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ABSTRACT: The present study was designed to investigate (i) the role of AMPK activation in inducing autophagy in androgen-dependent prostate cancer cells subjected to androgen deprivation and hypoxia, and (ii) whether autophagy offers a survival advantage under these harsh conditions. Low androgen and low oxygen are two co-existing conditions frequently found in prostate cancer tissue following surgical or medical castration. In LNCaP cells, androgen deprivation and hypoxia together boosted AMPK activation to a higher level than that seen with either condition alone. The augmented AMPK response was associated with improved viability and the induction of autophagy. These observations suggest that a threshold of AMPK activity has to be attained in order to trigger autophagy, since neither androgen deprivation nor hypoxia by itself was capable of pushing AMPK activity past that threshold. Beclin-1 was identified as a potential downstream target of AMPK in turning on the autophagic cascade. If autophagy was blocked by chemical inhibition or RNA interference of key regulators, e.g., AMPK or beclin-1, more cells would die by apoptosis. The occurrence of autophagy is thus a survival mechanism for androgen-dependent prostate cancer cells to escape from an androgen-deprived and hypoxic subsistence.
Cellular signalling 09/2011; 23(9):1466-72. · 4.09 Impact Factor
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Li Tang,
Song Yao,
Cathee Till,
Phyllis J Goodman,
Catherine M Tangen, Yue Wu,
Alan R Kristal,
Elizabeth A Platz,
Marian L Neuhouser,
Frank Z Stanczyk,
Juergen K V Reichardt,
Regina M Santella,
Ann Hsing,
Ashraful Hoque,
Scott M Lippman,
Ian M Thompson,
Christine B Ambrosone
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ABSTRACT: The etiology of prostate cancer remains elusive, although steroid hormones probably play a role. Considering the carcinogenic potential of estrogen metabolites as well as altered intraprostatic estrogen biosynthesis during the development of prostate cancer, we investigated associations between repeat polymorphisms of three key estrogen-related genes (CYP11A1, CYP19A1, UGT1A1) and risk of prostate cancer in the Prostate Cancer Prevention Trial (PCPT), designed to test finasteride versus placebo as a chemoprevention agent. Using data and specimens from 1154 cases and 1351 controls who were frequency matched on age, family history of prostate cancer and PCPT treatment arm, we used logistic regression to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs) separately in the placebo and finasteride arms. Among men in the placebo arm, CYP19A1 7/8 genotype carriers had a significantly higher risk of prostate cancer compared with those with the 7/7 genotype (OR = 1.70, 95% CI = 1.16-2.5), regardless of Gleason grade. This genotype was also associated with elevated serum estrogen levels. For the (TA)(n) repeat polymorphism in UGT1A1, the heterozygous short (<7 repeats)/long (≥7 repeats) genotype was significantly associated with the risk of low-grade prostate cancer (OR = 1.34, 95% CI = 1.05-1.70) compared with the short/short genotype. No significant association was found with CYP11A1. These associations were not observed among men in the finasteride arm. The results indicate that repeat polymorphisms in genes involved in estrogen biosynthesis and metabolism may influence risk of prostate cancer but that their effects may be modified by factors altering hormone metabolism, such as finasteride treatment.
