Squamocin modulates histone H3 phosphorylation levels and induces G₁ phase arrest and apoptosis in cancer cells

Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), trypan blue, penicillin G, and streptomycin were obtained from GIBCO BRL (Gaithersburg, MD, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), ribonuclease (RNase), and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). An Annexin V-FITC Staining Kit was purchased from Strong Biotech (Taipei, Taiwan). Antibodies against aurora B, H3S10p, and H3S28p were purchased from Abcam (Cambridge, UK). Antibodies against pERK, pMSK1, caspase-3, caspase-8, caspase-9, and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-PARP was purchased from Upstate Biotechnology (Charlottesville, VA, USA). Anti-mouse and anti-rabbit immunoglobulin G (IgG) peroxidase-conjugated secondary antibodies were purchased from Pierce (Rockford, IL, USA). Polyvinylidene difluoride (PVDF) membranes and an enhanced chemiluminescence (ECL) Western blotting detection kit were obtained from Amersham Life Science (Buckinghamshire, UK).

Squamocin was provided by Prof. Yang-Chang Wu, Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan. The structure of this compound was verified by means of mass spectrometry and spectroscopic techniques [16]. Squamocin was dissolved in DMSO (< 0.01%) and made up immediately prior to the experiments.

The GBM8401, Huh-7, and SW620 cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and are derived from brain, liver and colon cancers, respectively. Cells were maintained in DMEM which was supplemented with 10% FBS, 2 mM glutamine, and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) at 37°C in a humidified atmosphere of 5% CO₂.

Cell viability was determined by an MTT assay, and results are presented as a percentage of the control. For the MTT assay, 10 μl of MTT (5 mg/ml) dye was directly added to the cell cultures. The medium was removed 2 h later, and cells were lysed with 100 μl of DMSO. The absorbance at 570 nm was read on a microplate reader.

Externalization of phosphatidylserine (PS) and the membrane integrity were quantified using an Annexin V-FITC Staining Kit. Cells were washed twice with phosphate-buffered saline (PBS), and collected by centrifugation at 200 × g for 5 min at 25°C. Cells were resuspended in 100 μl of binding buffer and labeled with 2 μl of annexin V-FITC and PI for 15 min at 25°C. After labeling, cells were resuspended in 500 μl of binding buffer and detected on a flow cytometer using 488-nm excitation and a 515-nm band-pass filter for fluoresce detection and a filter > 600 nm for PI detection. To analyze the cell cycle distribution, cells were washed twice with PBS, collected by centrifugation at 200 × g for 5 min at 4°C, and fixed in 70% (v/v) ethanol at 4°C for 30 min. After fixation, cells were treated with 0.2 ml of the DNA extraction buffer (0.2 M Na₂HPO₄ and 0.1 M citric acid buffer; pH 7.8) for 30 min, centrifuged, and then resuspended in 1 ml of PI staining buffer (0.1% TritonX-100, 100 μg/ml RNase A, and 500 μg/ml PI in PBS) at 37°C for 30 min. Cells were detected using a flow cytometer and analyzed by FACScan and the Cell Quest program (Becton Dickinson, San Jose, CA, USA).

Total proteins were extracted as previously described [17]. Proteins were extracted from the experimental and control samples and analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) as follows: after electrophoresis, proteins were transferred from the gel onto PVDF membranes. The membranes were blocked with a skim milk solution (5% skim milk in PBS) and agitated for 30 min at room temperature. Membranes were exposed to the primary antibody and agitated for 1 h at room temperature before being washed three times for 10-min periods with PBST (0.05% Tween 20 in PBS), and then incubated for 1 h with the secondary antibody at a 1:2500 dilution. After incubation with the antibody, the membranes were washed three times with PBST for 10 min each and then immersed in an ECL solution (combining solutions A and B of the ECL kit in a 1:1 ratio) with agitation for 1 min. After washing, the blots were developed by ECL.

RNA was isolated from cultured cells, and the analysis was performed as previously described [18]. The PCR was performed in a final volume of 20 μl using a LightCycler instrument (Roche Diagnostics) according to the manufacturer's recommendations. The amounts of complementary (c)DNA were normalized to the housekeeping gene, GAPDH, to calculate the relative expressions of aurora B and MSK1 RNA (Table 1). Primers used to detect these genes were designed using ProbeFinder software http://​www.​roche.​com and were synthesized by custom oligonucleotide synthesis (Genomics, Taipei, Taiwan). The qRT-PCR cycling parameters were set as follows: 40 cycles of 95°C for 10 s (denaturation), followed by 60°C for 30 s (annealing), and 72°C for 1 s (extension).

Results from multiple experiments are expressed as the mean ± standard error. The difference between the treatment and control groups was analyzed by Student's t-test. A probability (p) value of < 0.05 was considered significant.

