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

Abstract

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.

The dietary flavonoid isoliquiritigenin is a potent cytotoxin for human neuroblastoma cells

“…Flavonoids have received significant attention in this regard, not only as preventative strategies, but also as potential chemotherapeutic agents [16,17]. In particular, isoliquiritigenin (ISLQ), a chalcone-derived flavonoid naturally found in liquorice and shallots [18], has been investigated for its anticancer properties due to its potent inhibition of cell proliferation and viability in a range of cancer cell types [19][20][21][22][23][24][25][26][27][28][29]. The anticancer effects of ISLQ on NB are yet to be studied, however ISLQ has recently been found to induce cytotoxicity in the pheochromocytoma (PC-12) cell line, which, like NB, has a neural crest origin [30], suggesting that ISLQ may have anticancer effects in NB cells.…”

Isoliquiritigenin Suppresses Osteosarcoma U2OS Cell Proliferation and Invasion by Regulating the PI3K/Akt Signalling Pathway

“…We observed that ISL suppressed osteosarcoma cell proliferation in a time-and dose-dependent manner. Additionally, 20 μM ISL significantly inhibited cell growth at 72 h. Si et al [20] had also observed that cell proliferation decreased in human bladder cancer T24 cells after treatment with ISL. The inhibi- tory influence of ISL on cell migration or invasion has been described in melanoma cells [21], prostate cancer cells, and breast cancer cells [22,23].…”

“…ISL-induced apoptosis has been observed in bladder cancer T24 cells, human lung cancer cells, and endometrial cancer cells [18,20,27]. Si et al [20] found that ISL induced apoptosis in bladder cancer T24 cells by enhancing the protein expression level of Bax and Caspase-3 and decreasing the level of antiapoptotic protein Bcl-2. Moreover, Wu et al [18] reported that ISL markedly induced apoptosis by upregulating the cleavage of Caspase-3 and -7 and PARP expression in endometrial cancer, Ishikawa, or HEC-1A cells.…”

“…It is also a vital performer of apoptosis, which degrades proteins that relate to cell structure, cell-signalling pathways, cell-cycle regulation, and DNA repair, resulting in cell death [25,26]. ISL-induced apoptosis has been observed in bladder cancer T24 cells, human lung cancer cells, and endometrial cancer cells [18,20,27]. Si et al [20] found that ISL induced apoptosis in bladder cancer T24 cells by enhancing the protein expression level of Bax and Caspase-3 and decreasing the level of antiapoptotic protein Bcl-2.…”

“…The Bcl-2 family includes antiapoptotic proteins (such as Bcl-2 and Bcl-XL) and proapoptotic proteins (such as Bak and Bax) [24]. The balance between antiapoptotic and proapoptotic proteins determines the status of the cell as alive or dead [20]. Caspase-3 is a protein that plays a crucial role in apoptotic signal transduction.…”