Carcinogenesis 07/2011; 32(10):1500-6. · 5.70 Impact Factor
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ABSTRACT: Finasteride is known to inhibit Type 2 5α-reductase and thus block the conversion of testosterone to dihydrotestosterone (DHT). The structural similarity of finasteride to DHT raises the possibility that finasteride may also interfere with the function of the androgen receptor (AR). Experiments were carried out to evaluate the antiandrogenic effect of finasteride in LNCaP, C4-2 and VCaP human prostate cancer cells. Finasteride decreased DHT binding to AR, and DHT-stimulated AR activity and cell growth in LNCaP and C4-2 cells, but not in VCaP cells. LNCaP and C4-2 (derived from castration-resistant LNCaP) cells express the T877A mutant AR, while VCaP cells express the wild type AR. When PC-3 cells, which are AR-null, were transfected with either the wild type or the T877A mutant AR, only the mutant AR-expressing cells were sensitive to finasteride inhibition of DHT binding. Peroxiredoxin-1 (Prx1) is a novel endogenous facilitator of AR binding to DHT. In Prx1-rich LNCaP cells, the combination of Prx1 knockdown and finasteride was found to produce a greater inhibitory effect on AR activity and cell growth than either treatment alone. The observation suggests that cells with a low expression of Prx1 are likely to be more responsive to the antiandrogenic effect of finasteride. Additional studies showed that the efficacy of finasteride was comparable to that of bicalutamide (a widely used non-steroidal antiandrogen). The implication of the above findings is discussed in the context of developing strategies to improve the outcome of androgen deprivation therapy.
Cancer biology & therapy 05/2011; 11(10):902-9. · 2.64 Impact Factor
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ABSTRACT: Signaling between androgen receptor (AR) and mTOR may be crucial for prostate cancer cells to endure the low androgen and suboptimal nutrient conditions produced by androgen deprivation therapy.
AR and mTOR cross-talk was examined in LNCaP cells exposed to either high or low testosterone. AR and mTOR activities were modified separately using either siRNA knockdown or specific chemical inhibitor. The biological significance of the reciprocal communication was assessed by susceptibility to glucose deprivation-induced cell death.
AR positively regulated mTOR activity in both low and high testosterone levels. TSC1 and TSC2, the two negative regulators of mTOR, may be involved since both were up-regulated by AR knockdown. Sub-baseline mTOR increased AR protein levels. However, this effect only occurred with low testosterone. More cells underwent apoptosis if AR function was inhibited during glucose deprivation, which significantly depressed mTOR activity.
The compensatory increase of AR function due to a repressed mTOR signal is advantageous for survival. Disrupting this loop at the time of initiation of androgen deprivation therapy may delay, or even prevent, the recurrence of prostate cancer.
Anticancer research 10/2010; 30(10):3895-901. · 1.73 Impact Factor
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ABSTRACT: Androgen-independent prostate cancer usually develops as a relapse following androgen ablation therapy. Removing androgen systemically causes vascular degeneration and nutrient depletion of the prostate tumor tissue. The fact that the malignancy later evolves to androgen-independence suggests that some cancer cells are able to survive the challenge of energy/nutrient deprivation. AMP-activated protein kinase (AMPK) is an important manager of energy stress. The present study was designed to investigate the role of AMPK in contributing to the survival of the androgen-independent phenotype. Most of the experiments were carried out in the androgen-dependent LNCaP cells and the androgen-independent C4-2 cells. These two cell lines have the same genetic background, since the C4-2 line is derived from the LNCaP line. Glucose deprivation (GD) was instituted to model energy stress encountered by these cells. The key findings are as follows. First, the activation of AMPK by GD was much stronger in C4-2 cells than in LNCaP cells, and the robustness of AMPK activation was correlated favorably with cell viability. Second, the response of AMPK was specific to energy deficiency rather than to amino acid deficiency. The activation of AMPK by GD was functional, as demonstrated by appropriate phosphorylation changes of mTOR and mTOR downstream substrates. Third, blocking AMPK activation by chemical inhibitor or dominant negative AMPK led to increased apoptotic cell death. The observation that similar results were found in other androgen-independent prostate cancer cell lines, including CW22Rv1 abd VCaP, provided further assurance that AMPK is a facilitator on the road to androgen-independence of prostate cancer cells.
Cellular signalling 10/2010; 22(10):1554-61. · 4.09 Impact Factor
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ABSTRACT: Previous studies have demonstrated that physiological concentrations of methylseleninic acid (MSA) inhibits the growth of prostate cancer cells. The growth inhibitory effect could be attributed to cell cycle block and apoptosis induction. The current study was designed to investigate the involvement of forkhead box O1 (FOXO1) in the anticancer effect of MSA.
