Silvia D. Stan

Sulforaphane inhibits prostate carcinogenesis and pulmonary metastasis in TRAMP mice in association with increased cytotoxicity of natural killer cells.

The present study shows that oral gavage of 6 mumol d,l-sulforaphane (SFN), a synthetic analogue of cruciferous vegetable-derived L isomer, thrice per week beginning at 6 weeks of age, significantly inhibits prostate carcinogenesis and pulmonary metastasis in TRAMP mice without causing any side effects. The incidence of the prostatic intraepithelial neoplasia and well-differentiated (WD) carcinoma were approximately 23% to 28% lower (P < 0.05 compared with control by Mann-Whitney test) in the dorsolateral prostate (DLP) of SFN-treated mice compared with controls, which was not due to the suppression of T-antigen expression. The area occupied by the WD carcinoma was also approximately 44% lower in the DLP of SFN-treated mice relative to that of control mice (P = 0.0011 by Mann Whitney test). Strikingly, the SFN-treated mice exhibited approximately 50% and 63% decrease, respectively, in pulmonary metastasis incidence and multiplicity compared with control mice (P < 0.05 by t test). The DLP from SFN-treated mice showed decreased cellular proliferation and increased apoptosis when compared with that from control mice. Additionally, SFN administration enhanced cytotoxicity of cocultures of natural killer (NK) cells and dendritic cells (DC) against TRAMP-C1 target cells, which correlated with infiltration of T cells in the neoplastic lesions and increased levels of interleukin-12 production by the DC. In conclusion, the results of the present study indicate that SFN administration inhibits prostate cancer progression and pulmonary metastasis in TRAMP mice by reducing cell proliferation and augmenting NK cell lytic activity.

Bioactive food components and cancer risk reduction.

Research over the last three decades has provided convincing evidence to support the premise that diets rich in fruits and vegetables may be protective against the risk of different types of cancers. Initial evidence for protective effect of fruits and vegetables against cancer risk came from population‐based case‐control studies, which prompted intense research aimed at (a) identification of bioactive component(s) responsible for the anticancer effects of fruits and vegetables, (b) elucidation of the mechanisms by which bioactive food components may prevent cancer, and (c) determination of their efficacy for prevention of cancer in animal models. The bioactive components responsible for cancer chemopreventive effects of various edible plants have now been identified. For instance, anticancer effect of Allium vegetables including garlic is attributed to organosulfur compounds (e.g., diallyl trisulfide). Interestingly, unlike cancer chemotherapy drugs, many bioactive food components selectively target cancer cells. Molecular basis for selectivity of anticancer bioactive food components towards cancer cells remains elusive, but these agents appear promiscuous and target multiple signal transduction pathways to inhibit cancer cell growth in vitro and in vivo. Despite convincing observational and experimental evidence, however, limited effort has been directed towards clinical investigations to determine efficacy of bioactive food components for prevention of human cancers. This article reviews current knowledge on cancer chemopreventive effects of a few highly promising dietary constituents, including garlic‐derived organosulfides, berry compounds, and cruciferous vegetable‐derived isothiocyanates, and serves to illustrate complexity of the signal transduction mechanisms in cancer chemoprevention by these promising bioactive food components. J. Cell. Biochem. 104: 339–356, 2008.

Withaferin A Causes FOXO3a- and Bim-Dependent Apoptosis and Inhibits Growth of Human Breast Cancer Cells In Vivo

