Thomas T.Y. Wang

Inhibition of Prostate Cancer Growth by Muscadine Grape Skin Extract and Resveratrol through Distinct Mechanisms

The phytochemical resveratrol contained in red grapes has been shown to inhibit prostate cancer cell growth, in part, through its antioxidant activity. Muscadine grapes contain unique phytochemical constituents compared with other grapes and are potentially a source for novel compounds with antitumor activities. We compared the antitumor activities of muscadine grape skin extract (MSKE), which we show contains no resveratrol, with that of resveratrol using primary cultures of normal prostate epithelial cells (PrEC) and the prostate cancer cell lines RWPE-1, WPE1-NA22, WPE1-NB14, and WPE1-NB26, representing different stages of prostate cancer progression. MSKE significantly inhibited tumor cell growth in all transformed prostate cancer cell lines but not PrEC cells. Prostate tumor cell lines, but not PrEC cells, exhibited high rates of apoptosis in response to MSKE through targeting of the phosphatidylinositol 3-kinase-Akt and mitogen-activated protein kinase survival pathways. The reduction in Akt activity by MSKE is mediated through a reduction in Akt transcription, enhanced proteosome degradation of Akt, and altered levels of DJ-1, a known regulator of PTEN. In contrast to MSKE, resveratrol did not induce apoptosis in this model but arrested cells at the G(1)-S phase transition of the cell cycle associated with increased expression of p21 and decreased expression of cyclin D1 and cyclin-dependent kinase 4 proteins. These results show that MSKE and resveratrol target distinct pathways to inhibit prostate cancer cell growth in this system and that the unique properties of MSKE suggest that it may be an important source for further development of chemopreventive or therapeutic agents against prostate cancer.

Low‐dose genistein induces cyclin‐dependent kinase inhibitors and G1 cell‐cycle arrest in human prostate cancer cells

Genistein, a naturally occurring isoflavone found chiefly in soy products, reportedly has antiprostate cancer effects, but the mechanisms underlying these effects are unknown. We studied the antiproliferative and apoptosis‐inducing effects of genistein in the androgen‐sensitive human prostate cancer cell line LNCaP. Viable cell number was assessed by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay; cell‐cycle progression and apoptosis were evaluated by flow cytometry; apoptosis was also assessed by a histone enzyme‐linked immunosorbent assay; and the expression of several cell‐cycle– and apoptosis‐related genes and their gene products was determined by northern blot analysis, western blot analysis, and/or assays based on polymerase chain reaction. Physiologic concentrations of genistein (≤ 20 μM) decreased LNCaP viable cell number in a dose‐dependent manner, induced a G1 cell‐cycle block, decreased prostate‐specific antigen mRNA expression, and increased p27KIP1 and p21WAF1 (mRNA and protein) but had no effect on apoptosis or the mRNA expression of the apoptosis‐ and cell‐cycle–related markers bcl‐2, bax, Rb, and proliferating cell nuclear antigen. Higher concentrations of genistein (> 20 μM) did induce apoptosis. We conclude that genistein (at physiologic concentrations) exerts potent antiproliferative effects on LNCaP cells by inducing a G1 cell‐cycle block. The antiproliferative effects of genistein may be mediated by increased levels of p27KIP1 and p21WAF1, which are negative cell‐cycle regulators that act as cyclin‐dependent kinase inhibitors and that have been recently linked with prostate carcinogenesis. These findings may provide insights into the mechanisms underlying the apparent antiprostate cancer effects of soy consumption observed in epidemiologic studies. Mol. Carcinog. 29:92–102, 2000.

Differential effects of resveratrol and its naturally occurring methylether analogs on cell cycle and apoptosis in human androgen-responsive LNCaP cancer cells.

