Unique Formosan Mushroom Antrodia camphorata Differentially Inhibits Androgen-Responsive LNCaP and -Independent PC-3 Prostate Cancer Cells

Antrodia camphorata (AC), a precious and unique folkloric medicinal mushroom enriched in polyphenolics, isoflavonoids, triterpenoids, and polysaccharides, has been diversely used in Formosa (Taiwan) since the 18th century. In this study, prostate cancer (PCa) cell lines PC-3 (androgen independent) and LNCaP (androgen responsive) were treated with AC crude extract (ACCE) at 50–200 μ g/mL, respectively, for 48 h. At the minimum effective dose 150 μ g/mL, LNCaP showed a G ₁ /S phase arrest with significant apoptosis. Such dose-dependent behavior of LNCaP cells in response to ACCE was confirmed to proceed as Akt → p53→ p21→ CDK4/cyclin D1→ G ₁ /S-phase arrest→ apoptosis, which involved inhibiting cyclin D1 activity and preventing pRb phosphorylation. In contrast, being without p53, PC-3 cells showed a G ₂ /M-phase arrest mediated through pathway p21→ cyclin B1/Cdc2→ G ₂ /M-phase arrest, however, with limited degree of apoptosis, implicating that ACCE is able to differentially inhibit the growth of different PCa cells by modulating different cell cycle signaling pathways. We conclude that this unique Formosan mushroom, A. camphorata, due to its nontoxicity, might be used as a good adjuvant anticancer therapy for prostate cancers despite its androgen-responsive behaviors, which has long been a serious drawback often encountered clinically in hormonal refractory cases treated by antihormonal therapies and chemotherapeutics.     

Effects of phenylethyl isothiocyanate and its metabolite on cell-cycle arrest and apoptosis in LNCaP human prostate cancer cells

Abstract

Cruciferous vegetable consumption is associated with decreased risk of several cancers, including prostate cancer. Gluconasturtiin, one of the predominant glucosinolates in cruciferous vegetables, is hydrolyzed to yield phenylethyl isothiocyanate (PEITC). PEITC absorption and metabolism in humans involves glutathione conjugation followed by conversion via the mercapturic acid pathway to an N-acetylcysteine (NAC) conjugate that is excreted in the urine. We observed an inhibitory effect of PEITC and its metabolite, NAC-PEITC, on cancer cell proliferation, cell-cycle progression, and apoptosis in LNCaP human prostate cancer cells. PEITC and NAC?PEITC suppressed LNCaP cell proliferation in a dose-dependent manner, and exposure to 5 μM PEITC or NAC-PEITC reduced cell proliferation by 25% and 30%, respectively. Cell-cycle analysis revealed that cells treated with 5 μM PEITC or NAC-PEITC arrested at the G₂/M phase. In addition, the percentage of cells in the S phase decreased from 46% to 25% following 48 h of incubation with PEITC or NAC-PEITC. The G₂/M-phase cell-cycle arrest of LNCaP cells grown in the presence of PEITC or NAC-PEITC is correlated with the downregulation of Cdk1 and cyclin B₁ protein expression. Apoptosis was observed at the later stages of 24-h and 48-h treatments with 5 μM PEITC and NAC-PEITC. In conclusion, PEITC and NAC-PEITC are potential chemopreventive/chemotherapeutic agents against LNCaP human prostate cancer cells.     

The Effect of Different Treatments of (–)-Epigallocatechin-3-Gallate on Colorectal Carcinoma Cell Lines

Backgroud: (-)-Epigallocatechin-3-gallate (EGCG), the major component of green tea, is well documented to induce apoptosis and cell cycle arrest in cancer by targeting multiple signal transduction pathways. However, EGCG is extremely unstable in general culture conditions and rapidly degraded. So, to what extent EGCG or which degradation products of EGCG play a role in anti-tumor is still unknown. In this study, we evaluated the effect of different treatments of EGCG on HCT116 cells.

Design: MTT assay was applied to evaluated the inhibitory effect of different treatments of EGCG on HCT116 cells. Cell cycle and apoptosis were performed by flow cytometry. Finally, western blot analysis was used to elucidate the molecular mechanism associated with cell cycle arrest and apoptosis.

