Notch Activation by Phenethyl Isothiocyanate Attenuates Its Inhibitory Effect on Prostate Cancer Cell Migration

Phenethyl isothiocyanate (PEITC) is a promising cancer chemopreventive component of edible cruciferous vegetables with in vivo efficacy against prostate cancer in experimental rodents. Cancer chemopreventive response to PEITC is characterized by its ability to inhibit multiple oncogenic signaling pathways, including nuclear factor-κB, Akt, and androgen receptor. The present study demonstrates, for the first time, that PEITC treatment activates Notch signaling in malignant as well as normal human prostate cells. Exposure of human prostate cancer cells (LNCaP, PC-3, and DU145) and a normal human prostate epithelial cell line (PrEC) to PEITC resulted in cleavage (active form) of Notch1 and Notch2, and increased transcriptional activity of Notch. In PC-3 and LNCaP cells, PEITC treatment caused induction of Notch ligands Jagged1 and Jagged2 (PC-3), overexpression of γ-secretase complex components Presenilin1 and Nicastrin (PC-3), nuclear enrichment of cleaved Notch2, and/or up-regulation of Notch1, Notch2, Jagged1, and/or Jagged2 mRNA. PEITC-induced apoptosis in LNCaP and PC-3 cells was significantly attenuated by RNA interference of Notch2, but not by pharmacological inhibition of Notch1. Inhibition of PC-3 and LNCaP cell migration resulting from PEITC exposure was significantly augmented by knockdown of Notch2 protein as well as pharmacological inhibition of Notch1 activation. Nuclear expression of cleaved Notch2 protein was significantly higher in PC-3 xenografts from PEITC-treated mice and dorsolateral prostates from PEITC-fed TRAMP mice compared with respective control. Because Notch signaling is implicated in epithelial-mesenchymal transition and metastasis, the present study suggests that anti-metastatic effect of PEITC may be augmented by a combination regimen involving a Notch inhibitor

Benzyl Isothiocyanate Causes FoxO1-Mediated Autophagic Death in Human Breast Cancer Cells

Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, inhibits growth of breast cancer cells but the mechanisms underlying growth inhibitory effect of BITC are not fully understood. Here, we demonstrate that BITC treatment causes FoxO1-mediated autophagic death in cultured human breast cancer cells. The BITC-treated breast cancer cells (MDA-MB-231, MCF-7, MDA-MB-468, BT-474, and BRI-JM04) and MDA-MB-231 xenografts from BITC-treated mice exhibited several features characteristic of autophagy, including appearance of double-membrane vacuoles (transmission electron microscopy) and acidic vesicular organelles (acridine orange staining), cleavage of microtubule-associated protein 1 light chain 3 (LC3), and/or suppression of p62 (p62/SQSTM1 or sequestosome 1) expression. On the other hand, a normal human mammary epithelial cell line (MCF-10A) was resistant to BITC-induced autophagy. BITC-mediated inhibition of MDA-MB-231 and MCF-7 cell viability was partially but statistically significantly attenuated in the presence of autophagy inhibitors 3-methyl adenine and bafilomycin A1. Stable overexpression of Mn-superoxide dismutase, which was fully protective against apoptosis, conferred only partial protection against BITC-induced autophagy. BITC treatment decreased phosphorylation of mTOR and its downstream targets (P70s6k and 4E-BP1) in cultured MDA-MB-231 and MCF-7 cells and MDA-MB-231 xenografts, but activation of mTOR by transient overexpression of its positive regulator Rheb failed to confer protection against BITC-induced autophagy. Autophagy induction by BITC was associated with increased expression and acetylation of FoxO1. Furthermore, autophagy induction and cell growth inhibition resulting from BITC exposure were significantly attenuated by small interfering RNA knockdown of FoxO1. In conclusion, the present study provides novel insights into the molecular circuitry of BITC-induced cell death involving FoxO1-mediated autophagy

RNA interference of Notch2 augments phenethyl isothiocyanate (PEITC)-mediated inhibition of LNCaP and PC-3 cell migration.

<p>Representative images (Boyden chamber assay) depicting migration of (A) LNCaP and (C) PC-3 cells transfected with a control (non-specific) siRNA or a Notch2-targeted siRNA and treated for 24 hours with dimethyl sulfoxide (DMSO) or 5 µM PEITC at 200× magnification. Quantitation of migrated (B) LNCaP cells and (D) PC-3 cells from experiments shown in panels A and C. Three to four fields on each filter were scored for migrated cells under an inverted microscope at 200× magnification. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026615#s2" target="_blank">Results</a> shown are mean ± SD (n = 3). *Significantly different (<i>P</i><0.05) between the indicated groups by one-way ANOVA followed by Bonferroni's multiple comparison test. Each experiment was repeated twice, and representative data from one such experiment are shown.</p

Phenethyl isothiocyanate (PEITC)-mediated inhibition of prostate cancer cell migration is augmented by a γ-secretase inhibitor.

