Mitochondria-mediated apoptosis by diallyl trisulfide in human prostate cancer cells is associated with generation of reactive oxygen species and regulated by Bax/Bak.

Garlic constituent diallyl trisulfide (DATS) inhibits growth of cancer cells in vitro and in vivo by causing apoptosis, but the sequence of events leading to cell death is not fully understood. We now show that DATS treatment triggers mitochondria-mediated apoptosis program in human prostate cancer cells (LNCaP, LNCaP-C81, LNCaP-C4-2) irrespective of their androgen responsiveness. Interestingly, a normal prostate epithelial cell line (PrEC) is significantly more resistant to apoptosis induction by DATS compared with prostate cancer cells. The DATS-induced apoptosis in LNCaP cells correlated with the collapse of mitochondrial membrane potential, modest increase in protein level of Bak, and down-regulation of Bcl-2 and Bcl-xL protein levels. The DATS-induced apoptosis was significantly attenuated by knockdown of Bax and Bak proteins, but not by ectopic expression of either Bcl-2 or Bcl-xL. The DATS treatment caused generation of reactive oxygen species (ROS) in LNCaP cells, but not in PrEC, which was attenuated by pretreatment with antioxidant N-acetylcysteine. The N-acetylcysteine pretreatment conferred significant protection against DATS-mediated disruption of the mitochondrial membrane potential and apoptosis. In conclusion, the present study reveals that the mitochondria-mediated cell death by DATS is associated with ROS generation and regulated by Bax/Bak but independent of Bcl-2 or Bcl-xL.

Induction of p21 protein protects against sulforaphane-induced mitotic arrest in LNCaP human prostate cancer cell line.

Previous studies have indicated that d,l-sulforaphane (SFN), a synthetic cancer chemopreventive analogue of cruciferous vegetable-derived isomer (-)-1-isothiocyanato-(4R)-(methylsulfinyl)-butane, activates a checkpoint kinase 2 (Chk2)-dependent G(2)-M phase cell cycle arrest in p53-deficient human prostate cancer cells. Because p53 is a downstream target of Chk2 kinase and known to regulate G(2)-M transition by transcriptional regulation of cyclin-dependent kinase (Cdk) inhibitor p21(Cip1/Waf1) (p21), the present study was undertaken to determine the role of p21 in SFN-induced cell cycle arrest using wild-type p53-expressing cell line LNCaP. The SFN treatment caused a modest increase in S phase fraction and a marked increase in G(2)-M fraction in LNCaP cells in a concentration- and time-dependent manner. The SFN-induced S phase arrest correlated with a reduction in protein levels of cyclin D1, cyclin E, Cdk4, and Cdk6, whereas activation of the G(2)-M checkpoint was accompanied by induction of cyclin B1 and down-regulation of Cdk1 and Cdc25C protein levels. The SFN-treated LNCaP cells were also arrested in mitosis as revealed by immunofluorescence microscopy and increased Ser(10) phosphorylation of histone H3, a sensitive marker for mitotic cells. The SFN treatment increased activating phosphorylation of Chk2 (Thr(68)) that was accompanied by induction of p53 and p21. The SFN-induced mitotic arrest was statistically significantly increased by small interfering RNA-based knockdown of p21. However, p21 protein knockdown did not have any appreciable effect on SFN-induced cytoplasmic histone-associated DNA fragmentation (apoptosis). In conclusion, the present study indicates that induction of p21 protects against SFN-induced mitotic arrest in LNCaP cells.

D,L-Sulforaphane-induced cell death in human prostate cancer cells is regulated by inhibitor of apoptosis family proteins and Apaf-1.

