In Con8 rat mammary epithelial tumor cells, indirect immunofluorescence revealed that Sgk (Serum- and Glucocorticoid-regulated kinase) and Erk/MAPK (Extracellular signal-Regulated protein kinase/Mitogen Activated protein kinase) co-localized to the nucleus in serum treated cells and to the cytoplasmic compartment in cells treated with the synthetic glucocorticoid dexamethasone. Moreover, the subcellular distribution of the importin-alpha nuclear transport protein was similarly regulated in a signal dependent manner. In vitro GST-pull down assays revealed the direct interaction of importin-alpha with either Sgk or Erk/MAPK, while RNA interference knockdown of importin-alpha expression disrupted the localization of both Sgk and Erk into the nucleus of serum-treated cells. Wild type or kinase dead forms of Sgk co-immunoprecipitated with Erk/MAPK from either serum- or dexamethasone-treated mammary tumor cells, suggesting the existence of a protein complex containing both kinases. In serum treated cells, nucleus residing Sgk and Erk/MAPK were both hyperphosphorylated, indicative of their active states, whereas, in dexamethasone treated cells Erk/MAPK, but not Sgk, was in its inactive hypophosphorylated state. Treatment with a MEK inhibitor, which inactivates Erk/MAPK, caused the relocalization of both Sgk and ERK to the cytoplasm. We therefore propose that the signal dependent co-localization of Sgk and Erk/MAPK mediated by importin-alpha represents a new pathway of signal integration between steroid and serum/growth factor regulated pathways.
BZL101 is an aqueous extract from the Scutellaria barbata plant shown to have anticancer properties in a variety of human cancers. In order to determine its efficacy on human reproductive cancers, we assessed the responses of two human breast cancer cell lines, estrogen sensitive MCF7 and estrogen insensitive MDA-MB-231, and of two human prostate cancer cell lines, androgen sensitive LNCaP and androgen insensitive PC3 which are human cell lines that represent early and late stage reproductive cancers. BZL101 inhibited reproductive cancer growth in all cell lines by regulating expression levels of key cell cycle components that differ with respect to the cancer cell phenotypes. In early stage estrogen sensitive MCF7 cells, BZL101 induced a G1 cell cycle arrest and ablated expression of key G1 cell cycle regulators Cyclin D1, CDK2 and CDK4, as well as growth factor stimulatory pathways and estrogen receptor-α expression. Transfection of luciferase reporter plasmids revealed that the loss of CDK2, CDK4 and estrogen receptor-α transcript expression resulted from the BZL-dependent ablation of promoter activities. BZL101 growth arrests early stage androgen sensitive LNCaP cells in the G2/M phase with corresponding decreases in Cyclin B1, CDK1 and androgen receptor expression. In late stage hormone insensitive breast (MDA-MB-231) and prostate (PC3) cancer cells, BZL101 induced an S phase arrest with corresponding ablations in Cyclin A2 and CDK2 expression. Our results demonstrate that BZL101 exerts phenotype specific anti-proliferative gene expression responses in human breast and prostate cancer cells, which will be valuable in the potential development of BZL-based therapeutic strategies for human reproductive cancers.
Scutellaria barbata; BZL101; cell cycle; CDK gene expression; breast cancer; prostate cancer; steroid receptors
We have established in human breast cancer cells that indole-3-carbinol (I3C), a promising anti-cancer phytochemical from Brassica vegetables, ablates ERα expression by stimulating the Rbx-1 E3 ligase mediated degradation of ERα protein and disruption of a cross-regulatory positive feedback loop involving ERα and the GATA3 transcription factor.
