Functional Significance of Selective Expression of ERα and ERß in Mammary Gland Organ Culture.

Thoracic pair of mammary glands from steroid hormone-pretreated mice respond to hormones structurally and functionally in organ culture. A short exposure of glands for 24 h to 7,12 Dimethylbenz(a)anthracene (DMBA) during a 24-day culture period induced alveolar or ductal lesions. Methods: To differentiate the functional significance of ERα and ERß, we employed estrogen receptor (ER) knockout mice. We compared the effects of DMBA on the development of preneoplastic lesions in the glands in the absence of ERα (αERKO) and ERß (ßERKO) using an MMOC protocol. Glands were also subjected to microarray analyses. We showed that estradiol can be replaced by EGF for pretreatment of mice. The carcinogen-induced lesions developed under both steroids and EGF pretreatment protocols. The glands from αERKO did not develop any lesions, whereas in ßERKO mice in which ERα is intact, mammary alveolar lesions developed. Comparison of microarrays of control, αERKO and ßERKO mice showed that ERα was largely responsible for proliferation and the MAP kinase pathways, whereas ERß regulated steroid metabolism-related genes. The results indicate that ERα is essential for the development of precancerous lesions. Both subtypes, ERα and Erß, differentially regulated gene expression in mammary glands in organ cultures.

Biofunctional significance of multi-herbal combination against paracetamol-induced hepatotoxicity in Wistar rats.

A multi-herbal combination (MHC) of five herbs, namely Punica granatum L., Putranjiva roxburghii Wall., Swertia chirata Buch.-Ham., Tinospora cordifolia (Willd.) Miers and Trigonella corniculata L. was assessed against the paracetamol-induced acute hepatotoxicity in female Wistar rats. The animals were randomly assorted into seven groups with six animals in each group. The rats were pre-treated with MHC (50, 100, and 200 mg/kg bw) and silymarin (50 mg/kg bw) once daily for seven consecutive days via oral route followed by administration of paracetamol (3 g/kg bw) on day 7, an hour after the last administration of MHC and silymarin. It was observed that MHC administration significantly (p ≤ 0.05) overturned the paracetamol-induced increase in serum liver function biomarkers (serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase, and total bilirubin), phase I reaction enzymes (NADPH-cytochrome P450 reductase and NADH-cytochrome b5 reductase), and oxidant biomarkers (lactate dehydrogenase, lipid peroxidation, lipid hydroperoxides, and protein content). MHC administration also reinstated the paracetamol-induced significant decrease (p ≤ 0.05) in haematological indices (haematocrit, haemoglobin, red and white blood cells, and platelets), phase II reaction enzymes (glutathione-S-transferase and DT-diaphorase), membrane-bound enzymes (Na+/K+-ATPase, Ca2+-ATPase, and Mg2+-ATPase), and antioxidant biomarkers (reduced glutathione, superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase). Overall, MHC at 200 mg/kg bw dose significantly (p ≤ 0.05) sheltered the red blood cells from the assault of free radicals, stabilized the structural and functional integrity of hepatocytes, hindered acetaminophen (APAP) biotransformation to its toxic metabolites, and endorsed conjugating abilities to detoxify toxic entities. Furthermore, MHC significantly (p ≤ 0.05) activated enzymatic machinery to scavenge/inhibit the formation of reactive oxygen species, regulated nucleic acid metabolism, surface potential, and membrane fluidity, attenuated tissue breakdown, quenched peroxyl radicals, and provided protection against tissue injury. The necroinflammatory scores revealed strong evidence of MHC (200 mg/kg bw) effectiveness against the paracetamol-induced hepatotoxicity in rats at p ≤ 0.05. The synergistic effect of major inherent phytoconstituents (kaempferol, ellagic acid, and gallic acid), detected by HPLC-PDA, in MHC might have overturned the paracetamol-induced biochemical toxic alterations in rat liver.

A novel and cost-effective ex vivo orthotopic model for the study of human breast cancer in mouse mammary gland organ culture.

