Tumor promoters--microcystin-LR, nodularin and TNF-α and human cancer development

Microcystin-LR and nodularin, along with okadaic acid, are potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A). The mechanisms of action of microcystin-LR and nodularin in the liver and that of okadaic acid, a potent tumor promoter on mouse skin, have attracted the attention of the scientists. This paper reviews several topics: new inhibitors of PP1 and PP2A with new chemical structures, structure-function relationships for both receptor binding and inhibition of protein phosphatases, the crystal structure of PP1 or PP2A-toxin complex, induction of gene expression and apoptosis. These subjects were studied by using in vitro and in vivo experimental systems. Two-stage carcinogenesis experiments with microcystin-LR and nodularin for the first time demonstrated that microcystin-LR is a new tumor promoter in rat liver initiated with diethylnitrosamine (DEN), and that nodularin is a potent tumor promoter associated with weak initiating activity in rat liver initiated with DEN. A working group of WHO (IARC) concluded that microcystin-LR is "possibly carcinogenic to humans" and that nodularin is "not classifiable as to carcinogenicity". Our studies revealed that chemical tumor promoters are inducers of TNF-α in the cells of target tissues and that TNF-α is an endogenous tumor promoter. This advance in carcinogenesis made it possible to look for the link between chemical tumor promoters and endogenous tumor promoters, such as TNF-α and IL-1. The carcinogenic features of TNF-α are described in this review, and the TNF-α inducing protein (Tipα) of Helicobacter pylori genome is presented as an example of a tumor promoter of human stomach cancer development.     

A mechanistic perspective,clinical applications,and phage-display-assisted discovery of TNFα inhibitors

TNFα participates in a variety of physiological processes, but at supra-physiological concentrations it has been implicated in the pathology of inflammatory and autoimmune diseases. Therefore, much attention has been devoted to the development of strategies that overcome the effects of aberrant TNFα concentration. Promising strategies include drugs that destabilize the active (trimeric) form of TNFα and antagonists of TNFα receptor type I. Underpinning these strategies is the successful application of phage-display technology to identify anti-TNFα peptides and antibodies. Here, we review the development of inhibitors of the TNFα–TNF receptor system, with particular focus on the phage-display-assisted identification of molecules that interfere with this system by acting as inhibitors of TNFα or by sequestering TNFα away from its receptor.     

p38 MAPK inhibitors, IKK2 inhibitors, and TNFα inhibitors in COPD

COPD represents a major respiratory disorder, causing significant morbidity and mortality throughout the world. While therapies exist for COPD, they are not always effective, and many patients experience exacerbations and morbidity despite current therapies. Study of the molecular mechanisms involved in the underlying physiological manifestations of COPD has yielded multiple new targets for therapeutic intervention. In this review, we discuss signaling pathways involved in COPD pathogenesis and review clinical studies of p38 MAPK inhibitors, TNFα inhibitors, and IKK2 inhibitors as potential COPD therapies.     

Involvement of Bcl-2, Src, and ERα in gossypol-mediated growth inhibition and apoptosis in human uterine leiomyoma and myometrial cells

Aim:

To investigate the effect of gossypol on the growth of cultured human uterine leiomyoma and myometrial cells, the level of Bcl-2 and the activity of Src and estrogen receptor (ERα).

Methods:

Human uterine leiomyoma and adjacent normal myometrial cells were cultured in vitro. Both cell types were treated with a graded concentration of gossypol. Cell viability was assayed using CCK-8. Morphological change was observed with optical and electronic microscopy. Apoptosis was evaluated using TUNEL assay. Levels of Bcl-2, ERα and Src were analyzed using Western blotting.