Stilbenes are phytoalexins that become activated when plants are stressed. These compounds exist in foods and are widely consumed. Resveratrol is a grape-derived stilbene, which possesses a wide range of health-promoting activities, including anticancer properties. Several other stilbenes structurally similar to resveratrol are also available in food, but their biological activities remain largely unknown. In this study, we compared the effects of resveratrol and its natural derivatives pterostilbene, trans-resveratrol trimethylether, trans-pinostilbene and trans-desoxyrhapontigenin on androgen-responsive human prostate cancer LNCaP cells. We found that these compounds exert differential effects on LNCaP cell growth, cell cycle and apoptosis. Trans-resveratrol trimethylether appeared to be the most potent compound among the stilbenes tested. Treatment of LNCaP cells with trans-resveratrol trimethylether resulted in G2/M blockage while other compounds, including resveratrol, induced G1/S arrest. Moreover, different from other compounds, trans-resveratrol trimethylether induced apoptosis. At the molecular level, the effects of these compounds on cell cycle correlated with induction of the cyclin-dependent kinase inhibitor 1A and B mRNA levels. Additionally, these compounds also inhibited both androgen- as well as estrogen-mediated pathways. These results provide mechanistic information on how resveratrol and its methylether analogs may act to contribute to potential antiprostate cancer activity.

Cellular uptake and transport of zein nanoparticles: effects of sodium caseinate.

Cellular evaluation of zein nanoparticles has not been studied systematically due to their poor redispersibility. Caseinate (CAS)-stabilized zein nanoparticles have been recently developed with better redispersibility in salt solutions. In this study, zein-CAS nanoparticles were prepared with different zein/CAS mass ratios. The prepared nanoparticles demonstrated good stabilities to maintain particle size (120-140 nm) in cell culture medium and HBSS buffer at 37 °C. The nanoparticles showed no cytotoxicity for Caco-2 cells for 72 h. CAS not only significantly enhanced cell uptake of zein nanoparticles in a concentration- and time-dependent manner but also remarkably improved epithelial transport through Caco-2 cell monolayer. The cell uptake of zein-CAS nanoparticles indicated an energy-dependent endocytosis process as evidenced by cell uptake under blocking conditions, that is, 4 °C, sodium azide, and colchicine. Fluorescent microscopy clearly showed the internalization of zein-CAS nanoparticles. This study may shed some light on the cellular evaluations of hydrophobic protein nanoparticles.

Differential effects of chemotherapeutic agents on the Bcl-2/Bax apoptosis pathway in human breast cancer cell line MCF-7

The present study explored the effects of three commonly used chemotherapeutic agents on the Bcl‐2/Bax apoptosis pathway and the interaction of these chemotherapeutic drugs with the estradiol‐mediated regulation of this pathway. Our results showed that: (1) Treatment of MCF‐7 cells with Adriamycin resulted in time‐ and concentration‐dependent decreases in Bcl‐2 and increases in Bax mRNA and protein levels. (2) Camptothecin elicited similar trends on Bcl‐2 and Bax as Adriamycin, while etoposide, at 50–100 fold (1–5 μM) the effective concentration of Adriamycin and camptothecin, only resulted in an increase in Bax mRNA levels. (3) Adriamycin and camptothecin, but not etoposide, were effective in suppressing estradiol‐stimulated increases in Bcl‐2 mRNA levels. Our study provides evidence that the Bcl‐2/Bax apoptosis pathway may be differentially regulated by chemotherapeutic agents. In addition, interaction between these agents and estradiol on the Bcl‐2/Bax apoptosis pathway may also exist.

Using DNA microarray analyses to elucidate the effects of genistein in androgen-responsive prostate cancer cells: identification of novel targets.

Many studies have correlated the consumption of soy‐rich diets with a decreased risk of developing hormone‐dependent cancers, including prostate cancer. Genistein is a candidate prostate cancer preventive phytochemical found at high levels in soybean and soy foods. To better understand the molecular mechanisms underlying the beneficial effects of genistein on prostate cancer prevention, we used a DNA microarray approach to examine the effects of genistein at concentrations in the physiologic range on global gene expression patterns in androgen‐responsive cancer cells. Microarray analyses were performed on androgen‐responsive LNCaP human prostate cancer cells exposed to 0, 1, 5, or 25 μM genistein. We found a concentration‐dependent modulation of multiple cellular pathways that are important in prostate carcinogenesis. Interestingly, the androgen receptor (AR)‐mediated pathways, in particular, appeared to be modulated by genistein at the lowest concentrations. Based on these results, we propose that the regulation of AR‐mediated pathways is potentially the most relevant chemopreventive mechanism for genistein administered at physiologic levels. Published 2004 Wiley‐Liss, Inc.

