Hong Jin Lee

Dietary intake of pterostilbene, a constituent of blueberries, inhibits the β-catenin/p65 downstream signaling pathway and colon carcinogenesis in rats

Stilbenes are phytochemicals present in grapes, berries, peanuts and red wine. A widely studied stilbene, resveratrol (trans-3,5,4-trihydroxystilbene), has been shown to exert antioxidant, anti-inflammatory, chemopreventive and antiaging effects in a number of biological systems. We reported earlier that pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene), a structurally related stilbene found in blueberries, was effective in reducing the incidence and multiplicity of aberrant crypt foci formation in the colon of rats injected with azoxymethane (AOM). Our present study was to identify the chemopreventive potential of pterostilbene with colonic tumor formation as an end point and further to evaluate the mechanistic action of pterostilbene during colon carcinogenesis. F344 rats were given two AOM injections subcutaneously when they were 7 and 8 weeks old and continuously fed the control or 40 p.p.m. pterostilbene diet for 45 weeks. Overall analyses indicated that pterostilbene reduced colon tumor multiplicity of non-invasive adenocarcinomas, lowered proliferating cell nuclear antigen and downregulated the expression of beta-catenin and cyclin D1. Pterostilbene decreased mucosal levels of the proinflammatory cytokines, tumor necrosis factor-alpha, interleukin (IL)-1beta and IL-4. Colon tumors from pterostilbene-fed animals showed reduced expression of inflammatory markers as well as nuclear staining for phospho-p65, a key molecule in the nuclear factor-kappaB pathway. In HT-29 cells, pterostilbene reduced the protein levels of beta-catenin, cyclin D1 and c-MYC, altered the cellular localization of beta-catenin and inhibited the phosphorylation of p65. Our data with pterostilbene in suppressing colon tumorigenesis, cell proliferation as well as key inflammatory markers in vivo and in vitro suggest the potential use of pterostilbene for colon cancer prevention.

In vitro and in vivo studies on stilbene analogs as potential treatment agents for colon cancer

Based on the potential of resveratrol as a colon cancer chemopreventive agent, a set of 26 stilbenes were synthesized and tested against the colon cancer cell lines HT-29 and Caco-2. (Z)-4-(3,5-Dimethoxystyryl)aniline (4), (Z)-methyl-4-(3,5-dimethoxystyryl)benzoate (6), and (Z)-1,3-dimethoxy-5-(4-methoxystyryl)benzene (10) showed strong inhibitory activity in vitro. In vivo studies using HT-29 xenografts in immunodeficient mice were conducted with 4, 6 and 10, together with their corresponding trans isomers (3, 5, and 9, respectively), at the dose of 10 mg/kg body weight. Tumor volume was significantly lowered in 3-, 4-, and 9-treated groups. The cis- and trans-amino analogs (4 and 3, respectively) had similar effect on tumor growth, a 40% decrease compared to the control. Analysis of the serum revealed that 4 isomerized to 3, which may explain their similar effects in SCID mice. Stilbenes 5, 6, 9, and 10 retained their configurations in the serum. Stilbenes 6 and 10 lacked tumor-suppressive effect in SCID mice; the serum levels of these analogs were low (18.8 and 15.5 ng/mL, respectively). Stilbene 9, while weakly active in vitro demonstrated good activity in vivo, was found at higher levels in the serum (69.9 ng/mL) compared to 10. The anti-tumorigenic activity of these stilbene analogs may be partly linked to their effects on proteins involved in cell proliferation, as observed by lowered expression of proliferating cell nuclear antigen and upregulation of the cyclin-dependent kinase inhibitor, p27, in the tumor tissues. Overall, identification of the anti-tumorigenic potential of these compounds provides opportunities for their use against colorectal cancer.

