Salidroside overcomes dexamethasone resistance in T-acute lymphoblastic leukemia cells.

The aim of the present study was to analyze whether the use of salidroside (SAL) could overcome dexamethasone (DEX) resistance in T-acute lymphocytic leukemia cells. The human T-ALL DEX-resistant cell line, CEM-C1 and the DEX-sensitive cell line, CEM-C7 were used in the current study. The proliferation inhibition rates in these cells, treated with SAL and DEX alone, and in combination were detected using a Cell Counting Kit-8 assay, while the morphological changes of the cells were observed using an inverted microscope. Reverse transcription-quantitative PCR was used to detect the mRNA expression levels of the c-Myc and LC3 genes, while flow cytometry was used to detect the cell cycle distribution and the rate of apoptosis. In addition, western blot analysis was used to detect the protein expression levels of c-Myc, BCL-2, Bax, cleaved PARP and LC3. and acridine orange staining was used to detect the changes in acidic autophagy vesicles. It was found that SAL could effectively inhibit cell proliferation and induce apoptosis in the CEM-C1 and CEM-C7 cells. In addition, SAL promoted the induction of autophagy. The protein expression levels of c-Myc in the CEM-C1 cells were significantly higher compared with that in the CEM-C7 cells. SAL downregulated the mRNA expression levels of the c-Myc gene and protein in a dose-dependent manner. This suggested that SAL could inhibit the proliferation of the CEM-C1 and CEM-C7 cells, induce apoptosis and autophagy and overcome DEX resistance in the CEM-C1 cells. The mechanism may be associated with the downregulation of c-Myc. Copyright: © Niu et al.

Introduction

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant tumor, that originates from T-cell precursors and has a high degree of genetic, immune phenotypic and clinical heterogeneity (1,2). It accounts for ~15% childhood ALL and 25% adult ALL worldwide (3). Administration of glucocorticoids (GC) is an important part of T-ALL treatment. GCs enter the cell by passive diffusion, where they bind to the GC receptor (GR; encoded by the NR3C1 gene), which is a member of the nuclear receptor family of ligand-dependent transcription factors (4-6). The activated receptor is then translocated to the nucleus, where it activates target genes, including NR3C1 itself, BCL-2, glucocorticoid-induced leucine zipper, Kruppel-like factor-13, NFKB inhibitor a and period 1, with assistance from chaperone and transporter proteins, and binds to GR elements (GREs) (7). GR-induced activation or repression of gene transcription controls apoptosis of normal and malignant lymphocytes (8). In lymphoid cells, GR induces the mRNA expression level of BCL2L11, which encodes the proapoptotic BH3-only factor, BIM, triggering apoptosis (9). In dexamethasone (DEX)-resistant ALL, the activated GR cannot bind to the BIM intronic region to trigger apoptosis (10). Therefore, resistance to GC is one of the most common causes of T-ALL treatment failure or relapse (11).

Salidroside (SAL) is the main active ingredient of Rhodiola. It is the glycoside of a phenolic compound. Several studies have shown that SAL has a potential anti-cancer effect (12-17). Therefore, SAL has become potential drug candidate for cancer treatment. Recently, another study has shown that SAL could improve the microenvironment of hypoxic tumors and reverse the resistance to platinum drugs in hepatocellular carcinoma (18). Thus, the human T-ALL GC DEX-resistant cell line, CEM-C1 and the DEX-sensitive cell line, CEM-C7 were selected as cell lines to investigate reversal of tumor resistance caused by SAL.

The proto-oncogene, c-Myc is a transcription factor, which belongs to the helix-loop helix-leucine zipper protein family, and functions primarily to maintain cell proliferation, differentiation, apoptosis and normal cell cycle (19). It has been found that c-Myc was associated with acute myeloid leukemia drug resistance (20). Mounting evidence also suggests that downregulation of c-Myc mRNA expression may increase the sensitivity of tumor cells to chemotherapeutic agents, including enhancing the sensitivity of breast cancer cells to palbociclib (21), the sensitivity of human glioblastoma cells to temozolomide (22), and the sensitivity of malignant mesothelioma cells to the p21-activated kinase blockage-induced cytotoxicity (23). In the present study, it was found that CEM-C1 cells exhibited higher protein expression levels of c-Myc compared with those in CEM-C7 cells. Since c-Myc has been associated with drug resistance in various studies (24-27), the present study aimed to reveal the anti-leukemic effect and reversal resistance effect of SAL, and to investigate c-Myc in T-ALL cells and its association with DEX resistance.

