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7-epi-nemorosone from Clusia rosea induces apoptosis, androgen receptor down-regulation and dysregulation of PSA levels in LNCaP prostate carcinoma cells.

ARTICLE INFO

Keywords:

Clusia rosea and Clusia grandiflora

(Clusiaceae)

Antitumoral activity

Prostate carcinoma

Polyisoprenylated benzophenone

7-epi-nemorosone

Signal transduction analysis

MEK1/2

Akt/PKB

PSA

AR

ABSTRACT

The aim of this work was to characterize the antitumoral activity of the plant compound 7-epi-nemorosone in prostate carcinoma cell lines. Prostate cancer is the most frequently diagnosed malignancy and the second-leading cause of cancer death in men. In spite of the current therapeutic options for this cancer entity, many patients die due to metastases in distant organs and acquired chemotherapy resistance. Thus, approaches to provide improvements in outcome and quality of life for such patients are urgently needed. Recently, the polyisoprenylated benzophenone 7-epi-nemorosone, originally collected by honeybees from Clusia rosea and Clusia grandiflora (Clusiaceae), has been described to be a potent antitumoral agent. Here, its activity in prostate carcinoma is reported. 7-epi-nemorosone was isolated from Caribbean propolis employing RP-HPLC techniques. Its cytotoxicity was assessed using the MIT proliferation assay in human androgen-dependent prostate carcinoma LNCaP cells including an MDR1' sub-line. No cross-resistance was detected. FACS-based cell cycle analysis revealed a significant increase in the sub-GO/G1, Cl, and depletion in the S phase populations. A concomitant clown-regulation of cyclins D1/D3 and CDK 4/6 in LNCaP cells was detected by Western blot. Annexin-V-FITC labeling and caspase-3 cleavage assays showed that 7-epi-nemorosone induced apoptotic events. Major signal transduction elements such as p38 MAPK and Akt/PKB as well as androgen receptor AR and PSA production were found to be down-regulated after exposure to the drug. ERK1/2 protein levels and phosphorylation status were down-regulated accompanied by up-regulation but inhibition of the activity of their immediate upstream kinases MEK1/2. Additionally, Akt/PKB enzymatic activity was effectively inhibited at a similar concentration as for MEK1/2. Here, we demonstrate for the first time that 7-epi-nemorosone exerts cytotoxicity in an androgen-dependent prostate carcinoma entity by targeting the MEK1/2 signal transducer.

[c] 2012 Elsevier GmbH. All rights reserved.

Abbreviations: AR, androgen receptor; BrdU, 5-bromo-2'-deoxyuridine; CASE, Cellular Activation of Signaling; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; DU-145, prostate cancer cell line; ELISA, enzyme-linked immunosorbent assay; ETO, etoposide; FACS, fluorescence-activated cell sorting; FCS, fetal calf serum; HAT, histone acetyl transferase; FIRPC, hormone-refractory PCa; LNCaP, lymph node carcinoma of prostate, prostate cancer cell line; MAPK, mitogen-activated protein kinases; MCF-7, Michigan Cancer Foundation-7, breast cancer cell line; MES, 2-(N-morpholino)ethanesullonic acid; MIT, 3-(4,5-climethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate buffered saline; PC-3, prostate cancer cell line; PCa, prostate carcinoma: PI, propidium iodide; PMSF, phenylmethylsulfonyl fluoride; PPAP, polyclyclic polyprenylated acylphloroglucinol; PSA, prostate specific antigen; RF, resistance factor; RIPA, radio-immunoprecipitation assay; RP-HPLC, reversed phase high-performance liquid chromatography; SD, standard deviation; SDS, sodium dodecyl sulfate; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; UPR, unfolded protein response; WT, wildtype.

Introduction

The first phytochemical studies on 7-epi-nemorosone (Fig. 1), a polycyclic polyprenylated acylphloroglucinol (PPAP) derivate, were reported by de Oliveira etal. (1996, 1999). PPAPs are characteristic secondary metabolites of the Clusiaceae or Guttiferae family, which comprise about 250 species distributed from southern Florida to southern Brazil. Thus, 7-epi-nemorosone has been isolated from the floral resins of Clusia rosea, Clusia nemorosa and Clusia grandiflora (de Oliveira et al. 1996, 1999). In addition, 7-epi-nemorosone was found in propolis from the Caribbean area (Cuesta-Rubio et al. 2002; Diaz-Carballo et al. 2008a,b) and Brazil (de Castro Ishida et al. 2011). The structure of 7-epi-nemorosone has been subjected to revisions during the last years by different chemists. The most exact proposition of its chemical structure, attending its stereochemistry was suggested by Bittrich et al. (2003) based on spectroscopic analyses. The very first works reporting the antitumoral activity of nemorosone, were performed by the group of Diaz-Carballo (Diaz-Carballo et al. 2003). This small molecule has been reported in recent years to be a potent cytotoxic agent particularly in aggressive cancer models characteristic for their highly chemoresistance. More interestingly, these compounds are less cytotoxic in normal, non-tumorigenic cells (Diaz-Carballo et al. 2008a,b; Holtrup et al. 2011; Popolo et al. 2011a). It could be demonstrated that nemorosone inhibits Akt/PKB activity, activates p300/03P HAT, acts as a protonophoric mitochondrial decoupler, and induces apoptosis in different cancer entities by activation of DNA damage inducible transcript 3 and enhancing unfolded protein response (UPR) (Dal Piaz et al. 2010; Holtrup et al. 2011; Pardo-Andreu et al. 2011). In light of their challenging structures and promising biological activities, the synthetic chemistry community has shown significant attention to the PPAP family. Up today, the most elegant synthesis of 7-epi-nemorosone was developed by the group of Porco (Zhang and Porco 2012).