Mouse mammary organ culture (MMOC) is used to evaluate the efficacy of chemopreventive agents against the development of carcinogen-induced preneoplastic lesions and is highly correlative to in vivo carcinogenesis models. Here, we developed a new ex vivo MMOC model, by introducing human breast cancer cells into the mouse mammary gland. This novel model, termed human breast cancer in MMOC (BCa-MMOC), mimics in vivo orthotopic breast cancer mouse models. To develop this model, estradiol- and progesterone-sensitized female mice were injected with letrozole-sensitive and -resistant T47D breast cancer cells in the mammary glands and then euthanized. The glands were cultured in vitro with hormone-supplemented media. On day 25, the glands were fixed and processed by histopathology and immunohistochemistry to evaluate for the presence of T47D cells, growth pattern, cancer markers and estradiol responsiveness. Histopathological analyses demonstrated an identical pattern of growth between the breast cancer cells injected ex vivo and in vivo Interestingly, clusters of cancer cells in the mammary gland stroma appeared similar to those observed in human breast tumors. The injected T47D cells survived and proliferated for 15 days maintaining expression of estrogen receptor alpha (ER), progesterone receptor (PR), epidermal growth factor receptor (EGFR), and aromatase. The aromatase-overexpressing T47D grown in the BCa-MMOC sufficiently metabolized estrogen, resulting in enhanced cell proliferation, induction of estrogen target genes (i.e. ER and PR-B), and showed typical changes to estrogenic milieu. In summary, here we show a novel, inexpensive ex vivo model, to potentially study the effects of therapeutic agents on cancer cells grown in an orthotopic micromilieu.This article has an associated First Person interview with the first author of the paper.

Development of aqueous-based multi-herbal combination using principal component analysis and its functional significance in HepG2 cells.

BACKGROUND: The present study was carried out to prepare multi-herbal combination via comparing antioxidant activity and polyphenolic composition of five medicinal plant extracts of Punica granatum L., Putranjiva roxburghii Wall., Swertia chirata Buch.-Ham., Tinospora cordifolia (Willd.) Miers and Trigonella corniculata L. METHODS: The herbs were individually evaluated using in vitro antioxidant assays and analyzed by HPLC-PDA. The resultant data was examined using principal component analysis (PCA). Further, herbal combination was prepared on the basis of PCA. RESULTS: The PCA divided the plants into three groups. The leading or primary group contained P. granatum and P. roxburghii with the highest antioxidant activity strongly correlated with high amount of kaempferol. S. chirata was acknowledged as nourisher herb in one and T. cordifolia and T. corniculata were identified as stimulator herbs in other group. The herbal combination exhibited high antioxidant activity as compared to the individual plants. The combination revealed good antiproliferative efficacy against hepatocellular carcinoma (HepG2) cells with IC50 of 75.864 µg/ml. CONCLUSIONS: The activity observed in vitro with HepG2 cells suggests that the herbal combination can provide therapeutic activity in vivo in future. The study may provide information regarding precise preparation of multi-herbal formulations using PCA as a tool in pharmaceutical industries.

Progression of conventional hepatic cell culture models to bioengineered HepG2 cells for evaluation of herbal bioactivities.

Cancer cell lines of human tissue origin have been extensively used to investigate antiproliferative activity and toxicity of herbal extracts, isolated compounds, and anticancer drugs. These cell lines are genetically and/or epigenetically well characterized to determine the altered expression of proteins within given cellular pathways and critical genes in cancer. Human derived hepatoma (HepG2) cell line has been extensively exploited to examine cytoprotective, antioxidative, hepatoprotective, anti-hepatoma, hypocholesterolemic, anti-steatosis, bioenergetic homeostatic and anti-insulin resistant properties. Moreover, mechanism of action of various botanicals and bioactive constituents has been reported using these cells. HepG2 cells have significant differences as compared to primary hepatocytes with respect to expression of cytochrome P450 enzymes and xenobiotic receptors in conventional in vitro culture conditions. Therefore, strategies have been employed to overcome limitations of two dimensional (2D) in vitro HepG2 cell culture in order to recognize functional biomarkers more accurately and to boost its predictive value in clinical research. In consequence, three dimensional (3D) human hepatoma cell culture models are being developed as a resource to achieve these goals of simulating the in vivo tumor microenvironment. It is assumed that bioengineered 3D hepatoma cell culture models can provide significant assistance in scrutinizing the molecular response of herbal natural products to recognize novel prognostic targets and crucial biomarkers in treatment strategies for cancer patients in near future.