Dietary tocopherols inhibit cell proliferation, regulate expression of ERα, PPARγ, and Nrf2, and decrease serum inflammatory markers during the development of mammary hyperplasia

Previous clinical and epidemiological studies of vitamin E have used primarily α‐tocopherol for the prevention of cancer. However, γ‐tocopherol has demonstrated greater anti‐inflammatory and anti‐tumor activity than α‐tocopherol in several animal models of cancer. This study assessed the potential chemopreventive activities of a tocopherol mixture containing 58% γ‐tocopherol (γ‐TmT) in an established rodent model of mammary carcinogenesis. Female ACI rats were utilized due to their sensitivity to 17β‐estradiol (E2) to induce mammary hyperplasia and neoplasia. The rats were implanted subcutaneously with sustained release E2 pellets and given dietary 0.3% or 0.5% γ‐TmT for 2 or 10u2009wk. Serum E2 levels were significantly reduced by the treatment with 0.5% γ‐TmT. Serum levels of inflammatory markers, prostaglandin E2 and 8‐isoprostane, were suppressed by γ‐TmT treatment. Histology of mammary glands showed evidence of epithelial hyperplasia in E2‐treated rats. Immunohistochemical analysis of the mammary glands revealed a decrease in proliferating cell nuclear antigen (PCNA), cyclooxygenase‐2 (COX‐2), and estrogen receptor α (ERα), while there was an increase in cleaved‐caspase 3, peroxisome proliferator‐activated receptor γ (PPARγ), and nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) in γ‐TmT‐treated rats. In addition, treatment with γ‐TmT resulted in a decrease in the expression of ERα mRNA, whereas mRNA levels of ERβ and PPARγ were increased. In conclusion, γ‐TmT was shown to suppress inflammatory markers, inhibit E2‐induced cell proliferation, and upregulate PPARγ and Nrf2 expression in mammary hyperplasia, suggesting that γ‐TmT may be a promising agent for human breast cancer prevention.

Gemini Vitamin D Analog Suppresses ErbB2-Positive Mammary Tumor Growth via Inhibition of ErbB2/AKT/ERK Signaling

Numerous synthetic vitamin D analogs have been studied for their effects on the prevention and treatment of breast cancer. However, the inhibitory effects of naturally occurring 1alpha,25-dihydroxyvitamin D3 or its synthetic analogs on ErbB2 overexpressing mammary tumorigenesis have not been reported. Gemini vitamin D analogs are novel synthetic vitamin D derivatives with a unique structure of two six-carbon chains at C-20. We have previously shown that Gemini vitamin D analogs significantly inhibited carcinogen-induced estrogen receptor (ER)-positive mammary tumorigenesis and reduced ER-negative MCF10DCIS.com xenograft tumor growth without hypercalcemic toxicity. In the present study, we have determined the inhibitory effect of a potent Gemini vitamin D analog BXL0124 (1alpha,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol) on the ErbB2/Her-2/neu overexpressing mammary tumorigenesis. The Gemini BXL0124 inhibits ErbB2-positive mammary tumor growth and down-regulates the phosphorylation of ErbB2, ERK and AKT in tumors of MMTV-ErbB2/neu transgenic mice. These effects of Gemini BXL0124 in vivo were confirmed by using the ErbB2 overexpressing tumor cells derived from the mammary tumors of MMTV-ErbB2/neu mice. In conclusion, the Gemini vitamin D analog BXL0124 inhibits the growth of ErbB2 overexpressing mammary tumors through regulating the ErbB2/AKT/ERK signaling pathways, suggesting that Gemini vitamin D analog may be considered for translational studies.

Structure–function study of gemini derivatives with two different side chains at C-20, Gemini-0072 and Gemini-0097

Derivatives of vitamin D(3) containing a second side-chain emanating at C-20 are known as gemini and act as vitamin D receptor agonists. Recently, two of these, namely Gemini-0072 and the epimeric Gemini-0097, were selected for further studies in view of their high biological activities and lack of hypercalcemic effects. We now show that the two analogs recruit coactivator SRC-1 better than the parental gemini and act as VDR superagonists. The crystal structures of complexes of zVDR with Gemini-0072 and Gemini-0097 indicate that these ligands induce an extra cavity within the ligand-binding pocket similar to gemini and that their superagonistic activity is due to an increased stabilization of helix H12.

A novel vitamin D derivative activates bone morphogenetic protein signaling in MCF10 breast epithelial cells.

