Isoliquiritigenin Inhibits Cell Proliferation and Induces Apoptosis in Human Hepatoma Cells

• Abstract
• Introduction
• Materials and Methods
• Reagents and materials
• Preparation of ISL and 5-Fu
• Cell culture
• Growth inhibition and XTT assay
• Detection of apoptosis
• The activity of caspase-3
• The activity of NF-κB
• Western blot
• Statistical analysis
• Results
• Discussion
• References

Abstract

Isoliquiritigenin (4,2′,4′-trihydroxychalcone, ISL) is a natural pigment with a simple chalcone structure. In this study, we report the ISL-induced inhibition on the growth of human hepatoma cells (Hep G2) for the first time. The cell growth inhibition achieved by ISL treatment resulted in programmed cell death in a caspase activation-dependent manner, with an IC₅₀ of 10.51 μg/mL. Outcomes of ISL treatment included the up-regulation of IκBα expression in the cytoplasm, and the decrease of NF-κB level as well as its activity in the nucleus. In addition, ISL also suppressed the expression of Bcl-X_L and c-IAP1/2 protein, the downstream target molecule of NF-κB. These results demonstrated that ISL treatment inhibited the NF-κB cell survival-signaling pathway and induced apoptotic cell death in Hep G2 cells.

Introduction

Apoptosis is a multi-step and multi-pathway programmed cell death that is inherent in every cell of the body. In tumors, the ratio of apoptosis to cell proliferation is unbalanced, which leads to an increase of malignant tissue [1]. Many studies have demonstrated that cancer treatment by chemotherapy and γ-irradiation kills target cells primarily by the induction of apoptosis [1]. Nuclear factor-κB (NF-κB) is an important transcriptional factor that participates in the regulation of inflammatory, immune and apoptotic responses [2], [3]. A variety of external stimuli induce phosphorylation and subsequent degradation of IκB inhibitory proteins, thereby releasing NF-κB protein for translocation to the nucleus to function as transcription factor [2], [3]. NF-κB signaling may contribute to apoptosis or may be dispensable for apoptosis, and may even inhibit apoptosis to promote proliferation and differentiation [4]. The antiapoptotic molecules that are regulated by NF-κB include c-IAPs (cellular inhibitors of apoptosis), and members of the Bcl-2 family proteins (A1/BFL1 and Bcl-X_L) [2], [3]. NF-κB can also decrease the induction of apoptosis mediated by genotoxic chemotherapeutic agents and ionizing radiation. Cancer cells in which NF-κB is constitutively active are highly resistant to anticancer agents or ionizing radiation, and inhibition of NF-κB activity in these cells greatly enhances their sensitivity to anticancer treatment [2], [3], [4].

Isoliquiritigenin (4,2′,4′-trihydroxychalcone, ISL, Fig. [1]), a flavonoid found in licorice (Glycyrrhiza glabra L.) and shallot (Allium ascalonicum Hort), is a potent antioxidant with anti-inflammatory, anti-platelet aggregation and cancer-preventing properties [5], [6], [7], [8]. It exhibits an inhibitory effect on carcinogenesis in skin and colon, and antiproliferation activity in pulmonary, prostate, breast, gastric and melanoma cancer cells [9], [10], [11], [12], [13]. In this study, we used an in vitro human hepatoma cancer model system, the Hep G2 cell line, to evaluate the potential of ISL as a chemopreventive agent against liver cancer. Here, we report on the effects and molecular mechanisms of action of ISL in Hep G2 cells that are mediated through down-regulation of NF-κB signaling pathway.

Materials and Methods

Reagents and materials

Fetal bovine serum (FBS), penicillin G, streptomycin, amphotericin B and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from GIBCO BRL (Gaithersburg, MD). Isoliquiritigenin (According to the certificate of analysis, the purity was 98 % by GC), 5-Fluorouracil (5-FU), dimethyl sulfoxide (DMSO), Dulbecco’s modified Eagle’s medium (DMEM), ribonuclease (RNase), and propidium iodide were purchased from Sigma Chemical (St. Louis, MO). XTT was obtained from Roche Diagnostics GmbH (Mannheim, Germany). Nucleosome ELISA kit, and Bcl-X_L antibody were purchased from Calbiochem (Cambridge, MA). The antibodies to IκBα, phospho-IκBα and c-IAP1/2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Preparation of ISL and 5-Fu

The stock solution of ISL or 5-FU was prepared at concentration 2 mg/mL of DMSO. It was then stored at -20 °C until use. Amber lights were used to ensure ISL stability. For all experiments, the final concentrations of the test compound were prepared by diluting the stock with DMEM. Control cultures received the carrier solvent (0.1 % DMSO).