Suppression of Breast Cancer Cell Proliferation by Selective Single-Domain Antibody for Intracellular STAT3.

BACKGROUND: The serendipitous discovery of heavy-chain antibodies devoid of light chains in camelids and the subsequent development of VHHs (variable region of camelid heavy chain) have provided a very important tool for research and possibly for therapeutics. In this study, we synthesized single-domain 15-kDa antibody SBT-100 (anti-STAT3 B VHH13) against human STAT3 (signal transducer and activator of transcription) that binds selectively to STAT3 and suppresses the function of phosphorylated STAT3 (p-STAT3). METHODS: Single-chain VHH nanobodies were generated by immunizing camelid with humanized STAT3. Commercially available breast cancer cell lines including MDA-MB-231, MDA-MB-468, MDA-MB-453, MCF-7, and BT474 were used. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The association of anti-STAT3 B VHH13 with STAT3 and p-STAT3 was determined by immunoprecipitation and Western blot analyses. The efficacy of SBT-100 on the growth of MDA-MB-231 xenografts in vivo was determined using athymic mice. Statistical significance for cell proliferation was determined using analysis of variance. If a significant difference (P < .05) was observed, then Tukey-Kramer multiple comparison test was conducted. RESULTS: SBT-100 suppressed cell proliferation of triple-negative breast cancer cells (P < .01) as well as provided significant inhibition of tumor growth (P < .05) in a xenograft model without any toxicity. Results are presented to show that anti-STAT3 B VHH13 selectively binds to STAT3 suggesting that the effects were mediated by inhibiting STAT3. CONCLUSIONS: A very large number of human malignancies and benign diseases have constitutive STAT3 activation. Therefore, the results described here suggest that anti-STAT3 B VHH13 can be developed for therapeutic intervention for cancer cells expressing STAT3 or p-STAT3.

Crosstalk between the vitamin D receptor (VDR) and miR-214 in regulating SuFu, a hedgehog pathway inhibitor in breast cancer cells.

The vitamin D receptor (VDR), and its ligand 1α,25-dihydroxyvitamin D3 (1,25D3) prevent breast cancer development and progression, yet the molecular mechanisms governing this are unclear. MicroRNAs (miRNAs) on the other hand, promote or inhibit breast cancer growth. To understand how VDR regulates miRNAs, we compared miRNA expression of wild-type (WT) and VDR knockout (VDRKO) breast cancer cells by a Mouse Breast Cancer miRNA PCR array. Compared to VDR WT cells, expressions of miR-214, miR-199a-3p and miR-199a-5p of the miR-199a/miR-214 cluster were 42, 15, and 10 fold higher in VDRKO cells respectively. Overexpression of VDR in breast cancer cells reduced the miR-199a/miR-214 cluster expression by 30%. VDR status also negatively correlated with Dnm3os expression, a non-coding RNA transcript of the dynamin-3 gene encoding the miR-199a/miR-214 cluster, suggesting that VDR represses this cluster through Dnm3os. Conversely, overexpression of miR-214 in MCF-7 and T47D cells antagonized VDR mediated signaling. Furthermore, there was a positive correlation between VDR status and the expression of Suppressor of fused gene (SuFu), a hedgehog pathway inhibitor. miR-214 on the other hand suppressed SuFu protein expression. These findings suggest a crosstalk between VDR and miR-214 in regulating hedgehog signaling in breast cancer cells, providing new therapies for breast cancer.

LiSIs: An Online Scientific Workflow System for Virtual Screening.