Results: Compared with control, both EGCG and O-EGCG (i.e., EGCG being pre-incubated at 37°C for 3 h) significantly inhibited HCT116 cells growth. Surprisingly, we found that the inhibitory effect of O-EGCG was stronger than that of EGCG. The IC₅₀ values of EGCG and O-EGCG were 8.75 and 5.40 μM, respectively. Cell cycle analysis showed that 20 μM of EGCG simultaneously caused cell cycle arrest at G1 and G2 phase in HCT116 cells, differing from O-EGCG which exclusively caused cell cycle arrest at G2. This result suggested that parent EGCG at the early treatment might cause cell cycle arrest at G1. As time went on, EGCG disappeared and degraded products of EGCG were formed which might cause cell cycle arrest at G2. Further studies revealed that EGCG induced cell cycle arrest at G1 by downregulation of cyclin E and cyclin D1 and upregulation of p21. On the other hand, O-EGCG induced HCT116 cells apoptosis mainly by increasing the expression of p53 and cleaved caspase-3, which might be the underlying reason why O-EGCG had stronger inhibitory effect on HCT116 cells line than EGCG.

Conclusions: The pretreatment of EGCG may be an effective way to enhance its antitumor effect.     

Resveratrol Potentiates Growth Inhibitory Effects of Rapamycin in PTEN-deficient Lipoma Cells by Suppressing p70S6 Kinase Activity

Patients with phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome and germline mutations in PTEN frequently develop lipomatosis, for which there is no standard treatment. Rapamycin was shown to reduce the growth of lipoma cells with heterozygous PTEN deficiency in vitro, but concomitantly induced an upregulation of AKT phosphorylation. Since it was shown that resveratrol stabilizes PTEN, we asked whether co-incubation with resveratrol could suppress the rapamycin-induced AKT phosphorylation in PTEN-deficient lipoma cells.

Resveratrol incubation resulted in decreased lipoma cell viability by inducing G1-phase cell cycle arrest and apoptosis. PTEN expression and AKT phosphorylation were not significantly changed, whereas p70S6 kinase (p70S6K) phosphorylation was reduced in PTEN-deficient lipoma cells after resveratrol incubation. Rapamycin/resveratrol co-incubation significantly decreased viability further at lower doses of resveratrol and resulted in decreased p70S6K phosphorylation compared to rapamycin incubation alone, suggesting that resveratrol potentiated the growth inhibitory effects of rapamycin by reducing p70S6K activation. Both viability and p70S6K phosphorylation of primary PTEN wild-type preadipocytes were less affected compared to PTEN-deficient lipoma cells by equimolar concentrations of resveratrol. These results support the concept of combining chemopreventive natural compounds with mammalian target of rapamycin (mTOR) inhibitors to increase the efficacy of chemotherapeutic drugs for patients suffering from overgrowth syndromes.     

Methyl selenium-induced vascular endothelial apoptosis is executed by caspases and principally mediated by p38 MAPK pathway

The induction of vascular endothelial cell apoptosis and inhibition of tumor-associated angiogenesis by selenium may contribute to its cancer chemopreventive effects. Here we examined the stress-activated/mitogen-activated protein kinases (p38 MAPK, ERK1/2) and protein kinase B/AKT as potential signaling mediators for apoptosis induction by a methylselenol precursor methylseleninic acid (MSeA) in human umbilical vein endothelial cells (HUVEC). Time course experiments showed that p38 MAPK hyperphosphorylation and ERK1/2 dephosphorylation occurred before the cleavage of procaspase-3 and poly(ADP-ribose) polymerase (PARP), whereas AKT dephosphorylation occurred after caspase activation. The p38 MAPK inhibitor SB202190 attenuated the MSeA-induced morphological changes and decreased DNA fragmentation and the cleavage of procaspase-3 and PARP in concordant proportions. The general caspase inhibitor zVADfmk completely blocked the MSeA-induced PARP cleavage and DNA fragmentation, whereas zDEVDfmk, an inhibitor for caspase-3-like activities, was nearly as effective for inhibiting apoptosis. In comparison, apoptosis induced by selenite in HUVECs was observed in the complete absence of an activation of the major caspases. Taken together, the data support p38 MAPK as a key upstream mediator for the methylselenol-specific induction of vascular endothelial caspase-dependent apoptosis, which is principally executed by caspase-3-like activities.     

