All the solvents used in the experiments were of extra-pure grade. Hexane, ethyl acetate and acetonitrile were purchased from J.T. Baker (Phillipsburg, NJ, USA). Silica gel for thin layer chromatography and precoated silica gel plate (Kieselgel 60F254; Merck, NJ, USA) was used. Silica gel for silica gel column, Kieselgel 60 (70–230 mesh; Merck), was used to purify manumycin. CS392 was grown on a rotary shaker at 180 rpm in Emerson media for 2–3 days at 28°C. Culture broth (3L) was centrifuged at 6,000 rpm for 20 min. The supernatant was extracted two times with ethyl acetate (1:1, v/v). The extracted ethyl acetate fraction was evaporated and dried using a rotary evaporator at 50°C under reduced pressure. Purification of the antibiotic was carried out by silica gel column chromatography (0.8×15 cm). After washing the column with hexane, active material was eluted from the column with hexane-ethyl acetate (4:1). Active fractions were collected and rechromatographed, using a reverse phase-C18 silica gel column (1.0×15 cm) with 0.01% formic acid-acetonitrile (4:6) to isolate manumycin.

The human MPM MSTO-211H and H28 cells were purchased from the American Tissue Culture Collection (ATCC; Manassas, VA, USA). The MSTO-211H and H28 cells were grown in RPMI-1640 medium, supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 100 U/ml each of penicillin and streptomycin (Thermo Scientific, Logan, UT, USA) at 37°C in a humidified atmosphere of 5% CO₂ and 95% air.

Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) dye-reduction assay. MSTO-211H (3×10³) and H28 (2×10³) cells were seeded into 96-well plates for 24 and 48 h treated with various concentrations (2.5, 5 and 10 µM) of Manu A. The cells were incubated with MTS solution for 2 h. Absorbance was determined using an EnSpire Multimode plate reader (Perkin-Elmer, Waltham, MA, USA) at 490 nm. Experiments were carried out in triplicates on different days. The percentage of viability was calculated as: Viable cells (%) = (Manu A-treated cell to measure the absorbance - Manu A-treated blank wells (no cells) to measure the background absorbance)/absorbance of the untreated sample × 100.

MSTO-211H and H28 cells treated with Manu A were harvested by trypsin and fixed in 100% methanol at room temperature (RT) for 2 h. The cells were spread on slides, stained with DAPI solution (2 µg/ml) and analyzed under a FluoView confocal laser microscope (Fluoview FV10i; Olympus Corporation, Tokyo, Japan).

The cells were seeded on 6-well plates for MSTO-211H, H28 and treated with various concentrations of Manu A for 48 h (2.5, 5 and 10 µM). The cells were harvested by trypsinization and were incubated with Annexin V/7-aminoactinomycin (7-AAD) for 20 min at RT in the dark for detection of apoptosis. Apoptotic and necrotic cells were analyzed by Muse Cell analyzer (Merck Millipore, Billerica, MA, USA) using the Muse Annexin V & Dead Cell kit (MCH100105; Merck Millipore). The experiment was performed in triplicate independently.

Total RNA was extracted from the cells using TRIzol^® reagent (Life Technologies, Carlsbad, CA, USA), and 2.5 µg of RNA was used to synthesize cDNA using the HelixCript™ first Strand cDNA Synthesis kit (NanoHelix, Korea). Amplimers were obtained by PCR using β-actin-specific and Sp1-specific primers as described below under following PCR conditions (25 cycles: 1 min at 95°C, 1 min at 56°C, and 1 min at 72°C). The β-actin primers used were; forward, 5′-GTG GGG CGC CCC AGG CAC CA-3′ and reverse, 5′-CTC CTT AAT GTC ACG CAC GAT TTC-3′; and the Sp1 primers were; forward, 5′-ATG CCT AAT ATT CAG TAT CAA GTA-3′ and reverse, 5′-CCC TGA GGT GAC AGG CTG TGA-3′. PCR products were analyzed by 2% agarose gel electrophoresis.

Western blot analyses were performed using cell lysates. Lysates of the treated cells were prepared using PRO-PREP™ protein extraction solution (iNtRON Biotechnology, Korea), followed by centrifugation and supernatant collection. Protein samples were separated by 10 or 15% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Darmstadt, Germany) using standard procedures. The primary antibodies used were: Sp1, actin, poly (ADP-ribose) polymerase (PARP), cyclin D1, survivin, BID, Bcl-xL, Bax, CHOP, DR4 and DR5 (Santa Cruz Biotechonology, Santa Cruz, CA, USA) antibodies. The protein bands were detected using ECL Plus Western Blotting detection system (Santa Cruz Biotechnology).

To investigate the mitochondrial membrane permeability, the control and Manu A-treated (2.5, 5 and 10 µM) cells were harvested. After washing with phosphate-buffered saline (PBS), the cells were dissociated with trypsin. For detection of the depolarized mitochondria of cells undergoing apoptosis, MitoPotential working solution was added to the MPM cell culture and then reaction was carried out for 20 min in the dark. Muse 7-AAD was added to each well and samples were incubated in the dark at RT for 5 min. The experiment was analyzed by Muse cell analyzer.