LNCaP and LAPC-4 cells were treated with 10 microM MSA for various time points, and the expression of FOXO1 was analyzed by qRT-PCR and Western blotting. FOXO1 activity was determined by a luciferase construct containing FOXO binding sites. The trans-activation activity of the androgen receptor (AR) was determined by the ARE-luciferase assay. FOXO1 gene silencing was achieved by using a small interfering RNA (siRNA).
MSA treatment led to a rapid and robust increase of FOXO1 expression, as well as an increase of the FOXO1 transcriptional activity. Blocking FOXO1 activation by gene silencing abolished apoptosis induction by MSA, suggesting FOXO1 plays a critical role in mediating the apoptotic effect of MSA. Recent studies have shown that FOXO1 and AR antagonize the actions of each other. We examined the consequence of FOXO1 induction on AR activity. Consistent with previous reports, we found that ectopic expression of FOXO1 suppressed the transcriptional activity of AR. Furthermore, FOXO1 silencing attenuated MSA suppression of AR activity, suggesting that FOXO1 induction contributes to suppression of AR signaling by MSA.
In prostate cancer cells, MSA activates the FOXO1 signaling pathway. FOXO1 activation is critical for the anticancer effects of MSA.
The Prostate 09/2010; 70(12):1265-73. · 3.48 Impact Factor
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ABSTRACT: The progression of prostate cancer to the castration-recurrent phenotype remains a major problem medically. The present study examined the transcriptomics of de novo androgen synthesis as a potential mechanism to escape from dependence on circulating androgen. VCaP, LNCaP and LAPC4 cells were acclimated to 1 nM testosterone for five generations before subjecting them to a reduced level of 0.03 nM testosterone. Changes in gene expression were quantified using qRT-PCR. Analyses of the cholesterol biosynthesis pathway and the Δ4, Δ5 and backdoor steroidogenic pathways were carried out. VCaP cells showed no change in the transcriptome of cholesterol biosynthesis. However, several receptors for cholesterol transport were upregulated. The Δ4 and Δ5 steroidogenic pathways, but not the backdoor pathway, were stimulated. Additionally, androgen receptor (AR) expression was increased. Taken together, the above changes might allow recovery of AR activity to a near normal level. In contrast, LNCaP cells showed only minimal adjustment in the transcriptome of steroidogenesis. LAPC4 cells were equally unresponsive to boosting the machinery of androgen production. In brief, our results suggest that the VCaP model is an appropriate model for further investigation of targeting the androgen-AR axis to block the emergence of castration-resistant prostate cancer.
Cancer biology & therapy 06/2010; 9(12):1033-42. · 2.64 Impact Factor
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ABSTRACT: Androgen receptor (AR) signaling plays a critical role in the development and progression of prostate cancer. It has been reported previously that peroxiredoxin-1 (Prx1), a member of a novel family of peroxidases, interacts physically with AR to enhance AR transactivation of target genes. In the present study, we evaluated the biological significance of Prx1 in modulating dihydrotestosterone (DHT)-stimulated growth and AR target gene expression of prostate cancer cells. We also investigated the mechanism by which Prx1 might potentiate AR signaling. The contribution of Prx1 was assessed mainly by using the approach of stable Prx1 knockdown. The major observations are as follows: (a) A low level of Prx1 desensitizes cells to growth stimulation and AR target gene induction by DHT, such that exposure to a higher level of DHT is required to reach the same magnitude of response when Prx1 is depressed; (b) Prx1 increases the affinity of AR to DHT and decreases the rate of DHT dissociation from the occupied receptor; (c) Prx1 enhances the NH2 terminus and COOH terminus interaction of AR; a stronger N-C interaction is consistent with a more robust AR activation signal by keeping DHT tight in the ligand-binding pocket; (d) the stimulatory effects of Prx1 on AR ligand binding affinity and AR N-C interaction are manifested regardless of a wild-type or mutant AR. The above findings led us to believe that Prx1 may be a therapeutic target in blocking the transition of prostate cancer from an androgen-dependent to an androgen-refractory phenotype.
