1-Benzyl-indole-3-carbinol is a highly potent new small molecule inhibitor of Wnt/ß-catenin signaling in melanoma cells that coordinately inhibits cell proliferation and disrupts expression of microphthalmia-associated transcription factor isoform-M.

1-Benzyl-indole-3-carbinol (1-benzyl-I3C), a synthetic analogue of the crucifer-derived natural phytochemical I3C, displayed significantly wider sensitivity and anti-proliferative potency in melanoma cells than the natural compound. Unlike I3C, which targets mainly oncogenic BRAF-expressing cells, 1-benzyl-I3C effectively inhibited proliferation of melanoma cells with a more extensive range of mutational profiles, including those expressing wild-type BRAF. In both cultured melanoma cell lines and in vivo in melanoma cell-derived tumor xenografts, 1-benzyl-I3C disrupted canonical Wnt/ß-catenin signaling that resulted in the downregulation of ß-catenin protein levels with a concomitant increase in levels of the ß-catenin destruction complex components such as glycogen synthase kinase-3ß (GSK-3ß) and Axin. Concurrent with the inhibition of Wnt/ß-catenin signaling, 1-benzyl-I3C strongly downregulated expression of the melanoma master regulator, microphthalmia-associated transcription factor isoform-M (MITF-M) by inhibiting promoter activity through the consensus lymphoid enhancer factor-1 (LEF-1)/T-cell transcription factor (TCF) DNA-binding site. Chromatin immunoprecipitation revealed that 1-benzyl-I3C downregulated interactions of endogenous LEF-1 with the MITF-M promoter. 1-Benzyl-I3C ablated Wnt-activated LEF-1-dependent reporter gene activity in a TOP FLASH assay that was rescued by expression of a constitutively active form of the Wnt co-receptor low-density lipoprotein receptor-related protein (LRP6), indicating that 1-benzyl-I3C disrupts Wnt/ß-catenin signaling at or upstream of LRP6. In oncogenic BRAF-expressing melanoma cells, combinations of 1-benzyl-I3C and Vemurafenib, a clinically employed BRAF inhibitor, showed strong anti-proliferative effects. Taken together, our observations demonstrate that 1-benzyl-I3C represents a new and highly potent indolecarbinol-based small molecule inhibitor of Wnt/ß-catenin signaling that has intriguing translational potential, alone or in combination with other anti-cancer agents, to treat human melanoma.

Inhibition of oncogenic BRAF activity by indole-3-carbinol disrupts microphthalmia-associated transcription factor expression and arrests melanoma cell proliferation.

Indole-3-carbinol (I3C), an anti-cancer phytochemical derived from cruciferous vegetables, strongly inhibited proliferation and down-regulated protein levels of the melanocyte master regulator micropthalmia-associated transcription factor (MITF-M) in oncogenic BRAF-V600E expressing melanoma cells in culture as well as in vivo in tumor xenografted athymic nude mice. In contrast, wild type BRAF-expressing melanoma cells remained relatively insensitive to I3C anti-proliferative signaling. In BRAF-V600E-expressing melanoma cells, I3C treatment inhibited phosphorylation of MEK and ERK/MAPK, the down stream effectors of BRAF. The I3C anti-proliferative arrest was concomitant with the down-regulation of MITF-M transcripts and promoter activity, loss of endogenous BRN-2 binding to the MITF-M promoter, and was strongly attenuated by expression of exogenous MITF-M. Importantly, in vitro kinase assays using immunoprecipitated BRAF-V600E and wild type BRAF demonstrated that I3C selectively inhibited the enzymatic activity of the oncogenic BRAF-V600E but not of the wild type protein. In silico modeling predicted an I3C interaction site in the BRAF-V600E protomer distinct from where the clinically used BRAF-V600E inhibitor Vemurafenib binds to BRAF-V600E. Consistent with this prediction, combinations of I3C and Vemurafenib more potently inhibited melanoma cell proliferation and reduced MITF-M levels in BRAF-V600E expressing melanoma cells compared to the effects of each compound alone. Thus, our results demonstrate that oncogenic BRAF-V600E is a new cellular target of I3C that implicate this indolecarbinol compound as a potential candidate for novel single or combination therapies for melanoma. © 2016 Wiley Periodicals, Inc.

