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1.  Diindolylmethane suppresses ovarian cancer growth and potentiates the effect of cisplatin in tumor mouse model by targeting signal transducer and activator of transcription 3 (STAT3) 
BMC Medicine  2012;10:9.
Signal transducer and activator of transcription 3 (STAT3) is activated in majority of ovarian tumors and confers resistance to cisplatin treatment in patients with ovarian cancer. We have reported previously that diindolylmethane (DIM) inhibits the growth of ovarian cancer cells. However, to date the exact mechanism by which DIM induces growth suppressive effects has not been clear. In this report the mode of action of DIM is investigated.
Six human ovarian cancer cell lines and an ovarian tumor xenograft animal model were used to study the effect of diindolylmethane alone or in combination with cisplatin.
Diindolylmethane treatment induced apoptosis in all six ovarian cancer cell lines. Phosphorylation of STAT3 at Tyr-705 and Ser-727 was reduced by DIM in a concentration-dependent manner. In addition, diindolylmethane treatment inhibited nuclear translocation, DNA binding, and transcriptional activity of STAT3. Interleukin (IL)-6-induced phosphorylation of STAT3 at Tyr-705 was significantly blocked by DIM. Overexpression of STAT3 by gene transfection blocked DIM-induced apoptosis. In addition, DIM treatment reduced the levels of IL-6 in ovarian cancer cells and in the tumors. DIM treatment also inhibited cell invasion and angiogenesis by suppressing hypoxia-inducible factor 1α (HIF-1α) and vascular epithelial growth factor (VEGF). Importantly, diindolylmethane treatment potentiated the effects of cisplatin in SKOV-3 cells by targeting STAT3. Oral administration of 3 mg diindolylmethane per day and subsequent administration of cisplatin substantially inhibited in vivo tumor growth. Western blotting analysis of tumor lysates indicated increased apoptosis and reduced STAT3 activation.
These findings provide a rationale for further clinical investigation of DIM alone or in combination for chemoprevention and/or chemotherapy of ovarian cancer.
PMCID: PMC3298725  PMID: 22280969
apoptosis; angiogenesis; cisplatin; diindolylmethane; STAT3
2.  Activation of Hif1α by the Prolylhydroxylase Inhibitor Dimethyoxalyglycine Decreases Radiosensitivity 
PLoS ONE  2011;6(10):e26064.
Hypoxia inducible factor 1α (Hif1α) is a stress responsive transcription factor, which regulates the expression of genes required for adaption to hypoxia. Hif1α is normally hydroxylated by an oxygen-dependent prolylhydroxylase, leading to degradation and clearance of Hif1α from the cell. Under hypoxic conditions, the activity of the prolylhydroxylase is reduced and Hif1α accumulates. Hif1α is also constitutively expressed in tumor cells, where it is associated with resistance to ionizing radiation. Activation of the Hif1α transcriptional regulatory pathway may therefore function to protect normal cells from DNA damage caused by ionizing radiation. Here, we utilized the prolylhydroxylase inhibitor dimethyloxalylglycine (DMOG) to elevate Hif1α levels in mouse embryonic fibroblasts (MEFs) to determine if DMOG could function as a radioprotector. The results demonstrate that DMOG increased Hif1α protein levels and decreased the sensitivity of MEFs to ionizing radiation. Further, the ability of DMOG to function as a radioprotector required Hif1α, indicating a key role for Hif1α's transcriptional activity. DMOG also induced the Hif1α -dependent accumulation of several DNA damage response proteins, including CHD4 and MTA3 (sub-units of the NuRD deacetylase complex) and the Suv39h1 histone H3 methyltransferase. Depletion of Suv39h1, but not CHD4 or MTA3, reduced the ability of DMOG to protect cells from radiation damage, implicating increased histone H3 methylation in the radioprotection of cells. Finally, treatment of mice with DMOG prior to total body irradiation resulted in significant radioprotection of the mice, demonstrating the utility of DMOG and related prolylhydroxylase inhibitors to protect whole organisms from ionizing radiation. Activation of Hif1α through prolylhydroxylase inhibition therefore identifies a new pathway for the development of novel radiation protectors.
PMCID: PMC3189247  PMID: 22016813
3.  B-DIM Impairs Radiation-Induced Survival Pathways Independently of Androgen Receptor Expression and Augments Radiation Efficacy in Prostate Cancer 
Cancer Letters  2011;318(1):86-92.
Increased consumption of cruciferous vegetables is associated with decreased risk in prostate cancer (PCa). The active compound in cruciferous vegetables appears to be the self dimerized product [3,3’-diindolylmethane (DIM)] of indole-3-carbinol (I3C). Nutritional grade B-DIM (absorption-enhanced) has proven safe in a Phase I trial in PCa. We investigated the anti-cancer activity of B-DIM as a new biological approach to improve the effects of radiotherapy for hormone refractory prostate cancer cells, which were either positive or negative for androgen receptor (AR) expression. B-DIM inhibited cell growth in a dose-dependent manner in both PC-3 (AR−) and C4-2B (AR+) cell lines. B-DIM was effective at increasing radiation-induced cell killing in both cell lines, independently of AR expression. B-DIM inhibited NF-κB and HIF-1α DNA activities and blocked radiation-induced activation of these transcription factors in both PC-3 and C4-2B cells. In C4-2B (AR+) cells, AR expression and nuclear localization were significantly increased by radiation. However, B-DIM abrogated the radiation–induced AR increased expression and trafficking to the nucleus, which was consistent with decreased PSA secretion. In vivo, treatment of PC-3 prostate tumors in nude mice with B-DIM and radiation resulted in significant primary tumor growth inhibition and control of metastasis to para-aortic lymph nodes. These studies demonstrate that B-DIM augments radiation-induced cell killing and tumor growth inhibition. B-DIM impairs critical survival signaling pathways activated by radiation, leading to enhanced cell killing. These novel observations suggest that B-DIM could be used as a safe compound to enhance the efficacy of radiotherapy for castrate-resistant PCa.
PMCID: PMC3288414  PMID: 22155105
radiation; B-DIM; prostate cancer; androgen receptor
4.  CDK1 stabilizes HIF-1α via direct phosphorylation of Ser668 to promote tumor growth 
Cell Cycle  2013;12(23):3689-3701.
