Andrographolide down-regulates hypoxia-inducible factor-1α in human non-small cell lung cancer A549 cells

Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess multiple pharmacological activities. In our previous study, Andro had been shown to inhibit non-small cell lung cancer (NSCLC) A549 cell migration and invasion via down-regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Here we demonstrated that Andro inhibited the expression of hypoxia-inducible factor-1α (HIF-1α) in A549 cells. HIF-1α plays an important role in tumor growth, angiogenesis and lymph node metastasis of NSCLC. The Andro-induced decrease of cellular protein level of HIF-1α was correlated with a rapid ubiquitin-dependent degradation of HIF-1α, and was accompanied by increased expressions of hydroxyl-HIF-1α and prolyl hydroxylase (PHD2), and a later decrease of vascular endothelial growth factor (VEGF) upon the treatment of Andro. The Andro-inhibited VEGF expression appeared to be a consequence of HIF-1α inactivation, because its DNA binding activity was suppressed by Andro. Molecular data showed that all these effects of Andro might be mediated via TGFβ1/PHD2/HIF-1α pathway, as demonstrated by the transfection of TGFβ1 overexpression vector and PHD2 siRNA, and the usage of a pharmacological MG132 inhibitor. Furthermore, we elucidated the involvement of Andro in HIF-1α transduced VEGF expression in A549 cells and other NSCLC cell lines. In conclusion, these results highlighted the potential effects of Andro, which may be developed as a chemotherapeutic or an anti-angiogenesis agent for NSCLC in the future.     

DNA strand breaks and hypoxia response inhibition mediate the radiosensitisation effect of nitric oxide donors on prostate cancer under varying oxygen conditions

Prostate cancer cells can exist in a hypoxic microenvironment, causing radioresistance. Nitric oxide (NO) is a radiosensitiser of mammalian cells. NO-NSAIDs are a potential means of delivering NO to prostate cancer cells. This study aimed to determine the effect and mechanism of action of NO-sulindac and radiation, on prostate cancer cells and stroma, under normoxia (21% oxygen) and chronic hypoxia (0.2% oxygen). Using clonogenic assays, at a surviving fraction of 10% the sensitisation enhancement ratios of radiation plus NO-sulindac over radiation alone on PC-3 cells were 1.22 and 1.42 under normoxia and hypoxia, respectively. 3D culture of PC-3 cells revealed significantly reduced sphere diameter in irradiated spheres treated with NO-sulindac. Neither NO-sulindac nor sulindac radiosensitised prostate stromal cells under normoxia or hypoxia. HIF-1α protein levels were reduced by NO-sulindac exposure and radiation at 21 and 0.2% oxygen. Alkaline Comet assay analysis suggested an increased rate of single strand DNA breaks and slower repair of these lesions in PC-3 cells treated with NO-sulindac prior to irradiation. There was a higher level of γ-H2AX production and hence double strand DNA breaks following irradiation of NO-sulindac treated PC-3 cells. At all radiation doses and oxygen levels tested, treatment of 2D and 3D cultures of PC-3 cells with NO-sulindac prior to irradiation radiosensitised PC-3, with minimal effect on stromal cells. Hypoxia response inhibition and increased DNA double strand breaks are potential mechanisms of action. Neoadjuvent and concurrent use of NO-NSAIDs have the potential to improve radiotherapy treatment of prostate cancer under normoxia and hypoxia.     

Berberine inhibits HIF-1alpha expression via enhanced proteolysis

We have studied the antiangiogenic property of berberine. We showed that berberine could directly inhibit in vitro human umbilical vein endothelial cell (HUVEC) tube formation and migration. In addition, to determine whether berberine could influence the cross-talk between the gastric adenocarcinoma cell line SC-M1 and vascular endothelial cells, we performed modified confrontation culture experiments and showed that berberine (7.5 microM, 16 h) could inhibit the capacity of hypoxic SC-M1 cells to stimulate HUVEC migration. These results demonstrated berberine's antiangiogenic property and its clinical potential as an inhibitor of tumor angiogenesis. Parallel Western blot analyses revealed that berberine prevented hypoxic SC-M1 cultures from expressing vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1alpha, two key factors in mediating tumor angiogenesis. However, overexpression of HIF-1alpha in SC-M1 cells dramatically reversed the inhibitory effect of berberine on SC-M1-induced in vitro HUVEC migration. These data indicated that HIF-1alpha repression is a critical step in the inhibitory effect of berberine on tumor-induced angiogenesis. Northern blot analyses plus pulse-chase assays revealed that berberine did not down-regulate HIF-1alpha mRNA but destabilized HIF-1alpha protein. We found that berberine-induced HIF-1alpha degradation was blocked by a 26S proteasome inhibitor. Moreover, immunoprecipitation and Western blot analyses showed that berberine increased the lysine-acetylated HIF-1alpha in hypoxic SC-M1 cultures. These data indicated that a proteasomal proteolytic pathway and lysine acetylation were involved in berberine-triggered HIF-1alpha degradation. In conclusion, our data provided molecular evidence to support berberine as a potent antiangiogenic agent in cancer therapy.     

