The simplified code name and structure of MHY-449 [(±)-(R^*)-5-methoxy-11-methyl-2-((R^*)-2-methyloxiran-2-yl)-1,2-dihydrobenzofuro[4,5-b][1,8]naphthyridin-6(11H)-one] used in this study is shown in Fig. 1. Detailed method for the design and synthesis of this compound is described elsewhere (9). The compound was dissolved in dimethylsulfoxide (DMSO) and stored at −20°C before the experiments and dilutions were made in culture medium. The maximal concentration of DMSO did not exceed 0.1% (v/v) in the treatment range, where there was no influence on the cell growth. Antibodies specific for Bax, Bcl-2, caspase-3, -8, poly (ADP-ribose) polymerases (PARP), Akt, p-Akt, Forkhead-Box Class O (FoxO) 1, and p-FoxO1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against for extracellular signal-regulated kinases (ERK), phospho(p)-ERK, c-Jun N-terminal kinases (JNK), p-JNK, p38 mitogen-activated protein kinases (MAPK) and p-p38 MAPK were purchased from Cell Signaling (Beverly, MA, USA). PD98059 was from Santa Cruz Biotechnology.

The human prostate cancer cell lines p53-null PC3 and p53-wt LNCaP cells were cultured in RPMI-1640 (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, HyClone), 2 mM glutamine (Sigma-Aldrich Co., St. Louis, MO, USA), 100 U/ml penicillin (HyClone), and 100 μg/ml streptomycin (HyClone) at 37°C in a humidified 5% CO₂. Cell viability was determined by MTT assay. For the MTT assay, PC3 and LNCaP cells were seeded in a 24-well culture plate, cultured for 24 h in the growth media, and then treated with or without various reagents for the indicated concentrations. The cells were incubated with 0.5 mg/ml MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma-Aldrich) at 37°C for 2 h. The formazan granules generated by the live cells were dissolved in DMSO, and the absorbance at 540 nm was monitored by using a multi-well reader.

Cells were washed with PBS and fixed with 3.7% paraformaldehyde (Sigma-Aldrich) in PBS for 10 min at room temperature. Fixed cells were washed with PBS, and stained with 4 μg/ml Hoechst 33342 (Invitrogen, Eugene, OR, USA) for 20 min at room temperature. The cells were washed two more times with PBS and analyzed via a fluorescent microscope.

Cells were lysed in a buffer, containing 5 mM Tris-HCl (pH 7.5), 5 mM EDTA, and 0.5% Triton X-100, for 30 min on ice. Lysates were vortexed and cleared by centrifugation at 27,000 × g for 20 min. Fragmented DNA in the supernatant was treated with RNase, followed by proteinase K digestion, phenol/chloroform/isoamyl alcohol mixture (25:24:1, v/v/v) extraction and isopropanol precipitation. DNA was separated through a 1.6% agarose gel, and stained with 0.1 μg/ml ethidium bromide (EtBr, Fluka), and then visualized by UV source.

The DNA content was measured following the staining of the cells with propidium iodide (PI, Sigma-Aldrich). The cells were treated under the appropriate conditions for 24 h, subsequently trypsinized, washed once in cold PBS, and then fixed in 70% ethanol at −20°C overnight. The fixed cells were pelleted and stained in cold PI solution (50 μg/ml in PBS) at room temperature for 30 min in the dark. The stained cells were analyzed by flow cytometry (FC500, Beckman Coulter, Istanbul, Turkey).

The cells were harvested and washed with cold PBS. Total cells were lysed with the lysis buffer [40 mM Tris (pH 8.0), 120 mM, NaCl, 0.5% NP-40, 0.1 mM sodium orthovanadate, 2 μg/ml aprotinin, 2 μg/ml leupeptin and 100 μg/ml phenymethylsulfonyl fluoride (PMSF)] at 4°C for 30 min. The lysed cells were centrifuged at 10,000 × g for 2 min, and 100 μg of protein was incubated with 100 μl of reaction buffer and 10 μl of colorimetric tetrapeptides, Z-DEVDpNA for caspase-3, Z-IETD-pNA for caspase-8, and Ac-LEHD-pNA for caspase-9, respectively. The reaction mixture was incubated at 37°C for 30 min and liberated p-nitroaniline (pNA) was measured at 405 nm using a multi-well reader.

