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1.  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.
doi:10.1371/journal.pone.0034975
PMCID: PMC3325915  PMID: 22514694
2.  Pharmacokinetic evaluation and In Vitro–In Vivo Correlation (IVIVC) of novel methylene-substituted 3,3′ diindolylmethane (DIM) 
Purpose
3,3′-Diindolylmethane (DIM) is the major in vivo product of the acid-catalyzed oligomerization of indole-3-carbinol present in cruciferous vegetables. 1, 1-bis (3′-indolyl)-1-(p-substituted phenyl) methanes [C-substituted diindolylmethanes (C-DIMs)] are a new class of anticancer compounds derived from indole 3-carbinol. Despite rapidly increasing knowledge regarding mechanisms responsible for the chemopreventive properties of DIM-C-pPhC6H5, there have been relatively few studies determining the absorption and pharmacokinetic properties of DIM-C-pPhC6H5 to explore its clinical utility.
Methods
In this study, we assessed the solubility, lipophilicity and Caco-2 cell permeability of methylene-substituted DIM. Pharmacokinetic properties in rats were determined following i.v. and oral administration of a novel analog of DIM. Pharmacokinetic parameters were determined using noncompartmental and compartmental techniques with WinNonlin® 5.0 software. To explore potential In Vitro–In Vivo Correlation (IVIVC) between the in vitro permeability values, and the oral absorption pharmacokinetics, we employed deconvolution of i.v. and oral data using a three compartment Exact Loo–Riegelman method.
Results
The oral absorption and disposition were described by a three compartment model with combined zero-order/Michaelis–Menten limited systemic uptake using differential equations, at physiologically relevant doses. The saturation model obtained accounts for a nonlinear change in Cmax/Dose, and the absolute bioavailability (0.13 ± 0.06) was also dose dependent. The absorption rate profile of DIMC-pPhC6H5 across Caco-2 cells was significantly different than in vivo. Conclusions: The pharmacokinetic absorption model presented represents a useful basis for obtaining plasma level predictability for poorly bioavailable, highly lipophilic drugs, such as the DIM analog DIM-C-pPhC6H5.
doi:10.1016/j.ejps.2012.01.012
PMCID: PMC3374645  PMID: 22342559
Anticancer; Pharmacokinetic model; Chemoprevention; DIM; Anticarcinogen
3.  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.
doi:10.1371/journal.pone.0026064
PMCID: PMC3189247  PMID: 22016813
4.  Induction of G1 and G2/M cell cycle arrests by the dietary compound 3,3'-diindolylmethane in HT-29 human colon cancer cells 
BMC Gastroenterology  2009;9:39.
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-230X-9-39
PMCID: PMC2700124  PMID: 19480695
5.  3,3′-diindolylmethane, but not indole-3-carbinol, inhibits histone deacetylase activity in prostate cancer cells 
Toxicology and applied pharmacology  2012;263(3):345-351.
Increased consumption of cruciferous vegetables is associated with a reduced risk of developing prostate cancer. Indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) are phytochemicals derived from cruciferous vegetables that have shown promise in inhibiting prostate cancer in experimental models. Histone deacetylase (HDAC) inhibition is an emerging target for cancer prevention and therapy. We sought to examine the effects of I3C and DIM on HDACs in human prostate cancer cell lines: androgen insensitive PC-3cells and androgen sensitive LNCaP cells. I3C modestly inhibited HDAC activity in LNCaP cells by 25% but no inhibition of HDAC activity was detected in PC-3 cells. In contrast, DIM significantly inhibited HDAC activity in both cell lines by as much as 66%. Decreases in HDAC activity correlated with increased expression of p21, a known target of HDAC inhibitors. DIM treatment caused a significant decrease in the expression of HDAC2 protein in both cancer cell lines but no significant change in the protein levels of HDAC1, HDAC3, HDAC4, HDAC6 or HDAC8 were detected. Taken together, these results show that inhibition of HDAC activity by DIM may contribute to the phytochemicals anti-proliferative effects in the prostate. The ability of DIM to target aberrant epigenetic patterns, in addition to its effects on detoxification of carcinogens, may make it an effective chemopreventive agent by targeting multiple stages of prostate carcinogenesis.
doi:10.1016/j.taap.2012.07.007
PMCID: PMC3428467  PMID: 22800507
3,3′-diindolylmethane; histone deacetylase; indole-3-carbinol; prostate cancer; chemoprevention; epigenetics
6.  Modulation of Aflatoxin B1–Mediated Genotoxicity in Primary Cultures of Human Hepatocytes by Diindolylmethane, Curcumin, and Xanthohumols 
Toxicological Sciences  2009;112(2):303-310.