Cell culture

Hep G2 cells (ATCC HB8065) were maintained in a monolayer culture at 37 °C and 5 % CO₂ in DMEM supplemented with 10 % FBS, 100 units/mL of penicillin G, 100 μg/mL of streptomycin, and 0.25 μg/mL of amphotericin B.

Growth inhibition and XTT assay

Inhibition of cell proliferation by ISL was measured by XTT {sodium 3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate} assay. Briefly, cells were plated in 96-well culture plates (1 × 10⁴ cells/well). After 24 h incubation, the cells were treated with ISL (0, 1, 5, 10 and 20 μg/mL) for 12, 24, 48, and 72 h. Fifty μL of XTT test solution, which was prepared by mixing 5 mL of XTT-labeling reagent with 100 μL of electron coupling reagent, was then added to each well. After 6 h incubation, the absorbance was measured on an ELISA reader (Multiskan EX, Labsystems) at a test wavelength of 492 nm and a reference wavelength of 690 nm.

Detection of apoptosis

Cells (4 × 10⁶) were treated with vehicle alone (0.1 % DMSO) or various concentrations of ISL for 48 h, and were subsequently collected and lysed by DNA lysis buffer (10 mM Tris, pH 7.5, 400 mM EDTA, and 1 % Triton X-100). After centrifugation, the supernatant was incubated overnight with proteinase K (0.1 mg/mL) and then with RNase (0.2 mg/mL) for 2 h at 37 °C. After extraction with phenol-chloroform (1 : 1), the DNA was separated in 2 % agarose gel and visualized by UV after staining with ethidium bromide.

Quantitative assessment of oligonucleosomal DNA fragmentation was assayed using the Nucleosome ELISA kit. This kit uses a photometric enzyme immunoassay that quantitatively determines the formation of cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) after apoptotic cell death. For determination of apoptosis by ELISA, the cells were treated with vehicle alone (0.1 % DMSO) or ISL (10 and 20 μg/mL) for the indicated time. The induction of apoptosis was evaluated by assessing the enrichment of nucleosome in the cytoplasm, and determined exactly as described in the manufacturer’s protocol.

The activity of caspase-3

The activity of caspase-3 was determined using the CaspACE assay kit (Promega Corporation, Wisconsin). These assays measure the cleavage of a specific colorimetric caspase substrate, DEVD-pNA. pNA (p-nitroaniline) is released from the substrate upon cleavage by caspase. Free pNA produces a yellow color that is monitored by a spectrophotometer at 405 nm. The detailed analysis procedure is described in the manufacturer’s protocol.

The activity of NF-κB

The activity of NF-κB was determined by an ELISA Trans-AM kit. The analytical procedure was performed according to the manufacturer’s specifications (Active Motif, Carlsbad, CA). Briefly, the transcriptional factor of nuclear extracts, which were prepared by the Nuclear Extract kit (Active Motif, Carlsbad, CA), were captured by binding to a consensus oligonucleotide (5′-GGGACTTTCC-3′) immobilized on a 96-well plate. The p65 subunit of NF-κB was determined in a colorimetric reaction using a specific primary antibody and a secondary horseradish peroxidase-conjugated antibody. Spectrophotometric data were expressed as the ratio of absorbance of each experimental condition compared with control cells exposed to vehicle alone [14].

Western blot

Cells (8 × 10⁶/dish) were seeded in a 10 cm dish and incubated with 20 μg/mL of ISL for the indicated time. Twenty μg of total cell proteins were separated by electrophoresis on 10 - 12 % SDS-PAGE and transferred to a PVDF membrane. Detection was conducted by immuno-staining using specific primary antibodies and horseradish peroxidase-conjugated anti-IgG antibody. The proteins bands were visualized by the enhanced chemiluminescence (ECL) detection system (Amersham, USA).

The following antibodies and working dilutions were used for the Western blots: mouse monoclonal antibodies against human IκBα (1 : 1000), NF-κB (1 : 1500), Bcl-X_L (1 : 1000), β-actin (1 : 3000); goat polyclonal antibody against c-IAP1/2 (1 : 1000); rabbit anti-mouse and mouse anti-goat immunoglobulin G (IgG)-horseradish peroxidase conjugate (1 : 2000).

Statistical analysis

Data are expressed as means ± SD. Statistical comparisons of the results were made using analysis of variance (ANOVA). Significant differences (p < 0.05) between the means of control and ISL-treated cells were analyzed by Dunnett’s test.

Results

We tested the antiproliferative effect of ISL in Hep G2 cells. As shown in Fig. [2] A, ISL reduced the proliferation of Hep G2 cells in a dose- and time-dependent manner. Twenty μg/mL of ISL inhibited proliferation in 73.92 % of Hep G2 cells, and the IC₅₀ value was 10.51 μg/mL at 48 h. In comparison, 5-FU was used as a positive control. Two hundred μg/mL of 5-FU was shown to possess only a 50 % inhibition rate against the proliferation of Hep G2 and increasing the concentration of the drug did not significantly further increase its antiproliferative effect (Fig. [2] B). This result was similar to that of a previous report [15].

