Curcumin enhances the apoptosis-inducing potential of TRAIL in prostate cancer cells: molecular mechanisms of apoptosis, migration and angiogenesis-2

<p><b>Copyright information:</b></p><p>Taken from "Curcumin enhances the apoptosis-inducing potential of TRAIL in prostate cancer cells: molecular mechanisms of apoptosis, migration and angiogenesis"</p><p>http://www.jmolecularsignaling.com/content/2/1/10</p><p>Journal of Molecular Signaling 2007;2():10-10.</p><p>Published online 4 Oct 2007</p><p>PMCID:PMC2082014.</p><p></p>alysis

Curcumin enhances the apoptosis-inducing potential of TRAIL in prostate cancer cells: molecular mechanisms of apoptosis, migration and angiogenesis-5

<p><b>Copyright information:</b></p><p>Taken from "Curcumin enhances the apoptosis-inducing potential of TRAIL in prostate cancer cells: molecular mechanisms of apoptosis, migration and angiogenesis"</p><p>http://www.jmolecularsignaling.com/content/2/1/10</p><p>Journal of Molecular Signaling 2007;2():10-10.</p><p>Published online 4 Oct 2007</p><p>PMCID:PMC2082014.</p><p></p> fluorometric assay as per manufacturer's instructions. (C and D), PC-3 and LNCaP cells were treated with curcumin (0–40 μM), in the presence or absence of TRAIL, and caspase-8 activity was measured by fluorometric assay as per manufacturer's instructions. (E and F), PC-3 and LNCaP cells were treated with curcumin (0, 10 or 20 μM), in the presence or absence of TRAIL (25 nM for PC-3, and 50 nM for LNCaP), and the cleavage of caspase-3, caspase-9, caspase-8 and PARP was measured by the Western blot analysis. β-actin was used as a loading control

Interactive effects of resveratrol and TRAIL on caspase activation and PARP cleavage

<p><b>Copyright information:</b></p><p>Taken from "Sensitization of TRAIL-resistant LNCaP cells by resveratrol (3, 4', 5 tri-hydroxystilbene): molecular mechanisms and therapeutic potential"</p><p>http://www.jmolecularsignaling.com/content/2/1/7</p><p>Journal of Molecular Signaling 2007;2():7-7.</p><p>Published online 24 Aug 2007</p><p>PMCID:PMC2018690.</p><p></p> (A), Effects of resveratrol and/or TRAIL on caspase-3 activity in LNCaP cells. Cells were treated with resveratrol (0–30 μM) in the presence or absence of TRAIL (50 nM) for 24 h. At the end of incubation period, caspase-3 activity was measured by flurometric assay. (B), Effects of resveratrol and/or TRAIL on caspase-8 activity. LNCaP cells were treated with resveratrol (0–30 μM) in the presence or absence of TRAIL (50 nM) for 24 h. At the end of incubation period, caspase-8 activity was measured by flurometric assay. (C), Effects of resveratrol and/or TRAIL on cleavage of pro-caspase-8, pro-caspase-3, pro-caspase-9 and PARP. LNCaP cells were pretreated with resveratrol (0, 10 or 20 μM) for 24 h followed by treatment with or without TRAIL (50 nM) for 24 h. At the end of incubation period, cells were harvested, and the Western blot analysis was performed to measure the expression of pro-caspase-8, cleaved-caspase-3, cleaved-caspase-9 and PARP. β-actin was used as a loading control

Effects of resveratrol and/or TRAIL on mitochondrial membrane potential (Δψ)

<p><b>Copyright information:</b></p><p>Taken from "Sensitization of TRAIL-resistant LNCaP cells by resveratrol (3, 4', 5 tri-hydroxystilbene): molecular mechanisms and therapeutic potential"</p><p>http://www.jmolecularsignaling.com/content/2/1/7</p><p>Journal of Molecular Signaling 2007;2():7-7.</p><p>Published online 24 Aug 2007</p><p>PMCID:PMC2018690.</p><p></p> (A), Resveratrol induces drop in Δψ. LNCaP cells were treated with or without resveratrol (20 μM) for 0–24 h. Cells were stained with JC1 dye, and Δψwas measured by a fluorometer as per manufacturer's instructions. (B), Interactive effects of resveratrol and TRAIL on Δψ. LNCaP cells were treated with resveratrol (20 μM) in the presence or absence of TRAIL (50 nM) for 1, 2, 8 and 16 h. Cells were stained with JC1 dye, and Δψwas measured