Hypoxia-inducible factor 1 (HIF-1) is a major mediator of tumor physiology, and its activation is correlated with tumor progression, metastasis, and therapeutic resistance. HIF-1 is activated in a broad range of solid tumors due to intratumoral hypoxia or genetic alterations that enhance its expression or inhibit its degradation. As a result, decreasing HIF-1α expression represents an attractive strategy to sensitize hypoxic tumors to anticancer therapies. Here, we show that cyclin-dependent kinase 1 (CDK1) regulates the expression of HIF-1α, independent of its known regulators. Overexpression of CDK1 and/or cyclin B1 is sufficient to stabilize HIF-1α under normoxic conditions, whereas inhibition of CDK1 enhances the proteasomal degradation of HIF-1α, reducing its half-life and steady-state levels. In vitro kinase assays reveal that CDK1 directly phosphorylates HIF-1α at a previously unidentified regulatory site, Ser668. HIF-1α is stabilized under normoxic conditions during G2/M phase via CDK1-mediated phosphorylation of Ser668. A phospho-mimetic construct of HIF-1α at Ser668 (S668E) is significantly more stable under both normoxic and hypoxic conditions, resulting in enhanced transcription of HIF-1 target genes and increased tumor cell invasion and migration. Importantly, HIF-1α (S668E) displays increased tumor angiogenesis, proliferation, and tumor growth in vivo compared with wild-type HIF-1α. Thus, we have identified a novel link between CDK1 and HIF-1α that provides a potential molecular explanation for the elevated HIF-1 activity observed in primary and metastatic tumors, independent of hypoxia, and offers a molecular rationale for the clinical translation of CDK inhibitors for use in tumors with constitutively active HIF-1.
PMCID: PMC3903720  PMID: 24189531
CDK1; HIF-1α; angiogenesis; cell cycle; hypoxia
5.  STAT3 and HIF1α cooperatively activate HIF1 target genes in MDA-MB-231 and RCC4 cells 
Oncogene  2013;33(13):1670-1679.
Solid tumors often exhibit simultaneously inflammatory and hypoxic microenvironments. The ‘signal transducer and activator of transcription-3’ (STAT3)-mediated inflammatory response and the hypoxia-inducible factor (HIF)-mediated hypoxia response have been independently shown to promote tumorigenesis through the activation of HIF or STAT3 target genes and to be indicative of a poor prognosis in a variety of tumors. We report here for the first time that STAT3 is involved in the HIF1, but not HIF2-mediated hypoxic transcriptional response. We show that inhibiting STAT3 activity in MDA-MB-231 and RCC4 cells by a STAT3 inhibitor or STAT3 small interfering RNA significantly reduces the levels of HIF1, but not HIF2 target genes in spite of normal levels of hypoxia-inducible transcription factor 1α (HIF1α) and HIF2α protein. Mechanistically, STAT3 activates HIF1 target genes by binding to HIF1 target gene promoters, interacting with HIF1α protein and recruiting coactivators CREB binding protein (CBP) and p300, and RNA polymerase II (Pol II) to form enhanceosome complexes that contain HIF1α, STAT3, CBP, p300 and RNA Pol II on HIF1 target gene promoters. Functionally, the effect of STAT3 knockdown on proliferation, motility and clonogenic survival of tumor cells in vitro is phenocopied by HIF1α knockdown in hypoxic cells, whereas STAT3 knockdown in normoxic cells also reduces cell proliferation, motility and clonogenic survival. This indicates that STAT3 works with HIF1 to activate HIF1 target genes and to drive HIF1-depedent tumorigenesis under hypoxic conditions, but also has HIF-independent activity in normoxic and hypoxic cells. Identifying the role of STAT3 in the hypoxia response provides further data supporting the effectiveness of STAT3 inhibitors in solid tumor treatment owing to their usefulness in inhibiting both the STAT3 and HIF1 pro-tumorigenic signaling pathways in some cancer types.
PMCID: PMC3868635  PMID: 23604114
cotranscriptional activation; HIF; hypoxia; STAT3; transcription
6.  HIF-1α: a Valid Therapeutic Target for Tumor Therapy 
Hypoxia plays a major role in the induction of angiogenesis during tumor development. One mechanism by which tumor cells respond to a reduced oxygen level is via the activation of hypoxia-inducible factor-1 (HIF-1). HIF-1 is an oxygen-dependent transcriptional activator that plays crucial roles in the angiogenesis of tumors and mammalian development. HIF-1 consists of a constitutively expressed HIF-1β subunit and the highly regulated HIF-1α subunits. The stability and activity of HIF-1α are regulated by various post-translational modifications, hydroxylation, acetylation, phosphorylation and sumoyaltion. Therefore, HIF-1α interacts with several protein factors including PHD, pVHL, ARD-1, SUMO and p300/CBP. Under normoxia, the HIF-1α subunit is rapidly degraded via the von Hippel-Lindau tumor suppressor gene product (pVHL)-mediated ubiquitin/proteasome pathway. The association of pVHL and HIF-1α under normoxic conditions is triggered by the hydroxylation of prolines and the acetylation of lysine within a polypeptide segment known as the oxygen-dependent degradation (ODD) domain. On the contrary, under the hypoxia condition, the HIF-1α subunit becomes stable and interacts with coactivators such as p300/CBP to modulate its transcriptional activity. Under hypoxic conditions, HIF-1 eventually acts as a master regulator of numerous hypoxia-inducible genes. The target genes of HIF-1 are especially related to angiogenesis, cell proliferation and survival, and to glucose and iron metabolism. Moreover, it was reported that the activation of HIF-1α is closely associated with a variety of tumors and oncogenic pathways. Hence, the blocking of HIF-1α itself or the blocking of HIF-1α interacting proteins inhibits tumor growth. Based on these findings, HIF-1 can be a prime target for anticancer therapies. Therefore, this review summarizes the molecular mechanism of HIF-1α stability, the biological functions of HIF-1 and its potential applications for cancer therapies.
