Capsaicin is the major pungent ingredient in red peppers. Here, we report that it has a profound antiproliferative effect on prostate cancer cells, inducing the apoptosis of both androgen receptor (AR)-positive (LNCaP) and -negative (PC-3, DU-145) prostate cancer cell lines associated with an increase of p53, p21, and Bax. Capsaicin down-regulated the expression of not only prostate-specific antigen (PSA) but also AR. Promoter assays showed that capsaicin inhibited the ability of dihydrotestosterone to activate the PSA promoter/enhancer even in the presence of exogenous AR in LNCaP cells, suggesting that capsaicin inhibited the transcription of PSA not only via downregulation of expression of AR, but also by a direct inhibitory effect on PSA transcription. Capsaicin inhibited NF-K activation by preventing its nuclear migration. In further studies, capsaicin inhibited tumor necrosis factor-A-stimulated degradation of IKBA in PC-3 cells, which was associated with the inhibition of proteasome activity. Taken together, capsaicin inhibits proteasome activity which suppressed the degradation of IKBA, preventing the activation of NF-KB. Capsaicin, when given orally, significantly slowed the growth of PC-3 prostate cancer xenografts as measured by size [75 F 35 versus 336 F 123 mm 3 (FSD); P = 0.017] and weight [203 F 41 versus 373 F 52 mg (FSD); P = 0.0006; capsaicin-treated versus vehicletreated mice, respectively]. In summary, our data suggests that capsaicin, or a related analogue, may have a role in the management of prostate cancer. (Cancer Res 2006; 66(6): 3222-9)

ABSTRACT

Prostaglandin E 2 plays a growth-stimulatory role in breast cancer, and the rate-limiting enzyme in its synthesis, cyclooxygenase-2, is often overexpressed in these cancers. Little is known about the role of the key prostaglandin catabolic enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in breast cancer pathogenesis. Using a pharmacologically based screen for epigenetically silenced genes, we found low levels of 15-PGDH in MDA-MB-231 cells [estrogen receptor (ER) negative] but high levels in MCF-7 cells (ER positive) and observed its up-regulation following demethylation treatment. Further analysis revealed methylation of the 15-PGDH promoter in one breast cancer cell line and 30% of primary tumors. Analysis of 15-PGDH expression revealed low levels in 40% of primary breast tumors and identified a correlation between 15-PGDH and ER expression. Transfection assays showed that transient up-regulation of 15-PGDH levels in MDA-MB-231 cells resulted in a decreased clonal growth, and stable up-regulation significantly decreased the ability of these cells to form tumors in athymic mice. In contrast, transient silencing of 15-PGDH in MCF-7 cells resulted in their enhanced proliferation, and a stable silencing in these cells enhanced cell cycle entry in vitro and tumorigenicity in vivo. Forced expression of 15-PGDH inhibited the ER pathway and silencing of 15-PGDH up-regulated expression of aromatase. In addition, 15-PGDH levels were down-regulated by estrogen but up-regulated by the tumor suppressor gene CAAT/ enhancer binding protein a. Our results indicate for the first time that 15-PGDH may be a novel tumor suppressor gene in breast cancer, and suggest that this enzyme can modulate the ER pathway. (Cancer Res 2006; 66(15): 7818-23)