Materials and methods

Reagents

SAL (purity, >99%) was purchased from Chengdu Ruifensi Biotechnology Co., Ltd. RPMI 1640 culture medium was purchased from HyClone (GE Healthcare Life Sciences), while fetal bovine serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd., and penicillin-streptomycin was purchased from Beyotime Institute of Biotechnology. Cell Counting Kit (CCK)-8 assay kit was purchased from Dojindo Molecular Technologies Inc., while DEX (Chinese medicine standard, H41020036) was purchased from Shanghai Shyndec Pharmaceutical Co., Ltd., and the cell cycle detection kit was purchased from Nanjing KeyGen Biotech Co., Ltd., and the Annexin V-FITC/PI apoptosis kit was purchased from BD Biosciences. The total RNA extraction kit was purchased from Tiangen Biotech Co., Ltd., while the reverse transcription and quantitative PCR (qPCR) kits were purchased from Toyobo Life Science, and the acridine orange stain was purchased from Biotopped Life Sciences. The rabbit anti-human c-Myc and GAPDH antibodies were purchased from ProteinTech Group, Inc., while the rabbit anti-human LC3A/B, Bax, BCL-2 and cleaved PARP antibodies were purchased from Cell Signaling Technology, Inc., and the goat anti-rabbit IgG-HRP antibody was purchased from BIOSS. Lastly, the PCR primers were synthesized by Shanghai Shenggong Biology Engineering Technology Service, Ltd.

Cell lines and culture

The CEM-C1 and CEM-C7 cell lines were donated by Professor Ma Zhigui (Department of Pediatric Hematology and Oncology, West China Second Hospital of Sichuan University, Chengdu, China) and were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 µg/ml streptomycin and 100 U/ml penicillin at 37˚C in a humidified incubator with 5% CO2. The medium was changed every 2 to 3 days and the cells were passaged once before the start of the experiments.

Drug dissolution

SAL (1 g) was dissolved in sterile PBS (3 ml), made into a liquid and frozen in aliquots at -20˚C. The compound was diluted in RPMI 1640 medium to the required concentration prior to the experiment.

CCK8 assay

The CEM-C7 and CEM-C1 cells were used in the logarithmic growth phase and plated in 96-well microplates (1.5x105 cells/well), then different concentrations of SAL (5.0, 7.5, 10.0, 12.5 and 15.0 mg/ml) were added. At the same time, the blank group (containing only culture medium and no cells) and the control group (containing only cells and culture medium) were prepared. A total of 4 replicate wells were used for each group. Following incubation for 20, 44 and 68 h, 10 µl CCK8 solution was added to each well, then the cells were incubated for another 4 h, after which time the optical density (OD) was measured using a microplate reader at 450 nm. The experiment was repeated 3 times. The percentage cell inhibition rate (%) was calculated using the following formula: Cell inhibition=(OD value of control group-OD value of experimental group)/(OD value of control group-OD value of blank group) x100%.

The CEM-C7 and CEM-C1 cells were used in the logarithmic growth phase and plated in 96-well microplates (1.5x105 cells/well), then they were treated with different concentrations of DEX. The CEM-C7 cells were treated with 0.25, 0.5, 1.0, 1.5 and 2.0 µg/ml DEX with or without 1.5 mg/ml SAL (cell inhibition rate <4%), while the CEM-C1 cells were treated with 25, 50, 100, 150 and 200 µg/ml DEX with or without 1.5 mg/ml SAL (cell inhibition rate <4%). Following incubation for 44 h, 10 µl CCK8 solution was added to each well, then the cells were incubated for another 4 h, after which time the OD was measured using a microplate reader at 450 nm. The experiment was repeated 3 times. The half inhibitory concentration IC50 was calculated using the GraphPad Prism v8.0.2 software (GraphPad Software, Inc.). The resistance index (RI) was calculated using the following equation: RI=IC50 of resistant cells/IC50 of sensitive cells. The reversal fold (RF) was calculated as follows: RF=IC50 of resistant cells/IC50 following addition of the reversal agent.

Observation of cell morphology

The CEM-C1 and CEM-C7 cells, in the logarithmic growth phase, were treated with 1.5 µg/ml DEX for 48 h, then the morphological changes in the cells were observed under a light microscope and images were captured (magnification, x400).