PMCID: PMC2843877  PMID: 20368827
ARD1; Angiogenesis; Anticancer therapy; Cell proliferation/survival; Glucose metabolism; HIF-1; Iron metabolism; PHD; SUMO; pVHL; p300/CBP; Transcription factor
7.  Regulation of hypoxia inducible factor-1α expression by the alteration of redox status in HepG2 cells 
Hypoxia inducible factor-1 (HIF-1) has been considered as a critical transcriptional factor in response to hypoxia. It can increase P-glycoprotein (P-Gp) thus generating the resistant effect to chemotherapy. At present, the mechanism regulating HIF-1α is still not fully clear in hypoxic tumor cells. Intracellular redox status is closely correlated with hypoxic micro-environment, so we investigate whether alterations in the cellular redox status lead to the changes of HIF-1α expression. HepG2 cells were exposed to Buthionine sulphoximine (BSO) for 12 h prior to hypoxia treatment. The level of HIF-1α expression was measured by Western blot and immunocytochemistry assays. Reduce glutathione (GSH) concentrations in hypoxic cells were determined using glutathione reductase/5,5'-dithiobis-(2-nitrob-enzoic acid) (DTNB) recycling assay. To further confirm the effect of intracellular redox status on HIF-1α expression, N-acetylcysteine (NAC) was added to culture cells for 8 h before the hypoxia treatment. The levels of multidrug resistance gene-1 (MDR-1) and erythropoietin (EPO) mRNA targeted by HIF-1α in hypoxic cells were further determined with RT-PCR, and then the expression of P-Gp protein was observed by Western blotting. The results showed that BSO pretreatment down-regulated HIF-1α and the effect was concentration-dependent, on the other hand, the increases of intracellular GSH contents by NAC could partly elevate the levels of HIF-1α expression. The levels of P-Gp (MDR-1) and EPO were concomitant with the trend of HIF-1α expression. Therefore, our data indicate that the changes of redox status in hypoxic cells may regulate HIF-1α expression and provide valuable information on tumor chemotherapy.
PMCID: PMC3113749  PMID: 21595915
Hypoxia; Redox; Multidrug resistance; HepG2
8.  Hypoxia and hypoglycaemia in Ewing's sarcoma and osteosarcoma: regulation and phenotypic effects of Hypoxia-Inducible Factor 
BMC Cancer  2010;10:372.
Hypoxia regulates gene expression via the transcription factor HIF (Hypoxia-Inducible Factor). Little is known regarding HIF expression and function in primary bone sarcomas. We describe HIF expression and phenotypic effects of hypoxia, hypoglycaemia and HIF in Ewing's sarcoma and osteosarcoma.
HIF-1α and HIF-2α immunohistochemistry was performed on a Ewing's tumour tissue array. Ewing's sarcoma and osteosarcoma cell lines were assessed for HIF pathway induction by Western blot, luciferase assay and ELISA. Effects of hypoxia, hypoglycaemia and isoform-specific HIF siRNA were assessed on proliferation, apoptosis and migration.
17/56 Ewing's tumours were HIF-1α-positive, 15 HIF-2α-positive and 10 positive for HIF-1α and HIF-2α. Expression of HIF-1α and cleaved caspase 3 localised to necrotic areas. Hypoxia induced HIF-1α and HIF-2α in Ewing's and osteosarcoma cell lines while hypoglycaemia specifically induced HIF-2α in Ewing's. Downstream transcription was HIF-1α-dependent in Ewing's sarcoma, but regulated by both isoforms in osteosarcoma. In both cell types hypoglycaemia reduced cellular proliferation by ≥ 45%, hypoxia increased apoptosis and HIF siRNA modulated hypoxic proliferation and migration.
Co-localisation of HIF-1α and necrosis in Ewing's sarcoma suggests a role for hypoxia and/or hypoglycaemia in in vivo induction of HIF. In vitro data implicates hypoxia as the primary HIF stimulus in both Ewing's and osteosarcoma, driving effects on proliferation and apoptosis. These results provide a foundation from which to advance understanding of HIF function in the pathobiology of primary bone sarcomas.
PMCID: PMC2918574  PMID: 20637078
9.  Tight Control of Hypoxia-inducible Factor-α Transient Dynamics Is Essential for Cell Survival in Hypoxia 
The Journal of Biological Chemistry  2014;289(9):5549-5564.
Background: Hypoxia inducible factor-α (HIF-α) is the main transcription factor activated in low oxygen conditions.
Results: Single cell imaging reveals pulses in nuclear levels of HIF-α.
Conclusion: The transient nature of the HIF-α nuclear accumulation is required to avoid cell death.
Significance: The duration of HIF-α response depends on cellular oxygenation, and can encode information and dictate cell fate.
Intracellular signaling involving hypoxia-inducible factor (HIF) controls the adaptive responses to hypoxia. There is a growing body of evidence demonstrating that intracellular signals encode temporal information. Thus, the dynamics of protein levels, as well as protein quantity and/or localization, impacts on cell fate. We hypothesized that such temporal encoding has a role in HIF signaling and cell fate decisions triggered by hypoxic conditions. Using live cell imaging in a controlled oxygen environment, we observed transient 3-h pulses of HIF-1α and -2α expression under continuous hypoxia. We postulated that the well described prolyl hydroxylase (PHD) oxygen sensors and HIF negative feedback regulators could be the origin of the pulsatile HIF dynamics. We used iterative mathematical modeling and experimental analysis to scrutinize which parameter of the PHD feedback could control HIF timing and we probed for the functional redundancy between the three main PHD proteins. We identified PHD2 as the main PHD responsible for HIF peak duration. We then demonstrated that this has important consequences, because the transient nature of the HIF pulse prevents cell death by avoiding transcription of p53-dependent pro-apoptotic genes. We have further shown the importance of considering HIF dynamics for coupling mathematical models by using a described HIF-p53 mathematical model. Our results indicate that the tight control of HIF transient dynamics has important functional consequences on the cross-talk with key signaling pathways controlling cell survival, which is likely to impact on HIF targeting strategies for hypoxia-associated diseases such as tumor progression and ischemia.