Results:

Gossypol significantly inhibited growth and promoted apoptosis in cultured human uterine leiomyoma cells with the IC₅₀ value and its corresponding 95% confidence intervals (CI) of 6.5 (4.0–10.5), 9.0 (4.9–16.5), and 7.5 (4.0–14.1) μmol/L at 20, 40, and 60 h, respectively. Gossypol exerted inhibitory effects on the myometrial cells with the IC₅₀ value and its 95% CI of 49.1 (28.3–85.0), 14.5 (7.7–27.4), and 2.6 (1.2–5.6) μmol/L at 20, 40, and 60 h, respectively. Compared with control, gossypol 0.1-3.0 μmol/L markedly decreased the protein expression of Bcl-2 (P<0.05) in both leiomyoma and myometrial cells in a concentration-dependent manner, and significantly suppressed the level of phospho-Tyr416Src (P<0.05) in both cell types at 3.0 μmol/L without obvious alteration of c-Src and phospho-Tyr527Src levels (P>0.05). In addition, gossypol markedly reduced both the expression of ERα (P<0.05) at the low concentration of 0.1 μmol/L in the myometrial cells and the level of phospho-ser167ERα (P<0.05) at the high concentration of 3.0 μmol/L in the leiomyoma cells.

Conclusion:

Gossypol inhibits proliferation and induces apoptosis in human uterine leiomyoma and myometrial cells. It is likely that the mechanisms of action involve reducing the protein level of Bcl-2 and the activity of Src and ERα.     

PPARα activation by culinary herbs and spices

Hyper- and dyslipidemia are risk factors for cardiovascular disease, the primary cause of death in industrialized countries. Peroxisome proliferators-activated receptor (PPAR)α activation is involved in various mechanisms that improve the lipid profile. We tested various plant extracts and their compounds to determine whether they stimulated PPARα activity in vitro. Out of 34 tested plant extracts, nine exhibited low to moderate PPARα transactivation, including caraway, chili pepper, nutmeg, licorice, black and white pepper, paprika, coriander, saffron, and stevia tea. The active components of black pepper and chili pepper, piperine, and capsaicin exerted the highest transactivational activities with EC?? values of 84 μM and 49 μM, respectively. The chalcones, including 2-hydroxychalcone, 2'-hydroxychalcone, 4-hydroxychalcone, and 4-methoxychalcone, moderately transactivated PPARα. Resveratrol and apigenin only slightly transactivated PPARα. These results suggest that a diet rich in fruit, herbs, and spices provides a number of PPARα agonists that might contribute to an improved lipid profile.     

Expression and clinical significance of HER-2,TOPO-Ⅱα and PI3K in ovarian epithelial cancer

目的 探讨原癌基因人类表皮生长因子受体2(HER-2)、拓扑异构酶Ⅱ(Topo-Ⅱα)和磷脂酰肌醇3激酶(PI3K)在卵巢上皮癌组织中的表达、相互关系及临床意义.方法 采用免疫组化SP方法检测58例卵巢上皮癌(卵巢癌组)、20例卵巢良性上皮性肿瘤(良性组)及10例卵巢正常组织标本(对照组)中HER-2、TOPO-Ⅱα及PI3K的表达情况.结果 HER-2在对照组、良性组中的阳性表达率分别为10.0%、15.0%,显著低于卵巢癌组的46.6%;卵巢癌组中TOPO-Ⅱα的阳性表达率(53.4%)显著高于对照组(10.0%)和良性组(15.0%)(P<0.05);PI3K在卵巢癌组的阳性表达率(69.0%)明显高于对照组(0.0%)和良性组(20.0%) (P<0.05);而良性组和对照组间比较差异无统计学意义(P＞0.05).HER-2、TOPO-Ⅱα及PI3K的表达与卵巢上皮癌临床分期、细胞组织学分化有显著相关性(P＜0.05),而与年龄、组织学类型无显著相关性(P＞0.05);卵巢上皮癌中HER-2分别与TOPO-Ⅱα、PI3K 的表达之间均呈正相关(P＜0.05),TOPO-Ⅱα与PI3K的表达呈正相关(P＜0.05).结论 HER-2、TOPO-Ⅱα和PI3K的表达均与卵巢上皮癌的发生、发展及侵袭、转移有关,并对卵巢上皮癌的发展起协同作用,对临床治疗及预后判断具有重要的指导意义.     

Inhibitors of cytosolic phospholipase A2α with carbamate structure: synthesis,biological activity,metabolic stability,and bioavailability

Cytosolic phospholipase A₂α is a serine hydrolase involved in the generation of pro-inflammatory lipid mediators. Here, we describe structure–activity relationship studies on a series of carbamate-substituted indazole-5-carboxylic acid inhibitors of this enzyme. Bioavailability experiments in mice with 1-(3-(4-butylphenoxy)-2-{[(ethylthio)carbonyl]amino}propyl)indazole-5-carboxylic acid (14) revealed a high rate of glucuronidation and biliary excretion of this compound. Replacement of the rigid aromatic carboxylic acid system by a more flexible aliphatic one led to substances with a significantly increased in vitro stability against metabolic glucuronidation.     

