Expression and significance of IGF-Ⅱand VEGF in hepatocellular carcinoma

目的 检测肝癌(HCC)患者组织中IGF-Ⅱ、血管内皮生长因子 (VEGF)的表达及临床意义.方法 采用免疫组织化学染色方法 检测HCC及癌旁组织中IGF-Ⅱ、VEGF蛋白表达水平.结果 HCC及癌旁组织IGF-Ⅱ蛋白阳性表达率分别为55%(22/40)、12.5%(5/40),VEGF蛋白阳性表达率分别为62.5%(25/40)、12.5%(5/40),HCC组织中IGF-Ⅱ、VEGF蛋白表达均显著高于癌旁组织(P<0.05),且IGF-Ⅱ、VEGF表达呈明显正相关(r=0.59,P<0.05).IGF-Ⅱ、VEGF表达与患者的年龄、性别、肿瘤大小无关,但与肿瘤的组织学分级、是否侵及包膜及有无癌栓密切相关.结论 与癌旁组织比较,HCC组织中IGF-Ⅱ、VEGF呈现高表达,提示IGF-Ⅱ、VEGF参与了HCC的发生发展过程,可能与HCC的生长和增殖有关.     

Altered liver α1-adrenoceptor density and phospholipase C activity in the human hepatocellular carcinoma

The human hepatocellular carcinoma (HCC) is a common cancer with high mortality rate. We examined the density and coupling to phospholipase C (PLC) of the α₁-adrenoceptors. In HCC liver, the α₁-adrenoceptor density – as assessed by [³H]-Prazosin binding – was significantly reduced to about 75% when compared to non-adjacent non-tumorous liver (NA-NL) (P = 0.0002). The decrease in maximal α₁-adrenoceptor concentration (B_max) was accompanied by a significant reduction in noradrenaline-stimulated PLC activity (P < 0.032 versus NA-NL) (assessed by [³H]-PIP₂ hydrolysis). GTPγS-stimulated PLC activity in HCC livers did not statistically differ from NA-NL livers. NaF, which activates all G-proteins, stimulated PLC in both HCC and NA-NL livers to a similar extent. The altered noradrenaline-induced functional responsiveness of HCC livers was not reflected by changes in the binding affinity of [³H]-Prazosin for α₁-adrenoceptors (NA-NL: 0.066 ± 0.010 pmol/l; tumour: 0.067 ± 0.020 pmol/l). These results demonstrate that human HCC causes profound alteration of the hepatic α₁-adrenoceptor signal transduction pathway and may account for a negative cancer related metabolism of carbohydrates and wasting syndrome in tumour patients.     

Comparison of the densities of 5-HT4 receptors, β1- and β2-adrenoceptors in human atrium: functional implications

We measured in human atrium the density of 5-HT₄ receptors, labelled with [¹²⁵I]-SB 207710 (1-butyl-4-piperidinyl) methyl 8-amino-7-iodo-1, (4-benzodioxan-5-carboxylate), and compared it with the density of ₁- and ₂-adrenoceptors, labelled with (–)-[¹²⁵I]-cyanopindolol. [¹²⁵I]-SB 207710 (5–1200 pmol/l) labelled a small population of saturable binding sites (B _max 4 fmol/mg protein) with a pK_D of 9.7 and with 5-HT₄ receptor characteristics, as assessed with competing ligands. The density of atrial binding sites with 5-HT₄ receptor characteristics was 10 and 5 times lower, respectively, than the density of ₁- and ₂-adrenoceptors. We suggest that the small 5-HT₄ receptor population may in part explain why the positive inotropic effects of 5-HT are smaller than those of catecholamines mediated through ₁- and ₂-adrenoceptors.     