Tetramethylpyrazine inhibits hypoxia-induced pulmonary vascular leakage in rats via the ROS-HIF-VEGF pathway

Tetramethylpyrazine (TMP) is a reactive oxygen species (ROS) antagonist that has potent properties for the treatment of a variety of vascular diseases, such as ischemic stroke and pulmonary hypertension secondary to chronic obstructive pulmonary diseases. However, there are few data about the role of TMP in hypoxia-induced pulmonary vascular leakage. This study examined the effect of TMP on hypoxia-induced pulmonary vascular leakage and the underlying mechanisms. Rat pulmonary microvascular endothelial cells (RPMVECs) treated with TMP or not were subjected to hypoxic or normoxic conditions for 24 h, and the monolayer permeability, intracellular ROS, hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) proteins levels were determined. Additionally, rats administrated TMP were exposed to hypobaric hypoxia to evaluate the effect of TMP in vivo by measuring lung water content, pulmonary vascular leakage into the lungs and immunohistochemistry for HIF-1α and VEGF. Hypoxia was found to cause a significant increase in RPMVEC monolayer permeability and intracellular ROS, HIF-1α and VEGF protein levels. Treatment with TMP decreased the hypoxia-induced RPMVEC monolayer permeability and attenuated the elevation of ROS, HIF-1α and VEGF protein levels. TMP-treated animals showed less pulmonary vascular leakage and HIF-1α and VEGF expression compared with those exposed to hypoxia alone. These observations supported that TMP inhibited the increase in pulmonary vascular permeability induced by hypoxia. The underlying mechanisms may be related to the scavenging of intracellular ROS and the suppression of hypoxia-induced upregulation of HIF-1α and VEGF proteins.     

Inhibitory effect of YC-1 on the hypoxic induction of erythropoietin and vascular endothelial growth factor in Hep3B cells

YC-1 is a newly developed agent that inhibits platelet aggregation and vascular contraction. Although its effects are independent of nitric oxide (NO), it mimics some of the biological actions of NO. For example, it stimulates soluble guanylate cyclase (sGC) and increases intracellular cGMP concentration. Here, we tested the possibility that YC-1 inhibits hypoxia-inducible factor (HIF)-1-mediated hypoxic responses, as does NO. Hep3B cells were used during the course of this work to observe hypoxic induction of erythropoietin (EPO) and vascular endothelial growth factor (VEGF), and the effects of YC-1 were compared with those of a NO donor, sodium nitropurruside (SNP). In hypoxic cells, YC-1 blocked the induction of EPO and VEGF mRNAs, and inhibited the DNA-binding activity of HIF-1. It suppressed the hypoxic accumulation of HIF-1alpha, but not its mRNA level. It also reduced HIF-1alpha accumulation induced by cobalt and desferrioxamine. Treatment with antioxidants did not recover the HIF-1alpha suppressed by YC-1. We examined whether these effects of YC-1 are related to the sGC/cGMP signal transduction system. Two sGC inhibitors examined failed to block the effects of YC-1, and 8-bromo-cGMP did not mimic actions of YC-1. The effects of YC-1 on the hypoxic responses were comparable with those of SNP. These results suggest that YC-1 and SNP suppressed the hypoxic responses by post-translationally inhibiting HIF-1alpha accumulation. The YC-1 effect may be linked with the metal-related oxygen sensing pathway, and is not due to the stimulation of sGC. This observation implies that the inhibitory effects of YC-1 on hypoxic responses can be developed to suppress EPO-overproduction by tumor cells and tumor angiogenesis.     

Dauricine inhibits insulin-like growth factor-I-induced hypoxia inducible factor 1α protein accumulation and vascular endothelial growth factor expression in human breast cancer cells

Aim:

To investigate the effects of dauricine (Dau) on insulin-like growth factor-I (IGF-I)-induced hypoxia inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression in human breast cancer cells (MCF-7).

