HIF-1α acts downstream of TNF-α to inhibit vasodilator-stimulated phosphoprotein expression and modulates the adhesion and proliferation of breast cancer cells

Metastasis is the leading cause of death in breast cancer patients. Recent evidence suggests that inflammation-related cytokine tumor necrosis factor-alpha (TNF-α) is implicated in tumor invasion and metastasis, but the mechanism of its involvement remains elusive. In this study, we employed MCF-7 breast cancer cells as an experimental model to demonstrate that TNF-α inhibits breast cancer cell adhesion and cell proliferation through hypoxia inducible factor-1alpha (HIF-1α) mediated suppression of vasodilator-stimulated phosphoprotein (VASP). We observed that TNF-α treatment attenuated the adhesion and proliferation of MCF-7 cells it also dramatically increased HIF-1α expression and decreased VASP expression. Through a variety of approaches, including promoter assay, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP), we identified VASP as a direct target gene of HIF-1α. In addition, we confirmed that HIF-1α mediated the repression of VASP expression by TNF-α in MCF-7 cells. We also demonstrated that exogenous VASP expression or knockdown of HIF-1α relieved TNF-α induced inhibition of cell adhesion and proliferation. We identified a novel TNF-α/HIF-1α/VASP axis in which HIF-1α acts downstream of TNF-α to inhibit VASP expression and modulate the adhesion and proliferation of breast cancer cells. These data provide new insight into the potential anti-tumor effects of TNF-α.     

A novel regulation of VEGF expression by HIF-1α and STAT3 in HDM2 transfected prostate cancer cells

On the basis of increasing roles for HDM2 oncoprotein in cancer growth and progression, we speculated that HDM2 might play a major role in hypoxia-induced metastatic process. For verification of this hypothesis, wild-type LNCaP prostate cancer cells and HDM2 transfected LNCaP-MST (HDM2 stably transfected) cells were studied. The data obtained from our experiments revealed that the HDM2 transfected LNCaP-MST cells possessed an ability to multiply rapidly and show distinct morphological features compared to non-transfected LNCaP cells. During exposures to hypoxia HDM2 expression in the LNCaP and LNCaP-MST cells was significantly higher compared to the normoxic levels. The LNCaP-MST cells also expressed higher levels of HIF-1α (hypoxia-inducible factor-1α) and p-STAT3 even under the normoxic conditions compared to the non-transfected cells. The HIF-1α and p-STAT3 expressions were increased several fold when the cells were subjected to hypoxic conditions. The HIF-1α and p-STAT3 protein expressions observed in HDM2 transfected LNCaP-MST cells were 20 and 15 folds higher, respectively, compared to the non-transfected wild-type LNCaP cells. These results demonstrate that HDM2 may have an important regulatory role in mediating the HIF-1α and p-STAT3 protein expression during both normoxic and hypoxic conditions. Furthermore, the vascular endothelial growth factor (VEGF) expression that is typically regulated by HIF-1α and p-STAT3 was also increased significantly by 136% (P < 0.01) after HDM2 transfection. The overall results point towards a novel ability of HDM2 in regulating HIF-1α and p-STAT3 levels even in normoxic conditions that eventually lead to an up-regulation of VEGF expression.     

The increased expression of DEC1 gene is related to HIF-1α protein in gastric cancer cell lines

Overexpression of differentiated embryo chondrocyte 1 (DEC1) has been reported to contribute to the cellular differentiation, proliferation, and apoptosis of various cancers. Our previous studies have shown that DEC1 was highly expressed in gastric cancer (GCa) tissues. However, there is no report about the expression of DEC1 in GCa cell lines until now. In this study, We evaluated the mRNA and protein expression of DEC1 and hypoxia-inducible factor 1α (HIF-1α) under normoxic and hypoxic conditions in six GCa cell lines: BGC-823, MGC80-3, MKN1, AGS, FU97 and SGC-7901. An HIF-1α protein inhibitor was used to analyze the association of DEC1 and HIF-1α expression. Under normoxia, the mRNA expression of both HIF-1α and DEC1 was moderate, whereas the protein expression of DEC1 was higher than that of HIF-1α. Hypoxia induced the mRNA expression of DEC1 and the protein expression of HIF-1α and DEC1 in a time-dependent manner but had no effect on the mRNA expression of HIF-1α. Furthermore, inhibition of HIF-1α protein expression resulted in a significant decrease in both the mRNA and protein expression of DEC1. Taken together, DEC1 expression is correlated with HIF-1α protein in GCa cell line, blockage of HIF-1α protein led to reduced DEC1 expression. The efficacy of inhibiting HIF-1α and DEC1 expression should be tested in clinical trials as possible treatment for GCa.     