Withaferin A (WA) is derived from the medicinal plant Withania somnifera, which has been safely used for centuries in Indian Ayurvedic medicine for treatment of different ailments. We now show, for the first time, that WA exhibits significant activity against human breast cancer cells in culture and in vivo. The WA treatment decreased viability of MCF-7 (estrogen-responsive) and MDA-MB-231 (estrogen-independent) human breast cancer cells in a concentration-dependent manner. The WA-mediated suppression of breast cancer cell viability correlated with apoptosis induction characterized by DNA condensation, cytoplasmic histone-associated DNA fragmentation, and cleavage of poly-(ADP-ribose)-polymerase. On the other hand, a spontaneously immortalized normal mammary epithelial cell line (MCF-10A) was relatively more resistant to WA-induced apoptosis compared with breast cancer cells. The WA-mediated apoptosis was accompanied by induction of Bim-s and Bim-L in MCF-7 cells and induction of Bim-s and Bim-EL isoforms in MDA-MB-231 cells. The cytoplasmic histone-associated DNA fragmentation resulting from WA exposure was significantly attenuated by knockdown of protein levels of Bim and its transcriptional regulator FOXO3a in both cell lines. Moreover, FOXO3a knockdown conferred marked protection against WA-mediated induction of Bim-s expression. The growth of MDA-MB-231 cells implanted in female nude mice was significantly retarded by 5 weekly i.p. injections of 4 mg WA/kg body weight. The tumors from WA-treated mice exhibited reduced cell proliferation and increased apoptosis compared with tumors from control mice. These results point toward an important role of FOXO3a and Bim in regulation of WA-mediated apoptosis in human breast cancer cells.

Chemoprevention strategies for pancreatic cancer.

Pancreatic cancer has a poor prognosis and is often diagnosed at an advanced stage, which makes it difficult to treat. The low survival rate of patients with pancreatic cancer points towards an increased need for novel therapeutic and chemopreventive strategies and also early detection of this disease. Increased consumption of fruits and vegetables has been associated with a reduced risk of pancreatic cancer. Synthetic and natural, diet-derived bioactive compounds have been evaluated as pancreatic cancer chemopreventive agents and have demonstrated various degrees of efficacy in cellular and in vivo animal models. Some chemopreventive agents (for example, curcumin or resveratrol) have also been reported to sensitize pancreatic cancer cells to standard chemotherapeutic drugs (for example, gemcitabine or erlotinib), which suggests that chemopreventive agents could potentially be used as potentiators of standard chemotherapy. Few clinical trials of pancreatic cancer chemopreventive agents have been completed and some are in early phases. Further development of pancreatic cancer chemopreventive agents may prove to be tremendously valuable for individuals at high risk of developing pancreatic cancer and patients who present with premalignant lesions. This Review discusses the current state of the pancreatic cancer chemoprevention field and highlights the challenges ahead.

Ayurvedic Medicine Constituent Withaferin A Causes G2 and M Phase Cell Cycle Arrest in Human Breast Cancer Cells

Withaferin A (WA) is derived from the medicinal plant Withania somnifera that has been safely used for centuries in the Indian Ayurvedic medicine for treatment of various ailments. We now demonstrate that WA treatment causes G2 and mitotic arrest in human breast cancer cells. Treatment of MDA-MB-231 (estrogen-independent) and MCF-7 (estrogen-responsive) cell lines with WA resulted in a concentration- and time-dependent increase in G2-M fraction, which correlated with a decrease in levels of cyclin-dependent kinase 1 (Cdk1), cell division cycle 25C (Cdc25C) and/or Cdc25B proteins, leading to accumulation of Tyrosine15 phosphorylated (inactive) Cdk1. Ectopic expression of Cdc25C conferred partial yet significant protection against WA-mediated G2-M phase cell cycle arrest in MDA-MB-231 cells. The WA-treated MDA-MB-231 and MCF-7 cells were also arrested in mitosis as judged by fluorescence microscopy and analysis of Ser10 phosphorylated histone H3. Mitotic arrest resulting from exposure to WA was accompanied by an increase in the protein level of anaphase promoting complex/cyclosome substrate securin. In conclusion, the results of this study suggest that G2-M phase cell cycle arrest may be an important mechanism in antiproliferative effect of WA against human breast cancer cells.

Garlic Constituent Diallyl Trisulfide Prevents Development of Poorly Differentiated Prostate Cancer and Pulmonary Metastasis Multiplicity in TRAMP Mice

