Curcumin sensitizes TRAIL-resistant xenografts: molecular mechanisms of apoptosis, metastasis and angiogenesis

<p>Abstract</p> <p>Background</p> <p>We have recently shown that curcumin (a diferuloylmethane, the yellow pigment in turmeric) enhances apoptosis-inducing potential of TRAIL in prostate cancer PC-3 cells, and sensitizes TRAIL-resistant LNCaP cells <it>in vitro </it>through multiple mechanisms. The objectives of this study were to investigate the molecular mechanisms by which curcumin sensitized TRAIL-resistant LNCaP xenografts <it>in vivo</it>.</p> <p>Methods</p> <p>Prostate cancer TRAIL-resistant LNCaP cells were implanted in Balb c nude mice to examine the effects of curcumin and/or TRAIL on tumor growth and genes related to apoptosis, metastasis and angiogenesis.</p> <p>Results</p> <p>Curcumin inhibited growth of LNCaP xenografts in nude mice by inducing apoptosis (TUNEL staining) and inhibiting proliferation (PCNA and Ki67 staining), and sensitized these tumors to undergo apoptosis by TRAIL. In xenogrfated tumors, curcumin upregulated the expression of TRAIL-R1/DR4, TRAIL-R2/DR5, Bax, Bak, p21^/WAF1, and p27^/KIP1, and inhibited the activation of NFκB and its gene products such as cyclin D1, VEGF, uPA, MMP-2, MMP-9, Bcl-2 and Bcl-X_L. The regulation of death receptors and members of Bcl-2 family, and inactivation of NFκB may sensitize TRAIL-resistant LNCaP xenografts. Curcumin also inhibited number of blood vessels in tumors, and circulating endothelial growth factor receptor 2-positive endothelial cells in mice.</p> <p>Conclusion</p> <p>The ability of curcumin to inhibit tumor growth, metastasis and angiogenesis, and enhance the therapeutic potential of TRAIL suggests that curcumin alone or in combination with TRAIL can be used for prostate cancer prevention and/or therapy.</p

Resveratrol Induces Growth Arrest and Apoptosis through Activation of FOXO Transcription Factors in Prostate Cancer Cells

Resveratrol, a naturally occurring phytopolyphenol compound, has attracted extensive interest in recent years because of its diverse pharmacological characteristics. Although resveratrol possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. The present study was carried out to examine whether PI3K/AKT/FOXO pathway mediates the biological effects of resveratrol.Resveratrol inhibited the phosphorylation of PI3K, AKT and mTOR. Resveratrol, PI3K inhibitors (LY294002 and Wortmannin) and AKT inhibitor alone slightly induced apoptosis in LNCaP cells. These inhibitors further enhanced the apoptosis-inducing potential of resveratrol. Overexpression of wild-type PTEN slightly induced apoptosis. Wild type PTEN and PTEN-G129E enhanced resveratrol-induced apoptosis, whereas PTEN-G129R had no effect on proapoptotic effects of resveratrol. Furthermore, apoptosis-inducing potential of resveratrol was enhanced by dominant negative AKT, and inhibited by wild-type AKT and constitutively active AKT. Resveratrol has no effect on the expression of FKHR, FKHRL1 and AFX genes. The inhibition of FOXO phosphorylation by resveratrol resulted in its nuclear translocation, DNA binding and transcriptional activity. The inhibition of PI3K/AKT pathway induced FOXO transcriptional activity resulting in induction of Bim, TRAIL, p27/KIP1, DR4 and DR5, and inhibition of cyclin D1. Similarly, resveratrol-induced FOXO transcriptional activity was further enhanced when activation of PI3K/AKT pathway was blocked. Over-expression of phosphorylation deficient mutants of FOXO proteins (FOXO1-TM, FOXO3A-TM and FOXO4-TM) induced FOXO transcriptional activity, which was further enhanced by resveratrol. Inhibition of FOXO transcription factors by shRNA blocked resveratrol-induced upregulation of Bim, TRAIL, DR4, DR5, p27/KIP1 and apoptosis, and inhibition of cyclin D1 by resveratrol.These data suggest that FOXO transcription factors mediate anti-proliferative and pro-apoptotic effects of resveratrol, in part due to activation of extrinsic apoptosis pathway