PMCID: PMC3937633  PMID: 24394419
Cell Death; Hypoxia; Hypoxia-inducible Factor; Imaging; Mathematical Modeling; Negative Feedback Loop; p53; Prolyl Hydroxylase
10.  Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp, Ctenopharyngodon idellus 
Hypoxia-inducible factors (HIFs) are involved in adaptive and survival responses to hypoxic stress in mammals. In fish, very little is known about the functions of HIFs.
We have cloned and characterized two distinct HIF-alpha cDNAs – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp. The deduced gcHIF-1alpha protein is highly similar to the HIF-1alphas (57–68%) from various vertebrate species, while gcHIF-4alpha is a novel isoform, and shows an equivalent degree of amino acid identity (41–47%) to the HIF-1alpha, HIF-2alpha and HIF-3alpha proteins so far described. Parsimony analysis indicated that gcHIF-4alpha is most closely related to the HIF-3alpha proteins. Northern blot analysis showed that mRNA levels of gcHIF-1alpha and gcHIF-4alpha differ substantially under normoxic and hypoxic conditions, while Western blot studies demonstrated that the endogenous protein levels for both gcHIF-1alpha and gcHIF-4alpha are similarly responsive to hypoxia. Our findings suggest that both gcHIF-1alpha and gcHIF-4alpha are differentially regulated at the transcriptional and translational levels. HRE-luciferase reporter assays show that both proteins function as transcription activators and play distinct roles in modulating the hypoxic response in grass carp.
There are at least two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – in the hypoxia-tolerant grass carp, which are differentially expressed and regulated in different fish organs in response to hypoxic stress. Overall, the results suggest that unique molecular mechanisms operate through these two HIF-alpha isoforms, which underpin the hypoxic response in the hypoxia-tolerant grass carp.
PMCID: PMC1473195  PMID: 16623959
11.  Identification of small molecule compounds that inhibit the HIF-1 signaling pathway 
Molecular Cancer  2009;8:117.
Hypoxia-inducible factor-1 (HIF-1) is the major hypoxia-regulated transcription factor that regulates cellular responses to low oxygen environments. HIF-1 is composed of two subunits: hypoxia-inducible HIF-1α and constitutively-expressed HIF-1β. During hypoxic conditions, HIF-1α heterodimerizes with HIF-1β and translocates to the nucleus where the HIF-1 complex binds to the hypoxia-response element (HRE) and activates expression of target genes implicated in cell growth and survival. HIF-1α protein expression is elevated in many solid tumors, including those of the cervix and brain, where cells that are the greatest distance from blood vessels, and therefore the most hypoxic, express the highest levels of HIF-1α. Therapeutic blockade of the HIF-1 signaling pathway in cancer cells therefore provides an attractive strategy for development of anticancer drugs. To identify small molecule inhibitors of the HIF-1 pathway, we have developed a cell-based reporter gene assay and screened a large compound library by using a quantitative high-throughput screening (qHTS) approach.
The assay is based upon a β-lactamase reporter under the control of a HRE. We have screened approximate 73,000 compounds by qHTS, with each compound tested over a range of seven to fifteen concentrations. After qHTS we have rapidly identified three novel structural series of HIF-1 pathway Inhibitors. Selected compounds in these series were also confirmed as inhibitors in a HRE β-lactamase reporter gene assay induced by low oxygen and in a VEGF secretion assay. Three of the four selected compounds tested showed significant inhibition of hypoxia-induced HIF-1α accumulation by western blot analysis.
The use of β-lactamase reporter gene assays, in combination with qHTS, enabled the rapid identification and prioritization of inhibitors specific to the hypoxia induced signaling pathway.
PMCID: PMC2797767  PMID: 20003191
12.  Hypoxia-inducible factor-1 (HIF-1) is involved in the regulation of hypoxia-stimulated expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and MCP-5 (Ccl12) in astrocytes 
Neuroinflammation has been implicated in various brain pathologies characterized by hypoxia and ischemia. Astroglia play an important role in the initiation and propagation of hypoxia/ischemia-induced inflammation by secreting inflammatory chemokines that attract neutrophils and monocytes into the brain. However, triggers of chemokine up-regulation by hypoxia/ischemia in these cells are poorly understood. Hypoxia-inducible factor-1 (HIF-1) is a dimeric transcriptional factor consisting of HIF-1α and HIF-1β subunits. HIF-1 binds to HIF-1-binding sites in the target genes and activates their transcription. We have recently shown that hypoxia-induced expression of IL-1β in astrocytes is mediated by HIF-1α. In this study, we demonstrate the role of HIF-1α in hypoxia-induced up-regulation of inflammatory chemokines, human monocyte chemoattractant protein-1 (MCP-1/CCL2) and mouse MCP-5 (Ccl12), in human and mouse astrocytes, respectively.
Primary fetal human astrocytes or mouse astrocytes generated from HIF-1α+/+ and HIF-1α+/- mice were subjected to hypoxia (<2% oxygen) or 125 μM CoCl2 for 4 h and 6 h, respectively. The expression of HIF-1α, MCP-1 and MCP-5 was determined by semi-quantitative RT-PCR, western blot or ELISA. The interaction of HIF-1α with a HIF-1-binding DNA sequence was examined by EMSA and supershift assay. HIF-1-binding sequence in the promoter of MCP-1 gene was cloned and transcriptional activation of MCP-1 by HIF-1α was analyzed by reporter gene assay.
Sequence analyses identified HIF-1-binding sites in the promoters of MCP-1 and MCP-5 genes. Both hypoxia and HIF-1α inducer, CoCl2, strongly up-regulated HIF-1α expression in astrocytes. Mouse HIF-1α+/- astrocytes had lower basal levels of HIF-1α and MCP-5 expression. The up-regulation of MCP-5 by hypoxia or CoCl2 in HIF-1α+/+ and HIF-1α+/- astrocytes was correlated with the levels of HIF-1α in cells. Both hypoxia and CoCl2 also up-regulated HIF-1α and MCP-1 expression in human astrocytes. EMSA assay demonstrated that HIF-1 activated by either hypoxia or CoCl2 binds to wild-type HIF-1-binding DNA sequence, but not the mutant sequence. Furthermore, reporter gene assay demonstrated that hypoxia markedly activated MCP-1 transcription but not the mutated MCP-1 promoter in transfected astrocytes.
