Genoveva Murillo

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

ABSTRACTSince 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.

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

25‐Hydroxyvitamin D3 (25(OH)D3) is a prohormone and a major vitamin D metabolite. The discovery of (25(OH)D3) 1α‐hydroxylase in many vitamin D target organs has yielded an increased interest in defining the role(s) of 25(OH)D3 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)D3 protects breast epithelial cells from cellular stress using an established breast epithelial cell line MCF12F. To better elucidate the role of 25(OH)D3 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)D3 (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)D3 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)D3. 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)D3 effectively reversed this alteration. These data suggest that there is a significant protective role for 25(OH)D3 against cellular stress in the breast epithelial cells and these effects may be mediated by altered miRNA expression. J. Cell. Biochem. 110: 1324–1333, 2010.

Actions of vitamin D are mediated by the TLR4 pathway in inflammation-induced colon cancer.

Many chronic inflammatory diseases are associated with increased risk of developing cancer. In the colon, strong support for a link between chronic inflammation and cancer extends, in part, from population-based studies of persons with inflammatory bowel disease (IBD). Patients with IBD are at increased risk of developing colorectal cancer (CRC). The general consensus is that IBD results from the combined effects of genetics and environment factors known to affect the immune system. Vitamin D, an important regulator of the immune system, has been linked to IBD. Despite the strong potential reported for 1,25-dihydroxyvitamin D (1,25-OH)2D), its effects on calcium metabolism limits its application. Recently, less active vitamin D metabolites, cholecalciferol and 25-hydroxyvitamin D (25(OH)D), have gained considerable attention as promising agents against IBD-related colon cancer. Yet, their anti-proliferative properties and mechanism of action remain to be better defined. We present several signaling pathways commonly regulated by vitamin D compounds and highlight their regulation on TLR4. The efficacy of 25(OH)D and 1alpha-hydroxyviatmin D5 are evaluated using the azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced IBD-related colon carcinogenesis model. In summary, vitamin D supplementation may provide a cost-effective approach to reduce IBD related colon cancer.

Chemopreventive Efficacy of 25-Hydroxyvitamin D3 in Colon Cancer

Recently, it has been reported that 25-hydroxyvitamin D3-1alpha-hydroxylase [1alpha(OH)ase, CYP27B1], required to convert non-toxic 25-hyxdroxyvitamin D3 [25(OH)D(3)] to its active metabolite [1alpha,25(OH)(2)D(3)], is present in the epithelial cells of the human colon. In the present study, the potential chemoprotective role of 25(OH)D(3) was evaluated for colon cancer using the HT-29, human colon cancer cell line. Colon cancer cells were treated with 25(OH)D(3) (500nM or 1muM), 1alpha,25(OH)(2)D(3) (500nM), cholecalciferol (D3, 1muM) or vehicle and cell number determined at days 2 and 5 post-treatment. Results showed that both 25(OH)D(3) and 1alpha,25(OH)(2)D(3) induced dose- and time-dependent anti-proliferative effects on the HT-29 cells, with maximum inhibition noted at day 5. Western blot analyses revealed an up-regulation of VDR and 1alpha(OH)ase expression following 24h of treatment with 25(OH)D(3), and 1alpha,25(OH)(2)D(3). These results are consistent with the expression of VDR and 1alpha(OH)ase in samples of normal colonic tissue, aberrant crypt foci (ACFs) and colon adenocarcinomas. The VDR expression was sequentially increased from normal to pre-cancerous lesions to well-differentiated tumors and then decreased in poorly differentiated tumors. Expression of 1alpha(OH)ase was equally expressed in normal, pre-cancerous lesions and malignant human colon tissues. The increased expression of 1alpha(OH)ase in colon cancer cells treated with the pro-hormone and its anti-proliferative effects, suggest that 25(OH)D(3) may offer possible therapeutic and chemopreventive option in colon cancer.

Estrogen Receptor-β Protects Against Colitis-Associated Neoplasia in Mice

Estrogen receptor‐beta (ERβ) 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 ERβ in CAC has not yet been tested. We assessed the effects of ERβ deficiency in the azoxymethane (AOM)/dextran sodium sulfate (DSS)‐induced CAC model using ERβ knockout (βERKO) 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. βERKO 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. ERβ‐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, β‐catenin, proliferating cell nuclear antigen, mucin‐1 and significantly lower caveolin‐1 and mucin‐2 protein levels were shown in βERKO mice compared to WT mice. These data suggest a possible anti‐inflammatory and anti‐neoplastic mechanism of action of ERβ in CAC. These results demonstrate for the first time that ERβ provides protection in the AOM/DSS‐induced CAC model in mice, suggesting a preventive and/or therapeutic potential for the use of ERβ‐selective agonists in IBD.

