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.
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.
3,3'-Diindolylmethane (DIM), an indole derivative produced in the stomach after the consumption of broccoli and other cruciferous vegetables, has been demonstrated to exert anti-cancer effects in both in vivo and in vitro models. We have previously determined that DIM (0 – 30 μmol/L) inhibited the growth of HT-29 human colon cancer cells in a concentration-dependent fashion. In this study, we evaluated the effects of DIM on cell cycle progression in HT-29 cells.
HT-29 cells were cultured with various concentrations of DIM (0 – 30 μmol/L) and the DNA was stained with propidium iodide, followed by flow cytometric analysis. [3H]Thymidine incorporation assays, Western blot analyses, immunoprecipitation and in vitro kinase assays for cyclin-dependent kinase (CDK) and cell division cycle (CDC)2 were conducted.
The percentages of cells in the G1 and G2/M phases were dose-dependently increased and the percentages of cells in S phase were reduced within 12 h in DIM-treated cells. DIM also reduced DNA synthesis in a dose-dependent fashion. DIM markedly reduced CDK2 activity and the levels of phosphorylated retinoblastoma proteins (Rb) and E2F-1, and also increased the levels of hypophosphorylated Rb. DIM reduced the protein levels of cyclin A, D1, and CDK4. DIM also increased the protein levels of CDK inhibitors, p21CIP1/WAF1 and p27KIPI. In addition, DIM reduced the activity of CDC2 and the levels of CDC25C phosphatase and cyclin B1.
Here, we have demonstrated that DIM induces G1 and G2/M phase cell cycle arrest in HT-29 cells, and this effect may be mediated by reduced CDK activity.
This study employed cultured human primary hepatocytes to investigate the ability of the putative chemopreventive phytochemicals curcumin (CUR), 3,3′-diindolylmethane (DIM), isoxanthohumol (IXN), or 8-prenylnaringenin (8PN) to reduce DNA adduct formation of the hepatocarcinogen aflatoxin B1 (AFB). Following 48 h of pretreatment, DIM and 8PN significantly increased AFB-DNA adduct levels, whereas CUR and IXN had no effect. DIM greatly enhanced the transcriptional expression of cytochrome P450 (CYP) 1A1 and CYP1A2 mRNA. Glutathione S-transferase mRNAs were not increased by any of the treatments. In vitro enzyme activity assays demonstrated that 8PN and DIM, but not CUR or IXN, inhibited human CYP1A1, CYP1A2, and CYP3A4 activities. To distinguish between treatment effects on transcription versus direct effects on enzyme activity for DIM, we evaluated the effects of pretreatment alone (transcriptional activation) versus cotreatment alone (enzyme inhibition). The results demonstrated that effects on gene expression, but not catalytic activity, are responsible for the observed effects of DIM on AFB-DNA adduct formation. The increase in AFB-DNA damage following DIM treatment may be explained through its substantial induction of CYP1A2 and/or its downregulation of GSTM1, both of which were significant. The increase in DNA damage by DIM raises potential safety risks for dietary supplements of DIM and its precursor indole-3-carbinol.
phytochemicals; biotransformation; hepatocytes; human; CYP
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.
diabetic retinopathy; VEGF; erythropoietin; hypoxia‐inducible factor; proline hydroxylase
Hypoxia-inducible factor 1 (HIF-1) activates the transcription of a wide range of genes related to oxygen delivery and metabolic adaptation under hypoxic (low-oxygen) conditions. HIF-1 is, in fact, a heterodimer of two subunits, HIF-1α and HIF-1β. The only analytical methods available for measuring HIF-1α levels in tumors are immunohistochemistry and Western blotting. Immunohistochemistry has the advantage of allowing the identification and direct examination of HIF-1α-expressing cells, but has the intrinsic limitation, as for Western blotting, of being nonquantitative. We developed and validated an enzyme-linked immunosorbent assay (ELISA) approach to measure HIF-1α levels in cultured tumor cell lines in vitro. HIF-1α was expressed in thirteen tumor cell lines grown under hypoxic conditions; however, the levels differed strongly between cell lines. These data point to intrinsic differences between cell lines for the induction of HIF-1α under hypoxic conditions. The ELISA developed in the present study is thus an interesting alternative to other analytical methods used to measure HIF-1α protein levels and should be useful in preclinical pharmacological studies targeting HIF-1α.