Resveratrol Enhances Antitumor Activity of TRAIL in Prostate Cancer Xenografts through Activation of FOXO Transcription Factor

Resveratrol (3, 4', 5 tri-hydroxystilbene), a naturally occurring polyphenol, exhibits anti-inflammatory, antioxidant, cardioprotective and antitumor activities. We have recently shown that resveratrol can enhance the apoptosis-inducing potential of TRAIL in prostate cancer cells through multiple mechanisms in vitro. Therefore, the present study was designed to validate whether resveratrol can enhance the apoptosis-inducing potential of TRAIL in a xenograft model of prostate cancer.Resveratrol and TRAIL alone inhibited growth of PC-3 xenografts in nude mice by inhibiting tumor cell proliferation (PCNA and Ki67 staining) and inducing apoptosis (TUNEL staining). The combination of resveratrol and TRAIL was more effective in inhibiting tumor growth than single agent alone. In xenografted tumors, resveratrol upregulated the expressions of TRAIL-R1/DR4, TRAIL-R2/DR5, Bax and p27(/KIP1), and inhibited the expression of Bcl-2 and cyclin D1. Treatment of mice with resveratrol and TRAIL alone inhibited angiogenesis (as demonstrated by reduced number of blood vessels, and VEGF and VEGFR2 positive cells) and markers of metastasis (MMP-2 and MMP-9). The combination of resveratrol with TRAIL further inhibited number of blood vessels in tumors, and circulating endothelial growth factor receptor 2-positive endothelial cells than single agent alone. Furthermore, resveratrol inhibited the cytoplasmic phosphorylation of FKHRL1 resulting in its enhanced activation as demonstrated by increased DNA binding activity.These data suggest that resveratrol can enhance the apoptosis-inducing potential of TRAIL by activating FKHRL1 and its target genes. The ability of resveratrol to inhibit tumor growth, metastasis and angiogenesis, and enhance the therapeutic potential of TRAIL suggests that resveratrol alone or in combination with TRAIL can be used for the management of prostate cancer

Anticonvulsant and Sedative-Hypnotic Activities of N-Acetyl / Methyl Isatin Derivatives

A series of N-methyl/acetyl 5-(un)-substituted isatin-3-semicarbazones were screened for anticonvulsant and sedative-hypnotic activities. The results revealed that protection was obtained in all the screens i.e., Maximal electroshock, (MES) subcutaneous pentylene tetrazole (scPTZ) and subcutaneous strychnine (scSTY) screens. Three compounds (2a,2e and 2i) possessed anti-MES activity and all the compounds were less neurotoxic than phenytoin, carbamazepine and phenobarbital. All the compounds were completely non-toxic at 4h when compared to phenytoin, carbamazepine and phenobarbital, which were toxic at 100 and 300 mg/kg respectively. Compounds 2a, 2b, 2e, 2g and 2i emerged as the active compounds in oral MES screen. Selected compounds were evaluated for quantification studies in MES, scPTZ and neurotoxicity screens after i. p (2b, 2i) and oral administration (2a, 2g) in rats. Among all the compounds 2a, 2b and 2g emerged as broad-spectrum compounds as indicated by their protection in MES, scSTY and scPTZ screens. All the compounds except compound 2b showed significant sedative-hypnotic activity

UXT, a novel MDMX-binding protein, promotes glycolysis by mitigating p53-mediated restriction of NF-κB activity

The importance of stress-induced p53 activation has been extensively investigated and well established. How the basal activity of p53 prevents carcinogenesis, however, remains incompletely understood. We report the identification of a novel p53 inhibitor, UXT, which binds to MDMX and suppresses the basal activity of p53. Interestingly, human TCGA database indicates that the UXT gene is frequently amplified in human sarcoma where p53 mutation is rare. We thus used sarcoma as a model to show that UXT acts as an oncogene promoting cell proliferation in vitro and tumor progression in vivo. A screening of 10 major cellular pathways uncovered that UXT-mediated p53 inhibition results in an activation of NF-κB, leading to induction of glycolysis. While elevated glycolytic metabolism provides growth advantage it also renders UXT expressing sarcoma cells heightened sensitivity to glycolysis inhibition. Altogether, our data demonstrate a crucial role for the basal activity of p53 in restriction of NF-κB. By impeding such an activity of p53, UXT unleashes the oncogenic activity of NF-κB resulting in induction of glycolysis fueling carcinogenesis

