Luteolin was purchased from Sigma-Aldrich (Oakville, ON, USA) and dissolved in dimethyl sulfoxide to stock concentration of 100 mM at −20°C. The Cell Counting kit-8 (CCK-8) and Hoechst 33258 were purchased from Beyotime Institute of Biotechnology (Beijing, China). Lipofectamine RNAiMAX, TRIzol and RT reagents were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). SYBR Green Supermix was purchased from Takara Bio, Inc. (Otsu, Japan). An Annexin V-FITC/propidium iodide (PI) double staining kit was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Antibodies were purchased against the phosphorylated forms of RSK1 (Ser221; ab10695; Abcam, Cambridge, MA, USA), B-cell lymphoma (Bcl)-2-associated death promoter (BAD) (Ser112; 9291; Cell Signaling Technology, Inc., Danvers, MA, USA), kidney/brain protein (KIBRA) (Ser947; ab107637; Abcam) and GAPDH (ab8245; Abcam), and were diluted at 1:1,000.

A total of 30 patients with primary newly-diagnosed acute myeloid leukemia (AML) were enrolled into the present study. The patients underwent consecutive chemotherapy at the Department of Hematology of the Sun Yat-Sen Memorial Hospital (Sun Yat-Sen University, Guangzhou, China) between July 2011 and July 2014. The samples were obtained by bone marrow aspiration prior to initiation of the therapy and after finishing 6 cycles of daunorubicin and cytarabine (DA) therapy, which was defined as the first complete remission (CR) stage (CR1). Each chemotherapy cycle included standard dose cytarabine (100–200 mg/m²) as a continuous infusion for 7 days with daunorubicin hydrochloride (40–60 mg/m²) ×3 once every 28 days. Informed consent was obtained from all patients following a protocol approved by the Ethics Committee of the Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University. AML was evaluated by the World Health Organization classification (19). CR was defined as a bone marrow sample with <5% blast cells and a neutrophil count of >10,000 cells.

RSK1 level was detected in MOLM-13, Kasumi-1 and primary AML cells. The primary AML cells were isolated from from 2 ml bone marrow using lymphocyte separation liquid by a density gradient centrifugation method (20). For each clinical sample, mononuclear cells were isolated from 2 ml bone marrow using lymphocyte separation liquid by a density gradient centrifugation method. In order to compare the expression of RSK1 in leukemic cell line, mononuclear cells were isolated from 2 ml peripheral blood of 3 cases using the seam method, and these samples were used as the control group. Total RNA was extracted from the samples using TRIzol reagent, according to the manufacturer's instructions. The cDNA was synthesized using the M-MLV reverse transcriptase kit (Invitrogen; Thermo Fisher Scientific, Inc.). Quantitative analysis of RSK1 mRNA expression was evaluated by qPCR using SYBR^® Green One-Step qRT-PCR kit (11736059; Invitrogen; Thermo Fisher Scientific, Inc.), and β-actin was used as an endogenous control. The sequences of the qPCR primers were as follows: RSK1 forward, 5′-GGTGGTCCTATGGGGTGTTG-3′, and reverse, 5′-TCGCCTTCAGAATCAGTGTCA-3′; and β-actin forward, 5′-TGAAGTGTGACGTGGACATC-3′, and reverse, 5′-GGAGGAGCAATGATCTTGAT-3′. All the reactions were performed in a 20 µl reaction volume in triplicate. The thermal cycling conditions were as follows: A denaturation step of 31 cycles at 90°C for 20 sec, an annealing step at 52°C for 25 sec and synthesis at 72°C for 20 sec. The fold changes were calculated through relative quantification with the 2^−∆∆Cq method.

MOLM-13 and Kasumi-1 cells (Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% heat-inactivated fetal bovine serum (10437028; Gibco; Thermo Fisher Scientific, Inc.) in a 95% humidified incubator with 5% CO₂ at 37°C. The cultures were split every second day by dilution to a concentration of 2×10⁵ cells/ml.

The full-length human RSK1 cDNA sequence was amplified from the cDNA library of MOLM-13 cells by PCR using the following two primers: Sense, 5′-AAGGTACCACCATGGAGCAGGATCCCAAGC-3′, and antisense, 5′-CTCTCGAGTCACAGGGTGGTGGATGGC-3′. Taq DNA polymerase (Thermo Fisher Scientific, Inc.) was used for amplification. The RSK1 cDNA sequencing was inserted into the pcDNA3.1 plasmid (Invitrogen; Thermo Fisher Scientific, Inc.). In order to silence RSK1 expression by siRNA, 21 nt complementary RNA with symmetrical 2 nt overhangs was obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). The DNA sequence was 5′-CCCAACATCATCACTCTGAAA-3′. The siRNA-RSK1 NC was also transferred into leukemic cells, which had the same number and type of bases as the siRNA-RSK1, but the arrangement was different. The DNA sequence was 5′-CCCAACCTAATCTACCTGAAA-3′. The plasmid and siRNA was transferred into MOLM-13 and Kasumi-1 cells, respectively.