Estrogen Receptor-β Mediates the Inhibition of DLD-1 Human Colon Adenocarcinoma Cells by Soy Isoflavones

To understand the relationship between the role of soy isoflavones and estrogen receptor (ER)-β in colon tumorigenesis, we investigated the cellular effects of soy isoflavones (composed of genistein, daidzein, and glycitein) in DLD-1 human colon adenocarcinoma cells with or without ER-β gene silencing by RNA interference (RNAi). Soy isoflavones decreased the expression of proliferating cell nuclear antigen (PCNA), extracellular signal-regulated kinase (ERK)-1/2, AKT, and nuclear factor (NF)-κB. Soy isoflavones dose-dependently caused G2/M cell cycle arrest and downregulated the expression of cyclin A. This was associated with inhibition of cyclin dependent kinase (CDK)-4 and up-regulation of its inhibitor p21^cip1 expressions. ER-β gene silencing lowered soy isoflavone-mediated suppression of cell viability and proliferation. ERK-1/2 and AKT expressions were unaltered and NF-κB was modestly upregulated by soy isoflavones after transient knockdown of ER-β expression. Soy isoflavone-mediated arrest of cells at G2/M phase and upregulation of p21^cip1 expression were not observed when ER-β gene was silenced. These findings suggest that maintaining the expression of ER-β is crucial in mediating the growth-suppressive effects of soy isoflavones against colon tumors. Thus upregulation of ER-β status by specific food-borne ER-ligands such as soy isoflavones could potentially be a dietary prevention or therapeutic strategy for colon cancer.     

Genistein‐induced upregulation of p21 WAF1 , downregulation of cyclin B,and induction of apoptosis in prostate cancer cells

Increased soy consumption in Asian diets, resulting in increased serum isoflavone levels, has been associated with a decreased risk for prostate adenocarcinoma (PCa). The isoflavone genistein is believed to be the anticancer agent found in soy, and significant levels of genistein have been detected in human prostatic fluid, implicating the role of genistein in PCa prevention. Recent studies have demonstrated genistein's ability to inhibit cell growth and induce apoptosis in several cell lines; however, the molecular mechanisms of genistein's effect are not known. We have evaluated the mechanism by which genistein may inhibit PCa cell growth. Here we report that genistein inhibits PCa cell growth in culture in a dose‐dependent manner, which is accompanied by a G₂/M cell cycle arrest. Cell growth inhibition was observed with concomitant downregulation of cyclin B, upregulation of the p21^WAF1 growth‐inhibitory protein, and induction of apoptosis. Collectively, these results provide experimental evidence for a novel effect of genistein on cell cycle gene regulation, resulting in the inhibition of cell growth and ultimate demise of tumor cells.     

Tomato and soy polyphenols reduce insulin-like growth factor-I-stimulated rat prostate cancer cell proliferation and apoptotic resistance in vitro via inhibition of intracellular signaling pathways involving tyrosine kinase

We examined the ability of polyphenols from tomatoes and soy (genistein, quercetin, kaempferol, biochanin A, daidzein and rutin) to modulate insulin-like growth factor-I (IGF-I)-induced in vitro proliferation and apoptotic resistance in the AT6.3 rat prostate cancer cell line. IGF-I at 50 micro g/L in serum-free medium produced maximum proliferation and minimized apoptosis. Polyphenols exhibited different abilities to modulate IGF-I-induced proliferation, cell cycle progression (flow cytometry) and apoptosis (Annexin V/propidium iodide and terminal deoxynucleotidyltransferase-mediated deoxyuridine 5'-triphosphate nick end labeling). Genistein, quercetin, kaempferol and biochanin A exhibited dose-dependent inhibition of growth with a 50% inhibitory concentration (IC(50)) between 25 and 40 micro mol/L, whereas rutin and daidzein were less potent with an IC(50) of >60 micro mol/L. Genistein and kaempferol potently induced G(2)/M cell cycle arrest. Genistein, quercetin, kaempferol and biochanin A, but not daidzein and rutin, counteracted the antiapoptotic effects of IGF-I. Human prostate epithelial cells grown in growth factor-supplemented medium were also sensitive to growth inhibition by polyphenols. Genistein, biochanin A, quercetin and kaempferol reduced the insulin receptor substrate-1 (IRS-1) content of AT6.3 cells and prevented the down-regulation of IGF-I receptor beta in response to IGF-I binding. IGF-I-stimulated proliferation was dependent on activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and phosphatidylinositide 3-kinase pathways. Western blotting demonstrated that ERK1/2 was constitutively phosphorylated in AT6.3 cells with no change in response to IGF-I, whereas IRS-1 and AKT were rapidly and sensitively phosphorylated after IGF-I stimulation. Several polyphenols suppressed phosphorylation of AKT and ERK1/2, and more potently inhibited IRS-1 tyrosyl phosphorylation after IGF-I exposure. In summary, polyphenols from soy and tomato products may counteract the ability of IGF-I to stimulate proliferation and prevent apoptosis via inhibition of multiple intracellular signaling pathways involving tyrosine kinase activity.     

