Improved anticancer activity of betulinic acid on breast cancer through a grafted copolymer-based micelles system.

Betulinic acid (3ß-Hydroxy-20(29)-lupaene-28-oic acid, BA) has excellent anti-cancer activity but poor solubility and low bioavailability. To improve the antitumor activity of BA, a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol (PVCL-PVA-PEG) graft copolymer (Soluplus) encapsulated BA micelle (Soluplus-BA) was fabricated. The Soluplus-BA micelles presented a mean size of 54.77 ± 1.26 nm and a polydispersity index (PDI) of 0.083. The MTT assay results showed that Soluplus-BA micelles increased the inhibitory effect of BA on MDA-MB-231 cells, mainly due to the enhanced accumulation of reactive oxygen species (ROS) and the destruction of mitochondrial membrane potential (MMP). Soluplus-BA micelles induced the DNA double-strand breaks (DSBs) as the γH2AX foci increased. Moreover, Soluplus-BA also inhibited the tube formation and migration of human umbilical vein endothelial cells (HUVECs), and inhibited the neovascularization of the chicken chorioallantoic membrane (CAM). This angiogenesis inhibitory effect may be accomplished by regulating the HIF-1/VEGF-FAK signaling pathway. The in vivo study confirmed the improved anti-tumor effect of Soluplus-BA and its inhibitory effect on angiogenesis, demonstrating the possibility of Soluplus-BA as an effective anti-breast cancer drug delivery system.

Apatinib Inhibits Angiogenesis in Intrahepatic Cholangiocarcinoma by Regulating the Vascular Endothelial Growth Factor Receptor-2/Signal Transducer and Activator of Transcription Factor 3/Hypoxia Inducible Factor 1 Subunit Alpha Signaling Axis.

INTRODUCTION: Intrahepatic cholangiocarcinoma (ICC), which is difficult to diagnose and is usually fatal due to its late clinical presentation and a lack of effective treatment, has risen over the past decades but without much improvement in prognosis. OBJECTIVE: The study aimed to investigate the role of apatinib that targets vascular endothelial growth factor receptor-2 (VEGFR2) in ICC. METHODS: MTT assays, cell scratch assays, and tube formation assays were used to assess the effect of apatinib on human ICC cell line (HuCCT-1) and RBE cells proliferation, migration, and angiogenic capacity, respectively. Expression of vascular endothelial growth factor (VEGF), VEGFR2, signal transducer and activator of transcription factor 3 (STAT3), pSTAT3, and hypoxia inducible factor 1 subunit alpha (HIF-1α) pathway proteins was assessed using Western blotting and mRNA expression analysis in HuCCT-1 was performed using RT-qPCR assays. The pcDNA 3.1(-)-VEGFR2 and pcDNA 3.1(-)-HIF-1α were transfected into HuCCT-1 and RBE cells using Lipofectamine 2,000 to obtain overexpressed HuCCT-1 and RBE cells. RESULTS: We found that apatinib-inhibited proliferation, migration, and angiogenesis of HuCCT-1 and RBE cells in vitro in a dose-dependent manner. We also proved that apatinib effectively inhibits angiogenesis in tumor cells by blocking the expression of VEGF and VEGFR2 in these cells. In addition, we demonstrated that apatinib regulates the expression of STAT3 phosphorylation by inhibiting VEGFR2. Finally, we showed that apatinib regulates ICC angiogenesis and HIF-1α/VEGF expression via STAT3. CONCLUSIONS: Based on the above findings, we conclude that apatinib inhibits HuCCT-1 and RBE cell proliferation, migration, and tumor angiogenesis by inhibiting the VEGFR2/STAT3/HIF-1α axis signaling pathway. Apatinib can be a promising drug for ICC-targeted molecular therapy.

Sevoflurane Exerts Protective Effects in Murine Peritonitis-induced Sepsis via Hypoxia-inducible Factor 1α/Adenosine A2B Receptor Signaling.