Estrogen receptor (ER)α is a critical target of therapeutic strategies to control the proliferation of hormone-dependent breast cancers. Preferred clinical options have significant adverse side effects that can lead to treatment resistance due to the persistence of active estrogen receptors. We have established the cellular mechanism by which indole-3-carbinol (I3C), a promising anticancer phytochemical from Brassica vegetables, ablates ERα expression, and we have uncovered a critical role for the GATA3 transcription factor in this indole-regulated cascade. I3C-dependent activation of the aryl hydrocarbon receptor (AhR) initiates Rbx-1 E3 ligase-mediated ubiquitination and proteasomal degradation of ERα protein. I3C inhibits endogenous binding of ERα with the 3′-enhancer region of GATA3 and disrupts endogenous GATA3 interactions with the ERα promoter, leading to a loss of GATA3 and ERα expression. Ectopic expression of GATA3 has no effect on I3C-induced ERα protein degradation but does prevent I3C inhibition of ERα promoter activity, demonstrating the importance of GATA3 in this I3C-triggered cascade. Our preclinical results implicate I3C as a novel anticancer agent in human cancers that coexpress ERα, GATA3, and AhR, a combination found in a large percentage of breast cancers but not in other critical ERα target tissues essential to patient health.
The two Rho kinase isoforms ROCK1 and ROCK2 are downstream effectors of the small GTPase RhoA, although relatively little is known about potential isoform specific functions or the selective control of their cellular activities. Using Con8 rat mammary epithelial cells, we show that the synthetic glucocorticoid dexamethasone strongly stimulates the level of ROCK2 protein, which accounts for the increase in total cellular ROCK2 activity, whereas, steroid treatment down-regulated ROCK1 specific kinase activity without altering ROCK1 protein levels. In Con8 cells, the glucocorticoid induced formation of tight junctions requires the steroid-mediated down-regulation RhoA and function of the RhoA antagonist Rnd3. Treatment with the ROCK inhibitor Y-27632 ablated both the glucocorticoid-induced and Rnd3-mediated stimulation in tight junction sealing. Taken together, our results demonstrate that the expression and activity of ROCK1 and ROCK2 can be uncoupled in a signal-dependent manner, and further implicate a new function for ROCK2 in the steroid control of tight junction dynamics.
Rho kinase isoforms; ROCK1; ROCK2; glucocorticoids; differential regulation; tight junction dynamics
Indole-3-carbinol (I3C), a naturally occurring hydrolysis product of glucobrassicin from cruciferous vegetables such as broccoli, cabbage and Brussels sprouts, is an anticancer phytochemical that triggers complementary sets of antiproliferative pathways to induce a cell cycle arrest of estrogen-responsive MCF7 breast cancer cells. I3C strongly downregulated transcript expression of the catalytic subunit of the human telomerase (hTERT) gene, which correlated with the dose-dependent indole-mediated G1 cell cycle arrest without altering the transcript levels of the RNA template (hTR) for telomerase elongation. Exogenous expression of hTERT driven by a constitutive promoter prevented the I3C-induced cell cycle arrest and rescued the I3C inhibition of telomerase enzymatic activity and activation of cellular senescence. Time course studies showed that I3C downregulated expression of estrogen receptor-alpha (ERα) and cyclin-dependent kinase-6 transcripts levels (which is regulated through the Sp1 transcription factor) prior to the downregulation of hTERT suggesting a mechanistic link. Chromatin immunoprecipitation assays demonstrated that I3C disrupted endogenous interactions of both ERα and Sp1 with an estrogen response element–Sp1 composite element within the hTERT promoter. I3C inhibited 17β-estradiol stimulated hTERT expression and stimulated the production of threonine-phosphorylated Sp1, which inhibits Sp1–DNA interactions. Exogenous expression of both ERα and Sp1, but not either alone, in MCF7 cells blocked the I3C-mediated downregulation of hTERT expression. These results demonstrate that I3C disrupts the combined ERα- and Sp1-driven transcription of hTERT gene expression, which plays a significant role in the I3C-induced cell cycle arrest of human breast cancer cells.