Liuwei Dihuang-active fraction combination,LW-AFC,improved spatial learning and memory impairment induced by TNF-α in mice

OBJECTIVE Tumor necrosis factor-α(TNF-α) plays a vital role in cognitive dysfunction caused by stress.In our previous study, Liuwei Dihuang-active fraction combination(LW-AFC) could attenuate the effects of mental stress and non-psychotic stress in mice. The aim of this study is to investigate the effects of LW-AFC on cognitive dysfunction caused by TNF-α in mice. METHODS 40 male BALB/c mice were divided into 4 groups according to their body weight,including control, TNF-α model, LW-AFC treatment and Etanercept(TNF-α antagonist) treatment groups. LW-AFC(1.6 or 3.2 g·kg~(-1) per day) were orally administrated for 7 consecutive days before TNF-α administration. Etanercept was injected subcutaneously at 30 mg·kg~(-1) the day before TNF-α administration. One hour before the behavioral test, TNF-αwere injected intraperitoneally at 0.2 mg·kg~(-1) to mice. RESULTS Compared with control group, the time of mice stayed in the target quadrant and the number of mice crossing the plate significantly decreased after TNF-α injection, suggested that the spatial learning and memory ability of the mice were impaired. LW-AFC administration could increase the time of mice stayed in the target quadrant and the number of mice crossing the plate significantly at 1.6 g·kg~(-1), indicated that LW-AFC could improve spatial learning and memory in TNF-α treated mice. CONCLUSION LW-AFC can improve spatial learning and memory impairment induced by TNF-α in mice, the further mechanism still need to be clarified.     

Syzygium cumini ameliorates insulin resistance and β-cell dysfunction via modulation of PPAR, dyslipidemia, oxidative stress, and TNF-α in type 2 diabetic rats

Syzygium cumini (SC) is well known for its anti-diabetic potential, but the mechanism underlying its amelioration of type 2 diabetes is still elusive. Therefore, for the first time, we investigated whether SC aqueous seed extract (100, 200, or 400 mg/kg) exerts any beneficial effects on insulin resistance (IR), serum lipid profile, antioxidant status, and/or pancreatic β-cell damage in high-fat diet / streptozotocin-induced (HFD-STZ) diabetic rats. Wistar albino rats were fed with HFD (55% of calories as fat) during the experiment to induce IR and on the 10th day were injected with STZ (40 mg/kg, i.p.) to develop type 2 diabetes. Subsequently, after confirmation of hyperglycemia on the 14th day (fasting glucose level > 13.89 mM), diabetic rats were treated with SC for the next 21 days. Diabetic rats showed increased serum glucose, insulin, IR, TNF-α, dyslipidemia, and pancreatic thiobarbituric acid-reactive substances with a concomitant decrease in β-cell function and pancreatic superoxide dismutase, catalase, and glutathione peroxidase antioxidant enzyme activities. Microscopic examination of their pancreas revealed pathological changes in islets and β-cells. These alterations reverted to near-normal levels after treatment with SC at 400 mg/kg. Moreover, hepatic tissue demonstrated increased PPARγ and PPARα protein expressions. Thus, our study demonstrated the beneficial effect of SC seed extract on IR and β-cell dysfunction in HFD-STZ-induced type 2 diabetic rats.     

AVE8134, a novel potent PPARα agonist, improves lipid profile and glucose metabolism in dyslipidemic mice and type 2 diabetic rats

Aim:

AVE8134 is a structurally novel potent PPARα agonist. The aim of this study is to investigate the efficacy of AVE8134 on lipid profile and glucose metabolism in dyslipidemic mice and type 2 diabetic rats.

Methods:

A cell based PPAR Gal4 transactivation assay was constructed for testing the activities of AVE8134 at 3 different PPAR isoforms in vitro. Transgenic human Apo A1 (hApo A1) mice and insulin-resistant ZDF rats were used to evaluate the effects of AVE8134 in vivo.