We investigated the action of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], a novel Gemini vitamin D3 analog Ro-438-3582 [1α,25-dihydroxy-20S-21(3-hydroxy-3-methyl-butyl)-23-yne-26,27-hexafluorocholecalciferol (Ro3582)], and a classic vitamin D3 analog Ro-26-2198 [1α,25-dihydroxy-16,23(Z)-diene-26,27-hexafluoro-19-nor-cholecalciferol (Ro2198)] in modulating the transforming growth factor-β (TGF-β)/bone morphogenetic protein (BMP) system in MCF10 immortalized breast epithelial cells. We found that 1α,25(OH)2D3, Ro3582, and Ro2198 all enhanced BMP/Smad signaling by increasing the phosphorylation of receptor-regulated Smads. Ro3582 was more active than Ro2198, but both were considerably more active than 1α,25(OH)2D3. Ro3582 enhanced BMP/Smad signaling by 1) inducing the phosphorylation of receptor-regulated Smads (Smad1/5), 2) increasing the accumulation of phosphorylated Smad1/5 in the nucleus, and 3) activating BMP-mediated transcription in MCF10 breast epithelial cells. Furthermore, Ro3582 induced the synthesis of BMP-2 and BMP-6 mRNA and protein, and the expression of Smad6 mRNA in MCF10 breast epithelial cells was inhibited by Ro3582. The induction of phospho-Smad1/5 by Ro3582 was inhibited by treatment with the BMP antagonist Noggin, whereas neutralizing antibody to TGF-β did not block the induction of phospho-Smad1/5 by Ro3582. Treatment with Noggin also blocked the effect of Ro3582 on nuclear accumulation of phospho-Smad1/5 and the induction of BMP-2 and BMP-6 mRNA synthesis. These results indicate that the activation of BMP/Smad signaling by the Gemini vitamin D3 analog Ro3582 may be through the production of BMP ligands, including BMP-2 and BMP-6, and/or down-regulation of the inhibitory Smad6. This is the first report to show that 1α,25(OH)2D3 and its derivatives activate BMP/Smad-specific signaling in human breast epithelial cells.

Structural analysis and biological activities of BXL0124, a gemini analog of vitamin D

Gemini analogs of calcitriol, characterized by the extension of the C21-methyl group of calcitriol with a second chain, act as agonists of the vitamin D receptor (VDR). This second side chain of gemini is accommodated in a new cavity inside the VDR created by the structural rearrangement of the protein core. The resulting conformational change preserves the active state of the receptor and bestows gemini compounds with biological activities that exceed those of calcitriol. Of particular interest are geminis anti-cancer properties, and in this study we demonstrate anti-proliferative and tumor-reducing abilities of BXL0124 and BXL0097, differing only by the presence or absence, respectively, of the methylene group on the A ring. BXL0124 acts as a more potent VDR agonist than its 19-nor counterpart by activating VDR-mediated transcription at lower concentrations. In a similar manner, BXL0124 is more active than BXL0097 in growth inhibition of breast cancer cells and reduction of tumor volume. Structural comparisons of BXL0097 and BXL0124, as their VDR complexes, explain the elevated activity of the latter.

Abstract 833: Combination of atorvastatin with sulindac or naproxen profoundly inhibits colonic adenocarcinomas by suppressing the p65/beta-catenin/cyclin D1 signaling pathway in rats