The process was carried out as instructed in the Muse Multi-Caspase kit (Merck Millipore). Each group, including negative and positive controls was harvested to quantitatively measure caspase activation and cell permeability. Cell samples in 1X caspase buffer with Muse Multi-Caspase reagent working solution were incubated at 37°C for 30 min. Then, 7-AAD working solution was added to each triplicate sample and samples were analyzed by Muse Cell analyzer.

Data are presented as mean ± SD of at least three independent experiments performed in triplicate. Data were analyzed for statistical significance using a one-way analysis of variance. A value of p<0.05 was considered significant.

To examine the cytotoxic effect of Manu A on the MSTO-211H (Fig. 1A) and H28 (Fig. 1B) MPM cell lines, MTS assay was carried out after treatment with Manu A at various concentrations (2.5, 5 and 10 µM) for different times (24 and 48 h). The IC₅₀ of Manu A for cytotoxicity in the MSTO-211H and H28 cells was 8.3 and 4.3 µM, respectively. Furthermore, to confirm the inhibitory effects of Manu A on cancer cell proliferation through apoptosis, we investigated morphological nuclear changes through DAPI staining. Manu A treatment effectively induced nuclear changes in the MPM cells in a dose-dependent manner. To quantify the cell death induced by Manu A, MPM cells were treated with Manu A at various concentrations (2.5, 5 and 10 µM), and then we measured the number of apoptotic and necrotic cells by flow cytometry after staining with Annexin V and 7-AAD. The numbers of live cell were significantly reduced while cells undergoing apoptosis were increased in a concentration-dependent manner. In the MSTO-211H (Fig. 1E) and H28 cells (Fig. 1F), the total apoptotic cell population was increased from 11.15±0.9 to 95.80±0.6% (MSTO-211H) and from 0.93±0.9 to 90.25±1.2% (H28), respectively.

Sp1 plays an important role in oncogenesis, regulation of cell survival and death (13). To demonstrate the link between Sp1 and apoptosis, we investigated the expression level of Sp1 by RT-PCR and western blotting after treatment of the MPM cells with Manu A (2.5, 5 and 10 µM) for 48 h. Sp1 mRNA expression levels in the MSTO-211H (Fig. 2A) and H28 (Fig. 2B) cells were reduced by Manu A in a dose-dependent manner. Consistent with mRNA levels, the Sp1 protein levels in the MSTO-211H and H28 cells were decreased in a dose-dependent manner (Fig. 2C and D). Sp1 is a transcription factor that modulates cell cycle regulation, anti-proliferative, and apoptosis cell death by regulating the promoter of target genes (14,32). Moreover, Sp1 regulates expression of many proteins such as p21, p27, cyclin D1, Mcl-1 and survivin (33). Among a variety of genes involved, in this experiment, cyclin D1, Mcl-1 and survivin were investigated. After treatment of the cells with Manu A for 48 h, Sp1 target proteins in the MSTO-211H (Fig. 3A) and H28 cells (Fig. 3B) were downregulated.

There are two pathways which execute cell apoptosis; that is, the extrinsic pathway through activation of cell death receptors and the intrinsic pathway through mitochondrial damage (34,35). To confirm whether the mitochondrial pathway is activated by Manu A, we measured the degree of mitochondrial membrane potential. This was measured by staining with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide reagent in the MPM cells. As a result, MMP was significantly decreased in a concentration-dependent manner. Total depolarized MSTO-211H cell populations were 38.7±1.3, 51.9±1.2 and 72.1±1.7% and those of H28 (Fig. 4B) were 2.73±1.2, 86.0±0.5 and 93.9±0.9% at increasing doses, respectively. To confirm whether Manu A could kill cells by inducing mitochondrial damage, we treated MPM cells with Manu A (2.5, 5 and 10 µM) for 48 h and then carried out western blot analysis. CHOP is increased by ER stress and the cell death receptors such as DRs (DR4 and DR5) are upregulated by cell stress (36,37). Based on our results, CHOP, DR4 and DR5 were increased in a concentration-dependent manner (Fig. 4C and D).

Treatment of the MSTO-211H and H28 cells with Manu A regulated the expression levels of apoptosis-related proteins. To clarify the association between Manu A and Sp1-mediated apoptosis, we performed western blot analysis. When MPM cells were treated with Manu A, Bax expression was increased in a concentration-dependent manner, while the expression of Bcl-xL, an apoptosis inhibitory protein, was reduced in a concentration-dependent manner (Fig. 5A and B). PARP that interferes with the apoptosis of cancer cells is decreased (38). In this experiment, PARP was decreased in both the MPM cell lines following treatment with Manu A (Fig. 5A and B). When cells are exposed to internal and external stimuli, apoptotic signals are transmitted to induce activation of caspase-8 and -9. Then, procaspase-3 is cleaved into caspase-3, and inactivates PARP finally inducing apoptosis (35). To examine whether Manu A-mediated apoptosis could be associated with caspases, the caspase activity was measured using a multi-caspase kit. Total multi-caspase activities in the MSTO-211H cells (Fig. 5C) were 23.7±1.3, 43.3±3.4 and 62.2±1.1%, and those in the H28 cells (Fig. 5D) were 15.8±0.7, 19.0±0.5 and 78.9±0.5% at increasing doses. From the above results, we confirmed that Manu A regulated the expression of apoptosis-related proteins in the MPM cells.