Modern methods of drug discovery and development in recent years make a wide use of computational algorithms. These methods utilise Virtual Screening (VS), which is the computational counterpart of experimental screening. In this manner the in silico models and tools initial replace the wet lab methods saving time and resources. This paper presents the overall design and implementation of a web based scientific workflow system for virtual screening called, the Life Sciences Informatics (LiSIs) platform. The LiSIs platform consists of the following layers: the input layer covering the data file input; the pre-processing layer covering the descriptors calculation, and the docking preparation components; the processing layer covering the attribute filtering, compound similarity, substructure matching, docking prediction, predictive modelling and molecular clustering; post-processing layer covering the output reformatting and binary file merging components; output layer covering the storage component. The potential of LiSIs platform has been demonstrated through two case studies designed to illustrate the preparation of tools for the identification of promising chemical structures. The first case study involved the development of a Quantitative Structure Activity Relationship (QSAR) model on a literature dataset while the second case study implemented a docking-based virtual screening experiment. Our results show that VS workflows utilizing docking, predictive models and other in silico tools as implemented in the LiSIs platform can identify compounds in line with expert expectations. We anticipate that the deployment of LiSIs, as currently implemented and available for use, can enable drug discovery researchers to more easily use state of the art computational techniques in their search for promising chemical compounds. The LiSIs platform is freely accessible (i) under the GRANATUM platform at: http://www.granatum.org and (ii) directly at: http://lisis.cs.ucy.ac.cy.

Differential roles of ERα and ERß in normal and neoplastic development in the mouse mammary gland.

The present experiments were performed to determine the roles of estrogen receptors α and ß (ERα and ERß) in normal and neoplastic development in the mouse mammary gland. In wild-type mice, in vivo administration of estradiol (E) + progesterone (P) stimulated mammary ductal growth and alveolar differentiation. Mammary glands from mice in which the ERß gene has been deleted (ßERKO mice) demonstrated normal ductal growth and differentiation in response to E + P. By contrast, mammary glands from mice in which the ERα gene has been deleted (αERKO mice) demonstrated only rudimentary ductal structures that did not differentiate in response to E + P. EGF demonstrates estrogen-like activity in the mammary glands of αERKO mice: treatment of αERKO mice with EGF + P (without E) supported normal mammary gland development, induced expression of progesterone receptor (PR), and increased levels of G-protein-coupled receptor (GPR30) protein. Mammary gland development in ßERKO mice treated with EGF + P was comparable to that of wild-type mice receiving EGF + P; EGF had no statistically significant effects on the induction of PR or expression of GPR30 in mammary glands harvested from either wild-type mice or ßERKO mice. In vitro exposure of mammary glands to 7,12-dimethylbenz[a]anthracene (DMBA) induced preneoplastic mammary alveolar lesions (MAL) in glands from wild-type mice and ßERKO mice, but failed to induce MAL in mammary glands from αERKO mice. Microarray analysis of DMBA-treated mammary glands identified 28 functional pathways whose expression was significantly different in αERKO mice versus both ßERKO and wild-type mice; key functions that were differentially expressed in αERKO mice included cell division, cell proliferation, and apoptosis. The data demonstrate distinct roles for ERα and ERß in normal and neoplastic development in the mouse mammary gland, and suggest that EGF can mimic the ERα-mediated effects of E in this organ.

Inhibition of proliferation and induction of autophagy by atorvastatin in PC3 prostate cancer cells correlate with downregulation of Bcl2 and upregulation of miR-182 and p21.

The epidemiologic association between statin use and decreased risk of advanced prostate cancer suggests that statins may inhibit prostate cancer development and/or progression. Studies were performed to determine the effects of a model statin, atorvastatin (ATO), on the proliferation and differentiation of prostate cancer cells, and to identify possible mechanisms of ATO action. ATO inhibited the in vitro proliferation of both LNCaP and PC3 human prostate cancer cells in a dose- and time-dependent fashion. The greater inhibitory activity of ATO in PC3 cells was associated with induction of autophagy in that cell line, as demonstrated by increased expression of LC3-II. miR-182 was consistently upregulated by ATO in PC3 cells, but not in LNCaP cells. ATO upregulation of miR-182 in PC3 cells was p53-independent and was reversed by geranylgeraniol. Transfection of miR-182 inhibitors decreased expression of miR-182 by >98% and attenuated the antiproliferative activity of ATO. miR-182 expression in PC3 cells was also increased in response to stress induced by serum withdrawal, suggesting that miR-182 upregulation can occur due to nutritional stress. Bcl2 and p21 were identified to be potential target genes of miR-182 in PC3 cells. Bcl2 was downregulated and p21 was upregulated in PC3 cells exposed to ATO. These data suggest that miR-182 may be a stress-responsive miRNA that mediates ATO action in prostate cancer cells.

