Cytotoxic metabolites from an Indonesian sponge Lendenfeldia sp

The lipid extract of an Indonesian Lendenfeldia sp. sponge inhibited hypoxia-induced hypoxia-inducible factor-1 (HIF-1) activation in T47D breast tumor cells. Chromatographic separation yielded the new substituted naphthalene dimer 1, the new furanolipid 2, and three known homoscalarane sesterterpenes, 3-5. Compounds 1 and 3-5 inhibited hypoxia-induced HIF-1 activation (IC50 values: 0.64-6.9 microM), but also reduced the viability of T47D and MDA-MB-231 breast tumor cells. Compound 4 was the most potent and showed a unique tumor cell line selectivity in the NCI 60-cell line panel. The general cytotoxicity of these compounds precluded their further consideration as HIF-1 inhibitors.     

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.     

Natural and semisynthetic mammea-type isoprenylated dihydroxycoumarins uncouple cellular respiration

In an effort to identify natural product-based molecular-targeted antitumor agents, mammea-type coumarins from the tropical/subtropical plant Mammea americana were found to inhibit the activation of HIF-1 (hypoxia-inducible factor-1) in human breast and prostate tumor cells. In addition to the recently reported mammea E/BB (15), bioassay-guided fractionation of the active extract yielded 14 mammea-type coumarins including three new compounds, mammea F/BB (1), mammea F/BA (2), and mammea C/AA (3). The absolute configuration of C-1' in 1 was determined by the modified Mosher's method on a methylated derivative. These coumarins were evaluated for their effects on mitochondrial respiration, HIF-1 signaling, and tumor cell proliferation/viability. Acetylation of 1 afforded a triacetoxylated product (A-2) that inhibited HIF-1 activation with increased potency in both T47D (IC(50) 0.83 μM for hypoxia-induced) and PC-3 cells (IC(50) 0.94 μM for hypoxia-induced). Coumarins possessing a 6-prenyl-8-(3-methyloxobutyl) substituent pattern exhibited enhanced HIF-1 inhibitory effects. The O-methylated derivatives were less active at inhibiting HIF-1 and suppressing cell proliferation/viability. Mechanistic studies indicate that these compounds act as anionic protonophores that potently uncouple mitochondrial electron transport and disrupt hypoxic signaling.     

Abietane diterpenes from Salvia miltiorrhiza inhibit the activation of hypoxia-inducible factor-1

The hypoxia-inducible factor-1 (HIF-1) has been known to be correlated to the adaptation and proliferation of tumor cells; therefore HIF-1 has become an important target in the development of anticancer drugs. A phytochemical study of the CHCl3-soluble fraction of Salvia miltiorrhiza, which strongly inhibited hypoxia-induced reporter gene expression, led to the isolation of 12 abietane-type diterpenes. Of these compounds, sibiriquinone A (1), sibiriquinone B (2), cryptotanshinone (3), and dihydrotanshinone I (4) potently inhibited hypoxia-induced luciferase expression with IC50 values of 0.34, 3.36, 1.58, and 2.05 microM on AGS cells, a human gastric cancer cell line, and 0.28, 3.18, 1.36, and 2.29 microM on Hep3B cells, a human hepatocarcinoma cell line, respectively. Consistently, 1 and 4 dose-dependently suppressed the HIF-1alpha accumulation and 1 inhibited mRNA expression of vascular endothelial growth factor (VEGF) under hypoxia. These results suggest that the anticancer activity of tanshinones is likely at least in part associated with their inhibition of HIF-1 accumulation.     

Phenanthroquinolizidine alkaloids from the roots of Boehmeria pannosa potently inhibit hypoxia-inducible factor-1 in AGS human gastric cancer cells

A bioassay-guided phytochemical investigation on the methanol extract of Boehmeria pannosa, using a HIF-1-mediated reporter gene assay, led to the isolation of two phenanthroquinolizidine alkaloids, (-)-cryptopleurine (1) and (-)-(15R)-hydroxycryptopleurine (2). The structure of the new compound 2 was determined by spectroscopic methods. Compounds 1 and 2 potently inhibited the hypoxia-induced expression of a reporter gene under the control of a hypoxia response element (HRE) with IC(50) values of 8.7 and 48.1 nM, respectively. Furthermore, 1 and 2 suppressed the accumulation of HIF-1alpha protein in a dose-dependent manner, but not the HIF-1beta protein and inhibited expression of vascular endothelial growth factor (VEGF) by hypoxia.     

