Isoliquiritigenin induces apoptosis of human bladder cancer T24 cells via a cyclin-dependent kinase-independent mechanism.

The aim of the present study was to investigate whether an increase in cyclin-dependent kinase 2 (CDK2) activity is involved in apoptosis of human bladder cancer T24 cells induced by isoliquiritigenin (ISL). The viability of T24 cells was estimated using a sulforhodamine B assay. Cell morphological changes were examined using Hoechst 33258 staining. The apoptotic rate was determined by staining cells with Annexin V-fluorescein isothiocyanate and propidium iodide labeling. The mitochondrial membrane potential (ΔΨm) was measured using 5,5,6,6-tetrachloro-1,1, 3,3-tetraethyl benzimidazole carbocyanine iodide. Alterations in the apoptosis-related regulators B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Bcl-2-interacting mediator of cell death (Bim), apoptotic protease-activating facter-1 (Apaf-1), caspase-9 and caspase-3 were determined using reverse transcription-polymerase chain reaction (PCR) and quantitative PCR methods. Western blot analysis was used to detect the expression of Bcl-2, Bax and caspase-3. CDK2 activity was measured using a spectrometric assay. Following treatment with ISL (between 30 and 70 µg/ml) for 24 h, typical apoptotic morphological changes were observed in T24 cells, exhibiting an edge set of chromosomes, nuclear condensation, nuclear fragmentation and other morphological features. Treatment with ISL increased the apoptotic ratio of T24 cells in a concentration-dependent manner and induced a decrease in the ΔΨm in a time-dependent manner. Treatment with ISL upregulated the expression of Bax, Bim, Apaf-1, caspase-9 and caspase-3, downregulated the expression of Bcl-2, and increased CDK2 activity. MK-8776 (an inhibitor of CDK2) antagonized the apoptosis induced by ISL, and, compared with treatment with ISL alone, pretreatment with MK-8776 inhibited the decrease in ΔΨm, downregulated the mRNA expression of Bax, Bim, Apaf-1, caspase-9 and caspase-3, and upregulated Bcl-2 mRNA expression. Western blot analysis demonstrated that, with increasing ISL concentration, the Bcl-2 expression level was significantly decreased (P<0.05), whereas caspase-3 and Bax expression levels were significantly increased (P<0.01). These results indicated that ISL treatment caused a significant decrease in the proliferation rate and increase in apoptosis of T24 cells. The mechanism by which ISL induces T24 cell apoptosis in vitro may be associated with an increase in CDK2 activity, downregulation of the ΔΨm and activation of caspase-3/caspase-9-mediated mitochondrial apoptotic signaling pathways.

Introduction

To the best of our knowledge, bladder cancer is currently the fourth most common cancer worldwide (1). In China, the incidence of bladder cancer had increased from 1991 to 2009, with >20,000 mortalities in 2009 (2,3). In the United States, there were ~76,000 new cases of bladder cancer and 16,000 bladder cancer-associated mortalities in 2016 (4,5). Currently, although surgical therapies associated with adjuvant chemotherapy following surgery have made marked advances in the treatment of bladder cancer, the rate of recurrence remains high (6). Furthermore, chemotherapy has a high incidence of side effects. Consequently, research into and development of highly efficient and minimally toxic novel drugs is urgently required (7).

Cyclin-dependent kinases (CDKs) are a family of serine/threonine kinases that are required for cell cycle progression, particularly CDK2. CDK2 serves an important role in G1/S phase transition and S phase progression (8,9). In previous studies, it was also demonstrated that cyclin A-CDK2 activity was markedly upregulated in the early stages of apoptosis and that this upregulation was required for the progression of apoptosis induced by treatment with ginsenoside Rh2 (10). Nevertheless, the mechanism by which CDK2 functions in the progression of apoptosis in T24 cells induced by isoliquiritigenin (ISL) remains unknown.