Prostate carcinoma (PCa) is the most frequently diagnosed malignancy and the second-leading cause of cancer death in men, predominantly in persons over 50 years of age (lemal et al. 2010), typically progressing at a slow rate (Sciarra et at. 2008). Therapeutic options currently available for the treatment of PCa are rather palliative than curative. These include brachytherapy, androgen ablation, radical prostatectomy, castration and chemotherapy (Kao et at. 2011; Mottet et al. 2011). Novel strategies comprise the approach to target specific molecular structures in combination with conventional chemotherapeutics like docetaxel (Sonpavde and Sternberg 2010; Zhang et at. 2007).

PSA serum levels are used as a clinical marker for disease prognosis and outcome (Alapont Alacreu et at. 2008; Takizawa et at. 2010). Androgen ablation in metastatic PCa alleviates the clinical symptoms and leads to a decrease of prostate specific antigen (PSA) levels and tumor regression in most patients. Unfortunately, after a short period of remission, the majority of patients progress to androgen independence, hormone-refractory PCa (HRPC) with progressive clinical deterioration, and ultimately die (de Wit 2008; Fitzpatrick et al. 2008). In recent years, it has become clear that in anti-androgen-resistant cancer, the signaling cascades downstream of the androgen receptor (AR) are permanently activated, promoting tumor growth (Agoulnik and Weigel 2008; Lavery and Bevan 2011). Clinical and experimental evidence suggests that PCa progression occurs through alteration of the normal androgen axis by dysregulation of the AR-associated signal transduction cascades (Chen et at. 2008; Zhu and Kyprianou 2008), amplification of the AR gene (Koivisto and Helin 1999), changes in the expression of AR coregulators, and mutations of AR itself that enhance its transcriptional activity in response to the binding of ligands such as testosterones (Hull and Bostwick 2008). Consequently, there is increased interest in developing small molecules that interfere with the AR signaling pathway (Chen et at. 2008; Wu et at. 2011), like abiraterone acetate, a specific inhibitor of CYP17, a key to androgen and estrogen synthesis, and MDV3100, an AR antagonist, both currently being tested in Phase Ill clinical trials in castration-resistant prostate cancer (Attard et al. 2011).

Relatively few therapeutic options exist for PCa patients with metastases in distant organs who have become resistant to androgen deprivation and chemotherapy. Immunotherapeutic approaches and new agents targeting angiogenesis and apoptosis signal transduction pathways are currently under investigation in an attempt to improve the outcome and quality of life of PCa patients (Attard et al. 2011; Chen et at. 2008; Jahnisch et al. 2010; Sinnathamby et al. 2011).

In this work, we examined the cytotoxicity of 7-epi-nemorosone in human wildtype and chemotherapy-refractory PCa cell lines and tried to define its mechanism of action particularly in the androgen-dependent LNCaP cells, as prerequisite for in vivo studies based on this system.

Materials and methods

General

- Cell cultures were incubated in the Heraeus cell incubator type Hera Cell.

- MIT assay was analyzed in the microtiter plate reader Infinite F200 Tecan, Berlin, Germany, at 570 nm. BrdU incorporation and DNA content were measured using a Coulter EPICS XL flow cytometer (Beckman Coulter, Krefeld, Germany) at an excitation wavelength of [lambda] = 488 nm.

- For analysis of Hoechst-33342 and Annexin-V-FITC cell labeling, the Olympus XB51 fluorescence microscope controlled by the Soft Imaging System AnalySIS using the filters appropriate for these fluorochromes was used.

- Densitometric analyses were performed using a Bio-Rad Molecular Imager ChemiDoc[TM] XR5+ Imaging System.

- For immunoblotting, a Bio-Rad Mini Protean[TM] Tetra Cell blotting system was used.

- Signaling ELISA CASE[TM] was measured using an ELISA reader MRXII (Dynex, Berlin, Germany) at 450 nm using a reference wavelength of [lambda] = 655 nm.