银杏内酯诱导嗜铬细胞瘤细胞表达低氧诱导因子-1α

 目的研究银杏内酯(ginkgolides,Gin)对神经生长因子(nerve growth factor,NGF)诱导分化的嗜铬细胞瘤(PC12)细胞的影响以及与其相关的信号通路。方法通过MTT比色法观察不同浓度Gin对PC12细胞活性的影响,RT-PCR和Western Blot法分析Gin对PC12细胞低氧诱导因子-1(αhypoxia inducible factor-1α,HIF-1α)表达以及MAPK信号通路的影响。结果一定浓度范围的Gin可促进PC12细胞的活性,在37.5 mg·L^-1作用24 h效果最明显。37.5 mg·L^-1 Gin单独处理PC12细胞24 h可诱导HIF-1αmRNA表达增强和蛋白水平上调。Gin还引起p-ERK水平的明显提高。Gin增加PC12细胞HIF-1α蛋白的稳定性存在一定的时间和剂量依赖关系,PD98059可部分抑制Gin的增强作用,金雀异黄素可完全阻断之;与此相对应的是,Gin以时间和剂量依赖方式诱导PC12细胞p-ERK水平的增高,PD98059和金雀异黄素均能完全阻断Gin的诱导作用。结论Gin可诱导PC12细胞表达HIF-1α,主要与MEK-ERK信号通路激活有关,可能是其促进分化的PC12生长的原因。     

Liquiritigenin inhibits serum-induced HIF-1α and VEGF expression via the AKT/mTOR-p70S6K signalling pathway in HeLa cells

Liquiritigenin (LQ) is a non-toxic dietary flavonoid with chemopreventive and anticancer properties. However, the mechanism of its antiangiogenesis remains unclear. Hypoxia-inducible factor-1α (HIF-1α) and its downstream target, vascular endothelial growth factor (VEGF), play a critical role in tumour angiogenesis and represent an attractive chemotherapeutic target. In this study, we investigated the effect of LQ on the molecular mechanism of angiogenesis. We found that LQ inhibited VEGF expression at both mRNA and protein levels. Liquiritigenin did not affect HIF-1α expression at the mRNA level, but it dramatically inhibited both serum- and mimicked hypoxic-induced HIF-1α protein accumulation in HeLa cells. Furthermore, we showed that LQ inhibited serum-induced expression of HIF-1α by reducing its stability and decreased the synthesis in a dose-dependent manner. Mechanistically, we demonstrated that LQ inhibited HIF-1α and VEGF expression involved in blocking the protein kinase B (PKB/Akt) signalling pathway, and the mechanisms correlated with dephosphorylation of the mammalian target of rapamycin (mTOR) and its effector ribosomal protein S6 kinase (p70S6K). In addition, LQ inhibited VEGF-induced formation of capillary-like structures in human umbilical vein endothelial cells (HUVEC). Taken together, our study provided valuable insights into the mechanism of antiangiogenic effect of LQ.     

Mammea E/BB, an isoprenylated dihydroxycoumarin protonophore that potently uncouples mitochondrial electron transport, disrupts hypoxic signaling in tumor cells

The mammea-type coumarin mammea E/BB (1) was found to inhibit both hypoxia-induced and iron chelator-induced hypoxia-inducible factor-1 (HIF-1) activation in human breast tumor T47D cells with IC(50) values of 0.96 and 0.89 μM, respectively. Compound 1 suppressed the hypoxic induction of secreted VEGF protein (T47D cells) and inhibited cell viability/proliferation in four human tumor cell lines. Compound 1 (at 5 and 20 μM) inhibited human breast tumor MDA-MB-231 cell migration. While the mechanisms that underlie their biological activities have remained unknown, prenylated mammea coumarins have been shown to be cytotoxic to human tumor cells, suppress tumor growth in animal models, and display a wide variety of antimicrobial effects. Mechanistic studies revealed that 1 appears to exert an assemblage of cellular effects by functioning as an anionic protonophore that potently uncouples mitochondrial electron transport and disrupts mitochondrial signaling in human tumor cell lines.     