Recently, natural products have become one of the important sources in modern drug discovery for its active components with anticancer potential and fewer side effects, particularly for anticancer drugs (6). Licorice is frequently used for culinary purposes in western countries (11–13), and is one of the most commonly used herbs in China for therapeutic effects on different diseases ranging from duodenal ulcers to cancers (14–16). ISL is a natural flavonoid isolated from the root of licorice (Glycyrrhiza uralensis). Previous studies have indicated that ISL possesses distinct biological properties, including anti-inflammatory, antioxidant, anti-platelet aggregation, vasorelaxant and estrogenic effects (17–19). A number of studies have demonstrated marked antitumor activities of ISL, including apoptosis induction, cell cycle arrest, migration inhibition and initiation of oxidative stress (20–22). Previous studies have also demonstrated that ISL possesses chemopreventive activities (23–26).

In view of previous studies suggesting an association between CDK2 and apoptosis, the aim of the present study was to determine the effect of MK-8776, an inhibitor of CDK2, on the ISL-induced apoptotic rate in T24 cells. In the present study, the potential underlying molecular mechanism of CDK2 in apoptosis induced by ISL in T24 cells was investigated.

Materials and methods

Reagents

ISL (purity, ≥98%) was obtained from Jiangxi Herb Tiangong Technology Co., Ltd. (Jiangxi, China). Culture medium (RPMI-1640), glutamine and dimethylsulfoxide (DMSO) were purchased from Sigma; Merck KGaA (Darmstadt, Germany). Fetal bovine serum (FBS) was purchased from Tianjin Hao Yang Biological Manufacture Co., Ltd. (Tianjin, China). Penicillin and streptomycin were obtained from Shandong Sunrise Pharmaceutical Co., Ltd. (Shandong, China). MK-8776 was obtained from Selleck Chemicals (Shanghai, China). The CDK2/Cyclin A Kinase Assay kit was obtained from Shanghai Genmed Gene Pharmaceutical Technology Co., Ltd. (Shanghai, China). Primary antibodies against β-actin (catalog no. sc-130300), B cell lymphoma 2 (Bcl-2) (catalog no. sc-130308), caspase-3 (catalog no. sc-56052) and Bcl-2 associated X factor (Bax; catalog no. sc-80658) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Unless indicated otherwise, other reagents were purchased from Sigma; Merck KGaA. All other chemicals were of analytical grade.

Cell culture and treatment

T24 cells were purchased from the Cell Bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were maintained in RPMI-1640 medium supplemented with 10% (v/v) FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Cells were allowed to attach for 24 h before treatment. ISL was dissolved in DMSO and diluted with fresh medium to achieve the desired concentration. The final concentration of DMSO was <0.2% in the fresh medium, and DMSO at this concentration exhibited no significant effect on cell viability.

Cell viability assay

The T24 cells were trypsinized and seeded into 96-well plates at 8×104 cells/ml. Subsequently, the cells were exposed to ISL (0, 15, 30, 45, 60 and 75 µg/ml) at 37°C for 24 h followed by further incubation in fresh medium at 37°C for another 24 h. The effect of ISL-induced cytotoxicity was evaluated using a sulforhodamine B (SRB) assay as described previously (27). The optical density (OD) was determined at a wavelength of 490 nm. The inhibition ratio was calculated as follows.

Trypan blue exclusion assay

The lethality of ISL on T24 cells was determined using a trypan blue exclusion assay. After 24 h of incubation with 0, 15, 30, 45, 60 and 75 µg/ml ISL at 37°C, T24 cells were removed from the culture medium and cells that excluded trypan blue were counted in a Neubauer chamber using a light microscope. The lethal ratio was calculated as follows.

