Eun-Ryeong Hahm

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.

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.

Atg5 Regulates Phenethyl Isothiocyanate–Induced Autophagic and Apoptotic Cell Death in Human Prostate Cancer Cells

Phenethyl isothiocyanate (PEITC) is a promising cancer chemopreventive agent but the mechanism of its anticancer effect is not fully understood. We now show, for the first time, that PEITC treatment triggers Atg5-dependent autophagic and apoptotic cell death in human prostate cancer cells. Exposure of PC-3 (androgen independent, p53 null) and LNCaP (androgen responsive, wild-type p53) human prostate cancer cells to PEITC resulted in several specific features characteristic of autophagy, including appearance of membranous vacuoles, formation of acidic vesicular organelles, and cleavage and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes. A normal human prostate epithelial cell line (PrEC) was markedly more resistant toward PEITC-mediated cleavage and recruitment of LC3 compared with prostate cancer cells. Although PEITC treatment suppressed activating phosphorylations of Akt and mammalian target of rapamycin (mTOR), which are implicated in regulation of autophagy by different stimuli, processing and recruitment of LC3 was only partially/marginally reversed by ectopic expression of constitutively active Akt or overexpression of mTOR-positive regulator Rheb. The PEITC-mediated apoptotic DNA fragmentation was significantly attenuated in the presence of a pharmacologic inhibitor of autophagy (3-methyl adenine). Transient transfection of LNCaP and PC-3 cells with Atg5-specific small interfering RNA conferred significant protection against PEITC-mediated autophagy as well as apoptotic DNA fragmentation. A xenograft model using PC-3 cells and Caenorhabditis elegans expressing a lgg-1:GFP fusion protein provided evidence for occurrence of PEITC-induced autophagy in vivo. In conclusion, the present study indicates that Atg5 plays an important role in regulation of PEITC-induced autophagic and apoptotic cell death.

Phenethyl isothiocyanate inhibits oxidative phosphorylation to trigger reactive oxygen species-mediated death of human prostate cancer cells

Phenethyl isothiocyanate (PEITC), a constituent of edible cruciferous vegetables such as watercress, not only affords significant protection against chemically induced cancer in experimental rodents but also inhibits growth of human cancer cells by causing apoptotic and autophagic cell death. However, the underlying mechanism of PEITC-induced cell death is not fully understood. Using LNCaP and PC-3 human prostate cancer cells as a model, we demonstrate that the PEITC-induced cell death is initiated by production of reactive oxygen species (ROS) resulting from inhibition of oxidative phosphorylation (OXPHOS). Exposure of LNCaP and PC-3 cells to pharmacologic concentrations of PEITC resulted in ROS production, which correlated with inhibition of complex III activity, suppression of OXPHOS, and ATP depletion. These effects were not observed in a representative normal human prostate epithelial cell line (PrEC). The ROS production by PEITC treatment was not influenced by cyclosporin A. The Rho-0 variants of LNCaP and PC-3 cells were more resistant to PEITC-mediated ROS generation, apoptotic DNA fragmentation, and collapse of mitochondrial membrane potential compared with respective wild-type cells. The PEITC treatment resulted in activation of Bax in wild-type LNCaP and PC-3 cells, but not in their respective Rho-0 variants. Furthermore, RNA interference of Bax and Bak conferred significant protection against PEITC-induced apoptosis. The Rho-0 variants of LNCaP and PC-3 cells also resisted PEITC-mediated autophagy. In conclusion, the present study provides novel insight into the molecular circuitry of PEITC-induced cell death involving ROS production due to inhibition of complex III and OXPHOS.

Withaferin A-Induced Apoptosis in Human Breast Cancer Cells Is Mediated by Reactive Oxygen Species

Withaferin A (WA), a promising anticancer constituent of Ayurvedic medicinal plant Withania somnifera, inhibits growth of MDA-MB-231 and MCF-7 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo in association with apoptosis induction, but the mechanism of cell death is not fully understood. We now demonstrate, for the first time, that WA-induced apoptosis is mediated by reactive oxygen species (ROS) production due to inhibition of mitochondrial respiration. WA treatment caused ROS production in MDA-MB-231 and MCF-7 cells, but not in a normal human mammary epithelial cell line (HMEC). The HMEC was also resistant to WA-induced apoptosis. WA-mediated ROS production as well as apoptotic histone-associated DNA fragment release into the cytosol was significantly attenuated by ectopic expression of Cu,Zn-superoxide dismutase in both MDA-MB-231 and MCF-7 cells. ROS production resulting from WA exposure was accompanied by inhibition of oxidative phosphorylation and inhibition of complex III activity. Mitochondrial DNA-deficient Rho-0 variants of MDA-MB-231 and MCF-7 cells were resistant to WA-induced ROS production, collapse of mitochondrial membrane potential, and apoptosis compared with respective wild-type cells. WA treatment resulted in activation of Bax and Bak in MDA-MB-231 and MCF-7 cells, and SV40 immortalized embryonic fibroblasts derived from Bax and Bak double knockout mouse were significantly more resistant to WA-induced apoptosis compared with fibroblasts derived from wild-type mouse. In conclusion, the present study provides novel insight into the molecular circuitry of WA-induced apoptosis involving ROS production and activation of Bax/Bak.