BACKGROUND: Sepsis is one of the leading causes of mortality in intensive care units, and sedation in the intensive care unit during sepsis is usually performed intravenously. The inhalative anesthetic sevoflurane has been shown to elicit protective effects in various inflammatory studies, but its role in peritonitis-induced sepsis remains elusive. The hypothesis was that sevoflurane controls the neutrophil infiltration by stabilization of hypoxia-inducible factor 1α and elevated adenosine A2B receptor expression. METHODS: In mouse models of zymosan- and fecal-induced peritonitis, male mice were anesthetized with sevoflurane (2 volume percent, 30 min) after the onset of inflammation. Control animals received the solvent saline. The neutrophil counts and adhesion molecules on neutrophils in the peritoneal lavage of wild-type, adenosine A2B receptor -/-, and chimeric animals were determined by flow cytometry 4 h after stimulation. Cytokines and protein release were determined in the lavage. Further, the adenosine A2B receptor and its transcription factor hypoxia-inducible factor 1α were evaluated by real-time polymerase chain reaction and Western blot analysis 4 h after stimulation. RESULTS: Sevoflurane reduced the neutrophil counts in the peritoneal lavage (mean ± SD, 25 ± 17 × 105vs. 12 ± 7 × 105 neutrophils; P = 0.004; n = 19/17) by lower expression of various adhesion molecules on neutrophils of wild-type animals but not of adenosine A2B receptor -/- animals. The cytokines concentration (means ± SD, tumor necrosis factor α [pg/ml], 523 ± 227 vs. 281 ± 101; P = 0.002; n = 9/9) and protein extravasation (mean ± SD [mg/ml], 1.4 ± 0.3 vs. 0.8 ± 0.4; P = 0.002; n = 12/11) were also lower after sevoflurane only in the wild-type mice. Chimeric mice showed the required expression of the adenosine A2B receptor on the hematopoietic and nonhematopoietic compartments for the protective effects of the anesthetic. Sevoflurane induced the expression of hypoxia-inducible factor 1α and adenosine A2B receptor in the intestine, liver, and lung. CONCLUSIONS: Sevoflurane exerts various protective effects in two murine peritonitis-induced sepsis models. These protective effects were linked with a functional adenosine A2B receptor.

Perinatal Hypoxia-Inducible Factor Stabilization Preserves Lung Alveolar and Vascular Growth in Experimental Bronchopulmonary Dysplasia.

Rationale: Antenatal inflammation with placental dysfunction is strongly associated with high bronchopulmonary dysplasia (BPD) risk in preterm infants. Whether antenatal or postnatal HIF (hypoxia-inducible factor) augmentation can preserve lung structure and function and prevent pulmonary hypertension after intrauterine inflammation is controversial.Objectives: To determine whether antenatal or postnatal prolyl-hydroxylase inhibitor (PHi) therapy increases lung HIF expression, preserves lung growth and function, and prevents pulmonary hypertension in a rat model of chorioamnionitis-induced BPD caused by antenatal inflammation.Methods: Endotoxin (ETX) was administered to pregnant rats by intraamniotic injection at Embryonic Day 20, and pups were delivered by cesarean section at Embryonic Day 22. Selective PHi drugs, dimethyloxalylglycine or GSK360A, were administered into the amniotic space at Embryonic Day 20 or after birth by intraperitoneal injection for 2 weeks. Placentas and lung tissue were collected at birth for morphometric and Western blot measurements of HIF-1a, HIF-2a, VEGF (vascular endothelial growth factor), and eNOS (endothelial nitric oxide synthase) protein contents. At Day 14, lung function was assessed, and tissues were harvested to determine alveolarization by radial alveolar counts, pulmonary vessel density, and right ventricle hypertrophy (RVH).Measurements and Main Results: Antenatal PHi therapy preserves lung alveolar and vascular growth and lung function and prevents RVH after intrauterine ETX exposure. Antenatal administration of PHi markedly upregulates lung HIF-1a, HIF-2a, VEGF, and eNOS expression after ETX exposure.Conclusions: HIF augmentation improves lung structure and function, prevents RVH, and improves placental structure following antenatal ETX exposure. We speculate that antenatal or postnatal PHi therapy may provide novel strategies to prevent BPD due to antenatal inflammation.

Hypoxic Environment Promotes Barrier Formation in Human Intestinal Epithelial Cells through Regulation of MicroRNA 320a Expression.