To explore the possibility that ISL could be associated with the proapoptotic activity, we assessed the DNA fragmentation in Hep G2 cells. The data from agarose gel electrophoresis (Fig. [3] A) at 48 h showed that ISL treatment resulted in the formation of DNA fragments in Hep G2. Similarly, a quantitative evaluation detecting the amount of histone-associated oligonucleosome fragments in cytoplasm has shown that DNA fragmentation of Hep G2 was exhibited at 24 h and maximized at 48 h after addition of 10 μg/mL ISL (Fig. [3] B). The induction of apoptosis by 20 μg/mL ISL was more pronounced and had an earlier appearance (12 h) than that of 10 μg/mL ISL treatment.

Hallmarks of the apoptotic process are the activation of caspases, which represent both initiators and executors of cell death. Fig. [4] A shows that the activity of caspase-3 in 20 μg/mL ISL treated cells was significantly higher than the control as early as 12 h post treatment. Furthermore, our results showed that the antiproliferative activity and induction of apoptosis by ISL were significantly decreased in the presence of an inhibitor of caspase-3 (DEVD-fmk) (Figs. [4] B and C).

Since NF-κB plays an important role in regulating cell growth and cell viability in many tumors, we determined the expression of IκBα and NF-κB/p65 activity in ISL-treated Hep G2 cells. Employing Western blot analysis, we found that ISL treatment resulted in an inhibition of NF-κB/p65 protein expression in the nuclear fractions of Hep G2 cells (Fig. [5] A). The inhibition of NF-κB/p65 in the nuclear fractions correlated with the enhancement of the protein expression of IκBα and the decrease of phospho-IκBα in the cytoplasmic extract. In addition, as shown in Fig. [5] B, the activity of NF-κB/p65 was decreased by 20.5 %, 34.7 %, 49.7 %, and 58.9 % at 3, 6, 12, and 24 h, in contrast to the control (20 μg/mL ISL).

To further explore the effect of ISL on NF-κB/p65 signaling pathway, we assessed the expression of Bcl-X_L and c-IAP1/2, which are regulated by the NF-κB transcription factor. As shown in Fig. [6], 20 μg/mL of ISL exhibited a significant decrease in the expression of Bcl-X_L and c-IAP1/2 proteins. These results suggested that NFκB/p65 suppression was associated with the induction of apoptosis in Hep G2 cells.

Discussion

Isoliquiritigenin (ISL), a chalcone that is isolated from licorice and shallot, has anti-tumor activity, including inhibition of colon carcinogenesis [8], inhibition of tumor promotion [9], and blockade of tumor metastasis [10]. ISL also inhibits prostate cancer cell proliferation through the induction of GADD153 mRNA [11]. In addition, ISL induces cell apoptosis in B16 mouse melanoma 4A5 cells, gastric and breast cancer cells [12], [13], [16]. However, the effect of ISL on liver cancer cells remains unknown. In this study, we examined the effect and mechanism of ISL on human hepatoma cell growth. Our results have shown that ISL inhibits the growth of Hep G2 cells. Treatment of the Hep G2 cells with ISL induced apoptosis and caspase-3 activation. Furthermore, the cell growth inhibition and apoptotic induction effects of ISL decreased in Hep G2 cells treated with caspase-3 inhibitor. Thus, our results have demonstrated that caspase-3 activation plays an important role in ISL-mediated Hep G2 cellular apoptosis.

Numerous studies have indicated that NF-κB activation can block apoptosis by several pathways [2], [3]. NF-κB activates TRAF 1 and 2, and c-IAP 1 and 2 to inhibit potential TNF-induced caspase-8 activation [2], [3], [4]. Other antiapoptotic molecules which have been shown to be increased by NF-κB include the Bcl-2 homologues A1/Bfl-1, Bcl-X_L, IEX-1, and XIAP [2], [3], [4]. In this study, we found that ISL suppressed constitutive NF-κB activation in Hep G2 cells. The result was in concurrence with the up-regulation of IκBα through the decrease of phospho-IκBα in the cytoplasm followed by the down-regulation of the NF-κB amount in the nucleus after ISL treatment. Moreover, the suppression of Bcl-X_L and c-IAP1/2 was found in ISL-treated Hep G2 cells. These results have established that ISL inhibits that antiapoptotic activity of NF-κB signaling pathway in Hep G2 cells.