Regional distribution of β1- and β2-adrenoceptors in the failing and nonfailing human heart

Summary Total -adrenoceptor density and ₁- and ₂-subtype distribution in right and left atria and in different ventricular regions from 14 failing and seven nonfailing human hearts have been compared. End-stage heart failure was due to idiopathic dilated cardiomyopathy (n=8) or ischaemic cardiomyopathy (n=6).In nonfailing hearts the total -adrenoceptor density was similar in the right and left atria and in all the ventricular regions studied (about 70 to 80 fmol/mg protein). The ₁:₂-adrenoceptor ratio in both nonfailing atria was similar (about 70:30%) and was significantly smaller than in the different regions of both ventricles (about 80:20%). The ₁-subtype density was similar in nonfailing atria and ventricles (about 55 fmol/mg protein). The ₂-subtype density was significantly higher in the right and left atrium (about 25 fmol/mg protein) than in both ventricles (about 15 fmol/mg protein).In patients with end-stage heart failure due to idiopathic dilated cardiomyopathy or ischaemic cardiomyopathy the total -adrenoceptor density was reduced by 50–60% in all regions. On the other hand, the ₁- and ₂-subtype distribution differed with the cause of heart failure. In patients with idiopathic dilated cardiomyopathy, the ₁-adrenoceptor density was lower in all regions, but the ₂-adrenoceptor density was not significantly reduced. In patients with ischaemic cardiomyopathy both ₁- and ₂-adrenoceptors were reduced in all regions.It is concluded that downregulation of -adrenoceptors in patients with end-stage idiopathic dilated cardiomyopathy or ischaemic cardiomyopathy occurs uniformly throughout the heart. The results support the hypothesis that changes in -adrenoceptor subtypes may be related to the cause of heart failure.     

L-Asparaginase and inhibitors of glutamine synthetase disclose glutamine addiction of β-catenin-mutated human hepatocellular carcinoma cells

Selected oncogenic mutations support unregulated growth enhancing glutamine availability but increasing the dependence of tumor cells on the amino acid. Data from literature indicate that a subset of HepatoCellular Carcinomas (HCC) is characterized by mutations of β-catenin and overexpression of Glutamine Synthetase (GS). To assess if this phenotype may constitute an example of glutamine addiction, we treated four human HCC lines with the enzyme L-Asparaginase (ASNase), a glutaminolytic drug. ASNase had a significant antiproliferative effect only in the β-catenin mutated HepG2 cells, which were partially rescued by the anaplerotic intermediates pyruvate and α-ketoglutarate. The enzyme severely depleted cell glutamine, caused eIF2α phosphorylation, inhibited mTOR activity, and increased autophagy in both HepG2 and in the β-catenin wild type cell line Huh-7. When used with ASNase, the GS inhibitor methionine sulfoximine (MSO) emptied cell glutamine pool, arresting proliferation in ASNase-insensitive Huh-7 cells and activating caspase-3 and apoptosis in HepG2 cells. Compared with Huh-7 cells, HepG2 cells accumulated much higher levels of glutamine and MSO, due to the higher expression and activity of SNAT2, a concentrative transporter for neutral amino acids, but were much more sensitive to glutamine withdrawal from the medium. In the presence of ASNase, MSO caused a paradoxical maintenance of rapamycin-sensitive mTOR activity in both HepG2 and Huh-7 cells. β-catenin silencing lowered ASNase sensitivity of HepG2 cells and of Huh-6 cells, another β-catenin-mutated cell line, which also exhibited high sensitivity to ASNase. Thus, β-catenin mutated HCC cells are more sensitive to glutamine depletion and accumulate higher levels of GS inhibitors. These results indicate that glutamine deprivation may constitute a targeted therapy for β-catenin-mutated HCC cells addicted to the amino acid.     

Expression and function of β-arrestin 2 stimulated by IL-1β in human fibroblast-like synoviocytes and the effect of paeoniflorin

The aim of this study was to explore the expression and function of β-arrestin 2 in human fibroblast-like synoviocytes (FLS) stimulated by IL-1β and the effect of paeoniflorin (Pae). We isolated and cultured human FLS, which were stimulated by IL-1β. The FLS proliferations were detected by [3H] thymidine incorporation. The level of cAMP stimulated by IL-1β on different times was investigated by radioimmunoassay, and the activity of PKA was measured by luminescent kinase assay. The expression of β-arrestin 2 was measured by western blot. We found that the human FLS proliferation increased apparently in 24 h, and the activities of PKA and cAMP accumulation increased significantly in 6 h after stimulated by IL-1β, while cAMP accumulation and the activities of PKA decreased especially in 24 h when the expression of β-arrestin 2 increased significantly. Decreased cAMP accumulation and the increased expression of β-arrestin 2 may reveal a positive correlation with the FLS proliferation. Pae (10^- 5, 10^- 6, 10-7 mol?L^- 1) in vitro could suppress the FLS proliferation and the high expression of β-arrestin 2. The expression of β-arrestin 2 may have a positive correlation with the human FLS proliferation, while the activities of PKA and cAMP accumulation have a negative correlation with the proliferation. The increased β-arrestin 2 may down-regulate cAMP-PKA signaling pathway and promote FLS proliferation. Pae may suppress the expression of β-arrestin 2 and up-regulate cAMP-PKA signaling, which may be one of the mechanisms for the effects of Pae on inhibiting human FLS proliferation.     

