钠钾泵抑制剂通过调节细胞周期相关蛋白的生成介导肝癌HepG2细胞周期S期阻滞与凋亡

目的研究钠泵抑制剂哇巴因(ouabain)和华蟾毒配基(cinobufogenin)对人肝癌HepG2细胞增殖的抑制作用及细胞周期的改变,初步分析其机制。方法以人肝癌细胞HepG2为靶细胞,MTT比色法检测哇巴因和华蟾毒配基对HepG2细胞增殖的影响;Hoechst 33342荧光染色检测细胞形态学变化;流式细胞术检测细胞周期;实时定量PCR和Western blot检测CyclinA1、CDK2、PCNA和p21CIP1表达的变化。结果哇巴因和华蟾毒配基可明显抑制HepG2细胞增殖,抑制作用呈时间-浓度依赖性。荧光染色显示药物处理24h后,细胞呈现典型的凋亡形态特征;细胞周期分析显示,实验组S期细胞比例升高,实时定量PCR和Western blot结果显示:哇巴因和华蟾毒配基可下调CyclinA1、CDK2和PC-NA的表达(P<0.05),上调p21CIP1的表达(P<0.05)。结论钠泵抑制剂可抑制肝癌HepG2细胞的增殖,引起细胞周期S期阻滞,诱导细胞凋亡,这与其调节细胞周期相关蛋白的生成关系密切。     

Characterization of differentiation-inducer-resistant HL-60 cells

Sub-lines of the cultured human promyelocytic leukemia cell line HL-60 were individually selected for their ability to sustain exponential growth in the presence of 3 structurally-unrelated inducers of granulocytic differentiation - retinoic acid (RA), dimethylsulfoxide (DMSO), and 6-thioguanine (6TG). Selections were made by step-wise augmentation to final drug concentrations of 10(-3)mM RA, 169mM (1.2%) DMSO and 0.12mM (20 micrograms ml-1) 6TG. In addition to growth resistance, cells in each sub-line displayed variable cytodifferentiation resistance to each of the 3 selective agents, which was quantitated as the ratio of the concentration of drug required to induce differentiation in 50% of the cells in each resistant sub-line versus comparably-passaged wild-type HL-60 cells. The levels of resistance/cross-resistance were as follows: RA-resistant (res) sub-line greater than 2700-fold to RA, 1.3-fold to DMSO and greater than 1.5-fold to hypoxanthine (HXN; the noncytotoxic purine base inducer analogue of 6TG); DMSO-res sub-line 2.5-fold to DMSO, 137-fold to RA and greater than 1.5-fold to HXN; and 6TG-res sub-line greater than 1.5-fold to HXN, 9-fold to RA and 1.6-fold to DMSO. These sub-lines were not cross-resistant to sodium butyrate (NaBut), a monocyte inducer, or to 12-0-tetradecanoylphorbol 13-acetate (TPA), a macrophage inducer. HL-60 sub-lines selected by exposure to a single high concentration of 5-bromo-2'-deoxyuridine (BUdR; 3.3 X 10(-2)mM) or oubain (Ou; 5 X 10(-3)mM) were not or were slightly cross-resistant to either granulocyte or monocyte inducers. Although some variations in line/sub-line phenotype were observed, this was minor compared to the quantitative variations in response to individual inducing agents. The RA-res and 6TG-res sub-lines contained numerous double minute chromosomes (indicators of amplified genes) which were either absent or present in much smaller numbers in the parental wild-type cells or in the other drug-resistant sub-lines. There was little change or a decrease in the amplification level of the known amplified oncogene c-myc in the various drug-resistant sub-lines compared to wild-type HL-60 cells. These results (a) confirm that the neutrophilic granulocytic and monocytic/macrophagic differentiation programs in HL-60 cells are mechanistically different and separable; (b) suggest that both agent-specific and common quantitative alterations contribute to the mechanism(s) for resistance to granulocyte differentiation; and (c) suggest that the latter quantitative defects could be related to amplification of genes other than c-myc.     