Artemisinin disrupts androgen responsiveness of human prostate cancer cells by stimulating the 26S proteasome-mediated degradation of the androgen receptor protein.

Androgen receptor (AR) expression and activity is highly linked to the development and progression of prostate cancer and is a target of therapeutic strategies for this disease. We investigated whether the antimalarial drug artemisinin, which is a sesquiterpene lactone isolated from the sweet wormwood plant Artemisia annua, could alter AR expression and responsiveness in cultured human prostate cancer cell lines. Artemisinin treatment induced the 26S proteasome-mediated degradation of the receptor protein, without altering AR transcript levels, in androgen-responsive LNCaP prostate cancer cells or PC-3 prostate cancer cells expressing exogenous wild-type AR. Furthermore, artemisinin stimulated AR ubiquitination and AR receptor interactions with the E3 ubiquitin ligase MDM2 in LNCaP cells. The artemisinin-induced loss of AR protein prevented androgen-responsive cell proliferation and ablated total AR transcriptional activity. The serine/threonine protein kinase AKT-1 was shown to be highly associated with artemisinin-induced proteasome-mediated degradation of AR protein. Artemisinin treatment activated AKT-1 enzymatic activity, enhanced receptor association with AKT-1, and induced AR serine phosphorylation. Treatment of LNCaP cells with the PI3-kinase inhibitor LY294002, which inhibits the PI3-kinase-dependent activation of AKT-1, prevented the artemisinin-induced AR degradation. Furthermore, in transfected receptor-negative PC-3 cells, artemisinin failed to stimulate the degradation of an altered receptor protein (S215A/S792A) with mutations in its two consensus AKT-1 serine phosphorylation sites. Taken together, our results indicate that artemisinin induces the degradation of AR protein and disrupts androgen responsiveness of human prostate cancer cells, suggesting that this natural compound represents a new potential therapeutic molecule that selectively targets AR levels.

Therapeutic targeting of replicative immortality.

One of the hallmarks of malignant cell populations is the ability to undergo continuous proliferation. This property allows clonal lineages to acquire sequential aberrations that can fuel increasingly autonomous growth, invasiveness, and therapeutic resistance. Innate cellular mechanisms have evolved to regulate replicative potential as a hedge against malignant progression. When activated in the absence of normal terminal differentiation cues, these mechanisms can result in a state of persistent cytostasis. This state, termed "senescence," can be triggered by intrinsic cellular processes such as telomere dysfunction and oncogene expression, and by exogenous factors such as DNA damaging agents or oxidative environments. Despite differences in upstream signaling, senescence often involves convergent interdependent activation of tumor suppressors p53 and p16/pRB, but can be induced, albeit with reduced sensitivity, when these suppressors are compromised. Doses of conventional genotoxic drugs required to achieve cancer cell senescence are often much lower than doses required to achieve outright cell death. Additional therapies, such as those targeting cyclin dependent kinases or components of the PI3K signaling pathway, may induce senescence specifically in cancer cells by circumventing defects in tumor suppressor pathways or exploiting cancer cells' heightened requirements for telomerase. Such treatments sufficient to induce cancer cell senescence could provide increased patient survival with fewer and less severe side effects than conventional cytotoxic regimens. This positive aspect is countered by important caveats regarding senescence reversibility, genomic instability, and paracrine effects that may increase heterogeneity and adaptive resistance of surviving cancer cells. Nevertheless, agents that effectively disrupt replicative immortality will likely be valuable components of new combinatorial approaches to cancer therapy.