Reverse transcription-qPCR (RT-qPCR) analysis

The CEM-C1 and CEM-C7 cells, in the logarithmic growth phase, were seeded in a 6-well culture plate (5x106 cells/well). The following experimental groups were used: Control group (0 mg/ml SAL) and the experimental groups (5.0, 7.5 and 10.0 mg/ml SAL). The cells were cultured for 48 h, then RNA was extracted using TRIzol®, according to the manufacturer's instructions (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was generated using RT and the TOYOBO reverse transcriptase kit. The mRNA expression levels of c-Myc and the autophagy-related gene, LC3, were detected using SYBR®-Green I Supermix (Toyobo Life Science), according to the manufacturer's instructions. The primer sequences are shown in Table I. The thermocycling conditions were as follows: Initial denaturation at 95˚C for 30 sec, followed by 40 cycles of 95˚C for 5 sec, 60˚C for 10 sec and 72˚C for 30 sec. Using GAPDH as the internal reference gene, the relative expression levels of the target genes were expressed using the 2-∆∆Cq method (28). The experiment was repeated 3 times.

Table I

Sequences of the primers for quantitative PCR.

Primer name	Primer sequence
c-Myc	F: 5'-CTACCCTCTCAACGACAGCA-3'
	R: 5'-AGAGCAGAGAATCCGAGGAC-3'
LC3	F: 5'-CAGCGTCTCCACACCAATCT-3'
	R: 5'-TCTCCTGGGAGGCATAGACC-3'
GAPDH	F: 5'-CAATGACCCCTTCATTGACC-3'
	R: 5'-GACAAGCTTCCCGTTCTCAG-3’

F, forward; R, reverse.

Cell cycle analysis using flow cytometry

The CEM-C1 and CEM-C7 cells, in the logarithmic growth phase, were seeded in a 6-well culture plate (3x105 cells/well), cultured for 48 h, then the cells were collected and washed with PBS solution. The supernatant was discarded and 500 µl 70% cold ethanol was added. The cells were fixed overnight at 4˚C. Prior to staining, the ethanol was removed and the cells were washed with PBS and centrifuged at 300 x g at 4˚C for 5 min. A total of 500 µl PI/RNase A staining working solution was added to each well. The samples were protected from light and incubated at room temperature for 30 min. The red fluorescence was examined at an excitation wavelength of 488 nm. The experimental groups were the same as those in the aforementioned RT-qPCR subheading.

Detection of cell apoptosis using flow cytometry

The CEM-C1 and CEM-C7 cells, in the logarithmic growth phase, were seeded in a 6-well culture plate (3x105 cells/well), cultured for 48 h, then the cells were collected, washed twice with cold PBS and finally resuspended with 1X binding buffer, to adjust the cell density to 1x106 cells/ml. A total of 100 µl cell suspension was used in a 5 ml flow cytometer tube and 5 µl PI was mixed with 5 µl Annexin V-FITC and added to the cells. The samples were shaken and placed at room temperature for 25 min in the dark. Subsequently, 200 µl 1X binding buffer was added to the cells, and measured using flow cytometry within 1 h. The experiment was repeated 3 times. The experimental groups were the same as those in the aforementioned RT-qPCR subheading. Additionally, according to whether SAL was combined with DEX, CEM-C1 cells were divided into control group, SAL group (1.5 mg/ml), DEX group (100 µg/ml) and combination group (DEX 100 µg/ml + SAL 1.5 mg/ml).

Western blot analysis

The CEM-C1 and CEM-C7 cells, in the logarithmic growth phase, were seeded into a 6-well culture plate (5x106 cells/well) and the total protein from each group was extracted 48 h later using RIPA lysis buffer (Beyotime Institute of Biotechnology) for 30 min and the total protein concentration was determined using the BCA method. A total of 25 µg total protein was extracted and analyzed using SDS-PAGE, transferred to a PVDF membrane, blocked with 7% skimmed milk at room temperature for 1 h and incubated with the following primary antibodies anti-GAPDH (1:15,000 dilution; cat. no. 10494-1-AP), anti-Bax (1:1,000 dilution; cat. no. 5023), anti-BCL-2 (1:1,000 dilution; cat. no. 4223), anti-cleaved-PARP (1:1,000 dilution; cat. no. 9185), anti-LC3A/B (1:1,000 dilution; cat. no. 12741) and anti-c-Myc (1:2,000 dilution; cat. no. 10828-1-AP) overnight at 4˚C. The membrane was washed 3 times with PBS with 0.07% Tween-20 (PBST), then the secondary antibody (HRP-labeled goat anti-rabbit antibody; 1:2,000; cat. no. bs-0295G-HRP) was added and the membrane was incubated for 1 h at room temperature. The membrane was washed with PBST three times and developed using an enhanced chemiluminescence kit (EMD Millipore). The protein expression level was measured using densitometry of the bands with ImageJ v1.4.3.67 (National Institute of Health). The protein expression levels were normalized to GAPDH. The experiments were repeated three times. The experimental groups were the same as those in the aforementioned RT-qPCR subheading. Additionally, according to whether SAL was combined with DEX, CEM-C1 cells were divided into control group, SAL group (1.5 mg/ml), DEX group (100 µg/ml) and combination group (DEX 100 µg/ml + SAL 1.5 mg/ml).