D,L-Sulforaphane (SFN), a synthetic analogue of cruciferous vegetable-derived isomer l-SFN, suppresses proliferation of cancer cells by causing apoptosis but the mechanism of cell death is not fully understood. We used LNCaP (wild-type p53) and PC-3 (p53 deficient) human prostate cancer cells to gain further insights into the mechanism of SFN-induced apoptosis. The LNCaP cell line was relatively more sensitive to SFN-induced apoptosis compared with PC-3. The SFN treatment caused stabilization of p53 protein in LNCaP cells, but SFN-mediated apoptosis was not attenuated by knockdown of p53 protein. Instead, the differential sensitivity of these cells to SFN-induced apoptosis correlated with difference in kinetics of Bax conformational change. Ectopic expression of Bcl-2 failed to confer protection against SFN-induced cell death in LNCaP cells. Treatment of PC-3 cells with SFN resulted in a marked decrease in the levels of inhibitor of apoptosis (IAP) family proteins (cIAP1, cIAP2 and XIAP), which was accompanied by inhibition of nuclear translocation of p65-nuclear factor kappaB (NFkappaB). The effect of SFN on levels of IAP family proteins as well as transcriptional activity of NFkappaB was biphasic in LNCaP cells. The SFN-treated LNCaP and PC-3 cells exhibited a marked increase in protein level of Apaf-1, which was accompanied by an increase in transcriptional activity of E2F1. The SFN-induced apoptosis in both cell lines was significantly attenuated by Apaf-1 protein knockdown. In conclusion, the present study reveals a complex signaling mechanism involving Bax activation, downregulation of IAP family proteins and Apaf-1 induction in regulation of SFN-induced cell death.

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 micromoL, 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 approximately 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.

Diallyl trisulfide inhibits angiogenic features of human umbilical vein endothelial cells by causing Akt inactivation and down-regulation of VEGF and VEGF-R2.

We have shown recently that diallyl trisulfide (DATS), a cancer-chemopreventive constituent of garlic, inactivates Akt to trigger mitochondrial translocation of proapoptotic protein BAD in human prostate cancer cells. Because Akt activation is implicated in the promotion of endothelial cell survival and angiogenesis, we hypothesized that DATS may inhibit angiogenesis. In the present study, we tested this hypothesis using human umbilical vein endothelial cells (HUVECs) as a model. Survival of HUVECs was reduced significantly in the presence of DATS in a concentration-dependent manner, with an IC50 of approximately 4 microM. The DATS-mediated suppression of HUVEC survival was associated with apoptosis induction characterized by accumulation of subdiploid cells, cytoplasmic histone-associated DNA fragmentation, and cleavage of caspase-3 and poly-(ADP-ribose)-polymerase. The DATS-induced DNA fragmentation was significantly attenuated in the presence of pan-caspase inhibitor zVAD-fmk and specific inhibitors of caspase-9 (zLEHD-fmk) and caspase-8 (zIETD-fmk). DATS treatment inhibited the formation of capillary-like tube structure and migration by HUVECs in association with suppression of vascular endothelial growth factor (VEGF) secretion and VEGF receptor-2 protein level and inactivation of Akt kinase. DATS treatment also caused activation of extracellular signal-regulated kinase 1/2 (ERK1/2) but not c-Jun NH2-terminal kinase (JNK) or p38 mitogen-activated protein kinase (p38MAPK).DATS-mediatedapoptosis induction and inhibition of HUVEC tube formation was partially but statistically significantly attenuated by pharmacologic inhibition of ERK1/2 but not JNK or p38MAPK. The present study demonstrates, for the first time, that DATS has the ability to inhibit angiogenic features of human endothelial cells.

Phenethyl isothiocyanate-induced apoptosis in PC-3 human prostate cancer cells is mediated by reactive oxygen species-dependent disruption of the mitochondrial membrane potential.

The present study was undertaken to gain insights into the molecular mechanism of apoptosis induction by phenethyl isothiocyanate (PEITC), which is a cancer chemopreventive constituent of cruciferous vegetables, using PC-3 human prostate cancer cells as a model. The PEITC-induced cell death in PC-3 cells was associated with disruption of the mitochondrial membrane potential, release of apoptogenic molecules (cytochrome c and Smac/DIABLO) from mitochondria to the cytosol and generation of reactive oxygen species (ROS), which were blocked in the presence of a combined mimetic of superoxide dismutase and catalase (Euk134). Ectopic expression of Bcl-xL, whose protein level is reduced markedly on treatment of PC-3 cells with PEITC, conferred partial protection against PEITC-induced apoptosis only at higher drug concentrations (>10 microM). Administration of 12 micromol PEITC/day (Monday through Friday) by oral gavage significantly retarded growth of PC-3 xenografts in athymic mice. For instance, 31 days after the initiation of PEITC administration, the average tumor volume in control mice (721 +/- 153 mm3) was approximately 2-fold higher compared with mice receiving 12 micromol PEITC/day. The PEITC-mediated inhibition of PC-3 xenograft growth was associated with induction of Bax and Bid proteins. In conclusion, the present study indicates that the PEITC-induced apoptosis in PC-3 cells is mediated by ROS-dependent disruption of the mitochondrial membrane potential and regulated by Bax and Bid.

