Mitochondrial respiration inhibitors suppress protein translation and hypoxic signaling via the hyperphosphorylation and inactivation of translation initiation factor eIF2α and elongation factor eEF2

Over 20,000 lipid extracts of plants and marine organisms were evaluated in a human breast tumor T47D cell-based reporter assay for hypoxia-inducible factor-1 (HIF-1) inhibitory activity. Bioassay-guided isolation and dereplication-based structure elucidation of an active extract from the Bael tree (Aegle marmelos) afforded two protolimonoids, skimmiarepin A (1) and skimmiarepin C (2). In T47D cells, 1 and 2 inhibited hypoxia-induced HIF-1 activation with IC50 values of 0.063 and 0.068 μM, respectively. Compounds 1 and 2 also suppressed hypoxic induction of the HIF-1 target genes GLUT-1 and VEGF. Mechanistic studies revealed that 1 and 2 inhibited HIF-1 activation by blocking the hypoxia-induced accumulation of HIF-1α protein. At the range of concentrations that inhibited HIF-1 activation, 1 and 2 suppressed cellular respiration by selectively inhibiting the mitochondrial electron transport chain at complex I (NADH dehydrogenase). Further investigation indicated that mitochondrial respiration inhibitors such as 1 and rotenone induced the rapid hyperphosphorylation and inhibition of translation initiation factor eIF2α and elongation factor eEF2. The inhibition of protein translation may account for the short-term exposure effects exerted by mitochondrial inhibitors on cellular signaling, while the suppression of cellular ATP production may contribute to the inhibitory effects following extended treatment periods.     

Total synthesis of 2‴,5‴-diepisilvestrol and its c1‴ epimer: key structure activity relationships at c1‴ and c2‴

The first total synthesis of the low-abundance natural product 2?,5?-diepisilvestrol (4) is described. The key step involved a Mitsunobu coupling between cyclopenta[b]benzofuran phenol 7 and dioxane lactol 6. Deprotection then gave a 1:2.6 ratio of natural product 2?,5?-diepisilvestrol (4) and its C1 epimer 1?,2?,5?-triepisilvestrol (15) in 50% overall yield. An in vitro protein translation inhibition assay showed that 2?,5?-diepisilvestrol (4) was considerably less active than episilvestrol (2), while the unnatural isomer 1?,2?,5?-triepisilvestrol (15) was essentially inactive, showing that the configuration at C1? and C2? has a large effect on the biological activity.     

γ-Mangostin increases serotonin 2A/2C, muscarinic, histamine and bradykinin receptor mRNA expression

Aim of the study

γ-Mangostin is a xanthone found in the fruit hulls of Garcinia mangostana L., which have long been used in Southeast Asia as a traditional medicine for the treatment of abdominal pain, dysentery, wound infections, fever and convulsions. Recent studies have revealed that γ-mangostin exhibits a variety of pharmacological activities, including serotonin 2 (5-HT₂) receptor antagonism, anti-inflammatory effects and analgesic effects. To explore the mechanism of γ-mangostin responsible for these pharmacological activities, especially its effects on some related receptors, we investigated the effects of γ-mangostin on 5-HT₂, histamine (H₁) and bradykinin (BK₂) receptor gene expression in neuroblastoma (NG 108-15) cells in vitro. Additionally, to extend the study of the pharmacological properties, we examined the effect of γ-mangostin on the muscarinic (M₄) receptor.

Materials and methods

NG 108-15 cells were cultured in vitro and treated with γ-mangostin or a 5-HT₂ receptor antagonist (either imipramine or ketanserin). Then, the levels of mRNA for 5-HT_2A/2C receptors were evaluated by semi-quantitative RT-PCR. The preventive effect of serotonin on the enhancement effects was also revealed. Additionally, the effects of γ-mangostin on the muscarinic, histamine and bradykinin receptors were determined.

