Human AML KG-1a cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium containing 15% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin in a humidified incubator of 5% CO₂ at 37°C. SNX-2112 was synthesized as previously described (26) with a purity >98.0%, and 10 mM SNX-2112 stock solutions were prepared in dimethyl sulfoxide (DMSO) and stored at 4°C. 17-AAG and bardoxolone methyl (BAR; an NF-κB pathway inhibitor) were obtained from Selleck Chemicals (Houston, TX, USA). RPMI-1640 medium and FBS were purchased from Gibco (Grand Island, NY, USA). Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH), caspase-3, Bcl-xl, Bcl-2, Akt, p-Akt (Thr308), IKKα, IKKβ, p65, p-p65, IκB, and PU.1 were purchased from Cell Signaling Technology (Beverly, MA, USA), and cluster of differentiation (CD)11b was purchased from BD Biosciences (Franklin Lakes, NJ, USA).

Cell proliferation was evaluated using Cell Counting Kit-8 (CCK-8). Briefly, 1.0×10⁴ cells/well were seeded into 96-well plates and treated with SNX-2112 or 17-AAG for 24, 48 or 72 h. A 10-µl volume of CCK-8 working solution was added to each well for 2 h. The absorbance was assessed at 450 nm on a microplate reader (Bio-Rad, Hercules, CA, USA) and used to determine the drug concentration inhibiting growth by 50% (IC₅₀).

KG-1a cells were seeded at 1.0×10⁵ cells/ml and treated with SNX-2112 for 48 h. The cells were collected by centrifugation at 500 × g for 5 min; 70% ethanol was then added, followed by 2 washes with phosphate-buffered saline (PBS). Cells were resuspended in 1 ml PBS containing 2.5 µg/ml ribonuclease and 50 µg/ml propidium iodide (PI) (Beyotime Institute of Biotechnology, Shanghai, China) and incubated in the dark for 30 min at room temperature before analysis by flow cytometry (BD FACSCalibur, Lake Franklin, NJ, USA; BD Biosciences).

Cells were seeded at 1.0×10⁵ cells/ml and incubated with SNX-2112 for 48 h. The cells were collected by centrifugation at 500 × g for 5 min and washed with PBS twice. Then, a 20-µl volume of CD11b was added for 20 min in the dark and analyzed by flow cytometry.

Cells were seeded at 1.0×10⁵ cells/ml and incubated with SNX-2112/Bar for 48 h. Samples were prepared according to the instructions provided with the Annexin V-fluorescein isothiocyanate/PI staining kit (Beyotime Institute of Biotechnology) and analyzed by flow cytometry.

KG-1a cells were seeded at 1.0×10⁵ cells/ml, and incubated with DMSO or SNX-2112 for 48 h, and total RNA was immediately extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. For analysis of PU.1 and C/EBPα, 2 µg of total RNA was used to synthesize first-strand DNA with reverse transcriptase (Promega, Madison, WI, USA). qRT-PCR was then carried out using Green PCR Master Mix (Shine Co., Shanghai, China), 1 µl of cDNA, gene-specific primers (Table I), and the Hot Start Fluo-PCR Mix in a 20-µl reaction under the following cycling conditions: 95°C for 5 min, followed by 35 cycles at 95°C for 10 sec, 57°C for 15 sec, and 72°C for 20 sec. Each sample was prepared in triplicate and transcript levels were quantified using the 2^−ΔΔCt method (27).

KG-1a cells cultured in 6-well plates were treated with DMSO or SNX-2112 (0, 0.25, 0.5 or 1 µM) for 48 h. The cells were washed 3 times with ice-cold PBS and fixed with 4% paraformaldehyde. The cell membrane was permeabilized by adding 0.1% Triton X-100, and the cells were blocked with 5% bovine serum albumin (HyClone, Logan, UT, USA) and incubated for 2 h at room temperature with an antibody against NF-κB p65 (1:400) (Cell Signaling Technology), followed by incubation with a fluorophore-conjugated anti-rabbit antibody for 2 h at room temperature. The cells were then stained with PI (Beyotime Institute of Biotechnology) for 15 min, and subsequently washed and examined with an epifluorescence microscope (Zeiss, Jena, Germany).

KG-1a cells were washed twice in ice-cold PBS, lysed in radioimmunoprecipitation buffer for 30 min on ice at 4°C, and centrifuged at 12,000 × g for 15 min. The protein content in the supernatant was determined with the bicinchoninic acid assay. Equivalent amounts (30–50 µg) of protein were denatured in sodium dodecyl sulfate (SDS) sample buffer and resolved by 10–15% SDS-polyacrylamide gel electrophoresis, and then transferred to an Immobilon polyvinylidene difluoride membrane, which was blocked in 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 1 h and then probed with primary antibodies (1:1,000) overnight at 4°C. The membrane was washed 3 times for 10 min in TBST, and incubated with a secondary antibody (1:6,000–1:8,000) in TBST with 5% skimmed milk at room temperature for 1 h. After washing, immunoreactivity was detected with an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA). GAPDH served as a loading control.