Hoechst 33258 staining

The T24 cells were seeded in 6-well culture dishes (2×105 cells/well). Following treatment with ISL at 0, 30, 50 and 70 µg/ml at 37°C for 24 h, the cells were fixed with Carnoy's fixative consisting of methanol and glacial acetic acid (3:1, v/v), washed twice with PBS, stained with Hoechst 33258 (5 mg/ml) for 5 min in the dark, as described previously (28), and washed extensively three times with PBS. Nuclear staining was examined using a fluorescence microscope (Nikon LH-M100CB; Jirui Co., Ltd., Suzhou, China) and images were captured using Image-Pro Plus Premier (version 9.1.4; Media Cybernetics, Inc., Rockville, MD, USA). Cells that exhibited decreased nuclear size, chromatin condensation, intense fluorescence and nuclear fragmentation were considered apoptotic.

Analysis of apoptosis by Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI)

The cells (2×105 cells/well) were pretreated with 0.16 µM MK-8776 at 37°C for 1 h, incubated with ISL at 0, 30, 50 and 70 µg/ml at 37°C for 24 h and harvested. Specific binding of Annexin V-FITC was carried out by incubating the cells at room temperature for 15 min in a binding buffer containing a saturating concentration of Annexin V-FITC and PI, as described previously (29). Following incubation, the cells were quantified using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using CellQuest Pro acquisition software (version 4.0; FACS Calibur; BD Biosciences, San Jose, CA, USA).

Detection of mitochondrial membrane potential (ΔΨm) using 5,5,6,6-tetrachloro-1,1,3,3-tetraethyl benzimidazole carbocyanine iodide (JC-1)

Following reaching optimal confluence, cells were pretreated with 0.16 µM MK-8776 for 1 h, and incubated with 50 µg/ml ISL at 37°C for 24 h. In addition, the cells not pretreated with MK-8776 at 0.16 µM were incubated with ISL at 0, 30, 50 and 70 µg/ml at 37°C for 4, 8, 16 and 24 h washed with ice-cold PBS, exposed to 500 µl JC-1 dye and incubated in the dark at 37°C for 20 min. Following washing three times with incubation buffer (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), the cells were diluted in 500 µl incubation buffer and the fluorescence intensity of the cells was analyzed using a FACScan flow cytometer (BD Biosciences). Fluorescence at excitation/emission wavelengths of 485/580 nm (red) and 485/530 nm (green) was read using a fluorescence plate reader (Varioskan Flash 3001; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR)

RT-PCR and qPCR (30,31) were performed to examine the mRNA expression of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Bcl-2-interacting mediator of cell death (Bim), apoptotic protease-activating factor-1 (Apaf-1), caspase-9 and caspase-3. The primer sequences are presented in Table I. The cells were pretreated with 0.16 µM MK-8776 at 37°C for 1 h and incubated with 50 µg/ml ISL at 37°C for 24 h. The cells were further incubated with 0, 30, 50 and 70 µg/ml ISL at 37°C for 24 h and harvested. Total RNA was extracted from the T24 cells using TRIzol reagent (Shanghai Sangong Biotech Co., Ltd. (Shanghai, China) and quality was determined according to the A260/A280 ratio. cDNA synthesis was performed using a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Subsequently, the synthesized cDNA was amplified. The cycling conditions were set as indicated by Sangong Biotech Co. Ltd. The reaction mixture contained 12.5 µl 2X PCR Master mix (Sangong Biotech Co., Ltd.), 3 µl cDNA template and 0.5 µl each primer. The cycling conditions used were as follows: 95°C for 5 min, followed by 37 cycles of 94°C for 30 sec, heating at specified melting temperature (Tm, Table I) for 30 sec, 72°C for 30 sec and 72°C for 5 min. cDNA products were analyzed using 2% agarose gel electrophoresis. Three independent experiments were performed. Images were captured using Image-Pro Plus Premier (version 9.1.4; Media Cybernetics, Inc., Rockville, MD, USA).

Table I.

Primer sequences.

