20(S)-protopanaxadiol-aglycone downregulation of the full-length and splice variants of androgen receptor.

As a public health problem, prostate cancer engenders huge economic and life-quality burden. Developing effective chemopreventive regimens to alleviate the burden remains a major challenge. Androgen signaling is vital to the development and progression of prostate cancer. Targeting androgen signaling via blocking the production of the potent ligand dihydrotestosterone has been shown to decrease prostate cancer incidence. However, the potential of increasing the incidence of high-grade prostate cancers has been a concern. Mechanisms of disease progression after the intervention may include increased expression of androgen receptor (AR) in prostate tissue and expression of the constitutively active AR splice variants (AR-Vs) lacking the ligand-binding domain. Thus, novel agents targeting the receptor, preferentially both the full-length and AR-Vs, are urgently needed. In the present study, we show that ginsenoside 20(S)-protopanaxadiol-aglycone (PPD) effectively downregulates the expression and activity of both the full-length AR and AR-Vs. The effects of PPD on AR and AR-Vs are manifested by an immediate drop in proteins followed by a reduction in transcripts, attributed to PPD induction of proteasome-mediated degradation and inhibition of the transcription of the AR gene. We further show that although PPD inhibits the growth as well as AR expression and activity in LNCaP xenograft tumors, the morphology and AR expression in normal prostates are not affected. This study is the first to show that PPD suppresses androgen signaling through downregulating both the full-length AR and AR-Vs, and provides strong rationale for further developing PPD as a promising agent for the prevention and/or treatment of prostate cancer.

Prostate cancer cells differ in testosterone accumulation, dihydrotestosterone conversion, and androgen receptor signaling response to steroid 5α-reductase inhibitors.

BACKGROUND: Blocking 5α-reductase-mediated testosterone conversion to dihydrotestosterone (DHT) with finasteride or dutasteride is the driving hypothesis behind two prostate cancer prevention trials. Factors affecting intracellular androgen levels and the androgen receptor (AR) signaling axis need to be examined systematically in order to fully understand the outcome of interventions using these drugs. METHODS: The expression of three 5α-reductase isozymes, as determined by immunohistochemistry and qRT-PCR, was studied in five human prostate cancer cell lines. Intracellular testosterone and DHT were analyzed using mass spectrometry. A luciferase reporter assay and AR-regulated genes were used to evaluate the modulation of AR activity. RESULTS: Prostate cancer cells were capable of accumulating testosterone to a level 15-50 times higher than that in the medium. The profile and expression of 5α-reductase isozymes did not predict the capacity to convert testosterone to DHT. Finasteride and dutasteride were able to depress testosterone uptake in addition to lowering intracellular DHT. The inhibition of AR activity following drug treatment often exceeded the expected response due to reduced availability of DHT. The ability to maintain high intracellular testosterone might compensate for the shortage of DHT. CONCLUSIONS: The biological effect of finasteride or dutasteride appears to be complex and may depend on the interplay of several factors, which include testosterone turnover, enzymology of DHT production, ability to use testosterone and DHT interchangeably, and propensity of cells for off-target AR inhibitory effect.

The direct inhibitory effect of dutasteride or finasteride on androgen receptor activity is cell line specific.

BACKGROUND: Finasteride and dutasteride were developed originally as 5α-reductase inhibitors to block the conversion of testosterone to dihydrotestosterone (DHT). These drugs may possess off-target effects on the androgen receptor (AR) due to their structural similarity to DHT. METHODS: A total of four human prostate cancer cell models were examined: LNCaP (T877A mutant AR), 22Rv1 (H874Y mutant AR), LAPC4 (wild-type AR), and VCaP (wild-type AR). Cells were cultured in 10% charcoal-stripped fetal bovine serum, either with or without DHT added to the medium. AR activity was evaluated using the ARE-luciferase assay or the expression of AR regulated genes. RESULTS: Dutasteride was more potent than finasteride in interfering with DHT-stimulated AR signaling. Disruption of AR function was accompanied by decreased cell growth. Cells that rely on DHT for protection against death were particularly vulnerable to dutasteride. Different prostate cancer cell models exhibited different sensitivities to dutasteride and finasteride. LNCaP was most sensitive, LAPC4 and VCaP were intermediate, while 22Rv1 was least sensitive. Regardless of the AR genotype, if AR was transfected into drug-sensitive cells, AR was inhibited by drug treatment; and if AR was transfected into drug-resistant cells, AR was not inhibited. CONCLUSIONS: The direct inhibitory effect of dutasteride or finasteride on AR signaling is cell line specific. Mutations in the ligand binding domain of AR do not appear to play a significant role in influencing the AR antagonistic effect of these drugs. Subcellular constituent is an important factor in determining the drug effect on AR function.