Results

Chronic application of γ-mangostin at a concentration of 0.1 μM induced a significant increase in the level of 5-HT_2A/2C receptor mRNA. These effects were prevented by serotonin. Moreover, γ-mangostin up-regulated the M₄, H₁ and BK₂ receptors.

Conclusion

The ability of γ-mangostin to enhance the expression of 5-HT_2A/2C, muscarinic, histamine and bradykinin receptor mRNA suggests that this compound has antagonistic effects. These pharmacological properties may partly account for the benefits of using mangosteen in the treatment of inflammation, pain and neuropsychiatric symptoms.     

Aloe vera juice: IC₅₀ and dual mechanistic inhibition of CYP3A4 and CYP2D6

The aim of this study was to evaluate the inhibitory potency (IC₅₀ values) of ethanol extracts of two commercially available aloe vera juice (AVJ) products, on CYP3A4 and CYP2D6 activities in vitro and to determine if such inhibitions could be mechanism‐based. Recombinant human CYP3A4 and CYP2D6 enzymes were used and the activities were expressed by the metabolism of testosterone and dextromethorphan with ketoconazole and quinidine as positive inhibitor controls, respectively. The formed metabolites were quantified by validated HPLC techniques. Time‐ and NADPH‐ dependent inhibition assays were performed to evaluate a possible mechanism‐based inhibition. One of the AVJ extracts showed about twice the inhibitory potency towards both CYP enzymes over the other with IC₅₀ values of 8.35 ± 0.72 and 12.5 ± 2.1 mg/mL for CYP3A4 and CYP2D6, respectively. The AVJ was found to exert both CYP mediated and non‐CYP mediated inhibition of both CYP3A4 and CYP2D6. This dual mechanistic inhibition, however, seems to be governed by different mechanisms for CYP3A4 and CYP2D6. Estimated IC₅₀ inhibition values indicate no major interference of AVJ with drug metabolism in man, but the dual mechanistic inhibition of both enzymes might be of clinical significance. Copyright © 2011 John Wiley & Sons, Ltd.     

Triptolide,a Novel Diterpenoid Triepoxide From Tripterygium wilfordii Hook.f., Suppresses the Production and Gene Expression of Pro-Matrix Metalloproteinases 1 and 3 and Augments Those of Tissue Inhibitiors of Metalloproteinases 1 and 2 in Human

Ｍｅｔｈｏｄｓ.Ｈｕｍａｎｓｙｎｏｖｉａｌｆｉｂｒｏｂｌａｓｔｓａｎｄｍｏｕｓｅｍａｃｒｏｐｈａｇｅｓｗｅｒｅｃｕｌｔｕｒｅｄｗｉｔｈｉｎｔｅｒ ｌｅｕｋｉｎ 1α(ＩＬ 1α)ｏｒｌｉｐｏｐｏｌｙｓａｃｃｈａｒｉｄｅ(ＬＰＳ)ｉｎｔｈｅｐｒｅｓｅｎｃｅｏｒａｂｓｅｎｃｅｏｆｔｒｉｐｔｏｌｉｄｅ .Ｔｈｅｐｒｏ ｄｕｃｔｉｏｎｏｆｐｒｏＭＭＰｓ 1ａｎｄ 3 ,ＴＩＭＰｓ 1ａｎｄ 2 ,ｃｙｃｌｏｏｘｙｇｅｎａｓｅ 1 (ＣＯＸ 1 )ａｎｄＣＯＸ 2 ,ｐｒｏｓｔａｇｌａｎｄｉｎＥ2 (ＰＧＥ2 ) ,ＩＬ 1 β ,ａｎｄＩＬ 6ｗａｓａｓ…     

