Meridianin C and its derivatives (compounds 7a-j) were synthesized as previously described (27) and prepared as 10 mM stock solutions in DMSO. AZD1208 (cat. no. S7104) was purchased from Selleck Chemicals. RPMI-1640 (cat. no. LM011-01), fetal bovine serum (FBS; S001-01), and penicillin/streptomycin cocktail (cat. no. LS202-02) were purchased from Welgene, Inc. Anti-procaspase-9 antibody (cat. no. ADI-AAM-139) was purchased from Enzo Life Sciences, Inc. Anti-death receptor (DR)-5 antibody (cat. no. NBP1-45951) was purchased from Novus Biologicals. Anti-poly(ADP-ribose) polymerase (PARP) antibody (cat. no. 11835238001) was purchased from Roche Diagnostics; anti-β-actin antibody (cat. no. A5441) was obtained from Sigma-Aldrich; Merck KGaA; anti-human X-linked inhibitor of apoptosis protein (XIAP) antibody (cat. no. AF221) was purchased from R&D Systems, Inc. Anti-phosphorylated (p)-eukaryotic initiation factor (eIF)-2α (S51) antibody (cat. no. ab32157) was purchased from Abcam. Anti-p-S6 (S235/236; cat. no. 2211), anti-S6 (cat. no. 2317), anti-eIF-2α (cat. no. 9722), anti-p-extracellular signal-regulated kinase (ERK)-1/2 (T202/Y204; cat. no. 9101), and anti-ERK-1/2 (cat. no. 9102) antibodies were purchased from Cell Signaling Technology, Inc. Anti-DR-4 (cat. no. sc-8411), anti-PIM-1 (cat. no. sc-13513), anti-PIM-2 (cat. no. sc-271844), anti-PIM-3 (cat. no. sc-293237), anti-p-Bcl-2-associated death promoter (BAD) (Ser112) (cat. no. sc-7998), anti-BAD (cat. no. sc-8044), anti-glucose-regulated protein (GRP)-78 (cat. no. sc-13968), anti-Bcl-2 (cat. no. sc-509), anti-myeloid cell leukemia (Mcl)-1 (cat. no. sc-819), secondary goat anti-rabbit (cat. no. sc-2004), and goat anti-mouse IgG (cat. no. sc-2005) antibodies were purchased from Santa Cruz Biotechnology, Inc. z-VAD-fmk (627610) and protease inhibitor cocktail (PIC, 100X) (539134) were purchased from EMD Millipore. Super Signal™ West Pico PLUS Enhanced chemiluminescence (ECL; cat. no. 34080) was purchased from Thermo Fisher Scientific, Inc. Well plates (6- and 24-wells) were obtained from SPL Life Sciences.

MV4-11 human AML cells [CRL-9591™, American Type Culture Collection (ATCC)] and K562 human chronic myeloid leukemia (CML) cells (CCL-243™, ATCC) were grown in RPMI-1640 supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin at 37°C in humidified air (95% air and 5% CO₂). MV4-11 and K562 cells carried a Fms-like tyrosine kinase 3 internal tandem duplication and a breakpoint cluster region-Abelson mutation, respectively (29,30).

MV4-11 or K562 cells were treated with vehicle control (DMSO; 0.1%), meridianin C, meridianin C derivatives and/or AZD1208 at indicated concentrations (1, 5 and 10 µM) for different durations (24 and 48 h). The number of surviving cells was counted with the trypan blue exclusion method, which based on the principle that live cells have intact cell membranes and cannot be stained. Briefly, equal amounts (15 µl) of 0.4% trypan blue dye (cat. no. 15250-061, Gibco; Thermo Fisher Scientific, Inc.) were added to the cell suspension and mixed by pipetting up and down. The mixture was incubated for 1 min at room temperature (25-27°C), after which time 10 µl of the mixture was added to the hemocytometer and cells were counted under a phase-contrast microscope. Approximately 50 cells were counted in each evaluation.

MV4-11 cells were treated with vehicle control (DMSO; 0.1%), meridianin C, meridianin C derivatives and/or AZD1208 at indicated concentrations (1, 5 and 10 µM) for different durations (24 and 48 h). At the end of treatment, 20 µl of MTS solution was added to each well, and the plates were incubated at 37°C for 1 h. The absorbance of each well was measured at 490 nm using a microplate reader (SPECTRA max 340PC; Molecular Devices, LLC).