Gene	Primer	Tm, °C
Bcl-2	Forward: 5′-GGAAATATGGCGCACGCT-3′	59
	Reverse: 5′-TCACTTGTGGCCCA-3′
Bax	Forward: 5′-ACGAACTGGACAGTAACATGGAG-3′ 59
	Reverse: 5′-CAGTTTGCTGGCAAAGTAGAAAAG-3′
Bim	Forward: 5′-CACATGAGCACATTTCCCTCT-3′ 57
	Reverse: 5′-AAGGCACAAAACCTGCAGTAA-3′
Apaf-1	Forward: 5′-TGGAATGGCAGGCTGTGGGA-3′	62
	Reverse: 5′-TGCACTCCCCCTGGGAAACA-3′
Caspase-9	Forward: 5′-ACGCGTTACTGGCATTGAGG-3′	62
	Reverse: 5′-CAGTGGGCTCACTCTGAAGACC-3′
Caspase-3	Forward: 5′-CTGGACTGTGGCATTGAGAC-3′ 59
	Reverse: 5′-ACAAAGCGACTGGATGAACC-3′
GAPDH	Forward: 5′-CAAGGTCATCCATGACAACTTTG-3′ 57
	Reverse: 5′-GTCCACCACCCTGTTGCTGTAG-3′

Tm, melting temperature. Bcl-2, B-cell lymphoma-2; Bax, Bcl-2-associated X protein; Bim, Bcl-2-interacting mediator of cell death; Apaf-1, apoptotic protease-activating factor-1.

Relative gene expression was quantified using qPCR with a SYBR Green kit (Rotor-Gene Q; Qiagen, Inc. Valencia, CA, USA), enabling detection of PCR products, according to the manufacturer's protocol. The cDNA was amplified using qPCR with the primer pairs given in Table I (synthesized by Sangon Biotech Co., Ltd.). The cycling conditions were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 30 sec, Tm (see Table I) for 30 sec, and 72°C for 30 sec. A total of three independent experiments were performed. Quantitative data was analyzed using the Sequence Detection system software (version 1.0; Applied Biosystems; Thermo Fisher Scientific, Inc.). The relative quantity of target mRNA was determined using the 2−ΔΔCq method (32). Each sample was analyzed using GAPDH as an endogenous reference gene for mRNA normalization.

CDK2 activity assay

T24 cells were seeded at a density of 2.5×105 cells/ml in flasks and incubated at 37°C for 24 h. The cells were pretreated with 0.16 µM MK-8776 at 37°C for 1 h and incubated with 50 µg/ml ISL at 37°C for 24 h. The cells were further incubated with ISL at 0, 30, 50 and 70 µg/ml at 37°C for 4, 8, 16 and 24 h. Following the addition of 2 ml cleanup solution (CDK2/cyclin A kinase assay reagent A; Shanghai Genmed Gene Pharmaceutical Technology Co., Ltd., Shanghai, China) and protein extraction, the CDK2/cyclin A kinase assay kit (Shanghai Genmed Gene Pharmaceutical Technology Co., Ltd.) was used to determine CDK2 activity, according to the manufacturer's protocol. Experiments were performed in triplicate.

Western blot analysis

The cells were harvested, extracted with cell lysis buffer (catalog no. R0010; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and centrifuged at 12,000 × g for 5 min at 4°C. Protein concentration was determined using a bicinchoninic acid assay kit (catalog no. PC0020; Beijing Solarbio Science & Technology Co., Ltd.). The protein lysates were diluted to equal concentrations and heated at 100°C for 5 min. Soluble lysates (15 µl/lane) were subjected to SDS-PAGE (10% gel), transferred onto nitrocellulose membranes (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) and blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBST) for 2 h at room temperature. The membranes were incubated with the anti-β-actin (mouse anti-human; 1:5,000), anti-Bcl-2 (monoclonal, mouse anti-human; 1:500), anti-caspase-3 (monoclonal, mouse anti-human; 1:500) and anti-Bax (monoclonal, mouse anti-human; 1:400) at 4°C overnight and incubated with horseradish peroxidase-conjugated secondary antibody (goat anti-mouse, 1:5,000; catalog no. HS201; Beijing TransGen Biotech Co., Ltd., Beijing, China). Western blots were developed using enhanced chemiluminescence (ECL, Thermo Fisher Scientific, Inc.) and exposed on radiographic film (Kodak, Rochester, NY, USA). Densitometric analysis was performed using Image Quant LAS 4000 (Amersham, Buckinghamshire, UK), and the autoradiographs were quantified using ImageJ software (version 1.49n; National Institutes of Health, Bethesda, Maryland, USA). Three independent experiments were performed.