MOLM-13 and Kasumi-1 cells were seeded into 96-well culture plates at a density of 1.5×10⁴ cells/well in 100 µl medium and were treated with different concentrations (15–60 µM) of luteolin for 24, 48 and 72 h. The pcDNA3.1-RSK1 plasmid was initially constructed, which overexpressed RSK1. Following transfection of cells with pcDNA3.1-RSK1 or empty plasmid for 24 h, the cells were treated with 30 µM luteolin, which was the representable value near the half maximal inhibitory concentration (IC₅₀), for 24 h. Viable cells were then evaluated with the CCK-8 assay according to the manufacturer's instructions. Briefly, CCK-8 solution (50 µl/well) was added to the cells in 24-well plates (0.6×10⁴ cells/well), and the samples were incubated at 37°C for 4 h. Subsequently, the optical density of each well was read at 450 nm using a microplate reader (ELx800 absorbance reader; BioTek Instruments, Inc., Winooski, VT, USA). The viability was assessed with the following equation: Viability (%) = Experimental / Control × 100%.

Apoptotic and dead cell counts were performed using a FITC-labeled Annexin V and PI staining by flow cytometry. The cells were collected and resuspended in binding buffer at a concentration of 3×10⁶/ml. Next, 100 µl cell suspension was added to 5 µl Annexin V-FITC and 10 µl PI, and the sample was mixed for 15 min in the dark at room temperature. Subsequently, 400 µl phosphate-buffered saline was added to the solution. A FACScan instrument (BD Biosciences, Franklin Lakes, NJ, USA) was used to count the cells (1×10³) at an excitation wavelength of 490 nm and determine the cell apoptosis. CellQuest software version 5.1 (BD Biosciences) was used for data collection and processing.

SDS Lysis Buffer (P0013G; Beyotime Institute of Biotechnology) was added to MOLM-13 and Kasumi-1 cells to isolate the proteins. Protein concentration was detected using a bicinchoninic acid kit (P0009; Beyotime Institute of Biotechnology). For each western blot sample, 20 µg protein was load onto each lane and resolved through 12% SDS-PAGE, and then electrophoretically transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). Next, the membrane was probed with primary antibodies against human RSK1 (Ser221; ab10695, Abcam), BAD (Ser112; 9291; Cell Signaling Technology, Inc.) and KIBRA (Ser947; ab107637; Abcam), with GAPDH (ab8245; Abcam) used as the internal control. The antibodies were diluted 1:1,000 and incubated at room temperature for 2 h. Next, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (ab6721; Abcam; dilution, 1:5,000) at room temperature for 1.5 h. The membranes were visualized with SignalFire™ ECL Reagent (#6883; Cell Signaling Technology, Inc.), and the band intensity was measured by Quantity One software 3.0 (Bio-Rad Laboratories, Inc. Hercules, CA, USA).

The migration of MOLM-13 and Kasumi-1 cells was assayed using a Boyden chamber (Qilinbeier Bio Co., Haimen, China). Cells were treated with different concentrations of luteolin and then plated at 1×10⁶ cells/ml in the upper chamber, with 10% RPMI-1640 medium added to the lower chamber. After incubating for 24 h, non-migration cells were removed from the top well, while the bottom cells were collected and counted by trypan blue exclusion assay in triplicate.

All computations were carried out using the SPSS v18.0 for Windows (SPSS, Inc., Chicago, IL, USA). Data were expressed as mean ± standard deviation. One-way analysis of variance was used to analyze the differences, and Kaplan-Meier survival analysis was used to analyze patient survival rate. P<0.05 was considered to indicate a statistically significant difference.