Molecular Cancer Research 10/2009; 7(9):1543-52. · 4.29 Impact Factor
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ABSTRACT: Taxanes are first line drugs for treating prostate cancer recurrence after the failure of anti-androgen therapy. There is a need to make taxanes more effective since they only provide palliative benefit. Exploiting endoplasmic reticulum (ER) stress death signaling to enhance drug efficacy has not been delineated. Human PC-3 cells were used as a model of hormone refractory prostate cancer. Thapsigargin and methylseleninic acid (MSA) were examined as sensitizers. Thapsigargin is a classic ER stress inducer. The activity of MSA in inducing ER stress has recently been studied by our group. The efficacy of single drug and the various combinations was evaluated by measuring apoptosis with a cell death ELISA kit. Thapsigargin increased the cell killing potency of paclitaxel or docetaxel by 10- to 12-fold, while MSA caused a 5- to 8-fold increase. Since thapsigargin is not used clinically because of its toxicity, the follow-up experiments were done with MSA. To test the hypothesis that a threshold level of ER stress is crucial to chemotherapeutic sensitization, three different approaches designed to dampen the severity of ER stress induced by MSA were examined. Lowering ER stress consistently attenuated the efficacy of MSA/taxane. GADD153 is a pro-apoptotic transcription factor which is upregulated during ER stress. Knocking down GADD153 by siRNA also reduced the cell killing effect of MSA/taxane. Both the intrinsic and extrinsic apoptotic pathways were involved in the sensitization mechanism. Our study supports the idea that marshalling ER stress apoptotic response is conducive to chemotherapeutic sensitization.
Cancer biology & therapy 02/2009; 8(2):146-52. · 2.64 Impact Factor
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ABSTRACT: Conjugated linoleic acid (CLA) inhibits rat mammary carcinogenesis, in part by inducing apoptosis of preneoplastic and neoplastic mammary epithelial cells. The current study focused on the mechanism by which apoptosis is induced. In TM4t mammary tumor cells, trans-10,cis-12 (t10,c12)-CLA induced proapoptotic C/EBP-homologous protein (CHOP) concurrent with the cleavage of poly(ADP-ribose) polymerase. Knockdown of CHOP attenuated t10,c12-CLA-induced apoptosis. Furthermore, t10,c12-CLA induced the cleavage of endoplasmic reticulum (ER)-resident caspase-12, and a selective inhibitor of caspase-12 significantly alleviated t10,c12-CLA-induced apoptosis. Using electron microscopy, we observed that t10,c12-CLA treatment resulted in marked dilatation of the ER lumen. Together, these data suggest that t10,c12-CLA induces apoptosis through ER stress. To further explore the ER stress pathway, we examined the expression of the following upstream ER stress signature markers in response to CLA treatment: X-box binding protein 1 (XBP1) mRNA (unspliced and spliced), phospho-eukaryotic initiation factor (eIF) 2 alpha, activating transcription factor 4 (ATF4), and BiP proteins. We found that t10,c12-CLA induced the expression and splicing of XBP1 mRNA as well as the phosphorylation of eIF2 alpha. In contrast, ATF4 was induced modestly, but not significantly, and BiP was not altered. In summary, our data demonstrate that apoptosis induced by t10,c12-CLA is mediated, at least in part, through an atypical ER stress response that culminates in the induction of CHOP and the cleavage of caspase-12.
The Journal of Lipid Research 06/2008; 49(5):985-94. · 5.56 Impact Factor
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ABSTRACT: Previous reports showed that alpha-tocopheryl succinate (alphaTS) and methylseleninic acid (MSA) independently reduce the abundance of androgen receptor (AR) in prostate cancer cells. The response to MSA happens quickly, whereas the response to alphaTS takes much longer. The present study was designed to investigate whether a combination of alphaTS and MSA would produce an additive or a greater than additive effect in suppressing AR level, AR transactivation, and prostate-specific antigen (PSA).