Aurora B and MSK1 are thought to be involved in chromatin organization, gene expression, and carcinogenesis [14, 15]. More than 20 compounds with cytotoxic effects were screened, and we found a compound, squamocin, isolated from several genera of the plant family Annonaceae, which decreased (m)RNA expression levels of aurora B and MSK1 in cancer cells. The expressions of aurora B and MSK1 were significantly downregulated in squamocin-treated GBM8401, Huh-7, and SW620 cells compared to the control (Figure 2). Similarly, squamocin treatment decreased the protein expression levels of aurora B and phosphorylated MSK1 (pMSK1) (Figure 3). These results imply that squamocin regulates aurora B and MSK1 activities at the transcriptional and translation levels.

In eukaryotes, aurora B and MSK1 are linked to the phosphorylation of H3S10 and H3S28 [7‐9]. In order to investigate the effects of squamocin on H3S10p and H3S28p, cells were treated with squamocin for 24 h, and the protein expression levels were analyzed by Western blotting. The results showed that decreased H3S10p and H3S28p protein expression levels were detected in squamocin-treated cells (Figure 3). Our experiment revealed that squamocin treatment decreased phosphorylation of histone H3S10 and H3S28, as well as caused declines in the protein and RNA expression levels of aurora B and pMSK1. The modulation of H3S10 and H3S28 phosphorylation by aurora B and/or pMSK1 indicates that squamocin probably decreased the phosphorylation of H3S10 and H3S28 by downregulating aurora B and pMSK1 in cancer cells.

The growth-inhibitory activity of squamocin was assessed by an MTT assay. GBM8401, Huh-7, and SW620 cells were treated with different concentrations (15~120 μM) of squamocin for 24 h. The results showed that squamocin-treated cancer cells exhibited significant loss of viability in dose-dependent manners (Figure 4). The 50% inhibitory concentrations (IC₅₀) of GBM8401, Huh-7, and SW620 cells were 46.1, 39.4, and 40.4 μM, respectively.

To further evaluate the potential relevance of histone H3 phosphorylation in cancer therapy, we examined the effects of squamocin on cell growth and viability. Cells were treated with squamocin for 24 h, and the cell cycle distribution and apoptosis were measured by a flow cytometric analysis. Squamocin treatment significantly increased the population of G₁ phase cells (Figure 5). Also, high levels of apoptosis were detected in squamocin-treated cells (Figure 6). As shown in Figure 5, treatment of cells with 0, 15, 30, and 60 μM of squamocin resulted in G₁ phase accumulation of cells corresponding to 37.8%, 46.7%, 60.6% and 56.3%, respectively in GBM841 cells (Figure 5A), 41%, 59.7%, 53.6%, and 54.5%, respectively in Huh-7 cells (Figure 5B), and 53.2%, 64.9%, 59.1%, 54.5%, respectively in SW620 cells (Figure 5C). Moreover, squamocin-treated cells were stained with propidium iodide (PI) and annexin V to determine the apoptotic cells. Cells were differentiated among viable (annexin V, PI double negative), early-apoptotic (annexin V positive, PI negative) and late-apoptotic (annexin V, PI double positive) cells. Treatment of cells with 0, 15, 30, and 60 μM of squamocin increased the percentage of early apoptosis from 0.7% to 14.1%, 4.3%, and 5.3%, respectively and late apoptosis from 4.1% to 5.5%, 21.9%, and 49%, respectively in GBM8401 cells (Figure 6A), early apoptosis from 1.8% to 15.1%, 21%, and 7.6%, respectively and late apoptosis from 3.0% to 8.6%, 12.1%, and 62.9%, respectively in Huh-7 cells (Figure 6B), and early apoptosis from 3.0% to 21.2%, 20.1%, and 22.8%, respectively and late apoptosis from 1.2% to 2.4%, 20.2%, and 36.9%, respectively in SW620 cells (Figure 6C ). Further, we extended our study to apoptosis-associated molecules and found that increasing levels of caspase-3, -8, and -9 activities and cleavage of poly ADP-ribose polymerase (PARP) were observed in squamocin-induced apoptosis (Figure 7). From the results, it is evident that squamocin affected cell cycle progression and apoptosis.

The MAPK signaling pathway is implicated in a wide range of cellular functions, including cell proliferation, differentiation, survival, and apoptosis [19]. To assess whether activation of the MAPK signaling pathway is involved in squamocin-induced apoptosis, we investigated the activities of MAPK. The results showed that squamocin treatment significantly decreased ERK phosphorylation (pERK) levels and increased JNK phosphorylation (pJNK) levels (Figure 7). It was determined that activation of JNK affects members of the Bcl-2 family and activates caspases-3, -8, and -9 which results in apoptosis, whereas ERK is connected to cell survival [20, 21] Our results indicate that inhibition of ERK and activation of JNK may participate in squamocin-induced apoptosis.