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.
doi:10.1093/toxsci/kfp206
PMCID: PMC2777076  PMID: 19770484
phytochemicals; biotransformation; hepatocytes; human; CYP
7.  Cell Cycle-Dependent Effects of 3,3′-Diindolylmethane on Proliferation and Apoptosis of Prostate Cancer Cells 
Journal of cellular physiology  2009;219(1):94-99.
Epidemiological studies have shown that a diet rich in fruits and cruciferous vegetables is associated with a lower risk of prostate cancer. Indole-3-carbinol (I3C) and its dimeric product 3,3′-diindolylmethane (DIM) have been shown to exhibit anti-tumor activity both in vitro and in vivo. Recently, we have reported that a formulated DIM (B-DIM) induced apoptosis and inhibited growth, angiogenesis, and invasion of prostate cancer cells by regulating Akt, NF-κB, VEGF and the androgen receptor (AR) signaling pathway. However, the precise molecular mechanism(s) by which B-DIM inhibits prostate cancer cell growth and induces apoptosis have not been fully elucidated. Most importantly, it is not known how B-DIM affects cell cycle regulators and proteasome activity, which are critically involved in cell growth and apoptosis. In this study, we investigated the effects of B-DIM on proteasome activity and AR transactivation with respect to B-DIM-mediated cell cycle regulation and induction of apoptosis in both androgen-sensitive LNCaP and androgen-insensitive C4-2B prostate cancer cells. We believe that our results show for the first time the cell cycle-dependent effects of B-DIM on proliferation and apoptosis of synchronized prostate cancer cells progressing from G1 to S phase. B-DIM inhibited this progression by induction of p27Kip1 and down-regulation of AR. We also show for the first time that B-DIM inhibits proteasome activity in S phase, leading to the inactivation of NF-κB signaling and induction of apoptosis in LNCaP and C4-2B cells. These results suggest that B-DIM could be a potent agent for the prevention and/or treatment of both hormone sensitive as well as hormone-refractory prostate cancer.
doi:10.1002/jcp.21650
PMCID: PMC3785943  PMID: 19062173
8.  3,3′-diindolylmethane induces activating transcription factor 3 (ATF3) via ATF4 in human colorectal cancer cells 
A 3,3′-diindolylmethane (DIM) is a major in vivo condensation product of indole-3-carbinol (I3C), which are present in cruciferous vegetables. Although these compounds have been widely implicated in anti-tumorigenic and pro-apoptotic properties in animal as well as in vitro models of cancer, the underlying cellular mechanisms regulated by DIM are only partially understood. Activating transcription factor 3 (ATF3) is a member of the ATF/CREB subfamily of the basic-region leucine zipper (bZIP) family and has been known to induce apoptosis in human colorectal cancer cells. The present study was performed to elucidate the molecular mechanism of ATF3 induction by DIM in human CRC cells. The DIM treatment induced apoptosis and induced ATF3 gene expression at protein and mRNA levels. DIM increased ATF3 promoter activity and the region of −84 to +34 within ATF3 promoter was responsible for promoter activation by DIM. This region contained an ATF binding site. Deletion and point mutation of the ATF binding site (−23 to −16) abolished ATF3 promoter activation by DIM and overexpression of ATF4 enhanced ATF3 transactivation. Chromatin immunoprecipitation (ChIP) assay confirmed the binding of ATF4 in the ATF3 promoter. Inhibition of ATF4 expression by siRNA results in repression of DIM-induced ATF3 expression. The current study demonstrates that DIM stimulates ATF3 expression through ATF4-mediated pathway and subsequently induces apoptosis in human colorectal cancer cells.
doi:10.1016/j.jnutbio.2012.03.016
PMCID: PMC3481000  PMID: 22819556
DIM; ATF3; ATF4; colon cancer
9.  Hypoxia‐inducible factor expression in human RPE cells 
The British Journal of Ophthalmology  2007;91(10):1406-1410.