Phytochemical regulation of the tumor suppressive microRNA, miR-34a, by p53-dependent and independent responses in human breast cancer cells.

The tumor suppressive microRNA miR-34a is transcriptionally regulated by p53 and shown to inhibit breast cancer cell proliferation as well as being a marker of increased disease free survival. Indole-3-carbinol (I3C) derived from cruciferous vegetables, artemisinin, extracted from the sweet wormwood plant, and artesunate, a semi-synthetic derivative of artemisinin, are phytochemicals with anti-tumorigenic properties however, little is known about the role of microRNAs in their mechanism of action. Human breast cancer cells expressing wild-type (MCF-7) or mutant p53 (T47D) were treated with a concentration range and time course of each phytochemical under conditions of cell cycle arrest as detected by flow cytometry to examine the potential connection between miR-34a expression and their anti-proliferative responses. Real-time PCR and western blot analysis of extracted RNA and total protein revealed artemsinin and artesunate increased miR-34a expression in a dose-dependent manner correlating with down-regulation of the miR-34a target gene, CDK4. I3C stimulation of miR-34a expression required functional p53, whereas, both artemisinin and artesunate up-regulated miR-34a expression regardless of p53 mutational status or in the presence of dominant negative p53. Phytochemical treatments inhibited the luciferase activity of a construct containing the wild-type 3'UTR of CDK4, but not those with a mutated miR-34a binding site, whereas, transfection of miR-34a inhibitors ablated the phytochemical mediated down-regulation of CDK4 and induction of cell cycle arrest. Our results suggest that miR-34a is an essential component of the anti-proliferative activities of I3C, artemisinin, and artesunate and demonstrate that both wild-type p53 dependent and independent pathways are responsible for miR-34a induction.

Cooperative antiproliferative signaling by aspirin and indole-3-carbinol targets microphthalmia-associated transcription factor gene expression and promoter activity in human melanoma cells.

Antiproliferative signaling of combinations of the nonsteroidal anti-inflammatory drug acetylsalicylic acid (aspirin) and indole-3-carbinol (I3C), a natural indolecarbinol compound derived from cruciferous vegetables, was investigated in human melanoma cells. Melanoma cell lines with distinct mutational profiles were sensitive to different extents to the antiproliferative response of aspirin, with oncogenic BRAF-expressing G361 cells and wild-type BRAF-expressing SK-MEL-30 cells being the most responsive. I3C triggered a strong proliferative arrest of G361 melanoma cells and caused only a modest decrease in the proliferation of SK-MEL-30 cells. In both cell lines, combinations of aspirin and I3C cooperatively arrested cell proliferation and induced a G1 cell cycle arrest, and nearly ablated protein and transcript levels of the melanocyte master regulator microphthalmia-associated transcription factor isoform M (MITF-M). In melanoma cells transfected with a -333/+120-bp MITF-M promoter-luciferase reporter plasmid, treatment with aspirin and I3C cooperatively disrupted MITF-M promoter activity, which accounted for the loss of MITF-M gene products. Mutational analysis revealed that the aspirin required the LEF1 binding site, whereas I3C required the BRN2 binding site to mediate their combined and individual effects on MITF-M promoter activity. Consistent with LEF1 being a downstream effector of Wnt signaling, aspirin, but not I3C, downregulated protein levels of the Wnt co-receptor LDL receptor-related protein-6 and ß-catenin and upregulated the ß-catenin destruction complex component Axin. Taken together, our results demonstrate that aspirin-regulated Wnt signaling and I3C-targeted signaling pathways converge at distinct DNA elements in the MITF-M promoter to cooperatively disrupt MITF-M expression and melanoma cell proliferation.

Essential role of the cancer stem/progenitor cell marker nucleostemin for indole-3-carbinol anti-proliferative responsiveness in human breast cancer cells.