Annexin V-FITC is used to quantitatively determine the percentage of cells within a population that are actively undergoing apoptosis. The cells were treated under the appropriate conditions for 24 h, subsequently harvested, trypsinized, washed once in cold PBS, suspended the cells in 1X binding buffer (Becton-Dickinson, Annexin V-FITC Apoptosis Detection Kit). The counted cells were stained in PI and Annexin V-FITC solution (Becton-Dickinson, Annexin V-FITC Apoptosis Detection Kit) at room temperature for 15 min in the dark. The stained cells were analyzed by flow cytometry within 1 h.

The cells were treated under the appropriate conditions, harvested and washed with cold PBS. Total cells were lysed in lysis buffer [40 mM Tris (pH 8.0), 120 mM NaCl, 0.5% NP-40, 0.1 mM sodium orthovanadate, 2 μg/ml aprotinin, 2 μg/ml leupeptin and 100 μg/ml phenymethylsulfonyl fluoride (PMSF)]. Protein extracts were denatured by boiling at 100°C for 5 min in sample buffer (0.5 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.1% bromophenol blue, 10% β-mercaptoethanol). Equal amount of the total proteins were subjected to 6–15% SDS-PAGE and transferred to PVDF membrane. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with Tween-20 buffer (TBS-T; 20 mM Tris, 100 mM NaCl, pH 7.5 and 0.1% Tween-20) for 1 h at room temperature. Then, the membranes were incubated overnight at 4°C with the primary antibodies. The membranes were washed with TBS-T buffer and incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobin (Santa Cruz Biotechnology). The membranes were washed again with TBS-T buffer. Antigen-antibody complexes were detected by the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences Co., Little Chalfont, UK).

Results were expressed as the mean ± SD of three separate experiments and analyzed by Student’s t-test. Means were considered significantly different at ^*p<0.05 or ^**p<0.01.

To evaluate the effects of MHY-449 on cell growth of a human prostate cancer cell line, the MTT assay was performed. As shown in Fig. 2, treatment with MHY-449 showed concentration- and time-dependent cytotoxicity on p53-null PC3 and p53-wt LNCaP cells. However, p53-null PC3 cells were more sensitive to MHY-449 than p53-wt LNCaP cells. The IC₅₀ value of MHY-449 was approximately 0.75 and 0.5 μM at 24 and 48 h in PC3 cells, respectively, whereas the IC₅₀ value of MHY-449 was approximately 1 μM at 48 h in p53-wt LNCaP cells.

To investigate whether the growth inhibitory effects of MHY-449 were due to the induction of apoptosis in LNCaP and PC3 cells, micro-photographs were observed. LNCaP and PC3 cells treated with MHY-449 showed apparently distinct morphological changes compared with control (Fig. 3A). Although p53-wt LNCaP cells did not show significant decrease of cell numbers and morphological changes with MHY-449 treatment (Fig. 3Aa-d), p53-null PC3 cells showed prominent decrease of cell numbers and morphological changes concentration-dependently (Fig. 3Ae-h), and they were rounded and more dispersed with aggregation. Further morphological changes of cellular structures were assessed with Hoechst 33342 staining. As shown in Fig. 3Be-h, nuclei with chromatin condensation and formation of apoptotic bodies, which are characteristics of apoptosis, were seen in p53-null PC3 cells cultured with MHY-449 concentration-dependently, whereas the control cells maintained the nuclear structure intact. However, p53-wt LNCaP cells did not show any morphological changes of the nuclei after MHY-449 treatment. We also analyzed whether DNA fragmentation, another hallmark of apoptosis, was induced by MHY-449 treatment on LNCaP and PC3 cells. Following agarose gel electrophoresis of LNCaP and PC3 cells treated with MHY-449 for 24 h, a typical ladder pattern of internucleosomal fragmentation was observed in a concentration-dependent manner in p53-null PC3 cells, but not in p53-wt LNCaP cells (Fig. 4A). Polypeptide degradation, including poly(ADP-ribose) polymerase (PARP), was examined to see the possible involvement of apoptosis-associated protease during the growth inhibition of PCa cells. PARP cleavage was evident by the appearance of the p85 PARP cleavage fragment and clearly observed in the 0.25, 0.5 and 1.0 μM of MHY-449 treatment in p53-null PC3 cells (Fig. 4B, right panel). However, p53-wt LNCaP cells did not show any PARP cleavage after MHY-449 treatment (Fig. 4B, left panel). These results suggest that MHY-449 induced apoptotic cell death in p53-null PC3, not in p53-wt LNCaP cells. Therefore, p53-null PC3 cells were used for further studies.