Anticancer Activity and Molecular Mechanism of Polyphenol Rich Calophyllum inophyllum Fruit Extract in MCF-7 Breast Cancer Cells

This study was conducted to investigate the anticancer effects and mechanism of Calophyllum inophyllum fruit extract against MCF-7 cells. C. inophyllum fruit extract was found to have markedly cytotoxic effect against MCF-7 cells in a dose-dependent manner with the IC₅₀ for 24 h of 23.59 µg/mL. Flow cytometry analysis revealed that C. inophyllum fruit extract mediated cell cycle at G₀/G₁ and G₂/M phases, and MCF-7 cells entered the early phase of apoptosis. The expression of anti-apoptotic proteins Bcl-2 was decreased whereas the expression of the pro-apoptotic protein Bax, cytochrome C and p53 were increased after treatment. C. inophyllum fruit extract led to apoptosis in MCF-7 cells via the mitochondrial pathway in a dose dependent manner. This is evidenced by the elevation of intracellular ROS, the loss of mitochondria membrane potential (Δψm), and activation of caspase-3. Meanwhile, dose-dependent genomic DNA fragmentation was observed after C. inophyllum fruits extract treatment by comet assay. This study shows that C. inophyllum fruits extract-induced apoptosis is primarily p53 dependent and mediated through the activation of caspase-3. C. inophyllum fruit extract could be an excellent source of chemopreventive agent in the treatment of breast cancer and has potential to be explored as green anticancer agent.     

Lycopene differentially induces quiescence and apoptosis in androgen-responsive and -independent prostate cancer cell lines

BACKGROUND & AIMS: Lycopene has been credited with a number of health benefits including a decrease in prostate cancer risk. Our study investigates the molecular mechanism underlying anti-cancer activity of lycopene-based products in androgen-responsive (LNCaP) and androgen-independent (PC3) cells. METHODS: The effect of lycopene-based agents on prostate cancer growth and survival were examined using proliferation assays, bromodeoxyuridine incorporation and flow cytometric analysis of cellular DNA content. Biochemical effects of lycopene treatment were investigated by immunoblotting for changes in the absolute levels and phosphorylation states of cell cycle regulatory and signalling proteins. RESULTS: LNCaP and PC3 cells treated with the lycopene-based agents undergo mitotic arrest, accumulating in G0/G1 phase. Immunoblot screening indicated that lycopene's antiproliferative effects are likely achieved through a block in G1/S transition mediated by decreased levels of cyclins D1 and E and cyclin dependent kinase 4 and suppressed Retinoblastoma phosphorylation. These responses correlated with decreased insulin-like growth factor-I receptor expression and activation, increased insulin-like growth factor binding protein 2 expression and decreased AKT activation. Exposure to lycopene at doses as low as 10 nM for 48 h induced a profound apoptotic response in LNCaP cells. In contrast PC3 cells were resistant to apoptosis at doses up to 1 microM. CONCLUSIONS: Lycopene exposure can suppress phosphatidylinositol 3-kinase-dependent proliferative and survival signalling in androgen-responsive LNCaP and androgen-independent PC3 cells suggesting that the molecular mechanisms for the cytostatic and cytotoxic actions of lycopene involve induction of G0/G1 cell cycle arrest. This study supports further examination of lycopene as a potential agent for both the prevention and treatment of prostate cancer.     