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.
3,3′-Diindolylmethane (DIM) is a natural component of cruciferous plants. It has strong antioxidant and anti-angiogenic effects and promotes the apoptosis of a variety of tumor cells. However, little is known about the critical role of DIM on cardiac hypertrophy. In the present study, we investigated the effects of DIM on cardiac hypertrophy.
Multiple molecular techniques such as Western blot analysis, real-time PCR to determine RNA expression levels of hypertrophic, fibrotic and oxidative stress markers, and histological analysis including H&E for histopathology, PSR for collagen deposition, WGA for myocyte cross-sectional area, and immunohistochemical staining for protein expression were used.
In pre-treatment and reverse experiments, C57/BL6 mouse chow containing 0.05% DIM (dose 100 mg/kg/d DIM) was administered one week prior to surgery or one week after surgery, respectively, and continued for 8 weeks after surgery. In both experiments, DIM reduced to cardiac hypertrophy and fibrosis induced by aortic banding through the activation of 5′-adenosine monophosphate-activated protein kinase-α2 (AMPKα2) and inhibition of mammalian target of the rapamycin (mTOR) signaling pathway. Furthermore, DIM protected against cardiac oxidative stress by regulating expression of estrogen-related receptor-alpha (ERRα) and NRF2 etc. The cardioprotective effects of DIM were ablated in mice lacking functional AMPKα2.
DIM significantly improves left ventricular function via the activation of AMPKα2 in a murine model of cardiac hypertrophy.
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 μM) 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 μM) blocked the induction of HIF-1α 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-1α 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.
There is a large body of scientific evidence suggesting that 3,3′-Diindolylmethane (DIM), a compound derived from the digestion of indole-3-carbinol, which is abundant in cruciferous vegetables, harbors anti-tumor activity in vitro and in vivo. Accumulating evidence suggests that AMP-activated protein kinase (AMPK) plays an essential role in cellular energy homeostasis and tumor development and that targeting AMPK may be a promising therapeutic option for cancer treatment in the clinic. We previously reported that a formulated DIM (BR-DIM; hereafter referred as B-DIM) with higher bioavailability was able to induce apoptosis and inhibit cell growth, angiogenesis, and invasion of prostate cancer cells. However, the precise molecular mechanism(s) for the anti-cancer effects of B-DIM have not been fully elucidated. In the present study, we investigated whether AMP-activated protein kinase (AMPK) is a molecular target of B-DIM in human prostate cancer cells. Our results showed, for the first time, that B-DIM could activate the AMPK signaling pathway, associated with suppression of the mammalian target of rapamycin (mTOR), down-regulation of androgen receptor (AR) expression, and induction of apoptosis in both androgen-sensitive LNCaP and androgen-insensitive C4-2B prostate cancer cells. B-DIM also activates AMPK and down-regulates AR in androgen-independent C4-2B prostate tumor xenografts in SCID mice. These results suggest that B-DIM could be used as a potential anti-cancer agent in the clinic for prevention and/or treatment of prostate cancer regardless of androgen responsiveness, although functional AR may be required.