Development and Application of Nanoparticles Synthesized with Folic Acid Conjugated Soy Protein

In this study, soy protein isolate (SPI) was conjugated with folic acid (FA) to prepare nanoparticles for target-specific drug delivery. Successful conjugation was evidenced by UV spectrophotometry and primary amino group analysis. An increase in count rate by at least 142% was observed in FA-SPI nanoparticles compared to the nonconjugated ones, whereas the particle size was decreased upon FA conjugation. This was probably attributed to the substitution of positively charged lysine residues by the FA backbone. The ζ-potential ranged from -36 to -42 mV depending on the conjugation degree, indicating desirable dispersion stability. Curcumin as a model drug was encapsulated successfully into FA-SPI nanoparticles, evidenced by X-ray diffraction study. The highest encapsulation and loading efficiencies were around 92.7% and 5.4%, respectively, which were significantly higher (P < 0.05) than those with nonconjugated SPI nanoparticles. In addition, a faster and more complete release of curcumin was observed for FA-SPI nanoparticles in PBS/Tween 20 buffer. Cell culture study showed that conjugation of FA resulted in an increase in cellular uptake by at most 93% in Caco-2 cells. These results suggested that FA-SPI is a potential wall material for encapsulation and enhanced delivery of anticancer drugs.

Phenolic composition and nutraceutical properties of organic and conventional cinnamon and peppermint

Conventional and organic cinnamon and peppermint were investigated for their phenolic profile, anti-proliferative, anti-inflammatory, and antioxidant properties. Accelerated solvent extraction with 75% acetone was used to extract samples. Caffeic acid was the most abundant phenolic acid in peppermint. Catechin, (-)-epigallocatechin gallate, syringic acid, gallic acid, vanillic acid, and p-coumaric acid were also detected in both spices. There was no significant difference between conventional and organic spices in the composition of most individual phenolics. All conventional and organic peppermint and cinnamon extracts exhibited strong anti-proliferative and anti-inflammatory properties. Cinnamon was more efficient in inhibiting IL-1β and COX-2 expression, while peppermint showed better inhibitory effect on IL-6 and MCP-1. This study indicates that cinnamon and peppermint may potentially be used as dietary sources of bioactive phytochemicals for improving health.

Chemical Composition of Five Commercial Gynostemma pentaphyllum Samples and Their Radical Scavenging, Antiproliferative, and Anti-inflammatory Properties

Five Gynostemma pentaphyllum (GP) samples were investigated and compared for their chemical compositions and their antioxidant, antiproliferative, and anti-inflammatory effects. Extracts (50% acetone, 75% ethanol, and 100% ethanol) of the five GP samples (GP1-5) differed in their total phenolic, saponin, and flavonoid contents and in their rutin and quercetin concentrations. The highest level of total flavonoids was 63.5 mg of rutin equiv/g in GP4, and the greatest total phenolic content was 44.3 mg of gallic acid equiv/g in GP1 with 50% acetone as the extraction solvent. GP2 had the highest total saponin content of 132.6 mg/g with 100% ethanol as the extraction solvent. These extracts also differed in their scavenging capacity against DPPH and hydroxyl radicals, although they all showed significant radical scavenging capacity. The 100% ethanol extracts also showed dose-dependently strong inhibition on IL-6 and Ptgs2 mRNA expression and weak inhibition on TNF-α mRNA expression. In addition, GP1 had the highest antiproliferative activity at 3.2 mg equiv/mL concentration in HT-29 human colon cancer cells. The results from this study will be used to promote the application of G. pentaphyllum for improving human health.

Effect of N-(4-hydroxyphenyl)retinamide on apoptosis in human breast cancer cells

The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) has been used in breast cancer prevention and treatment. However, the molecular mechanisms mediating the effects of 4-HPR remain elusive. In the present study, we examined the effects of 4-HPR on components of apoptosis pathway (i.e Bcl-2 and Bax) and apoptotic death in both estrogen receptor-positive and estrogen receptor-negative cell lines. We found that: (1) 4-HPR treatment resulted in decreased Bcl-2 mRNA but not Bax mRNA levels; (2) the effect of 4-HPR on Bcl-2 mRNA level was different from other retinoids; (3) 4-HPR treatment induced apoptosis in both estrogen receptor-positive and -negative cells. Hence, the breast cancer chemopreventive properties of 4-HPR may involve modulation of apoptosis pathways.