Effect of Moxibustion on IL-1β and IL-2 in Rat Models of Rheumatoid Arthritis

目的：观察艾灸“肾俞”、“足三里”穴对类风湿关节炎大鼠细胞因子IL-1D、IL-2的影响,探讨艾灸效应的作用机制。方法：将50Wistar大鼠随机分为正常组、模型组、药物组、艾灸组及激光组,每组10只。除正常组大鼠外,其他4组大鼠复制类风湿关节炎大鼠模型,观察各组大鼠治疗前后体重、趾围的变化,ELISA法检测大鼠血液中IL-1β、IL-2的含量。结果：治疗后,艾灸组大鼠体重、跖围有显著改善,与模型组相比有非常显著性差异（P〈0．01）,其中跖围改善与药物组及激光组比较有显著性差异（P〈0．05）;艾灸组及激光组均能下调类风湿关节炎大鼠血清中IL-1β、IL-2含量,与模型组比较,差异有统计学意义（P〈0．05）,与药物纽比较,无显著性差异。结论：艾灸能显著改善类风湿关节炎大鼠的足跖肿胀,效果优于10．6μmCO2激光照射。在降低促炎症细胞因子IL—1β、IL-2含量方面,10．6μmCO2激光照射具有与艾灸相似的效应。     

cience and TechnologyInner

a Agricultural Science and TechnologyInner Mongolia Agricultural Science and TechnologyInner Mongolia Agricultural Science and TechnologyInner Mongolia Agricultural Science and TechnologyInner Mongolia Agricultural Science and TechnologyInner Mongolia Agricultural Science and TechnologyIn诸多不足,有待进一步提高。     

γ-Sitosterol from Acacia nilotica L. induces G2/M cell cycle arrest and apoptosis through c-Myc suppression in MCF-7 and A549 cells

Ethnopharmacological relevance

Acacia nilotica is widely distributed in Asia. In India, it occupies an important place in the indigenous system of medicine against anti-inflammatory, antioxidant, cancers, and/or tumors.

Aim of the study

The purpose of this study is to investigate the inhibitory effect of Acacia nilotica leaves extract and γ-Sitosterol on cell proliferation, the apoptotic effect and cell cycle arrest in breast and lung cancer cells.

Materials and methods

GC–MS and HPLC were used to determine the chemical constituents of this extract and γ-Sitosterol respectively. Human MCF-7 and A549 cell lines were treated with Acacia nilotica extract and γ-Sitosterol. Cell viability was determined by MTT assay. Cell proliferation was determined by BrdU incorporation assay. Apoptosis was detected by cell morphologic observation through AO/EtBr staining, cell cycle analysis, and immunoblot analysis on the expression of protein associated with cell cycle arrest.

Results

Experimental results of bioactive compound analysis indicate that γ-Sitosterol, bioactive ingredients of Acacia nilotica extract. The IC₅₀ value of extract on MCF-7 and A549 cancer cells was 493.3 ± 15.2 and 696.6 ± 11.5 μg/ml, respectively. Acacia nilotica extract and γ-Sitosterol were inhibited the cell proliferation by 54.34 ± 1.8 and 42.18 ± 3.9% for MCF-7 and 58.26 ± 1.5 and 44.36 ± 3.05% for A549 cells. The percentage of apoptotic cells observed in the MCF-7 and A549 cell lines were increased to 42.46 and 36.8% of extract; 46.68 and 43.24% for γ-Sitosterol respectively. Flow cytometric analysis results demonstrate that cells were arrested at the G2/M phase and decrease the c-Myc expression.

Conclusions

This study demonstrates in vitro results, which support the ethnomedical use of γ-Sitosterol against cancer. Experimental results of this study suggest that γ-Sitosterol exerts potential anticancer activity through the growth inhibition, cell cycle arrest and the apoptosis on cancer cells.     