Intestinal epithelial cells (IECs) are exposed to the low-oxygen environment present in the lumen of the gut. These hypoxic conditions on one hand are fundamental for the survival of the commensal microbiota and, on the other hand, favor the formation of a selective semipermeable barrier, allowing IECs to transport essential nutrients/water while keeping the sterile internal compartments separated from the lumen containing commensals. The hypoxia-inducible factor (HIF) complex, which allows cells to respond and adapt to fluctuations in oxygen levels, has been described as a key regulator in maintaining IEC barrier function by regulating their tight junction integrity. In this study, we sought to better evaluate the mechanisms by which low oxygen conditions impact the barrier function of human IECs. By profiling miRNA expression in IECs under hypoxia, we identified microRNA 320a (miRNA-320a) as a novel barrier formation regulator. Using pharmacological inhibitors and short hairpin RNA-mediated silencing, we could demonstrate that expression of this microRNA (miRNA) was HIF dependent. Importantly, using overexpression and knockdown approaches of miRNA-320a, we could confirm its direct role in the regulation of barrier function in human IECs. These results reveal an important link between miRNA expression and barrier integrity, providing a novel insight into mechanisms of hypoxia-driven epithelial homeostasis.

Preconditioning against renal ischaemia reperfusion injury: the failure to translate to the clinic.

Acute kidney injury (AKI) as a result of ischaemia-reperfusion represents a major healthcare burden worldwide. Mortality rates from AKI in hospitalized patients are extremely high and have changed little despite decades of research and medical advances. In 1986, Murry et al. demonstrated for the first time the phenomenon of ischaemic preconditioning to protect against ischaemia-reperfusion injury (IRI). This seminal finding paved the way for a broad body of research, which attempted to understand and ultimately harness this phenomenon for human application. The ability of preconditioning to limit renal IRI has now been demonstrated in multiple different animal models. However, more than 30 years later, a safe and consistent method of protecting human organs, including the kidneys, against IRI is still not available. This review highlights agents which, despite strong preclinical data, have recently failed to reduce AKI in human trials. The multiple reasons which may have contributed to the failure to translate some of the promising findings to clinical therapies are discussed. Agents which hold promise in the clinic because of their recent efficacy in preclinical large animal models are also reviewed.

Atorvastatin attenuates paraquat poisoning-induced epithelial-mesenchymal transition via downregulating hypoxia-inducible factor-1 alpha.

AIM: This study investigated the effects of atorvastatin (ATS) on the paraquat (PQ)-induced epithelial-mesenchymal transition (EMT) and the potential mechanism through hypoxia-inducible factor-1 alpha (HIF-1α). MAIN METHODS: Sprague-Dawley (SD) rats were randomly divided into a control group (n = 5), PQ group (n = 20), PQ + ATS L group (n = 20, ATS 20 mg/kg daily) and PQ + ATS H group (n = 20, ATS 40 mg/kg daily). All treated rats were given a 20% PQ solution (50 mg/kg) once by gavage and then sacrificed 12, 24, 72 and 168 h after PQ exposure. The A549 and RLE-6TN cell lines were treated with ATS, PQ or both for 24 h. Mesenchymal (α-SMA and vimentin) and epithelial (E-cadherin and ZO-1) cell marker expression was tested both in vivo and in vitro. The effects of ATS on HIF-1α and ß­catenin expression were also evaluated. KEY FINDINGS: ATS alleviated PQ poisoning-induced lung injury and pulmonary fibrosis in vivo. This effect was dose-dependent. ATS treatment attenuated the EMT by increasing the levels of the epithelial markers E-cadherin and ZO-1 and by decreasing the expression of the mesenchymal markers α-SMA and vimentin in both lung tissues and in vitro cell culture. In addition, ATS treatment may decrease the HIF-1α and ß­catenin levels both in vivo and in vitro. SIGNIFICANCE: In conclusion, ATS can attenuate PQ-induced pulmonary fibrosis. The mechanism may involve the downregulation of the HIF-1α/ß­catenin pathway and the inhibition of the PQ-induced EMT by ATS. ATS may be considered as a therapeutic agent for PQ poisoning-induced pulmonary fibrosis.

Hypoxia-independent downregulation of hypoxia-inducible factor 1 targets by androgen deprivation therapy in prostate cancer.