LNCaP cells were treated with alphaTS alone for 31 hours, MSA alone for 3 hours, or alphaTS first for 28 hours and alphaTS/MSA together for the last 3 hours. AR and PSA mRNA levels were quantitated by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). AR transactivation was determined by the ARE-luciferase reporter assay. Both cellular and secretory PSA was also measured by the enzyme-linked immunosorbent assay (ELISA) method.
Different doses of alphaTS were evaluated in combination with MSA. Some striking results are highlighted below for alphaTS alone, MSA alone, or alphaTS/MSA (presented in that order). AR mRNA level was depressed by 0%, 20%, or 60%, respectively; AR transactivation was inhibited by 35%, 10%, or 60%, respectively; whereas the PSA mRNA level was decreased by 40%, 60%, or 90%, respectively. Interestingly, secretory PSA was consistently reduced to a greater extent than cellular PSA.
A combination of alphaTS/MSA produced a greater than additive effect in suppressing AR signaling compared with the single agent. Decreased AR abundance is a major factor, but not necessarily the sole factor, in diminishing the transcriptional activity of AR by alphaTS or MSA.
Cancer 01/2007; 107(12):2942-8. · 4.77 Impact Factor
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ABSTRACT: The up-regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is prevalent in many cancers. This phenomenon makes PI3K and Akt fruitful targets for cancer therapy and/or prevention because they are mediators of cell survival signaling. Although the suppression of phospho-Akt by selenium has been reported previously, little information is available on whether selenium modulates primarily the PI3K-phosphoinositide-dependent kinase 1 (PDK1) side of Akt phosphorylation or the phosphatase side of Akt dephosphorylation. The present study was aimed at addressing these questions in PC-3 prostate cancer cells which are phosphatase and tensin homologue-null. Our results showed that selenium decreased Akt phosphorylation at Thr308 (by PDK1) and Ser473 (by an unidentified kinase); the Thr308 site was more sensitive to selenium inhibition than the Ser473 site. The protein levels of PI3K and phospho-PDK1 were not affected by selenium. However, the activity of PI3K was reduced by 30% in selenium-treated cells, thus discouraging the recruitment of PDK1 and Akt to the membrane due to low phosphatidylinositol-3,4,5-trisphosphate formation by PI3K. Consistent with the above interpretation, the membrane localization of PDK1 and Akt was significantly diminished as shown by Western blotting. In the presence of a calcium chelator or a specific inhibitor of calcineurin (a calcium-dependent phosphatase), the suppressive effect of selenium on phospho-Akt(Ser473) was greatly reduced. The finding suggests that selenium-mediated dephosphorylation of Akt via calcineurin is likely to be an additional mechanism in regulating the status of phospho-Akt.
Molecular Cancer Therapeutics 03/2006; 5(2):246-52. · 5.23 Impact Factor
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ABSTRACT: A monomethylated selenium metabolite called methylseleninic acid (MSA) has recently been shown to cause global thiol redox
modification of proteins. These changes represent a form of cellular stress due to protein misfolding or unfolding. When this
occurs in the endoplasmic reticulum (ER), a process known as unfolded protein response (UPR) is orchestrated to repair the
damage or commit the cells to apoptosis if the rescue effort becomes inadequate. Treatment of PC-3 human prostate cancer cells
with MSA leads to activation of three signature ER stress transducers and increased expression of UPR target genes, as exemplified
by GRP78 and GADD153. GRP78 is part of the damage control mechanism, while GADD153 is a transcription factor associated with
growth arrest and apoptosis. Transfection with GRP78 largely negates the apoptotic effect of MSA and GADD 153 induction because
an abundance of GRP78 allows cells to cope better with ER stress. Conversely, knocking down GRP78 by siRNA magnifies the cell
growth arrest effect of MSA and GADD153 expression. Collectively, the research findings support the idea that UPR plays an
important role in mediating the anticancer activities of selenium.