These findings suggest that both MCP-1 and MCP-5 are HIF-1 target genes and that HIF-1α is involved in transcriptional induction of these two chemokines in astrocytes by hypoxia.
PMCID: PMC1872020  PMID: 17474992
13.  3,3′-Diindolylmethane Induces G1 Arrest and Apoptosis in Human Acute T-Cell Lymphoblastic Leukemia Cells 
PLoS ONE  2012;7(4):e34975.
Certain bioactive food components, including indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) from cruciferous vegetables, have been shown to target cellular pathways regulating carcinogenesis. Previously, our laboratory showed that dietary I3C is an effective transplacental chemopreventive agent in a dibenzo[def,p]chrysene (DBC)-dependent model of murine T-cell lymphoblastic lymphoma. The primary objective of the present study was to extend our chemoprevention studies in mice to an analogous human neoplasm in cell culture. Therefore, we tested the hypothesis that I3C or DIM may be chemotherapeutic in human T-cell acute lymphoblastic leukemia (T-ALL) cells. Treatment of the T-ALL cell lines CCRF-CEM, CCRF-HSB2, SUP-T1 and Jurkat with DIM in vitro significantly reduced cell proliferation and viability at concentrations 8- to 25-fold lower than the parent compound I3C. DIM (7.5 µM) arrested CEM and HSB2 cells at the G1 phase of the cell cycle and 15 µM DIM significantly increased the percentage of apoptotic cells in all T-ALL lines. In CEM cells, DIM reduced protein expression of cyclin dependent kinases 4 and 6 (CDK4, CDK6) and D-type cyclin 3 (CCND3); DIM also significantly altered expression of eight transcripts related to human apoptosis (BCL2L10, CD40LG, HRK, TNF, TNFRSF1A, TNFRSF25, TNFSF8, TRAF4). Similar anticancer effects of DIM were observed in vivo. Dietary exposure to 100 ppm DIM significantly decreased the rate of growth of human CEM xenografts in immunodeficient SCID mice, reduced final tumor size by 44% and increased the apoptotic index compared to control-fed mice. Taken together, our results demonstrate a potential for therapeutic application of DIM in T-ALL.
PMCID: PMC3325915  PMID: 22514694
14.  Prostate Cancer Cell Lines under Hypoxia Exhibit Greater Stem-Like Properties 
PLoS ONE  2011;6(12):e29170.
Hypoxia is an important environmental change in many cancers. Hypoxic niches can be occupied by cancer stem/progenitor-like cells that are associated with tumor progression and resistance to radiotherapy and chemotherapy. However, it has not yet been fully elucidated how hypoxia influences the stem-like properties of prostate cancer cells. In this report, we investigated the effects of hypoxia on human prostate cancer cell lines, PC-3 and DU145. In comparison to normoxia (20% O2), 7% O2 induced higher expressions of HIF-1α and HIF-2α, which were associated with upregulation of Oct3/4 and Nanog; 1% O2 induced even greater levels of these factors. The upregulated NANOG mRNA expression in hypoxia was confirmed to be predominantly retrogene NANOGP8. Similar growth rates were observed for cells cultivated under hypoxic and normoxic conditions for 48 hours; however, the colony formation assay revealed that 48 hours of hypoxic pretreatment resulted in the formation of more colonies. Treatment with 1% O2 also extended the G0/G1 stage, resulting in more side population cells, and induced CD44 and ABCG2 expressions. Hypoxia also increased the number of cells positive for ABCG2 expression, which were predominantly found to be CD44bright cells. Correspondingly, the sorted CD44bright cells expressed higher levels of ABCG2, Oct3/4, and Nanog than CD44dim cells, and hypoxic pretreatment significantly increased the expressions of these factors. CD44bright cells under normoxia formed significantly more colonies and spheres compared with the CD44dim cells, and hypoxic pretreatment even increased this effect. Our data indicate that prostate cancer cells under hypoxia possess greater stem-like properties.
PMCID: PMC3247249  PMID: 22216200
15.  Suppression of Hypoxia-Inducible Factor 1α (HIF-1α) by Tirapazamine Is Dependent on eIF2α Phosphorylation Rather Than the mTORC1/4E-BP1 Pathway 
PLoS ONE  2010;5(11):e13910.
Hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor that mediates the adaptation of tumor cells and tissues to the hypoxic microenvironment, has attracted considerable interest as a potential therapeutic target. Tirapazamine (TPZ), a well-characterized bioreductive anticancer agent, is currently in Phase II and III clinical trials. A major aspect of the anticancer activity of TPZ is its identity as a tumor-specific topoisomerase IIα inhibitor. In the study, for the first time, we found that TPZ acts in a novel manner to inhibit HIF-1α accumulation driven by hypoxia or growth factors in human cancer cells and in HepG2 cell-derived tumors in athymic nude mice. We investigated the mechanism of TPZ on HIF-1α in HeLa human cervical cancer cells by western blot analysis, reverse transcription-PCR assay, luciferase reporter assay and small interfering RNA (siRNA) assay. Mechanistic studies demonstrated that neither HIF-1α mRNA levels nor HIF-1α protein degradation are affected by TPZ. However, TPZ was found to be involved in HIF-1α translational regulation. Further studies revealed that the inhibitory effect of TPZ on HIF-1α protein synthesis is dependent on the phosphorylation of translation initiation factor 2α (eIF2α) rather than the mTOR complex 1/eukaryotic initiation factor 4E-binding protein-1 (mTORC1/4E-BP1) pathway. Immunofluorescence analysis of tumor sections provide the in vivo evidences to support our hypothesis. Additionally, siRNA specifically targeting topoisomerase IIα did not reverse the ability of TPZ to inhibit HIF-1α expression, suggesting that the HIF-1α inhibitory activity of TPZ is independent of its topoisomerase IIα inhibition. In conclusion, our findings suggest that TPZ is a potent regulator of HIF-1α and provide new insight into the potential molecular mechanism whereby TPZ serves to reduce HIF-1α expression.