Activation of FOXO1 is critical for the anticancer effect of methylseleninic acid in prostate cancer cells.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: In prostate cancer cells, MSA activates the FOXO1 signaling pathway. FOXO1 activation is critical for the anticancer effects of MSA.

Human prx1 gene is a target of Nrf2 and is up-regulated by hypoxia/reoxygenation: implication to tumor biology.

Peroxiredoxin 1 (Prx1) has been found to be elevated in several human cancers. The cell survival-enhancing function of Prx1 is traditionally attributed to its reactive oxygen species-removing capacity, although the growth-promoting role of Prx1 independent of this antioxidant activity is increasingly gaining attention. Although much progress has been made in understanding the behavior of Prx1, little information is available on the mechanism responsible for the abnormal elevation of Prx1 level in cancer. We hypothesized that the hypoxic and unstable oxygenation microenvironment of a tumor might be crucial for prx1 up-regulation. In this study, we cloned the human prx1 promoter and identified nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) as a key transcription factor. Hypoxia/reoxygenation, an in vitro condition suited to mimic changes of oxygenation, increased Nrf2 nuclear localization and its binding to the electrophile-responsive elements located at the proximal (-536 to -528) and distal (-1429 to -1421) regions of the prx1 promoter. A significant reduction of both steady-state and hypoxia/reoxygenation-mediated prx1 gene expression was shown in Nrf2 knock-out cells. Our results indicated that decreased Kelch-like ECH-associated protein, Keap1, might be an important mechanism for the increased nuclear translocation and activation of Nrf2 in response to hypoxia/reoxygenation. A constitutive elevation of prx1 mRNA and protein was observed in Keap1 knock-out cells. The above information suggests that the Nrf2-Prx1 axis may be a fruitful target for intervention with respect to inhibiting the malignant progression and/or reducing the treatment resistance of cancer cells.

Peroxiredoxin 1 interacts with androgen receptor and enhances its transactivation.

Although hypoxia is accepted as an important microenvironmental factor influencing tumor progression and treatment response, it is usually regarded as a static global phenomenon. Consequently, less attention is given to the impact of dynamic changes in tumor oxygenation in regulating the behavior of cancer cells. Androgen receptor (AR) signaling plays a critical role in prostate cancer. We previously reported that hypoxia/reoxygenation, an in vitro condition used to mimic an unstable oxygenation climate in a tumor, stimulates AR activation. In the present study, we showed that peroxiredoxin 1 (Prx1), a member of the peroxiredoxin protein family, acts as a key mediator in this process. We found that the aggressive LN3, C4-2, and C4-2B prostate cancer cell lines derived from LNCaP possess constitutively elevated Prx1 compared with parental cells, and display greater AR activation in response to hypoxia/reoxygenation. Although the cell survival-enhancing property of Prx1 has traditionally been attributed to its antioxidant activity, the reactive oxygen species-scavenging activity of Prx1 was not essential for AR stimulation because Prx1 itself was oxidized and inactivated by hypoxia/reoxygenation. Increased AR transactivation was observed when wild-type Prx1 or mutant Prx1 (C52S) lacking antioxidant activity was introduced into LNCaP cells. Reciprocal immunoprecipitation, chromatin immunoprecipitation, and in vitro pull-down assays corroborated that Prx1 interacts with AR and enhances its transactivation. We also show that Prx1 is capable of sensitizing a ligand-stimulated AR. Based on the above information, we suggest that disrupting the interaction between Prx1 and AR may serve as a fruitful new target in the management of prostate cancer.