Diindolylmethane (DIM), a biologically active congener of indole-3-carbinol (I3C) derived from cruciferous vegetables, is a promising agent for the prevention of estrogen-sensitive cancers. Both DIM and estrogen affect transcription of genes by binding receptors, such as aryl hydrocarbon receptor (AhR) or estrogen receptors (ER). Gene regulation by DIM and estradiol (E2) can be very complex. While DIM typically binds the AhR, this complex can directly associate with the ER, recruit co-activators that bind to estrogen-responsive promoters, and activate transcription. Alternately, DIM can bind the ER directly. In this study, we have analyzed gene expression using microarray profiling and quantitative real time–polymerase chain reaction in MCF7 breast cancer cells treated with E2 (1 nM) or DIM (25 μM) alone or in combination for 16 h. The interplay of E2 and DIM was reflected in the expression of a subset of genes (<90) in which the combination of E2 and DIM acted either additively or antagonistically to alter gene expression.
3,3′-Diindolylmethane (DIM) is a potential chemopreventive phytochemical derived from Brassica vegetables. In this study we characterized the effect of DIM on cell cycle regulation in both androgen dependent LNCaP and androgen receptor negative-p53 mutant DU145 human prostate cancer cells. DIM had an antiproliferative effect on both LNCaP and DU145 cells, as it significantly inhibited [3H]-thymidine incorporation. FACS analysis revealed a DIM mediated G1 cell cycle arrest. DIM strongly inhibited the expression of cdk2 and cdk4 protein and increased expression of the cell cycle inhibitor p27Kip1 protein in LNCaP and DU145 cells. Promoter deletion studies with p27Kip1 reporter gene constructs showed that this DIM-mediated increase in p27Kip1 was dependent on the Sp1 transcription factor. Moreover, using a dominant negative inhibitor of p38 MAPK, we showed that the induction of p27Kip1 and subsequent G1 arrest by DIM involves activation of the p38 MAPK pathway in the DU145 cells. Taken together, our results indicate that DIM is able to stop the cell cycle progression of human prostate cancer cells regardless of their androgen-dependence and p53 status, by differentially modulating cell cycle regulatory pathways. The Sp1 and p38 MAPK pathways mediate the DIM cell cycle regulatory effect in DU145 cells.
3; 3′-Diinolylmethane; Prostate cancer; Cell cycle arrest; p27Kip1; p38 MAPK; Cancer
The importance of hypoxia-inducible factor (HIF) in promoting angiogenesis and vasculogenesis during wound healing has been demonstrated. It is widely accepted that HIF activity can be promoted by many factors, including hypoxia in the wound or cytokines from inflammatory cells infiltrating the wound. However, there has not been a systematic exploration of the relationship between HIF activity and hypoxia in the burn wound. The location of the hypoxic tissue has not been clearly delineated. The time course of the appearance of hypoxia and the increased activity of HIF and appearance of HIF’s downstream transcription products has not been described. The aim of this study was to utilize pimonidazole, a specific tissue hypoxia marker, to characterize the spatial and temporal course of hypoxia in a murine burn model and correlate this with the appearance of HIF-1α and its important angiogenic and vasculogenic transcription products VEGF and SDF-1. Hypoxia was found in the healing margin of burn wounds beginning at 48 hours after burn and peaking at day 3 after burn. On sequential sections of the same tissue block, positive staining of HIF-1α, SDF-1, and VEGF all occurred at the leading margin of the healing area and peaked at day 3, as did hypoxia. Immunohistochemical analysis was used to explore the characteristics of the hypoxic region of the wound. The localization of hypoxia was found to be related to cell growth and migration, but not to proliferation or inflammatory infiltration.
ypoxia; Hypoxia-inducible factor -10α; Burn; Wound Healing; Ki67 Cell Proliferation; Keratin17
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.
Hypoxia; Redox; Multidrug resistance; HepG2
Thyroid cancer is the most common endocrine related cancer with increasing incidences during the past five years. Current treatments for thyroid cancer, such as surgery or radioactive iodine therapy, often require patients to be on lifelong thyroid hormone replacement therapy and given the significant recurrence rates of thyroid cancer, new preventive modalities are needed. The present study investigates the property of a natural dietary compound found in cruciferous vegetables, 3,3′-diindolylmethane (DIM), to target the metastatic phenotype of thyroid cancer cells through a functional estrogen receptor.