Identification of agents that are nontoxic but can delay onset and/or progression of prostate cancer, which is the second leading cause of cancer-related deaths among men in the United States, is highly desirable. We now show that p.o. gavage of garlic constituent diallyl trisulfide (DATS; 1 and 2 mg/day, thrice/week for 13 weeks beginning at age 8 weeks) significantly inhibits progression to poorly differentiated prostate carcinoma and pulmonary metastasis multiplicity in transgenic adenocarcinoma of mouse prostate (TRAMP) mice without any side effects. There was a trend of a decrease in average wet weights of the urogenital tract and prostate gland in 1 and 2 mg DATS-treated mice compared with controls ( approximately 25-46% decrease in DATS-treated mice compared with controls). The incidence and the area of the dorsolateral prostate occupied by the poorly differentiated carcinoma were significantly lower in both 1 and 2 mg DATS-treated mice compared with control mice. In addition, DATS administration resulted in a statistically significant decrease in pulmonary metastasis multiplicity compared with controls (P = 0.002). The dorsolateral prostate from DATS-treated TRAMP mice exhibited decreased cellular proliferation in association with induction of cyclinB1 and securin protein levels, and suppression of the expression of neuroendocrine marker synaptophysin. However, DATS administration did not have any appreciable effect on apoptosis induction, angiogenesis, or natural killer and dendritic cell function. In conclusion, the results of the present study show, for the first time, that DATS administration prevents progression to invasive carcinoma and lung metastasis in TRAMP mice.

Activation of a novel ataxia-telangiectasia mutated and Rad3 related/checkpoint kinase 1-dependent prometaphase checkpoint in cancer cells by diallyl trisulfide, a promising cancer chemopreventive constituent of processed garlic

Diallyl trisulfide (DATS), a cancer chemopreventive constituent of garlic, inhibits growth of cancer cells by interfering with cell cycle progression, but the mechanism is not fully understood. Here, we show the existence of a novel ataxia-telangiectasia mutated and Rad3 related (ATR)/checkpoint kinase 1 (Chk1)–dependent checkpoint partially responsible for DATS-mediated prometaphase arrest in cancer cells, which is different from the recently described γ irradiation–induced mitotic exit checkpoint. The PC-3 human prostate cancer cells synchronized in prometaphase by nocodazole treatment and released to DATS-containing medium remained arrested in prometaphase, whereas the cells released to normal medium exited mitosis and resumed cell cycle. The mitotic arrest was maintained even after 4 h of culture of DATS-treated cells (4-h treatment) in drug-free medium. The DATS-arrested mitotic cells exhibited accumulation of anaphase-promoting complex/cyclosome (APC/C) substrates cyclin A and cyclin B1 and hyperphosphorylation of securin, which was accompanied by increased phosphorylation of the APC/C regulatory subunits Cdc20 and Cdh1. The DATS-mediated accumulation of cyclin B1 and hyperphosphorylation of securin, Cdc20, and Cdh1 were partially but markedly attenuated by knockdown of Chk1 or ATR protein. The U2OS osteosarcoma cells expressing doxycycline-inducible kinase dead ATR were significantly more resistant not only to DATS-mediated prometaphase arrest but also to the accumulation of cyclin B1 and hyperphosphorylation of securin, Cdc20, and Cdh1 compared with cells expressing wild-type ATR. However, securin protein knockdown failed to rescue cells from DATS-induced prometaphase arrest. In conclusion, the present study describes a novel signaling pathway involving ATR/Chk1 in the regulation of DATS-induced prometaphase arrest. [Mol Cancer Ther 2007;6(4):1249–61]

Transcriptional Repression and Inhibition of Nuclear Translocation of Androgen Receptor by Diallyl Trisulfide in Human Prostate Cancer Cells

Purpose: The present study was undertaken to determine the effect of diallyl trisulfide (DATS), a promising cancer chemopreventive constituent of garlic, on androgen receptor (AR) protein expression and function using prostate cancer cells. Experimental Design: The protein levels of AR and prostate-specific antigen (PSA) were determined by immunoblotting. The effect of DATS treatment on AR mRNA level and AR promoter activity was determined by quantitative reverse transcription-PCR and luciferase reporter assay, respectively. Expression of AR protein in poorly differentiated carcinoma and normal prostate of transgenic adenocarcinoma of mouse prostate (TRAMP) mice was determined by immunohistochemistry. Confocal microscopy was done to determine nuclear translocation of AR. Cell viability was determined by trypan blue dye exclusion assay. Results: Exposure of prostate cancer cells (LNCaP, C4-2, and TRAMP-C1) to DATS resulted in a concentration-dependent decrease in protein level of AR, which was accompanied by suppression of intracellular and secreted levels of PSA. Structure-activity studies revealed critical roles for allyl groups and the oligosulfide chain length in DATS-mediated down-modulation of AR protein. Quantitative reverse transcription-PCR showed a dose-dependent decrease in AR mRNA level, which correlated with inhibition of AR promoter activity. DATS treatment inhibited synthetic androgen (R1881)-stimulated nuclear translocation of AR in LNCaP/C4-2 cells and proliferation of LNCaP cells. Oral gavage of 2 mg/day DATS (three times per week for 13 weeks) markedly suppressed AR protein level in poorly differentiated prostate cancer in TRAMP mice. Conclusion: The present study shows, for the first time, that DATS treatment suppresses AR function in prostate cancer cells.

