Bladder cancer (BLCA) is one of the most common cancers worldwide and is also the main cause of cancer morbidity and mortality involving the urinary system (Yu
Epigenetic-based biomarkers have been studied in an attempt to better understand the molecular mechanisms involved in BLCA. Histone acetylation, as a fully characterized epigenetic modification, is conducted by HATs and HDACs. Human KAT8/MOF is a highly conserved member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of HATs that was originally discovered in a study of X-chromosome dosage compensation in Drosophila (Hilfiker
Numerous studies have shown that MOF plays pivotal roles in various cellular functions, including genome stability, gene transcription, DNA damage repair, cell cycle regulation, and early embryonic development (Rea
In this study, T24 human urinary BLCA cells were employed as an experimental model to investigate the effects of GEM on BLCA. Our results showed a decrease in the MOF protein expression and its corresponding histone H4K16ac in T24 cells exposed to GEM, suggesting the potential interaction between MOF and GEM. To explore the potential MOF-mediated molecular mechanisms of GEM in T24 BLCA cells, we conducted a series of biochemical and molecular biological experiments such as the cell viability assay, flow cytometry, colony formation assay, and wound healing assay.
Anti-mouse IgG-HRP (IH-0031) and anti-rabbit IgG-HRP (IH-0011) were obtained from Beijing Dingguo Changsheng Biotechnology Co. Ltd (Beijing, China). Anti-Flag (M2) (F3165) monoclonal antibody and anti-H4K16ac (H9164) polyclonal antibody were from Sigma-Aldrich (St. Louis, MO, USA). Anti-MOF (A02757) monoclonal antibody got from BosterBio (Wuhan, China). Antibodies for H4K5ac (07-327), H4K8ac (07-328), H3K4me1 (07-436), and H3K4me2 (07-030) were purchased from Merck Millipore (Darmstadt, Germany). Antibodies for H4 (16047-1-AP) and HDAC2 (12922-3-AP) were purchased from Proteintech Group (Wuhan, China). Antibodies for H2AX (CBS-PA15429A0R6) was from Cusabio Technology (Wuhan, China). Anti-γH2AX (07-164) monoclonal antibody was purchased from Merck Millipore. Antibodies for H3K4me3 (RLM3104), H3K9me2 (RLM3108), Parp1 (RLM3145), HDAC1 (RLT2145) and E-cadherin (RLT1453) were obtained from Ruiying (Suzhou, China). Antibodies for N-cadherin (ab18203) and Vimentin (ab45939) were from Abcam (Cambridge, UK). While anti-GAPDH (NM_002046, full length) rabbit polyclonal antibody was raised against bacterially expressed proteins (Jilin University, Changchun, China). The chemotherapy drug GEM was kindly gifted by Dr. Yong Wang (Urology Department, Jilin province People’s Hospital, Changchun, China).
T24 and 5637 human urinary BLCA cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 medium (Gibco Life TechnologiesTM, Waltham, MA, USA) containing 10% fetal bovine serum (KY-01003, Kang Yuan Biology, Beijing, China) and 1% penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C in the presence of 5% CO2. The cells were treated with GEM according to the experimental design.
The pLVX-shRNA system was used to knockdown endogenous MOF. Two shRNA sequences (shMOF-1, GTGATCCAGTCTCGAGTGA; shMOF-2, CGAAATTGATGCCTGGTAT), which targeted the coding DNA sequence of MOF, were introduced into the pLVX vector. The pLVX-shRNA plasmids were transiently transfected using polyethylenimine (PEI, 23966) (Polysciences, Shenzhen, China) according to the manufacturer’s recommendation.
Cells were cultured at a density of 3×103/well in 96-well plates and treated with GEM. Then, cells were incubated with CCK-8 reagent (017319, Promega Corporation, Madison, WI, USA) for 1 h. The absorbance at a wavelength of 450 nm was measured using a microplate reader (Infinite F200 Pro, TECAN, Shanghai, China).
T24 cells were cultured in 6-well plates as ~90% confluent monolayers, and wounds were introduced by scraping the cells with the tip of a 10-μL pipette. Floating cells were removed and medium without serum was added. To analyze scratch wound closure, optical images were captured at 0 and 24 h time points using a microscope (Olympus IX73 microscope; Olympus Corporation, Tokyo, Japan). The wound area was analyzed with ImageJ (bundled with 64-bit Java 1.8.0_112; National Institutes of Health, MD, USA).
Cells (2×103) were seeded into a 12-well plate. After seven days of culture, formed colonies were stained with 0.1% crystal violet. Colonies were photographed by Gel Imaging System (Liuyi Instrument Plant, Beijing, China).