Deguelin action involves c-Met and EGFR signaling pathways in triple negative breast cancer cells.

BACKGROUND: Treatment of breast cancer patients with antiestrogens and aromatase inhibitor(s) or Herceptin have shown significant success in steroid receptor positive or Her-2+ breast cancers respectively. However, choice of treatments for breast cancer patients with negative status for estrogen, progesterone receptors and HER2/neu is limited. As a result, search for appropriate therapy regimen for these triple negative breast cancers (TNBC) has become a major focus of investigations for many laboratories. Recently, Deguelin, a natural product isolated from African plant Mundulea sericea (Leguminossae) has shown both antiproliferative actions in various cancers including breast as well as chemoprenventive activity against carcinogen induced experimental cancers. In this report we evaluated efficacy and mechanism of action of Deguelin in triple negative breast cancer cell lines. METHODS/FINDINGS: In vitro, Deguelin in a dose and time dependent manner inhibited the growth of MDA-MB-231, MDA-MB-468, BT-549 and BT-20 cells. Deguelin (2 or 4 mg/kg body weight), when injected intraperitoneally, reduced the in vivo tumor growth of MDA-MB-231 cells transplanted subcutaneously in athymic mice. Moreover it was nontoxic as evident from daily observations on mobility, food and water consumption and comparison of bodyweight and other visceral organ weights with those in control animals at the termination of the study. The western blot analyses and immunostaining studies indicated that the deguelin effects may be mediated through EGFR-PAKT/c-Met p-ERK and NF-κB by down regulating their downstream targets such as p-STAT3, c-Myc, Survivin. CONCLUSION/SIGNIFICANCE: These results suggest that Deguelin may have a significant therapeutic value for the treatment of TNBC patients.

Efficacy and mechanism of action of Deguelin in suppressing metastasis of 4T1 cells.

Cancer related deaths in breast cancer patients are due to metastasis of the disease. Murine 4T1 cells (Murine mammary cancer cell line developed from 6-thioguanine resistant tumor) provide an excellent research tool for metastasis related studies because these cells are highly aggressive and readily metastasize to the lungs. In this study we determined the effect of Deguelin on in vivo/vitro growth and metastasis of 4T1 cells. Deguelin inhibited the in vitro growth of 4T1 cells in a time and dose dependent manner accompanied with reduced nuclear PCNA immunostaining. In cells treated with Deguelin, reduced expression of nuclear c-Met, and its downstream targets such p-ERK and p-AKT was observed. Deguelin reduced the cell migration in 4T1 cells as determined by scratch wound assay. Combined treatment with Deguelin + ERK or PI3K/AKT inhibitor had no additional effect on cell migration. These results indicated that the action of Deguelin on cell migration may be mediated by AKT and ERK mediated signaling pathways. In vivo, Deguelin treatment significantly inhibited growth of 4T1 cells. Deguelin also reduced the occurrence of metastatic lung lesions by 33 % when cells were injected intravenously into Balb/c female mice. There was no difference in the body weight, nor was there a difference in liver and spleen weights between vehicle treated-control and Deguelin-treated animals, which indicated that Deguelin was nontoxic at the dose used in the present study. These results provide rationale for developing Deguelin as a chemotherapeutic agent for triple negative breast cancer patients.

PPARγ antagonist GW9662 induces functional estrogen receptor in mouse mammary organ culture: potential translational significance.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) plays a central role in regulating metabolism, including interaction with the estrogen receptor-α (ERα). Significantly, PPARγ activity can be modulated by small molecules to control cancer both in vitro and in vivo (Yin et al., Cancer Res 69:687-694, 2009). Here, we evaluated the effects of the PPARγ agonist GW7845 and the PPARγ antagonist GW9662 on DMBA-induced mammary alveolar lesions (MAL) in a mouse mammary organ culture. The results were as follows: (a) the incidence of MAL development was significantly inhibited by GW 7845 and GW 9662; (b) GW9662 but not GW7845, in the presence of estradiol, induced ER and PR expression in mammary glands and functional ERα in MAL; (c) while GW9662 inhibited expression of adipsin and ap2, GW 7845 enhanced expression of these PPARγ-response genes; and (d) Tamoxifen caused significant inhibition of GW9662 treated MAL, suggesting that GW9662 sensitizes MAL to antiestrogen treatment, presumably through rendering functional ERα and induction of PR. The induction of ERα by GW9662, including newer analogs, may permit use of anti-ER strategies to inhibit breast cancer in ER- patients.