Phenethyl isothiocyanate inhibits proliferation and induces apoptosis in pancreatic cancer cells in vitro and in a MIAPaca2 xenograft animal model.

Pancreatic cancer is often diagnosed at an advanced stage and it has a poor prognosis that points to an increased need to develop effective chemoprevention strategies for this disease. We examined the ability of phenethyl isothiocyanate (PEITC), a naturally occurring isothiocyanate found in cruciferous vegetables, to inhibit the growth of pancreatic cancer cells in vitro and in a MIAPaca2 xenograft animal model. Exposure to PEITC inhibited pancreatic cancer cell growth in a dose-dependent manner, with an IC50 of approximately 7 μmol/L. PEITC treatment induced G2/M phase cell cycle arrest, downregulated the antiapoptotic proteins Bcl-2 and Bcl-XL, upregulated the proapoptotic protein Bak, and suppressed Notch 1 and 2 levels. In addition, treatment with PEITC induced cleavage of poly-(ADP-ribose) polymerase and led to increased cytoplasmic histone-associated DNA fragmentation and subdiploid (apoptotic) fraction in pancreatic cancer cells. Oral administration of PEITC suppressed the growth of pancreatic cancer cells in a MIAPaca2 xenograft animal model. Our data show that PEITC exerts its inhibitory effect on pancreatic cancer cells through several mechanisms, including G2/M phase cell cycle arrest and induction of apoptosis, and supports further investigation of PEITC as a chemopreventive agent for pancreatic cancer.

Abstract 4596: Phenethyl isothiocyanate inhibits growth of MIAPaca2 human pancreatic cancer xenograft in vivo in association with apoptosis induction

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Pancreatic cancer is the fourth leading cause of cancer-related deaths in the U.S. with a 5-year survival rate of less than 5%. Low survival rate for patients with pancreatic cancer points towards an increased need for novel chemoprevention, early detection, and therapeutic strategies for this disease. This study was undertaken to determine the efficacy of phenethyl isothiocyanate (PEITC), a naturally occurring isothiocyanate found in cruciferous vegetables, against pancreatic cancer cells in vitro and in vivo. Exposure of human pancreatic cancer cells (MIAPaca2, PL-45, and BxPC3) to PEITC resulted in growth inhibition, which was accompanied by apoptotic cell death regardless of the K-ras status. Apoptotic cell death by PEITC treatment was characterized by an increase in cytoplasmic histone-associated DNA fragmentation and enrichment of sub-diploid fraction. PEITC-induced apoptosis correlated with induction of pro-apoptotic protein Bak, down-regulation of anti-apoptotic proteins Bcl-2 and Bcl-xL, and cleavage of caspase-3 and PARP. Oral administration of 12 µmol PEITC/mouse (five times per week) significantly retarded the growth of MIAPaca2 xenografts in male nude mice without causing weight loss or any other signs of toxicity. PEITC administration significantly reduced proliferation as determined by PCNA and Ki-67 stainings and increased apoptosis in tumors as shown by TUNEL assay. In conclusion, the present study demonstrates, for the first time, that PEITC administration retards the growth of human pancreatic cancer xenografts by inducing apoptotic cell death. The present study provides pre-clinical evidence for development of PEITC as a chemopreventive agent for pancreatic cancer. This investigation was supported in part by the Wayne Fusaro Pancreatic Cancer Research Fund (DCW), the Shirley Hobbs Martin Memorial Fund (REB), and the NCI grants CA101753 and CA113363 (SVS). Citation 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 4596. doi:10.1158/1538-7445.AM2011-4596