BMP2 sensitizes glioblastoma stem-like cells to Temozolomide by affecting HIF-1�� stability and MGMT expression

Glioblastoma multiforme (GBM) is the most common brain tumour, characterized by a central and partially necrotic (i.e., hypoxic) core enriched in cancer stem cells (CSCs). We previously showed that the most hypoxic and immature (i.e., CSCs) GBM cells were resistant to Temozolomide (TMZ) in vitro, owing to a particularly high expression of O6-methylguanine-DNA-methyltransferase (MGMT), the most important factor associated to therapy resistance in GBM. Bone morphogenetic proteins (BMPs), and in particular BMP2, are known to promote differentiation and growth inhibition in GBM cells. For this reason, we investigated whether a BMP2-based treatment would increase TMZ response in hypoxic drug-resistant GBM-derived cells. Here we show that BMP2 induced strong differentiation of GBM stem-like cells and subsequent addition of TMZ caused dramatic increase of apoptosis. Importantly, we correlated these effects to a BMP2-induced downregulation of both hypoxia-inducible factor-1α (HIF-1α) and MGMT. We report here a novel mechanism involving the HIF-1α-dependent regulation of MGMT, highlighting the existence of a HIF-1α/MGMT axis supporting GBM resistance to therapy. As confirmed from this evidence, over-stabilization of HIF-1α in TMZ-sensitive GBM cells abolished their responsiveness to it. In conclusion, we describe a HIF-1α-dependent regulation of MGMT and suggest that BMP2, by down-modulating the HIF-1α/MGMT axis, should increase GBM responsiveness to chemotherapy, thus opening the way to the development of future strategies for GBM treatment.     

Hypoxic preconditioning advances CXCR4 and CXCR7 expression by activating HIF-1α in MSCs

Recent evidence indicated that sublethal hypoxic preconditioning (HP) of bone marrow-derived mesenchymal stem cells (MSCs) before transplantation could ameliorate their capacity to survive and engraft in the target tissue through yet undefined mechanisms. In this study, we demonstrated that HP (3% oxygen) induced the high expression of both chemokine stromal-derived factor-1 (SDF-1) receptors, CXCR4 and CXCR7, in MSCs. HP also improved in vitro migration, adhesion and survival of MSCs. Although SDF-1-induced migration of HP-MSCs was only abolished by an anti-CXCR4 antibody, both CXCR4 and CXCR7 were responsible for elevated adhesion of HP-MSCs. Moreover, CXCR7 but not CXCR4 was essential for the resistance to oxidative stress of HP-MSC. In addition, HP also evoked an increase in expression of hypoxia-inducible factor-1 (HIF-1α) and phosphorylation of Akt. The chemical inducers of HIF-1α, desferrioxamine (DFX) and cobalt chloride (CoCl₂), induced upregulation of CXCR4 and CXCR7 expression in MSCs under normoxic conditions. Contrarily, blockade of HIF-1α by siRNA and inhibition of Akt by either wortmannin or LY294002 abrogated upregulation of HP-induced CXCR4 and CXCR7 in MSCs. Collectively, these findings provide evidence for a crucial role of PI3K/Akt-HIF-1α-CXCR4/CXCR7 pathway on enhanced migration, adhesion and survival of HP-MSCs in vitro.     

Concerted inhibition of HIF-1α and -2α expression markedly suppresses angiogenesis in cultured RPE cells

HIF-1α is known to play an important role in the induction of VEGF by hypoxia in retinal pigment epithelial (RPE) cells. However, the involvement of the other isoform, HIF-2α, in RPE cells remains unclear. Thus, the purpose of present study was to clarify the role of HIF-2α during induction of angiogenic genes in hypoxic RPE cells. When human RPE cells (ARPE-19) were cultured under hypoxic conditions, HIF-1α and HIF-2α proteins increased. This induced an increase in mRNA for VEGF, causing secretion of VEGF protein into the medium. This conditioned medium induced tube formation in human vascular endothelial cells (HUVEC). The increased expression of mRNA for VEGF in hypoxic RPE cells was partially inhibited by HIF-1α siRNA, but not by HIF-2α siRNA. However, co-transfection of HIF-1α siRNA and HIF-2α siRNA augmented downregulation of VEGF mRNA and protein in hypoxic RPE cells and inhibited formation of tube-like structures in HUVEC. GeneChip and PCR array analyses revealed that not only VEGF, but also expression of other angiogenic genes were synergistically downregulated by co-transfection of hypoxic RPE cells with HIF-1α and HIF-2α siRNAs. These findings suggest an important compensatory role for the HIF-2α isoform in the regulation of angiogenic gene expression. Thus, suppression of angiogenic genes for HIF-1α and HIF-2α may be a possible therapeutic strategy against retinal angiogenesis in Age-related macular degeneration (ARMD).     