- Measurement of PSA levels was performed on a Cobas Core analyzer (Hoffmann-La Roche, Basel, Switzerland).

- For statistical analysis Sigmastat 3.0 software was used.

Cell culture and proliferation assay

Human PCa cell lines DU-145, PC-3, and LNCaP were adapted and routinely cultured in DMEM medium (lnvitrogen, Karlsruhe, Germany) containing 10% heat-inactivated fetal calf serum (FCS) and 15[micro]/m1 ciprofloxacin (Bayer AG, Wuppertal, Germany). The I[C.sub.50] values of all drugs studied were determined using the MIT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] proliferation assay (Bono et al. 2008) and reported as the mean of three independent experiments. Briefly, cells in exponential growth phase were harvested, washed with medium, and seeded at appropriate densities according to their growth kinetics in 96-well plates. After a conditioning period of 24h, cells were exposed to increasing concentrations of etoposide or 7-epi-nemorosone for 24h, respectively. The cultures were then incubated with Mil' (Sigma-Aldrich, Munich, Germany) dissolved in PBS at a final concentration of 1 mg/m1 for 4h at 37[degrees]C in an atmosphere of 5% [CO.sub.2]. Supernatants were aspirated and the purple formazan crystals were dissolved in 10011I of solubilization solution (10% SDS in DMSO, Sigma-Aldrich, Munich, Germany). The absorbance was measured in a microtiter plate reader (Infinite F200 Tecan, Berlin, Germany) at 570nm.

Generation of etoposide-resistant cell lines

Etoposide resistance was developed in parental LNCaP, PC-3 and DU-145 cell lines by exposing cell cultures to increasing etoposide concentrations. The characterization of the MDR-phenotype and the resistance factor (RF) of generated resistant cells were performed in the same way as recently described (Azu ma et al. 2008; Diaz-Carballo et al. 2008a).

Chemical isolation

7-epi-nemorosone was initially isolated in very little quantities from floral resins of Clusia rosea collected in Florida, USA. Instead Caribbean propolis contents large amounts of this compound. Consequently, we used this source for the semi-preparative isolation of this small molecule. Thus, 7-epi-nemorosone was isolated from the methanolic extracts of Caribbean propolis using sequential reverse phase high pressure liquid chromatography (RP-HPLC) techniques employing a Waters Separation Module Alliance 2695 HPLC system, detection was performed with a Waters 2998 PDA detector, both controlled by the Empower Pro software (Waters GmbH, Eschborn, Germany). Chemical fractionation was performed on a 250mm [x] 10mm semi-preparative column packed with Nucleosil 100-7C18 (Macherey-Nagel, Dueren, Germany) at a controlled temperature of 40[degrees]C, using a gradient system starting at time point zero with a mixture of ammonium formiate (0.01M, pH 7.0), methanol and acetonitrile (60:30:10, v/v/v). The composition of this mixture changed linearly within 120 min to ammonium formiate:methanol:acetonitrile = 5:85:10 (v/v/v) at a flow rate of 2ml/min. The purity (98% HPLC) of 7-epi-nemorosone was further analyzed employing a Symmetry C18 column of 150mm x 2.1mm (Waters GmbH, Eschborn, Germany) using the same chromatographic conditions as described above at a flow rate of 0.5ml/min.

Cell cycle analysis

Cell cycle analyses were performed combining propidium iodide (PI, Sigma-Aldrich, Munich, Germany) staining and 5-bromo-2'-deoxyuridine (BrdU, Sigma-Aldrich, Munich, Germany) incorporation as described previously (Tsugita et al. 1991). 106 LNCaP wildtype cells in exponential growth phase were incubated with 2 [x] I[C.sub.50] of 7-epi-nemorosone for 24h. Anti-BmIU FITC-conjugated mAb was purchased from PharMingen, Heidelberg, Germany. BrdU incorporation and DNA content were measured using a Coulter EPICS XL flow cytometer (Beckman Coulter, Krefeld, Germany) at an excitation of [lambda]= 488 nm.

Analysis of apoptosis induction by fluorescence microscopy

The early stage of apoptosis was examined by means of fluorescence microscopy, combining Hoechst-33342 and Annexin-V-FITC labeling (BD Biosciences PharMingen, Heidelberg, Germany) performed as described previously (Di Lorenzo et al. 2007). Briefly, [10.sup.6] LNCaP wr cells were incubated with 8 [micro]M (2 [x] I[C.sub.50]) of 7-epi-nemorosone for 6h. After this time, cells were trypsinized, centrifuged and rinsed with binding buffer 1 [x] (PBS, 0.1% Tween 20), in which the cells were incubated with Annexin-V-FITC and Hoechst-33342 for 20 min. Cells were washed with PBS lx, mounted on a slide and analyzed under a fluorescence microscope (Olympus XB51, controlled by the Soft Imaging System AnalySIS) using the filters appropriate for these fluorochromes, and maintaining the same light intensity in all cases (200 ISO).