Prx1 suppresses radiation-induced c-Jun NH2-terminal kinase signaling in lung cancer cells through interaction with the glutathione S-transferase Pi/c-Jun NH2-terminal kinase complex.

Radiotherapy is one of the major treatment modalities for lung cancer. Cell killing by ionizing radiation is mediated primarily through the reactive oxygen species (ROS) and ROS-driven oxidative stress. Prx1, a peroxiredoxin family member, was shown to be frequently elevated in lung cancer cells and tissues. Although the antioxidant function of Prx1 is expected to affect the radiotherapy response of lung cancer, the physiologic significance of its peroxidase activity in irradiated cells is unclear because the catalytic Cys52 is easily inactivated by ROS due to its overoxidation to sulfinic or sulfonic acid. In this study, we investigated the role of Prx1 in radiation sensitivity of human lung cancer cells, with special emphasis on the redox status of the catalytic Cys52. We found that overexpression of Prx1 enhances the clonogenic survival of irradiated cells and suppresses ionizing radiation-induced c-Jun NH2-terminal kinase (JNK) activation and apoptosis. The peroxidase activity of Prx1, however, is not essential for inhibiting JNK activation. The latter effect is mediated through its association with the glutathione S-transferase pi (GSTpi)-JNK complex, thereby preventing JNK release from the complex. Reduced JNK activation is observed when the peroxidase activity of Prx1 is compromised by Cys52 overoxidation or in the presence of the Cys52 to Ser52 mutant (Prx1C52S) lacking peroxidase activity. We show that both Prx1 and Prx1C52S interact with the GSTpi-JNK complex and suppress the release of JNK from the complex. Our study provides new insight into the antiapoptotic function of Prx1 in modulating radiosensitivity and provides the impetus to monitor the influence of Prx1 levels in the management of lung cancer.

Hypoxia increases androgen receptor activity in prostate cancer cells.

Recent studies show that prostate cancer cells are able to survive in a hypoxic tumor environment, and the extent of tumor hypoxia correlates with poor clinical outcome. Androgen deprivation, the most common form of prostate cancer therapy, was itself shown to induce a state of transient hypoxia at the microenvironmental level. Because androgen receptor (AR) signaling plays a critical role in prostate cancer, we investigated the effect of hypoxia in regulating AR function. We found that in LNCaP prostate cancer cells, AR binding to the androgen-responsive element (ARE), prostate-specific antigen accumulation, and ARE-reporter gene activity were increased after hypoxia treatment. Hypoxia-enhanced AR function was also observed when AR was exogenously introduced into AR-null DU145 cells. Confocal microscopy and chromatin immunoprecipitation assays showed that AR translocation to the nucleus and AR recruitment to the prostate-specific antigen promoter were facilitated after hypoxia treatment. The AR stimulatory effect seemed to be ligand-dependent because it was abrogated when cells were cultured in an androgen-depleted medium, but was restored with the addition of R1881, a synthetic androgen. The sensitivity of AR activation to R1881 was also increased after hypoxia treatment. Although concentrations of <1 nmol/L R1881 did not induce ARE reporter activity under normoxic conditions, exposure to hypoxia greatly potentiated the AR response to low levels of R1881. Collectively, our results provide compelling evidence that changes in hypoxia/reoxygenation stimulate AR trans-activation and sensitization. The AR-stimulatory effect of an unstable tissue oxygenation milieu of a tumor is likely to contribute to treatment resistance and the emergence of recurrent prostate cancer.

Selenium sensitizes MCF-7 breast cancer cells to doxorubicin-induced apoptosis through modulation of phospho-Akt and its downstream substrates.