VPA inhibits renal cancer cell migration by targeting HDAC2 and down-regulating HIF-1α

Cell migration plays major roles in human renal cancer-related death, but the molecular mechanisms remain unclear. Valproic acid (VPA) is a broad-spectrum inhibitor of class I and II histone deacetylases and shows great anticancer activity in a variety of human cancers. In this study, we found that VPA significantly inhibited cell migration but not proliferation of human renal cancer ACHN cells. Mechanistic studies found that VPA significantly inhibited the expression of HIF-1α. Knockdown of HIF-1α could obviously inhibited cell migration, while over-expression of HIF-1α markedly rescued the inhibition of VPA on cell migration. Further studies found that knockdown of HDAC2 completely mimicked the effects of VPA on HIF-1α and cell migration, and over-expression of HIF-1α could also rescue the effects of HDAC2 knockdown on cell migration. Collectively, these results indicated that the potential of specific inhibition of HDAC2 by small molecular chemicals may lead to future therapeutic agents in human renal cancer treatment.     

MicroRNA-382 induced by HIF-1α is an angiogenic miR targeting the tumor suppressor phosphatase and tensin homolog

Recent studies have revealed that microRNAs (miRs) play important roles in the regulation of angiogenesis. In this study, we have characterized miR-382 upregulation by hypoxia and the functional relevance of miR-382 in tumor angiogenesis. miRs induced by hypoxia in MKN1 human gastric cancer cells were investigated using miRNA microarrays. We selected miR-382 and found that the expression of miR-382 was regulated by HIF-1α. Conditioned media (CM) from MKN1 cells transfected with a miR-382 inhibitor (antagomiR-382) under hypoxic conditions significantly decreased vascular endothelial cell (EC) proliferation, migration and tube formation. Algorithmic programs (Target Scan, miRanda and cbio) predicted that phosphatase and tensin homolog (PTEN) is a target gene of miR-382. Deletion of miR382-binding sequences in the PTEN mRNA 3′-untranslated region (UTR) diminished the luciferase reporter activity. Subsequent study showed that the overexpression of miR-382 or antagomiR-382 down- or upregulated PTEN and its downstream target AKT/mTOR signaling pathway, indicating that PTEN is a functional target gene of miR-382. In addition, PTEN inhibited miR-382-induced in vitro and in vivo angiogenesis as well as VEGF secretion, and the inhibition of miR-382 expression reduced xenograft tumor growth and microvessel density in tumors. Taken together, these results suggest that miR-382 induced by hypoxia promotes angiogenesis and acts as an angiogenic oncogene by repressing PTEN.     

Quercetin metabolites inhibit MMP-2 expression in A549 lung cancer cells by PPAR-γ associated mechanisms

Our previous study demonstrated that quercetin-metabolite-enriched plasma (QP) but not quercetin itself upregulates peroxisome proliferator-activated receptor gamma (PPAR-γ) expression to induce G₂/M arrest in A549 cells. In the present study, we incubated A549 cells with QP as well as quercetin-3-glucuronide (Q3G) and quercetin-3′-sulfate (Q3′S), two major metabolites of quercetin, to investigate the effects of quercetin metabolites on cell invasion and migration, the possible mechanisms and the role of PPAR-γ. We also compared the effects of QP with those of quercetin and troglitazone (TGZ), a PPAR-γ ligand. The results showed that QP significantly suppressed cell invasion and migration, as well as matrix metalloproteinases (MMPs)-2 activity and expression in a dose-dependent manner. The effects of 10% QP on those parameters were similar to those of 10 μM quercetin and 20 μM TGZ. However, QP and TGZ rather than quercetin itself increased the expressions of nm23-H1 and tissue inhibitor of metalloproteinase (TIMP-2). Furthermore, we demonstrated that Q3G and Q3′S also inhibited the protein expression of MMP-2. GW9662, a PPAR-γ antagonist, significantly diminished such an effect of Q3G and Q3′S. Silencing PPAR-γ expression in A549 cells also significantly diminished the suppression effect of Q3G and Q3′S on MMP-2 expression. Taken together, our study demonstrated that QP inhibited cell invasion and migration through nm23-H1/TIMP-2/MMP-2 associated mechanisms. The upregulation of PPAR-γ by quercetin metabolites such as Q3G and Q3′S could play an important role in the effects of QP.     