Acridine orange staining

The CEM-C1 and CEM-C7 cells, in the logarithmic growth phase, were seeded in a 6-well culture plate (3x105 cells/well), cultured for 48 h, then washed with PBS, and stained with acridine orange staining solution (10 µg/ml) for 30 min in the dark at room temperature. The cells were observed and images were captured using a fluorescence microscope (magnification, x400). The experimental groups were the same as those in the aforementioned RT-qPCR subheading.

Statistical analysis

The SPSS v23.0 software (IBM Corp.) was used for data analysis. The quantitative data are presented as the mean ± SD. Comparisons between two groups was performed using an independent Student's t-test, while one-way ANOVA was used for the comparison of multiple groups. Tukey's post hoc test was used when the homogeneity of variance was equal, while the Tamhane's T2 test was used when the variance was unequal. P<0.05 was considered to indicate a statistically significant difference.

Results

SAL inhibits the proliferation of the T-ALL cells

To investigate the anti-proliferative activity of SAL on the T-ALL cells, cell proliferation was determined using a CCK8 assay. As depicted in Fig. 1A, SAL effectively inhibited the proliferation of the CEM-C1 and CEM-C7 cells in a dose-and time-dependent manner. The IC50 of the CEM-C1 cells at 24, 48 and 72 h was 11.26, 6.69 and 6.45 mg/ml, respectively, while the IC50 of the CEM-C7 cells at 24, 48 and 72 h was 11.42, 8.03 and 7.73 mg/ml, respectively (data not shown). No significant difference was found in the IC50 values between the 48 and 72 h time points (P>0.05). Based on this finding, 48 h was selected as the intervention time point. In subsequent experiments, different concentrations of SAL (5.0, 7.5 and 10.0 mg/ml) to treat the cells were selected to detect the effect on cell cycle, apoptosis, and autophagy. The results also showed that SAL was more effective at inhibiting CEM-C1 cell viability compared with that in the CEM-C7 cells, which indicated that the DEX-resistant cells were more sensitive to SAL, as shown in Fig. 1B.

Figure 1

Effects of different concentrations of SAL and at different time periods on the CEM-C1 and CEM-C7 cells. (A) The CEM-C1 and CEM-C7 cells were treated with different concentrations of SAL for 24, 48 and 72 h, then cell viability was measured. *P<0.05, **P<0.01, ***P<0.001 vs. 24 h. #P<0.05 vs. 48 h. (B) The CEM-C1 and CEM-C7 cells were treated with 5.0 mg/ml SAL for the indicated time periods and subsequently incubated with Cell Counting Kit-8 solution. The data are presented as the mean ± SD. *P<0.05, **P<0.01. SAL, salidroside.

Effect of DEX on the morphology of the CEM-C1 and CEM-C7 cells

The CEM-C1 and CEM-C7 cells were treated with 1.5 µg/ml DEX for 48 h and cellular morphology was assessed using a light microscope. As shown in Fig. 2, the morphology of the DEX-resistant, CEM-C1 cells changed from slender and irregular shapes to round shapes and no notable reduction in cell viability was noted using microscopy compared with that in the control group. However, the DEX-sensitive CEM-C7 cells showed a large number of cell fragments and increased cell death compared with that in the control cells. It was suggested that CEM-C1 cells exhibited strong resistance to DEX.

Figure 2

Cell morphology was observed using a light microscope. The changes in cell morphology were monitored following treatment of the DEX-resistant, CEM-C1 and DEX-sensitive, CEM-C7 cell lines with 1.5 µg/ml DEX for 48 h. Compared with that in the control group, the morphology of the CEM-C1 cells changed to round shapes and no notable reduction in cell viability. CEM-C7 cells showed plenty of cell fragments and cell death were apparent, as shown by the black arrows. Magnification, x400. DEX, dexamethasone.