Immunohistochemical examination of Bcl-2 family members and death receptors

<p><b>Copyright information:</b></p><p>Taken from "Curcumin sensitizes TRAIL-resistant xenografts: molecular mechanisms of apoptosis, metastasis and angiogenesis"</p><p>http://www.molecular-cancer.com/content/7/1/16</p><p>Molecular Cancer 2008;7():16-16.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2249593.</p><p></p> Immunohistochemistry was performed to measure the expression of Bak, Bax, Bcl-2, Bcl-X, TRAIL-R1/DR4 and TRAIL-R2/DR5 in tumor tissues derived from control and/or treated mice on week 6

Effects of curcumin and/TRAIL on markers of phospho-p65NFκB, Cox-2, and IL-8

<p><b>Copyright information:</b></p><p>Taken from "Curcumin sensitizes TRAIL-resistant xenografts: molecular mechanisms of apoptosis, metastasis and angiogenesis"</p><p>http://www.molecular-cancer.com/content/7/1/16</p><p>Molecular Cancer 2008;7():16-16.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2249593.</p><p></p> (A), Immunohistochemistry was performed to measure the expression of phospho-p65NFκB, Cox-2 and IL-8 in tumor tissues derived from control and/or treated mice on week 6. (B), Expression of phospho-p65NFκB, Cox-2, IL-8 and β-actin in tumor tissues derived on week 6 were measured by the Western blot analysis. (C), NFκB-DNA binding activity. Nuclear extracts were prepared from tumor tissues derived from different treatment groups on week 6. NFκB-DNA binding activity was measured by Gelshift assay as described in Materials and Methods. The relative nuclear NFκB-DNA binding activities were quantified by scanning densitometry

Effects of FOXO transcription factors on the regulation of antiproliferative and proapoptotic effects of resveratrol.

<p>(A), Inhibition of FOXO transcription factor by shRNA blocks anti-proliferative effects of resveratrol. LNCaP cells were transiently transfected with plasmids expressing FKHR shRNA, FKHRL1 shRNA, AFX shRNA or respective scrambled control and treated with resveratrol (20 µM) for 48 h, and cell viability was measured. (B), Inhibition of FOXO transcription factors or ROS (reactive oxygen species) by NAC blocks resveratrol-induced apoptosis. LNCaP cells were transfected with a mixture of plasmids expressing FKHR shRNA, FKHRL1 shRNA plus AFX shRNA or scrambled control. After transfection, the culture medium was changed and cells were pretreated with NAC for 2 h, and treated with resveratrol (20 µM) for 48 h. At the end of incubation period, the apoptosis was measured by TUNEL assay. (C), Inhibition of FOXO transcription factors or ROS by NAC blocks resveratrol-induced caspase-3 activity. LNCaP cells were transfected with a mixture of plasmids expressing FKHR shRNA, FKHRL1 shRNA plus AFX shRNA or scrambled control. After transfection, the culture medium was changed and cells were pretreated with NAC for 2 h, and treated with resveratrol (20 µM) for 48 h. At the end of incubation period, the caspase-3 activity was measured as per manufacturer instructions.</p

Activation of transcription factor FKHRL1 by resveratrol.

<p>(A), Inhibition of FKHRL1 phosphorylation by resveratrol. Western blot analysis was performed to measure the expression of phospho-FKHRL1 in tumor tissues derived from control and/or drug treated mice on week 6. (B), Immunohistochemical examination of phospho-FKHRL1. Immunohistochemistry was performed to measure the expression of phospho-FKHRL1 in tumor tissues derived from control, resveratrol and/or TRAIL treated mice on week 6. (C), Quantification of FKHRL-positive cells in tumor tissues. (D), FKHRL1-DNA binding activity. Nuclear extracts were prepared from tumor tissues derived from control and drug-treated mice. Gelshift assay was performed to measure the FKHRL1-DNA binding activity as described in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015627#s4" target="_blank">Methods</a>.</p