Methods:

Serum-starved MCF-7 cells were pretreated for 1 h with different concentrations of Dau, followed by incubation with IGF-I for 6 h. HIF-1α and VEGF protein expression levels were analyzed by Western blotting and ELISA, respectively. HIF-1α and VEGF mRNA levels were determined by real-time PCR. In vitro angiogenesis was observed via the human umbilical vein endothelial cell (HUVEC) tube formation assay. An in vitro invasion assay on HUVECs was performed.

Results:

Dau significantly inhibited IGF-I-induced HIF-1α protein expression but had no effect on HIF-1α mRNA expression. However, Dau remarkably suppressed VEGF expression at both protein and mRNA levels in response to IGF-I. Mechanistically, Dau suppressed IGF-I-induced HIF-1α and VEGF protein expression mainly by blocking the activation of PI-3K/AKT/mTOR signaling pathway. In addition, Dau reduced IGF-I-induced HIF-1α protein accumulation by inhibiting its synthesis as well as by promoting its degradation. Functionally, Dau inhibited angiogenesis in vitro. Moreover, Dau had a direct effect on IGF-I-induced invasion of HUVECs.

Conclusion:

Dau inhibits human breast cancer angiogenesis by suppressing HIF-1α protein accumulation and VEGF expression, which may provide a novel potential mechanism for the anticancer activities of Dau in human breast cancer.     

A novel fusicoccin derivative preferentially targets hypoxic tumor cells and inhibits tumor growth in xenografts

Malignant cells in solid tumors survive under prolonged hypoxia and can be a source of resistance to current cancer therapies. Tumor hypoxia is also associated with a more malignant phenotype and poor survival in cancer patients. Recent progress in our understanding of the biology of tumor cells under hypoxia has led to increased attention on targeting hypoxia for cancer therapy. We report here that a novel fusicoccin derivative (ISIR-042), but not its parent or related compounds such as fusicoccin A and cotylenin A, is more cytotoxic to hypoxic cells than to normoxic cells. The hypoxia-induced accumulation of hypoxia-inducible factor (HIF)-1α and the phosphorylation of Akt were effectively inhibited by treatment with ISIR-042, suggesting that the preferential cytotoxicity toward hypoxic cells is associated with a reduction of HIF-1α and Akt activation. ISIR-042 inhibited the growth of human pancreatic cancer MIAPaCa-2 cells while sparing normal endothelial cells, and significantly inhibited the growth of MIAPaCa-2 cells as xenografts without apparent adverse effects. Pancreatic cancer cells expressing CD24 and CD44 exhibited characteristics of stem cells. Treatment with gemcitabine increased this stem cell-enriched population, and this effect was significantly inhibited by ISIR-042, suggesting that ISIR- 042 preferentially inhibits stem/progenitors in pancreatic cancer cell lines compared with chemotherapeutic agents. These results suggest that ISIR-042 may be a potential therapeutic agent for hypoxic tumors such as pancreatic cancer.     

Involvement of hypoxia-inducible factor-1α in the oxidative stress induced by advanced glycation end products in murine Leydig cells

Hyperglycemia increases the formation of advanced glycation end products (AGEs), triggers oxidative impairments and influences inducible factor (HIF)-1α protein levels and transactivation function. Compromised HIF-1α in testis leads to male infertility. The aim of the study was to investigate the role of HIF-1α in oxidative stress induced by AGEs in murine Leydig TM3 cells. TM3 cells were treated with 50 μg/ml of AGEs, or HIF-1α siRNA or 500 μM of DMOG (dimethyloxalylglycine) respectively. The cells were also pretreated with HIF-1α siRNA or 500 μM of DMOG and then were treated with 50 μg/ml of AGEs. The formation of reactive oxygen species (ROS) and cell apoptosis was evaluated. The expression of caspase-3, Heme oxygenase (HO)-1, steroidogenic acute regulatory protein (StAR) and cytochrome P450 17α polypeptide 1 (CYP17A1) was examined by Western blotting. AGEs increased ROS production, induced apoptosis and activated HIF-1α and HO-1 in TM3 cells. HIF-1α attenuated the AGE-induced ROS formation and promoted apoptosis via the upregulation of caspase-3. Knockdown of HIF-1α inhibited the expression of CYP17A1 and StAR, and enhanced the inhibition of StAR and CYP17A1 by AGEs. These findings indicate that attenuated HIF-1α exacerbates the oxidative stress injury by AGEs in murine Leydig cells, and contributes to diabetic male infertility.     