Curcumin enhances the apoptosis-inducing potential of TRAIL in prostate cancer cells: molecular mechanisms of apoptosis, migration and angiogenesis-0

<p><b>Copyright information:</b></p><p>Taken from "Curcumin enhances the apoptosis-inducing potential of TRAIL in prostate cancer cells: molecular mechanisms of apoptosis, migration and angiogenesis"</p><p>http://www.jmolecularsignaling.com/content/2/1/10</p><p>Journal of Molecular Signaling 2007;2():10-10.</p><p>Published online 4 Oct 2007</p><p>PMCID:PMC2082014.</p><p></p>h. Cell viability was measured at the end of 48 h by XTT assay. (B), LNCaP cells were treated with various concentrations of curcumin (0–30 μM) for 24 h, followed by treatment with TRAIL (50 nM) for another 24 h. Cell viability was measured at the end of 48 h by XTT assay. (C), PC-3 cells were seeded in soft agar and treated with curcumin (5–40 μM) in the presence or absence of TRAIL (25 nM). After three weeks, no of colonies were counted. Data represent mean ± SE. (D), LNCaP cells were seeded in soft agar and treated with curcumin (5–40 μM) in the presence or absence of TRAIL (50 nM). After three weeks, no of colonies were counted. Data represent mean ± SE. (E and F), Effects of dominant negative FADD on curcumin and/or TRAIL-induced apoptosis. PC-3 and LNCaP cells were transiently transfected with either control plasmid or plasmid expressing dominant negative FADD (DN-FADD) along with plasmid (pCMV-LacZ) encoding the β-galactosidase (β-Gal) enzyme. There was no difference in transfection efficiency among groups. Transfected cells were treated with curcumin (0, 10 or 20 μM) in the presence or absence of TRAIL (25 nM for PC-3 cells or 50 nM for LNCaP) for 48 h. Apoptosis was measured by DAPI staining. Data represent mean ± SE. * = significantly different from respective control; # and % = treatment groups were significantly different, P < 0.05

Involvement of reactive oxygen species in sensitization of TRAIL-resistant LNCaP cells by resveratrol

<p><b>Copyright information:</b></p><p>Taken from "Sensitization of TRAIL-resistant LNCaP cells by resveratrol (3, 4', 5 tri-hydroxystilbene): molecular mechanisms and therapeutic potential"</p><p>http://www.jmolecularsignaling.com/content/2/1/7</p><p>Journal of Molecular Signaling 2007;2():7-7.</p><p>Published online 24 Aug 2007</p><p>PMCID:PMC2018690.</p><p></p> (A), Generation of ROS by resveratrol. LNCaP cells were seeded in 96-well plates, loaded with 5 μM CM-HDCFDA dye for 30 min, and treated with either resveratrol (20 μM) or N-acetylcysteine (NAC) (50 mM) plus resveratrol (20 μM) for 0–360 min. Fluorescence was measured by a fluorometer as per manufacturer's instructions (EMD Biosciences/Molecular Probes). * = significantly different from respective controls, P < 0.05. (B), Inhibition of resveratrol-induced caspase-3 activity by NAC. LNCaP cells were pretreated with 50 mM NAC for 2 h followed by treatment with resveratrol (10, 20 or 30 μM) for 12 h, and caspase-3 activity was measured by a fluorometer as per manufacturer's instructions. * = significantly different from respective control; %, # or $ = significantly different from each other, P < 0.05. (C), Inhibition of resveratrol-induced apoptosis by NAC. LNCaP cells were pretreated with 50 mM NAC for 2 h followed by treatment with resveratrol (10, 20 or 30 μM) for 48 h, and apoptosis was measured by TUNEL assay. a, c and e were significantly different from b, d and f, respectively, P < 0.05. (D), Interactive effects of resveratrol and TRAIL on ROS production. LNCaP cells were pretreated with NAC (50 mM) for 2 h followed by treatment with resveratrol (20 μM) with or without TRAIL (50 nM) for 120 min., and ROS production was measured. * = significantly different from respective controls, P < 0.05; # or % = significantly different between groups, P < 0.05. (E), Interactive effects of resveratrol and TRAIL on caspase-3 activity. LNCaP cells were pretreated with 50 mM NAC for 2 h followed by treatment with resveratrol (20 μM) for 12 h, and caspase-3 activity was measured. * = significantly different from respective controls, P < 0.05; # or % = significantly different between groups, P < 0.05. (F), Interactive effects of resveratrol and TRAIL on apoptosis. LNCaP cells were pretreated with 50 mM NAC for 2 h followed by treatment with resveratrol (20 μM) with or without TRAIL (50 nM) for 48 h, and apoptosis was measured by TUNEL assay. * = significantly different from respective controls, P < 0.05; # = significantly different between groups, P < 0.05