Caspase-dependent apoptosis induction by phenethyl isothiocyanate, a cruciferous vegetable-derived cancer chemopreventive agent, is mediated by Bak and Bax.

PURPOSE: The present study was undertaken to gain insights into the molecular mechanism of apoptosis induction by phenethyl isothiocyanate (PEITC) using prostate cancer cell lines derived from transgenic adenocarcinoma mouse prostate (TRAMP) mice (TRAMP-C1 and TRAMP-C2). EXPERIMENTAL DESIGN AND RESULTS: The viability of TRAMP-C1 and TRAMP-C2 cells was reduced significantly in the presence of PEITC in a concentration-dependent manner as determined by sulforhodamine B and trypan blue dye exclusion assays. Treatment of TRAMP-derived cells with PEITC revealed features characteristic of apoptosis induction, including appearance of subdiploid cells (determined by flow cytometry), cytoplasmic histone-associated DNA fragmentation (determined by an ELISA assay), and cleavage of caspase-3 (determined by immunoblotting). The PEITC-induced apoptosis in TRAMP-derived cells was associated with a marked increase in the level of proapoptotic protein Bak and/or a decrease in the levels of antiapoptotic protein Mcl-1 or Bcl-xL and disruption of mitochondrial membrane potential. The SV40 immortalized mouse embryonic fibroblasts derived from Bak and Bax double knockout mice were significantly more resistant to PEITC-induced DNA fragmentation compared with wild-type or Bak-/- mouse embryonic fibroblasts. The PEITC-induced apoptosis in both cell lines was significantly attenuated in the presence of caspase inhibitors zVAD-fmk, zLEHD-fmk, and zIETD-fmk. Oral administration of PEITC (9 or 12 micromol PEITC/d, Monday-Friday) significantly retarded growth of TRAMP-C1 xenografts in nude mice without causing weight loss or any other side effects. CONCLUSION: The results of the present study indicate that caspase-dependent apoptosis by PEITC is mediated by Bak and Bax proteins.

Sulforaphane-induced cell death in human prostate cancer cells is initiated by reactive oxygen species.

We have shown previously that sulforaphane (SFN), a constituent of many edible cruciferous vegetables including broccoli, suppresses growth of prostate cancer cells in culture as well as in vivo by causing apoptosis, but the sequence of events leading to cell death is poorly defined. Using PC-3 and DU145 human prostate cancer cells as a model, we now demonstrate, for the first time, that the initial signal for SFN-induced apoptosis is derived from reactive oxygen species (ROS). Exposure of PC-3 cells to growth-suppressive concentrations of SFN resulted in ROS generation, which was accompanied by disruption of mitochondrial membrane potential, cytosolic release of cytochrome c, and apoptosis. All these effects were significantly blocked on pretreatment with N-acetylcysteine and overexpression of catalase. The SFN-induced ROS generation was significantly attenuated on pretreatment with mitochondrial respiratory chain complex I inhibitors, including diphenyleneiodonium chloride and rotenone. SFN treatment also caused a rapid and significant depletion of GSH levels. Collectively, these observations indicate that SFN-induced ROS generation is probably mediated by a nonmitochondrial mechanism involving GSH depletion as well as a mitochondrial component. Ectopic expression of Bcl-xL, but not Bcl-2, in PC-3 cells offered significant protection against the cell death caused by SFN. In addition, SFN treatment resulted in an increase in the level of Fas, activation of caspase-8, and cleavage of Bid. Furthermore, SV40-immortalized mouse embryonic fibroblasts (MEFs) derived from Bid knock-out mice displayed significant resistance toward SFN-induced apoptosis compared with wild-type MEFs. In conclusion, the results of the present study indicate that SFN-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic caspase cascades contribute to the cell death caused by this highly promising cancer chemopreventive agent.

Sulforaphane-induced G2/M phase cell cycle arrest involves checkpoint kinase 2-mediated phosphorylation of cell division cycle 25C.