Expression and significance of β-catenin and MMP-7 in esophagus squamous cell carcinoma

目的 探讨β-catenin和MMP-7蛋白在食管癌组织中的表达变化及与病理学分级、浸润深度和淋巴结转移的关系.方法 应用免疫组化方法检测β-catenin和MMP-7蛋白在55例食管鳞癌组织及15例食管正常组织中的表达.结果 (1)β-catenin在食管鳞状细胞癌组织和食管正常组织中异常表达率分别为61.82%和6.67%;MMP-7在食管鳞状细胞癌组织和食管正常组织中异常表达率分别为69.09%和13.33%,两者比较差异有统计学意义(P<0.05);(2)β-catenin在食管癌组织中异常表达与细胞分化程度及淋巴结转移有关(P<0.05),但与患者的性别、年龄和浸润深度无关(P>0.05);MMP-7蛋白在食管癌组织中异常表达与细胞分化程度及淋巴结转移和浸润深度有关(P<0.05),但与患者的性别、年龄无关(P>0.05);(2)β-catenin的异常表达与MMP-7的表达有一定的相关性(r=0.487,P<0.05).结论 β-catenin和MMP-7在食管鳞状细胞癌组织中阳性表达可能在食管癌的发生发展及转移中起重要作用.     

Distinct down-regulation of cardiac β1- and β2-adrenoceptors in different human heart diseases

Summary Cardiac -adrenoceptor density and ₁- and ₂-subtype distribution were examined in human left ventricular myocardium from transplant donors serving as controls and from patients with mitral valve stenosis, aortic valve stenosis, idiopathic dilated cardiomyopathy, and ischaemic cardiomyopathy respectively. The total -adrenoceptor density was similar in transplant donors and patients with moderate heart failure (NYHA II–III) due to mitral valve stenosis, but was markedly reduced in all forms of severe heart failure (NYHA III–IV) studied. A reduction of both ₁- and ₂-adrenoceptors was found in patients with severe heart failure due to mitral valve stenosis or ischaemic cardiomyopathy. In contrast, a selective down-regulation of ₁-adrenoceptors with unchanged ₂-adrenoceptors and hence a relative increase in the latter was observed in idiopathic dilated cardiomyopathy and aortic valve stenosis. It is concluded that the extent of total -adrenoceptor down-regulation is related to the degree of heart failure. Selective loss of ₁-adrenoceptors is not specific for idiopathic dilated cardiomyopathy but also occurs in aortic valve stenosis. Changes in ₁- and ₂-subtype distribution are rather related to the aetiology than to the clinical degree of heart failure. Send offprint requests to M. Steinfath at the above address     

Protection by dexamethasone of the functional desensitization to β2-adrenoceptor-mediated responses in human lung mast cells