Epicatechin Gallate Induces Cell Death via p53 Activation and Stimulation of p38 and JNK in Human Colon Cancer SW480 Cells

The tea flavonoid epicatechin gallate (ECG) exhibits a wide range of biological activities. In this study, the in vitro anticancer effects of ECG on SW480 colon cancer cell line was investigated by analyzing the cell cycle, apoptosis, key proteins involved in cellular survival/proliferation, namely AKT/phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinases (MAPKs), and the role of p53 in these processes. ECG induced cell cycle arrest at the G0/G1-S phase border associated with the stimulation of p21, p-p53, and p53 and the suppression of cyclins D1 and B1. Exposure of SW480 cells to ECG also led to apoptosis as determined by time-dependent changes in caspase-3 activity, MAPKs [extracellular regulated kinase (ERK), p38, and c-jun amino-terminal kinase (JNK)], p21 and p53 activation, and AKT inhibition. The presence of pifithrin, an inhibitor of p53 function, blocked ECG-induced apoptosis as was manifested by restored cell viability and caspase-3 activity to control values and reestablished the balance among Bcl-2 anti- and proapoptotic protein levels. Interestingly, ECG also inhibited p53 protein and RNA degradation, contributing to the stabilization of p53. In addition, JNK and p38 have been identified as necessary for ECG-induced apoptosis, upon activation by p53. The results suggest that the activation of the p53-p38/JNK cascade is required for ECG-induced cell death in SW480 cells.     

L-Carvone Induces p53, Caspase 3 Mediated Apoptosis and Inhibits the Migration of Breast Cancer Cell Lines

A wide variety of natural compounds exists that possesses significant cytotoxic as well as chemopreventive activity through induction of apoptosis in cancer cells. The antiproliferative and apoptotic effect of L-carvone, an active component of spearmint (Mentha spicata) was studied on breast cancer (MCF 7 and MDA MB 231) and normal (MCF 10A) cell lines, and insight into its mechanism of action was attained. L-carvone inhibited proliferation of MCF 7 (IC₅₀ 1.2 mM) and MDA MB 231 cells (IC₅₀ 1.0 mM) and inhibited the migration of breast cancer cell lines. L-carvone induced apoptosis as observed by nuclei fragmentation and the presence of apoptotic bodies in DAPI, AnnexinV/propidium iodide, and TUNEL assays. L-carvone exposure arrested MCF 7 cells in S phase of the cell cycle. DNA damage caused by L-carvone was apparent from the increased tail moment in COMET assay, which could be induced by an increase in ROS that was measured using a fluorescence probe. Glutathione levels were also increased. The increased level of p53, Bad, cleaved caspase 3, and cleaved PARP explained p53 and caspase-mediated apoptosis.     

Genistein-induced cell cycle arrest and apoptosis in a head and neck squamous cell carcinoma cell line.

Epidemiological studies have shown a lower incidence of breast, prostate, and colon cancers in Asian countries, particularly China and Japan, than in the United States. It is believed that genistein, a natural tyrosine kinase inhibitor and a metabolite of soy products, may be responsible for the protection from these cancers. Genistein was shown to inhibit cell proliferation and to induce cell cycle arrest at the G2-M phase in breast, prostate, and jurkat T cell leukemia cell lines. However, such studies have not been reported in squamous cell cancers of the head and neck. In this report, we show that genistein inhibits proliferation of a squamous cell carcinoma cell line HN4. Additionally, genistein caused cell cycle arrest at the S/G2-M phase and induced programmed cell death (apoptosis) in these cells. These effects appear to be dose and time dependent, irreversible, persisting when the cells were recultured in genistein-free medium for up to 72 hours, and specific for tumor cells, because genistein did not affect normal keratinocytes. These results suggest that if genistein shows similar results in clinical trials, it can be a potential chemopreventive/chemotherapeutic agent for cancers of the head and neck.     

