BH3 mimetic ABT-737 and a proteasome inhibitor synergistically kill melanomas through Noxa-dependent apoptosis

The Bcl-2 family is important in modulating sensitivity to anticancer drugs in many cancers, including melanomas. The BH3 mimetic ABT-737 is a potent small molecule inhibitor of the anti-apoptotic proteins Bcl-2/Bcl-X(L)/Bcl-w. In this report, we examined whether ABT-737 is effective in killing melanoma cells in combination with the proteasome inhibitor MG-132, and further evaluated the mechanisms of action. Viability, morphological, and Annexin V apoptosis assays showed that ABT-737 alone exhibited little cytotoxicity, yet it displayed strong synergistic lethality when combined with MG-132. In addition, the detection of Bax/Bak activation indicated that the combination treatment synergistically induced mitochondria-mediated apoptosis. Furthermore, mechanistic analysis revealed that this combination treatment induced expression of the pro-apoptotic protein Noxa- and caspase-dependent degradation of the anti-apoptotic protein, Mcl-1. Finally, siRNA-mediated inhibition of Mcl-1 expression significantly increased sensitivity to ABT-737 in these cells, and knocking down Noxa expression protected the cells from cytotoxicity induced by the combination treatment. These findings demonstrate that ABT-737 combined with MG-132 synergistically induced Noxa-dependent mitochondrial-mediated apoptosis. In summary, this study indicates promising therapeutic potential of targeting anti-apoptotic Bcl-2 family members in treating melanoma, and it validates rational molecular approaches that target anti-apoptotic defenses when developing cancer treatments.     

雷公藤三萜酸C的分离与结构鉴定

雷公藤总甙系雷公藤(Tripterygium wilfordii Hook.f)去皮根心经提取后得到的有效组分,它具有很强的抗炎免疫抑制作用。作者从雷公藤总甙中分离出5个三萜类化合物T_1,T_2,T_3,T_6,T_(28)。本文报道T_(28)化合物的分子结构,其他待另文发表。T_(28)是一个新的乌     

Beneficial effects of danshensu, an active component of Salvia miltiorrhiza, on homocysteine metabolism via the trans-sulphuration pathway in rats

Background and purpose:

Elevated plasma total homocysteine (tHcy) level has been established as an independent risk factor for cardiovascular diseases. Danshensu, an active ingredient of Salvia miltiorrhiza, shows wide cardiovascular benefit. However, in terms of its own methylation, danshensu could elevate tHcy level, which would act against its cardiovascular benefit, thus posing a ‘therapeutic paradox’. As this paradox has not been fully assessed, we have evaluated the effects of danshensu on tHcy levels to uncover the underlying mechanisms.

Experiment approach:

We evaluated the influence of danshensu on homocysteine (Hcy) metabolism in rats with normal tHcy levels and in rat models of elevated tHcy (single intravenous methionine loading model and a hyperhomocysteinemic model after 3 weeks methionine dosing, with and without 3 weeks of danshensu treatment). We also quantified some metabolic intermediates (S-adenosyl methionine, S-adenosyl-l-homocysteine, cysteine and glutathione) relevant to Hcy metabolism in rat liver and kidney.

Key results:

Acute treatment with a single dose of danshensu in rats with normal tHcy did not change plasma tHcy. In contrast, danshensu significantly lowered tHcy in rats with elevated tHcy. The relatively higher cysteine and glutathione levels after treatment with danshensu indicated that its tHcy-lowering effect was via increased activity of the trans-sulphuration pathway.

Conclusions and implications:

Our results suggested that danshensu may act both acutely to increase trans-sulphuration and after chronic exposure to up-regulate the activity of the trans-sulphuration enzymes. The tHcy-lowering effect of danshensu is another cardiovascular benefit provided by S. miltiorrhiza and suggests a potential tHcy-lowering therapy.     

Interaction between udenafil and tamsulosin in rats: non-competitive inhibition of tamsulosin metabolism by udenafil via hepatic CYP3A1/2

Background and purpose:

Orthostatic hypotension has been observed when PDE 5 (cGMP-specific phosphodiesterase type 5) inhibitors are co-administered with α-adrenoceptor antagonists. Here we assessed the pharmacokinetic and haemodynamic interactions between udenafil and tamsulosin in rats, as both drugs are metabolized via rat hepatic cytochrome P450 3A1/2.

Experimental approach:

Interactions between the two drugs were evaluated in rats after simultaneous 1 or 15 min i.v. infusion or after p.o. administration of udenafil (30 mg·kg⁻¹) and/or tamsulosin (1 mg·kg⁻¹). In vitro metabolism of tamsulosin with udenafil was measured to obtain the inhibition constant (K_i) and [I]/K_i ratio of udenafil.

Key results:

The total area under the plasma concentration–time curve from time zero to time infinity (AUC)s (or AUC_0–4h) of tamsulosin were significantly greater after 15 min of i.v. infusion or after oral administration with udenafil, compared with tamsulosin alone. The hepatic first-pass metabolism of tamsulosin was inhibited by udenafil, and the inhibition in vitro was in a non-competitive mode. The arterial systolic blood pressure was significantly lower at 5, 10 and 60 min after oral co-administration of the drugs.

Conclusions and implications:

The significantly greater AUC of tamsulosin after i.v. and p.o. administration of both drugs may be attributable to non-competitive inhibition of cytochrome P450 3A1/2-mediated hepatic tamsulosin metabolism by udenafil. The inhibition was also observed in human liver S9 fractions, suggesting that a reassessment of the oral dosage of tamsulosin is necessary when udenafil and tamsulosin are co-administered to patients with benign prostatic hyperplasia.     