1. The β-adrenoceptor agonist, isoprenaline, inhibited the IgE-mediated release of histamine from human lung mast cells (HLMC) in a dose-dependent manner. Maximal inhibitory effects were obtained with 0.1 μM isoprenaline. However, the inhibition of histamine release from HLMC by isoprenaline (0.1 μM) was highly variable ranging from 33 to 97% inhibition (mean, 59±3%, n=27).
2. Long-term (24 h) incubation of HLMC with isoprenaline led to a subsequent reduction in the ability of a second exposure of isoprenaline to inhibit IgE-mediated histamine release from HLMC. The impairment in the ability of isoprenaline (0.1 μM) to inhibit histamine release following desensitizing conditions (1 μM isoprenaline for 24 h) was highly variable amongst HLMC preparations ranging from essentially negligible levels of desensitization in some preparations to complete abrogation of the inhibitory response in others (mean, 65±6% desensitization, n=27).
3. The ability of HLMC to recover from desensitization was investigated. Following desensitizing conditions (1 μM isoprenaline for 24 h), HLMC were washed and incubated for 24 h in buffer and the effectiveness of isoprenaline (0.1 μM) to inhibit IgE-mediated histamine release from HLMC was assessed. The extent of recovery was highly variable with some HLMC preparations failing to recover and others displaying a complete restoration of responsiveness to isoprenaline (mean, 40±6% recovery, n=23).
4. The effects of the glucocorticoid, dexamethasone, were also investigated. Long-term (24–72 h) treatments with dexamethasone (0.1 μM) had no effect on IgE-mediated histamine release from HLMC. Additionally, long-term (24–72 h) treatments with dexamethasone (0.1 μM) had no effect on the effectiveness of isoprenaline to inhibit histamine release. However, long-term (24–72 h) treatments with dexamethasone (0.1 μM) protected against the functional desensitization induced by incubation (24 h) of HLMC with isoprenaline (1 μM). The protective effect was time-dependent and pretreatment of HLMC with dexamethasone for either 24, 48 or 72 h prevented desensitization by either 15±7, 19±5 or 51±10%, respectively (n=5–7).
5. HLMC preparations which were relatively refractory to isoprenaline even after withdrawal (24 h) from desensitizing conditions responded more effectively to isoprenaline (0.1 μM) if dexamethasone (0.1 μM) was also included during the recovery period (19±9% recovery after 24 h in buffer; 50±8% recovery after 24 h with dexamethasone, n=5).
6. These data indicate that the responses of different HLMC preparations to isoprenaline, the susceptibility of HLMC to desensitization and the ability of HLMC to recover from desensitizing conditions varies markedly. Dexamethasone, which itself has no direct effects on IgE-mediated histamine release from HLMC, protected HLMC from the functional desensitization to β-adrenoceptor agonists. Because β₂-adrenoceptor agonists and glucocorticoids are important in the therapeutic management of asthma and as the HLMC is probably important in certain types of asthma, these findings may have wider clinical implications.

     

Are there functional β3-adrenoceptors in the human heart?

β₃-Adrenoceptor mRNA is expressed in the human heart, but corresponding receptor protein has not yet consistently been demonstrated. Furthermore, their physiological role remains highly controversial. For example, in human atria these receptors apparently do not promote cAMP formation. Evidence presented in this issue of the BJP suggests that a previously reported β₃-adrenoceptor-mediated stimulation of Ca²⁺ channels at room temperature is absent at physiological temperatures, and that β₃-adrenoceptors have no effect on atrial contraction. Drugs classified as β₃-adrenoceptor agonists cause contraction in human atria but in most cases this involves β₁- and/or β₂-adrenoceptors. In contrast, in human ventricles β₃-adrenoceptor agonists can exhibit negative inotropic effects, potentially involving Pertussis toxin-sensitive G-proteins and activation of a NO synthase. However, firmer pharmacological evidence is required that these effects indeed occur via β₃-adrenoceptors. Whether the expected future use of β₃-adrenoceptor agonists in the treatment of urinary bladder dysfunction is associated with adverse events related to cardiac function remains to be determined from clinical studies.

LINKED ARTICLE

This article is a commentary on Christ et al., pp. 823–839 of this issue. To view this paper visit http://dx.doi.org/10.1111/j.1476-5381.2010.00996.x     

The role of cyclic AMP in the positive inotropic effect mediated by β1- and β2-adrenoceptors in isolated human right atrium

Summary The time course of the effects of isoprenaline (3 × 10^–7 mol/l) on contractile force and on the cyclic AMP level was studied in the electrically driven isolated muscle strip of the human right atrium. Isoprenaline produced a rise in cyclic AMP content (maximum increase after 60 s) preceding the increase in contractile force. The effects of isoprenaline on contractile force and on the intracellular level of cyclic AMP were enhanced in the presence of the phosphodiesterase inhibitor papaverine (10^–5 mol/l). On the other hand, the -adrenoceptor antagonist propranolol (10^–7 mol/l) suppressed isoprenaline-induced cyclic AMP increases, but reduced the increase in force of contraction by only 35%. In addition, both the ₁-selective antagonist bisoprolol (3 × 10^–9 × 10^–8 mol/l) and the ₂-selective antagonist ICI 118,551 (3 × 10^–9 × 10^–8 mol/l) inhibited the isoprenaline-induced cyclic AMP increase concentration-dependently; ICI 118,551 produced more pronounced inhibition than bisoprolol. It is concluded that cyclic AMP is involved in the positive inotropic action of isoprenaline evoked by ₁- and ₂-adrenoceptor stimulation in isolated human right atrium; however, an additional cyclic AMP independent mechanism cannot be ruled out. Send offprint requests to O.-E. Brodde at the above address     