Previously, we showed that sulforaphane (SFN), a naturally occurring cancer chemopreventive agent, effectively inhibits proliferation of PC-3 human prostate cancer cells by causing caspase-9- and caspase-8-mediated apoptosis. Here, we demonstrate that SFN treatment causes an irreversible arrest in the G(2)/M phase of the cell cycle. Cell cycle arrest induced by SFN was associated with a significant decrease in protein levels of cyclin B1, cell division cycle (Cdc) 25B, and Cdc25C, leading to accumulation of Tyr-15-phosphorylated (inactive) cyclin-dependent kinase 1. The SFN-induced decline in Cdc25C protein level was blocked in the presence of proteasome inhibitor lactacystin, but lactacystin did not confer protection against cell cycle arrest. Interestingly, SFN treatment also resulted in a rapid and sustained phosphorylation of Cdc25C at Ser-216, leading to its translocation from the nucleus to the cytoplasm because of increased binding with 14-3-3beta. Increased Ser-216 phosphorylation of Cdc25C upon treatment with SFN was the result of activation of checkpoint kinase 2 (Chk2), which was associated with Ser-1981 phosphorylation of ataxia telangiectasia-mutated, generation of reactive oxygen species, and Ser-139 phosphorylation of histone H2A.X, a sensitive marker for the presence of DNA double-strand breaks. Transient transfection of PC-3 cells with Chk2-specific small interfering RNA duplexes significantly attenuated SFN-induced G(2)/M arrest. HCT116 human colon cancer-derived Chk2(-/-) cells were significantly more resistant to G(2)/M arrest by SFN compared with the wild type HCT116 cells. These findings indicate that Chk2-mediated phosphorylation of Cdc25C plays a major role in irreversible G(2)/M arrest by SFN. Activation of Chk2 in response to DNA damage is well documented, but the present study is the first published report to link Chk2 activation to cell cycle arrest by an isothiocyanate.

Sulforaphane induces caspase-mediated apoptosis in cultured PC-3 human prostate cancer cells and retards growth of PC-3 xenografts in vivo.

Sulforaphane (SFN), a constituent of cruciferous vegetables, is highly effective in affording protection against chemically induced cancers in animal models. Here, we report that SFN inhibited proliferation of cultured PC-3 human prostate cancer cells by inducing apoptosis that was characterized by appearance of cells with sub-G0/G1 DNA content, formation of cytoplasmic histone associated DNA fragments and cleavage of poly(ADP-ribose)polymerase (PARP). SFN-induced apoptosis was associated with up-regulation of Bax, down-regulation of Bcl-2 and activation of caspases-3, -9 and -8. SFN-induced apoptosis, and cleavage of procaspase-3 and PARP were blocked upon pre-treatment of cells with pan caspase inhibitor z-VADfmk, and specific inhibitors of caspase-9 (z-LEHDfmk) and caspase-8 (z-IETDfmk) suggesting involvement of both caspase-9 and caspase-8 pathways in SFN-induced cell death. Oral administration of SFN (5.6 micro mol, 3 times/week) significantly inhibited growth of PC-3 xenografts in nude mice. For instance, 10 days after starting therapy, the average tumor volumes in control and SFN-treated mice were 170 +/- 13 and 80 +/- 14 mm3, respectively, reflecting a >50% reduction in tumor volume due to SFN administration. To the best of our knowledge, the present study is the first published report to document in vivo anticancer activity of SFN in a tumor xenograft model.

Allyl isothiocyanate, a constituent of cruciferous vegetables, inhibits growth of PC-3 human prostate cancer xenografts in vivo.

We have shown previously that allyl isothiocyanate (AITC), a constituent of cruciferous vegetables, significantly inhibits survival of PC-3 and LNCaP human prostate cancer cells in culture, whereas proliferation of a normal prostate epithelial cell line is minimally affected by AITC even at concentrations that are highly cytotoxic to the prostate cancer cells. The present studies were designed to test the hypothesis that AITC administration may retard growth of human prostate cancer xenografts in vivo. Bolus i.p. injection of 10 micromol AITC, three times per week (Monday, Wednesday and Friday) beginning the day of tumor cell implantation, significantly inhibited the growth of PC-3 xenograft (P < 0.05 by two-way ANOVA). For example, 26 days after tumor cell implantation, the average tumor volume in control mice (1025 +/- 205 mm3) was approximately 1.7-fold higher compared with AITC-treated mice. Histological analysis of tumors excised at the termination of the experiment revealed a statistically significant increase in number of apoptotic bodies with a concomitant decrease in cells undergoing mitosis in the tumors of AITC-treated mice compared with that of control mice. Western blot analysis indicated an approximately 70% reduction in the levels of anti-apoptotic protein Bcl-2 in the tumor lysate of AITC-treated mice compared with that of control mice. Moreover, the tumors from AITC-treated mice, but not control mice, exhibited cleavage of BID, which is known to promote apoptosis. Statistically significant reduction in the expression of several proteins that regulate G2/M progression, including cyclin B1, cell division cycle (Cdc)25B and Cdc25C (44, 45 and 90% reduction, respectively, compared with control), was also observed in the tumors of AITC-treated mice relative to control tumors. In conclusion, the results of the present study indicate that AITC administration inhibits growth of PC-3 xenografts in vivo by inducing apoptosis and reducing mitotic activity.