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1136/bjo.2007.123125
PMCID: PMC2001032  PMID: 17567660
diabetic retinopathy; VEGF; erythropoietin; hypoxia‐inducible factor; proline hydroxylase
10.  Enzyme-Linked Immunosorbent Assay for Pharmacological Studies Targeting Hypoxia-Inducible Factor 1α 
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.
doi:10.1128/CDLI.12.5.660-664.2005
PMCID: PMC1112088  PMID: 15879029
11.  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.
doi:10.1016/j.canlet.2011.12.006
PMCID: PMC3288414  PMID: 22155105
radiation; B-DIM; prostate cancer; androgen receptor
12.  3,3′-Diindolylmethane Protects against Cardiac Hypertrophy via 5′-Adenosine Monophosphate-Activated Protein Kinase-α2 
PLoS ONE  2013;8(1):e53427.
Purpose
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.
Methods
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.
Results
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.
Conclusion
DIM significantly improves left ventricular function via the activation of AMPKα2 in a murine model of cardiac hypertrophy.
doi:10.1371/journal.pone.0053427
PMCID: PMC3541184  PMID: 23326427
13.  Identification of small molecule compounds that inhibit the HIF-1 signaling pathway 
Molecular Cancer  2009;8:117.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1476-4598-8-117
PMCID: PMC2797767  PMID: 20003191
14.  The Caulerpa Pigment Caulerpin Inhibits HIF-1 Activation and Mitochondrial Respiration 
Journal of natural products  2009;72(12):2104-2109.
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.
doi:10.1021/np9005794
PMCID: PMC2798910  PMID: 19921787
15.  Activation of AMP-Activated Protein Kinase by 3,3′-Diindolylmethane (DIM) Is Associated with Human Prostate Cancer Cell Death In Vitro and In Vivo 
PLoS ONE  2012;7(10):e47186.
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.
doi:10.1371/journal.pone.0047186
PMCID: PMC3467201  PMID: 23056607
16.  Interplay of Genes Regulated by Estrogen and Diindolylmethane in Breast Cancer Cell Lines 
Molecular Medicine  2007;13(1-2):69-78.
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.
doi:10.2119/2006-00038.Mulvey
PMCID: PMC1869626  PMID: 17515958
17.  Stathmin Regulates Hypoxia-Inducible Factor-1α Expression through the Mammalian Target of Rapamycin Pathway in Ovarian Clear Cell Adenocarcinoma 
ISRN Pharmacology  2013;2013:279593.
Stathmin, a microtubule-destabilizing phosphoprotein, is highly expressed in ovarian cancer, but the pathophysiological significance of this protein in ovarian carcinoma cells remains poorly understood. This study reports the involvement of stathmin in the mTOR/HIF-1α/VEGF pathway in ovarian clear cell adenocarcinoma (CCA) during hypoxia. HIF-1α protein and VEGF mRNA levels were markedly elevated in RMG-1 cells, a CCA cell line, cultured under hypoxic conditions. Rapamycin, an inhibitor of mTOR complex 1, reduced the level of HIF-1α and blocked phosphorylation of ribosomal protein S6 kinase 1 (S6K), a transcriptional regulator of mTOR, demonstrating that hypoxia activates mTOR/S6K/HIF-1α signaling in CCA. Furthermore, stathmin knockdown inhibited hypoxia-induced HIF-1α and VEGF expression and S6K phosphorylation. The silencing of stathmin expression also reduced Akt phosphorylation, a critical event in the mTOR/HIF-1α/VEGF signaling pathway. By contrast, stathmin overexpression upregulated hypoxia-induced HIF-1α and VEGF expression in OVCAR-3 cells, another CCA cell line. In addition, suppression of Akt activation by wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, decreased HIF-1α and VEGF expression. These results illustrate that regulation of HIF-1α through the PI3K/Akt/mTOR pathway is controlled by stathmin in CCA. Our findings point to a new mechanism of stathmin regulation during ovarian cancer.
doi:10.1155/2013/279593
PMCID: PMC3683482  PMID: 23819061
18.  3,3′-Diindolylmethane Induces a G1 Arrest in Human Prostate Cancer Cells Irrespective of Androgen Receptor and p53 Status 
Biochemical pharmacology  2009;78(5):469-476.
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.
doi:10.1016/j.bcp.2009.05.008
PMCID: PMC2706920  PMID: 19433067
3; 3′-Diinolylmethane; Prostate cancer; Cell cycle arrest; p27Kip1; p38 MAPK; Cancer
19.  Inhibition of ARNT severely compromises endothelial cell viability and function in response to moderate hypoxia 
Angiogenesis  2012;15(3):409-420.