Functional effects of polyamines via activation of human β1- and β2-adrenoceptors stably expressed in CHO cells

Polyamines mediate acute metabolic effects and cardiac hypertrophy associated with β-adrenoceptor stimulation. They may also modulate β-adrenoceptors, causing functional responses in rat atria and tracheal smooth muscle. The aim of this study was to determine whether polyamines interact with human β(1)- and β(2)-adrenoceptors and the functional consequences of such an interaction. Chinese hamster ovary (CHO) cells stably transfected with human β(1)- and β(2)-adrenoceptors were used to evaluate the effect of polyamines binding to β-adrenoceptors, cAMP production and morphological changes, which were pharmacologically validated by investigating the effects of the β-adrenoceptor agonists, isoproterenol and salbutamol. Polyamines interacted with human β(1)- and β(2)-adrenoceptors, as shown by the displacement of [(125)I]iodocyanopindolol in the binding assay. Putrescine showed higher affinity to β(1)- than β(2)-adrenoceptors. Spermidine and spermine produced partial displacement (approximately 50%) and, at the highest concentration, the effect was reversed. Putrescine and spermine acutely increased cAMP and, in a serum-free medium, induced a stellate-like form in cells, which was inhibited by propranolol, a β-blocker. A 10 to 15 h incubation with putrescine produced a spindle-like form and spatial organization via β-adrenoceptor activation, evidenced by the antagonizing effect by propranolol and lack of effect in wild-type CHO cells. Additionally, it decreased cell proliferation independently of β-adrenoceptor activation. Spermine caused cell death via fetal bovine serum-dependent and -independent mechanisms. The results suggest that putrescine may act as a non-selective and low affinity agonist of human β(1)- and β(2)-adrenoceptors, eliciting morphological changes. These findings may be of importance in physiology and in diseases involving β-adrenoceptor functionality.     

Comparison of the in vitro binding characteristics of the β-carbolines harman and norharman in rat brain and liver and in bovine adrenal medulla

The in vitro binding of the naturally occurring -carbolines harman and norharman in their tritium-labelled forms to cell membranes from the rat brain and liver and from bovine adrenal medulla was investigated. Displacement of the specific [³H]harman binding in bovine adrenal medulla and rat liver by several -carbolines and monoamine oxidase (MAO) inhibitors revealed the pharmacological profile of a single, high-affinity binding site (K _D 4.92±0.43 nmol/l, B_max 8.47±0.17 pmol/mg protein; adrenal medulla) which corresponded to the active site of MAO type A (MAO-A). Similar characteristics have previously been found for brain tissue from rat, marmoset and pig. In order to determine the temperature dependence of the [³H]harman binding, the K _D and B_max values for rat cerebral cortex were calculated from the results of saturation experiments at 5 temperatures (range: 0°C–37°C). Whereas the B_max values under all conditions were – 4 pmol/mg protein, the K _D values, with increasing temperature, ranged from 3 nmol/l to 30 nmol/l. The calculated linear van't Hoff plot (-In K _D against 1/T) suggested an enthalpy-driven binding of [³H]harman to MAO-A.At least three different [³H]norharman-binding sites were detected. In the rat forebrain, 85% of the specific binding (at about 2 nmol/l of [³H]norharman) can be attributed to a MAO binding site of type B: the binding is displaceable, in nmol/l concentrations by the potent and selective MAO-B inhibitors MDL 72,974A, R(–)-deprenyl and pargyline and, in mol/l concentrations, by S(+)-deprenyl and the potent and selective MAO-A inhibitors clorgyline, harmine, harman, harmaline, brofaromine 5-F--methyltryptamine. After suppression of the MAO binding sites with 1 mol/l clorgyline and mol/l R(–)-deprenyl, a second binding site was found. However, the binding at this site was biphasically displaceable by harman and norharman (Hill-slopes about 0.5 and 0.6, curvilinear Rosenthal plots) suggesting the presence of negative co-operativity or of two binding sites (states). A similar clorgyline/R(–)-deprenyl resistent single (Hill-slopes of displacement by norharman, harman and 6-hydroxy--carboline about unity; linear Rosenthal plots) high affinity binding site (K _D 7.5±2 nmol/l, B_max 130±30 fmol/mg protein) was found in bovine adrenal medullary cell membranes. A third quite different clorgyline/R(–)-deprenyl resistent high-affinity (K _D14 nmol/l) and high-density (B_max 10–30 pmol/mg protein) binding site was detected in the liver. The specific binding at this site was not displaceable by harman or most other substituted -carbolines or by tetrahydro--carbolines, but was displaced by norharman and several newly synthesized 6-substituted aromatic -carbolines (e.g. F-, CH₃-, CH₃O-, HO-). The [³H]norharman binding site in the liver is certainly not identical with any of the binding sites for MAO-inhibitors, benzodiazepines or sigma receptor ligands and is slightly enriched in the microsomal (P₃) fraction whereas most of the specific [³H]harman binding was detected in the crude mitochondrial (P₂) fraction. Correspondence to: T. May at the above address     