Thyroid cancer cell lines were treated with estrogen and/or DIM and subjected to in vitro adhesion, migration and invasion assays to investigate the anti-metastatic and anti-estrogenic effects of DIM. We observed that DIM inhibits estrogen mediated increase in thyroid cell migration, adhesion and invasion, which is also supported by ER-α downregulation (siRNA) studies. Western blot and zymography analyses provided direct evidence for this DIM mediated inhibition of E2 enhanced metastasis associated events by virtue of targeting essential proteolytic enzymes, namely MMP-2 and MMP-9.
Our data reports for the first time that DIM displays anti-estrogenic like activity by inhibiting estradiol enhanced thyroid cancer cell proliferation and in vitro metastasis associated events, namely adhesion, migration and invasion. Most significantly, MMP-2 and MMP-9, which are known to promote and enhance metastasis, were determined to be targets of DIM. This anti-estrogen like property of DIM may lead to the development of a novel preventive and/or therapeutic dietary supplement for thyroid cancer patients by targeting progression of the disease.
Tumor hypoxia induces the up-regulation of Hif-1alpha which in turn induces the expression of genes including VEGF to recruit new blood vessel outgrowth, enabling tumor growth and metastasis. Interference with the Hif-1 pathway and neoangiogenesis is an attractive anti-tumor target. The hydroxylation of Hif-1alpha by PHD proteins during normoxia serves as a recognition motif for its proteasomal degradation. However, under hypoxic conditions, hydroxylation is inhibited and furthermore, PHD proteins are themselves poly-ubiquitylated and degraded by Siah ubiqiuitin ligases. Our data demonstrate for the first time that inhibition of the interaction between Siah and PHD proteins using a peptide derived from a Drosophila protein interferes with the PHD degradation. Furthermore, cells stably expressing the inhibitor display reduced up-regulation of Hif-1alpha protein levels and Hif-1 mediated gene expression under hypoxia. In a syngeneic mouse model of breast cancer, the inhibitor reduced tumor growth and neoangiogenesis and prolonged survival of the mice. In addition, levels of Hif-1alpha and its target Glut-1 are reduced in the inhibitor expressing tumors. These data demonstrate, in a proof-of-principle study, that Siah protein, the most upstream component of the hypoxia pathway yet identified, is a viable drug target for anti-tumor therapies.
Hypoxia inducible factor-1 (HIF-1) promotes tumor cell adaptation to microenvironmental stress. HIF-1 is up-regulated in irradiated tumors and serves as a promising target for radiosensitization. We initially confirmed that the orally bioavailable HIF-1 inhibitor PX-478 reduces HIF-1 protein levels and signaling in vitro in a dose-dependent manner and provides direct radiosensitization of hypoxic cancer cells in clonogenic survival assays using C6 glioma, HN5 and UMSCCa10 squamous cells, and Panc-1 pancreatic adenocarcinoma cell lines. However, PX-478 yields striking in vivo tumor sensitization to single-dose irradiation, which cannot be explained by incremental improvement in direct tumor cell killing. We show that PX-478 prevents postradiation HIF-1 signaling and abrogates downstream stromal adaptation in C6 and HN5 reporter xenografts as measured by serial ultrasound, vascular magnetic resonance imaging, and hypoxia response element–specific micro–positron emission tomography imaging. The primacy of indirect PX-478 in vivo effects was corroborated by our findings that (a) either concurrent or early postradiation sequencing of PX-478 provides roughly equivalent sensitization and (b) constitutive vascular endothelial growth factor expression maintains refractory tumor vessel function and progression following combined radiation and PX-478. These results confirm that disruption of postradiation adaptive HIF-1 signaling by PX-478 imparts increased therapeutic efficacy through blockade of HIF-1–dependent reconstitution of tumor stromal function. Successful translation of targeted HIF-1 radiosensitization to the clinical setting will require specific consideration of tumor microenvironmental effects and mechanisms.