Cells were treated with SNX-2112 or 17-AAG for 2 days, and then washed twice with PBS. The cells were seeded (1×10³ cells/dish) in 1.5 ml of 0.30–0.35% soft agar (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 20% FBS and cultured for 12 days at 37°C and 5% CO₂. After 12 days, the colonies were counted under a light microscope.

All data were confirmed by at least 3 independent experiments. Data are expressed as the mean ± SD. Statistical significance was determined by one-way analysis of variance (ANOVA) and covariance calculated using GraphPad 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). Differences between 2 groups were detected using two-tailed Student's t-test. P<0.05 and P<0.01 were considered to indicate a statistically significant result.

The effects of SNX-2112 and 17-AAG on the viability of KG-1a cells were evaluated using CCK-8 assay. Both compounds showed dose- and time-dependent inhibition of KG-1a cells proliferation after 24, 48 and 72 h. The IC₅₀ values of SNX-2112 were 1.02, 0.29 and 0.22 µM, respectively. These were lower than the IC₅₀ values of 17-AAG, which were 209.6, 30.27 and 44.75 µM, respectively (Fig. 1A and B), indicating that SNX-2112 inhibits cell growth to a greater degree.

To determine whether cell growth inhibition by SNX-2112 is associated with cell cycle arrest, the cell cycle distribution was analyzed using flow cytometry. KG-1a cells treated with SNX-2112 were arrested in the G2/M phase after 48 h (Fig. 1C); the proportion of arrested cells was 22.5, 41.2 and 28.7% at concentrations of 0.25, 0.5 and 1 µM, respectively, as compared to 17% in the control group (Fig. 1D).

To determine whether KG-1a cells differentiation was affected by SNX-2112 treatment, we analyzed the expression of the differentiation marker CD11b by flow cytometry at 24 and 48 h. CD11b expression was increased by treatment with SNX-2112 from 3.32 to 19.18% at 24 h (data not shown), and from 4.99 to 50.8% at 48 h (Fig. 2A and B).

Since the aim of AML differentiation therapy is to suppress the growth of AML cells, we performed a colony formation assay to determine whether SNX-2112 induced irreversible growth arrest. KG-1a cells were exposed to the drug for 2 days, and after washing, an equal number of viable cells were plated on soft agar. A marked decrease in the number of colonies formed was observed upon treatment with SNX-2112 (Fig. 2C), indicating that SNX-2112 can cause KG-1a cells growth arrest, and that SNX-2112 was more potent than 17-AAG.

To determine whether SNX-2112 inhibits KG-1a cells growth by inducing apoptosis, the treated cells were labeled with Annexin V/PI and analyzed by flow cytometry. Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺ cells were designated as early apoptotic and necrotic cells, respectively. The number of apoptotic cells was increased from 3.93% in the control cells to 14.82, 29.5 and 35.2% by treatment with 0.25, 0.5 and 1 µM SNX-2112, respectively, for 48 h (Fig. 3A and B). Moreover, the expression of the apoptosis-related protein cleaved caspase-3 was increased in the presence of SNX-2112, although that of B cell lymphoma (Bcl)-2 and Bcl-2-associated X protein were unaltered (Fig. 3C).

Hsp90 inhibition has been demonstrated to block PI3K and IKK signaling pathways (9). We therefore investigated whether SNX-2112 treatment affected the expression of Akt and IKK by western blotting. Previous studies revealed that Hsp90 was overexpressed in many types of cancer (3,5). Our results revealed that Hsp90 was highly expressed in KG-1a cells, and Hsp90 client proteins IKKα, IKKβ and Akt expression levels were downregulated upon treatment with SNX-2112 for 48 h (Fig. 4A). Consistent with previous studies (28), NF-κB pathway inhibitor Bar, inhibited KG-1a cell proliferation and induced apoptosis (Fig. 4B-D). Bar is an IKKβ-inhibitor which inhibited the expression of downstream protein p65 but did not directly interfere with the expression of IKK after treatment with Bar (0.5 µM) for 48 h (Fig. 4E), as Bar could possibly block the upstream kinases that have been implicated in the activation of IKK (29). SNX-2112 treatment resulted in the upregulation of NF-κB inhibitors (i.e., IκB) and downregulation of p65 and p-p65 after 48 h (Fig. 4F and G). Since C/EBPα and PU.1 play key roles in myeloid differentiation and NF-κB has been demonstrated to regulate PU.1 (30–32), we evaluated their expression after 48 h by qRT-PCR. The levels of both transcripts were increased by >2-fold in the presence of SNX-2112 (Fig. 5A and B). The increase in PU.1 expression was confirmed by immunofluorescence analysis (Fig. 5C). These results indicate that inhibition of Hsp90 by SNX-2112 suppresses the expression of Akt and IKK and induces that of C/EBPα and PU.1 via inhibition of NF-κB signaling in KG-1a cells.