Doxorubicin is an effective drug against breast cancer. However, the favorable therapeutic response to doxorubicin is often associated with severe toxicity. The present research was aimed at developing a strategy of increasing doxorubicin sensitivity so that lower doses may be used without compromising efficacy. The MCF-7 human breast cancer cell line currently in use in our laboratory did not respond to doxorubicin cell killing during a 24-h treatment period. By combining doxorubicin with selenium, we were successful in producing a brisk enhancement of apoptosis. We examined the effects of these two agents on Akt activation and found that selenium was capable of depressing doxorubicin-induced Akt phosphorylation. Several lines of evidence converged to support the notion that this effect is important in mediating the synergy between selenium and doxorubicin. Selenium was no longer able to sensitize cells to doxorubicin under a condition in which Akt was constitutively activated. Increased Akt phosphorylation following treatment with doxorubicin was accompanied by increased phosphorylation of glycogen synthase kinase 3beta (GSK3beta) and FOXO3A, which are substrates of Akt (both GSK3beta and FOXO3A lose their proapoptotic activities when they are phosphorylated). Selenium reduced the abundance of phospho-GSK3beta induced by doxorubicin, whereas chemical inhibition of GSK3beta activity muted the apoptotic response to the selenium/doxorubicin combination. Additional experiments showed that selenium increased the transactivation activity of FOXO3A, as evidenced by a reporter gene assay, as well as by the elevated expression of Bim (a target gene of FOXO3A). The functional significance of Bim was confirmed by the observation that RNA interference of Bim markedly reduced the potency of selenium/doxorubicin to induce apoptosis.

High superoxide dismutase and low glutathione peroxidase activities in red blood cells predict susceptibility of lung cancer patients to radiation pneumonitis.

Radiation pneumonitis is an unpredictable complication of radiotherapy for lung cancer and a condition which can cause significant morbidity. The ability to identify patients at a high risk of developing pneumonitis is critical, since it will enable the individualization of the treatment plan. Because the cytotoxic effect of radiation is propagated through reactive oxygen species (ROS) and ROS-driven oxidative stress, the role of antioxidant defense systems in radiation pneumonitis was investigated. Using the pneumonitis-sensitive C3H/HeN mice as a model, we demonstrated that the antioxidant response of the lung correlated well with that of red blood cells (RBC). We then proceeded to test whether differences of RBC antioxidant response would predict the pneumonitis development in patients. Superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) activities and glutathione in RBC were measured at baseline and then weekly for 6 weeks of treatment in 15 eligible patients receiving concurrent chemo-radiotherapy for unresectable stage III NSCLC. Striking differences were found in the antioxidant activities of RBC with respect to the pneumonitis development. Those who developed pneumonitis showed higher SOD and lower GPX activities at baseline compared to those who did not (3.7 vs 6.8 units/mg for median SOD, 16.5 vs 10.7 nmol/min/mg for median GPX). The functional imbalance of SOD and GPX was displayed consistently throughout the treatment period. The sensitivity and specificity of pneumonitis prediction were further increased when the GPX/SOD ratio was analyzed (pretreatment P = 0.0046). Our results provide a strong rationale to monitor SOD and GPX activities of RBC to identify patients who are at risk of developing pneumonitis, and to implement a strategy of increasing the GPX/SOD ratio in order to lower the risk.

Doxorubicin and selenium cooperatively induce fas signaling in the absence of Fas/Fas ligand interaction.

BACKGROUND: The synergistic effect of doxorubicin and selenium in apoptosis induction in MCF-7 breast cancer cells has been previously reported. Mitochondrial activation of caspase-9 is in part responsible for the synergy. The present study aimed at examining the death receptor pathway in activating caspase-8 by the two-drug combination. MATERIALS AND METHODS: We determined the expression of TRAIL and FasL signaling molecules and monitored activated caspase-8 in response to neutralizing/blocking antibodies against ligands/receptors. RESULTS: Our data suggest that TRAIL signaling might not play a role. With respect to the Fas pathway, it was found that doxorubicin enhanced Fas oligomerization (i.e. activation) independent of FasL-Fas interaction. Selenium, on the other hand, increased the expression of FADD, a key adaptor molecule responsible for recruitment of caspase-8 to the Fas oligomer. The significance of the above changes was confirmed by the detection of considerably more caspase-8 in both the Fas or FADD immunoprecipitate obtained from cells treated with the doxorubicin/selenium combination. CONCLUSION: Doxorubicin and selenium cooperatively activate Fas signaling by targeting key regulatory steps.