Western blot analysis

5 [x] 106 cells in exponential growth phase were incubated with 2-8[micro]M (0.5-2 [x] I[C.sub.50]) of 7-epi-nemorosone for 24h. The medium was removed and the cells were pelleted, washed once with cold PBS and then lyzed in RIPA buffer (150mM NaCI, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50mM Tris-HCI pH 7.4) in the presence of a proteinase inhibitor cocktail according to the manufacturer's instructions (Roche Diagnostics GmbH, Mannheim, Germany) for 30 min on ice and subsequently centrifuged at 14,000 [x] g at 4[degrees]C for 20min. Protein concentrations in supernatants were measured using the Bradford assay (Bio-Rad, Munich, Germany). Protein extracts (40[micro]g per slot) were resolved by SDS-PAGE in a gradient gel of 4-12% (Invitrogen, Karlsruhe, Germany) using MES (2-morpholino ethanesulfonic acid) buffer (Sigma-Aldrich, Munich, Germany), and transferred overnight onto Hybond nitrocellulose paper (Amersham, Freiburg, Germany). Primary and secondary antibodies were used following the recommendations of the respective manufacturers. Antibodies for caspase-3, cyclins D1, D3, CDK4, CDK6, p38 MAPK, ERK1/2, AR, and [beta]-actin were purchased from Cell Signaling (Frankfurt, Germany), anti-mouse IgG HRP-linked whole antibody from Amersham Biosciences, UK. Proteins were detected using an Enhanced Chemi-Luminescence kit (Amersham, Freiburg, Germany) according to the manufacturer's instructions. The densitometric analyses of the intensity of the bands were performed using the free version of the NIH-Imagei program and reported in arbitrary units.

MEK1/2 activity assay

The influence of 7-epi-nemorosone on the kinase activity of MEK1/2 was measured using the MEK1/2 Kinase Assay Kit (New England BioLabs GmbH, Frankfurt, Germany) as per the manufacturer's instructions. 2 x 107 untreated LNCaP cells in exponential growth phase were used as source of activated MEK1/2 (Ser 217/221) kinase. Cells were lyzed in lx Cell Lysis Buffer (20 mM Tris pH 7.5, 150mM NaCI, 1mM EDTA, 1mM EGTA, 1%Triton X-100, 2.5mM sodium pyrophosphate, 1mM [beta]-glycerol phosphate, 1mM [Na.sub.3VO.sub.4], 1[micro]g/m1 leupeptin, 1mM PMSF). The cell lysates were incubated with phospho-MEK1/2 antibody with gentle rocking overnight at 4[degrees]C. The immunocomplexes were then retrieved using protein A-sepharose CL-4B (Sigma-Aldrich, Munich, Germany) by incubation with gentle rocking for 3h at 4[degrees]C. The enzymatic activity was measured as follows: ppt.s were resuspended in 1 [x] kinase buffer (25mM Tris pH 7.5, 5mM 13-glycerol phosphate, 2mM MT, 0.1mM [Na.sub.3VO/sub.4], 10mM [MgC1.sub.2], 200[micro]M ATP), 2[micro]g of inactive p42 (ERK2), MAPK protein and increasing concentrations of 7-epi-nemorosone (0.5-4[micro]M) diluted in lx kinase buffer were added, and the mix was incubated for 30 min at 30[degrees]C. Finally, the phosphorylation status of p42 MAPK was qualitatively analyzed by immunoblotting as described above, employing an anti-phospho-p44/42 MAPK (Thr202/Tyr204) monoclonal antibody.

Akt/PKB activity assay

Akt/PKB kinase activity was measured in a cell free system using the Ala Kinase BioAssay purchased from US Biological (MA, USA) as described previously (Azuma et al. 2008; Diaz-Carballo et al. 2008a). [10.sup.7] LNCaP cells in exponential growth phase were used as source of Alct/PKB. 7-epi-nemorosone was diluted in lx kinase buffer as described above, and the influence on the phosphorylation of the GSK-3 fusion protein was visualized by Western blot as described above.

Cellular Activation of Signaling ELISA assay

The phosphorylation status of cytosolic proteins (e.g. extracellular signal-regulated kinase, ERK1 /2) involved in signal transduction pathways was monitored using the Cellular Activation of Signaling ELISA CASE[TM] kit (SuperArray, Frederick, MD, USA). The kit uses two antibodies, the first one recognizing the protein to be investigated regardless of its phosphorylation status and the second one exclusively detecting the phosphorylated form. Briefly, 2 x [10.sup.3] LNCaP cells/well were seeded in a 96-well plate and after 24 h, cells were treated with 4 [micro]M (2 x [IC.sup.50]) of 7-epi-nemorosone for an additional 24 h. The experiments were evaluated comparing the absorbances determined on an ELISA reader MRXII (Dynex, Berlin, Germany) at 450 nm using a reference wavelength of [lambda] = 655 nm.

