MAP30 was a gift from School of Medical Laboratory Science, Chengdu Medical College. N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), rapamycin and bafilomycin A1 (baf A1) were purchased from Selleck Chemicals (Houston, TX, USA), and C646 was purchased from Sigma-Aldrich (St. Louis, MO, USA), which were dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO used did not exceed 0.1%, which had no adverse effect on the cell cultures. The antibodies against LC3, p62, beclin 1, caspase-3, caspase-8, caspase-9, PARP, survivin, Bcl-2, Bax, GAPDH, acetylated-histone H3, or histone H3 were provided by Cell Signaling Technology (Beverly, MA, USA).

HL-60 and THP-1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (both from Gibco, Grand Island, NY, USA) at 37°C in 5% CO₂. Primary AML cells were isolated from bone marrow aspirates of five newly diagnosed and untreated patients with AML [M3, 1; M4, 4; the diagnosis and classification was established according to the world health organization criteria (27)] using Ficoll-hypaque, and cultured in RPMI-1640 medium supplemented with 20% fetal bovine serum at 37°C in 5% CO₂. All protocols and experiments were approved by The First Affiliated Hospital of Wenzhou Medical university Institutional Review Board for clinical experiments and use of human samples; written consents were obtained from all subjects participated in this study in accordance with the Declaration of Helsinki protocol.

A Cell Counting Kit-8 (CCK-8) was used to assess the cytotoxicity of MAP30 on AML cells according to the manufacturer's protocol (Dojindo, Kumamoto, Japan). Briefly, AML cells were diluted and seeded at a density of 4×10³/well in 96-well plates. The cells were subsequently exposed to MAP30 at different concentrations for 48 and 72 h. Then, the CCK-8 was added, and absorbance (A) was measured at 450 nm using an ELISA reader (ELx800; Bio-Tek Instruments, Winooski, VT, USA). Cell viability rate (%) = A_{450, MAP30}/A₄₅₀, control × 100%.

Apoptosis was determined using the Annexin V-FITC/propidium iodide (PI) Detection kit (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China) according to the manufacturer's instruction. Briefly, after treatment with MAP30 for 48 h, the cells were collected rapidly and washed by cold PBS and subsequently incubated with FITC-labeled Annexin V and PI for 15 min at room temperature in the dark and then analyzed with CellQuest software on a flow cytometry (FACSCalibur; BD Biosciences, Mountain View, CA, USA).

After treatment with or without MAP30 at different concentrations, the cells were collected and lysed immediately using RIPA lysis buffer (Beyotime Institute of Biotechnology) supplemented with PMSF and Halt protease and phosphatase inhibitor cocktail (Pierce, Rockford, IL, USA). The protein was boiled for 5 min in 1× loading buffer and subjected to western blot analysis according to our previously described method (28). Bands were visualized by enhanced chemiluminescence reagents (Thermo Fisher, Fremont, CA, USA) and the optical densities of the bands were analyzed using ImageJ software (NIH, Bethesda, MD, USA).

Quantitative real-time PCR was used to evaluate the expression of p300 and a reference gene GAPDH according to our previously described method (29). The sequences of specific primers see Table I.

The data are presented as mean ± SEM and analyzed by one-way ANOVA followed by a post hoc Turkey's test to determine the differences between the groups. Differences were considered significant at P<0.05.

We first examined the effects of MAP30 on the proliferation of AML cell lines HL-60 and THP-1, and primary AML cells. As shown in Fig. 1A, MAP30 inhibited the proliferation of both HL-60 and THP-1 cells in a dose- and time-dependent manner, with IC₅₀ at 48 h of 2.6 and 9.2 µM, respectively. Furthermore, MAP30 also inhibited the proliferation of primary AML cells, with IC₅₀ at 48 h of 4.7 to 8.1 µM (Fig. 1A). Since the induction of apoptosis is a leading cause for MAP30-induced cytotoxicity against liver cancer cell line HepG2 (11), we next investigate whether apoptosis also occurs when AML cells are inhibited by MAP30. As shown in Fig. 1B, MAP30 (0–2 µM) dose-dependently induced apoptosis of AML HL-60 and THP-1 cells as well as primary AML cells by flow cytometric analysis using Annexin V/PI staining. As expected, high concentrations of MAP30 (8 µM, 20 µM) strongly induced apoptosis in all AML cells tested (data not shown). MAP30-induced apoptosis was further confirmed using western blot analysis of two apoptosis-related proteins PARP and survivin. After treatment with MAP30 for 48 h, cleaved-PARP was markedly increased and conversely, survivin was decreased (Fig. 1C). Taken together, these data suggested that MAP30 exhibits cytotoxicity in AML cells through inducing apoptosis.