Diallyl Trisulfide Suppresses Growth of PC-3 Human Prostate Cancer Xenograft In vivo in Association with Bax and Bak Induction

Purpose: The present study was undertaken to determine the effect of garlic constituent diallyl trisulfide (DATS) on growth of PC-3 human prostate cancer xenograft in vivo. Experimental Design: DATS was given orally (6 μmoL, thrice weekly) to male athymic mice s.c. implanted with PC-3 cells. Tumor sections from control and DATS-treated mice were examined for apoptotic bodies by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay. Protein levels of apoptosis and cell cycle regulating proteins in tumor tissues of control and DATS-treated mice were determined by immunoblotting. The effect of DATS treatment on in vivo angiogenesis was determined by immunohistochemical analysis of CD31 in tumors. Results: Oral gavage of DATS significantly retarded growth of PC-3 xenografts in athymic mice without causing weight loss. For instance, 20 days after starting therapy, the average tumor volume in control mice was ∼3-fold higher compared with DATS-treated mice. Tumors from DATS-treated mice exhibited a markedly higher count of apoptotic bodies compared with control tumors. Consistent with the results in cultured PC-3 cells, the DATS-mediated suppression of PC-3 xenograft growth correlated with induction of proapoptotic proteins Bax and Bak. Although DATS treatment inhibited migration of cultured PC-3 cells in association with down-regulation of vascular endothelial growth factor receptor-2 protein, formation of new blood vessels was comparable in tumors of control and DATS-treated mice as judged by CD31 immunostaining. Conclusions: The present study indicates that DATS administration inhibits growth of PC-3 xenografts in vivo in association with induction of Bax and Bak.

Honokiol causes G0-G1 phase cell cycle arrest in human prostate cancer cells in association with suppression of retinoblastoma protein level/phosphorylation and inhibition of E2F1 transcriptional activity.

The present study was undertaken to gain insights into the mechanism of cell cycle arrest caused by honokiol, a constituent of oriental herb Magnolia officinalis. The honokiol treatment decreased the viability of PC-3 and LNCaP human prostate cancer cells in a concentration- and time-dependent manner, which correlated with G0-G1 phase cell cycle arrest. The honokiol-mediated cell cycle arrest was associated with a decrease in protein levels of cyclin D1, cyclin-dependent kinase 4 (Cdk4), Cdk6, and/or cyclin E and suppression of complex formation between cyclin D1 and Cdk4 as revealed by immunoprecipitation using anti–cyclin D1 antibody followed by immunoblotting for Cdk4 protein. The honokiol-treated PC-3 and LNCaP cells exhibited a marked decrease in the levels of total and phosphorylated retinoblastoma protein (Rb), which correlated with the suppression of transcriptional activity of E2F1. Exposure of PC-3 and LNCaP cells to honokiol resulted in the induction of p21 (PC-3 and LNCaP) and p53 protein expression (LNCaP). However, small interfering RNA (siRNA)–mediated knockdown of either p21 (PC-3 and LNCaP) or p53 (LNCaP) protein failed to confer any protection against honokiol-induced cell cycle arrest. The honokiol treatment caused the generation of reactive oxygen species (ROS), and the cell cycle arrest caused by honokiol was partially but significantly attenuated in the presence of antioxidant N-acetylcysteine. In conclusion, the present study reveals that the honokiol-mediated G0-G1 phase cell cycle arrest in human prostate cancer cells is associated with the suppression of protein level/phosphorylation of Rb leading to inhibition of transcriptional activity of E2F1. [Mol Cancer Ther 2007;6(10):2686–95]

Chemopreventative Potential of the Cruciferous Vegetable Constituent Phenethyl Isothiocyanate in a Mouse Model of Prostate Cancer