SAL enhances the sensitivity of the CEM-C1 cells to DEX

To verify the resistance of the CEM-C1 cells to DEX, the cytotoxic effect of DEX on DEX-sensitive CEM-C7 cells and DEX-resistant CEM-C1 cells was determined using a CCK-8 assay. Fig. 3A demonstrated that the IC50 in the CEM-C1 and CEM-C7 cells, treated with DEX and without SAL was 111.83±2.87 and 0.67±0.02 µg/ml, respectively, whereas the RI was 166.92 (data not shown). Our preliminary drug concentration screening results showed that the cell proliferation inhibition rate on the CEM-C1 and CEM-C7 cells treated with 1.5 mg/ml SAL was <4% (Fig. S1). Therefore, 1.5 mg/ml SAL was selected, combined with DEX, to culture the cells for 48 h. Fig. S2A indicated that the IC50 in the CEM-C1 cells treated with DEX + SAL was significantly decreased to 35.59±3.73 µg/ml. The RF was 3.14 (data not shown). In contrast to this finding, the DEX + SAL group exhibited no significant effect on the IC50 value in the CEM-C7 cells compared with that in the cells treated with DEX alone (Fig. S2B; P>0.05).

Figure 3

SAL enhances the chemosensitivity of the CEM-C1 cells to DEX. (A) The CEM-C1 and CEM-C7 cells were treated with DEX at various concentrations alone or in combination with 1.5 mg/ml SAL for 48 h, then cell viability was determined using a Cell Counting Kit-8 assay. The data are presented as the mean ± SD. ***P<0.001. (B) Flow cytometry using annexin V/PI staining was used to determine the rate of apoptosis after the cells were treated with 1.5 mg/ml SAL, 100 µg/ml DEX or in combination. ***P<0.001 vs. control group. ###P<0.001 vs. DEX group. (C) The expression level of the apoptotic-associated proteins in the CEM-C1 cells following treatment with 1.5 mg/ml SAL, 100 µg/ml DEX or in combination. *P<0.05, **P<0.01, ***P<0.001 vs. control group. #P<0.05, ##P<0.01 vs. DEX group. (D) The protein expression level of LC3 in the CEM-C1 cells following treatment with 1.5 mg/ml SAL, 100 µg/ml DEX or in combination. ***P<0.001 vs. control group. ###P<0.001 vs. DEX group. (E) The protein expression level of c-Myc in the CEM-C1 cells following treatment with 1.5 mg/ml SAL and 100 µg/ml DEX or in combination. ***P<0.001 vs. control group. ##P<0.01 vs. DEX group. SAL, salidroside; DEX, dexamethasone.

To determine whether SAL could enhance the sensitivity of the CEM-C1 cells to DEX, the CEM-C1 cells were treated with SAL (1.5 mg/ml), DEX (100 µg/ml) or in combination for 48 h. Flow cytometry analysis showed that a combination of SAL and DEX increased the apoptotic rate of the CEM-C1 cells from 10.65 to 26.35% compared with that in the DEX only group (Fig. 3B). Subsequently, western blot analysis showed that the combination treatment induced the activation of cleaved-PARP and Bax, and decreased the protein expression of BCL-2 (Fig. 3C). Notably, in the combination treatment group, there was also a significant increase in LC3 protein expression level when compared with that in the DEX or SAL only groups (Fig. 3D). Furthermore, Fig. 3E showed that the DEX alone group inhibited the protein expression level of c-Myc in the CEM-C1 cells and the combination of the two drugs was the most effective and statistically significant. The data suggested that SAL increased the sensitivity of the CEM-C1 cells to DEX.

Effect of SAL on the cell cycle in T-ALL cells

To investigate whether SAL could affect the cell cycle in the T-ALL cells, the CEM-C1 and CEM-C7 cell lines were treated with different concentrations of SAL for 48 h and subsequently stained with PI (Fig. 4A and B). Following an increase in SAL concentration, the percentage of the cells in the G0/G1 phase in the CEM-C7 cells was significantly decreased (F, 11.93; P<0.01), whereas the percentage of the cells in the S phase was significantly increased (F, 9.30; P<0.01). No significant change was noted with respect to the G2/M phase (P>0.05), indicating that SAL blocked the CEM-C7 cells in the S phase (Fig. 4D). However, SAL exhibited no significant difference in the cell cycle of the CEM-C1 cells (P>0.05; Fig. 4C).