Indole-3-carbinol (I3C), a naturally occurring component of Brassica vegetables, such as cabbage, broccoli, and Brussels sprouts, induces a G1 cell cycle arrest of human breast cancer cells. Structure-activity relationships of I3C that mediate this anti-proliferative response were investigated using synthetic and natural I3C derivatives that contain substitutions at the indole nitrogen. Nitrogen substitutions included N-alkoxy substituents of one to four carbons in length, which inhibit dehydration and the formation of the reactive indolenine. Analysis of growth and cell cycle arrest of indole-treated human breast cancer cells revealed a striking increase in efficacy of the N-alkoxy I3C derivatives that is significantly enhanced by the presence of increasing carbon lengths of the N-alkoxy substituents. Compared to I3C, the half maximal growth arrest responses occurred at 23-fold lower indole concentration for N-methoxy-I3C, 50-fold lower concentration for N-ethoxy-I3C, 217-fold lower concentration for N-propoxy-I3C, and 470-fold lower concentration for N-butoxy-I3C. At these lower concentrations, each of the N-alkoxy substituted compounds induced the characteristic I3C response in that CDK6 gene expression, CDK6 promoter activity, and CDK2 specific enzymatic activity for its retinoblastoma protein substrate were strongly down-regulated. 3-Methoxymethylindole and 3-ethoxymethylindole were approximately as bioactive as I3C, whereas, both tryptophol and melatonine failed to induce the cell cycle arrest, showing the importance of the C-3 hydroxy methyl substituent on the indole ring. Taken together, our study establishes the first I3C structure activity relationship for cytostatic activities, and implicates I3C-based N-alkoxy derivatives as a novel class of potentially more potent experimental therapeutics for breast cancer.
I3C; synthetic derivatives; N-alkoxy constituents; breast cancer cells; cell cycle arrest
Indole-3-carbinol (I3C), a natural autolysis product of a gluccosinolate present in Brassica vegetables such as broccoli and cabbage, has anti-proliferative and anti-estrogenic activities in human breast cancer cells. A new and significantly more potent I3C analogue, 1-benzyl-I3C was synthesized, and in comparison to I3C, this novel derivative displayed an approximate 1000-fold enhanced potency in suppressing the growth of both estrogen responsive (MCF-7) and estrogen independent (MDA-MB-231) human breast cancer cells (I3C IC50 of 52 μM, and 1-benzyl-I3C IC50 of 0.05 μM). At significantly lower concentrations, 1-benzyl-I3C induced a robust G1 cell cycle arrest and elicited the key I3C-specific effects on expression and activity of G1 acting cell cycle genes including the disruption of endogenous interactions of the Sp1 transcription factor with the CDK6 promoter. Furthermore, in estrogen responsive MCF-7 cells, with enhanced potency 1-benzyl-I3C down regulated production of estrogen receptor-alpha protein, acts with tamoxifen to arrest breast cancer cell growth more effectively than either compound alone, and inhibited the in vivo growth of human breast cancer cell-derived tumor xenografts in athymic mice. Our results implicate 1-benzyl-I3C as a novel, potent inhibitor of human breast cancer proliferation and estrogen responsiveness that could potentially be developed into a promising therapeutic agent for the treatment of indole-sensitive cancers.
Indole-3-carbinol; 1-benzyl-indole-3-carbinol; breast cancer cells; anti-proliferative signaling; disrupted estrogen receptor responsiveness; high potency indole derivatives
Selective estrogen receptor modulators (SERMs), such as tamoxifen and raloxifene can act as estrogen receptor (ER) antagonists or agonists depending on the cell type. The antagonistic action of tamoxifen has been invaluable for treating breast cancer, whereas the agonist activity of SERMs also has important clinical applications as demonstrated by the use of raloxifene for osteoporosis. Whereas the mechanism whereby SERMs function as antagonists has been studied extensively very little is known about how SERMs produce agonist effects in different tissues with the two ER types; ERα and ERβ. We examined the regulation of 32 SERM-responsive regions with ERα and ERβ in transiently transfected MCF-7 breast, Ishikawa endometrial, HeLa cervical and WAR-5 prostate cancer cells. The regions were regulated by tamoxifen and raloxifene in some cell types, but not in all cell lines. Tamoxifen activated similar number of regions with ERα and ERβ in the cell lines, whereas raloxifene activated over twice as many regions with ERβ compared to ERα. In Ishikawa endometrial cancer cells, tamoxifen activated 17 regions with ERα, whereas raloxifene activated only 2 regions, which might explain their different effects on the endometrium. Microarray studies also found that raloxifene regulated fewer genes than tamoxifen in U2OS bone cancer cells expressing ERα, whereas tamoxifen was equally effective at regulating genes with ERα and ERβ. Our studies indicate that tamoxifen is a non-selective agonist, whereas raloxifene is a relative ERβ-selective agonist, and suggest that ERβ-selective SERMs might be safer for treating clinical conditions that are dependent on the agonist property of SERMs.