Statistical analysis

All results were obtained by ≥3 independent experiments. The data are expressed as the mean ± standard deviation. One-way analysis of variance was performed, and the mean values were compared using Fisher's multiple comparison test. All statistical analyses were performed using Minitab for Windows (version 16; Minitab Inc., PA, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

ISL treatment inhibits proliferation of T24 cells

The effect of ISL-induced cytotoxicity was determined using an SRB assay. At 24 h following ISL treatment, cell proliferation decreased with increasing concentrations of ISL, and the half-maximal inhibitory concentration was ~50 µg/ml (Fig. 1A). However, the SRB assay did not reveal whether the decrease in cell proliferation was associated with cell death or an apparent loss of viability. To solve this problem, a trypan blue exclusion test was performed on T24 cells treated with ISL. ISL (<75 µg/ml) did not significantly affect the lethality ratio of T24 cells (Fig. 1B), which indicated that the inhibitory effect of ISL on cell proliferation was not due to direct killing of T24 cells and identified that no significant cytotoxicity was observed compared with the control.

Figure 1.

Effect of ISL on T24 cell proliferation and survival. (A) The inhibition ratio of cell proliferation was determined using a sulforhodamine B assay and (B) the lethal ratio was determined using a trypan blue exclusion test following incubation with ISL at the concentrations indicated for 24 h. Results are presented as the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01 vs. untreated control group cells. ISL, isoliquiritigenin.

Nuclear morphology

Morphological changes were examined by Hoechst 33258 staining. T24 cells were treated with ISL for 24 h, and evident apoptotic morphological changes were observed. In the control group, the nuclei of T24 cells were round and homogeneously stained (Fig. 2), whereas ISL-treated cells exhibited evident apoptotic characteristics including cell shrinkage, loss of membrane integrity or deformation, nuclear fragmentation and chromatin compaction of late apoptotic appearance.

Figure 2.

Hoechst 33258 staining of T24 cells following treatment with isoliquiritigenin at the concentrations indicated for 24 h. Fluorescence images were captured following Hoechst 33258 staining (magnification, ×200).

Assessment of apoptotic cells

Apoptosis was determined by staining cells with Annexin V-FITC and PI. A concentration-dependent increase in the apoptotic ratio of T24 cells was demonstrated by Annexin V and PI staining using flow cytometry (Fig. 3). After 24 h of incubation, there were limited numbers of apoptotic cells in the control group (5.57±2.89%), whereas in the 30, 50 and 70 µg/ml ISL treatment groups, the proportion of apoptotic cells was 19.72±2.03, 35.3±1.93 and 59.77±3.09%, respectively.

Figure 3.