Endoplasmic reticulum stress signal mediators are targets of selenium action.

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.

Selenium disrupts estrogen signaling by altering estrogen receptor expression and ligand binding in human breast cancer cells.

Cancer prevention studies suggest that selenium is effective in reducing the incidence of cancers including prostate, colon, and lung cancers. Previous reports showed that selenium inhibits premalignant human breast MCF-10AT1 and MCF10AT3B cell growth in vitro and reduces mammary tumor incidence after exposure to carcinogens in tumor models. Because estrogen is critical to the development and differentiation of estrogen target tissues, including the breast, the present study was designed to examine the effect of selenium on estrogen receptor (ER) expression and activation using methylseleninic acid (MSA), an active form of selenium in vitro. Selenium decreased the levels of expression of ERalpha mRNA and protein and reduced the binding of labeled estradiol to estrogen receptor in MCF-7 cells. Selenium inhibited the trans-activating activity of estrogen receptor in MCF-7 cells expressing functional estrogen receptor using a luciferase reporter construct linked to estrogen responsive element. Selenium decreased the binding of estrogen receptor to the estrogen responsive element site using an electrophoretic mobility gel shift assay. Selenium suppressed estrogen induction of the endogenous target gene c-myc. In contrast to the effect on ERalpha in MCF-7 cells, selenium increased ERbeta mRNA expression in MDA-MB231 human breast cancer cells. Thus, differential regulation of ERalpha and ERbeta in breast cancer cells may represent a novel mechanism of selenium action and provide a rationale for selenium breast cancer prevention trial.

Delineating the mechanism by which selenium deactivates Akt in prostate cancer cells.

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.

Methylseleninic acid potentiates apoptosis induced by chemotherapeutic drugs in androgen-independent prostate cancer cells.

PURPOSE: To test whether and how selenium enhances the apoptosis potency of selected chemotherapeutic drugs in prostate cancer (PCA) cells. EXPERIMENTAL DESIGN: DU145 and PC3 human androgen-independent PCA cells were exposed to minimal apoptotic doses of selenium and/or the topoisomerase I inhibitor 7-ethyl-10-hydroxycamptothecin (SN38), the topoisomerase II inhibitor etoposide or the microtubule inhibitor paclitaxel/taxol. Apoptosis was measured by ELISA for histone-associated DNA fragments, by flow cytometric analysis of sub-G(1) fraction, and by immunoblot analysis of cleaved poly(ADP-ribose)polymerase. Pharmacologic inhibitors were used to manipulate caspases and c-Jun-NH(2)-terminal kinases (JNK). RESULTS: The methylselenol precursor methylseleninic acid (MSeA) increased the apoptosis potency of SN38, etoposide, or paclitaxel by several folds higher than the expected sum of the apoptosis induced by MSeA and each drug alone. The combination treatment did not further enhance JNK1/2 phosphorylation that was induced by each drug in DU145 cells. The JNK inhibitor SP600125 substantially decreased the activation of caspases and apoptosis induced by MSeA combination with SN38 or etoposide and completely blocked these events induced by MSeA/paclitaxel. The caspase-8 inhibitor zIETDfmk completely abolished apoptosis and caspase-9 and caspase-3 cleavage, whereas the caspase-9 inhibitor zLEHDfmk significantly decreased caspase-3 cleavage and apoptosis but had no effect on caspase-8 cleavage. None of these caspase inhibitors abolished JNK1/2 phosphorylation. A JNK-independent suppression of survivin by SN38 and etoposide, but not by paclitaxel, was also observed. In contrast to MSeA, selenite did not show any enhancing effect on the apoptosis induced by these drugs. CONCLUSIONS: MSeA enhanced apoptosis induced by cancer therapeutic drugs in androgen-independent PCA cells. In DU145 cells, the enhancing effect was primarily through interactions between MSeA and JNK-dependent targets to amplify the caspase-8-initiated activation cascades. The results suggest a novel use of methyl selenium for improving the chemotherapy of PCA.

Synergy between selenium and vitamin E in apoptosis induction is associated with activation of distinctive initiator caspases in human prostate cancer cells.

