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.

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.

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.

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.

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.

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.

Protection against cellular stress by 25-hydroxyvitamin D3 in breast epithelial cells.

25-Hydroxyvitamin D(3) (25(OH)D(3)) is a prohormone and a major vitamin D metabolite. The discovery of (25(OH)D(3)) 1 alpha-hydroxylase in many vitamin D target organs has yielded an increased interest in defining the role(s) of 25(OH)D(3) in these tissues. The etiology of cancer appears to be complex and multi-factorial. Cellular stress (e.g., DNA damage, hypoxia, oncogene activation) has been identified as one of the key factors responsible for initiating the carcinogenesis process. In this study, we investigated whether 25(OH)D(3) protects breast epithelial cells from cellular stress using an established breast epithelial cell line MCF12F. To better elucidate the role of 25(OH)D(3) in the stress response, we used multiple in vitro stress models including serum starvation, hypoxia, oxidative stress, and apoptosis induction. Under all these stress conditions, 25(OH)D(3) (250 nmol/L) treatment significantly protected cells against cell death. Low-serum stress induced p53 expression accompanied with downregulation of PCNA, the presence of 25(OH)D(3) consistently inhibited the alteration of p53 and PCNA, suggesting that these molecules were involved in the stress process and may be potential target genes of 25(OH)D(3). miRNA microarray analysis demonstrated that stress induced by serum starvation caused significant alteration in the expression of multiple miRNAs including miR182, but the presence of 25(OH)D(3) effectively reversed this alteration. These data suggest that there is a significant protective role for 25(OH)D(3) against cellular stress in the breast epithelial cells and these effects may be mediated by altered miRNA expression.

Functionality of unliganded VDR in breast cancer cells: repressive action on CYP24 basal transcription.

It is well-established that CYP24, an immediate target gene of VDR is upregulated by VDR ligands. This study is focused on the functional role of unliganded VDR by investigating the correlation between the expression of VDR protein and basal mRNA levels of CYP24 in breast cancer cell lines. Analyses of multiple breast cancer cell lines demonstrated an inverse correlation between VDR protein expression and CYP24 mRNA expression levels; while in the presence of ligand, VDR protein level was positively correlated with CYP24 expression. In MCF-7 cells, VDR was mainly distributed in the nuclei in the absence of ligand. VDR overexpression in MCF-7 cells and MDA-MB231 cells decreased CYP24 mRNA expression levels and CYP24 promoter activity. Conversely, knock-down of VDR using siRNA techniques in MCF-7 and T47D cells significantly increased CYP24 mRNA expression. We also found that overexpression of VDR with a polymorphic site (FokI-FF) at its AF-1 domain, which makes VDR shorter by three amino acids, failed to repress CYP24 promoter activity. This report provides conclusive evidence for the repressive action of unliganded VDR on the expression of its target gene CYP24 and the importance of an intact VDR AF-1 domain for its repressive action.

Cancer chemoprevention by natural products: how far have we come?

Since ancient times, natural products, herbs and spices have been used for preventing several diseases, including cancer. The term chemoprevention was coined in the late 1970s and referred to the prevention of cancer by selective use of phytochemicals or their analogs. The field utilizes experimental carcinogenesis models to examine the efficacy of chemopreventive agents in a stage-specific manner. The concept of using naturally derived chemicals as potential chemopreventive agents has advanced the field dramatically. Throughout the years, a vast number of chemopreventive agents present in natural products have been evaluated using various experimental models. A number of them have progressed to early clinical trials. More recently, the focus has been directed towards molecular targeting of chemopreventive agents to identify mechanism(s) of action of these newly discovered bioactive compounds. Moreover, it has been recognized that single agents may not always be sufficient to provide chemopreventive efficacy, and, therefore, the new concept of combination chemoprevention by multiple agents or by the consumption of "whole foods" has become an increasingly attractive area of study. Novel technologies, such as nanotechnology, along with a better understanding of cancer stem cells, are certain to continue the advancement of the field of cancer chemoprevention in years to come.

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.

25-Hydroxyvitamin D3 is a natural chemopreventive agent against carcinogen induced precancerous lesions in mouse mammary gland organ culture.