Effect of ISL on apoptotic rates in T24 cells. Cells were treated with ISL at 0, 30, 50 and 70 µg/ml for 24 h, permeabilized, stained with Annexin V, and kept on ice until analysis using flow cytometry. (A) Cells in the lower left quadrant were alive, cells in the lower right quadrant were in early apoptosis, cells in the upper right quadrant were in late apoptosis and cells in the upper left quadrant were dead. (B) Quantification of apoptotic rates. Results are presented as the mean ± standard deviation of three separate experiments. **P<0.01 vs. untreated control group cells. ISL, isoliquiritigenin; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Effects of ISL on the ΔΨm of the cells

Mitochondria serve an important role in the regulation of apoptosis, and apoptosis mediated by the mitochondrial signaling pathway is often associated with a decrease in ΔΨm. The ΔΨm changes in T24 cells were determined by staining with JC-1 dye following various treatment periods, and detection using flow cytometry and fluorescence microscopic analysis. The decrease in intensity of JC-1 dye staining reflected a decrease in the ΔΨm. As presented in Fig. 4, a concentration- and time-dependent decrease in ΔΨm was observed in ISL-treated cells.

Figure 4.

ISL decreases the mitochondrial membrane potential in T24 cells. Cells were incubated with ISL at 0, 30, 50 and 70 µg/ml for various periods of time, and treated with JC-1 dye. (A) The retention of JC-1 dye was determined using flow cytometry. (B) Quantitative analysis of cell cycle distribution. Results are presented as the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01 vs. untreated control group cells. ISL, isoliquiritigenin.

ISL increases the mRNA expression of Bax, Bim, Apaf-1, caspase-9 and caspase-3, and decreases the mRNA expression of Bcl-2 in T24 cells

The mRNA expression levels of Bax, Bim, Apaf-1, caspase-9 and caspase-3 were significantly increased, and that of Bcl-2 was significantly decreased in ISL-treated T24 cells in a concentration-dependent manner (P<0.05 or <0.01; Fig. 5).

Figure 5.

mRNA expression of Bcl-2, Bax, Bim, Apaf-1, caspase-9 and caspase-3 in T24 cells treated with isoliquiritigenin at the concentrations indicated (µg/ml). mRNA expression was determined using the reverse transcription-polymerase chain reaction and agarose gel electrophoresis. Quantification results are presented as the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01 vs. untreated control group cells. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; Bim, Bcl-2-interacting mediator of cell death; Apaf-1, apoptotic protease-activating factor-1.

ISL increases the activity of CDK2 in T24 cells

As presented in Fig. 6, CDK2 activity was increased significantly following treatment with ISL compared with that of the control (P<0.05). The maximum enhancement in CDK2 activity was observed following treatment with 70 µg/ml ISL at all time points.

Figure 6.

Effect of isoliquiritigenin on cyclin-dependent kinase 2 activity. Results are presented as the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01 vs. untreated control group cells.

MK-8776 reverses the ISL-induced increase in CDK2 activity in T24 cells

The cells were pretreated with 0.16 µM MK-8776 at 37°C for 1 h and incubated with ISL at 50 µg/ml at 37°C for 24 h. CDK2 activity was significantly increased in ISL-treated cells (P<0.01), whereas CDK2 activity in ISL-treated cells was significantly decreased by pretreatment with MK-8776 for 1 h compared with ISL-treated cells without MK-8776 pre-treatment (P<0.05; Fig. 7).

Figure 7.

Effect of MK-8776 on CDK2 activity in ISL-treated T24 cells. T24 cells pretreated or not with MK-8776 were treated with ISL at 50 µg/ml and CDK2 activity was determined. Results are the mean ± standard deviation of three independent experiments. **P<0.01 vs. untreated control group cells; #P<0.05 vs. 50 µg/ml ISL-treated group cells. CDK2, cyclin-dependent kinase 2; ISL, isoliquiritigenin; MK, MK-8776.

MK-8776 decreases the apoptotic ratio induced by ISL in T24 cells

The cells were pretreated with 0.16 µM MK-8776 at for 1 h and incubated with 50 µg/ml ISL for 24 h. The apoptotic ratio of T24 cells was significantly increased by ISL treatment at 50 µg/ml for 24 h, whereas the apoptotic ratio of ISL-treated cells was significantly decreased by pretreatment with MK-8776 for 1 h compared with ISL-treated cells not pretreated with MK-8776 (P<0.01; Fig. 8).