<p>Representative images (Boyden chamber assay) depicting migration of (A) LNCaP and (C) PC-3 cells after 24-hour treatment with dimethyl sulfoxide (DMSO) or 5 µM PEITC in the absence or presence of 50 µM DAPT at 200× magnification. Quantitation of migrated (B) LNCaP cells and (D) PC-3 cells after 24-hour treatment with DMSO or 5 µM PEITC and/or 50 µM DAPT. Three to four fields on each filter were scored for migrated cells under an inverted microscope. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026615#s2" target="_blank">Results</a> shown are mean ± SD (n = 3). *Significantly different (<i>P</i><0.05) between the indicated groups by one-way ANOVA followed by Bonferroni's multiple comparison test. Each experiment was repeated twice, and representative data from one such experiment are shown.</p

Phenethyl isothiocyanate (PEITC) increases nuclear levels of cleaved Notch2 protein in LNCaP and PC-3 cells.

<p>(A) Immunofluorescence microscopic images depicting nuclear levels of cleaved Notch2 protein in LNCaP and PC-3 cells after 8-hour treatment with dimethyl sulfoxide (DMSO) or 5 µM PEITC at 100× objective magnification. Quantitation of <i>Notch1</i>, <i>Notch2</i>, <i>Jagged1</i>, and <i>Jagged2</i> mRNA levels by real-time RT-PCR in (B) LNCaP and (C) PC-3 cells after 8-hour treatment with DMSO (control) or PEITC (2.5 or 5 µM). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026615#s2" target="_blank">Results</a> shown are mean ± SD (n = 3). *Significantly different (<i>P</i><0.05) compared with DMSO-treated control by one-way ANOVA with Dunnett's adjustment. Each experiment was repeated at least twice.</p

Notch2 knockdown confers protection against phenethyl isothiocyanate (PEITC)-induced apoptosis in LNCaP and PC-3 cells.

<p>(A) Immunoblotting for cleaved Notch2 protein using lysates from LNCaP and PC-3 cells transiently transfected with a control (non-specific) siRNA or a Notch2-targeted siRNA and treated for 24 hours with dimethyl sulfoxide (DMSO) or 5 µM PEITC. Numbers above bands represent changes in protein levels relative to control siRNA-transfected cells treated with DMSO. Arrow (PC-3) identifies cleaved Notch2, the lower band is non-specific. (B) Histone-associated DNA fragment release into the cytosol (a measure of apoptosis) in LNCaP and PC-3 cells transiently transfected with a control siRNA or a Notch2-targeted siRNA and treated for 24 hours with DMSO (control) or 5 µM PEITC. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026615#s2" target="_blank">Results</a> are expressed as apoptosis enrichment relative to control siRNA-transfected cells treated with DMSO. (C) Immunoblotting for cleaved Notch1 and cleaved Notch2 proteins using lysates from LNCaP and PC-3 cells treated for 8 hours or 24 hours with DMSO (control) or 5 µM PEITC in the absence or presence of 50 µM DAPT. Numbers above bands represent changes in protein levels relative to corresponding DMSO-treated control. (D) Histone-associated apoptotic DNA fragment release into the cytosol in LNCaP and PC-3 cells treated for 24 hours with DMSO (control) or 5 µM PEITC in the absence or presence of 50 µM DAPT. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026615#s2" target="_blank">Results</a> are expressed as apoptosis enrichment relative to DMSO-treated control. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026615#s2" target="_blank">Results</a> (panels B and D) are mean ± SD (n = 3). *Significantly different (<i>P</i><0.05) between the indicated groups by one-way ANOVA followed by Bonferroni's multiple comparison test. Each experiment was repeated twice, and representative data from one such experiment are shown.</p

Phenethyl isothiocyanate (PEITC) increases levels of cleaved Notch1 and cleaved Notch2 in prostate cancer cells.

<p>Immunoblotting for cleaved Notch1, cleaved Notch2, Jagged1, Jagged2, Presenilin1, and Nicastrin using lysates from (A) LNCaP, (B) PC-3, and (C) LNCaP−C4-2 cells after 8-, 16-, or 24-hour treatment with dimethyl sulfoxide (DMSO) or PEITC (2.5 or 5 µM). Arrow in panel B identifies cleaved Notch2, the lower band is non-specific based on siRNA results shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026615#pone-0026615-g004" target="_blank">Fig. 4A</a>. Blots were stripped and re-probed with anti-actin antibody. Immunoblotting for each protein was done at least twice using independently prepared lysates. Numbers above band represent changes in protein levels relative to corresponding DMSO-treated control.</p