Total PSA measurement

PSA serum concentrations were measured using an enzyme-immunoassay (Hoffmann-La Roche, Basel, Switzerland), using anti-PSA monoclonal antibody on a Cobas Core analyzer (Hoffmann-La Roche, Basel, Switzerland). The standards used where those included in the commercial kit. The detection limit of the assay was 0.2 ng/ml. Coefficients of variation of intra and inter assay (n= 10) were established by analyzing two control levels (Bioref[R], Wombris, Germany, Level 1:4.7 ng/ml [range 2.5-6.7], Level II: 69.0 ng/ml [range 48.0-89.01) and pooled serum. Total PSA values were represented in percentages.

Statistical analysis

The Sigmastat 3.0 software was used for the statistical analyses. All data are given as mean [+ or -] standard deviation (SD). Student's t-test was selected for the statistical analysis for comparison of two groups. The comparison of the PSA data between control and treated groups was performed by one-way analysis of variance (ANOVA) using the Bonferroni post hoc test corrections. Statistical significance was accepted when p [less than or equal to] 0.05.

Results

7-epi-nemorosone is cytotoxic in prostate carcinoma cells and shows no cross-resistance The cytotoxicity of 7-epi-nemorosone was analyzed in a set of well established PCa cell lines which differ in their molecular biology and thus represent different clinical manifestations of PCa. In order to elucidate the mechanism of action of the compound, we first examined LNCaP (lymph node carcinoma of prostate), a well known androgen-dependent cell line producing high enough PSA levels to be detectable in cell culture supernatants and in the serum of xenograft mouse models, which, in combination with tumor growth assessment, allows for the evaluation of the therapeutic index.

Etoposide is rarely used in clinical protocols due to its poor therapeutic outcome. But cell lines with acquired chemoresistance to etoposide show cross-resistance to cytostatics currently used in standard chemotherapy regimens for the treatment of PCa like mitoxantrone, taxanes, adriamycin, vinblastine, etc. The resistance is correlated with the over-expression of MDR1, a mechanism known to play a major role in chemotherapy resistance in PCa therapy. Thus, the use of etoposide for the development of chemotherapy-refractory cell lines is a rapid method to produce permanent MDR-phenotypes, and the resistant cell lines provide a useful tool to identify new therapeutics with the potential to overcome the resistance mechanism. Table 1 shows the cytotoxic spectrum of 7-epi-nemorosone in a range of PCa cell lines. No cross-resistance was found in cell lines with a high etoposide resistance factor.

TABLE 1
Cytotoxicity spectrum of 7-epi-nemorosone in a panel of
prostate carcinoma cell lines including etoposide-resistant
sub-lines. The [IC.sub.50] values were determined after
2411 of incubation with 7-epi-nemorosone using the MIT
proliferation assay. No cross-resistance was detected
in the cells with high resistance factor. Values
represent the mean [+ or -] standard deviation of at
least three independent experiments. RF, resistance
factor: [IC.sub.50] resistant/[IC.sub.50] parental
or wildtype (WT).

Cell line     Resistant  Resistance         ([micro]M)
                  to       factor (RF)   7-epi-nemorosone

LNCaP WT        -              -       4.12 [+ or -] 0.19

LNCaP, ETO   Etoposide      ~112       4.81 [+ or -] 0.68
[MDRI.sup.+]

PC-3 WC          -             -       5.01 [+ or -] 0.07

PC-3 ETO    Etoposide       ~150         5.1 [+ or -] 0.1
[MDRI.sup.+]

DU-145         -              -         7.3 [+ or -] 0.07

DU-145     Eroposide        ~120        6.8 [+ or -] 0.45
[MDRI.sup.+]


7-epi-nernorosone modulates the cell cycle distribution

The influence of 7-epi-nemorosone on cell cycle progression was studied in LNCaP parental cells by exposure to 4 [micro]M of the drug for 24 h (1 x [IC.sub.50]). 7-epi-nemorosone induced a significant increase in the sub-GO/G1 population which is generally accepted as the apoptotic fraction. A highly significant accumulation in GO/G1 correlating with S phase depletion was also noted. No changes were detected in the G2/M phase, as reflected in Fig. 2.

Cell cycle control elements are down-regulated by 7-epi-nemorosone

With the aim to further investigate the GO/G1 cell cycle arrest in LNCaP parental cells, some control elements which govern the progression from GO/G1 to S phase were investigated. After 24h of incubation, protein levels of cyclins D1 /D3 and CDK4/6 were found down-regulated in LNCaP parental cells at concentrations of 0.5-1 x [IC.50], as shown in Fig. 3. These events might account for the cell cycle arrest in GO/G1.