Estrogen receptor; Selective estrogen receptor modulator; Gene regulation; Tamoxifen; Raloxifene; Estradiol; Microarray
Indole-3-carbinol (I3C), a phytochemical derived from cruciferous vegetables such as broccoli and Brussels sprouts, has potent anti-proliferative effects in human breast cancer cells and has been shown to decrease metastatic spread of tumors in experimental animals. Using chemotaxis and fluorescent-bead cell motility assays, we demonstrated that I3C significantly decreased the in vitro migration of MDA-MB-231 cells, a highly invasive breast cancer cell line. Immunofluorescence staining of the actin cytoskeleton revealed that concurrent with the loss of cell motility, I3C treatment significantly increased stress fiber formation. Furthermore, I3C induced the localization of the focal adhesion component vinculin and tyrosine-phosphorylated proteins to the cell periphery, which implicates an indole-dependent enhancement of focal adhesions within the outer boundary of the cells. Co-immunoprecipitation analysis of focal adhesion kinase demonstrated that I3C stimulated the dynamic formation of the focal adhesion protein complex without altering the total level of individual focal adhesion proteins. The RhoA-Rho kinase pathway is involved in stress fiber and focal adhesion formation, and I3C treatment stimulated Rho kinase enzymatic activity, and cofilin phosphorylation, which is a downstream target of Rho kinase signaling, but did not increase the level of active GTP-bound RhoA. Exposure of MDA-MB-231 cells to the Rho kinase inhibitor Y-27632, or expression of dominant negative RhoA ablated the I3C induced formation of stress fibers and of peripheral focal adhesions. Expression of constitutively active RhoA mimicked the I3C effects on both processes. Taken together, our data demonstrate that I3C induces stress fibers and peripheral focal adhesions in a Rho kinase-dependent manner that leads to an inhibition of motility in human breast cancer cells.
Indole-3-carbinol; breast cancer; migration; focal adhesions; RhoA; Rho kinase
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
Approximately 600 million people chew Betel nut, making this practice the fourth most popular oral habit in the world. Arecoline, the major alkaloid present in betel nut is one of the causative agents for precancerous lesions and several cancers of mouth among those who chew betel nut. Arecoline can be detected in the human embryonic tissue and is correlated to low birth weight of newborns whose mothers chew betel nut during pregnancy, suggesting that arecoline can induce many systemic effects. However, few reports exist as to the effects of arecoline in human tissues other than oral cancer cell lines. Furthermore, in any system, virtually nothing is known about the cellular effects of arecoline treatment on membrane associated signaling components of human cancer cells.
Using the human Ishikawa endometrial cancer cell line, we investigated the effects of arecoline on expression, localization and functional connections between the ZO-1 tight junction protein and the HER2 EGF receptor family member. Treatment of Ishikawa cells with arecoline coordinately down-regulated expression of both ZO-1 and HER2 protein and transcripts in a dose dependent manner. Biochemical fractionation of cells as well as indirect immunofluorescence revealed that arecoline disrupted the localization of ZO-1 to the junctional complex at the cell periphery. Compared to control transfected cells, ectopic expression of exogenous HER2 prevented the arecoline mediated down-regulation of ZO-1 expression and restored the localization of ZO-1 to the cell periphery. Furthermore, treatment with dexamethasone, a synthetic glucocorticoid reported to up-regulate expression of HER2 in Ishikawa cells, precluded arecoline from down-regulating ZO-1 expression and disrupting ZO-1 localization.
Arecoline is known to induce precancerous lesions and cancer in the oral cavity of betel nut users. The arecoline down-regulation of ZO-1 expression and subcellular distribution suggests that arecoline potentially disrupts cell-cell interactions mediated by ZO-1, which may play a role in arecoline-mediated carcinogenesis. Furthermore, our study has uncovered the dependency of ZO-1 localization and expression on HER2 expression, which has therefore established a new cellular link between HER2 mediated signaling and apical junction formation involving ZO-1.