Unique formosan mushroom Antrodia camphorata differentially inhibits androgen-responsive LNCaP and -independent PC-3 prostate cancer cells

Antrodia camphorata (AC), a precious and unique folkloric medicinal mushroom enriched in polyphenolics, isoflavonoids, triterpenoids, and polysaccharides, has been diversely used in Formosa (Taiwan) since the 18th century. In this study, prostate cancer (PCa) cell lines PC-3 (androgen independent) and LNCaP (androgen responsive) were treated with AC crude extract (ACCE) at 50-200 microg/mL, respectively, for 48 h. At the minimum effective dose 150 microg/mL, LNCaP showed a G1/S phase arrest with significant apoptosis. Such dose-dependent behavior of LNCaP cells in response to ACCE was confirmed to proceed as Akt-->p53-->p21-->CDK4/cyclin D1-->G1/S-phase arrest-->apoptosis, which involved inhibiting cyclin D1 activity and preventing pRb phosphorylation. In contrast, being without p53, PC-3 cells showed a G2/M-phase arrest mediated through pathway p21-->cyclin B1/Cdc2-->G2/M-phase arrest, however, with limited degree of apoptosis, implicating that ACCE is able to differentially inhibit the growth of different PCa cells by modulating different cell cycle signaling pathways. We conclude that this unique Formosan mushroom, A. camphorata, due to its nontoxicity, might be used as a good adjuvant anticancer therapy for prostate cancers despite its androgen-responsive behaviors, which has long been a serious drawback often encountered clinically in hormonal refractory cases treated by antihormonal therapies and chemotherapeutics.     

Inhibition of proliferation and induction of apoptosis by γ-tocotrienol in human colon carcinoma HT-29 cells

Objectiveγ-Tocotrienol is a major component of the tocotrienol-rich fraction of palm oil, but there is limited evidence that it has antitumor activity. In particular, the effects of γ-tocotrienol on human colon carcinoma cells have not been reported. To investigate the chemopreventive effects of γ-tocotrienol on colon cancer, we examined its capacity to inhibit proliferation and induce apoptosis in HT-29 cells and explored the mechanism underlying these effects.MethodsWe cultured HT-29 cells in the presence of γ-tocotrienol. The effect of γ-tocotrienol on cell proliferation was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, mitotic index, and colony formation. The cell-cycle distribution was investigated by flow cytometry. We measured apoptosis by nuclear staining, transmission electron microscopy, and DNA fragmentation. Apoptosis-related proteins and the nuclear factor-κB p65 protein were determined by western blotting and immunofluorescence.Resultsγ-Tocotrienol inhibited cell growth and arrested HT-29 cells in G₀/G₁ phase. The 50% inhibitory concentration was 31.7 μmol/L (48 h). γ-Tocotrienol–induced apoptosis in HT-29 cells was accompanied by downregulation of Bcl-2, upregulation of Bax, and activation of caspase-3. Furthermore, we found that γ-tocotrienol reduced the expression level of total nuclear factor-κB p65 protein and inhibited its nuclear translocation.ConclusionThe results indicated that γ-tocotrienol inhibits cell proliferation and induces apoptosis in HT-29 cells in a time- and dose-dependent manner, and that this process is accompanied by cell-cycle arrest at G₀/G₁, an increased Bax/Bcl-2 ratio, and activation of caspase-3. Our data also indicated that nuclear factor-κB p65 protein may be involved in these effects.     

A comparative study of growth-inhibitory effects of isoflavones and their metabolites on human breast and prostate cancer cell lines

The possible growth-inhibitory properties of the recently synthesized novel metabolite 1-(2,4-dihydrobenzoyl)-1-(4-hydroxyphenyl)ethylene (2-de-O-DMA) and six other metabolites of isoflavones were investigated and compared with those of the major isoflavones genistein, daidzein, and glycitein on human breast noncancer and breast and prostate cancer cell lines in vitro. The novel metabolite 2-de-O-DMA was found to be a more potent inhibitor than genistein on human breast cancer MCF-7, MDA-MB-468, and SK-BR-3 cells and breast noncancer MCF-10A cells. In prostate cancer cell lines, LNCaP and DU145, 2-de-O-DMA elicited a six- to sevenfold more potent inhibition than genistein. Flow cytometric analysis showed that 2-de-O-DMA and genistein blocked cells at the G2/M phase of the cell cycle. Genistein and 2-de-O-DMA led to apoptosis of a variety of cancer cell lines. The rapid response of growth inhibition induced by 2-de-O-DMA compared with genistein strongly suggests that the observed antiproliferation effects elicited by this novel metabolite are mediated via a biological pathway different from that induced by genistein. 2-de-O-DMA, a novel metabolite of isoflavone, could have a potential role in chemopreventive and chemotherapeutic treatment of hormonal breast and prostate cancers.     

