepatoma HepG2 cell line to induce apoptosis and cell cycle arresting.

Recent research has shown that the Na(+)/K(+)-ATPase alpha1 subunit is a novel anti-cancer target, which plays pivotal roles in malignant cell ion transport, metabolism, migration and signal transduction. The purpose of the present study was to investigate the anti-cancer effects of ouabain and Na(+)/K(+)-ATPase alpha1 small interfering ribonucleic acid (siRNA) on HepG2 cell proliferation, apoptosis and cell cycle, and to explore the molecular mechanisms. The expression of Na(+)/K(+)-ATPase alpha1 subunit in human hepatocellular carcinoma (HCC), normal liver tissues and human HCC line (HepG2, SMMC-7721 and Bel-7402) has been investigated. Using the ouabain and Na(+)/K(+)-ATPase alpha1 subunit siRNA, which target the Na(+)/K(+)-ATPase, we have evaluated the effects of inhibiting Na(+)/K(+)-ATPase alpha1 in human HepG2 cells with respect to cell proliferation, morphology, cell cycle, impact on intracellular Ca2++, reactive oxygen species (ROS) concentration, and correlated gene expression level on messenger ribonucleic acid (mRNA) and protein. Our data showed that the expression Na(+)/K(+)-ATPase alpha1 subunit in HCC tissues is higher than that in normal liver tissues. Ouabain and Na(+)/K(+)-ATPase alpha1 siRNA could inhibit HepG2 cell proliferation. Ouabain could induce HepG2 cell apoptosis and generate S phase arrest, and siRNA could enhance the anti-cancer effect of ouabain that induced HepG2 cells apoptosis via an intracellular Ca(2+) and ROS increase-mediated, and generated cell cycle S phase arresting by decreasing the CyclinA1/cyclin-dependent kinase 2 (CDK2)/proliferating cell nuclear antigen (PCNA) complex product and increasing the expression of cyclin-dependent kinase inhibitor 1A (P21(CIP1)). We believe that targeting of the Na(+)/K(+)-ATPase alpha1 subunit in human HCC cells could provide new sight into the treatment of HCC.

Sphingosine-1-phosphate induces human endothelial VEGF and MMP-2 production via transcription factor ZNF580: novel insights into angiogenesis.

Sphingosine-1-phosphate (S1P)-induced migration and proliferation of endothelial cells are critical for angiogenesis. C2H2-zinc finger (ZNF) proteins usually play an essential role in altering gene expression and regulating the angiogenesis. The aim of this study is to investigate whether a novel human C2H2-zinc finger gene ZNF580 (Gene ID: 51157) is involved in the migration and proliferation of endothelial cells stimulated by S1P. Our study shows that EAhy926 endothelial cells express S1P1, S1P3 and S1P5 receptors. Furthermore, S1P upregulates both ZNF580 mRNA and protein levels in a concentration- and time-dependent manner. SB203580, the specific inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK) pathway, blocks the S1P-induced upregulation of ZNF580. Moreover, overexpression/downexpression of ZNF580 in EAhy926 cells leads to the enhancement/decrease of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) expression as well as the migration and proliferation of EAhy926 endothelial cells. These results elucidate the important role that ZNF580 plays in the process of migration and proliferation of endothelial cells, which provides a foundation for a novel approach to regulate angiogenesis.

[Protective effects of ouabain conjugated peptide from Ph.D.-7 Library on vascular endothelial cell].

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 alpha1-subunit and beta1-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 (3)H-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 alpha1-subunit and down-regulated expression of Na(+)-K(+)-ATPase beta1-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.

[The role of ouabain in cell death of vascular endothelial cells ECV304 and the changes of expression of Na+, K(+)-ATPase alpha1, beta1-subunit].

AIM: To study the effect of Na+, K(+)-ATPase inhibition by ouabain on growth and death of vascular endothelial cells ECV304 and involved mechanisms. METHODS: Growth inhibition of ouabain on ECV304 cells was analyzed using MTT assay. The feature of cell death was studied by Hoechst 33342/PI staining, transmission electron microscopy and DNA agarose gel electrophoresis in ECV304 cells treated with ouabain. The mRNA expression of Na+, K(+)-ATPase alpha1, beta1-subunit was examined by reverse transcription PCR (RT-PCR). RESULTS: Ouabain inhibited the growth of ECV304 cells in a dose and time-dependent manner. 10 micromol/L ouabain treated for 24 hours could stimulate the necrosis of ECV304 cells; When treated with 0.1 micromol/L ouabain for 24-48 hours, the cells showed obviously defluxion, the loss of cell-cell contacts, nuclear chromatin condensation, chromatin margination and DNA fragmentation. Na+, K(+)-ATPase alpha1-subunit mRNA expression was significantly up-regulated in ECV304 cells treated with ouabain while the beta1-subunit expression conversely showed a significant decrease. CONCLUSION: Ouabain could up-regulate Na+, K(+)-ATPase alpha1-Subunit expression and reduce beta1-Subunit expression which mediated signal transduction and decreased cell-cell adhesions and induced ECV304 cells death.

[Inducement effect of 9-cis retinoic acid on cell cycle arrest and apoptosis of gastric carcinoma cell line MGC803 and its mechanism].

BACKGROUND & OBJECTIVE: Antitumor effect of 9-cis retinoic acid (9-cis RA) on gastric carcinoma is unclear yet. This study was to explore the inducement effect of 9-cis RA on cell cycle arrest and apoptosis of gastric carcinoma cell line MGC803 and its mechanism. METHODS: The expression of RXRalpha, Cyclin D1, and CDK4 in MGC803 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR). Cell cycle was detected by flow cytometry. The growth inhibition was analyzed by MTT assay. The apoptosis was detected by agarose gel electrophoresis and Hoechst33342/PI staining. The expression of apoptosis-associated gene Bcl-2 was detected by SP immunocytochemistry. RESULTS: When treated with 0.1-10 micromol/L 9-cis RA for 96 h, the proliferation of MGC803 cells was significantly inhibited. The proportion of MGC803 cells at G1 phase was significantly increased when treated with 10 micromol/L 9-cis RA for 48, 72, and 96 h, and showed an apparent G1 phase arrest. When treated with 9-cis RA for 72 h, typical apoptotic changes, such as chromatin condensation and DNA ladder, were observed in MGC803 cells. The expression of Bcl-2 was significantly decreased in MGC803 cells when treated with 10 micromol/L 9-cis RA for 48 h. RXRalpha expression was at a low level in MGC803 cells and up-regulated when treated with 10 micromol/L 9-cis RA for 48 h (P<0.01). The expression of Cyclin D1 and CDK4 in MGC803 cells was both significantly down-regulated when treated with 10 micromol/L 9-cis RA for 96 h (P<0.01). CONCLUSION: 9-Cis RA could induce G1 phase arrest and apoptosis in MGC803 cells through down-regulating the expression of cell cycle factors Cyclin D1 and CDK4.