Measurement of DNA fragmentation was conducted as previously described (21). Briefly, MV4-11 cells were seeded into 6-well plates at a density of 2×10⁵ cells/ml with a volume of 2 ml in each well on the day prior to treatment. Cells were treated with vehicle control (DMSO) or compound 7a (1, 5 and 10 µM) and/or z-VAD-fmk (50 µM) at the indicated concentrations for 24 and 48 h. MV4-11 cells were then harvested, washed and lysed in a lysis buffer [50 mM Tris (pH 8.0), 0.5% sarkosyl, 0.5 mg/ml proteinase K and 1 mM EDTA] at 55°C for 3 h. Subsequently, RNase A (0.5 µg/ml) was added and the lysate was further incubated at 55°C for 18 h. Finally, the lysate was centrifuged at 10,000 × g at 4°C for 20 min. Genomic DNA was extracted and analyzed via electrophoresis at 100 V on a 1.8% agarose gel for 20 min. The DNA was visualized and photographed under UV illumination after staining with ethidium bromide (0.1 µg/ml; Sigma-Aldrich; Merck KGaA) using a gel documentation system (Gel Doc-XR; Bio-Rad Laboratories, Inc.).

MV4-11 cells were grown in 6-well plates at a density of 0.25×10⁶ cells/ml on the day prior to treatment. After treatment and at each time point, the cells were washed twice with PBS, and proteins were extracted using modified RIPA buffer [50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 0.1% sodium dodecyl sulfate, 0.25% sodium deoxycho-late, 1% Triton X-100, 1% Nonidet P-40, 1 mM EDTA, 1 mM EGTA and PIC (1X)]. Cell lysates were collected and centrifuged at 12,074 × g for 20 min at 4°C. Protein concentration in the supernatant was determined by using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.).

Western blotting was performed as previously described (10,21). Briefly, equal amounts of protein (50 µg) were separated via 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (EMD Millipore) by electroplating. The membranes were washed with Tris-buffered saline (TBS; 10 mM Tris, 150 mM NaCl, pH 7.5) supplemented with 0.05% (v/v) Tween-20 (TBS-T), followed by blocking with TBS-T containing 5% (w/v) non-fat dried milk. The membranes were probed overnight using antibodies against PIM-1 (1:2,000), PIM-2 (1:2,000), PIM-3 (1:2,000), p-BAD (1:2,000), T-BAD (1:2,000), procaspase-9 (1:2,000), procaspase-3 (1:2,000), DR-4 (1:2000), DR-5 (1:2,000), PARP (1:2,000), XIAP (1:1,000), Bcl-2 (1:1,000), Mcl-1 (1:1,000), p-eIF-2α (1:2,000), T-eIF-2α (1:2,000), GRP-78 (1:1,000), p-S6 (1:3,000), S6 (1:3,000), p-ERK-1/2 (1:2,000), T-ERK-1/2 (1:2,000) or β-actin (1:10,000) at 4°C, followed by incubation with secondary antibodies conjugated to horseradish peroxidase at room temperature for 2 h. The membranes were washed, and immune reactivities were detected by Super Signal™ West Pico PLUS ECL (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Equal protein loading was assessed via β-actin expression levels.

Total cellular RNA from conditioned MV4-11 cells was isolated using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RT-PCR was performed as previously described (10,21). Briefly, equal amounts of total RNA (5 µg) were reverse-transcribed in a 40-µl reaction mixture containing 8 µl Molony Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) 5X buffer, 3 µl 10 mM dNTPs, 0.45 µl 40 U/µl RNase inhibitor, 0.3 µl 200 U/µl M-MLV RT (Promega Corporation) and 3.75 µl 20 µM oligo dT (Bioneer Corporation). Single-stranded cDNA was amplified by PCR using 4 µl 5X Green Go-Taq^® Flexi reaction buffer, 0.4 µM 10 mM dNTPs, 0.1 µl 5 U/µl Taq polymerase, 1.2 µl 25 mM MgCl₂ (Promega Corporation), and 0.4 µl primer (20 pM/µl). The following primer pairs were used: DR-4 sense, 5′-CTGAGCAACGCAGACTCGCTGTCCAC-3′ and antisense, 5′-AAGGACACGGCAGAGCCTGTGCCAT-3′; DR-5 sense, 5′-AGCCGCTCATGAGGAAGTTGG-3′ and antisense, 5′-GGCAAGTCTCTCTCCCAGCGTCTC-3′ Mcl-1 sense, 5′-ATCTCTCGGTACCTTCGGGAG-3′ and antisense, 5′-ACCAGCTCCTACTCCAGCAAC-3′; Bcl-2 sense, 5′-GTGGAGGAGCTCTTCAGGGA-3′ and antisense, 5′-AGGCACCCAGGGTGATGCAA-3′; XIAP sense, 5′-CGTCGATTTTGTGCTCGTCAG-3′ and antisense, 5′-GAAGCATTTATCAGGGTTATTGTCTCATG-3′; and actin sense, 5′-TCAAGATCATTGCTCCTCCTG-3′ and antisense, 5′-CTG CTTGCTGATCCACATCTG-3′. The PCR conditions were as follows: For DR-4 and DR-5: 35 cycles of denaturation at 95°C for 30 sec, annealing at 63°C for 30 sec and extension at 72°C for 30 sec; for Mcl-1: 25 cycles of denaturation at 95°C for 45 sec, annealing at 56°C for 45 sec and extension at 72°C for 45 sec; for Bcl-2: 30 cycles of denaturation at 95°C for 45 sec, annealing at 56°C for 45 sec and extension at 72°C for 45 sec; for XIAP: 30 cycles of denaturation at 95°C for 35 sec, annealing at 54°C for 40 sec and extension at 72°C for 30 sec; and for β-actin: 25 cycles of denaturation at 95°C for 30 sec, annealing at 56°C for 30 sec and extension at 72°C for 30 sec. β-actin was used as an internal control to evaluate the relative expressions of DR-4, DR-5, Mcl-1, Bcl-2 and XIAP.