Results:

AVE8134 was a full PPARα dominated PPAR agonist (the values of EC₅₀ for human and rodent PPARα receptor were 0.01 and 0.3 μmol/L, respectively). AVE8134 was not active at PPARδ receptor. In female hApo A1 mice, AVE8134 (1–30 mg·kg⁻¹·d⁻¹, po for 12 d) dose-dependently lowered the plasma triglycerides, and increased the serum HDL-cholesterol, hApo A1 and mouse Apo E levels. In female ZDF rats, AVE8134 (3–30 mg·kg⁻¹·d⁻¹ for 2 weeks) improved insulin-sensitivity index. In pre-diabetic male ZDF rats (at the age of 7 weeks), AVE8134 (10 mg·kg⁻¹·d⁻¹ for 8 weeks) produced an anti-diabetic action comparable to rosiglitazone, without the PPARγ mediated adverse effects on body weight and heart weight. In male ZDF rats (at the age of 6 weeks), AVE8134 (20 mg·kg⁻¹·d⁻¹ for 12 weeks) increased mRNA levels of the target genes LPL and PDK4 about 20 fold in the liver, and there was no relevant effect with rosiglitazone.

Conclusion:

AVE8134 improves lipid profile and glucose metabolism in dyslipidemic mice and type 2 diabetic rats.     

Arylalkyl ketones, benzophenones, desoxybenzoins and chalcones inhibit TNF-α induced expression of ICAM-1: structure-activity analysis

The interaction between leukocytes and the vascular endothelial cells (EC) via cellular adhesion molecules plays an important role in the pathogenesis of various inflammatory and autoimmune diseases. Small molecules that block these interactions have been targeted as potential therapeutic agents against acute and chronic inflammatory diseases. In an effort to identify potent intercellular cell adhesion molecule-1 (ICAM-1) inhibitors, a large number of arylalkyl ketones, benzophenones, desoxybenzoins and chalcones and their analogs (54 in total) have been synthesized and screened for their ICAM-1 inhibitory activity. The structure-activity relationship studies of these compounds identified three potent chalcone derivatives and also demonstrated the possible mechanism for their ICAM-1 inhibitory activities. The most active compound was found to be 79.     

Update upon efficacy and safety of TNF-α inhibitors

The ongoing progresses in the knowledge of the pathogenic mechanisms of various immune-mediated and inflammatory diseases as well as the availability of innovative biotechnological approaches have led to the development of new drugs that add to conventional treatments. Among these, tumor necrosis factor (TNF)-α inhibitors, that is, infliximab, adalimumab, etanercept, golimumab and certolizumab pegol, are now available for clinical use. This editorial discusses the recent indications of TNF-α inhibitors, the pretreatment considerations, the reported adverse events and, finally, the recommendations for its use in pregnancy.     

Metabolic fate of sipoglitazar, a novel oral PPAR agonist with activities for PPAR-γ, -α and -δ, in rats and monkeys and comparison with humans in vitro

Sipoglitazar is a novel anti-diabetic agent with triple agonistic activities on the human peroxisome proliferator-activated receptors, hPPAR-γ, -α, and -δ. The bioavailability for sipoglitazar was 95.0% and 72.6% in rats and monkeys respectively and sipoglitazar is hardly subject to first pass metabolism in either species. Following oral administration of [1?C]sipoglitazar to rats, sipoglitazar and its metabolites were distributed to the rat tissues with relatively high concentrations in the liver and also to the target tissue, the adipose tissue. The major component was sipoglitazar in the plasma of rats and monkeys. In rats, sipoglitazar was mainly excreted into the feces via biliary excretion as sipoglitazar-G, while the major component was M-I-G in the urine and M-I in the feces of monkeys. In hepatocytes, the metabolism was not extensively advanced in rats and the main metabolites were M-I and sipoglitazar-G in humans, similar to the metabolic profile in monkeys. There was no metabolite specific for humans in vitro. In conclusion, the formation of M-I, M-I-G and sipoglitazar-G is considered to be crucial and sipoglitazar is presumed to be cleared primarily by oxidation and glucuronidation in humans, when examined in vivo and in vitro.     

