Diallyl Trisulfide Induces Apoptosis in Breast Ductal Carcinoma In Situ Derived and Minimally Invasive Breast Cancer Cells.

Breast ductal carcinoma in situ (DCIS) is a localized form of breast cancer that can progress to invasive breast cancer. Diallyl trisulfide (DATS) is a bioactive compound from Allium vegetables reported to induce anticancer effects in several cancer models. The objective of this study was to characterize DATS-induced apoptosis in breast DCIS and minimally invasive breast cancer cells. Breast DCIS cells SUM 102PT (ductal carcinoma in situ with areas of micro-invasion) and SUM 225CWN (chest wall recurrence of ductal carcinoma in situ) were used in this study. DATS induced a dose-dependent reduction in the colony formation ability of breast DCIS cells. DATS inhibited DCIS cell growth by inducing apoptosis as shown by a dose-dependent increase in cytoplasmic histone-associated DNA fragmentation. Induction of apoptosis was more pronounced in SUM 102PT cells than in SUM 225CWN cells at similar concentrations of DATS. DATS-induced apoptosis was characterized by a dose-dependent increase in cleaved poly-ADP ribose polymerase (PARP). DATS treatment resulted in an increase in the cytochrome c levels and cleavage of caspases 3, 7, and 9. This study shows that DATS inhibits cell proliferation and induces apoptosis in breast DCIS derived and minimally invasive breast cancer cells, and supports further investigation of DATS as a potential chemopreventive agent for DCIS.

Modulation of Notch Signaling Pathway by Bioactive Dietary Agents.

Notch signaling is often aberrantly activated in solid and hematological cancers and regulates cell fate decisions and the maintenance of cancer stem cells. In addition, increased expression of Notch pathway components is clinically associated with poorer prognosis in several types of cancer. Targeting Notch may have chemopreventive and anti-cancer effects, leading to reduced disease incidence and improved survival. While therapeutic agents are currently in development to achieve this goal, several researchers have turned their attention to dietary and natural agents for targeting Notch signaling. Given their natural abundance from food sources, the use of diet-derived agents to target Notch signaling offers the potential advantage of low toxicity to normal tissue. In this review, we discuss several dietary agents including curcumin, EGCG, resveratrol, and isothiocyanates, which modulate Notch pathway components in a context-dependent manner. Dietary agents modulate Notch signaling in several types of cancer and concurrently decrease in vitro cell viability and in vivo tumor growth, suggesting a potential role for their clinical use to target Notch pathway components, either alone or in combination with current therapeutic agents.

Dietary Bioactive Diallyl Trisulfide in Cancer Prevention and Treatment.

Bioactive dietary agents have been shown to regulate multiple cancer hallmark pathways. Epidemiologic studies have linked consumption of Allium vegetables, such as garlic and onions, to decreased incidence of cancer. Diallyl trisulfide (DATS), a bioactive compound derived from Allium vegetables, has been investigated as an anti-cancer and chemopreventive agent. Preclinical studies provide ample evidence that DATS regulates multiple cancer hallmark pathways including cell cycle, apoptosis, angiogenesis, invasion, and metastasis. DATS has been shown to arrest cancer cells at multiple stages of the cell cycle with the G2/M arrest being the most widely reported. Additionally, increased pro-apoptotic capacity as a result of regulating intrinsic and extrinsic apoptotic pathway components has been widely reported following DATS treatment. Invasion, migration, and angiogenesis represent emerging targets of DATS and support its anti-cancer properties. This review summarizes DATS mechanisms of action as an anti-cancer and chemopreventive agent. These studies provide rationale for future investigation into its use as a cancer chemopreventive agent.

Diallyl trisulfide, a chemopreventive agent from Allium vegetables, inhibits alpha-secretases in breast cancer cells.

Breast cancer affects one in eight women throughout the course of their lifetime creating a demand for novel prevention strategies against this disease. The Notch signaling pathway is often aberrantly activated in human malignancies including breast cancer. Alpha secretases, including ADAM (A Disintegrin and Metalloprotease) -10 and -17, are proteases that play a key role in the cleavage of cell surface molecules and subsequent ligand-mediated activation of Notch signaling pathway. High expression levels of ADAM10 and 17 have been clinically associated with a lower disease-free survival in breast cancer patients. This study was undertaken to determine the effect of diallyl trisulfide (DATS), a bioactive organosulfide found in garlic and other Allium vegetables, on alpha secretases in breast cancer cells. Here we report for the first time that DATS inhibits the expression of ADAM10 and ADAM17 in estrogen-independent MDA-MB-231 and estrogen-dependent MCF-7 breast cancer cells, and in Harvey-ras (H-Ras) transformed MCF10A-H-Ras breast epithelial cells. We also show that DATS induces a dose-dependent reduction in colony formation ability of MDA-MB-231 and MCF-7 cells, suggesting a long-term effect of DATS on growth inhibition of breast cancer cells. Furthermore, we show that DATS inhibits the Notch ligands Jagged-1 and Jagged-2 involved in activation of Notch signaling pathway. Collectively, these findings show that DATS targets Notch pathway components overexpressed in breast cancer tumors and may serve as a functionally relevant bioactive for breast cancer prevention.