PURPOSE: We explored changes in hypoxia-inducible factor 1 (HIF1) signaling during androgen deprivation therapy (ADT) of androgen-sensitive prostate cancer xenografts under conditions in which no significant change in immunostaining of the hypoxia marker pimonidazole had occurred. METHODS AND MATERIALS: Gene expression profiles of volume-matched androgen-exposed and androgen-deprived CWR22 xenografts, with similar pimonidazole-positive fractions, were compared. Direct targets of androgen receptor (AR) and HIF1 transcription factors were identified among the differentially expressed genes by using published lists. Biological processes affected by ADT were determined by gene ontology analysis. HIF1α protein expression in xenografts and biopsy samples from 35 patients receiving neoadjuvant ADT was assessed by immunohistochemistry. RESULTS: A total of 1344 genes showed more than 2-fold change in expression by ADT, including 35 downregulated and 5 upregulated HIF1 targets. Six genes were shared HIF1 and AR targets, and their downregulation was confirmed with quantitative RT-PCR. Significant suppression of the biological processes proliferation, metabolism, and stress response in androgen-deprived xenografts was found, consistent with tumor regression. Nineteen downregulated HIF1 targets were involved in those significant biological processes, most of them in metabolism. Four of these were shared AR and HIF1 targets, including genes encoding the regulatory glycolytic proteins HK2, PFKFB3, and SLC2A1. Most of the downregulated HIF1 targets were induced by hypoxia in androgen-responsive prostate cancer cell lines, confirming their role as hypoxia-responsive HIF1 targets in prostate cancer. Downregulation of HIF1 targets was consistent with the absence of HIF1α protein in xenografts and downregulation in patients by ADT (P<.001). CONCLUSIONS: AR repression by ADT may lead to downregulation of HIF1 signaling independently of hypoxic fraction, and this may contribute to tumor regression. HIF1α expression is probably not a useful hypoxia biomarker during ADT in prostate cancer.

Inducers of hypoxic response: marine sesquiterpene quinones activate HIF-1.

The hypoxia-inducible factor-1 (HIF-1) transcription factor regulates cellular oxygen homeostasis. Agents that activate HIF-1 and downstream HIF targets represent potential drug leads for the prevention and/or treatment of ischemic disorders. In a search for small-molecule HIF-1 activators, 1936 marine invertebrate and algal extract samples (U.S. National Cancer Institute's Open Repository) were evaluated for HIF-1 activation activity in a cell-based reporter assay. Bioassay-guided fractionation of two active extracts of the sponge Dactylospongia elegans afforded four new sesquiterpene quinones (2-5), one new sesquiterpene phenol (6), the known Golgi disruptor ilimaquinone (1), and three previously reported ilimaquinone analogues (7-9). While antiproliferative activity was observed at higher concentrations, the sesquiterpene quinones (1-3) possessing a 2-hydroxy-5-methoxy-1,4-benzoquinone moiety activated HIF-1 and increased the expression of HIF-1 target gene vascular endothelial growth factor (VEGF) in T47D cells.

Quercetin enhances hypoxia-mediated apoptosis via direct inhibition of AMPK activity in HCT116 colon cancer.

Tumor hypoxia is considered the best validated target in clinical oncology because of its significant contribution to chemotherapy failure and drug resistance. As an approach to target hypoxia, we assessed the potential of quercetin, a flavonoid widely distributed in plants, as a anticancer agent under hypoxic conditions and examined its pharmacological mechanisms by primarily focusing on the role of AMP-activated protein kinase (AMPK). Quercetin significantly attenuated tumor growth in an HCT116 cancer xenograft in vivo model with a substantial reduction of AMPK activity. In a cell culture system, quercetin more dramatically induced apoptosis of HCT116 cancer cells under hypoxic conditions than normoxic conditions, and this was tightly associated with inhibition of hypoxia-induced AMPK activity. An in vitro kinase assay demonstrated that quercetin directly inhibits AMPK activity. Inhibition of AMPK by expressing a dominant-negative form resulted in an increase of apoptosis under hypoxia, and a constitutively active form of AMPK effectively blocked quercetin-induced apoptosis under hypoxia. Collectively, our data suggest that quercetin directly inhibits hypoxia-induced AMPK, which plays a protective role against hypoxia. Quercetin also reduced the activity of hypoxia-inducible factor-1 (HIF-1), a major transcription factor for adaptive cellular response to hypoxia. Moreover, quercetin sensitized HCT116 cancer cells to the anticancer drugs cisplatin and etoposide under hypoxic conditions. Our findings suggest that AMPK may serve as a novel target for overcoming tumor hypoxia-associated negative aspects.