Effects of β2-agonist- and dexamethasone-treatment on relaxation and regulation of β-adrenoceptors in human bronchi and lung tissue

1. Long-term treatment with β₂-adrenoceptor agonists can lead to a decreased therapeutic efficacy of bronchodilatation in patients with obstructive pulmonary disease. In order to examine whether or not this is due to β-adrenoceptor desensitization, human bronchial muscle relaxation was studied in isolated bronchial rings after pretreatment with β₂-adrenoceptor agonists. Additionally, the influence of pretreatment with dexamethasone on desensitization was studied.
2. The effect of β₂-agonist incubation alone and after coincubation with dexamethasone on density and affinity of β-adrenoceptors was investigated by radioligand binding experiments.
3. In human isolated bronchi, isoprenaline induces a time- and concentration-dependent β-adrenoceptor desensitization as judged from maximal reduction in potency by a factor of 7 and reduction of 73±4% in efficacy of isoprenaline to relax human bronchial smooth muscle.
4. After an incubation period of 60 min with 100 μmol l⁻¹ terbutaline, a significant decline in its relaxing efficacy (81±8%) and potency (by a factor 5.5) occurred.
5. Incubation with 30 μmol l⁻¹ isoprenaline for 60 min did not impair the maximal effect of a subsequent aminophylline response but led to an increase in potency (factor 4.4).
6. Coincubation of dexamethasone with isoprenaline (120 min; 30 μmol l⁻¹) preserved the effect of isoprenaline on relaxation (129±15%).
7. In radioligand binding experiments, pretreatment of lung tissue for 60 min with isoprenaline (30 μmol l⁻¹) resulted in a decrease in β-adrenoceptor binding sites (B_max) to 64±1.6% (P<0.05), while the antagonist affinity (K_D) for [³H]-CGP-12177 remained unchanged.
8. In contrast, radioligand binding studies on lung tissue pretreated with either dexamethasone (30 μmol l⁻¹) or isoprenaline (30 μmol l⁻¹) plus dexamethasone (30 μmol l⁻¹) for 120 min did not lead to a significant change of B_max (160±22.1% vs 142.3±28.7%) or K_D (5.0 nmol l⁻¹ vs 3.5 nmol l⁻¹) compared to the controls.
9. In conclusion, pretreatment of human bronchi with β-adrenoceptor agonists leads to functional desensitization and, in lung tissue, to down-regulation of β-adrenoceptors. This effect can be counteracted by additional administration of dexamethasone. Our model of desensitization has proved useful for the identification of mechanisms of β-adrenoceptor desensitization and could be relevant for the evaluation of therapeutic strategies to counteract undesirable effects of long-term β-adrenoceptor stimulation.