BACKGROUND: Nucleostemin is a nucleolus residing GTPase that is considered to be an important cancer stem/progenitor cell marker protein due to its high expression levels in breast cancer stem cells and its role in tumor-initiation of human mammary tumor cells. It has been proposed that nucleostemin may represent a valuable therapeutic target for breast cancer; however, to date evidence supporting the cellular mechanism has not been elucidated. RESULTS: Expression of exogenous HER2, a member of the EGF receptor gene family, in the human MCF-10AT preneoplastic mammary epithelial cell line formed a new breast cancer cell line, 10AT-Her2, which is highly enriched in cells with stem/progenitor cell-like character. 10AT-Her2 cells display a CD44+/CD24-/low phenotype with high levels of the cancer stem/progenitor cell marker proteins nucleostemin, and active aldehyde dehydrogenase-1. The overall expression pattern of HER2 protein and the stem/progenitor cell marker proteins in the 10AT-Her2 cell population is similar to that of the luminal HER2+ SKBR3 human breast cancer cell line, whereas, both MCF-7 and MDA-MB-231 cells display reduced levels of nucleostemin and no detectable expression of ALDH-1. Importantly, in contrast to the other well-established human breast cancer cell lines, 10AT-Her2 cells efficiently form tumorspheres in suspension cultures and initiate tumor xenograft formation in athymic mice at low cell numbers. Furthermore, 10AT-Her2 cells are highly sensitive to the anti-proliferative apoptotic effects of indole-3-carbinol (I3C), a natural anti-cancer indolecarbinol from cruciferous vegetables of the Brassica genus such as broccoli and cabbage. I3C promotes the interaction of nucleostemin with MDM2 (Murine Double Mutant 2), an inhibitor of the p53 tumor suppressor, and disrupts the MDM2 interaction with p53. I3C also induced nucleostemin to sequester MDM2 in a nucleolus compartment, thereby freeing p53 to mediate its apoptotic activity. siRNA knockdown of nucleostemin functionally documented that nucleostemin is required for I3C to trigger its cellular anti-proliferative responses, inhibit tumorsphere formation, and disrupt MDM2-p53 protein-protein interactions. Furthermore, expression of an I3C-resistant form of elastase, the only known target protein for I3C, prevented I3C anti-proliferative responses in cells and in tumor xenografts in vivo, as well as disrupt the I3C stimulated nucleostemin-MDM2 interactions. CONCLUSIONS: Our results provide the first evidence that a natural anti-cancer compound mediates its cellular and in vivo tumor anti-proliferative responses by selectively stimulating cellular interactions of the stem/progenitor cell marker nucleostemin with MDM2, which frees p53 to trigger its apoptotic response. Furthermore, our study provides a new mechanistic template that can be potentially exploited for the development of cancer stem/progenitor cell targeted therapeutic strategies.

Artemisinin triggers a G1 cell cycle arrest of human Ishikawa endometrial cancer cells and inhibits cyclin-dependent kinase-4 promoter activity and expression by disrupting nuclear factor-κB transcriptional signaling.

Relatively little is known about the antiproliferative effects of artemisinin, a naturally occurring antimalarial compound from Artemisia annua, or sweet wormwood, in human endometrial cancer cells. Artemisinin induced a G1 cell cycle arrest in cultured human Ishikawa endometrial cancer cells and downregulated cyclin-dependent kinase-2 (CDK2) and CDK4 transcript and protein levels. Analysis of CDK4 promoter-luciferase reporter constructs showed that the artemisinin ablation of CDK4 gene expression was accounted for by the loss of CDK4 promoter activity. Chromatin immunoprecipitation demonstrated that artemisinin inhibited nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) subunit p65 and p50 interactions with the endogenous Ishikawa cell CDK4 promoter. Coimmunoprecipitation revealed that artemisinin disrupts endogenous p65 and p50 nuclear translocation through increased protein-protein interactions with IκB-α, an NF-κB inhibitor, and disrupts its interaction with the CDK4 promoter, leading to a loss of CDK4 gene expression. Artemisinin treatment stimulated the cellular levels of IκB-α protein without altering the level of IκB-α transcripts. Finally, expression of exogenous p65 resulted in the accumulation of this NF-κB subunit in the nucleus of artemisinin-treated and artemisinin-untreated cells, reversed the artemisinin downregulation of CDK4 protein expression and promoter activity, and prevented the artemisinin-induced G1 cell cycle arrest. Taken together, our results demonstrate that a key event in the artemisinin antiproliferative effects in endometrial cancer cells is the transcriptional downregulation of CDK4 expression by disruption of NF-κB interactions with the CDK4 promoter.