Despite the role of vitamin D(3) endocrine system in prevention of mammary gland transformation in animal models, use of 1,25(OH)(2)D(3 )in clinical settings is precluded due to its toxicity in vivo. Therefore much effort has been placed in developing relatively non-toxic vitamin D analogs. Recently, with the discovery of the expression of 25-hydroxy vitamin D(3) 1alpha-hydroxylase (CYP27B1) in multiple extrarenal organs, the functional role of prohormone, 25-hydroxyvitamin D(3) [25(OH)D(3)], has been redefined. Since 25(OH)D(3) does not cause hypercalcemia and maintains relative high concentration in serum, it is possible that the prohormone can be converted to active hormone in mammary epithelial cells to provide chemopreventive effects. In the present study, we evaluated its functional significance using mouse mammary organ culture (MMOC) system. We first showed that 25(OH)D(3) 1alpha-hydroxylase is extensively expressed in mammary ductal epithelial cells at both protein and mRNA levels, which is a prerequisite for 25(OH)D(3) to function in an autocrine/paracrine manner. However, we also observed that clotrimazol (1alpha-hydroxylase inhibitor) enhanced 25(OH)D(3) -induced CYP24 expression in breast cancer cells. In mammary glands derived from 1alpha-hydroxylase knockout mice, 25(OH)D(3) treatment in organ culture significantly induced CYP24 expression, indicating a potential direct effect of 25(OH)D(3). In MMOC, 100-250 nM 25(OH)D(3) suppressed both ovarian hormone-dependent and -independent mammary precancerous lesions (induced by DMBA) by more than 50%, while the active hormone 1,25(OH)(2)D(3) (positive control) at 100 nM suppressed alveolar lesions by more than 80%. The inactive vitamin D(3) (negative control) at 100 nM suppressed alveolar lesions by only 20% (P>0.05). We found that 25(OH)D(3) inhibits DMBA-induced mammary alveolar lesions (MAL) in a stage-specific manner: 25(OH)D(3) mainly inhibits the promotion stage of lesion formation. We conclude that 25(OH)D(3) could serve as a non-toxic natural chemopreventive agent for further development for breast cancer prevention.

Comprehensive review of cancer chemopreventive agents evaluated in experimental carcinogenesis models and clinical trials.

Cancer chemoprevention refers to the use of pharmacological agents to inhibit, delay or reverse the multi-step process of carcinogenesis. The last two decades in particular have witnessed explosive growth in this emerging field of cancer chemoprevention. Extensive efforts to evaluate possible application of various chemopreventive agents, in individuals at high risk of neoplastic development have been carried out. Epidemiological studies suggest a protective role of several agents in reducing the risk of cancer. The protective action of all these agents is explained as a combination of various proposed mechanisms involving anti-oxidant, anti-inflammatory, immunomodulatory action, apoptosis induction, molecular association with carcinogen, cell cycle arrest, cell differentiation induction, antimicrobial effect, and anti- angiogenesis etc. Large numbers of candidate substances such as phytochemicals and their synthetic derivatives have been identified by a combination of in vitro and in vivo studies in a wide range of biological assays. However, a comprehensive description of these chemopreventive agents has not been extensively reviewed. In this review we discuss cancer chemopreventive agents in relation to their source, efficacy in cancer chemopreventive action in vivo and epidemiological data. The experimental carcinogenesis studies in different biological models, in addition to the contribution from our laboratory are summarized.

Efficacy of chemopreventive agents in mouse mammary gland organ culture (MMOC) model: a comprehensive review.

Currently, breast cancer is considered as one of the leading causes for death in women in the United States. Consumption of natural products has received considerable attention in recent years as a possible approach for cancer prevention in general population. There are numerous cancer preventive agents present in the natural products, which may contribute to their chemopreventive properties. During the past two decades, numerous chemopreventive agents have been isolated and/or synthesized and evaluated for their efficacy in a variety of biological assays. To this end, we have established and utilized mouse mammary gland organ culture model (MMOC) as a bioassay for identifying chemopreventive agents. Mammary glands respond to growth promoting hormones and the physiological differentiation can be reproduced in MMOC in chemically defined medium by altering hormonal milieu. Both estrogen and progesterone dependent (mammary ductal lesions, MDL) and independent (mammary alveolar lesions, MAL) precancerous lesions can be induced in response to a 24 hour exposure to DMBA in MMOC. Suppression of the incidence and multiplicity of these lesions by a possible chemopreventive agent can serve as a tool to evaluate efficacy of potential experimental agents. Using this approach, we have evaluated more than 200 synthetic and natural product-derived chemopreventive agents in this model as a part of the National Cancer Institute-supported projects. Many of these chemopreventive agents expressing significant activity have progressed to the in vivo experimental mammary carcinogenesis studies. Thus, this bioassay has proven to be a valuable tool for screening cancer chemopreventive agents for breast cancer prevention and for understanding molecular mechanism(s) of action of these agents. In this comprehensive review, we provide a complete list of chemopreventive agents evaluated for the efficacy against development of mammary alveolar lesions (MAL) in MMOC along with the recent developments in this area. The structure-activity relationships for many chemopreventive agents evaluated in the MMOC model have been discussed.

Antiviral activity of phytochemicals: a comprehensive review.

Numerous numbers of biologically active agents have been identified for their diverse therapeutic functions. Detailed investigations of phytochemicals for antiviral activities have assumed greater importance in the last few decades. A wide variety of active phytochemicals, including the flavonoids, terpenoids, organosulfur compounds, limonoids, lignans, sulphides, polyphenolics, coumarins, saponins, chlorophyllins, furyl compounds, alkaloids, polyines, thiophenes, proteins and peptides have been found to have therapeutic applications against different genetically and functionally diverse viruses. The antiviral mechanism of these agents may be explained on basis of their antioxidant activities, scavenging capacities, inhibiting DNA, RNA synthesis, inhibition of the viral entry, or inhibiting the viral reproduction etc. Large number candidate substances such as phytochemicals and their synthetic derivatives have been identified by a combination of in vitro and in vivo studies in different biological assays. In this article we have made attempts to extensively review and provide comprehensive description of different phyto-antiviral agents. We have examined the recent developments in the field of plant derived antiviral agents. The major advances in the field of viral interactions in various biological assays have been summarized. In addition sources of origin, major viral studies mechanistic action and phase trials of various phytoantiviral agents have been included in the review.