Figure 4

Effects of SAL on cell cycle progression. Flow cytometry was used to detect the cell cycle distribution following treatment of the (A) CEM-C1 and the (B) CEM-C7 cells with SAL (0, 5.0, 7.5 or 10.0 mg/ml) for 48 h, and the data was subsequently (C and D) quantified, respectively. The data are presented as the mean ± SD. *P<0.05, **P<0.01 vs. control group. SAL, salidroside.

Effect of SAL on the induction of apoptosis in the T-ALL cells

To investigate whether SAL could induce apoptosis in the T-ALL cells, the CEM-C1 and CEM-C7 cell lines were treated with SAL at different concentrations. The results indicated that SAL could increase the early, late and total apoptotic rate of the CEM-C1 and CEM-C7 cells (Fig. 5A and C). Following an increase in the concentration of SAL, CEM-C1 cells underwent apoptosis. The total apoptotic rate was significantly increased from 5.06±0.66% in the control group to 10.18±0.87% in cells treated with 7.5 mg/ml SAL (P<0.01), whereas treatment with 10.0 mg/ml SAL increased the total apoptotic rate to 15.34±1.45%, which was significantly higher compared with that in the control group (P<0.001; Fig. 5B). In the CEM-C7 cells, the total apoptotic rate following 10.0 mg/ml SAL treatment was 16.62±3.44%, which was significantly higher compared with that in the control group 3.43±0.46% (P<0.001; Fig. 5D). This suggested that SAL could induce apoptosis in the human T-ALL cell lines.

Figure 5

Effect of SAL on the rate of apoptosis in the CEM-C1 and CEM-C7 cells. (A) The CEM-C1 cells were treated with different concentrations of SAL for 48 h and subsequently analyzed using flow cytometry using Annexin V-FITC/PI, then (B) statistically analyzed. (C) The CEM-C7 cells were treated with different concentrations of SAL for 48 h and subsequently analyzed using flow cytometry, then (D) statistically analyzed. The data are presented as the mean ± SD. **P<0.01, ***P<0.001 vs. control group. SAL, salidroside.

Effect of SAL on the expression level of apoptosis-associated proteins

To further investigate the molecular mechanism of SAL in promoting apoptosis of the T-ALL cell lines, the expression level of the pro-apoptotic and anti-apoptotic proteins was determined. Western blot analysis indicated that there was an increase in the expression levels of Bax and cleaved-PARP proteins following treatment with different concentrations of SAL. There was also inhibition in the protein expression level of BCL-2 in the CEM-C1 and CEM-C7 cells, in a dose-dependent manner (Fig. 6).

Figure 6

Effect of SAL on the expression of the apoptotic-associated proteins in the CEM-C1 and CEM-C7 cells. (A) Western blot analysis was used to determine the relative expression levels of the apoptotic-associated proteins in the CEM-C1 cells, then the data was (B) quantitatively analyzed. (C) Western blot analysis was used to determine the relative expression levels of the apoptotic-associated proteins in the CEM-C7 cells, then (D) quantitatively analyzed. The data are presented as the mean ± SD. *P<0.05, **P<0.01, ***P<0.001 vs. control group. SAL, salidroside.

SAL induces autophagy in the T-ALL cells

Autophagy is characterized by the formation of acidic autophagy vesicles in the cells and can be determined using acridine orange staining (29). Acridine orange is a fluorescent dye used for detecting the structure of acid vesicles that produces green fluorescence following binding to the nucleoli and the cytoplasm, and red fluorescence following binding to autophagic lysosomes (30). The results of acridine orange staining indicated that the number of orange fluorescent organelles in the CEM-C1 and CEM-C7 cells, corresponding to the number of acidic autophagy vesicles, was notably increased compared with that in the control group. This suggested that SAL promoted autophagy in the human T-ALL cell lines (Fig. 7A and B).

Figure 7

Acridine orange staining for the detection of autophagy in the CEM-C1 and CEM-C7 cells. The (A) CEM-C1 and (B) CEM-C7 were treated with different concentrations of SAL then stained with acridine orange. Green fluorescence indicates staining of the nucleoli and the cytoplasm, while red fluorescence indicates staining of the autophagic lysosomes and orange fluorescence indicates staining of the acidic autophagy vesicles (white arrows). Magnification, x400. SAL, salidroside.