Requirement of a carbon spacer in benzyl isothiocyanate-mediated cytotoxicity and MAPK activation in head and neck squamous cell carcinoma.

Cruciferous vegetable-derived isothiocyanates (ITCs; chemical structure: R-N=C=S) are highly effective in affording protection against chemically induced cancers in animal models. Here, we studied the antitumor effects of benzyl isothiocyanate (BITC; Ph-CH2-N=C=S), the predominant ITC compound in broccoli, on head and neck squamous cell carcinoma (HNSCC) cell lines. Proliferation, apoptosis and immunoblotting assays were used to determine the effects and mechanism of several ITCs on HNSCC cells. The IC50 for BITC (24 h treatment) in two of the HNSCC cell lines was approximately 22 and 17 micro M, respectively. Interestingly, phenyl isothiocyanate (PITC; Ph-N=C=S), which is a close structural analog of BITC but lacks a -CH2- spacer that links the aromatic ring to N=C=S moiety, did not result in significant killing of the HNSCC cells in this dose range. BITC (but not PITC) caused activation of caspase 3 and PARP cleavage. Within 20 min of treatment, BITC (but not PITC) induced a rapid activation of p38 MAPK. In addition, BITC (but not PITC) treatment resulted in the activation of p44/42 MAPK. Co-treatment with a specific p38 MAPK inhibitor, SB203580, or an inhibitor of the MEK/MAPK pathway, U0126, partially rescued cells from BITC-induced killing. Our results show that minor structural differences in ITCs can be crucial for the antiproliferative activity of ITCs and that BITC may be a promising chemopreventive as well as therapeutic agent in HNSCC.

Allyl isothiocyanate, a constituent of cruciferous vegetables, inhibits proliferation of human prostate cancer cells by causing G2/M arrest and inducing apoptosis.

Dietary isothiocyanates (ITCs) are highly effective in affording protection against chemically induced cancers in laboratory animals. In the present study, we demonstrate that allyl isothiocyanate (AITC), a constituent of cruciferous vegetables, significantly inhibits proliferation of cultured PC-3 (androgen-independent) and LNCaP (androgen-dependent) human prostate cancer cells in a dose-dependent manner with an IC(50) of approximately 15-17 micro M. On the other hand, survival of a normal prostate epithelial cell line (PrEC) was minimally affected by AITC even at concentrations that were highly cytotoxic to the prostate cancer cells. Reduced proliferation of PC-3 as well as LNCaP cells in the presence of AITC correlated with accumulation of cells in G(2)/M phase and induction of apoptosis. In contrast, AITC treatment failed to induce apoptosis or cause G(2)/M phase arrest in PrEC cells. A 24 h treatment of PC-3 and LNCaP cells with 20 micro M AITC caused a significant decrease in the levels of proteins that regulate G(2)/M progression, including Cdk1 (32-50% reduction), Cdc25B (44-48% reduction) and Cdc25C (>90% reduction). A significant reduction in the expression of cyclin B1 protein (approximately 45%) was observed only in LNCaP cells. A 24 h exposure of PC-3 and LNCaP cells to an apoptosis-inducing concentration of AITC (20 micro M) resulted in a significant decrease (31-68%) in the levels of anti-apoptotic protein Bcl-2 in both cell lines, and approximately 58% reduction in Bcl-X(L) protein expression in LNCaP cells. In conclusion, it seems reasonable to hypothesize that AITC, and possibly other ITCs, may find use in the treatment of human prostate cancers.