7-epi-nemorosone triggers apoptosis in LNCaP cells

Since the increased sub-G1 population may represents apop-totic cells, the occurrence of apoptotic events was analyzed in LNCaP parental cells. We first assessed the early stages of apoptosis induced by 7-epi-nemorosone by fluorescence microscopy-based visualization of changes in the cell membrane integrity through binding of extracellular Annexin V to phosphatidyl serine residues (Fig. 4A). Caspase-3 is considered to be a central apoptosis executor because it cleaves other proteins that are essential in late apoptosis. Cleaved - and thus activated - caspase-3 was detected by Western blot after exposure to 8 [micro]M (2 x [IC.sub.50]) of the drug for 24 h (Fig. 4B).

Signal transduction elements are deregulated by 7-epi-nemorosone

The influence of 7-epi-nemorosone on some pivotal cellular signal transducers was investigated. The protein expression levels of elements of different cascades related to cell cycle progression, like Akt/PKB, MEK1 /2, and ERK1 /2 were found to be changed as depicted in Fig. 5. Protein levels of Akt/PKB were down-regulated at concentrations near to 2 x [IC.sub.50] (8 [micro]M) for LNCaP cells after 24 h of exposure. Contrary to this, an up-regulation of both the protein levels and the phosphorylation status of MEK1 /2 were observed. ERK1 /2 protein levels were down-regulated, an effect which was more pronounced for the p44 (ERK1 ) isoform. Moreover, both ERK1 and ERK2 were found to be less phosphorylated after 7-epi-nemorosone treatment.

Influence of 7-epi-nemorosone on the phosphorylation status of ERK1/2 MAPK

The phosphorylation status of ERK1/2 MAPK was analyzed in LNCaP parental cells employing the semi-quantitative CASE technique. About 40% of ERK1/2 protein was phosphorylated in untreated LNCaP cells, and 7-epi-nemorosone induced a reduction of ERK1/2 phosphorylation - and thus deactivation - by more than 50% (Fig. 6).

In vitro inhibition of Akt/PKB and MEK1/2 by 7-epi-nemorosone

Active Akt/PKB is known to play a role in cell cycle progression leading to proliferation and survival. Previous reports described the inhibition of Akt/PKB kinase activity by 7-epi-nemorosone in neuroblastoma and leukemia cell lines to be correlated with disruption of the cell cycle (Diaz-Carballo et al. 2008a,b). The protein levels of Akt/PKB were down-regulated in LNCaP parental cells after 7-epi-nemorosone exposure in a concentration-dependent manner as shown in Fig. 5. On the other hand, as observed in other cell systems, the phosphorylation status of Akt/PKB increased in LNCaP parental cells as shown by CASE assay (data not shown). In order to investigate this phenomenon, we analyzed the enzymatic activity of Akt/PKB in a cell-free system by analyzing its ability to phosphorylate one of its substrates, GSK-3. Fig. 7A shows the total inhibition of Akt/PKB kinase activity in vitro at 2 x IC50 in LNCaP cells, suggesting that this pathway can be effectively deactivated by 7-epi-nemorosone.

Interestingly, 7-epi-nemorosone treatment also yielded contradictory results regarding ERK1 /2 and their upstream elements MEK1/2: while MEK1/2 protein levels and phospho-MEK1/2 levels were found to be up-regulated, phosphorylation of its substrates ERK1/2 decreased upon drug treatment (Fig. 5). This led us to hypothesize that MEK1/2 kinase activity may be inhibited by 7-epi-nemorosone, which could be confirmed in an in vitro system using MEK1/2 downstream elements ERK1/2 as substrates (Fig. 7B).

AR and PSA expression are modulated by 7-epi-nemorosone

Since it has been described that stress signaling kinases like p38 MAPK and MEK1 /2 regulate the phosphorylation, localization, and transcription of the nuclear receptor AR (Gioeli et al. 2006), the protein levels of AR were studied. Fig. 8 shows the down-regulation of AR in LNCaP parental cells in a concentration-dependent manner. AR gene expression analysis in LNCaP parental cells treated with 7-epi-nemorosone revealed a strong down-regulation of AR mRNA (data not shown).

Prostate-specific antigen (PSA) expression is often used to measure AR activity in prostate cells as well as PCa progression in patients. A correlation between regulation of AR, Akt/PKB and PSA expression has been described in PCa (Mikhailova et al. 2008). Since 7-epi-nemorosone was found to down-regulate both AR and Akt/PKB and inhibit the enzymatic activity of Akt/PKB, its influence on the protein level of PSA was studied. Fig. 9 shows the reduction in PSA protein levels in the supernatant of LNCaP cultures by increasing concentrations of 7-epi-nemorosone. This effect appears to be correlated to cell growth, as reflected.