PMCID: PMC2976688  PMID: 21085474
16.  BAY 87-2243, a highly potent and selective inhibitor of hypoxia-induced gene activation has antitumor activities by inhibition of mitochondrial complex I 
Cancer Medicine  2013;2(5):611-624.
The activation of the transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development, tumor progression, and resistance to chemo- and radiotherapy. In order to identify compounds targeting the HIF pathway, a small molecule library was screened using a luciferase-driven HIF-1 reporter cell line under hypoxia. The high-throughput screening led to the identification of a class of aminoalkyl-substituted compounds that inhibited hypoxia-induced HIF-1 target gene expression in human lung cancer cell lines at low nanomolar concentrations. Lead structure BAY 87-2243 was found to inhibit HIF-1α and HIF-2α protein accumulation under hypoxic conditions in non-small cell lung cancer (NSCLC) cell line H460 but had no effect on HIF-1α protein levels induced by the hypoxia mimetics desferrioxamine or cobalt chloride. BAY 87-2243 had no effect on HIF target gene expression levels in RCC4 cells lacking Von Hippel–Lindau (VHL) activity nor did the compound affect the activity of HIF prolyl hydroxylase-2. Antitumor activity of BAY 87-2243, suppression of HIF-1α protein levels, and reduction of HIF-1 target gene expression in vivo were demonstrated in a H460 xenograft model. BAY 87-2243 did not inhibit cell proliferation under standard conditions. However under glucose depletion, a condition favoring mitochondrial ATP generation as energy source, BAY 87-2243 inhibited cell proliferation in the nanomolar range. Further experiments revealed that BAY 87-2243 inhibits mitochondrial complex I activity but has no effect on complex III activity. Interference with mitochondrial function to reduce hypoxia-induced HIF-1 activity in tumors might be an interesting therapeutic approach to overcome chemo- and radiotherapy-resistance of hypoxic tumors.
PMCID: PMC3892793  PMID: 24403227
Antitumor activity; hypoxia; hypoxia-inducible factor-1; mitochondrial complex 1
17.  Bcl-2 Regulates HIF-1α Protein Stabilization in Hypoxic Melanoma Cells via the Molecular Chaperone HSP90 
PLoS ONE  2010;5(7):e11772.
Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a critical mediator of the cellular response to hypoxia. Enhanced levels of HIF-1α, the oxygen-regulated subunit of HIF-1, is often associated with increased tumour angiogenesis, metastasis, therapeutic resistance and poor prognosis. It is in this context that we previously demonstrated that under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth Factor (VEGF)-mediated tumour angiogenesis.
Methodology/Principal Findings
By using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1α protein stabilization as a key regulator for the induction of HIF-1 by bcl-2 under hypoxia. We also demonstrated that bcl-2-induced accumulation of HIF-1α protein during hypoxia was not due to an increased gene transcription or protein synthesis. In fact, it was related to a modulation of HIF-1α protein expression at a post-translational level, indeed its degradation rate was faster in the control lines than in bcl-2 transfectants. The bcl-2-induced HIF-1α stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1α degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1α protein. We also showed that bcl-2, HIF-1α and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1α protein in bcl-2 overexpressing clones under hypoxic conditions. Finally, by using genetic and pharmacological approaches we proved that HSP90 is involved in bcl-2-dependent stabilization of HIF-1α protein during hypoxia, and in particular the isoform HSP90β is the main player in this phenomenon.
We identified the stabilization of HIF-1α protein as a mechanism through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells involving the β isoform of molecular chaperone HSP90.
PMCID: PMC2910721  PMID: 20668552
18.  HIF-1α/GPER signaling mediates the expression of VEGF induced by hypoxia in breast cancer associated fibroblasts (CAFs) 
Carcinoma-associated fibroblasts (CAFs) play a pivotal role in cancer progression by contributing to invasion, metastasis and angiogenesis. Solid tumors possess a unique microenvironment characterized by local hypoxia, which induces gene expression changes and biological features leading to poor outcomes. Hypoxia Inducible Factor 1 (HIF-1) is the main transcription factor that mediates the cell response to hypoxia through different mechanisms that include the regulation of genes strongly associated with cancer aggressiveness. Among the HIF-1 target genes, the G-protein estrogen receptor (GPER) exerts a stimulatory role in diverse types of cancer cells and in CAFs.
We evaluated the regulation and function of the key angiogenic mediator vascular endothelial growth factor (VEGF) in CAFs exposed to hypoxia. Gene expression studies, Western blotting analysis and immunofluorescence experiments were performed in CAFs and breast cancer cells in the presence of cobalt chloride (CoCl2) or cultured under low oxygen tension (2% O2), in order to analyze the involvement of the HIF-1α/GPER signaling in the biological responses to hypoxia. We also explored the role of the HIF-1α/GPER transduction pathway in functional assays like tube formation in human umbilical vein endothelial cells (HUVECs) and cell migration in CAFs.
We first determined that hypoxia induces the expression of HIF-1α and GPER in CAFs, then we ascertained that the HIF-1α/GPER signaling is involved in the regulation of VEGF expression in breast cancer cells and in CAFs exposed to hypoxia. We also assessed by ChIP assay that HIF-1α and GPER are both recruited to the VEGF promoter sequence and required for VEGF promoter stimulation upon hypoxic condition. As a biological counterpart of these findings, conditioned medium from hypoxic CAFs promoted tube formation in HUVECs in a HIF-1α/GPER dependent manner. The functional cooperation between HIF-1α and GPER in CAFs was also evidenced in the hypoxia-induced cell migration, which involved a further target of the HIF-1α/GPER signaling like connective tissue growth factor (CTGF).
The present results provide novel insight into the role elicited by the HIF-1α/GPER transduction pathway in CAFs towards the hypoxia-dependent tumor angiogenesis. Our findings further extend the molecular mechanisms through which the tumor microenvironment may contribute to cancer progression.
PMCID: PMC3978922  PMID: 23947803
19.  The Hypoxia-Associated Factor Switches Cells from HIF-1α– to HIF-2α–Dependent Signaling Promoting Stem Cell Characteristics, Aggressive Tumor Growth and Invasion 
Cancer Research  2011;71(11):4015-4027.