MCF7 cells are an estrogen-responsive human breast cancer cell line that expresses both estrogen receptor (ER) α and ERβ. Treatment of MCF7 cells with artemisinin, an antimalarial phytochemical from the sweet wormwood plant, effectively blocked estrogen-stimulated cell cycle progression induced by either 17β-estradiol (E2), an agonist for both ERs, or by propyl pyrazole triol (PPT), a selective ERα agonist. Artemisinin strongly downregulated ERα protein and transcripts without altering expression or activity of ERβ. Transfection of MCF7 cells with ERα promoter-linked luciferase reporter plasmids revealed that the artemisinin downregulation of ERα promoter activity accounted for the loss of ERα expression. Artemisinin treatment ablated the estrogenic induction of endogenous progesterone receptor (PR) transcripts by either E2 or PPT and inhibited the estrogenic stimulation of a luciferase reporter plasmid driven by consensus estrogen response elements (EREs). Chromatin immunoprecipitation assays revealed that artemisinin significantly downregulated the level of endogeneous ERα bound to the PR promoter, whereas the level of bound endogeneous ERβ was not altered. Treatment of MCF7 cells with artemisinin and the pure antiestrogen fulvestrant resulted in a cooperative reduction of ERα protein levels and enhanced G1 cell cycle arrest compared with the effects of either compound alone. Our results show that artemisinin switches proliferative human breast cancer cells from expressing a high ERα:ERβ ratio to a condition in which ERβ predominates, which parallels the physiological state linked to antiproliferative events in normal mammary epithelium.
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
3,3′-Diindolylmethane (DIM) is a chemopreventive and chemotherapeutic phytochemical derived from the metabolism of indoles found at high concentrations in cruciferous vegetables. We have previously shown that DIM exhibits anti-angiogenic properties in cultured vascular endothelial cells and in Matrigel plug assays in rodents. In the present study, we demonstrate that DIM reduces the level of hypoxia-inducible factor (HIF)-1α in hypoxic tumor cell lines, as well as HIF-1 transcriptional activity as measured by a reporter assay. Moreover, DIM inhibited the expression of HIF-1-responsive endogenous genes, resulting in the reduced expression of key hypoxia responsive factors, VEGF, furin, enolase-1, glucose transporter-1 and phosphofructokinase. DIM reduced the level of HIF-1α in hypoxic cells by increasing the rate of the prolylhydroxylase- and proteosome-mediated degradation of HIF-1α, and by decreasing the rate of HIF-1α transcription. Using enzyme kinetics studies, we established that DIM interacts with the oligomycin-binding site on the F1 transmembrane component of mitochondrial F1F0-ATPase. The contributions of the resulting increases in levels of ROS and O2 in hypoxic cells to the inhibitory effects of DIM on HIF-1α expression are discussed. These studies are the first to show that DIM can decrease the accumulation and activity of the key angiogenesis regulatory factor, HIF-1α, in hypoxic tumor cells.
3,3′-Diindolylmethane; angiogenesis; hypoxia-inducible factor; cancer; ATPase; prolylhydroxylase
The transcriptionally regulated serum and glucocorticoid inducible protein kinase (Sgk) is localized to the nucleus in a serum-dependent manner, and a yeast two-hybrid genetic screen uncovered a specific interaction between Sgk and the importin-α nuclear import receptor. In vitro GST pull down assays demonstrated a strong and direct association of importin-α with endogenous Sgk and exogenously expressed HA-tagged Sgk, whereas both components coimmunoprecipitate and colocalize to the nucleus after serum stimulation. Consistent with an active mechanism of nuclear localization, the nuclear import of HA-Sgk in permeabilized cells required ATP, cytoplasm, and a functional nuclear pore complex. Ectopic addition of a 107 amino acid carboxy-terminal fragment of importin-α, which contains the Sgk binding region, competitively inhibited the ability of endogenous importin-α to import Sgk into nuclei in vitro. Mutagenesis of lysines by alanine substitution defined a KKAILKKKEEK sequence within the central domain of Sgk between amino acids 131–141 that functions as a nuclear localization signal (NLS) required for the in vitro interaction with importin-α and for nuclear import of full-length Sgk in cultured cells. The serum-induced nuclear import of Sgk requires the NLS-dependent recognition of Sgk by importin-α as well as the PI3-kinase–dependent phosphorylation of Sgk. Our results define a new role importin-α in the stimulus-dependent control of signal transduction by nuclear localized protein kinases.