Spheciosterol sulfates, PKCzeta inhibitors from a philippine sponge Spheciospongia sp

Three new sterol sulfates, spheciosterol sulfates A-C (1-3), and the known sterol sulfate topsentiasterol sulfate E (4) have been isolated from the sponge Spheciospongia sp., collected in the Philippines. Structures were assigned on the basis of extensive 1D and 2D NMR studies as well as analysis by HRESIMS. Compounds 1-4 inhibited PKCzeta with IC50 values of 1.59, 0.53, 0.11, and 1.21 microM, respectively. In a cell-based assay, 1-4 also inhibited NF-kappaB activation with EC50 values of 12-64 microM.     

Molecular-targeted antitumor agents. 19. Furospongolide from a marine Lendenfeldia sp. sponge inhibits hypoxia-inducible factor-1 activation in breast tumor cells

A natural product chemistry-based approach was employed to discover small-molecule inhibitors of the important tumor-selective molecular target hypoxia-inducible factor-1 (HIF-1). Bioassay-guided isolation of an active lipid extract of a Saipan collection of the marine sponge Lendenfeldia sp. afforded the terpene-derived furanolipid furospongolide as the primary inhibitor of hypoxia-induced HIF-1 activation (IC(50) 2.9 μM, T47D breast tumor cells). The active component of the extract also contained one new cytotoxic scalarane sesterterpene and two previously reported scalaranes. Furospongolide blocked the induction of the downstream HIF-1 target secreted vascular endothelial growth factor (VEGF) and was shown to suppress HIF-1 activation by inhibiting the hypoxic induction of HIF-1α protein. Mechanistic studies indicate that furospongolide inhibits HIF-1 activity primarily by suppressing tumor cell respiration via the blockade of NADH-ubiquinone oxidoreductase (complex I)-mediated mitochondrial electron transfer.     

The protective effects of total saponins from Ornithogalum saundersiae (Liliaceae) on acute hepatic failure induced by lipopolysaccharide and d-galactosamine in mice

Ethnopharmacological relevance

This study examined the protective effects of total saponins from Ornithogalum saundersiae (Liliaceae) on d-galactosamine (d-GalN) and lipopolysaccharide (LPS) - induced fulminant hepatic failure.

Materials and methods

Total saponins of Ornithogalum saundersiae (Liliaceae) (OC) were prepared with ethyl alcohol extract from bulbs of the plant. Mice were given an intraperitoneal injection of d-GalN (700 mg/kg)/LPS (10 μg/kg). OC (100 mg/kg, 200 mg/kg and 300 mg/kg) was administered orally for 3 days continuously, and at the last day at 1 h before the d-GalN/LPS injection. Mice were sacrificed at 8 h after the d-GalN/LPS injection. The liver injury was assessed biochemically, investigating aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA), glutathione (GSH) activities, and the expressions of caspase-3 and hypoxia inducible factor-1α (HIF-1α) as well. Tumor necrosis factor (TNF-α) content was measured after d-GalN/LPS induced 1 h by ELISA assay. The survival rates after application of OC in 24 h also were observed.

Results

d-GalN/LPS increased the serum aminotransferase levels and lipid peroxidation, while decreased the reduced glutathione level. The pretreatment with OC attenuated these changes in a dose-dependent manner. Elevation of TNF-α level and activation of caspase-3, HIF-1α were observed in the d-GalN/LPS group, which was attenuated by OC. The survival rate of the OC groups was significantly higher than that of the d-GalN/LPS group.

Conclusions

Protection afforded by OC against d-GalN/LPS-induced fulminant hepatic failure is the result of reduced oxidative stress, inhibited expression of caspase-3, HIF-1α, and anti-apoptotic activity.     