Protective effects of paeoniflorin against cobalt chloride-induced apoptosis of endothelial cells via HIF-1α pathway

Accumulating evidence has suggested the importance of hypoxia in the initiation and development of atherosclerotic lesion, and hypoxia has a profound impact on endothelial cell properties during cardiovascular disease processes. Paeoniflorin, isolated from the root of Paeonia lactiflora pall, can protect endothelial cells from hypoxic damage in a variety of ways, such as by enhancing the production of nitric oxide (NO) and decreasing the expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). This study evaluated the protective effects of paeoniflorin against cobalt chloride (CoCl₂, a hypoxia-mimicking agent)-induced apoptosis of endothelial cells (CRL-1730) and the underlying mechanisms in vitro. Endothelial cells were exposed to CoCl₂ with or without pre-treatment with different concentrations of paeoniflorin. After treated with 0.6 mM CoCl₂ for 24 h, endothelial cells showed significant decrease in cell viability and increased apoptosis rate, which could be reversed by pre-treatment with paeoniflorin. Similarly, pre-treatment with paeoniflorin could prevent CoCl₂-induced hypoxia-induced factor-1α (HIF-1α) accumulation and down-regulate the expressions of p53 and Bcl-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3). These findings indicate that paeoniflorin had effective protection against hypoxia-induced apoptosis of endothelial cells and that HIF-1α, p53 and BNIP3 might be involved in this process.     

NADPH oxidase-mitochondria axis-derived ROS mediate arsenite-induced HIF-1α stabilization by inhibiting prolyl hydroxylases activity

Arsenic exposure has been shown to induce hypoxia inducible factor 1α (HIF-1α) accumulation, however the underlying mechanism remains unknown. In the present study, we tested the hypothesis that arsenic exposure triggered the interaction between NADPH oxidase and mitochondria to promote reactive oxygen species (ROS) production, which inactivate prolyl hydroxylases (PHDs) activity, leading to the stabilization of HIF-1α protein. Exposure of human immortalized liver cell line HL-7702 cells to arsenite induced HIF-1α accumulation in a dose-dependent manner, which was abolished by SOD mimetic MnTMPyP. Inhibition of NADPH oxidase with diphenyleneiodonium chloride (DPI) or inhibition of mitochondrial respiratory chain with rotenone significantly blocked arsenite-induced ROS production, and the mitochondria appeared to be the major source of ROS production. Arsenite treatment inhibited HIF-1α hydroxylation by prolyl hydroxylases (PHDs) and increased HIF-1α stabilization, but did not affect HIF-1α mRNA expression and Akt activation. Supplementation of ascorbate or Fe(II) completely abolished arsenite-induced PHDs inhibition and HIF-1α stabilization. In conclusion, these results define a unique mechanism of HIF-1α accumulation following arsenic exposure, that is, arsenic activates NADPH oxidase–mitochondria axis to produce ROS, which deplete intracellular ascorbate and Fe(II) to inactivate PHDs, leading to HIF-1α stabilization.     

A quassinoid 6alpha-tigloyloxychaparrinone inhibits hypoxia-inducible factor-1 pathway by inhibition of eukaryotic translation initiation factor 4E phosphorylation

Hypoxia-inducible factor-1 (HIF-1) is the central mediator of cellular responses to low oxygen and vital to many aspects of cancer biology. In a search for HIF-1 inhibitors, we identified a quassinoid 6alpha-tigloyloxychaparrinone (TCN) as an inhibitor of HIF-1 activation from Ailantus altissima. We here demonstrated the effect of TCN on HIF-1 activation induced by hypoxia or CoCl(2). TCN showed the potent inhibitory activity against HIF-1 activation induced by hypoxia in various human cancer cell lines. This compound markedly decreased the hypoxia-induced accumulation of HIF-1alpha protein dose-dependently, whereas it did not affect the expressions of HIF-1beta and topoisomerase-I. Furthermore, TCN prevented hypoxia-induced expression of HIF-1 target genes for vascular endothelial growth factor (VEGF) and erythropoietin. Further analysis revealed that TCN strongly inhibited HIF-1alpha protein synthesis, without affecting the expression level of HIF-1alpha mRNA or degradation of HIF-1alpha protein. Moreover, the levels of phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2), mitogen-activated protein (MAP) kinase-interacting protein kinase-1 (MNK1) and eukaryotic initiation factor 4E (eIF4E) were significantly suppressed by the treatment of TCN, without changing the total levels of these proteins. Our data suggested that TCN may exhibit anticancer activity by inhibiting HIF-1alpha translation through the inhibition of eIF4E phosphorylation pathway and thus provide a novel mechanism for the anticancer activity of quassinoids. TCN could be a new HIF-1-targeted anticancer agent and be effective on mammalian target of rapamycin (mTOR)-targeted cancer therapy, in which mTOR inhibition increases eIF4E phosphorylation.