The influence of antibodies to TNF-α and IL-1β on haemodynamic responses to the cytokines,and to lipopolysaccharide,in conscious rats

1. Male, Long Evans rats (350–450 g) were anaesthetized and had pulsed Doppler probes and intravascular catheters implanted to allow monitoring of regional (renal, mesenteric and hindquarters) haemodynamics in the conscious state. Our main objectives were to:- assess the effects of administering human recombinant tumour necrosis factor (TNF)-α and human recombinant interleukin-1 (IL-1)β, alone and together; determine the influence of pretreatment with a mixture of antibodies to TNF-α and IL-1β on responses to co-administration of the cytokines; ascertain if pretreatment with a mixture of the antibodies to TNF-α and IL-1β had any influence on the responses to lipopolysaccharide (LPS).
2. TNF-α (10, 100 and 250 μg kg⁻¹, in separate groups, n=3, 9 and 8, respectively) caused tachycardia (maximum Δ, +101±9 beats min⁻¹) and modest hypotension (maximum Δ, −10±2 mmHg), accompanied by variable changes in renal and mesenteric vascular conductance, but clear increases in hindquarters vascular conductance; only the latter were dose-related (maximum Δ, +6±6, +27±9, and +61±12% at 10, 100 and 250 μg kg⁻¹, respectively).
3. IL-1β (1, 10, and 100 μg kg⁻¹ in separate groups, n=8, 8 and 9, respectively) evoked changes similar to those of TNF-α (maximum Δ heart rate, +69±15 beats min⁻¹; maximum Δ mean blood pressure, −14±2 mmHg; maximum Δ hindquarters vascular conductance, +49±17%), but with no clear dose-dependency.
4. TNF-α (250 μg kg⁻¹) and IL-1β (10 μg kg⁻¹) together caused tachycardia (maximum Δ, +76±15 beats min⁻¹) and hypotension (maximum Δ, −24±2 mmHg) accompanied by increases in renal, mesenteric and hindquarters vascular conductances (+52±6%, +23±8%, and +52±11%, respectively). Thereafter, blood pressure recovered, in association with marked reductions in mesenteric and hindquarters vascular conductances (maximum Δ, −50±3% and −58±3%, respectively). Although bolus injection of LPS (3.5 mg kg⁻¹) caused an initial hypotension (maximum Δ, −27±11 mmHg) similar to that seen with co-administration of the cytokines, it did not cause mesenteric or hindquarters vasodilatation, and there was only a slow onset renal vasodilatation. The recovery in blood pressure following LPS was less than after the cytokines, and in the former condition there was no mesenteric vasoconstriction. By 24 h after co-administration of TNF-α and IL-1β or after bolus injection of LPS, the secondary reduction in blood pressure was similar (−16±2 and −13±3 mmHg, respectively), but in the former group the tachycardia (+117±14 beats min⁻¹) and increase in hindquarters vascular conductance (+99±21%) were greater than after bolus injection of LPS (+54±16 beats min⁻¹ and +43±9%, respectively).
5. Pretreatment with antibodies to TNF-α and IL-1β (300 mg kg⁻¹) blocked the initial hypotensive and mesenteric and hindquarters vasodilator responses to co-administration of the cytokines subsequently. However, tachycardia and renal vasodilatation were still apparent. Premixing antibodies and cytokines before administration prevented most of the effects of the latter, but tachycardia was still present at 24 h.
6. Pretreatment with antibodies to TNF-α and IL-1β before infusion of LPS (150 μg kg⁻¹ h⁻¹ for 24 h) did not affect the initial fall in blood pressure, but suppressed the hindquarters vasodilatation and caused a slight improvement in the recovery of blood pressure. However, pretreatment with the antibodies had no effect on the subsequent cardiovascular sequelae of LPS infusion.
7. The results indicate that although co-administration of TNF-α and IL-1β can evoke cardiovascular responses which, in some respects, mimic those of LPS, and although antibodies to the cytokines can suppress most of the cardiovascular effects of the cytokines, the antibodies have little influence on the haemodynamic responses to LPS, possibly because, during infusion of LPS, the sites of production and local action of endogenous cytokines, are not accessible to exogenous antibodies.