Discussion

7-epi-nemorosone is a polyisoprenylated benzophenone that was first isolated from the floral resin of female Clusia grandiflora flowers and is a component of Caribbean propolis. It was shown to possess antimicrobial and antiviral activities (de Castro lshida et al. 2011: Serkedjieva et al. 1992). Recently, a study performed in an artificial retroviral system revealed that 7-epi-nemorosone inhibits HIV infectivity by more than 95% at 1 x [IC.sub.50] (Diaz-Carballo etal. 2010). Furthermore, the drug is cytotoxic in cancer cell lines derived from different tumor entities (Cuesta-Rubio et al. 2002; Diaz-Carballo et al. 2003, 2008a,b). Until now, only preliminary work has been published on the effect of 7-epi-nemorosoen on PCa (Cuesta-Rubio et al. 2002).

In tne present sway, t-epi-nemorosone was snown cytotoxicity in both androgen-dependent and independent PCa cell lines with [IC.sub.50] values between 4 and 7.5 [um]M. Like in leukemia and neuroblastoma, no cross-resistance was detected in chemotherapy-resistant cell lines. Thus, by overcoming chemoresistance which in PCa correlates with an [MDR1.sup.+] phenotype, 7-epi-nemorosone holds promise as a potential candidate for the treatment of chemorefractory PCa.

A variety of effects of 7-epi-nemorosone on cancer cells has been described, e.g. up-regulation of DNA damage inducible transcript 3 in pancreatic cancer cells leading to elevated activity of the unfolded protein response (UPR) network and apoptosis (Holtrup et al. 2011). or ATP depletion, [Ca.sup.++] release from [Ca.sup.++]-loaded mitochondria and protonophoric decoupling activity in HepG2 hepatocellular carcinoma cells leading to mitochondrial membrane potential dissipation and cell death (Pardo-Andreu et al. 2011). In addition, 7-epi-nemorosone binding to histone acetyl transferase (HAT) p300/CBP has bees described to enhance its enzymatic activity (Dal Piaz et al. 2010), which may be one facet of its cytotoxic effects seen in cancer cells. However, no direct mechanism of action could be inferred from these results.

In a first broad step toward understanding the cytotoxic effects of 7-epi-nemorosone, FACS analysis revealed a blockade of the cell cycle in GO/G1 phase, a concomitant depletion of the S phase population, and a partial induction of apoptosis in leukemia and neuroblastoma cells (Diaz-Carballo et al. 2008a,b; Popolo et al. 2011b). These effects could be confirmed for the androgen-dependent PCa cell line LNCaP. The detailed cell cycle analysis using the previously determined IC50 values as reference points showed that the GO/G1 arrest was reflected by the down-regulation of key regulators involved in cell cycle progression beyond the G1 /S checkpoint such as cyclins D1 /D3 and CDK 4/6. Again, like in neuroblastoma and leukemia cells, a significant increase in the sub-GO/G1 population was noted, suggesting apoptotic activity. The induction of apoptosis was further substantiated by Annexin-V-FITC binding to phosphatidyl serine which is transferred to the cell surface during membrane disintegration in early apoptosis, and by the activation of caspase-3, a potent effector of the cellular death program during late apoptosis, within 6 h of drug incubation (Vermes et al. 1995; Wilson 1998).

The clown-regulation of the cyclin-clepenclant louses 'mimeo in cell cycle progression led us to analyze specific pathways that promote survival and proliferation in cancer cells. We studied the possible influences of 7-epi-nemorosone on the MEK/ERK and Akt/PKB pathways, and the physiologic status of some of their key elements. Interestingly, MEK1 /2, a central member of the MEK/ERK pathway which is activated in a range of cancer entities was shown to be slightly up-regulated on the protein level and substantially hyperphosphorylated, suggesting its activation. At the same time, both the protein and phosphorylation levels of MEK1 /2 substrates ERK1 /2 were down-regulated in a differential manner, with the effect being more pronounced for ERK1 (p44) showing a differential influence of 7-epi-nemorosone on the functionality of ERK1 and ERK2, a hypothesis which is worth being studied in further experiments. In order to elucidate the ostensibly contradictory findings of MEK1 /2 hyperphosphorylation and decrease in substrate phosphorylation, the influence of 7-epi-nemorosone on the kinase activity of MEK1 /2 was studied in a cell-free system. The drug was found to substantially reduce the ability of MEK1 /2 to phosphorylate ERK1 /2 at 1 x [IC.sub.50]. This supports the hypothesis that 7-epi-nemorosone acts in part by targeting the kinase activity of MEK1 /2. Moreover, the fact that this small molecule acts synergistically with the Rafl inhibitor BAY 43-9006 in neuroblastoma cells supports the assumption that the RAS/RAF cascade is closely involved in mediating the cytotoxicity of 7-epi-nemorosone (Diaz-Carballo et al. 2008a). The up-regulation of MEK1 /2 protein and phosphorylation levels may be a cellular rescue response to overcome the enzymatic inhibition induced by 7-epi-nemorosone. This potential rescue response is not expected to have any significant downstream physiologic effects, though, because it may not override the enzymatic inhibition of MEK1 /2.