Efficacy and mechanism of action of Proellex, an antiprogestin in aromatase overexpressing and Letrozole resistant T47D breast cancer cells.

Aromatase inhibitors (AI) are considered as a first line therapy for ER+PR+ breast cancers. However, many patients acquire resistance to AI. In this study, we determined the response of antiprogestin CDB-4124 (Proellex) on the aromatase overexpressing and Letrozole resistant cell lines and also studies its mechanism of action in inhibition of breast cancer cell proliferation. For these studies we generated aromatase overexpressing T47D (T47Darom) and respective control (T47Dcon) breast cancer cell lines by stable transfection with plasmid containing CYP19A1 gene, or empty vector respectively. Letrozole resistant cell line (T47DaromLR) was generated by incubating T47Darom for 75 weeks in the presence of 10 µM Letrozole. Cell proliferation was determined by MTT or crystal violet assays. Gene expressions were quantified by QRT-PCR whereas proteins were identified by western blot analyses, flow cytometry and immunofluorescence staining. Aromatase activity was determined by estradiol ELISA. The effects of Proellex on the anchorage independent growth were measured by soft agar colony formation. Statistical differences between the various groups were determined by Student's 't' test or ANOVA followed by Bonferroni's post hoc test. Results showed that T47Darom and T47DaromLR cell lines had significantly higher aromatase expression (mRNA; 80-90 fold and protein) and as a result exhibited increased aromatization of testosterone to estradiol as compared to T47Dcon. Both these cell lines showed enhanced growth in the presence of Testosterone (50-60%). In T47DaromLR cells increased PR-B and EGFR expression as compared to T47Dcon cells was observed. Proellex and other known aromatase inhibitors (Letrozole, Anastrozole, and Exemestane) inhibited testosterone induced cell proliferation and anchorage independent growth of T47Darom cells. Cell growth inhibition was significantly greater when cells were treated with Proellex alone or in combination with other AIs as compared to AIs alone. Proellex inhibited mRNA and protein levels of PR-B, reduced PRB/p300 complex formation in the nuclei and significantly reduced EGFR expression in T47Darom cells. Our results in the present study indicate that antiproliferative effect of Proellex is probably due to PR-B/EGFR modulation in ER+PR+, aromatase expressing cells. Overall these results suggest that antiprogestin, Proellex can be developed as a possible treatment strategy for aromatase overexpressing ER+/PR+ breast cancer patients as well as for aromatase inhibitor resistant breast cancer patients.

Vitamin D and breast cancer: emerging concepts.

The benefit of vitamin D in cancer prevention and to certain extent therapy has been well recognized. The active form of vitamin D, 1,25-dihydroxycholecalciferol (1,25(OH)2 D3) is a natural ligand for vitamin D receptor (VDR). Since 1,25(OH)2D3 exerts toxic effects at a concentration that is beneficial, nearly 1500 analogs of vitamin D have been synthesized and evaluated for their efficacy in a variety of carcinogenesis and human cancer models both in vitro and in vivo. Among these only a handful of them have been approved for evaluation in clinical trials for leukemia, breast, prostate and colon cancers. The mechanism of vitamin D action is mediated by the nuclear VDR and the signaling cascade for its action is extensively reported. In this review we focus on the newer concepts for vitamin D action. These include (1) differential effects of vitamin D in maintaining cell proliferation when the cells are under stress but suppressing cell growth when the cells are transformed; (2) functional significance of VDR polymorphism in potential vitamin D responsiveness; (3) regulation of constitutive splicing of vitamin D target gene, CYP24a, by the hormone and its significance; and (4) regulation of microRNA by vitamin D in breast cancer. It is anticipated that the new work in these selective areas would expand the understanding of vitamin D in breast cancer prevention and therapy.