Twenty-four-well Transwell® chambers (Corning Inc., Corning, NY, USA) were used in the migration assay. Filters (8-μm pore size) were used for estimating cell migration. Cells in 0.2 mL of serum-free medium were placed into the upper chamber (2×104 cells). Medium containing 10% FBS was then added into the lower chamber. After incubation for 24 h, cells were fixed in methanol for 20 min, stained with 0.1% crystal violet for 10 min, and counted under a microscopy (Olympus Corporation, Miyazaki, Japan).
The MOF promoter region (1470 bp, –1464 to +6 bp) was introduced into the pGL4-Luc vector. The luciferase reporter assay was conducted as described (Su
Full-length cDNA encoding the hMOF (BC037773) protein was subcloned with Flag-tag into pcDNA3.1(-). Then, the T24 cells were transiently transfected with FLAG-MOF (Fl:MOF) plasmid using PEI according to the manufacturer’s instructions (PolyScience).
Cultured T24 cells were harvested by trypsinization. ~1×106 cells were suspended as single cell dispersions in 70% ethanol at –20°C for at least 4 h. After centrifugation at 300×g for 5 min, cells were washed twice with PBS and then re-suspended in 300 μl PBS containing 0.1% (v/v) Triton X-100, 0.3 mg/mL DNase-free, RNase A, 50 μg/mL propidium iodide, and then were incubated at 37°C for 1 h. Data collection was performed using the EPICS XLTM flow cytometer (Beckman Coulter, Brea, CA, USA). Acquired data were analyzed using ModFit LT software (Verity Software House, Topsham, ME, USA). The experiment was repeated three times under the same conditions.
T24 cells were cultured in RPMI-1640 medium with or without GEM (0.15 μM or 0.75 μM). After transfection for 48 h, cells were harvested and stained with an Annexin V-FITC/PI kit (KeyGEN Biotech, Nanjing, China). Propidium iodide (PI) was used to discriminate between apoptotic cells with membrane integrity (Annexin V+/PI–) and necrotic cells that lost membrane integrity (Annexin V+/PI+).
Statistical analysis was conducted using data from at least three independent experiments. All results are presented as mean ± standard deviation (SD). SPSS 20.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. Independent samples
Our initial research results suggest that the protein level of MOF in bladder cancer T24 and 5637 cells was suppressed in a dose-dependent manner after treatment with chemotherapeutic drugs, including GEM, mitomycin C, and camptothecin (data not shown). Thus, GEM was selected for the subsequent study on MOF-related mechanisms. To determine the appropriate concentrations of GEM for our experiments, the CCK-8 cell viability assay was first performed to verify their cytotoxicity in T24 cells. Fig. 1A shows that dose-dependent cytotoxicity of GEM in T24 cells was observed after treatment with gradient concentrations of GEM for 24 h, 36 h, and 48 h. Cytotoxicity increased with prolonged drug exposure time (48 h>36 h>24 h). Next, long-term clonogenic cell survival and cell migration ability were evaluated by colony formation and wound healing assays. Based on the CCK-8 cell viability assay, we selected 0.15 µM GEM for subsequent experiments because this drug concentration did not induce any cell death. Compared to the control group (No-drug group), the ability of cells to form colonies was significantly suppressed by GEM (Fig. 1B), and the progression of wound closure was much slower in GEM-exposed cells than in control cells (Fig. 1C). The normalized percentage of the scratch wound is shown in Fig. 1D. Next, FACS analysis was performed to verify the effects of GEM on T24 cell cycle progression. Fig. 1E shows that compared to the control group, GEM-exposed cells appeared to be arrested in G1/S phase (lower panel). The percentage of cells in the G1, S, and G2/M phases is shown in Fig. 1F. To further confirm this observation, T24 cells were treated with 1 mM hydroxyurea (HU) to block cells in the G1/S phase so that no new G2/M phase cells could be generated, and then released the cells at different time points (0, 2, 4, 8, 12, 16, and 20 h). As expected, 0.15 µM GEM-treated cells were completely arrested at the G1/S phase throughout the experiment (Fig. 1G).