Effects of resveratrol on the expressions of Bcl-2 family members and IAPs

<p><b>Copyright information:</b></p><p>Taken from "Sensitization of TRAIL-resistant LNCaP cells by resveratrol (3, 4', 5 tri-hydroxystilbene): molecular mechanisms and therapeutic potential"</p><p>http://www.jmolecularsignaling.com/content/2/1/7</p><p>Journal of Molecular Signaling 2007;2():7-7.</p><p>Published online 24 Aug 2007</p><p>PMCID:PMC2018690.</p><p></p> (A and B), Effects of resveratrol on protein expression of Bcl-2 family members. Cells were treated with resveratrol (0–20 μM) for 24 or 48 h. The expressions of Noxa, Bim, Bak, Bax, Bid, PUMA, Bcl-2, and Bcl-Xwere examined by Western blot analysis. Actin antibody was used as a loading control. Bim= Bim extra large, Bim= Bim large, Bim= Bim short. (C), Effects of resveratrol on the expressions of IAPs. Cells were treated with resveratrol (0–20 μM) for 24 or 48 h. Crude proteins were subjected to SDS-PAGE and immunoblotted with antibody specific for XIAP, survivin, cIAP1 or cIAP2. β-actin antibody was used as a loading control

Interactive effects of resveratrol and TRAIL on cell viability, colony formation and apoptosis in prostate cancer cells

<p><b>Copyright information:</b></p><p>Taken from "Sensitization of TRAIL-resistant LNCaP cells by resveratrol (3, 4', 5 tri-hydroxystilbene): molecular mechanisms and therapeutic potential"</p><p>http://www.jmolecularsignaling.com/content/2/1/7</p><p>Journal of Molecular Signaling 2007;2():7-7.</p><p>Published online 24 Aug 2007</p><p>PMCID:PMC2018690.</p><p></p> (A), Effects of resveratrol and/or TRAIL on cell viability in LNCaP cells. Cells were treated with various doses of resveratrol (0–30 μM) in the presence or absence of TRAIL (50 nM) for 48 h. Cell viability was measured by XTT assay as described in materials and methods. Data represent mean ± SD. * = Significantly different from respective control, P < 0.05. (B), Effects of resveratrol and/or TRAIL on apoptosis in human normal prostate epithelial cells (PrEC). PrEC were treated with resveratrol (20 μM) in the presence or absence of TRAIL (50 nM) for 48 h, and apoptosis was measured by TUNEL assay. (C), Effects of resveratrol and/or TRAIL on colony formation by LNCaP cells. Cells were treated with various doses of resveratrol (0–30 μM) in the presence or absence of TRAIL (50 nM). After three weeks, no of colonies were stained and counted. Data represent mean ± SD. * = Significantly different from respective control, P < 0.05. (D), Effects of different treatment combinations of resveratrol and TRAIL on apoptosis. LNCaP cells were treated with resveratrol (20 μM) in the presence or absence of TRAIL (50 nM). TRAIL was added simultaneously with resveratrol (cotreatment), before or after 24 h of resveratrol treatment. Apoptosis was measured by TUNEL assay. Data represent mean ± SD. * = Significantly different from respective control, P < 0.05