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.

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.

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.

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.

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.

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.

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.

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.

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 gamma 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.

Investigation of the presence of OH radicals in electrolyzed NaCl solution by electron spin resonance spectroscopy.

In the anode side of a two-chamber electrolyzer, electrolysis of a NaCl solution generates acidic electrolyzed oxidizing (EO) water, which exhibits bactericidal effects against a large number of pathogens. This study was undertaken to investigate whether OH radical species are present in EO water or are formed when EO water reacts with iron ions. Electron spin resonance spectroscopy (ESR) coupled with the spin trapping technique was used for the detection of free radicals. Samples of EO water were collected at 0.5, 1, 2, 3, and 5 min of electrolysis and immediately mixed with the spin trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The 5,5-dimethyl-2-hydroxypyrrolidine-N-oxyl (DMPO-OH) spin adduct, characteristic of OH radicals, was not observed. Starting with 2-min electrolysis, a seven-line spectrum characteristic of 5,5-dimethyl-2-pyrrolidone-N-oxyl (DMPOX) was formed. The reactions of EO water with Fe3+ and Fe2+ in the presence of DMPO yielded the spin adduct DMPO-OH. However, the addition of OH radical scavengers (ethanol and methanol) did not generate the characteristic DMPO-alkyl spin adducts. This indicated that the DMPO-OH spectrum was due to a nucleophilic addition of water to DMPO and not to trapping of OH radicals.

5,5-Dimethyl-2-pyrrolidone-N-oxyl formation in electron spin resonance studies of electrolyzed NaCl solution using 5,5-dimethyl-1-pyrroline-N-oxide as a spin trapping agent.

Electrolyzed oxidizing (EO) water has recently generated much interest as a disinfectant in the food industry. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) is a spin trapping agent widely used in the electron spin resonance (ESR) characterization of oxygen-centered free radicals. The reaction between electrolyzed water, collected from the anode side of a two-chamber electrolyzer, and DMPO was investigated by ESR spectroscopy. Addition of DMPO to EO water generated an ESR spectrum identical to that of 5,5-dimethyl-2-pyrrolidone-N-oxyl (DMPOX), suggesting that a compound from EO water oxidized DMPO with the formation of DMPOX. To further investigate the electrolytically generated compound that oxidized DMPO, aqueous solutions of different sodium salts (sodium chloride, sodium citrate, and sodium iodide) with similar conductivities were electrolyzed. The DMPOX signal was not detected in the electrolyzed sodium citrate sample, suggesting that DMPOX formation in the electrolyzed NaCl sample might be due to an electrolytically generated chlorine species. A low DMPOX signal was also observed from the electrolyzed NaI sample, suggesting that a similar species obtained through the electrolysis of I- can also oxidize DMPO. Hypochlorous acid is proposed to oxidize the spin trap DMPO with the formation of DMPOX. In a neutral pH environment, electrolyzed water also oxidized DMPO to DMPOX. This is consistent with the DMPOX formation in the reaction of chlorine water (containing HOCl and Cl2) or sodium hypochlorite with DMPO.

Reduction of Salmonella enterica on alfalfa seeds with acidic electrolyzed oxidizing water and enhanced uptake of acidic electrolyzed oxidizing water into seeds by gas exchange.

Alfalfa sprouts have been implicated in several salmonellosis outbreaks in recent years. The disinfectant effects of acidic electrolyzed oxidizing (EO) water against Salmonella enterica both in an aqueous system and on artificially contaminated alfalfa seeds were determined. The optimum ratio of seeds to EO water was determined in order to maximize the antimicrobial effect of EO water. Seeds were combined with EO water at ratios (wt/vol) of 1:4, 1:10, 1:20, 1:40, and 1:100, and the characteristics of EO water (pH, oxidation reduction potential [ORP], and free chlorine concentration) were determined. When the ratio of seeds to EO water was increased from 1:4 to 1:100, the pH decreased from 3.82 to 2.63, while the ORP increased from +455 to +1,073 mV. EO water (with a pH of 2.54 to 2.38 and an ORP of +1,083 to +1,092 mV) exhibited strong potential for the inactivation of S. enterica in an aqueous system (producing a reduction of at least 6.6 log CFU/ml). Treatment of artificially contaminated alfalfa seeds with EO water at a seed-to-EO water ratio of 1:100 for 15 and 60 min significantly reduced Salmonella populations by 2.04 and 1.96 log CFU/g, respectively (P < 0.05), while a Butterfield's buffer wash decreased Salmonella populations by 0.18 and 0.23 log CFU/g, respectively. After treatment, EO water was Salmonella negative by enrichment with or without neutralization. Germination of seeds was not significantly affected (P > 0.05) by treatment for up to 60 min in electrolyzed water. The uptake of liquid into the seeds was influenced by the internal gas composition (air, N2, or O2) of seeds before the liquid was added.