The ongoing Selenium and Vitamin E Chemoprevention Trial is designed to evaluate the efficacy of these two agents, either individually or in combination, in reducing the incidence of prostate cancer in healthy men over 55 years of age. Little information, however, is available on the potential synergy between vitamin E and selenium in chemoprevention. The present study was aimed at addressing this gap of knowledge with the use of the androgen-unresponsive, p53-null, PC-3 human prostate cancer cell line. The growth-inhibitory activity of vitamin E appeared to be dependent on the chemical form. In our hands, D-alpha-tocopheryl succinate (VES) was much more potent than either DL-alpha-tocopherol or D-alpha-tocopheryl acetate. Combining VES with methylseleninic acid (MSA), a selenium metabolite, produced a synergistic effect on cell growth suppression. The synergy was accounted for primarily by an augmented apoptotic response. Poly(ADP-ribose) polymerase cleavage and activation of specific caspases were confirmed by Western blot analysis. The caspases that were commonly modulated by either VES or MSA included initiator caspases-8 and -10, as well as executioner caspases-3, -6, and -7. In contrast, caspase-9 was activated only by VES, whereas caspases-1 and -12 were activated only by MSA. Based on the above information, it is proposed that the mitochondrial pathway and the endoplasmic reticulum stress/cytokine signaling pathway might be involved in apoptosis induction by VES and MSA, respectively. These two pathways may act in a cooperative manner to switch on the full force of the apoptotic machinery when cells are treated with both VES and MSA.

Identification of molecular targets associated with selenium-induced growth inhibition in human breast cells using cDNA microarrays.

Past research indicated that methylseleninic acid (MSA) is an excellent tool for investigating the cancer chemopreventive action of selenium in vitro. The present study was designed to examine the cellular and molecular effects of MSA in the MCF10AT1 and MCF10AT3B premalignant human breast cells. After exposure to MSA, both cell lines exhibited a dose- and time-dependent growth-inhibitory response as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay. Further characterization of cellular and molecular changes was carried out only with the MCF10AT1 cells. Flow cytometry analysis showed that MSA blocked cell cycle progression at the G(0)-G(1) phase. Induction of apoptosis was also observed with the use of either the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) or the annexin V binding method. cDNA microarray analyses with cell cycle- and apoptosis-targeted arrays were then applied to profile the gene expression changes mediating these two cellular events. The analyses were conducted at 6 and 12 h of MSA treatment using synchronized cells. The expression signals of 30 genes were found to be significantly altered by MSA. These genes fall into three categories: cell cycle checkpoint controllers (e.g., cyclins, cdcs, cdks, E2F family proteins, and serine/threonine kinases), apoptosis regulatory genes (e.g., Apo-3, c-jun, and cdk5/cyclin D1), and signaling molecules [e.g., mitogen-activated protein (MAP)/extracellular signal-regulated protein kinase (ERK) and phosphatidylinositol 3'-kinase (PI3k) cascade genes]. The expression changes of 15 genes were selected for verification by Western or semiquantitative reverse transcription-PCR analyses. An agreement rate of 60% (9 of 15) was obtained from these confirmation experiments. On the basis of the above findings, tentative signaling pathways mediating the outcome of selenium-induced cell cycle arrest and apoptosis are proposed. The present study thus demonstrated the feasibility of applying cDNA microarray technology in delineating the mechanisms of the action of selenium and in pinpointing molecular targets as potential biomarkers for evaluating the efficacy of selenium intervention.

Delineation of the molecular basis for selenium-induced growth arrest in human prostate cancer cells by oligonucleotide array.