Antiangiogenic activity of berberine is mediated through the downregulation of hypoxia-inducible factor-1, VEGF, and proinflammatory mediators.

Berberine, a naturally occurring isoquinoline alkaloid, is present in a number of important medicinal plants. Berberine has a wide range of biochemical and pharmacological effects, including anticancer effects. In this study, we elucidated the mechanism of antiangiogenic activity of berberine using in vivo and in vitro models. In vivo antiangiogenic activity was studied using B16F-10 melanoma cells and induced capillary formation in C57BL/6 mice. Berberine, at 10 mg/kg body weight, showed significant inhibition in tumor-directed capillary formation and in various proangiogenic factors, such as vascular endothelial growth factor (VEGF), and proinflammatory mediators, such as interleukin (IL)-1ß, IL-6, tumor necrosis factor alpha (TNF-α), and granulocyte macrophage colony-stimulating factor (GM-CSF), which are involved in tumor angiogenesis. At the same time, it could also increase antitumor factors, such as IL-2 and tissue-inhibitor metalloproteinase (TIMP) levels in the serum. Berberine could also inhibit endothelial motility, migration, tube formation, and vessel sprouting from rat aortic ring in vitro. Further, berberine inhibited various transcription factors involved in tumor development and angiogenesis, such as NF-ĸB, c-Fos, CREB, and ATF-2. mRNA expression levels of proangiogenic factors, such as cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and hypoxia-inducible factor (HIF), were also downregulated in tumor cells after treatment with berberine. Drastically elevated expressions of HIF and VEGF mRNA by tumor cells under hypoxic conditions were also decreased after treatment with berberine. This result clearly demonstrates that the antiangiogenic activity of berberine is mainly mediated through the inhibition of various proinflammatory and pro-angiogenic factors and the major ones are HIF, VEGF, COX-2, NO, NF-ĸB, and proinflammatory cytokines.

Impaired wound healing in hypoxic renal tubular cells: roles of hypoxia-inducible factor-1 and glycogen synthase kinase 3ß/ß-catenin signaling.

Wound and subsequent healing are frequently associated with hypoxia. Although hypoxia induces angiogenesis for tissue remodeling during wound healing, it may also affect the healing response of parenchymal cells. Whether and how wound healing is affected by hypoxia in kidney cells and tissues is currently unknown. Here, we used scratch-wound healing and transwell migration models to examine the effect of hypoxia in cultured renal proximal tubular cells (RPTC). Wound healing and migration were significantly slower in hypoxic (1% oxygen) RPTC than normoxic (21% oxygen) cells. Hypoxia-inducible factor-1α (HIF-1α) was induced during scratch-wound healing in normoxia, and the induction was more evident in hypoxia. Nevertheless, HIF-1α-null and wild-type cells healed similarly after scratch wounding. Moreover, activation of HIF-1α with dimethyloxalylglycine in normoxic cells did not suppress wound healing, negating a major role of HIF-1α in wound healing in this model. Scratch-wound healing was also associated with glycogen synthase kinase 3ß (GSK3ß)/ß-catenin signaling, which was further enhanced by hypoxia. Pharmacological inhibition of GSK3ß resulted in ß-catenin expression, accompanied by the suppression of wound healing and transwell cell migration. Ectopic expression of ß-catenin in normoxic cells could also suppress wound healing, mimicking the effect of hypoxia. Conversely, inhibition of ß-catenin via dominant negative mutants or short hairpin RNA improved wound healing and transwell migration in hypoxic cells. The results suggest that GSK3ß/ß-catenin signaling may contribute to defective wound healing in hypoxic renal cells and tissues.

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.

Oxidation of thiols and modification of redox-sensitive signaling in human lung epithelial cells exposed to Pseudomonas pyocyanin.