Aberrant RSK signaling is integral for several types of cancer, such as breast cancer and melanoma. In the present study, bone marrow samples from 30 AML patients (12 males and 18 females) at the newly-diagnosed and CR stages were analysed for RSK1 expression using the RT-qPCR method. The clinicopathological characteristics of the 30 AML patients are shown in Table I. The expression of RSK1 was found to be associated with the French-American-Britain (FAB) subtype and the percentage of minimal residual disease (MRD) cells. There were no statistically significant differences in other clinical features, including the patient gender and age, as well as the leukocyte, hemoglobin and platelet counts, between individuals with high and low RSK1 expression. Fig. 1A shows a markedly higher expression of RSK1 in newly diagnosed patients compared with that in patients at the CR1 stage. Furthermore, the prognostic significance of RSK1 expression was assessed in the 30 adult AML patients according to the clinical follow-up records. Kaplan-Meier survival analysis indicated that the high RSK1 expression group tended to have a shorter overall survival (OS) compared with that of the low RSK1 expression group (P=0.038; Fig. 1B).

RSKs serve a role in various cellular processes, including gene expression, cell survival, apoptosis and proliferation. The present study verified that, compared with normal mononuclear cells, RSK1 overexpression was observed in the leukemic cell lines MOLM-13 and Kasumi-1 (Fig. 2A). In order to investigate whether luteolin effects leukemic cells through RSK1, the cell viability of MOLM-13 and Kasumi-1 were first investigated upon treatment with luteolin. The luteolin concentration range used in the present experiment was 15–60 nM for 24, 48 and 72 h. The IC₅₀ for each concentration was determined and averaged for the triplicate experiments, and the final IC₅₀ values of luteolin on MOLM-13 and Kasumi-1 cells were found to be 34.75 and 34.05 µM, respectively. Luteolin treatment resulted in a marked reduction of cell proliferation in a dose-dependent manner (Fig. 2B).

To understand the mechanism by which luteolin caused viability loss in MOLM-13 and Kasumi-1 cells, apoptosis experiments were performed (Fig. 2C). The resultant viability loss was accompanied by decreased phosphorylation of proteins targeted by RSKs, such as BAD (21,22), which is a proapoptotic protein (Fig. 2D). Collectively, the current findings indicate that luteolin was able to induce the viability loss, while apoptosis of MOLM-13 and Kasumi-1 cells was associated with RSK1-associated apoptotic injury.

Leukocytes migrate into and out of blood vessels at multiple points during their development and maturation, and during immune surveillance. Migration is also an important part of leukemia progression. To determine the influence of luteolin on in vitro cell migration, the Boyden chamber assay was performed in MOLM-13 and Kasumi-1 cells. As shown in Fig. 3A, the results indicated that there was significantly less migration through the basement membrane following treatment with luteolin, in a dose-dependent manner. Cell migration of MOLM-13 and Kasumi-1 cells drove us to examine the possible biological functions of luteolin in leukemic cells, particularly the effect of this treatment on cell migration (Fig. 3B). The results demonstrated that the phosphorylated level of KIBRA also decreased in a dose-dependent manner.

To examine the role of RSK1 in the generation of luteolin-induced functional responses, MOLM-13 and Kasumi-1 leukemia cells were treated in the presence of luteolin with the RSK1-overexpressing plasmid or control plasmid. The overexpression of RSK1 mRNA and protein expression levels were evaluated by RT-qPCR and western blot analysis, respectively (Fig. 4A and B). As determined by RT-qPCR, the RSK1 mRNA level was upregulated by 15-fold, and the protein level in the RSK1 overexpression group was 3-fold greater than that of the control. The cell viability was also determined by CCK-8 assays, as shown in Fig. 4C. For the luteolin with RSK1-overexpressing plasmid group, transfection with the RSK1-overexpressing plasmid significantly counteracted the effect of luteolin on cell viability when compared with the luteolin alone group (Fig. 4C). Similarly, when induction of apoptosis was assessed, the apoptotic ratio of the luteolin and RSK1-overexpressing group was found to be significantly decreased (Fig. 4D), with a similar effect observed in the migration ability of cells (Fig. 4E).

To further verify whether RSK1 serves an important role in the luteolin-induced functional responses, the expression of RSK1 was blocked by siRNA transfection in MOLM-13 and Kasumi-1 leukemia cells. The siRNA silenced the expression of RSK1 on the mRNA and protein levels, as evaluated by RT-qPCR and western blot analysis, respectively (Fig. 5A and B). Following transfection with the siRNA-RSK1 in the leukemic cell lines, the cell viability was determined by CCK-8 assay. The results demonstrated that the viability of the siRNA-RSK1 group was significantly lower compared with that of the untreated control and NC groups (Fig. 5C). Similarly, when induction of apoptosis was assessed, the results identified that the apoptotic ratio of the siRNA group was significantly increased (Fig. 5D). By contrast, the migration ability decreased markedly upon silencing of RSK1 with siRNA (Fig. 5E).