Most solid tumors and their metastases experience periods of low oxygen or hypoxia, which is of major clinical significance as it promotes both tumor progression and resistance to therapy. Critical mediators of the hypoxic response are the hypoxia-inducible factors HIF-1α and HIF-2α. The HIFs are nonredundant and regulate both overlapping and unique downstream target genes. Here, we describe a novel mechanism for the switch between HIF-1α– and HIF-2α–dependent transcription during tumor hypoxia caused by the hypoxia associated factor (HAF). HAF is overexpressed in a variety of tumors and its levels are decreased during acute hypoxia, but increased following prolonged hypoxia. We have previously identified HAF as an E3 ubiquitin ligase that binds and ubiquitinates HIF-1α by an oxygen and pVHL-independent mechanism, thus targeting HIF-1α for proteasomal degradation. Here, we show that HAF also binds to HIF-2α, but at a different site than HIF-1α, and increases HIF-2α transactivation without causing its degradation. HAF, thus, switches the hypoxic response of the cancer cell from HIF-1α–dependent to HIF-2α–dependent transcription and activates genes involved in invasion such as MMP9, PAI-1, and the stem cell factor OCT-3/4. The switch to HIF-2α–dependent gene expression caused by HAF also promotes an enriched tumor stem cell population, resulting in highly aggressive tumors in vivo. Thus, HAF, by causing a switch from a HIF-1α– to HIF-2α–dependent response to hypoxia, provides a mechanism for more aggressive growth of tumors under prolonged hypoxia.
PMCID: PMC3268651  PMID: 21512133
20.  Differential hypoxic regulation of hypoxia-inducible factors 1α and 2α 
Molecular cancer research : MCR  2011;9(6):757-765.
The hypoxia-inducible transcription factors (HIF)-1α and -2α play a critical role in cellular response to hypoxia. Elevated HIF-α expression correlates with poor patient survival in a large number of cancers. Recent evidence suggests that HIF-2α appears to be preferentially expressed in neuronal tumor cells that exhibit cancer stem cell characteristics. These observations suggest that expression of HIF-1α and -2α is differentially regulated in the hypoxic tumor microenvironment. However, the underlying mechanisms remain to be fully investigated. In this study, we investigated the transcriptional regulation HIF-1α and -2α under different physiologically relevant hypoxic conditions. We found that transcription of HIF-2α was consistently increased by hypoxia, whereas transcription of HIF-1α showed variable levels of repression. Mechanistically, differential regulation of HIF-α transcription involved hypoxia-induced changes in acetylation of core histones H3 and H4 associated with the proximal promoters of the HIF-1α or HIF-2α gene. We also found that, although highly stable under acute hypoxia, HIF-1α and HIF-2α proteins become destabilized under chronic hypoxia. Our results have thus provided new mechanistic insights into the differential regulation of HIF-1α and -2α by the hypoxic tumor microenvironment. These findings also suggest an important role of HIF-2α in the regulation of tumor progression under chronic hypoxia.
PMCID: PMC3117969  PMID: 21571835
hypoxia; hypoxia-inducible factor; HIF-1α and HIF-2α; transcription; promoter
21.  Epstein-Barr Virus Latent Membrane Protein 1 Induces Synthesis of Hypoxia-Inducible Factor 1α 
Molecular and Cellular Biology  2004;24(12):5223-5234.
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor composed of HIF-1α and HIF-1β that is the central regulator of responses to hypoxia. The specific binding of HIF-1 to the hypoxia-responsive element (HRE) induces the transcription of genes that respond to hypoxic conditions, including vascular endothelial growth factor (VEGF). Here we report that expression of HIF-1α is increased in diverse Epstein-Barr virus (EBV)-infected type II and III cell lines, which express EBV latent membrane protein 1 (LMP1), the principal EBV oncoprotein, as well as other latency proteins, but not in the parental EBV-negative cell lines. We show first that transfection of an LMP1 expression plasmid into Ad-AH cells, an EBV-negative nasopharyngeal epithelial cell line, induces synthesis of HIF-1α protein without increasing its stability or mRNA level. The mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059 markedly reduces induction of HIF-1α by LMP1. Catalase, an H2O2 scavenger, strongly suppresses LMP1-induced production of H2O2, which results in a decrease in the expression of HIF-1α induced by LMP1. Inhibition of the NF-κB, c-jun N-terminal kinase, p38 MAPK, and phosphatidylinositol 3-kinase pathways did not affect HIF-1α expression. Moreover, LMP1 induces HIF-1 DNA binding activity and upregulates HRE and VEGF promoter transcriptional activity. Finally, LMP1 increases the appearance of VEGF protein in extracellular fluids; induction of VEGF is suppressed by PD98059 or catalase. These results suggest that LMP1 increases HIF-1 activity through induction of HIF-1α protein expression, which is controlled by p42/p44 MAPK activity and H2O2. The ability of EBV, and specifically its major oncoprotein, LMP1, to induce HIF-1α along with other invasiveness and angiogenic factors reported previously discloses additional oncogenic properties of this tumor virus.
PMCID: PMC419879  PMID: 15169887
22.  DNA Damage Is a Prerequisite for p53-Mediated Proteasomal Degradation of HIF-1α in Hypoxic Cells and Downregulation of the Hypoxia Marker Carbonic Anhydrase IX 
Molecular and Cellular Biology  2004;24(13):5757-5766.
We investigated the relationship between the tumor suppressor p53 and the hypoxia-inducible factor-1 (HIF-1)-dependent expression of the hypoxia marker, carbonic anhydrase IX (CAIX). MCF-7 (wt p53) and Saos-2 (p53-null) cells displayed similar induction of CAIX expression and CA9 promoter activity under hypoxic conditions. Activation of p53 by the DNA damaging agent mitomycin C (MC) was accompanied by a potent repression of CAIX expression and the CA9 promoter in MCF-7 but not in Saos-2 cells. The activated p53 mediated increased proteasomal degradation of HIF-1α protein, resulting in considerably lower steady-state levels of HIF-1α protein in hypoxic MCF-7 cells but not in Saos-2 cells. Overexpression of HIF-1α relieved the MC-induced repression in MCF-7 cells, confirming regulation at the HIF-1α level. Similarly, CA9 promoter activity was downregulated by MC in HCT 116 p53+/+ but not the isogenic p53−/− cells. Activated p53 decreased HIF-1α protein levels by accelerated proteasome-dependent degradation without affecting significantly HIF-1α transcription. In summary, our results demonstrate that the presence of wtp53 under hypoxic conditions has an insignificant effect on the stabilization of HIF-1α protein and HIF-1-dependent expression of CAIX. However, upon activation by DNA damage, wt p53 mediates an accelerated degradation of HIF-1α protein, resulting in reduced activation of CA9 transcription and, correspondingly, decreased levels of CAIX protein. A model outlining the quantitative relationship between p53, HIF-1α, and CAIX is presented.