Crosstalk between the peroxisome proliferator-activated receptor γ (PPARγ) and the vitamin D receptor (VDR) in human breast cancer cells: PPARγ binds to VDR and inhibits 1α,25-dihydroxyvitamin D3 mediated transactivation.

Heterodimerization and cross-talk between nuclear hormone receptors often occurs. For example, estrogen receptor alpha (ERα) physically binds to peroxisome proliferator-activated receptor gamma (PPARγ) and inhibits its transcriptional activity. The interaction between PPARγ and the vitamin D receptor (VDR) however, is unknown. Here, we elucidate the molecular mechanisms linking PPARγ and VDR signaling, and for the first time we show that PPARγ physically associates with VDR in human breast cancer cells. We found that overexpression of PPARγ decreased 1α,25-dihydroxyvitamin D(3) (1,25D(3)) mediated transcriptional activity of the vitamin D target gene, CYP24A1, by 49% and the activity of VDRE-luc, a vitamin D responsive reporter, by 75% in T47D human breast cancer cells. Deletion mutation experiments illustrated that helices 1 and 4 of PPARγ's hinge and ligand binding domains, respectively, governed this suppressive function. Additionally, abrogation of PPARγ's AF2 domain attenuated its repressive action on 1,25D(3) transactivation, indicating that this domain is integral in inhibiting VDR signaling. PPARγ was also found to compete with VDR for their binding partner retinoid X receptor alpha (RXRα). Overexpression of RXRα blocked PPARγ's suppressive effect on 1,25D(3) action, enhancing VDR signaling. In conclusion, these observations uncover molecular mechanisms connecting the PPARγ and VDR pathways.

Estrogen receptor-ß protects against colitis-associated neoplasia in mice.

Estrogen receptor-beta (ERß) has been suggested to exert anti-inflammatory and anti-tumorigenic effects in the colon, providing a translational potential to prevent and/or treat inflammatory bowel disease (IBD) and its progression to colitis-associated colorectal cancer (CAC). However, the specific direct role of ERß in CAC has not yet been tested. We assessed the effects of ERß deficiency in the azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CAC model using ERß knockout (ßERKO) mice and wild-type (WT) littermates. These mice were injected with AOM followed by 1 week of DSS treatment, and sacrificed on weeks 9 or 16. ßERKO mice developed more severe clinical colitis compared to WT mice, as evidenced by significantly higher disease activity index after DSS treatment, weight to length ratio of the colons, inflammation score and grade of dysplasia. ERß-deficient colons presented greater number and size of polyps at weeks 9 and 16, respectively, and were characterized by a significant increase in interleukin (IL)-6, IL-17, tumor necrosis factor alpha and interferon-gamma mRNA levels. Furthermore, higher protein expression levels of nuclear factor-kappa B, inducible nitric oxide synthase, ß-catenin, proliferating cell nuclear antigen, mucin-1 and significantly lower caveolin-1 and mucin-2 protein levels were shown in ßERKO mice compared to WT mice. These data suggest a possible anti-inflammatory and anti-neoplastic mechanism of action of ERß in CAC. These results demonstrate for the first time that ERß provides protection in the AOM/DSS-induced CAC model in mice, suggesting a preventive and/or therapeutic potential for the use of ERß-selective agonists in IBD.

Regulation of CYP24 splicing by 1,25-dihydroxyvitamin D3 in human colon cancer cells.

CYP24 is a well-established vitamin D receptor (VDR) target gene. The active VDR ligand 1,25(OH)2D3 regulates its own catabolism by increasing CYP24 expression. It is well known that in the presence of 1,25(OH)2D3, VDR binds to VDREs in the promoter region of CYP24 and initiates CYP24 transcription. However, little is known about the role of 1,25(OH)2D3 in the posttranscriptional modulation of CYP24. In this study, we investigated the functional significance of 1,25(OH)2D3 in CYP24 RNA splicing in colon cancer cells. Using RT-PCR, we found that 1,25(OH)2D3 actively induces CYP24 splicing in a time-dependent manner and CYP24 splicing pattern could be cell type or tissue specific. The induction of RNA splicing by 1,25(OH)2D3 was mainly CYP24 selective. Treatment of cells with parathyroid hormone inhibited basal CYP24 splicing, but failed to inhibit 1,25(OH)2D3-induced CYP24 splicing. Further experiments demonstrated that new RNA synthesis was required for the induction of CYP24 splicing by vitamin D. In addition, alteration of multiple signaling pathways also affected CYP24 splicing and cellular sensitivity in response to vitamin D appeared to correlate with the induction of CYP24 splicing. These results suggest that 1,25(OH)2D3 not only regulates CYP24 transcription, but also plays an important role in posttranscriptional modulation of CYP24 by inducing its splicing. Our findings reveal an additional regulatory step that makes the vitamin D mediated action more prompt and efficient.