Effect of SAL on autophagy-related protein expression levels

During the process of autophagy, LC3 is the membrane component of the autophagosome extension and LC3 is converted from LC3-I to LC3-II (31). Therefore, LC3-II can be used to quantify the number of intracellular autophagosomes (32). The results of western blot analysis showed that compared with that in the control group, the expression levels of the LC3-II protein in the CEM-C1 and CEM-C7 cells was significantly increased, and the protein expression ratio of LC3-II/LC3-I was also increased (F, 77.64 and 73.88, respectively, with 10.0 mg/ml SAL; both P<0.001; Fig. 8A-D). The mRNA expression level of LC3 was also found to be upregulated (F, 19.11 and 37.49, with 10.0 mg/ml SAL; P<0.05; Fig. 8E and F). This suggested that SAL could induce autophagy in the human T-ALL cell lines, CEM-C1 and CEM-C7.

Figure 8

Western blot analysis and reverse transcription-quantitative PCR was used to detect the protein and mRNA expression level of autophagy-related proteins in the CEM-C1 and CEM-C7 cells following SAL treatment. (A) The effects of SAL on LC3 protein expression level in the CEM-C1 cells following treatment with various concentrations of SAL for 48 h, then the results were (B) quantified. (C) The effects of SAL on LC3 protein expression level in the CEM-C7 cells following treatment with SAL at various concentrations for 48 h, then the data was subsequently (D) analyzed. Relative LC3 mRNA expression level in the (E) CEM-C1 and (F) CEM-C7 cells. The data are presented as the mean ± SD. *P<0.05, **P<0.01, ***P<0.001 vs. control group. SAL, salidroside.

Protein expression of c-Myc in the DEX-resistant CEM-C1 cells

To investigate the role of c-Myc in the DEX-resistant CEM-C1 cells, western blot analysis was used to detect the expression levels of the c-Myc protein in the CEM-C1 and CEM-C7 cells. The results indicated that the CEM-C1 cells expressed higher c-Myc protein levels compared with that in the CEM-C7 cells (Fig. 9A and B). High expression of c-Myc may reduce the sensitivity of the CEM-C1 cells to DEX, indicating that c-Myc could play an important role in the occurrence and development of tumor drug resistance.

Figure 9

High protein and mRNA c-Myc expression level in the DEX-resistant cell line, CEM-C1 (A) Western blot analysis was used to analyze the protein expression level of c-Myc in the CEM-C1 and CEM-C7 cells, then the data was subsequently statistically (B) quantified.

SAL overcomes DEX-resistance in the CEM-C1 cells by downregulating c-Myc protein and mRNA expression

Various studies have shown that high mRNA expression of c-Myc has been associated with drug resistance in pancreatic cancer and HPV-negative neck squamous cell carcinoma cells (33,34). It has also been shown that downregulation of c-Myc mRNA expression using siRNA could improve the efficacy of DEX in treatment of ALL (35). To investigate the mechanism in which the CEM-C1 cells could overcome DEX resistance following treatment with SAL, the CEM-C1 and CEM-C7 cells were treated with different concentrations of SAL for 48 h, and the protein and mRNA expression levels of c-Myc were determined. Western blot analysis indicated that the c-Myc protein expression level was decreased in a dose-dependent manner, in both cells, compared with that in the control group (F, 21.74 and 18.58, with 10.0 mg/ml SAL; P<0.001; Fig. 10A-D). The RT-qPCR results indicated that the c-Myc mRNA expression levels were also decreased in a dose-dependent manner compared with that in the control group (F, 43.14 and 161.0, with 10.0 mg/ml SAL; P<0.05; Fig. 10E and F). This suggested that SAL could reduce DEX resistance in the human T-ALL, CEM-C1 cells by downregulating c-Myc protein and mRNA expression.

Figure 10

Effect of SAL on c-Myc protein and mRNA expression levels in the CEM-C1 and CEM-C7 cells. (A) The CEM-C1 cells were treated with different concentrations of SAL for 48 h, then the c-Myc protein expression level was analyzed using western blot analysis, and the results were subsequently (B) quantified. (C) The CEM-C7 cells were treated with different concentrations of SAL for 48 h, then the c-Myc protein expression level was analyzed using western blot analysis, and the results were subsequently (D) quantified. The relative c-Myc mRNA expression levels in the (E) CEM-C1 and (F) CEM-C7 cells following treatment with different concentrations of SAL. The data are presented as the mean ± SD. *P<0.05, **P<0.01, ***P<0.001 vs. control group. SAL, salidroside.