Accumulating evidence suggests that hypoxia-inducible factor-2 (HIF-2) is important for the cellular response to hypoxia. However, it is not clear how HIF-2 is regulated under hypoxic conditions. We investigated kinetic changes in redox status and HIF-2α accumulation in hypoxic SH-SY5Y cells. Our results demonstrated that hypoxia causes a reducing environment and increases HIF-2α protein levels. Experiments with redox modulations (N-acetylcysteine and l-buthionine sulfoximine) confirmed that a reducing environment induced HIF-2α accumulation while an oxidizing environment decreased it. In addition, experiments with SOD mimic, catalase, and exogenous H2O2 provided evidence that the presence of H2O2 down-regulated the amount of HIF-2α protein. This study offers novel evidence supporting redox status regulation of HIF-2α accumulation under hypoxic conditions.
HIF-2α; Hypoxia; Redox status; SH-SY5Y; Stroke; Brain tumor
Hypoxia preconditioning protects corneal stromal cells from stress-induced death. This study determined whether the transcription factor HIF-1α (Hypoxia Inducible Factor) is responsible and whether this is promulgated by VEGF (Vascular Endothelial Growth Factor).
Cultured bovine stromal cells were preconditioned with hypoxia in the presence of cadmium chloride, a chemical inhibitor of HIF-1α, and HIF-1α siRNA to test if HIF-1α activity is needed for hypoxia preconditioning protection from UV-irradiation induced cell death. TUNEL assay was used to detect cell apoptosis after UV-irradiation. RT-PCR and western blot were used to detect the presence of HIF-1α and VEGF in transcriptional and translational levels.
During hypoxia (0.5% O2), 5 μM cadmium chloride completely inhibited HIF-1α expression and reversed the protection by hypoxia preconditioning. HIF-1α siRNA (15 nM) reduced HIF-1α expression by 90% and produced a complete loss of protection provided by hypoxia preconditioning. Since VEGF is induced by hypoxia, can be HIF-1α dependent, and is often protective, we examined the changes in transcription of VEGF and its receptors after 4 h of hypoxia preconditioning. VEGF and its receptors Flt-1 and Flk-1 are up-regulated after hypoxia preconditioning. However, the transcription and translation of VEGF were paradoxically increased by siHIF-1α, suggesting that VEGF expression in stromal cells is not down-stream of HIF-1α.
These findings demonstrate that hypoxia preconditioning protection in corneal stromal cells requires HIF-1α, but that VEGF is not a component of the protection.
Hypoxia may regulate the proliferation of diverse stem cells. Our previous study showed that hypoxia promoted the proliferation of embryonic neural stem/progenitor cells (NPCs) and that hypoxia inducible factor-1(HIF-1) was critical in this process. HIF-1 could be stabilized under hypoxic conditions, and heat shock protein 90 (HSP90) is an essential protein that controls the activity and stabilization of HIF-1α. In the present work, we investigate whether HSP90 is involved in proliferation of NPCs under hypoxia by regulating HIF-1α stabilization. Geldanamycin (GA), an HSP90 inhibitor, decreased the expression of HIF-1α in NPCs during hypoxia-driven proliferation and reduced the expression level of HIF-1α protein under hypoxia in a time-dependent manner. The proliferation of NPCs induced by hypoxia was inhibited after GA treatment for 24 h. Another HSP90 inhibitor, radicicol, had the same effect on NPCs as GA. Furthermore, the expression of erythropoietin (EPO) and vascular endothelial growth factor (VEGF) in NPCs under hypoxia was suppressed by GA. The above data indicated that HSP90 might be involved in regulation of hypoxia-driven proliferation.
Embryonic neural stem or progenitor cells; Geldanamycin; HIF-1α; HSP90; Hypoxia
The incidence of thyroid cancer is four to five times higher in women than in men, suggesting a role for estrogen (E2) in the pathogenesis of thyroid proliferative disease (TPD) that comprises cancer and goiter. The objective of this study was to investigate the antiestrogenic activity of 3,3′-diindolylmethane (DIM), a bioactive compound derived from cruciferous vegetables, in patients with TPD.