Modeling the cellular impact of nanoshell-based biosensors using mouse alveolar macrophage cultures.

In this study, the relative toxicity of native gold-silica nanoshells (NS) has been compared to nanoshells modified with poly(ethylene glycol)-thiol (PEG-SH) and a Raman-active PEG, p-mercaptoaniline-poly(ethylene glycol) (pMA-PEG), in mouse alveolar macrophage cell cultures (RAW 264.7). The results from toxicity profiling using an MTT assay demonstrate that cell viability post-particle exposure is a function of three factors: nanoshell concentration, surface functionalization, and incubation time. By minimizing particle concentrations and incubation times, cell cultures are able to recover within 24 h of nanoshell removal, indicative of nanoshells having more of a cytostatic versus cytotoxic effect on macrophage cells. The mechanism of the cytostatic effect has been investigated by imaging the presence of reactive oxygen species (ROS) using a fluorescence assay kit (Image-iT™ LIVE) after the introduction of NS to the cell cultures. Elevated ROS signals are seen in the cells containing higher concentration of NS, and indicate that the major reason of toxicity may due to the oxidative stress caused by excess NS particles. Raman imaging experiments with pMA-PEG coated nanoshells showed that cells exposed for even short exposure times (∼2 h) retained those particles up to 24 h after exposure, while migration experiments suggest that surviving cells retain their nanoshells and may reallocate them to progeny cells upon cell division.

Functional significance of vitamin D receptor FokI polymorphism in human breast cancer cells.

BACKGROUND: The FokI vitamin D receptor (VDR) polymorphism results in different translation initiation sites on VDR. In the VDRff variant, initiation of translation occurs at the first ATG site, giving rise to a full length VDR protein of 427 amino acids. Conversely, in the VDRFF variant, translation begins at the second ATG site, resulting in a truncated protein with three less amino acids. Epidemiological studies have paradoxically implicated this polymorphism with increased breast cancer risk. 1α,25 (OH)(2)D(3), the active metabolite of vitamin D, is known to inhibit cell proliferation, induce apoptosis and potentiate differentiation in human breast cancer cells. It is well documented that 1α,25 (OH)(2)D(3) downregulates estrogen receptor α expression and inhibits estrogen mediated signaling in these cells. The functional significance of the VDR FokI polymorphism in vitamin D action is undefined. METHODS/FINDINGS: To elucidate the functional role of FokI polymorphism in breast cancer, MCF-7-Vector, MCF-7-VDRff and MCF-7-VDRFF stable cell lines were established from parental MCF-7 cells as single-cell clones. In response to 1α,25 (OH)(2)D(3) treatments, cell growth was inhibited by 60% in VDRFF cells compared to 28% in VDRff cells. The induction of the vitamin D target gene CYP24A1 mRNA was 1.8 fold higher in VDRFF cells than in VDRff cells. Estrogen receptor-α protein expression was downregulated by 62% in VDRFF cells compared to 25% in VDRff cells. VDR protein stability was greater in MCF-7-VDRFF cells in the presence of cycloheximide. PCR array analyses of VDRff and VDRFF cells revealed increased basal expression levels of pro-inflammatory genes Cyclooxygenase-2, Interleukin-8 and Chemokine (C-C Motif) Ligand 2 in MCF-7-VDRff cells by 14, 52.7 and 5 fold, respectively. CONCLUSIONS/SIGNIFICANCE: These results suggest that a VDRff genotype may play a role in amplifying aggressive breast cancer, paving the way for understanding why some breast cancer cells respond inefficiently to vitamin D treatment.