Target protein interactions of indole-3-carbinol and the highly potent derivative 1-benzyl-I3C with the C-terminal domain of human elastase uncouples cell cycle arrest from apoptotic signaling.

Elastase is the only currently identified target protein for indole-3-carbinol (I3C), a naturally occurring hydrolysis product of glucobrassicin in cruciferous vegetables such as broccoli, cabbage, and Brussels sprouts that induces a cell cycle arrest and apoptosis of human breast cancer cells. In vitro elastase enzymatic assays demonstrated that I3C and at lower concentrations its more potent derivative 1-benzyl-indole-3-carbinol (1-benzyl-I3C) act as non-competitive allosteric inhibitors of elastase activity. Consistent with these results, in silico computational simulations have revealed the first predicted interactions of I3C and 1-benzyl-I3C with the crystal structure of human neutrophil elastase, and identified a potential binding cluster on an external surface of the protease outside of the catalytic site that implicates elastase as a target protein for both indolecarbinol compounds. The Δ205 carboxyterminal truncation of elastase, which disrupts the predicted indolecarbinol binding site, is enzymatically active and generates a novel I3C resistant enzyme. Expression of the wild type and Δ205 elastase in MDA-MB-231 human breast cancer cells demonstrated that the carboxyterminal domain of elastase is required for the I3C and 1-benzyl-I3C inhibition of enzymatic activity, accumulation of the unprocessed form of the CD40 elastase substrate (a tumor necrosis factor receptor family member), disruption of NFκB nuclear localization and transcriptional activity, and induction of a G1 cell cycle arrest. Surprisingly, expression of the Δ205 elastase molecule failed to reverse indolecarbinol stimulated apoptosis, establishing an elastase-dependent bifurcation point in anti-proliferative signaling that uncouples the cell cycle and apoptotic responses in human breast cancer cells.

Antiproliferative effects of artemisinin on human breast cancer cells requires the downregulated expression of the E2F1 transcription factor and loss of E2F1-target cell cycle genes.

Artemisinin, a sesquiterpene phytolactone derived from Artemisia annua, is a potent antimalarial compound with promising anticancer properties, although the mechanism of its anticancer signaling is not well understood. Artemisinin inhibited proliferation and induced a strong G1 cell cycle arrest of cultured MCF7 cells, an estrogen-responsive human breast cancer cell line that represents an early-stage cancer phenotype, and effectively inhibited the in-vivo growth of MCF7 cell-derived tumors from xenografts in athymic nude mice. Artemisinin also induced a growth arrest of tumorigenic human breast cancer cell lines with preneoplastic and late stage cancer phenotypes, but failed to arrest the growth of a nontumorigenic human mammary cell line. Concurrent with the cell cycle arrest of MCF7 cells, artemisinin selectively downregulated the transcript and protein levels of the CDK2 and CDK4 cyclin-dependent kinases, cyclin E, cyclin D1, and the E2F1 transcription factor. Analysis of CDK2 promoter-luciferase reporter constructs showed that the artemisinin ablation of CDK2 gene expression was accounted for by the loss of CDK2 promoter activity. Chromatin immunoprecipitation revealed that artemisinin inhibited E2F1 interactions with the endogenous MCF7 cell CDK2 and cyclin E promoters. Moreover, constitutive expression of exogenous E2F1 prevented the artemisinin-induced cell cycle arrest and downregulation of CDK2 and cyclin E gene expression. Taken together, our results demonstrate that the artemisinin disruption of E2F1 transcription factor expression mediates the cell cycle arrest of human breast cancer cells and represents a critical transcriptional pathway by which artemisinin controls human reproductive cancer cell growth.