PMCID: PMC480909  PMID: 15199132
23.  Hydrogen Sulfide Increases Hypoxia-inducible Factor-1 Activity Independently of von Hippel–Lindau Tumor Suppressor-1 in C. elegans 
Molecular Biology of the Cell  2010;21(1):212-217.
In C. elegans, hydrogen sulfide (H2S) exposure results in Hif-1 stabilization. hif-1 is required for survival in H2S and constitutive HIF-1 stabilization confers resistance to H2S. H2S-induced HIF-1 reporter activity appears to be independent of VHL-1, whereas VHL-1 is required for hypoxic regulation of HIF-1 reporter activity.
Rapid alteration of gene expression in response to environmental changes is essential for normal development and behavior. The transcription factor hypoxia-inducible factor (HIF)-1 is well known to respond to alterations in oxygen availability. In nature, low oxygen environments are often found to contain high levels of hydrogen sulfide (H2S). Here, we show that Caenorhabditis elegans can have mutually exclusive responses to H2S and hypoxia, both involving HIF-1. Specifically, H2S results in HIF-1 activity throughout the hypodermis, whereas hypoxia causes HIF-1 activity in the gut as judged by a reporter for HIF-1 activity. C. elegans require hif-1 to survive in room air containing trace amounts of H2S. Exposure to H2S results in HIF-1 nuclear localization and transcription of HIF-1 targets. The effects of H2S on HIF-1 reporter activity are independent of von Hippel–Lindau tumor suppressor (VHL)-1, whereas VHL-1 is required for hypoxic regulation of HIF-1 reporter activity. Because H2S is naturally produced by animal cells, our results suggest that endogenous H2S may influence HIF-1 activity.
PMCID: PMC2801715  PMID: 19889840
24.  Hypoxia‐inducible factor expression in human RPE cells 
The British Journal of Ophthalmology  2007;91(10):1406-1410.
Hypoxia‐inducible factor (HIF) is a common transcription factor for many angiogenic proteins. Retinal pigment epithelial (RPE) cells are an important source of angiogenic factors in the retina. The expression of HIF, its regulation by proline hydroxylase (PHD) enzymes, and its downstream regulation of angiogenic factors like vascular endothelial growth factor (VEGF) and erythropoietin (EPO) was studied in RPE cells in order to determine some of the molecular mechanisms underlying ischaemic retinal disease.
ARPE‐19 cells were cultured for various times under hypoxic conditions. Cellular HIF and PHD isoforms were analysed and quantified using western blot and densitometry. VEGF and EPO secreted into the media were assayed using enzyme‐linked immunosorbent assay (ELISA). Messenger RNA (mRNA) was quantified using real‐time quantitative reverse transcriptase polymerase chain reaction (qPCR). RNA interference was achieved using siRNA techniques.
HIF‐1α was readily produced by ARPE‐19 cells under hypoxia, but HIF‐2α and HIF‐3α could not be detected even after HIF‐1α silencing. HIF‐1α protein levels showed an increasing trend for the first 24 h while HIF‐1α mRNA levels fluctuated during this time. After 36 h HIF‐1α protein levels declined to baseline levels, a change that was coincident with a rise in both PHD2 and PHD3. Silencing HIF‐1α significantly decreased VEGF secretion. Significant production of EPO could not be detected at the protein or mRNA level.
HIF‐1α appears to be the main isoform of HIF functioning in ARPE‐19 cells. Under hypoxia, HIF‐1α levels are likely self‐regulated by a feedback loop that involves both transcriptional and post‐translational mechanisms. VEGF production by human RPE cells is regulated by HIF‐1α. EPO was not produced in significant amounts by RPE cells under hypoxic conditions, suggesting that other cells and/or transcription factors in the retina are responsible for its production.
PMCID: PMC2001032  PMID: 17567660
diabetic retinopathy; VEGF; erythropoietin; hypoxia‐inducible factor; proline hydroxylase
25.  HIF-1α and HIF-2α have divergent roles in colon cancer 
Hypoxia-inducible factor (HIF)-1 and HIF-2 are heterodimeric transcription factors that mediate the cellular response to hypoxia. Their key regulatory subunits, HIF-1α and HIF-2α, are induced similarly by hypoxia, but their functional roles in cancer may be distinct and isoform-specific. SW480 colon cancer cells with stable expression of siRNA to HIF-1α or HIF-2α or both were established. HIF-1α-deficient cells displayed lower rates of proliferation and migration, but HIF-2α-deficient cells exhibited enhanced anchorage independent growth in a soft agar assay. Xenograft studies revealed that HIF-1α deficiency inhibited overall tumor growth, whereas deficiency of HIF-2α stimulated tumor growth. In human colon cancer tissues, expression of HIF-1α and to a lesser extent, HIF-2α, was linked to upregulation of VEGF and tumor angiogenesis. However, loss of expression of HIF-2α but not HIF-1α was strongly correlated with advanced tumor stage. DNA microarray analysis identified distinct sets of HIF-1α and HIF-2α target genes that may explain these phenotypic differences. Collectively, these findings suggest that HIF isoforms may have differing cellular functions in colon cancer. In particular, HIF-1α promoted the growth of SW480 colon cancer cells but HIF-2α appeared to restrain growth. Consequently, therapeutic approaches that target HIF may need to consider these isoform-specific properties.
PMCID: PMC2682346  PMID: 19030186
colon cancer; HIF; angiogenesis

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