Transferrin receptor 2 (TfR2) is a membrane glycoprotein that mediates cellular iron uptake from holotransferrin. Homozygous mutations of this gene cause one form of hereditary hemochromatosis in humans. We recently reported that homozygous TfR2(Y245X) mutant mice, which correspond to the TfR2(Y250X) mutation in humans, showed a phenotype similar to hereditary hemochromatosis. In this study, we further analyzed the phenotype as well as iron-related gene expression in these mice by comparing the TfR2-mutant and wild-type siblings. Northern blot analyses showed that the levels of expression of hepcidin mRNA in the liver were generally lower, whereas those of duodenal DMT1, the main transporter for uptake of dietary iron, were higher in the TfR2-mutant mice as compared to the wild-type siblings. Expression of hepcidin mRNA in the TfR2 mutant mice remained low even after intraperitoneal iron loading. In isolated hepatocytes from both wild-type and TfR2 mutant mice, interleukin-6 and lipopolysaccharide each induced expression of hepcidin mRNA. These results suggest that up-regulation of hepcidin expression by inflammatory stimuli is independent of TfR2 and that TfR2 is upstream of hepcidin in the regulatory pathway of body iron homeostasis. IntroductionHereditary hemochromatosis (HH) is a group of genetic disorders that manifest iron deposition in a variety of organs such as the liver, pancreas, heart, and skin. If untreated, liver cirrhosis, heart failure, and diabetes can develop. Most HH is caused by mutations in the HFE gene. 1 The frequency of homozygous C282Y mutation of this gene is estimated to be 1 in 150 in people of northern European descent, 2 though its clinical penetrance is low. 3 Mutations in several other genes also produce an HH phenotype, including hemojuvelin (HFE2/HJV), 4 hepcidin (hepatic antimicrobial peptide: HAMP), 5 and transferrin receptor 2 (TfR2). 6 The phenotypes caused by mutations of these genes are similar, manifesting increased transferrin (Tf) saturations, periportal hepatic iron loading, and reticuloendothelial iron sparing. This observation suggests that the products of these genes are on a common pathway for regulation of iron homeostasis.TfR2 protein is a membrane glycoprotein that can interact with Tf. 7 Human TfR2 has at least 2 alternatively spliced transcripts, ␣ and ␤. TfR2-␣ is the membrane-bound form predominantly expressed in the liver, whereas TfR2-␤ is a form that consists of only the extracellular domain of TfR2-␣. Similar to TfR1, TfR2-␣ interacts with holo-Tf but not with apo-Tf at neutral pH. 8 Expression of TfR2 mRNA almost exclusively occurs in the liver and erythroid precursor cells. 9 Homozygosity for one of several mutations in the TfR2 gene, including the truncation mutation Y250X, has been associated with hereditary hemochromatosis in humans. 6 In addition, we recently reported that homozygous TfR2(Y245X) mutant mice, which correspond to the TfR2(Y250X) mutation in humans, showed hepatocellular iron deposition with elevated serum Tf saturations. 10 However,...

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Mantle cell lymphoma (MCL) is an aggressive form of B-cell non-Hodgkin's lymphoma, with a mean survival of only 3-5 years and suboptimal therapeutic options. MCL is characterized by a balanced translocation t(11;14)(q13;q32), resulting in overexpression of cyclin D1, a G 1 cyclin regulated by the PI3K/Akt/mammalian target of rapamycin (mTOR) signaling pathway. As improved therapy for MCL is required and the mTOR pathway may be involved in its pathophysiology, the antiproliferative effects of RAD001 (everolimus), an mTOR inhibitor, against three MCL cell lines were investigated. As a single agent, RAD001 inhibited proliferation in MCL cell lines (Jeko1, SP49 and NCEB1) approximately 40-65% compared to diluent control cells. This was associated with G 1 cell-cycle arrest and reduced phosphorylation of the mTOR downstream target, 4E-BP1. Furthermore, combination drug studies revealed predominantly synergistic cytotoxicity with RAD001 and several secondary agents, including doxorubicin, vincristine or rituximab (components of the standard MCL regimen), as well as paclitaxel, vorinostat and bortezomib. These data indicate that single agent RAD001 is effective in inhibiting growth of MCL cells in vitro and combination studies with secondary agents further demonstrate synergistic cytotoxicity. Thus, these findings support future clinical studies of RAD001 in the treatment of MCL.