BACKGROUND This study was undertaken to determine the chemopreventative efficacy of phenethyl isothiocyanate (PEITC), a bioactive constituent of many edible cruciferous vegetables, in a mouse model of prostate cancer, and to identify potential biomarker(s) associated with PEITC response. METHODS The chemopreventative activity of dietary PEITC was investigated in Transgenic Adenocarcinoma of Mouse Prostate mice that were fed a control diet or one containing 3 μmol PEITC/g (n = 21 mice per group) for 19 weeks. Dorsolateral prostate tissue sections were stained with hematoxylin and eosin for histopathologic evaluations and subjected to immunohistochemistry for analysis of cell proliferation (Ki-67 expression), autophagy (p62 and LC3 protein expression), and E-cadherin expression. Autophagosomes were visualized by transmission electron microscopy. Apoptotic bodies were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Plasma proteomics was performed by two-dimensional gel electrophoresis followed by mass spectrometry to identify potential biomarkers of PEITC activity. All statistical tests were two-sided. RESULTS Administration of PEITC (3 μmol/g diet) decreased incidence (PEITC diet vs control diet, mean = 21.65 vs 57.58%, difference = -35.93%, 95% confidence interval = -45.48% to -13.10%, P = .04) as well as burden (affected area) (PEITC diet vs control diet, mean = 18.53% vs 45.01%, difference = -26.48%, 95% confidence interval = -49.78% to -3.19%, P = .02) of poorly differentiated tumors in the dorsolateral prostate of transgenic mice compared with control mice, with no toxic effects. PEITC-mediated inhibition of prostate carcinogenesis was associated with induction of autophagy and overexpression of E-cadherin in the dorsolateral prostate. However, PEITC treatment was not associated with a decrease in cellular proliferation, apoptosis induction, or inhibition of neoangiogenesis. Plasma proteomics revealed distinct changes in the expression of several proteins (eg, suppression of clusterin protein) in the PEITC-treated mice compared with control mice. CONCLUSIONS In this transgenic model, dietary PEITC suppressed prostate cancer progression by induction of autophagic cell death. Potential biomarkers to assess the response to PEITC treatment in plasma were identified.

Withaferin A inhibits activation of signal transducer and activator of transcription 3 in human breast cancer cells

We have shown previously that withaferin A (WA), a promising anticancer constituent of Ayurvedic medicine plant Withania somnifera, inhibits growth of human breast cancer cells in culture and in vivo in association with apoptosis induction. The present study builds on these observations and demonstrates that WA inhibits constitutive as well as interleukin-6 (IL-6)-inducible activation of signal transducer and activator of transcription 3 (STAT3), which is an oncogenic transcription factor activated in many human malignancies including breast cancer. The WA treatment (2 and 4 μM) decreased constitutive (MDA-MB-231) and/or IL-6-inducible (MDA-MB-231 and MCF-7) phosphorylation of STAT3 (Tyr(705)) and its upstream regulator Janus-activated kinase 2 (JAK2; Tyr(1007/1008)) in MDA-MB-231, which was accompanied by suppression of their protein levels especially at the higher concentration. Exposure of MDA-MB-231 or MCF-7 cells to WA also resulted in suppression of (i) transcriptional activity of STAT3 with or without IL-6 stimulation in both cells; (ii) dimerization of STAT3 (MDA-MB-231) and (iii) nuclear translocation of Tyr(705)-phosphorylated STAT3 in both cells. To our surprise, the IL-6-stimulation, either before or after WA treatment, did not have an appreciable effect on WA-mediated apoptosis in MDA-MB-231 or MCF-7 cell line. The IL-6-stimulated activation of STAT3 conferred a modest protection against WA-mediated suppression of MDA-MB-231 cell invasion. General implication of these findings is that WA can trigger apoptosis and largely inhibit cell migration/invasion of breast cancer cells even after IL-6-induced activation of STAT3, which should be viewed as a therapeutic advantage for this agent.

Honokiol, a Constituent of Oriental Medicinal Herb Magnolia officinalis, Inhibits Growth of PC-3 Xenografts In vivo in Association with Apoptosis Induction

Purpose: This study was undertaken to determine the efficacy of honokiol, a constituent of oriental medicinal herb Magnolia officinalis, against human prostate cancer cells in culture and in vivo. Experimental Design: Honokiol-mediated apoptosis was assessed by analysis of cytoplasmic histone-associated DNA fragmentation. Knockdown of Bax and Bak proteins was achieved by transient transfection using siRNA. Honokiol was administered by oral gavage to male nude mice s.c. implanted with PC-3 cells. Tumor sections from control and honokiol-treated mice were examined for apoptotic bodies (terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling assay), proliferation index (proliferating cell nuclear antigen staining), and neovascularization (CD31 staining). Levels of Bcl-2 family proteins in cell lysates and tumor supernatants were determined by immunoblotting. Results: Exposure of human prostate cancer cells (PC-3, LNCaP, and C4-2) to honokiol resulted in apoptotic DNA fragmentation in a concentration- and time-dependent manner irrespective of their androgen responsiveness or p53 status. Honokiol-induced apoptosis correlated with induction of Bax, Bak, and Bad and a decrease in Bcl-xL and Mcl-1 protein levels. Transient transfection of PC-3 cells with Bak- and Bax-targeted siRNAs and Bcl-xL plasmid conferred partial yet significant protection against honokiol-induced apoptosis. Oral gavage of 2 mg honokiol/mouse (thrice a week) significantly retarded growth of PC-3 xenografts without causing weight loss. Tumors from honokiol-treated mice exhibited markedly higher count of apoptotic bodies and reduced proliferation index and neovascularization compared with control tumors. Conclusion: Our data suggest that honokiol, which is used in traditional oriental medicine for the treatment of various ailments, may be an attractive agent for treatment and/or prevention of human prostate cancers.