马齿苋酰胺E干预肾癌的作用机制研究

目的探讨马齿苋酰胺E对人肾癌786-O细胞增殖、迁移和侵袭的作用及机制。方法体外培养786-O细胞,给予马齿苋酰胺E(10、20、40μmol/L)进行干预,采用CCK-8法检测细胞增殖情况;采用划痕实验检测细胞迁移能力;采用Transwell小室法检测细胞侵袭能力;采用AO/EB试剂盒检测细胞凋亡率。建立裸鼠移植瘤肾癌模型,给予马齿苋酰胺E(3、15mg/kg)进行干预,测定各组裸鼠肿瘤质量及体积,采用苏木素-伊红(HE)染色法观察肿瘤组织病理变化;采用Westernblotting法检测786-O细胞和荷瘤裸鼠瘤组织中细胞增殖标志物如Ki67、增殖细胞核抗原(proliferatingcellnuclear antigen,PCNA)及凋亡标志物如活化半胱氨酸蛋白酶-3(Cleaved Caspase-3)、Cleaved Caspase-9及侵袭相关蛋白如基质金属蛋白酶-2(matrixmetalloproteinase-2,MMP-2)、MMP-9及希佩尔-林道抑癌基因(VonHippel-Lindau,VHL)/缺氧诱导因子-1α(hypoxia inducible fact...     

Calmodulin inhibitors from Leucophyllum ambiguum

Activity-directed fractionation of a CH(2)Cl(2)-MeOH (1:1) extract of Leucophyllum ambiguum led to the isolation of two new lignans designated with the trivial names of 2'-methoxykobusin (1) and 2'-methoxy-4' '-hydroxydemethoxykobusin (2). In addition, the known compounds kobusin (3), 2',2' '-dimethoxysesamin (4), trans-cinnamic acid, apigenin, and apigetrin were obtained. The identification of the novel analogues 1 and 2 was accomplished by spectral methods. The structure of 1 was unequivocally confirmed by X-ray analysis. Compounds 1-4 interacted with bovine-brain calmodulin and inhibited the activation of the calmodulin-dependent enzyme cAMP phosphodiesterase.     

Preconditioning with the traditional Chinese medicine Huang-Lian-Jie-Du-Tang initiates HIF-1α-dependent neuroprotection against cerebral ischemia in rats

Ethnopharmacological relevance

Huang-Lian-Jie-Du-Tang (HLJDT) is a classical heat-clearing and detoxicating formula of traditional Chinese medicine that is widely used to treat stroke. The present study was designed to investigate the effects of HLJDT preconditioning on neurons under oxygen and glucose deprivation (OGD) and rats subjected to middle cerebral artery occlusion (MCAO).

Materials and methods

A stroke model of rats was obtained through MCAO. Following HLJDT preconditioning, the cerebral infarction volume, cerebral water content, and neurological deficient score were determined. Cerebral cortical neurons cultured in vitro were preconditioned with HLJDT and then subjected to OGD treatment. The release of lactate dehydrogenase (LDH) from neurons was detected. The levels of hypoxia-inducible factor-1α (HIF-1α) and PI3K/Akt signaling were analyzed by western blotting, and the levels of erythropoietin (EPO) and vascular endothelial growth factor (VEGF) in the supernatant of the neurons and the plasma of MCAO rats were measured through a radioimmunological assay. The apoptosis and proliferation of neurons were analyzed by immunohistochemistry.

Results

HLJDT preconditioning significantly reduced the cerebral infarction volume and cerebral water content and ameliorated the neurological deficient score of MCAO rats. In addition, HLJDT preconditioning protected neurons against OGD. Increased HIF-1α, EPO, and VEGF levels and the activation of PI3K/Akt signaling were observed as a result of HLJDT preconditioning. Furthermore, HLJDT preconditioning was found to inhibit ischemia-induced neuron apoptosis and to promote neuron proliferation under conditions of ischemia/reperfusion.

Conclusion

Both rats and neurons subjected to HLJDT preconditioning were able to resist ischemia/reperfusion or hypoxia injury through the inhibition of apoptosis and the enhancement of proliferation, and these effects were primarily dependent on the activation of the PI3K/Akt signaling pathway and HIF-1α.