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is an ingredient of chili peppers with inhibitory effects against cancer cells of different origin. We examined the activity of capsaicin on breast cancer cells in vitro and in vivo. The drug potently inhibited growth of ER-positive (MCF-7, T47D, BT-474) and ER-negative (SKBR-3, MDA-MB231) breast cancer cell lines, which was associated with G 0 /G 1 cell-cycle arrest, increased levels of apoptosis and reduced protein expression of human epidermal growth factor receptor (EGFR), HER-2, activated extracellular-regulated kinase (ERK) and cyclin D1. In contrast, cell-cycle regulator p27 KIP1, caspase activity as well as poly-ADP ribose polymerase (PARP) cleavage were increased. Notably, capsaicin blocked breast cancer cell migration in vitro and decreased by 50% the size of MDA-MB231 breast cancer tumors growing orthotopically in immunodeficient mice without noticeable drug side effects. in vivo activation of ERK was clearly decreased, as well as expression of HER-2 and cyclin D1, whereas caspase activity and PARP cleavage products were increased in tumors of drug-treated mice. Besides, capsaicin potently inhibited the development of preneoplastic breast lesions by up to 80% without evidence of toxicity. Our data indicate that capsaicin is a novel modulator of the EGFR/HER-2 pathway in both ERpositive and -negative breast cancer cells with a potential role in the treatment and prevention of human breast cancer.

Cucurbitacins are a group of diverse triterpenoid substances isolated from plants with medicinal properties. One particularly potent family member is cucurbitacin B (CuB). The antiproliferative effects of CuB against human breast cancer cells were tested. Six human breast cancer cell lines were examined because they represent a diverse mix of breast cancer subtypes varying in expression of estrogen receptor (ER), Her2/neu, and p53 mutation. The antiproliferative effect of CuB were also studied in vivo. The effective dose inhibiting 50% growth (ED 50 ) was between 10 -8 M and 10 -7 M for this collection of breast cancer cell lines. These cells underwent rapid morphologic changes after 15-20 min exposure to CuB (5´10 -7 M), which was associated with disruption of the microtubules and F-actin, as observed by confocal microscopy. C ucurbitacins were originally isolated from Cucurbitaceae plants. They are a group of diverse triterpenoid molecules containing a cucurbitane skeleton characterized by a 19-(10→9β)-abeo-10α-lanost-5-ene (Fig. 1).(1) They have been reported to possess a broad range of biological effects such as chemoprevention and hepatoprotection, as well as anti-inflammatory, antimicrobial and antitumor activities.(2-10) Cucurbitacins have been reported to inhibit several types of cancers including those originating in the prostate, (9) lung and breast (11) as well as choriocarcinoma.(12) Among the various cucurbitacins, the most abundant is cucurbitacin B (CuB) and its analog dihydrocucurbitacin B.(13) Our preliminary data suggest that CuB is one of the most potent compounds in this family.Breast cancer afflicts about one in nine women, with approximately 43 000 deaths per year in the USA from this disease. Although great progress has been made in both controlling the disease and preventing recurrence, additional novel therapies are needed leading us to focus on investigating the effects of CuB on breast cancer in vitro and in vivo. Materials and Methods Cells and compounds. MCF-7, MDA-MB-231, MDA-MB-453, T47D, BT474, SK-BR-3 and ZR-75-1 cells (American TypeCulture Collection, Manassas, VA, USA) were maintained at 37°C in 5% CO 2 in Roswell Park Memorial Institute media (RPMI)-1640 (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Invitrogen), 10 U/mL penicillin and 10 mg/ mL streptomycin (Invitrogen). CuB was kindly provided by CK Life Sciences International Inc., Hong Kong.Cytoskeletal staining for b-tubulin and F-actin. After incubation with CuB, culture media were removed and cells were fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS), permeabilized with 0.1% Triton X-100 in PBS, and incubated with fluorescein isothyanate (FITC)-conjugated antiβ-tubulin antibody (Sigma Chemical, St. Louis, MO, USA) and/or rhodaminephalloidin (Invitrogen) to detect filamentous (F)-actin. Confocal images were collected on a Leica microscope (Leica Microsystems, Wetzlar, Germany).Animal experiments. MDA-MB-231 (1 × 10 6 ) breast cancer cells were resuspended in 100-μL matrigel and were inocul...