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnColorectal cancer is the third leading cause of death from cancer worldwide. Evidence support the protective role of non-steroidal anti-inflammatory drugs (NSAIDs) and statins. Experiments were designed to evaluate the efficacies atorvastatin and NSAIDs administered individually and in combination against colon adenocarcinoma (AdCa) formation. Also, we evaluated colonic AdCa inhibitory action of atorvastatin and NSAIDs in regulating the expression of key protein markers and cytokines. F344 rats were fed AIN-76A diet and colon AdCa were induced with azoxymethane (AOM). One week after AOM-treatment (adenoma-stage) groups of rats were fed AIN-76A diet containing atorvastatin (200 ppm), sulindac (100 ppm) or naproxen (150 ppm), or their combinations with atorvastatin (100 ppm) for 45 weeks. We found that rats fed experimental diets were comparable body weights and without any observable toxicity. Most of AOM-treated control-diet fed rats developed AdCa (74.2% incidence) at 45 weeks. Administration of sulindac and naproxen individually had modest inhibitory (∼25% incidence and ∼33% multiplicity) effect on colon AdCa. However, 200 ppm atorvastatin significantly suppressed both AdCa incidence (>52%, p<0.005) and multiplicity (59%, p<0.003). Importantly, colon AdCa incidence was significantly decreased when rats were given low-dose atorvastatin with either sulindac (p<0.001) or naproxen (p <0.0005). Also, colon AdCa multiplicities were profoundly reduced (80-85%, p<0.0001) when rats were given low-dose atorvastatin with either sulindac or naproxen. The staining of proliferation markers, PCNA, cyclin D1 and beta-catenin indicated that the tumors from the control group had the strong positive staining in the cells, whereas sulindac, naproxen, atorvastatin, and/or combinations had much weaker positive staining. Importantly, colon AdCa from atorvastatin and NSAIDs fed animals showed reduced expression of key inflammatory markers as well as nuclear staining for phospho-p65, a key molecule in the NF-kB pathway. In addition, atorvastatin and NSAIDs combination decreased mucosal and colonic tumor levels of the pro-inflammatory cytokines, TNF- alpha, IL-1 beta, IL-4 and IL-10. Overall, this is the first report on the combination treatment using low-dose atorvastatin with either low dose sulindac or naproxen, which greatly suppress the colon AdCa incidence and multiplicity. Also, our results suggest that decreased inflammatory cytokines and signaling molecules, particularly inhibition of nuclear p65, beta-catenin and cyclin D1 are responsible for suppression of colonic AdCa. In summary, low-dose atorvastatin with sulindac or naproxen might potential useful combinations for colon cancer prevention in humans. (Supported by NCI-N01-CN-53300 and R01-CA94962)nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 833. doi:10.1158/1538-7445.AM2011-833

Abstract 644: A novel Gemini vitamin D analogue represses the expression of a stem cell marker CD44 in breast cancer

Breast cancer stem cells are defined as cancer cells with self-renewal capacity. Cancer stem cells are resistant to current chemotherapy and radiation, making it difficult to investigate the mechanisms of resistance and the therapeutic targets of the tumor initiating cell population. Recently, CD44 has been recognized as a useful cell-surface marker for detecting breast cancer stem cells, and the CD44+/CD24- phenotype selects a population with stem cell properties similar to basal-like cancer cells. CD44 is a multi-functional transmembrane protein involved in cell proliferation, angiogenesis, invasion and metastasis. The CD44 positive subpopulation of breast cancer cells are enriched in residual breast cancer cell populations after conventional therapies, suggesting that CD44 positive cells may be responsible for the recurrence of cancer and therefore it may be an important therapeutic target. We have previously shown that novel Gemini vitamin D analogs inhibit the proliferation of breast cancer epithelial cells and suppress tumorigenesis in different animal models of breast cancer. Since therapies targeting CD44 by vitamin D have not been investigated, we examined whether a novel Gemini vitamin D analog regulates CD44 expression in cultured human breast cancer cells or in mammary tumors. In basal-like MCF10DCIS.com human breast cancer cells, the Gemini vitamin D analog BXL0124 significantly inhibited the expression of CD44 protein and reduced the transcriptional activity of the CD44 promoter in vitro. To evaluate the inhibitory effects of Gemini vitamin D on CD44 expression in vivo, MCF10DCIS.com cells were xenografted into immunodeficient mice. When administered intraperitoneally (0.1 microgram/kg body weight) or orally (0.3 microgram/kg body weight) once a day for 6 weeks, Gemini vitamin D BXL0124 inhibited tumor growth and markedly decreased the expression of CD44 in the xenograft tumors. To determine whether the repression of CD44 is dependent on the vitamin D receptor (VDR), siRNA VDR was employed. The repression of CD44 expression by BXL0124 was significantly blocked by siRNA VDR in MCF10DCIS.com cells, indicating that the regulation of CD44 expression by BXL0124 is a VDR-dependent event. Taken together, the novel Gemini vitamin D analog inhibits CD44 expression in MCF10DCIS.com cells in vitro and in xenograft tumors, suggesting an inhibitory role of Gemini vitamin D analogs on cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 644.