Cell count analysis and MTS assay were performed in triplicate and repeated three times. All other data were obtained via at least three independent measurements. Data are expressed as mean ± standard error of the mean. One-way ANOVA followed by Dunnett's post hoc test was performed using SPSS 11.5 software (SPSS, Inc). P<0.05 was considered to indicate statistically significant differences.

We first examined the effects of meridianin C and its derivatives (compounds 7a-j) at a concentration of 10 µM for 24 and 48 h on the survival and viability of MV4-11 cells. The parent compound meridianin C did not markedly affect cell growth. By contrast, some of its derivatives, such as compounds 7a, 7g and 7j, strongly reduced MV4-11 survival and viability after treatment for 24 and 48 h (Fig. 1A and B). We next investigated the effects of compounds 7a, 7g and 7j on MV4-11 cell survival and viability at different concentrations (1, 5 and 10 µM) and treatment durations (24 or 48 h) (Fig. 1C and D). The highest reduction in MV4-11 cell survival and viability was achieved by compound 7a in a concentration-dependent manner. The effects of compounds 7g and 7j were substantial as well. Compared with the other compounds, meridianin C exerted no or a very weak inhibitory effect on MV4-11 survival and viability within the tested concentration and for the specified treatment durations. Taking into consideration the PIM kinase inhibitory activities of meridianin C derivatives (27), we next investigated whether MV4-11 cells expressed PIM kinases and whether compound 7a affected PIM kinase expression and activity. PIM kinase activity was assessed by measuring the levels of phosphorylated BAD (Ser112), a downstream target of PIM kinases (31). High and low levels of PIM-3 and PIM-1/PIM-2, respectively, were detected in control MV4-11 cells (Fig. 1E). BAD phosphorylation was also observed. Treatment with compound 7a for 24 and 48 h inhibited PIM kinase activity in MV4-11 cells, as evidenced by reduced PIM kinase expression and decreased BAD phosphorylation levels. Densitometry data (PIM-3/actin or p-BAD/T-BAD ratio) were obtained by analyzing Fig. 1E using ImageJ software (version 1.8.0; National Institutes of Health) (Fig. 1F). The 2D structures of meridianin C and compound 7a are shown in Fig. 1G. Due to the stronger inhibitory effect of compound 7a on MV4-11 cell growth, we only focused on this particular meridianin C derivative in the following analyses.

Whether treatment with compound 7a induced apoptosis in MV4-11 cells was next determined by measuring the level of nuclear DNA fragmentation, a hallmark of apoptosis. Treatment with 5 and 10 µM compound 7a for 24 or 48 h resulted in marked accumulation of nuclear DNA fragments in MV4-11 cells (Fig. 2A). The expression and activation levels of caspases in MV4-11 cells treated with compound 7a were next determined in order to investigate a possible association between compound 7a-induced apoptosis and caspase activation. Treatment with 5 or 10 µM compound 7a increased and decreased the levels of upstream caspase-9 (proteolytically cleaved active caspase form) and downstream effector caspase-3 (inactive caspase proform) in MV4-11 cells, respectively (Fig. 2B). Caspase activation often leads to the proteolytic cleavage of several target proteins, such as PARP. PARP cleavage was observed in MV4-11 cells following treatment with compound 7a for 24 and 48 h. The densitometry data of Fig. 2B are shown in Fig. 2C-E. Finally, DR-4 and -5 expression levels were measured in MV4-11 cells treated with compound 7a for 24 h in order to assess whether compound 7a-induced apoptosis occurred via the extrinsic pathway. The protein and mRNA expression levels of DR-4 and -5 were unaffected in MV4-11 cells treated with 1, 5 and 10 µM of compound 7a (Fig. 2F and G). Actin expression levels (control) also remained constant.