Figure 8.

Effect of the CDK2 inhibitor MK-8776 on the apoptotic ratio. T24 cells pretreated or not with MK-8776 were treated with ISL at 50 µg/ml and CDK2 activity was determined. Results are the mean ± standard deviation of three independent experiments. **P<0.01 vs. untreated control group cells; ##P<0.01 vs. 50 µg/ml ISL-treated group cells. CDK2, cyclin-dependent kinase 2; ISL, isoliquiritigenin; MK, MK-8776.

MK-8776 reverses the ISL-induced decrease in the ΔΨm in T24 cells

ISL treatment at 50 µg/ml for 24 h led to a significant decrease in ΔΨm in T24 cells, whereas pretreatment with MK-8776 for 1 h significantly reversed the decrease in ΔΨm induced by ISL in T24 cells (P<0.01; Fig. 9).

Figure 9.

Effect of the cyclin-dependent kinase 2 inhibitor MK-8776 on ΔΨm. T24 cells pretreated or not with MK-8776 were treated with ISL at 50 µg/ml and the ΔΨm was determined. Results are presented as the mean ± standard deviation of three independent experiments. **P<0.01 vs. untreated control group cells; ##P<0.01 vs. 50 µg/ml ISL-treated group cells. ΔΨm, mitochondrial membrane potential; ISL, isoliquiritigenin; MK, MK-8776.

MK-8776 reverses the increases in Bax, Bim, Apaf-1, caspase-9 and caspase-3 mRNA expression, and the decrease in Bcl-2 mRNA expression in T24 cells induced by ISL treatment

Pretreatment with MK-8776 for 1 h led to a reversal of the decrease in the mRNA expression level of antiapoptotic Bcl-2, and of the increase in the mRNA expression of proapoptotic Bax, Bim, Apaf-1, caspase-9 and caspase-3 induced by treatment with ISL at 50 µg/ml (P<0.05 or <0.01; Fig. 10).

Figure 10.

MK-8776 antagonizes the increases in Bax, Bim, Apaf-1, caspase-9 and caspase-3 mRNA expression, and the decrease in Bcl-2 mRNA expression in T24 cells. T24 cells pretreated or not with MK-8776 were treated with ISL at 50 µg/ml and mRNA expression was determined using the reverse transcription-polymerase chain reaction and agarose gel electrophoresis. Quantification results are presented as the mean ± standard deviation of three independent experiments. **P<0.01 vs. untreated control group cells; #P<0.05, ##P<0.01 vs. 50 µg/ml ISL-treated group cells. Bax, Bcl-2-associated X protein; Bim, Bcl-2-interacting mediator of cell death; Apaf-1, apoptotic protease-activating factor-1; Bcl-2, B-cell lymphoma 2; ISL, isoliquiritigenin.

ISL increases the protein expression of Bax and caspase-3, and decreases the expression of Bcl-2 in T24 cells

To further investigate the potential underlying molecular mechanism of ISL-induced apoptosis, the protein levels of Bcl-2, Bax and caspase-3 were determined by western blot analysis. It was observed that the Bcl-2 expression level was decreased, and the Bax and caspase-9 expression levels were increased, in a ISL concentration-dependent manner. Compared with the ISL-treated cells alone, pretreatment with MK-8776 led to an increase in the level of antiapoptotic Bcl-2 mRNA, and a decrease in the level of the proapoptotic Bax and caspase-3 (active form with minor molecular mass) (Fig. 11).

Figure 11.

Effects of ISL on Bcl-2, Bax and caspase-3 protein expression. Protein levels of Bcl-2, caspase-3 and Bax following ISL treatment at the concentrations indicated (µg/ml) with or without MK-8776 pretreatment were examined by western blot analysis. β-actin was used as a loading control. ISL, isoliquiritigenin; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; MK, MK-8776.