     

Effects of {2-[（3-carboxy-l-oxoprogyl）amino]-2-deoxy-D-glucose} on human hepatocellular carcinoma cell line

Aim: To study the effects of {2-[(3-carboxy-1-oxoprogy 1)amino]-2-deoxy-D-glucose (COPADG) on cultured human hepatocellular carcinoma cells (HepG2).Methods: HepG2 cells were cultured in RPMI-1640 medium. Cell proliferation was determined by MTF assay. Apoptosis was determined by fluorescence microscopy,transmission electron microscopy, agarose gel electrophoresis of DNA fragmentation, and flow cytometry. Results: At the concentration ranging between 1-30μmol/L, COPADG potently inhibited the growth and induced apoptosis of HepG2 cells. Conclusion: COPADG could effectively induce apoptosis in human hepatocellular carcinoma cells. More investigations are warranted for the potential use of this compound as a new agent for the non-surgical management of human hepatocellular carcinoma.     

Verapamil regulates activity and mRNA-expression of human β-glucuronidase in HepG2 cells

A promising development in tumor therapy is the application of non-toxic prodrugs from which the active cytostatic is released by endogenous enzymes such as -glucuronidase (-gluc). Regulation of -gluc expression is one crucial factor modulating bioactivation of prodrugs. Recent experiments in rats indicate regulation of -gluc activity by the calcium channel blocker verapamil.To further explore this phenomenon, we investigated the effect of verapamil on -gluc enzyme activity, protein (western blot) and mRNA expression (RT-PCR) as well as the underlying mechanisms (effects of verapamil metabolites; promoter activity) in the human hepatoma cell line HepG2.Treatment of HepG2 cells with verapamil revealed down-regulation of -gluc activity, protein, and mRNA level down to 50% of the control with EC₅₀ values of 25 M. Effects were similar for both enantiomers. Moreover, it was demonstrated that reduced promoter activity contributes to the observed effects. In summary, our data demonstrate regulation of human -glucuronidase expression by verapamil. Based on our findings we hypothesize that coadministration of verapamil may effect cleavage of glucuronides by -glucuronidase.Abbreviations -gluc -Glucuronidase - MEM Minimal essential medium - DMEM Dubleccos modified Eagles medium - SD Standard deviation - RT-PCR Real-time PCR - AP2 Activating protein 2 - nt Nucleotide - NFB Nuclear factor B - SP1 Specificity protein 1 - HMR 1826 N-[4--glucuronyl-3-nitrobenzyloxycarbonyl]doxorubicin - bp Base pair - M6G Morphine-6-glucuronide - MUG 4-Methylumbelliferyl--D-glucuronide - MU 4-Methylumbelliferone - GAPDH Glyceraldehyde 3-phosphate dehydrogenase     

Both β1- and β2-adrenoceptors mediate catecholamine-evoked arrhythmias in isolated human right atrium

Summary The involvement of ₁- and ₂-adrenoceptors in catecholamine-evoked arrhythmias was investigated in isolated human right atrial appendages obtained from 22 patients chronically treated with blockers (usually ₁-selective) and 9 patients not treated with blockers. A simple experimental model that assesses the incidence of arrhythmic contractions as a function of heart rate (pacing) is introduced. ₁-adrenoceptors were activated by (–)-noradrenaline during ₂-adrenoceptor blockade with 50 nmol/l ICI 118551. ₂-adrenoceptors were activated by (–)-adrenaline during ₁-adrenoceptor blockade with 300 nmol/l CGP 20712A. Both (–)noradrenaline and (–)-adrenaline caused arrhythmic contractions whose incidence was greater at low than at high pacing rates. CGP 20712A (300 nmol/l) blocked the (–)-noradrenaline-evoked contractions in 1/1 atrial strip from 1/1 patient not treated with a blocker and 17/17 atrial strips from 15/15 patients chronically treated with blockers. ICI 118551 (50 nmol/l) blocked the (–)-adrenaline-evoked contractions in 3/4 atrial strips from 3/4 patients not treated with blockers and 17/20 atrial strips from 15/18 patients chronically treated with blockers. The incidence of arrhythmic contractions evoked by both (–)-noradrenaline and (–)-adrenaline was higher in chronically blocked patients than in non blocked patients. We conclude that both ₁- and ₂-adrenoceptors mediate atrial arrhythmias and that the generation of these arrhythmias is facilitated by chronic ₁-adrenoceptor blockade. Correspondence to: A. J. Kaumann at the Clinical Pharmacology Unit, University of Cambridge, as above