Panaxynol protects cortical neurons from ischemia-like injury by up-regulation of HIF-1α expression and inhibition of apoptotic cascade

Apoptosis is one of the major characteristics of delayed neuronal degeneration in neuronal injury following cerebral ischemia. Hypoxia-induced apoptosis may be co-regulated by HIF-1α as well as many other factors. In recent years, numerous studies concerning panaxynol (PNN) have been reported. However, whether PNN can show anti-hypoxia properties is still unknown. In this study, the protective effects of PNN on OGD-induced neuronal apoptosis and potential mechanisms were investigated. Pretreatment of the cells with PNN for 24 h following exposure to OGD resulted in a significant elevation of cell survival determined by MTT assay, LDH assay, Hoechst staining and flow cytometric assessment. In addition to enhancing the expression of HIF-1α, PNN also normalized the caspase-3 expression/activation and increased the Bcl-2/Bax ratio. In our study, the increased level of HIF-1α with decreased cellular apoptosis suggested an important role for HIF-1α in hypoxic neurons. These results indicated that the neuroprotective effects of PNN on hypoxic neurons were at least partly due to up-regulation of HIF-1α and raised the possibility that PNN might reduce neurodegenerative disorders and ischemic brain diseases.     

The expression of HIF-1α in primary hepatocellular carcinoma and its correlation with radiotherapy response and clinical outcome

The purpose of this study was to evaluate the relationship between hypoxia-inducible factor-1α (HIF-1α) protein expression in hepatocellular carcinoma (HCC), and responses of abdominal metastatic lymph nodes (LNs) from HCC patients treated with external beam radiotherapy (EBRT). HIF-1α immunohistochemical staining was performed on tissue microarrays (TMAs) of primary HCC specimens from 69 HCC patients with abdominal LN metastases. All patients received abdominal metastatic LN EBRT at the Department of Radiation Oncology at Zhongshan Hospital. A receiver-operating characteristic (ROC)-based approach and logistical regression analysis were used to determine the predictive value of HIF-1α expression in primary tumors with HCC metastatic LN EBRT response. Kaplan–Meier curves and log-rank tests were used to analyze patient survival. Cox proportional hazards regression model was used to analyze independent prognostic factors. HIF-1α expression was correlated with blood hemoglobin (Hb: r = −0.280, P = 0.020), response of abdominal metastatic LNs to EBRT (r = 0.286, P = 0.017), locoregional recurrence (r = 0.278, P = 0.021), and cancer-specific deaths (r = 0.298, P = 0.013). HIF-1α expression was predictive of EBRT response of metastatic LNs [area under the curve (AUC): 0.646; 95% confidence interval (CI): 0.499–0.793; P = 0.047], locoregional recurrence (AUC: 0.657; 95% CI: 0.509–0.805; P = 0.049) and cancer-specific deaths (AUC: 0.671; 95% CI: 0.531–0.812; P = 0.035). Patients with tumors exhibiting high HIF-1α expression had significantly poorer overall survival (OS) than those with low tumor expression of HIF-1α (P = 0.016). Multivariate analysis showed that Hb (P = 0.035), vascular invasion (P = 0.026), Child-Pugh score (P < 0.001), intrahepatic tumor control (P < 0.001), and HIF-1α (P = 0.020) were independent prognosis factors for OS of HCC patients after receiving abdominal metastatic LN EBRT. HIF-1α expression in primary HCCs was associated with EBRT response of abdominal metastatic LNs and poor prognosis.     

miR-155 inhibits the formation of hypertrophic scar fibroblasts by targeting HIF-1α via PI3K/AKT pathway

Hypertrophic scar (HS) is a serious skin fibrotic disease characterized by the excessive proliferation of fibroblasts and often considered as a kind of benign skin tumor. microRNA-155 (miR-155) is usually served as a promising marker in antitumor therapy. In view of the similarities of hypertrophic scar and tumor, it is predicted that miR-155 may be a novel therapeutic target in clinical trials. Here we found the expression levels of miR-155 was gradually down regulated and HIF-1α was upregulated in HS tissue and HS derived fibroblasts (HFs). And cell proliferation was inhibited when miR-155 was overexpressed or HIF-1α was silenced. Moreover, overexpression of miR-155 in HFs could reduce the expression of collagens in vitro and inhibit the collagen fibers arrangement in vivo, whereas miR-155 knockdown gave opposite results. Furthermore, we found that miR-155 directly targeted the HIF-1α, which could also independently inhibit the expression of collagens in vitro and obviously improved the appearance and architecture of the rabbit ear scar in vivo when it was silencing. Finally, we found that PI3K/AKT pathway was enrolled in these processes. Together, our results indicated that miR-155 was a critical regulator in the formation and development of hypertrophic scar and might be a potential molecular target for hypertrophic scar therapy.