AZD1208 is a highly selective pan-PIM kinase inhibitor (32). We herein evaluated a possible association between the anti-survival and pro-apoptotic effects of compound 7a and PIM kinase inhibition. For this purpose, MV4-11 cells were treated with compound 7a, meridianin C or AZD1208 at different concentrations (1, 5 and 10 µM), and cell survival, PARP cleavage and PIM kinase expression and activity levels were measured in the conditioned cells. As expected, compound 7a reduced the survival of MV4-11 cells in a concentration-dependent manner (Fig. 3A), and inhibited the expression and activity of PIM kinases (Fig. 3B). There was also slight and marked accumulation of cleaved PARP in MV4-11 cells treated with 5 µM and 10 µM compound 7a, respectively. However, AZD1208 and meridianin C exerted weaker inhibitory effects on MV4-11 survival compared with compound 7a within the tested concentration range. AZD1208 did not affect the expression of PIM kinases, but weakly inhibited BAD phosphorylation in MV4-11 cells. Moreover, PARP cleavage was not observed in MV4-11 cells treated with AZD1208. Meridianin C exerted little or no effect on PARP cleavage in MV4-11 cells, although it strongly inhibited the expression and activity of PIM kinases. Densitometry data of Fig. 3B are shown in Fig. 3C-E. Actin expression (control) was unchanged. Additionally, the viability of MV4-11 cells treated with combinations of compound 7a and AZD1208 was determined at different concentrations (1, 5 and 10 µM) for 24 h, and it was observed that the combination of AZD1208 and compound 7a did not markedly affect the viability of MV4-11 cells (Fig. 3F). These results indicate that AZD1208 does not affect the activity of compound 7a and the activity of compound 7a is not associated with any effects on PIM kinases.

The molecular signaling mechanisms underlying the growth-suppressive and apoptosis-inducing effects of compound 7a were investigated. For this purpose, MV4-11 cells were cultured with or without 5 µM compound 7a for 2, 4, 8 or 24 h, and the expression and phosphorylation levels of growth and apoptosis-related factors were measured. Compound 7a treatment led to early activation of caspase-9 and -3 in MV4-11 cells, as evidenced by increased and decreased expression levels of active caspase-9 and procaspase-3, respectively (Fig. 4A). Compound 7a treatment for 4 h also led to a marked decrease in PARP protein levels. Moreover, the expressions of Mcl-1 and XIAP proteins were strongly downregulated after 2 h of treatment with compound 7a in MV4-11 cells, followed by a further decline in levels of these proteins. eIF-2α phosphorylation increased, whereas S6 phosphorylation decreased in MV4-11 cells after 2 h of treatment with compound 7a. The expression of GRP-78, an endoplasmic reticulum (ER) stress marker, and the phosphorylation of ERK-1/2, a member of the MAPK family involved in cancer cell growth, remained unchanged in MV4-11 cells upon compound 7a treatment. Total eIF-2α, S6 and ERK-1/2 expression levels remained constant throughout the experiment, except for the significant decrease in the total eIF-2α and S6 expression levels at 24 h of compound 7a treatment. Treatment with compound 7a for 2, 4 or 8 h did not significantly affect Mcl-1 mRNA expression in MV4-11 cells; however, Mcl-1 transcript levels substantially decreased with 24 h of treatment (Fig. 4B). By contrast, the XIAP, DR-4 and DR-5 mRNA levels remained unchanged in compound 7a-treated MV4-11 cells for all treatment durations. Actin mRNA expression (control) also remained constant.

The role of caspases in compound 7a-induced apoptosis in MV4-11 cells was next investigated via treatment with the pan-caspase inhibitor z-VAD-fmk. Treatment with 50 µM z-VAD-fmk strongly inhibited compound 7a-induced DNA fragmentation (5 µM, Fig. 5A), caspase-9 and -3 activation and PARP cleavage (Fig. 5B). Moreover, z-VAD-fmk abolished the ability of compound 7a to induce cleaved Bcl-2 and Mcl-1 and XIAP downregulation. Actin protein expression (control) remained constant.

Finally, K562 CML cells were treated with compound 7a or meridianin C at different concentrations (1, 5 and 10 µM) for different durations (24 and 48 h), and K562 cell survival was measured in order to determine whether the growth inhibitory effect of compound 7a is limited to MV4-11 cells. Meridianin C and compound 7a weakly and strongly reduced the survival of K562 cells, respectively (Fig. 6A and B). In particular, treatment with 5 µM compound 7a, rather than meridianin C, for 24 and 48 h resulted in a markedly higher reduction of K562 survival.