Involvement of c-Jun N-terminal kinase in G2/M arrest and caspase-mediated apoptosis induced by sulforaphane in DU145 prostate cancer cells

Sulforaphane (SFN) is a major isothiocyanate compound in cruciferous vegetables such as broccoli, cauliflower, and Brussels sprouts. Preclinical animal models have recently shown that SFN and other isothiocyanates may be useful for prostrate cancer (PCa) chemoprevention. In this study we used a DU145 human PCa cell culture model to investigate the role of protein kinase signaling pathway(s) in SFN-induced cell cycle arrest and apoptosis and whether another chemopreventive agent selenium enhances the apoptosis potency of SFN. The results showed that SFN exposure for 24 h or longer significantly decreased the number of viable DU145 cells in a dose-dependent manner with an IC50 of asymptotically equal to 10 microM. The decreased cell number was associated with G2/M phase arrest and apoptotic cell death, with the latter being evidenced by caspase-mediated cleavage of poly(ADP-ribose) polymerase and increased release of histone-associated DNA fragments. A peptide inhibitor of caspase-8 completely blocked SFN-induced apoptosis and that for caspase-9 exerted a major protection; however, neither inhibitor attenuated SFN-induced G2/M arrest. Regarding potential mediators, SFN treatment induced a transient rise of reactive oxygen species (ROS) peaking within (1/2) h and the activation of JNK within 1 h but did not have any detectable effect on the phosphorylation of p38MAPK or ERK1/2 from 6 h to 24 h. Pretreatment of cells with N-acetylcysteine to enrich intracellular glutathione blocked SFN-induced ROS and apoptotic cell death. Inhibiting the JNK activity with a pharmacologic inhibitor SP600125 abolished the induction of G2/M arrest and apoptosis by SFN, whereas chemical inhibitors for p38MAPK and MEK1/2 did not have any modulating effect on SFN-induced apoptosis. Taken together, the data indicate that SFN decreased viable DU145 cell number in large part through the generation of ROS and JNK-mediated signaling to G2/M arrest and caspase-dependent apoptosis. Selenium in the form of inorganic sodium selenite salt or methylseleninic acid did not enhance SFN-induced apoptosis in this cell culture model.     

Protective effects of selenium against mercury toxicity in cultured Atlantic spotted dolphin (Stenella plagiodon) renal cells

Marine mammals are known for their low susceptibility to mercury toxicity, and selenium may play a role in this protection against mercury intoxication. To gain insight into mechanisms by which selenium might inhibit mercury toxicity in cetacean cells, we investigated the effects of sodium selenite on cell proliferation and cell death (including apoptosis, oncosis, and necrosis) of control and mercuric chloride–treated Atlantic spotted dolphin renal cells (Sp1K cells). Concurrent exposure to 80 μM Na₂SeO₃ provided full protection against the decrease in cell proliferation induced by 20 μM HgCl₂. Pretreatment with Na₂SeO₃ increased the protective effects of selenium administered later in conjunction with mercury, but pretreatment alone did not provide protection against mercury given alone. Furthermore, Na₂SeO₃ administered after the exposure to HgCl₂ did not protect cells. These data suggest that the coexistence of Na₂SeO₃ and HgCl₂ was essential for the protective effects of Na₂SeO₃ against the toxicity of HgCl₂ in Sp1K cells, and may involve selenium–mercury binding. This is supported by the results of an experiment in which earlier premixed mercury and selenium solutions were less cytotoxic than freshly mixed solutions. Furthermore, HgCl₂ induced apoptosis in Sp1K cells, as revealed by nuclear specific dye (7-AAD) incorporation and cell flow cytometry, and this was prevented by the concurrent exposure to Na₂SeO₃. Inhibition of mercury-induced apoptosis in marine mammal cells, provided by selenium, may contribute to the in vivo protection. This study is the first report that addresses the mechanism of mercury–selenium antagonism in cultured cetacean cells at the cellular level. Received: 28 July 2000/Accepted: 12 May 2001