In this limited phase I clinical trial study, patients found to have TPD were administered 300 mg of DIM per day for 14 days. Patients subsequently underwent a total or partial thyroidectomy, and tissue, urine, and serum samples were collected. Pre- and post-DIM serum and urine samples were analyzed for DIM levels as well as estrogen metabolites. DIM levels were also determined in thyroid tissue samples.
DIM was detectable in thyroid tissue, serum, and urine of patients after 14 days of supplementation. Urine analyses revealed that DIM modulated estrogen metabolism in patients with TPD. There was an increase in the ratio of 2-hydroxyestrones (C-2) to 16α-hydroxyestrone (C-16), consistent with antiestrogenic activity that results in more of C-2 product compared with C-16.
Our data suggest that DIM enhances estrogen metabolism in TPD patients and can potentially serve as an antiestrogenic dietary supplement to help reduce the risk of developing TPD. The fact that DIM is detected in thyroid tissue implicates that it can manifest its antiestrogenic activity in situ to modulate TPD.
► AAI increases whereas OTA decreases production of proangiogenic VEGF. ► The upregulation of VEGF expression by AAI is caused by induction of SP-1 and HIFs. ► Hypoxia prevents OTA-diminished VEGF production ► The effect of hypoxia on OTA-reduced VEGF is mediated by HIF-2α but not HIF-1α.
Aristolochic acid I (AAI) and ochratoxin A (OTA) cause chronic kidney diseases. Recently, the contribution of hypoxic injuries and angiogenic disturbances to nephropathies has been suggested, but underlying mechanisms have not been fully clarified yet.
In porcine kidney epithelial cell line, LLC-PK1 cells, treatment with non-toxic doses of AAI increased whereas with OTA decreased production of vascular endothelial growth factor (VEGF), the angiogenic factor with well-defined functions in kidney. Moreover, the activity of transcription factors regulating VEGF expression was differentially affected by examined compounds. Activity of hypoxia inducible factors (HIFs) and SP-1 was increased by AAI but diminished by OTA. Interestingly, AP-1 activity was inhibited while NFκB was not influenced by both toxins. Mithramycin A, a SP-1 inhibitor, as well as chetomin, an inhibitor of HIFs, reversed AAI-induced up-regulation of VEGF synthesis, indicating the importance of SP-1 and HIFs in this effect. Additionally, adenoviral overexpression of HIF-2α but not HIF-1α prevented OTA-diminished VEGF production suggesting the protective effect of this isoform towards the consequences exerted by OTA.
These observations provide new insight into complex impact of AAI and OTA on angiogenic gene regulation. Additionally, it adds to our understanding of hypoxia influence on nephropathies pathology.