Functional proteomic analysis of melanoma progression

Functional proteomics provides a powerful approach to screen for alterations in protein expression and posttranslational modifications under conditions of human disease. In this study, we use protein screening to examine markers of melanoma progression, by profiling melanocyte versus melanoma cell lines using two-dimensional electrophoresis and mass spectrometry. Eight candidate markers were identified as differentially regulated in transformed cells. In particular, hepatoma-derived growth factor (HDGF) and nucleophosmin B23 were strongly correlated with melanoma. Nucleophosmin B23 is a nucleolar and centrosome-associated protein, which has been implicated as a target for cyclin E/cyclin-dependent kinase 2 (CDK2) in modulating centrosome duplication and cell cycle control. Western blotting of one-dimensional and two-dimensional gels showed that the form of nucleophosmin B23 that is up-regulated in melanoma represents a posttranslationally modified form, most likely reflecting enhanced phosphorylation in the tumor-derived cells. In contrast, Western analysis of HDGF demonstrated increased expression of all forms in melanoma cell lines compared with melanocytes. Immunohistochemical analysis of human tissue biopsies showed strong expression of HDGF in early and late stage melanomas and low expression in melanocytes and nontumorigenic nevi. Interestingly, biopsies of nevi showed a graded effect in which HDGF immunoreactivity was reduced in nevoid nests penetrating deep into the dermis compared with nests at the epidermal-dermal junction, suggesting that HDGF expression in nevi is dependent on epidermal cell interactions. In contrast, biopsies of melanoma showed strong expression of HDGF throughout the tumor, including cells located deeply within dermis. Thus, expression of this antigen likely reports a reduced dependence of protein expression on epidermal interactions.     

Glucocorticoid receptor and heat shock protein 90 in peripheral blood mononuclear cells from asthmatics

目的探讨糖皮质激素受体(GR)和热休克蛋白90(HSP90)mRNA在糖皮质激素敏感型(SS)、依赖型(SD) 和抵抗型(SR)哮喘中的表达及其在SR哮喘发病中的作用。 方法采用反转录-聚合酶链(RT-PCR)的方法分别测定正常人(10例)、SS哮喘(10例)、SD哮喘(5例)和 SR哮喘(6例)的外周血单个核细胞(PBMC)中GR mRNA和HSP90 mRNA的表达,并在体外用IL-2、IL-4分 别、联合刺激上述细胞观察其受刺激后GR mRNA和HSP90 mRNA表达的改变情况。 结果 SR哮喘的GR和HSP90 mRNA表达水平最高(分别为0.730±0.171和1.122±0.165),SS哮喘次之 (分别为0.359±0.350和0.885±0.250),SD哮喘最低(分别为0.017±0.008和0.078±0.039)。正常人有 一定表达(分别为0.052±0.013和0.362±0.101)。GR和HSP90 mRNA的表达各组间相比P＜0.05。正 常人、SS、SD和SR哮喘HSP90/GR的比值分别为7.15±1.84、8.39±7.95、5.51±3.30、1.57±0.18,SR哮喘 HSP90/GR比值明显低于前三组(P＜0.05)。IL-2和IL-4单独刺激对SS、SD和SR哮喘的GR、HSP90 mRNA表达无明显影响,二者联合刺激可使SS、SD和SR哮喘GR mRNA表达以及SS、SD哮喘HSP90 mRNA 表达增强,但不能使SR哮喘HSP90 mRNA表达增强。 结论 SR哮喘中HSP90 mRNA表达相对不足造成HSP90/GR比值降低可能是SR哮喘形成的原因之一, IL-2+IL-4对GR和HSP90 mRNA表达的不同调节作用可能是形成SR哮喘HSP90/GR比值降低的原因之一。     

Loss of prdm1a accelerates melanoma onset and progression

Melanoma is an aggressive, deadly skin cancer derived from melanocytes, a neural crest cell derivative. Melanoma cells mirror the developmental program of neural crest cells in that they exhibit the same gene expression patterns and utilize similar cellular mechanisms, including increased cell proliferation, epithelial-mesenchymal transition, and migration. Here we studied the role of neural crest regulator PRDM1 in melanoma onset and progression. In development, Prdm1a functions to promote neural crest progenitor fate, and in melanoma, we found that PRDM1 has reduced copy number and is recurrently deleted in both zebrafish and humans. When examining expression of neural crest and melanocyte development genes, we show that sox10 progenitor expression is high in prdm1a^−/− mutants, while more differentiated melanocyte markers are reduced, suggesting that normally Prdm1a is required for differentiation. Data mining of human melanoma datasets indicates that high PRDM1 expression in human melanoma is correlated with better patient survival and decreased PRDM1 expression is common in metastatic tumors. When one copy of prdm1a is lost in the zebrafish melanoma model Tg(mitfa:BRAF^V600E);p53^−/−;prdm1a^+/−, melanoma onset occurs more quickly, and the tumors that form have a larger area with increased expression of sox10. These data demonstrate a novel role for PRDM1 as a tumor suppressor in melanoma.     

伯氨喹在乙醇—水混合溶剂中离解常数的测定及其与维生素C的配合比研究

本文推导出用pH滴定法测定三元弱酸离解解常数的方程,测得25±0.01℃时,伯氨喹在乙醇—水的体积比为1:1的混合溶剂中,离子强度范围5×10^-3～5×10^-2mol/L时逐级离解常数为Ka₁=(3.84±2.35)×10^-2(归属于仲铵基),Ka₂=(1.50±1.17)×10^-8(归属于叔铵基),Ka₃=(2.07±0.27)×10^-10(归属于伯铵基)。并用pH法及电导法测得伯氨喹与维生素C在上述溶剂中的配合比为1:1。