We further investigated the molecular mechanisms involved in MAP30-induced apoptosis. As shown in Fig. 2A, MAP30 treatment resulted in the cleavage of caspases, including the initiator caspase-8 and -9 and the effector caspase-3 in AML cell lines HL-60 and THP-1 and primary AML cells (Fig. 2A). These data suggested that MAP30 induces apoptosis of AML cells in a caspase-dependent manner, and both extrinsic and intrinsic apoptotic pathways are involved. Cleaved caspase-3 subsequently induced the cleavage of PARP, which ultimately led to apoptosis. Additionally, the expression of anti-apoptotic protein Bcl-2 was decreased and conversely, the expression of pro-apoptotic protein Bax was significantly increased in HL-60 cells treated by MAP30 (Fig. 2B), indicating that some Bcl-2 family members are also involved in caspase-dependent apoptosis induced by MAP30. Finally, a pan-caspase inhibitor z-VAD-fmk was employed to verify whether MAP30-induced apoptosis depends on caspases. As shown in Fig. 2C and D, z-VAD-fmk significantly reduced MAP30-induced apoptosis in HL-60 cells. Moreover, the growth inhibition of HL-60 cells induced by MAP30 was also partially rescued by z-VAD-fmk (Fig. 2E), further confirming MAP30 induces apoptosis in a caspase-dependent manner.

Low concentration of MAP30 (2 µM) exhibited strong cytotoxicities against AML cells (Fig. 1A), whereas only modest apoptosis was observed in AML cells induced by this concentration of MAP30 (Fig. 1B), suggesting that there are other mechanisms involved in MAP30-induced cytotoxicity besides the induction of apoptosis. Histone deacetylase inhibitors (HDACi) have been shown to kill leukemia cells while sparing normal cells by inhibiting autophagy (30,31). Accumulating evidence shows that one key aspect of the pro-survival function of autophagy is achieved through its ability to block necrotic cell death (32). To investigate whether MAP30 influences the autophagic flux of AML cells, the expression of autophage-related proteins, including LC3, p62, and beclin 1, were determined. As shown in Fig. 3, the expression of LC3II and beclin 1 were significantly decreased, whereas, p62 was increased in the two AML cell lines HL-60 and THP-1 as well as primary AML cells exposed to MAP30. These findings further supported the notion that MAP30 treatment inhibits the autophagic flux in AML cells.

Undoubtedly, MAP30 can inhibit autophagy, which prompted us to clarify whether the inhibition of autophagy plays an important role in antileukemic effects of MAP30. We used autophagy activator rapamycin, and another classical autophagy inhibitor baf A1, which inhibits the vacuolar ATPase required for the fusion between autophagosomes and lysosomes. Both rapamycin and baf A1 almost did not influence the cell viability and induce apoptosis of HL-60 cells and primary AML cells (Fig. 4), but at these same concentrations of rapamycin and baf A1 can activate and inhibit the autophagic flux of HL-60 cells, respectively (data not shown). As expected, rapamycin reduced the cytotoxicity and apoptosis in HL-60 cells induced by MAP30 (Fig. 4A, C and E), and conversely, baf A1 exacerbated the cytotoxicity and apoptosis in HL-60 cells induced by MAP30 (Fig. 4B, D and E). Similarly, rapamycin attenuated the cleavage of PARP and the inhibition of survivin, and baf A1 had a synergistic effect on the cleavage of PARP and the inhibition of survivin in primary AML cells induced by MAP30 (Fig. 4F). Taken together, these data further indicated that the inhibition of autophagy is implicated in MAP30-induced cytotoxicity and induction of apoptosis in AML cells.

Since MCP30, a mixture of MAP30 and α-MMC, was shown to promote histone H3 and H4 protein acetylation in our previous report (33), we next investigate whether the promotion of acetylation of MCP30 are attributed to its main ingredient MAP30. As shown in Fig. 5A and B, MAP30 treatment increased the mRNA and protein expression levels of p300 in HL-60 cells, which consequently promoted the acetylation of histone H3 (Fig. 5C). Similar results have been observed in primary AML cells treated by MAP30 (Fig. 5B and C). Accumulating evidence has shown that p300 is a major endogenous repressor of autophagy (23). To investigate whether the inhibition of autophagy of MAP30 is attributed to p300, C646, a pharmacological inhibitor of p300, was used. As shown in Fig. 5D and E, C646 almost completely reversed the acetylation of histone H3 and promoted the autophagic flux in the presence of MAP30 in HL-60 cells. Therefore, C646 partially rescued the cytotoxicity and apoptosis caused by MAP30 in HL-60 cells and primary AML cells (Fig. 5F and G). Taken together, these findings suggested that MAP30 inhibits the autophagic flux of AML cells by enhancing the acetyltransferase activity of p300.