Chemoprevention of chemically-induced mammary and colon carcinogenesis by 1α-hydroxyvitamin D5

Epidemiological data as well as experimental models yield evidence for a protective effect of vitamin D against the genesis of several types of cancers. Given its toxic properties at effective concentrations, numerous analogs of vitamin D have been developed. We synthesized an analog of vitamin D(5), 1alpha-hydroxy-24-ethylcholechalciferol (1alpha(OH)D(5)) and previously reported on its anti-proliferative activities against several cancer cell lines. To further examine its chemopreventive potential, experiments were conducted to investigate the in vivo effects of 1alpha(OH)D(5) using the N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis model. Results showed that 1alpha(OH)D(5) (25 and 50microg/kg diet) decreased the incidence and multiplicity of mammary tumors in female Sprague-Dawley rats. In a subsequent study, the stage specific inhibition was investigated using the 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis model. While supplementation with of 1alpha(OH)D(5) (40microg/kg diet) showed no significant effects during the initiation phase, tumor incidence during the promotional stage was significantly (p<0.05) decreased by 37.5%. In the colon, 1alpha(OH)D(5) (25microg/kg diet) was highly effective (p<0.001) in inhibiting the development of azoxymethane (AOM)-induced Aberrant crypt foci (ACF) in CF-1 mice. Studies on the stage specific inhibitory effects of 1alpha(OH)D(5) in the colon demonstrated that animals receiving 1alpha(OH)D(5) (25microg/kg diet) during the initiation, promotion, and entire period had a reduction in ACF number of 71, 80 and 82%, respectively. Immunohistochemistry studies comparing the colons of animals receiving control versus 1alpha(OH)D(5) supplemented diets showed that 1alpha(OH)D(5) partly mediates its effects by regulating members of the oncogenic beta-catenin pathway. 1Alpha(OH)D(5) inhibited expression of beta-catenin and peroxisome proliferator-activated receptor beta, a beta-catenin-TCF-4 responsive gene, whereas it induced expression of VDR. Cumulatively, these studies support the chemopreventive properties of 1alpha(OH)D(5) against the development of breast and colon cancers.

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.

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.

Ixocarpalactone A isolated from the Mexican tomatillo shows potent antiproliferative and apoptotic activity in colon cancer cells.

Physalis philadelphica Lam, commonly known as a tomatillo, is a staple of the Mesoamerican cuisine. In our laboratory, an ethyl acetate‐soluble extract and four withanolides [ixocarpalactone A (IxoA), ixocarpalactone B, philadelphicalactone B, and withaphysacarpin] were isolated. Studies conducted on Hepa‐1c1c7 hepatoma cells revealed that withanolides were potent inducers of quinone reductase, suggesting possible cancer chemoprotective activity. Here we evaluated the antiproliferative properties of the withanolides in SW480 human colon cancer cells. IxoA, which is present in the edible part of the tomatillo, was selected for further evaluation. SW480 cells treated with IxoA showed cell cycle arrest in the G2/M phase, up‐regulation of hyper‐phosphorylated retinoblastoma, and down‐regulation of E2F‐1 and DP‐1. On the basis of flow cytometry analysis, ethidium bromide/acridine orange, and 4′,6‐diamidino‐2‐phenylindole staining, it was found that IxoA induces apoptosis in SW480 cells. Moreover, increased concentrations of the pro‐apoptotic protein, BIM/BOD, were found by western blot analysis and immunocytochemistry. Morphological examination revealed vacuole formation in cells treated with IxoA, and Oil Red O staining showed that the vacuole content was nonlipid. Furthermore, immunocytochemistry demonstrated increased concentrations of mucin 3 in IxoA‐treated SW480 cells. These findings suggest that chemicals present in tomatillos (e.g. IxoA) may have cancer chemopreventive properties.

Resveratrol inhibits rhabdomyosarcoma cell proliferation

Rhabdomysarcoma is the most common soft tissue tumour in children under the age of 15. Although the introduction of multimodal treatment programmes, including chemotherapy, radiation therapy and excision have increased the overall survival, the chemotherapeutic agents currently used for the treatment of rhabdomyosarcoma exhibit considerable toxicity. The aim of this study was to investigate the effects and possible mechanism(s) of action of resveratrol on human embryonal rhabdomyosarcoma (RD) cells. Resveratrol is a natural polyphenolic compound produced in a number of edible plants and has received considerable attention as a potential chemopreventive and/or chemotherapeutic agent against various types of cancers. In the present study, resveratrol was shown to inhibit cell proliferation of RD cells in a dose-dependent manner with an IC50 of 48.1 μmol/l and induce an arrest in the S/G2 phase of the cell cycle. As evident from immunocytochemical data, resveratrol treatment increased the size of the RD cells. Furthermore, resveratrol treatment resulted in a significant downregulation of cyclin B expression as demonstrated by western blot analyses. In conclusion, the present study shows that resveratrol exerts a strong inhibition of rhabdomyosarcoma cell proliferation in part by arresting cells in S/G2 phase of the cell cycle. These findings warrant further investigation to establish potential use of resveratrol as a relatively non-toxic chemotherapeutic agent for the treatment of rhabdomyosarcoma.