Hypoxia Inducible Factor (HIF) is a master heterodimeric transcriptional regulator of oxygen (O2) homeostasis critical to proper angiogenic responses. Due to the distinctive coexpression of HIF-1α and HIF-2α subunits in endothelial cells, our goal was to examine the genetic elimination of HIF transcriptional activity in response to physiological hypoxic conditions by using a genetic model in which the required HIF-β subunit (ARNT, Aryl hydrocarbon Receptor Nuclear Translocator) to HIF transcriptional responses was depleted. Endothelial cells (ECs) and aortic explants were isolated from ArntloxP/loxP mice and infected with Adenovirus -Cre/GFP or control -GFP. We observed that moderate levels of 2.5% O2 promoted vessel sprouting, growth, and branching in control aortic ring assays while growth from Adenovirus -Cre infected explants was compromised. Primary Adenovirus -Cre infected EC cultures featured adverse migration and tube formation phenotypes. Primary pulmonary or cardiac ARNT-deleted ECs also failed to proliferate and survive in response to 8 or 2.5% O2 and hydrogen peroxide treatment. Our data demonstrates that ARNT promotes EC migration and vessel outgrowth and indispensible for the proliferation and preservation of ECs in response to the physiological environmental cue of hypoxia. Thus, these results demonstrate that ARNT plays a critical intrinsic role in ECs and support a critical role for the collaboration of HIF-1 and HIF-2 transcriptional activity in these cells.
doi:10.1007/s10456-012-9269-x
PMCID: PMC3661274  PMID: 22484908
Angiogenesis; ARNT; HIF; physiological hypoxia; endothelium
20.  Hypoxia and Hypoxia-Inducible Factor in the Burn Wound 
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.
doi:10.1111/j.1524-475X.2010.00656.x
PMCID: PMC3075089  PMID: 21362088
ypoxia; Hypoxia-inducible factor -10α; Burn; Wound Healing; Ki67 Cell Proliferation; Keratin17
21.  Hypoxic Regulation of VEGF, HIF-1(alpha) in Coronary Collaterals Development 
Background
The interindividual variability for the development of collaterals in coronary artery disease is dependent on the hypoxic induction level of VEGF. To determine whether the hypoxic induction of VEGF is controlled by the transcription of HIF-1 (alpha), the VEGF and HIF-1 (alpha) m-RNA levels were correlated to hypoxia in monocytes harvested from patients with coronary artery disease.
Methods
The collateral scoring system used was modified from the TIMI system. The mononuclear cell layer of the patients' blood was cultured in hypoxia (1% O2, 5% CO2, 94%N2) and normoxia (5% CO2, 95% room air) for 17 hours. The VEGF and HIF-1 (alpha) mRNA levels were measured using a RT-PCR technique. We calculated the fold inductionsof VEGF, HIF-1 (alpha) mRNA with hypoxia by dividing thehypoxic and the normoxic values.
Results
We found significantly higher hypoxic inductions of VEGF m-RNA in patients with collaterals compared to patients with no collaterals. However, there was no differencein the hypoxic inductions of HIF-1 (alpha) between the two groups (VEGF m-RNA mean fold inductions 3.71±3.30 versus 1.65±0.62, p=0.012, HIF-1(alpha) mRNA 1.42±0.58 versus 1.20±0.39, p=0.165).
Conclusions
We concluded that the interindividual variability in the hypoxic inductions of VEGF m-RNA in monocytes in patients is not controlled by the transcriptional levels of HIF-1 (alpha) with hypoxia. These findings suggest that a mechanism such as the post-transcriptional modification of HIF-1(alpha) is involved in the hypoxic inductions of VEGF.
doi:10.3904/kjim.2005.20.4.295
PMCID: PMC3891075  PMID: 16491827
VEGF; HIF-1(alpha); Collaterals
22.  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.
doi:10.1186/1756-9966-30-61
PMCID: PMC3113749  PMID: 21595915
Hypoxia; Redox; Multidrug resistance; HepG2
23.  Estrogen Induced Metastatic Modulators MMP-2 and MMP-9 Are Targets of 3,3′-Diindolylmethane in Thyroid Cancer 
PLoS ONE  2011;6(1):e15879.
Background
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.
Methodology/Principal Findings
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.
Conclusion/Significance
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.
doi:10.1371/journal.pone.0015879
PMCID: PMC3022654  PMID: 21267453
24.  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.
doi:10.1038/onc.2013.115
PMCID: PMC3868635  PMID: 23604114
cotranscriptional activation; HIF; hypoxia; STAT3; transcription
25.  Inhibition of Siah ubiquitin ligase function 
Oncogene  2008;28(2):289-296.
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.
doi:10.1038/onc.2008.382
PMCID: PMC3000903  PMID: 18850011

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