12/2005: pages 265-276;
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ABSTRACT: A monomethylated selenium metabolite, called methylseleninic acid (MSA), has recently been shown to cause global thiol redox modification of proteins. These changes represent a form of cellular stress due to protein misfolding or unfolding. An accumulation of aberrantly folded proteins in the endoplasmic reticulum (ER) triggers a defined set of transducers to correct the defects or commit the cells to apoptosis if the rescue effort is exhausted. Treatment of PC-3 human prostate cancer cells with MSA was found to induce a number of signature ER stress markers: (a) the survival/rescue molecules such as phosphorylated protein kinase-like ER-resident kinase (phospho-PERK), phosphorylated eukaryotic initiation factor-2alpha (phospho-eIF2alpha), glucose-regulated protein (GRP)-78, and GRP94; and (b) the apoptotic molecules such as caspase-12, caspase-7, and CAAT/enhancer binding protein homologous protein or growth arrest DNA damage-inducible gene 153 (CHOP/GADD153). Additional evidence suggested that CHOP/GADD153 might be an important transcription factor in apoptosis induction by MSA. In general, a higher concentration of MSA was required to elicit the apoptotic markers compared with the rescue markers. The apoptotic markers increased proportionally with the dose of MSA, whereas the rescue markers failed to keep pace with the increasing challenge from MSA. GRP78 is the rheostat of the ER stress transducers. In GRP78-overexpressing cells, the ability of MSA to up-regulate phospho-PERK, phospho-eIF2alpha, GRP94, caspase-12, caspase-7, and CHOP/GADD153 was significantly muted. A generous supply of GRP78 would allow cells to cope better with ER stress, thereby improving the odds for survival and negating the commitment to apoptotic death. The present study thus provides strong evidence to support an important role of ER stress response in mediating the anticancer effect of selenium.
Cancer Research 11/2005; 65(19):9073-9. · 7.86 Impact Factor
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ABSTRACT: alpha-Tocopherol and its synthetic derivative, a-tocopheryl succinate (alpha-TS), are known to inhibit proliferation of cancer cells. alpha-TS is considered a more desirable anticancer agent because of the ability to induce apoptosis. It has been established previously that the whole intact alpha-TS molecule is necessary for its pro-apoptotic activity. For this reason, alpha-TS is not suitable for oral use because the ester bond linking succinate to tocopherol is subject to hydrolysis by intestinal esterases. One approach to overcome this problem is to replace the ester bond with an ether bond, since the latter is resistant to esterase-mediated hydrolysis. alpha-Tocopheryloxybutyrate (alpha-TOB) is the ether analog of alpha-TS. In this study, we compared the potency of alpha-TS and alpha-TOB using a panel of bioassays: cell growth, TUNEL labelling for apoptosis, PARP cleavage, caspase-3 and caspase-9 activation, as well as Akt and JNK phosphorylation. The experiments were carried out in two human prostate cancer cell lines: LNCaP and PC-3. Our results showed that alpha-TOB was capable of inhibiting cell growth and inducing apoptosis, although alpha-TOB was less active than alpha-TS on an equimolar basis. In general, it took twice as much alpha-TOB as alpha-TS to achieve the same response. Nonetheless, these two compounds shared the same mechanism of targeting the Akt and JNK signaling pathways, and activating the intrinsic cell death mediators of caspase-9 and caspase-3. Cellular analysis of alpha-TS and alpha-TOB showed that alpha-TOB was taken up as efficiently as alpha-TS (if not more so), suggesting that the lower activity of alpha-TOB is an inherent property of the molecule and not due to impaired uptake. Additional evidence is provided to show that beta-TS may act at the membrane level to interfere with Akt phosphorylation, although the exact nature of this disruption remains unclear. The future design of new anticancer tocopherol analogs should incorporate the ether linkage of the side chain for esterase resistance as well as other structural modifications for enhanced blocking of membrane signaling.
Anticancer research 24(6):3795-802. · 1.73 Impact Factor