Indole-3-carbinol downregulation of telomerase gene expression requires the inhibition of estrogen receptor-alpha and Sp1 transcription factor interactions within the hTERT promoter and mediates the G1 cell cycle arrest of human breast cancer cells.

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 G(1) 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 17ß-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.

The dietary phytochemical indole-3-carbinol is a natural elastase enzymatic inhibitor that disrupts cyclin E protein processing.

Indole-3-carbinol (I3C), a naturally occurring component of Brassica vegetables, such as broccoli, cabbage, and Brussels sprouts, induces a G(1) cell-cycle arrest of human breast cancer cells, although the direct cellular targets that mediate this process are unknown. Treatment of highly invasive MDA-MB-231 breast cancer cells with I3C shifted the stable accumulation of cyclin E protein from the hyperactive lower-molecular-mass 35-kDa form that is associated with cancer cell proliferation and poor clinical outcomes to the 50-kDa cyclin E form that typically is expressed in normal mammary tissue. An in vitro cyclin E processing assay, in combination with zymography, demonstrated that I3C, but not its natural dimer, 3,3'-diindolylmethane, disrupts proteolytic processing of the 50-kDa cyclin E into the lower-molecular-mass forms by direct inhibition of human neutrophil elastase enzymatic activity. Analysis of elastase enzyme kinetics using either cyclin E or N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanalide as substrates demonstrated that I3C acts as a noncompetitive inhibitor of elastase activity with an inhibitory constant of approximately 12 microM. Finally, siRNA ablation of neutrophil elastase protein production in MDA-MB-231 cells mimicked the I3C-disrupted processing of the 50-kDa cyclin E protein and the indole-induced cell-cycle arrest. Taken together, our results demonstrate that elastase is the first identified specific target protein for I3C and that the direct I3C inhibition of elastase enzymatic activity implicates the potential use of this indole, or related compounds, in targeted therapies of human breast cancers where high elastase levels are correlated with poor prognosis.

Arecoline induced disruption of expression and localization of the tight junctional protein ZO-1 is dependent on the HER 2 expression in human endometrial Ishikawa cells.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Indole-3-carbinol inhibits MDA-MB-231 breast cancer cell motility and induces stress fibers and focal adhesion formation by activation of Rho kinase activity.

Indole-3-carbinol (I3C), a phytochemical derived from cruciferous vegetables such as broccoli and Brussels sprouts, has potent antiproliferative 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. Coimmunoprecipitation 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.

Anticancer activities of artemisinin and its bioactive derivatives.

Artemisinin, a sesquiterpene lactone derived from the sweet wormwood plant Artemisia annua, and its bioactive derivatives exhibit potent anticancer effects in a variety of human cancer cell model systems. The pleiotropic response in cancer cells includes growth inhibition by cell cycle arrest, apoptosis, inhibition of angiogenesis, disruption of cell migration, and modulation of nuclear receptor responsiveness. These effects of artemisinin and its derivatives result from perturbations of many cellular signalling pathways. This review provides a comprehensive discussion of these cellular responses, and considers the ramifications for the potential development of artemisinin-based compounds in anticancer therapeutic and preventative strategies.

Cell type- and estrogen receptor-subtype specific regulation of selective estrogen receptor modulator regulatory elements.