Role of Na+-K+ ATPase enzyme in vascular response of goat ruminal artery

Objective:

To study the role of Na⁺, K⁺- ATPase enzyme in the vascular response of goat ruminal artery.

Materials and Methods:

Ruminal artery was obtained in chilled aerated modified Krebs-Henseleit solution (KHS) from a local slaughterhouse and transported in ice for further processing. The endothelium intact arterial ring was mounted in a thermostatically controlled (37 ± 0.5°C) organ bath containing 20 ml of modified KHS (pH 7.4) bubbled with oxygen (95%) and CO₂ (5%) under 2g tension. An equilibration of 90 min was allowed before addition of drugs into the bath. The responses were recorded isometrically in an automatic organ bath connected to PowerLab data acquisition system. In order to examine intact functional endothelium, ACh (10 μM) was added on the 5-HT (1.0 μM) - induced sustained contractile response. Similarly, functional characterization of Na⁺, K⁺-ATPase activity was done by K⁺-induced relaxation (10 μM-10 mM) in the absence and presence of ouabain (0.1 μM/ 0.1 mM), digoxin (0.1 μM) and barium (30 μM).

Results:

ACh (10⁻⁵ M) did not produce any relaxing effect on 5-HT-induced sustained contractile response suggesting that vascular endothelium has no significant influence on the activation of sodium pump by extracellular K⁺ in ruminal artery. Low concentration of Ba²⁺ (30 μM) (IC₅₀: 0.479 mM) inhibited K⁺-induced relaxation suggesting K_ir (inward rectifier) channel in part had role in K⁺-induced vasodilatation in ruminal artery. Vasorelaxant effect of KCl (10 μM-10 mM) in K⁺-free medium is also blocked by ouabain (0.1 μM and 0.1 mM) (IC₅₀:0.398 mM and IC₃₅: 1.36 mM), but not by digoxin (0.1 μM) (IC₅₀ 0.234 mM) suggesting that ouabain sensitive Na⁺, K⁺-ATPase isoform is present in the ruminal artery.

Conclusion:

In the goat ruminal artery functional regulation of sodium pump is partly mediated by K⁺ channel and ouabain sensitive Na⁺, K⁺ ATPase.     

人红细胞膜钠,钾-ATP酶活性及其在疾病时的变化

用酶学比色法测定了冠心病、风湿热、白血病、再生障碍性贫血等48例患者及28例正常人红细胞膜Na~+,K~+-ATPase的活性。结果表明除再生障碍性贫血外,其余患者红细胞膜Na~+,K~+-ATPase活性均不同程度下降,随着病情恢复,酶活性又呈增高趋势。乌巴因对Na~+,K~+-ATPase活性抑制百分数在各疾病组改变不明显。     

Differential involvement of membrane (Na-K)atpase in thrombin- and trypsin-mediated platelet activation

The possibility that platelet activation may also involve membrane (Na-K)ATPase was investigated by testing the effects of both proteinases on platelet shape change and aggregation in the absence and presence of the specific (Na-K)ATPase inhibitor ouabain. Ouabain (8 to 80 μM) completely antagonized trypsin-induced platelet shape change and aggregation when it was preincubated with platelet suspension before the addition of trypsin. Unlike trypsin, thrombin-induced platelet activation was significantly enhanced by ouabain. It was also observed that on partially purified beef heart (Na-K)ATPase preparation, thrombin significantly enhanced the enzyme inhibition caused by submaximal inhibitory concentrations of ouabain.

Soybean trypsin inhibitor (4 μg/ml) employed as the agent capable to counteract proteinase effects on the (Na-K)ATPase, was shown both to prevent and antagonize the platelet activation induced by trypsin (0.3 to 1.5 μg/ml), but it failed to modify the responses evoked by thrombin.

It is concluded that membrane (Na-K)ATPase is involved differently in platelet activation by trypsin and thrombin probably because receptor sites to which either proteinase on the platelet surface binds, are distinct. Direct enzyme involvement is indeed apparent only in trypsin-induced platelet activation.