AA, aristolochic acid; AAI, aristolochic acid I; AAII, aristolochic acid II; AA-ATN, aristolochic acid-induced acute tubular necrosis; AAN, aristolochic acid-induced nephropathy; AdGFP, adenoviral vectors containing GFP cDNA; AdHIF-1,-2α, adenoviral vectors containing HIF-1,-2α cDNA; β-gal, β-galactosidase; BEN, Balkan endemic nephropathy; CKDs, chronic kidney diseases; EMT, epithelial to mesenchymal cell transformation; GFP, green fluorescent protein; HIF, hypoxia inducible factor; HRE, hypoxia responsive element; HRP, horseradish peroxidase; LDH, lactate dehydrogenase; LLC-PK1, porcine kidney epithelial cell line; IARC, The International Agency for Research on Cancer; OTA, ochratoxin A; ROS, reactive oxygen species; RT, room temperature; TGFβ, transforming growth factor β; VEGF, vascular endothelial growth factor; Nephropathy; Kidney diseases; Vascular endothelial growth factor; Angiogenesis; Hypoxia; LLC-PK1
The hypoxia-inducible factor (HIF) is a key regulator of the transcriptional response to hypoxia. While the mechanism underpinning HIF activation is well understood, little is known about its resolution. Both the protein and the mRNA levels of HIF-1α (but not HIF-2α) were decreased in intestinal epithelial cells exposed to prolonged hypoxia. Coincident with this, microRNA (miRNA) array analysis revealed multiple hypoxia-inducible miRNAs. Among these was miRNA-155 (miR-155), which is predicted to target HIF-1α mRNA. We confirmed the hypoxic upregulation of miR-155 in cultured cells and intestinal tissue from mice exposed to hypoxia. Furthermore, a role for HIF-1α in the induction of miR-155 in hypoxia was suggested by the identification of hypoxia response elements in the miR-155 promoter and confirmed experimentally. Application of miR-155 decreased the HIF-1α mRNA, protein, and transcriptional activity in hypoxia, and neutralization of endogenous miR-155 reversed the resolution of HIF-1α stabilization and activity. Based on these data and a mathematical model of HIF-1α suppression by miR-155, we propose that miR-155 induction contributes to an isoform-specific negative-feedback loop for the resolution of HIF-1α activity in cells exposed to prolonged hypoxia, leading to oscillatory behavior of HIF-1α-dependent transcription.
Human papillomaviruses (HPV) are the causative agents of cervical cancer and have been shown to increase expression of pro-angiogenic factors from infected cells. Many angiogenic factors are regulated by hypoxia inducible factor 1α (HIF-1α). We investigated whether HPV31 affects the levels of HIF-1α under normal and hypoxic conditions. Our studies indicate that cells containing complete HPV31 genomes showed enhanced levels of HIF-1α upon treatment with the hypoxia mimic DFO, which resulted from protein stabilization and lead to increased expression of some but not all HIF-1α target genes. Both HPV E6 and E7 were able independently to enhance induction of HIF-1α upon DFO treatment. Enhancement of HIF-1α stability was not restricted to high risk HPV types, as HPV11, a low risk HPV type, mediated a similar effect. These findings shed light on mechanisms by which HPV contributes to angiogenesis both in benign cervical lesions and in cervical cancers.
Papillomaviruses (HPV); Angiogenesis; Hypoxia; Keratinocytes; Hypoxia inducible factor-1 (HIF-1); Deferoxamine mesylate (DFO); Vascular Endothelial Growth Factor (VEGF); Carbonic Anhydrase (CAIX)
3,3′-Diindolylmethane (DIM), a major condensation product of indole-3-carbinol (I3C), exhibits chemopreventive properties in animal models of cancer. Recent studies have shown that DIM stimulates interferon-gamma (IFN-γ) production and potentiates the IFN-γ signaling pathway in human breast cancer cells via a mechanism that includes increased expression of the IFN-γ receptor. The goal of this study was to test the hypothesis that DIM modulates the murine immune function. Specifically, the effects of DIM were evaluated in a panel of murine immune function tests that included splenocyte proliferation, reactive oxygen species (ROS) generation, cytokine production and resistance to viral infection. DIM was found to induce proliferation of splenocytes as well as augment mitogen- and IL-2-induced splenocyte proliferation. DIM also stimulated the production of ROS by murine peritoneal macrophage cultures. Oral administration of DIM, but not intraperitoneal injection (i.p.), induced elevation of serum cytokines in mice, including interleukin (IL)-6, granulocyte-colony stimulating factor (G-CSF), IL-12 and IFN-γ. Finally, in a model of enteric virus infection, oral DIM administration to mice enhanced both clearance of reovirus from the GI tract and the subsequent mucosal IgA response. Thus, DIM is a potent stimulator of immune function. This property might contribute to the cancer inhibitory effects of this indole.
3,3′-diindolylmethane; Immune stimulation; Cytokine; Lymphocyte proliferation; reactive oxygen species