The PI3K signal transduction pathway governs anti-apoptotic events, promotes cell survival, and is found to be activated in many cancer entities. Like in leukemia and neuroblastoma, Akt/PKB, a key element of the P131< pathway, was shown to be down-regulated at the protein level in LNCaP PCa cells after exposure to 7-epi-nemorosone (Diaz-Carballo et al. 2008a,b). In addition, the ability of Akt/PKB to phosphorylate one of its substrates was strongly inhibited in a cell free system, as was the case for MEK1 /2. Interestingly, in estrogen receptor positive MCF-7 breast cancer cells, 7-epi-nemorosone lead to the inhibition of cell viability which correlated with the reduction of phospho-Akt/PKB and phospho-ERK1 /2 levels, the latter result corroborating the findings for LNCaP PCa cells (Popolo et al. 2011a).

Because Akt/PKB and MEK1 /2, among others, have been related to the transcription, localization and phosphorylation of AR (Benbrahim-Tallaa et al. 2007; Mikhailova et al. 2008), the physiologic status of AR was investigated in LNCaP cells. AR is a specific transducer involved in the tumorigenesis, adhesion and invasion of PCa and acts as a DNA binding transcription factor which regulates the expression of several genes (Mellado et al. 2009). AR activation controls G1 /S phase progression by enhancing translation of the D-type cyclins which in turn promote cell cycle progression through activation of CDK4/6 (Olshavsky et al. 2008). Furthermore, it has been shown that AR regulates the expression of the PSA gene (Kuroda et al. 2009; Mikhailova et al. 2008). Here, we show for the first time that 7-epi-nemorosone induces a drastic down-regulation of AR protein levels at less than 0.5 x [IC.sub.50], which correlates with the down-regulation of other signal transducers like ERK1 /2 and Akt/PKB. In accordance with the down-regulation of AR, 7-epi-nemorosone also blocked PSA production. Thus, the measurement of PSA levels in 7-epi-nemorosone-treated xenograft models may prove useful in assessing the therapeutic index of the drug (Graeser et al. 2008).

Taken together, we propose that 7-epi-nemorosone induces cell cycle disruption and apoptosis by targeting MEK1 /2 and Akt/PKB in the cell, which makes this compound a promising candidate for evaluation as an antitumoral drug in in vivo systems including chemoresistant [MDR1.sup.+] types, since both MEK1/2 and Akt/PKB are considered essential in PCa carcinogenesis and progression to more aggressive, androgen-independent entities (Mikhailova etal. 2008; Sundaram 2006; Tokunaga et al. 2008). Finally, future molecular biological experiments will aim at elucidating further targets in the cell, and in vivo studies will reveal the toxicity and antitumoral activity of 7-epi-nemorosone against PCa cells in animal models.

Conclusions

Here we demonstrate for the first time that 7-epi-nemorosone exerts cytotoxicity in an androgen-dependent PCa entity by down-regulation of AR protein levels, inhibition of PSA and targeting the MEK1/2 and Akt/PKB signal transducers.

Role of the funding sources

Grant support: Ministerium fur Innovation, Wissenschaft, Forschung und Technologie des Landes NRW (in frame of PURE-project) and Mildred Scheel Foundation (Grant: 108608); the foundations have no influence on the study design, the collection, the analysis and the interpretation of data, in the writing of the manuscript or in the decision to submit the manuscript for publication.

Acknowledgements

This investigation was supported by grants afforded by Min-isterium fur Innovation, Wissenschaft, Forschung uric' Technologie des Landes NRW (in frame of PURE-project) and the Mildred Scheel Foundation (Grant: 108608). Thanks to Jacqueline Klein, Ulrike Dembinski and Barbel Gobs-Hevelke for their excellent technical assistance.

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* Corresponding author. Tel.: +49 2323 499 1051: fax: +49 2323 499 1059. E-mail address: sebastian.gustmann@marienhospital-herne.de (S. Gustmann). 0944-7113/$ - see front matter [c] 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymec1.2012.08.004

David Diaz-Carballo (a), Sebastian Gustmann (a), (*), Ali Haydar Acikelli (a), Walter Bardenheuer (a), Helmut Buehler (a), Holgerfastrow (b), Suleyman Ergun (c), Dirk Strumberg (a)

(a.) Ruhr University of Bochum, Marienhospital Herne, HOlkeskainpring 40, D-44625 Herne, Germany

(b.) Division of Anatomy, University of Duisburg-Essen, School of Medicine, Hufelandstr. 55, D-45122 Essen, Germany

(c.) Institute of Anatomy and Cell Biology, Julius-Maximilians-University, Koellikerstr. 6, D-97070 Wurzburg Germany
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Author:Diaz-Carballo, David; Gustmann, Sebastian; Acikelli, Ali Haydar; Bardenheuer, Walter; Buehler, Helmu
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Date:Nov 15, 2012
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