COR and phorbol-12-myristate-13-acetate (PMA) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). COR was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA), and the proportion of DMSO in the culture medium was <0.05%. COR was stored at −20°C. Primary antibodies against phosphorylated (p)-ERK (Cat no. 4348S), total (t)-ERK (Cat no. 4695S), p-Akt (Cat no. 4060S), t-Akt (Cat no. 4685S), cleaved caspase-3 (Cat no. 1050S), β-actin (Cat no. 4970S) as well as Anti-rabbit Immunoglobulin G (IgG) secondary antibodies (Cat no. 5151S) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

THP-1 AMoL cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). THP-1 cells were cultured in RPMI 1640 medium (Hyclone; GE Healthcare, Little Chalfont, UK) containing 10% fetal bovine serum (Hyclone; GE Healthcare), 100 U/ml penicillin and 100 µg/ml streptomycin (Hyclone; GE Healthcare) in an incubator at 37°C with 5% CO₂. All cells we used at a passage of <20.

A Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) was used to assess the potential therapeutic effect of COR. For measurement of cell proliferation, THP-1 AMoL cells were seeded into each well of a 96-well plate at a density of 20,000 cells/well in 100 µl culture media containing 100 nM PMA to induce adherence, and treated with 0, 25, 50, 100, 150, 200 µmol/l COR for 96 h after adherence (22). Following treatment with COR for 24, 48, 72 and 96 h, 100 µl culture media and 10 µl CCK-8 solution were added to each well, followed by incubation at 37°C for 150 min. The optical density (OD) value at 450 nm was determined using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). Each experimental condition was repeated in three wells. The cell viability relative to that in the 0 µmol/l COR group (control) was calculated using the following equation: Cell viability (% of control group)=OD_{drug-treated group}/OD_{control group}.

THP-1 cells were incubated with various concentrations of COR for 24 h. Subsequently, the cells were washed twice with cold PBS and harvested. The cells were re-suspended in 1X Annexin-binding buffer, and stained with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI; BD Biosciences, Franklin Lakes, NJ, USA). The apoptotic rate was measured by flow cytometry using a BD FACSCalibur™ analyzer (BD Biosciences, Franklin Lakes, NJ, USA). The total apoptotic rate of cells was considered to be the early apoptotic rate (lower right quadrant in the PI vs. FITC dot plot) plus the late apoptotic rate (upper right quadrant in the dot plot).

THP-1 cells were cultured in complete medium in the presence of various concentrations of COR for 24 h. The total RNA of the THP-1 cells was then isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) following the manufacturer's instructions. For first-strand complementary DNA (cDNA) synthesis, RT was performed using the 1st Strand cDNA Synthesis kit (Takara Bio Inc., Dalian, China). The relative gene expression was determined by real-time PCR using the SYBR Premix Ex Taq kit (Takara Bio, Inc.) with the ABI Prism 7500 Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The real-time PCR thermocycling conditions were as follows: pre-degeneration at 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec and 60°C for 34 sec, and the dissociation stage was 34 sec at 95°C. The primers were designed and selected using the Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Gene expression was quantified using the 2^−∆∆Cq method (23). β-actin was used as the internal control. The primer sequences are listed in Table I.

THP-1 cells were treated as for the RT-qPCR analysis and then lysed with radioimmunoprecipitation assay buffer (Upstate Biotechnology, Inc., Lake Placid, NY, USA) supplemented with a protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). For western blot analysis, protein concentration was quantified using the BCA Protein Assay kit (Thermo Fisher Scientific, Inc.; catalogue no. 23250) according to manufacturer protocol. Samples were adjusted to the same protein concentration prior to loading (30 µg each sample) and separated by 10% SDS-PAGE for p-Akt, t-Akt, p-ERK, t-ERK and β-actin and 12.5% SDS-PAGE for cleaved caspase-3. Subsequently, proteins were transferred onto a nitrocellulose membrane (Merck Millipore, Billerica, MA, USA). Antibodies against p-Akt, t-Akt, p-ERK, t-ERK and cleaved caspase-3 were applied at the dilutions recommended by the manufacturer (1:1,000) at 4°C overnight. β-actin (1:1,000) was used as a loading control. After 37 min washes in Tris-buffered saline containing Tween-20 (TBST), the membranes were incubated with anti-rabbit second antibody (1:15,000; Cell Signalling Technology, Inc) for 1 h at room temperature. The target proteins were detected using the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA). The quantification of the bands was analyzed by the software of Odyssey Infrared Imaging System (Version 3.0).

All of the experiments were repeated 3 times. Values are expressed as the mean ± standard deviation. Statistical analysis was performed by one-way analysis of variance, followed by Duncan's post-hoc test using SPSS 19.0 (International Business Machines, Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

To assess the effects of COR on the proliferation of THP-1 cells, a CCK-8 assay using was performed. The inhibitory effect of COR on the proliferation of THP-1 cells was strengthened with the increase of the COR concentration after 24, 48, 72 and 96 h of treatment (Fig. 1). In conclusion, COR inhibited the proliferation of THP-1 cells in a dose- and time-dependent manner.

As presented in Fig. 2, flow cytometric analysis was employed to detect COR-induced apoptosis. Different concentrations of COR increased the total apoptotic rate in a dose-dependent manner (3.35% in the control vs. 11.48, 13.67 and 24.99% after treatment with COR at 100, 150 and 200 µM, respectively; P<0.05; Fig. 2A-G). Quantitative results on the early and late apoptotic rates were consistent with this phenomenon (Fig. 2H-I). To conclude, COR was found to exert its inhibitory function on THP-1 cells by induction of apoptosis, resulting in cell death.

To investigate the in-depth molecular mechanisms of the inhibitory effect of COR on THP-1 human monocytic leukemia cells, RT-qPCR was performed to examine its impact on the expression of apoptosis-associated genes. It was found that the expression levels of Bcl-2 were reduced in COR-treated THP-1 cells in a dose-dependent manner, while the expression of Bax was upregulated (Fig. 3). In addition, Akt signaling is crucial for the initiation and progression of AML and also regulates a number of downstream targets responsible for cell survival and proliferation (24,25). Therefore, the current study detected the mRNA level of three isoforms (Akt1, Akt2 and Akt3) of Akt. The cell survival-associated genes Akt1, Akt2 and Akt3 were downregulated after COR treatment (Fig. 4). Therefore, the phosphorylation of Akt was detected by western blot analysis based on the levels of mRNA (Fig. 4). These results indicated that these genes are potentially involved in signaling pathways downstream of those targeted by COR in monocytic leukemia cells.

To evaluate the potential involvement of the signal transduction pathways and the mechanisms of the effects of COR on THP-1 cells, the expression of various signaling proteins was determined in THP-1 cells by western blot analysis following treatment with different concentrations of COR. The results demonstrated that COR inhibited the protein expression of p-Akt and p-ERK in a concentration-dependent manner significantly (P<0.05; Fig. 5A-D). However, cleaved caspase-3 was significantly activated by increasing concentrations of COR (P<0.05; Fig. 5E and F). Taken together, these results suggested that Akt and ERK are potential downstream targets of COR in THP-1 cells, and that apoptosis is induced via associated signaling pathways.