Both BLCA T24 and 5637 cells treated with GEM showed downregulated endogenous levels of the HAT MOF protein, and this reduction was dose-dependent (Fig. 2A). Thus, we selected T24 cells for a series of subsequent experiments. At first, to explore whether GEM affects the transactivation of MOF, the MOF promoter region (–1,464 to +6 bp) was subcloned into the pGL4 luciferase vector (Fig. 2B, right upper). Then, the impact of GEM on MOF transactivation was estimated by measuring luciferase activity of pGL4-MOF in GEM- (0-375 nM) exposed cells. In contrast to basal level luciferase activity, a dose-dependent decrease in luciferase activity was observed in all concentrations of GEM treated cells (Fig. 2B), indicating the regulatory role of GEM on MOF transactivation. Considering that MOF acts as a catalytic subunit in cells, it can form 2 different protein complexes (NSL and MSL complexes) that are responsible for catalyzing the acetylation of histones H4K5, H4K8, and H4K16 (Cai
Double-strand DNA breaks (DSBs) often initiate phosphorylation of histone H2AX at serine 139, which is referred to as γH2AX, and the detection of γH2AX is a rapid method for assessing the effectiveness of chemotherapy drugs that induce DSBs (Wang
Our previous results confirmed that GEM targets and regulates intracellular MOF and its corresponding histone H4K5/K8/K16 acetylation levels, prompting us to further explore whether the regulatory effect of GEM on MOF is involved in its anti-cancer effect. To address this question, the viability of T24 cells treated with 0, 0.03, 0.15, 0.8 or 4.0 µM GEM for 24 h and 48 h in the presence or absence of exogenous MOF was assessed using the CCK-8 assay kit (017319, Promega Corporation). Compared with the pcDNA3.1-transfected control group, GEM dose-dependently inhibited cell viability at both 24 h and 48 h time points (Fig. 4A), suggesting that MOF facilitated the sensitivity of T24 cells to GEM. The expression level of Fl:MOF at 48 h after transfection is shown in Fig. 4B (lane 2). On the contrary, by knocking down the MOF with specific shRNA, cell viability increased regardless of whether the cells were treated with GEM for 24 h or 48 h (Fig. 4C). The protein level of endogenous MOF at 48 h after shMOF transfection was shown in Fig. 4D (lane 2). The results of the colony formation assay further support the previous results of cell viability. Fig. 4E shows that knocking down MOF with shRNA in T24 cells promotes colony formation. The number of colonies in the control and MOF-knockdown groups is shown in Fig. 4F. The above experimental results show the synergistic effects of GEM and MOF on BLCA tumorigenesis.
Cancer cells move within tissues during invasion and metastasis by their own motility (Yamazaki
Cell viability experiments suggest that MOF increases cell sensitivity to GEM. To further confirm whether MOF has an influence on GEM-induced cell apoptosis and necrosis, flow cytometry of Annexin V binding/PI uptake was assessed in cells exposed to increasing GEM concentrations with or without Fl:MOF transfection (Fig. 6A). Apoptosis and necrosis are depicted in Fig. 6B. Compared to the GEM-only treated cells, exogenous MOF significantly increased GEM-induced apoptosis (upper panel) and necrosis (lower panel) (**
Different histone post-translational modifications lead to distinct effects on chromatin architecture (Biswas and Rao, 2018). Among these, histone acetylation is controlled by HATs and HDACs. In cells, human HAT MOF is a catalytic subunit that can form at least 2 distinct multiprotein complexes (MSL and NSL). Both complexes can acetylate the histone H4K16 site; however, the H4K5 and H4K8 sites can also be acetylated by the NSL complex, suggesting the complexity of the functions of MOF (Cai
Subsequent research studies have shown that inhibiting the degradation of p300/CBP can enhance the cytotoxicity of 5-FU in colorectal cancer cells, whereas reducing the protein expression levels of p300/CBP by siRNA improves cellular resistance to 5-FU. Moreover, the downregulation of p300/CBP in colorectal carcinoma tissue is closely associated with poor clinical response to 5-FU-based chemotherapy (Du
In view of the research results described above, the role of MOF-mediated anti-cancer effects of GEM can be deduced. However, it is worth noting that the modulation of MOF expression by GEM not only directly affects the global intracellular acetylation of histone H4, but also may indirectly participate in the pathogenesis of cancer through acetylation of non-histone proteins. For instance, MOF is required for directly acetylating histone demethylase LSD1 in T47D, MDA-MB-468, BT474, and MCF7 cells, and increasing MOF expression can suppress the LSD1’s accessibility to chromatin, EMT, and invasion in A549 cells. In contrast, depletion of MOF promoted EMT and cell invasion, suggesting the function of acetylation of LSD1 by MOF in tumor malignant progression (Luo
In summary, our present findings uncovered novel mechanisms by which the anti-bladder cancer effect of GEM is at least partially achieved by inhibiting the expression of MOF and its corresponding histone H4 acetylation. Thus, our results suggest that human MOF or MOF-containing NSL complex may be potentially utilized as a target for the development of new drugs for the treatment of BLCA.
This work was supported by National Natural Science Foundation of China (31401086 and 31771421). In addition, we thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
The authors have no conflict of interest to declare.