Discussion

ALL is one of the most common malignancies, with the highest incidence rate in children, accounting for ~80% of leukemia cases. ALL is five times more common than acute myeloid leukemia (36). ALL can be divided into B-ALL and T-ALL. DEX is a synthetic GC, which has been used to treat patients with T-ALL (37). At present, resistance to DEX is one of the important reasons leading to treatment failure or recurrence. Therefore, it is important to clarify the mechanism of DEX resistance and overcome it.

Tumor cells are characterized by unrestricted proliferation. The two main pathways of tumor cell death are apoptosis and autophagy. Cell apoptosis and autophagy have been associated with tumorigenesis and cancer prevention (38). A previous study has shown that dysregulation of apoptosis promoted the survival of malignant cells and reduced the sensitivity of tumor cells to specific drugs in leukemia (39). Autophagy is an important intracellular process that causes the degradation of unnecessary or damaged cytoplasmic contents to maintain metabolism and homeostasis (40). Autophagy exhibits a dual function by promoting cell survival and cell death, and has been associated with tumorigenesis, metastasis and drug resistance (41). The induction of apoptosis and autophagy is an effective antitumor therapy strategy (42,43). Long et al (44) demonstrated that by promoting the induction of autophagy and apoptosis, this process could increase the sensitivity to GC treatment in human acute lymphoblastic leukemia cells.

SAL has been reported to have a wide range of pharmacological functions, including anti-tumor activity, that SAL-based activation of apoptosis and autophagy are the major mechanisms responsible for the anti-cancer activity of this compound (45). A previous study has shown that SAL induced apoptosis and autophagy in human colon cancer cells by inhibiting the PI3K/Akt/mTOR pathway (46). The therapeutic effect of SAL on a variety of tumors has been confirmed, including colorectal cancer (12), gastric cancer (47), bladder cancer (14), ovarian cancer (15), breast cancer (48) and Wilms' tumor (17); however, its role in promoting T-ALL apoptosis and autophagy and its molecular mechanism are not clear. In the present study, the protein expression levels of cleaved-PARP, Bax and LC3 were increased, while BCL-2 protein expression level was decreased in the CEM-C1 and CEM-C7 cells following treatment with SAL. This indicated that SAL could be a potential treatment for T-ALL. It was also found that DEX could induce apoptosis and autophagy in the CEM-C1 cells. In addition, when 1.5 mg/ml SAL (cell inhibition rate, <4%) was combined with DEX, the induction of apoptosis and autophagy was significantly increased (P<0.01) compared with that in the DEX group.

Previous studies have shown that DEX resistance was associated with upregulation of the oncogene c-Myc mRNA expression (10,49). In a separate study, Bhadri et al (50) demonstrated that in vivo DEX treatment in a DEX-sensitive ALL xenograft caused significant repression of c-Myc mRNA expression. In the present study, it was found that the CEM-C1 cells exhibited a higher protein expression level of c-Myc compared with that in the CEM-C7 cells. Long et al (51) demonstrated that imatinib-resistant K562/G cells exhibited high protein expression level of c-Myc compared with that in the parental K562 cells, and the c-Myc inhibitor 10058-F4 was found to reverse resistance caused by high expression level of c-Myc. It has also been shown that c-Myc inhibitors can produce synergistic anti-cancer effects with vincristine and sensitize pre-B-ALL cells to the anti-tumor effects of this chemotherapeutic drug by inducing apoptosis and autophagy (52). Sayyadi et al (53) demonstrated that c-Myc inhibition, using 10058-F4, increased the sensitivity of acute promyelocytic leukemia cells to arsenic trioxide. The results from the present study demonstrated that SAL could reduce c-Myc protein and mRNA expression levels. Notably, the combination treatment of SAL with DEX resulted in a more significant inhibition of c-Myc expression compared with that in the DEX group. Therefore, future studies should combine c-Myc inhibitors with SAL to verify their effects on apoptosis and autophagy, and the sensitivity to T-ALL cells to DEX.

In summary, the present study demonstrated the reversal effect of SAL on DEX resistance in the CEM-C1 cell line and confirmed that SAL exhibited an optimal effect on inhibiting proliferation, and induced apoptosis and autophagy in both the CEM-C1 and CEM-C7 cells. The CEM-C1 cells were more sensitive to SAL. SAL may overcome the resistance of the CEM-C1 cells to DEX by downregulating c-Myc protein and mRNA expression level. DEX resistance is a challenging problem for T-ALL chemotherapy. This provides a new treatment strategy for overcoming drug resistance and new evidence for clarifying the molecular mechanism of T-ALL-associated DEX resistance. The data further suggested that c-Myc may be a target for treating T-ALL resistance to DEX.