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; ERalpha and ERbeta. We examined the regulation of 32 SERM-responsive regions with ERalpha and ERbeta 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 ERalpha and ERbeta in the cell lines, whereas raloxifene activated over twice as many regions with ERbeta compared to ERalpha. In Ishikawa endometrial cancer cells, tamoxifen activated 17 regions with ERalpha, 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 ERalpha, whereas tamoxifen was equally effective at regulating genes with ERalpha and ERbeta. Our studies indicate that tamoxifen is a non-selective agonist, whereas raloxifene is a relative ERbeta-selective agonist, and suggest that ERbeta-selective SERMs might be safer for treating clinical conditions that are dependent on the agonist property of SERMs.

DEVD-NucView488: a novel class of enzyme substrates for real-time detection of caspase-3 activity in live cells.

Live-cell detection of intracellular enzyme activity requires that substrates are cell-permeable and that the generated products are easily detected and retained in cells. Our objective was to create a novel fluorogenic substrate that could be used for real-time detection of apoptosis in living cells. We have synthesized a highly cell-permeable caspase-3 substrate, DEVD-NucView488, by linking a fluorogenic DNA-binding dye to the caspase-3 recognition sequence that renders the dye nonfunctional. On substrate cleavage, the dye is released and becomes highly fluorescent on binding to DNA. DEVD-NucView488 detected caspase-3 activation within a live-cell population much earlier and with higher sensitivity compared with other apoptosis reagents that are currently available. Furthermore, cells incubated with DEVD-NucView488 exhibited no toxicity and normal apoptotic progression. DEVD-NucView488 is an ideal substrate for kinetic studies of caspase-3 activation because it detects caspase-3 activity in real-time and also efficiently labels DNA in nuclei of caspase-3-activated cells for real-time fluorescent visualization of apoptotic morphology. The strategy utilized in the design of this fluorogenic substrate can be applied in future endeavors to develop substrates for detecting real-time intracellular enzyme activity.

Artemisinin selectively decreases functional levels of estrogen receptor-alpha and ablates estrogen-induced proliferation in human breast cancer cells.

MCF7 cells are an estrogen-responsive human breast cancer cell line that expresses both estrogen receptor (ER) alpha and ERbeta. Treatment of MCF7 cells with artemisinin, an antimalarial phytochemical from the sweet wormwood plant, effectively blocked estrogen-stimulated cell cycle progression induced by either 17beta-estradiol (E(2)), an agonist for both ERs, or by propyl pyrazole triol (PPT), a selective ERalpha agonist. Artemisinin strongly downregulated ERalpha protein and transcripts without altering expression or activity of ERbeta. Transfection of MCF7 cells with ERalpha promoter-linked luciferase reporter plasmids revealed that the artemisinin downregulation of ERalpha promoter activity accounted for the loss of ERalpha expression. Artemisinin treatment ablated the estrogenic induction of endogenous progesterone receptor (PR) transcripts by either E(2) 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 ERalpha bound to the PR promoter, whereas the level of bound endogeneous ERbeta was not altered. Treatment of MCF7 cells with artemisinin and the pure antiestrogen fulvestrant resulted in a cooperative reduction of ERalpha protein levels and enhanced G(1) 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 ERalpha:ERbeta ratio to a condition in which ERbeta predominates, which parallels the physiological state linked to antiproliferative events in normal mammary epithelium.

N-Alkoxy derivatization of indole-3-carbinol increases the efficacy of the G1 cell cycle arrest and of I3C-specific regulation of cell cycle gene transcription and activity in 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 melatonin 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.

3,3'-Diindolylmethane stimulates murine immune function in vitro and in vivo.

3,3'-Diindolylmethane (DIM), a major condensation product of indole-3-carbinol, exhibits chemopreventive properties in animal models of cancer. Recent studies have shown that DIM stimulates interferon-gamma (IFN-gamma) production and potentiates the IFN-gamma signaling pathway in human breast cancer cells via a mechanism that includes increased expression of the IFN-gamma 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 interleukin (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, induced elevation of serum cytokines in mice, including IL-6, granulocyte colony-stimulating factor (G-CSF), IL-12 and IFN-gamma. 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.