To confirm apoptosis of MHY-449 in p53-null PC3 cells, flow cytometry analysis was performed. As shown in Fig. 5A, increases of early apoptosis (lower right quadrant) and late apoptosis/death (upper right quadrant) were clearly observed concentration-dependently. Moreover, the percentages of apoptotic cells, sub-G1 fraction, were significantly increased in p53-null PC3 cells treated with MHY-449 in a concentration-dependent manner (Fig. 5B).

To determine whether the expression levels of apoptosis-related proteins were modulated by MHY-449 in PC3 cells, western blot analysis was performed. The expression level of Bcl-2 protein was markedly downregulated, while Bax was upregulated in a concentration-dependent manner (Fig. 6A). The ratio between Bcl-2 and Bax has been suggested as a primary event in determining the susceptibility to apoptosis (10). These data suggest that MHY-449 induces apoptosis by the alteration in expression ratio of Bax/Bcl-2 protein. In an attempt to further characterize the mechanisms of apoptosis induced by MHY-449, the activities of caspase -3, -8 and -9 were determined by colorimetric assay. The activities of caspase-3 and -8 were increased with the treatment of MHY-449 concentration-dependently and there was little change in caspase-9 activity (Fig. 6B). Therefore, these results suggested that MHY-449 induce caspase-dependent apoptosis in p53-null PC3 cells.

To clarify the molecular mechanism of apoptotic cell death in p53-null PC3 cells, we hypothesized that MHY-449-induced apoptosis in p53-null PC3 cells was due to inhibition of Akt/FoxO pathway. To test our hypothesis, the level of Akt was first examined in whole lysate by western blot analysis. p53-null PC3 cells are also PTEN-negative via homozygous deletion (11). Therefore, deletion of PTEN in PC3 cells results in increased levels of phosphatidyl-inositol 3,4,5 triphosphate which increases activated Akt (12). As shown in Fig. 7A, phosphorylation level of Akt (p-Akt) was highly increased in vehicle-treated PC3 cells. However, p-Akt, not Akt, was drastically suppressed by MHY-449 treatment in a concentration-dependent manner. Next, we investigated the downstream molecules of Akt pathway such as FoxO1, which is known to be critical in carcino-genesis. Our results showed that phosphorylated level, but not protein level, of FoxO1 was also decreased in PC3 cells treated with MHY-449. This result clearly indicates that the reduced phosphorylated level of FoxO1 was not due to the alteration of protein expression of FoxO1 by MHY-449 treatment (Fig. 7A). Phosphorylation of FoxO1 by Akt leads to interaction with chaperone protein, such as 14-3-3, which serve as escort proteins for FoxO1 to move out of the nucleus (13) leading to transcriptional downregulation of several target proteins, such as Bim, p21WAF1/CIP1, and p27KIP1 (14). Thus, we assessed whether MHY-449 affects nuclear localization of FoxO1 by immunoblot analysis of nuclear and cytosolic fractions with FoxO1 antibody. Our results showed that FoxO1 protein level steadily increased in nuclear fraction and decreased in cytosolic fraction of MHY-449-treated p53-null and PTEN-negative PC3 cells (Fig. 7B).

To explore other intracellular signaling pathways that might be involved in MHY-449-induced apoptosis, we monitored activation of mitogen-activated protein kinases (MAPK), such as ERK, p38 MAPK and JNK by western blot analysis with phospho-specific antibodies. MHY-449 treatment of PC3 cells resulted in activation of ERK, but not in p38 MAPK and JNK, in a concentration-dependent manner (Fig. 8A). To determine whether MHY-449-induced ERK activation was required for cell toxicity, PC3 cells pretreated with PD98059, an ERK inhibitor, was subsequently exposed to MHY-449 for 24 h, and then cell death was measured by MTT assay. Pretreatment with the ERK inhibitor protected against MHY-449-induced cytotoxicity (Fig. 8B). Similarly, pretreatment of ERK inhibitor abolished MHY-449-induced downregulation of pro-caspase-8 and -3 and PARP cleavage (Fig. 8C). These results suggest that cell death by MHY-449 treatment requires at least activation of the ERK pathway.