LECTURES ON ACUPUNCTURE PartⅠFundamentals of Acupuncture and Moxibustion Lecture Eleven Acupuncture Techniques (2)

Besidesthetechniquesofthefiliformneedle,thereareothertherapiesalsousedintheclinic.Theyarethethree-edgedneedle,cutaneousneedle,intradermalneedle,electroacupuncture,auriculoacupunctureandscalpacupuncture.ⅡThree-edgedNeedle1.TheneedleThethree-edgedneedleisa…     

Calculus Bovis Sativus up-regulates hepatic protein 2(Mrp2) and Mrp4 in 17α-ethynylestradiol-induced cholestasis via a regulatory effect on ER signaling

OBJECTIVE:To investigate the pathway through which Calculus Bovis Sativus(CBS)up-regulates hepatic multidrug resistance-associated protein 2(Mrp2)and Mrp4 in 17 a-ethynylestradiol(EE)-induced cholestasis.METHODS:Five groups of rats were designed:control group,EE+ICI182780 group,EE group,EE+CBS50 mg/kg group and EE+CBS 150 mg/kg group.CBS(50 and 150 mg·kg~(-1)·d~(-1)) was orally given to rats by gavage for five consecutive days in coadministration with EE.The levels of cholestasis biomarkers,alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP)and total bilirubin(TBIL)were determined by biochemical methods.The bile flow was measured,The histopathology of the liver tissue was evaluated.The expression of Mrp2,Mrp3,Mrp4,estrogen receptor α(ERα)and ERB was determined by Western blotting.RESULTS:CBS markedly improved EE-induced cholestasis.EE exposure significantly reduced hepatic Mrp2 and Mrp4 expression compa red with the control group.EE also dramatically up-regulated the expression of Mrp3.Compared to the EE group,CBS notably up-regulated hepatic Mrp2 and Mrp4 but failed to influence the Mrp3 level significantly.ICI1 82780,an ER antagonist,showed similar beneficial effects as CBS.Decreased expression of Mrp2 and Mrp4 caused by EE was also restored by ICI1 82780.Additionally,EE significantly induced hepatic ERa expression,which was reversed by ICI1 82780 or CBS(1 50 mg/kg)treatment,suggesting that CBS exerted a moderate regulatory effect on ER signaling.CONCLUSION:CBS up-regulated hepatic Mrp2 and Mrp4 expression in EE-induced cholestasis,which might be associated with its regulation of ER signaling.     

Effect of Marsdenia tenacissima extract on G2/M cell cycle arrest by upregulating 14-3-3σ and downregulating c-myc in vitro and in vivo

Objective: Marsdenia tenacissima extract(MTE) is a traditional Chinese herbal medicine with anti-cancer activity. In some previous studies, different mechanism actions of the anti-cancer effect of MTE have been revealed. In this study, we first observed that MTE exhibited G2/M cell cycle arrest on two different human breast cancer cell lines, MDA-MB-231 and MCF-7 by mediating 14-3-3σ and c-myc.Methods: The effect of MTE on G2/M cell cycle arrest was evaluated in MDA-MB-231 and MCF-7 cell lines. MTT assay was done for evaluation of cell viability. Flow cytometry was employed for cell cycle analysis. Western blotting analysis and immunohistochemistry were performed to analyze the expression of G2/M cell cycle-related key protein in cells and tissue samples. Animal studies have been conducted to elucidate the anti-tumor effect of MTE.Results: Cell cycle is the backbone for developing cancer. Cell cycle proteins play a major role in the progression of cell cycle and cell proliferation. However, some key protein directly or indirectly modulate the action of cell cycle protein that highly affect cell cycle regulation. In order to investigate cellular proliferation of cancer, we observed that MTE induced the upregulation of 14-3-3σ and downregulation of c-myc,and then reduced the expression of G2/M cell cycle associated key protein, leading to the inhibition of cellular entry into mitosis phase. We also confirmed that MTE exerted a significant antitumor effect on the MDA-MB-231 xenograft model in vivo.Conclusion: G2/M cell cycle arrest occurred by the action of MTE, mediated by the upregulation of 14-3-3σ as well as downregulation of c-myc in MDA-MB-231 and MCF-7 cell lines.