Prohibitin is a novel target gene of vitamin D involved in its antiproliferative action in breast cancer cells.

Previously, we showed that N-methyl-N-nitrosourea-transformed MCF12F breast epithelial cells exhibited differential expression of several genes, including up-regulation of prohibitin and elevated sensitivity to a relatively noncalcemic vitamin D analogue, 1alpha-hydroxyvitamin D5 [1alpha(OH)D5]. In this report, we evaluated the functional significance of prohibitin in relation to the cellular response to vitamin D. The in silico screening for putative transcription factor binding sites identified two vitamin D receptor (VDR)/retinoid X receptor binding sites in the 1-kb promoter region of prohibitin. Prohibitin up-regulation by 1alpha(OH)D5 treatment at both transcriptional and translational levels was confirmed by real-time reverse transcription-PCR and Western blot analysis in breast cancer cells, identifying prohibitin as a vitamin D target gene. Confocal microscopic analysis showed that prohibitin was localized in the nuclei of MCF-7 cells and a portion of prohibitin was colocalized with VDR, but direct physical interaction between VDR and prohibitin in cell lysates was not detectable. In MCF-7 cells expressing tetracycline-inducible prohibitin (Tet-On model), the overexpression of prohibitin inhibited cell proliferation and enhanced vitamin D-induced antiproliferative activity. Knockdown of prohibitin was accompanied by increased number of cells incorporating bromodeoxyuridine in the whole population and increased cell distribution in the S phase of cell cycle. In addition, prohibitin level had no significant effect on the vitamin D-induced transactivation of CYP24, a VDR target gene. This is the first report to suggest that prohibitin serves as a novel vitamin D target gene, which is involved in the antiproliferative action of vitamin D without affecting CYP24 transactivation in breast cancer cells.

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.

Advanced rat mammary cancers are growth hormone dependent.

Epidemiological studies suggest that the GH/IGF-I axis may promote human cancers. Animal models in which the GH/IGF-I axis can be controlled may be helpful in elucidating the role of these hormones during mammary cancer progression. Beginning at 3 or 5 wk of age, spontaneous dwarf rats (Gh(dr/dr)), which lack GH and have very low serum IGF-I, were treated with either rat or bovine GH twice daily. Other Gh(dr/dr) rats received vehicle, and wild-type Sprague Dawley rats (Gh(+/+), parent strain to SDR) received vehicle. One week later, all rats were exposed to a single injection of N-methyl-N-nitrosourea. Body weight gain and serum IGF-I levels were similar in Gh(+/+) and GH-treated Gh(dr/dr) rats. Furthermore, mammary tumor incidence, latency, and multiplicity were similar in Gh(+/+) and GH-treated Gh(dr/dr) rats. Vehicle-treated Gh(dr/dr) rats developed no tumors. Once advanced (> or =1 cm(3)) mammary cancers were established in GH-treated Gh(dr/dr) rats, GH treatments were halted and nearly all tumors regressed completely within 2 wk. Tumor regression was associated with loss of phospho-signal transducer and activator of transcription-3, but not alterations in IGF-I, IGF-I receptor, or GH receptor. These results demonstrate that Gh(dr/dr) rats, which are nearly refractory to mammary carcinogenesis, can be made vulnerable by restoring GH and IGF-I. Furthermore, advanced rat mammary cancers are dependent on GH and/or IGF-I for their survival. Therefore, therapeutics that target either GH or IGF-I may be effective at treating even advanced mammary cancers.

Differential expression of prohibitin is correlated with dual action of Vitamin D as a proliferative and antiproliferative hormone in breast epithelial cells.

Our previous microarray analysis showed that N-methyl-N-nitrosourea (MNU) transformed MCF12F breast epithelial cells exhibited upregulation of several genes, including prohibitin, which was reversed by 1alpha-hydroxyvitamin D(5) (1alpha(OH)D(5)) treatment. The in silico screening for putative transcription factor binding sites identified two VDR/RXR binding sites in the 1kb promoter region of prohibitin. Other binding sites for EGR and GR which are also Vitamin D target genes were identified in this region, indicating that prohibitin is a potential target gene for Vitamin D. The combination of multiple binding sites also provides a basis for a possible dual regulation of prohibitin by Vitamin D. Prohibitin upregulation by 1alpha(OH)D(5) treatment at both transcription and translation level was observed in Vitamin D sensitive BT474 breast cancer cells, in which 1alpha(OH)D(5) significantly inhibited cell proliferation in normal culture condition. On the other hand, prohibitin down-regulation accompanied with Vitamin D mediated maintenance of proliferation of breast epithelial cells was observed under stressed condition. These results demonstrated that Vitamin D mediated antiproliferative activity in unstressed condition and growth maintaining activity under stressed condition involve differential expression of prohibitin.