Discussion

Although ISL has been implicated in the induction of apoptosis in a variety of cell types (33–37), the underlying molecular mechanism by which ISL induces apoptosis remains unclear. In particular, the signaling pathway by which ISL induces apoptosis in T24 cells remains unknown. The alteration in mitochondrial membrane depolarization is intimately associated with apoptosis, which indicates that the ISL-mediated arrest of proliferation has an association with T24 cell apoptosis. Prosurvival Bcl-2 family members localized in the outer membrane of the mitochondria are important regulatory factors in the process of cell apoptosis. Bcl-2 family members are divided into two groups: Antiapoptotic proteins, including Bcl-2, B-cell lymphoma extra large and B-cell lymphoma W, and proapoptotic proteins, including Bcl-2 antagonist/killer, Bax, Bcl-2-associated death promoter and Bim. The balance of these two groups determines the status of cells as alive or dead (38,39). First, proapoptotic proteins open the permeability transition pore on the mitochondrial membrane and promote the release of cytochrome c which combines with Apaf-1. Secondly, procaspase-9 is activated, and caspase-3, downstream of the activator caspases, is activated through the mitochondrial signaling pathway. Finally, activation of these signaling pathways leads to cell apoptosis. In the present study, following treatment of T24 cells with various concentrations of ISL, a decrease in the ΔΨm was induced in a time-dependent manner. In addition, ISL significantly upregulated the activities of the related apoptotic signaling molecules Bax, Bim, Apaf-1, caspase-9 and caspase-3, and downregulated the activation of Bcl-2. Furthermore, the results of the present study identified that ISL treatment upregulated the pro-apoptotic proteins Bax and caspase-3, and downregulated the anti-apoptotic protein Bcl-2. These results suggested that ISL induced apoptosis of T24 cells by a molecular mechanism responsible for depolarization of ΔΨm, and that ISL led to the apoptosis of T24 cells via mitochondrial signaling pathways.

The association of CDK2 and cyclin E/cyclin A allows progression through G1 phase and entry into DNA synthesis, which are associated with cell cycle regulation (39–41). Besides being important proteins of cell cycle regulation, CDK2 and cyclin A are involved in the apoptotic process. It has been demonstrated that upregulation of CDK2 activity is required for etoposide-induced apoptosis in human cervical carcinoma HeLa cells and mitochondrial translocation of Bax, as well as the decrease in ΔΨm (38). In order to determine whether CDK2 serves a vital role in ISL-induced apoptosis in T24 cells, T24 cells were treated with various concentrations of ISL, and the activity of CDK2 increased in a time-dependent manner, whereas the activity of CDK2 was decreased by pretreatment with MK-8776 in the ISL-treated cells.

In order to determine whether CDK2 is involved in ISL-induced apoptosis in T24 cells, the effect of MK-8776 on ISL-induced apoptosis was investigated. The apoptotic ratio was decreased markedly in the MK-8776 treatment group. Furthermore, MK-8776 inhibited the decrease in ΔΨm and downregulated the mRNA expression of Bax, Bim, Apaf-1, caspase-9 and caspase-3, and upregulated the expression of Bcl-2. In addition, compared with the ISL-treated alone, the level of antiapoptotic Bcl-2 mRNA increased, and the mRNA expression of proapoptotic protein Bax and caspase-3 was decreased by pretreatment with MK-8776, suggesting that CDK2 served an important role in ISL-induced apoptosis. The apoptotic mechanism may be associated with CDK2-mediated depolarization of ΔΨm (42–44), which initiates a caspase cascade by facilitating a decrease in ΔΨm and finally leading to mitochondrial apoptosis.

ISL induced apoptosis in T24 cells. CDK2 activation was the critical step, which induced irreversible apoptotic cell death in the cells. However, the regulatory molecular mechanism for CDK2 activity in association with ISL-induced apoptotic pathway remains unclear.