The aim of this study was to examine the effects of pyocyanin exposure on mitochondrial GSH, other cellular thiols (thioredoxin-1, Trx-1), and oxidant-sensitive signaling pathways hypoxia inducible factor (HIF-1α) and heme oxygenase (HO-1) in A549 and HBE cell lines. A549 human type II alveolar epithelial cells and human bronchial epithelial (HBE) cells were treated with varying concentrations of pyocyanin extracted from Pseudomonas aeruginosa bacteria. Cytoplasmic and mitochondrial thiols and oxidant sensitive signal transduction proteins (HIF-1α and HO-1) were measured. Exposure to pyocyanin generated reactive oxygen species (ROS) in cellular mitochondria and altered total cellular glutathione (GSH). Pyocyanin, at concentrations present in conditions in vivo, increased oxidized Trx-1 in A549 human type II alveolar epithelial cells and HBE cells by 184 and 74%, respectively. Oxidized mitochondrial glutathione (GSSG) was elevated more than twofold in both cell types. Pyocyanin also increased the cellular oxidant-sensitive proteins HIF-1α and HO-1. Data indicate that pyocyanin-induced alterations in mitochondrial and cytosolic thiols, as well as oxidant-sensitive proteins, may contribute to P. aeruginosa-mediated lung injury.

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.

The alternative medicine pawpaw and its acetogenin constituents suppress tumor angiogenesis via the HIF-1/VEGF pathway.

Products that contain twig extracts of pawpaw (Asimina triloba) are widely consumed anticancer alternative medicines. Pawpaw crude extract (CE) and purified acetogenins inhibited hypoxia-inducible factor-1 (HIF-1)-mediated hypoxic signaling pathways in tumor cells. In T47D cells, pawpaw CE and the acetogenins 10-hydroxyglaucanetin (1), annonacin (2), and annonacin A (3) inhibited hypoxia-induced HIF-1 activation with IC(50) values of 0.02 microg/mL, 12 nM, 13 nM, and 31 nM, respectively. This inhibition correlates with the suppression of the hypoxic induction of HIF-1 target genes VEGF and GLUT-1. The induction of secreted VEGF protein represents a key event in hypoxia-induced tumor angiogenesis. Both the extract and the purified acetogenins blocked the angiogenesis-stimulating activity of hypoxic T47D cells in vitro. Pawpaw extract and acetogenins inhibited HIF-1 activation by blocking the hypoxic induction of nuclear HIF-1alpha protein. The inhibition of HIF-1 activation was associated with the suppression of mitochondrial respiration at complex I. Thus, the inhibition of HIF-1 activation and hypoxic tumor angiogenesis constitutes a novel mechanism of action for these anticancer alternative medicines.

The Caulerpa pigment caulerpin inhibits HIF-1 activation and mitochondrial respiration.

The transcription factor hypoxia-inducible factor-1 (HIF-1) represents an important molecular target for anticancer drug discovery. In a T47D cell-based reporter assay, the Caulerpa spp. algal pigment caulerpin (1) inhibited hypoxia-induced as well as 1,10-phenanthroline-induced HIF-1 activation. The angiogenic factor vascular endothelial growth factor (VEGF) is regulated by HIF-1. Caulerpin (10 microM) suppressed hypoxic induction of secreted VEGF protein and the ability of hypoxic T47D cell-conditioned media to promote tumor angiogenesis in vitro. Under hypoxic conditions, 1 (10 microM) blocked the induction of HIF-1alpha protein, the oxygen-regulated subunit that controls HIF-1 activity. Reactive oxygen species produced by mitochondrial complex III are believed to act as a signal of cellular hypoxia that leads to HIF-1alpha protein induction and activation. Further mechanistic studies revealed that 1 inhibits mitochondrial respiration at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Under hypoxic conditions, it is proposed that 1 may disrupt mitochondrial ROS-regulated HIF-1 activation and HIF-1 downstream target gene expression by inhibiting the transport or delivery of electrons to complex III.

Inhibition of oxygen sensors as a therapeutic strategy for ischaemic and inflammatory disease.

Cells in the human body need oxygen to function and survive, and severe deprivation of oxygen, as occurs in ischaemic heart disease and stroke, is a major cause of mortality. Nevertheless, other organisms, such as the fossorial mole rat or diving seals, have acquired the ability to survive in conditions of limited oxygen supply. Hypoxia tolerance also allows the heart to survive chronic oxygen shortage, and ischaemic preconditioning protects tissues against lethal hypoxia. The recent discovery of a new family of oxygen sensors--including prolyl hydroxylase domain-containing proteins 1-3 (PHD1-3)--has yielded exciting novel insights into how cells sense oxygen and keep oxygen supply and consumption in balance. Advances in understanding of the role of these oxygen sensors in hypoxia tolerance, ischaemic preconditioning and inflammation are creating new opportunities for pharmacological interventions for ischaemic and inflammatory diseases.