Curcumin enhances the apoptosis-inducing potential of TRAIL in prostate cancer cells: molecular mechanisms of apoptosis, migration and angiogenesis-7

<p><b>Copyright information:</b></p><p>Taken from "Curcumin enhances the apoptosis-inducing potential of TRAIL in prostate cancer cells: molecular mechanisms of apoptosis, migration and angiogenesis"</p><p>http://www.jmolecularsignaling.com/content/2/1/10</p><p>Journal of Molecular Signaling 2007;2():10-10.</p><p>Published online 4 Oct 2007</p><p>PMCID:PMC2082014.</p><p></p>h. Cell viability was measured at the end of 48 h by XTT assay. (B), LNCaP cells were treated with various concentrations of curcumin (0–30 μM) for 24 h, followed by treatment with TRAIL (50 nM) for another 24 h. Cell viability was measured at the end of 48 h by XTT assay. (C), PC-3 cells were seeded in soft agar and treated with curcumin (5–40 μM) in the presence or absence of TRAIL (25 nM). After three weeks, no of colonies were counted. Data represent mean ± SE. (D), LNCaP cells were seeded in soft agar and treated with curcumin (5–40 μM) in the presence or absence of TRAIL (50 nM). After three weeks, no of colonies were counted. Data represent mean ± SE. (E and F), Effects of dominant negative FADD on curcumin and/or TRAIL-induced apoptosis. PC-3 and LNCaP cells were transiently transfected with either control plasmid or plasmid expressing dominant negative FADD (DN-FADD) along with plasmid (pCMV-LacZ) encoding the β-galactosidase (β-Gal) enzyme. There was no difference in transfection efficiency among groups. Transfected cells were treated with curcumin (0, 10 or 20 μM) in the presence or absence of TRAIL (25 nM for PC-3 cells or 50 nM for LNCaP) for 48 h. Apoptosis was measured by DAPI staining. Data represent mean ± SE. * = significantly different from respective control; # and % = treatment groups were significantly different, P < 0.05

Curcumin enhances the apoptosis-inducing potential of TRAIL in prostate cancer cells: molecular mechanisms of apoptosis, migration and angiogenesis-6

<p><b>Copyright information:</b></p><p>Taken from "Curcumin enhances the apoptosis-inducing potential of TRAIL in prostate cancer cells: molecular mechanisms of apoptosis, migration and angiogenesis"</p><p>http://www.jmolecularsignaling.com/content/2/1/10</p><p>Journal of Molecular Signaling 2007;2():10-10.</p><p>Published online 4 Oct 2007</p><p>PMCID:PMC2082014.</p><p></p>unted under a microscope. Data represent mean ± SD. * = significantly different from control, P < 0.05. (B), HUVECs were seeded in 24-well plates containing matrigel. Cells were pretreated with ERK inhibitor (10 μM) for 3 h, followed by treatment with curcumin (40 μM) for 24 h. Capillary tubes were counted under a microscope. Data represent mean ± SD. * = significantly different from control, P < 0.05. (C), Picture of capillary tube formation. HUVECs were treated as described in B. Pictures of capillary tubes were taken by a microscope. (D), HUVECs were treated with various concentrations of curcumin (20, 40 and 60 μM) or DMSO (control). Migration of HUVEC cells through the membrane was determined after 24 h of incubation at 37°C using Transwell Boyden chamber. Cells that had migrated to the lower chamber were fixed with 90% ethanol, stained with hematoxylin and eosin, quantified by counting the number of cells under a microscope. Data represent mean ± SD. * = significantly different from control, P < 0.05. (E), HUVECs were pretreated with ERK inhibitor (10 μM) for 3 h, followed by treatment with curcumin (40 μM) or DMSO (control) for 24 h at 37°C. Cells migrated to the lower chamber were fixed, stained and quantified