Despite the growing interest in selenium intervention of prostate cancer in humans, scanty information is currently available on the molecular mechanism of selenium action. Our past research indicated that methylseleninic acid (MSA) is an excellent reagent for investigating the anticancer effect of selenium in vitro. The present study was designed to examine the cellular and molecular effects of MSA in PC-3 human prostate cancer cells. After exposure to physiological concentrations of MSA, these cells exhibited a dose- and time-dependent inhibition of growth. MSA retarded cell cycle progression at multiple transition points without changing the proportion of cells in different phases of the cell cycle. Flow cytometric analysis of annexin V- and propidium iodide-labeled cells showed a marked induction of apoptosis by MSA. Array analysis with the Affymetrix human genome U95A chip was then applied to profile the gene expression changes that might mediate the effects of selenium. Gene profiling was done in a time course experiment (at 12, 24, 36, and 48 h) using synchronized cells. A large number of potential selenium-responsive genes with diverse biological functions were identified. These genes fell into 12 clusters of distinct kinetics pattern of modulation by MSA. The expression changes of 10 genes known to be critically involved in cell cycle regulation were selected for verification by Western analysis to determine the reliability of the array data. An agreement rate of 70% was obtained based on these confirmation experiments. The array data enabled us to focus on the role of potential key genes (e.g., GADD153, CHK2, p21(WAF1), cyclin A, CDK1, and DHFR) that might be targets of MSA in impeding cell cycle progression. The data also provide valuable insights into novel biological effects of selenium, such as inhibition of cell invasion, DNA repair, and stimulation of transforming growth factor beta signaling. The present study demonstrates the utility of a genome-wide analysis to elucidate the mechanism of selenium chemoprevention.

Prostate specific antigen expression is down-regulated by selenium through disruption of androgen receptor signaling.

A previous controlled intervention trial showed that selenium supplementation was effective in reducing the incidence of prostate cancer. Physiological concentrations of selenium have also been reported to inhibit the growth of human prostate cancer cells in vitro. The present study describes the observation that selenium was able to significantly down-regulate the expression of prostate-specific antigen (PSA) transcript and protein within hours in the androgen-responsive LNCaP cells. Decreases in androgen receptor (AR) transcript and protein followed a similar dose and time response pattern upon exposure to selenium. The reduction of AR and PSA expression by selenium occurred well before any significant change in cell number. With the use of a luciferase reporter construct linked to either the PSA promoter or the androgen responsive element, it was found that selenium inhibited the trans-activating activity of AR in cells transfected with the wild-type AR expression vector. Selenium also suppressed the binding of AR to the androgen responsive element site, as evidenced by electrophoretic mobility shift assay of the AR-androgen responsive element complex. In view of the fact that PSA is a well-accepted prognostic indicator of prostate cancer, an important implication of this study is that a selenium intervention strategy aimed at toning down the amplitude of androgen signaling could be helpful in controlling morbidity of this disease.

Inhibition of angiogenesis by the cancer chemopreventive agent conjugated linoleic acid.

Dietary conjugated linoleic acid (CLA) has been shown previously to inhibit rat mammary carcinogenesis. In addition to direct effects on mammary epithelial cells,including decreased proliferation and induction of apoptosis, CLA may exert its effects indirectly by inhibiting the differentiation of mammary stromal cells to an endothelial cell type. Specifically, CLA was found to decrease the ability of mammary stromal cells to form complex anastomosing microcapillary networks in vitro on Engelbreth-Holm-Swarm-derived reconstituted basement membrane. This suggested that CLA might inhibit angiogenesis in vivo. To test this possibility, CD2/F(1) mice were placed on synthetic diets containing 0, 1, or 2% CLA for 6 weeks, before angiogenic challenge by s.c. injection with an angiogenic gel substrate (Matrigel pellet assay). After 7 days, the pellets from animals fed the control diet were infiltrated by abundant branching networks of blood vessels with patent lumen-containing RBCs. In contrast, pellets from the CLA-fed animals contained fewer infiltrating cells, which formed limited branching cellular networks, the majority of which had collapsed lumen and no RBCs. Both levels of dietary CLA showed similar effects, with the number of RBC-containing vessels per 20x field decreased to a third of that seen in control. Dietary CLA decreased serum levels of vascular endothelial growth factor (VEGF) and whole mammary gland levels of VEGF and its receptor Flk-1. Both cis-9, trans-11 and trans-10, cis-12 CLA isomers were effective in inhibiting angiogenesis in vitro in a dose-dependent fashion. The ability of CLA to inhibit angiogenesis may contribute to its efficacy as a chemopreventive agent.