In summary, our results provide the first evidence that ISL suppresses proliferation and induces apoptosis via the caspase-dependent pathway in Hep G2 cells. The ISL treatment not only increases the IκBα molecules expression, but also decreases the NF-κB expression and activity. Moreover, both anti-apoptotic downstream targets of NF-κB, Bcl-X_L and c-IAP1/2 proteins were decreased by ISL treatment. Taken together, we have demonstrated that ISL has potential for development as a chemopreventive agent against liver cancer.

References

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• 12 Maggiolini M, Statti G, Vivacqua A, Gabriele S, Rago V, Loizzo M, Menichini F, Amdo S. Estrogenic and antiproliferative activities of isoliquiritigenin in MCF7 breast cancer cells.  J Steroid Biochem Mol Biol. 2002;  82 315-22
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• 16 Iwashita K, Kobori M, Yamaki K, Tsushida T. Flavonoids inhibit cell growth and induce apoptosis in B16 melanoma 4A5 cells.  Biosci Biotechnol Biochem. 2000;  64 1813-20
  CrossrefPubMedSearch in Google Scholar

Professor Chun-Ching Lin

Graduate Institute of Natural Products

College of Pharmacy

Kaohsiung Medical University

100 Shih-Chuan 1st Road

Kaohsiung 807

Taiwan

ROC

Phone: +886-7-3121101 ext. 2122

Fax: +886-7-3135215

Email: aalin@ms24.hinet.net

References

• 1 Evan G I, Vousden K H. Proliferation, cell cycle and apoptosis in cancer.  Nature. 2001;  411 342-8
  CrossrefPubMedSearch in Google Scholar
• 2 Herr I, Debatin K M. Cellular stress response and apoptosis in cancer therapy.  Blood. 2001;  98 2603-14
  CrossrefPubMedSearch in Google Scholar
• 3 Dixit V, Mak T W. NF-kappaB signaling. Many roads lead to Madrid.  Cell. 2002;  111 615-9
  CrossrefPubMedSearch in Google Scholar
• 4 Karin M, Cao Y, Greten F R, Li Z W. NF-kappaB in cancer: from innocent bystander to major culprit.  Nat Rev Cancer. 2002;  2 301-10
  CrossrefPubMedSearch in Google Scholar
• 5 Vaya J, Belinky P A, Aviram M. Antioxidant constituents from licorice roots: isolation, structure elucidation and antioxidative capacity toward LDL oxidation.  Free Radic Biol Med. 1997;  23 302-13
  CrossrefPubMedSearch in Google Scholar
• 6 Chan S C, Chang Y S, Wang J P, Chen S C, Kuo S C. Three new flavonoids and antiallergic, anti-inflammatory constituents from the heartwood of Dalbergia odorifera .  Planta Medica. 1998;  64 153-8
  PubMedSearch in Google Scholar
• 7 Tawata M, Aida K, Noguchi T, Ozaki Y, Kume S, Sasaki H, Chin M, Onaya T. Anti-platelet action of isoliquiritigenin, an aldose reductase inhibitor in licorice.  Eur J Pharmacol. 1992;  212 87-92
  CrossrefPubMedSearch in Google Scholar
• 8 Baba M, Asano R, Takigami I, Takahashi T, Ohmura M, Okada Y, Sugimoto H, Arika T, Nishino H, Okuyama T. Studies on cancer chemoprevention by traditional folk medicines XXV. Inhibitory effect of isoliquiritigenin on azoxymethane-induced murine colon aberrant crypt focus formation and carcinogenesis.  Biol Pharm Bull. 2002;  25 247-50
  CrossrefPubMedSearch in Google Scholar
• 9 Yamamoto S, Aizu E, Jiang H, Nakadate T, Kiyoto I, Wang J C, Kato R. The potent anti-tumor-promoting agent isoliquiritigenin.  Carcinogenesis. 1991;  12 317-23
  CrossrefPubMedSearch in Google Scholar
• 10 Yamazaki S, Morita T, Endo H, Hamamoto T, Baba M, Joichi Y, Kaneko S, Okada Y, Okuyama T, Nishino H, Tokue A. Isoliquiritigenin suppresses pulmonary metastasis of mouse renal cell carcinoma.  Cancer Lett. 2002;  183 23-30
  CrossrefPubMedSearch in Google Scholar
• 11 Kanazawa M, Satomi Y, Mizutani Y, Ukimura O, Kawauchi A, Sakai T, Baba M, Okuyama T, Nishino H, Miki T. Isoliquiritigenin inhibits the growth of prostate cancer.  Eur Urol. 2003;  43 580-6
  CrossrefPubMedSearch in Google Scholar
• 12 Maggiolini M, Statti G, Vivacqua A, Gabriele S, Rago V, Loizzo M, Menichini F, Amdo S. Estrogenic and antiproliferative activities of isoliquiritigenin in MCF7 breast cancer cells.  J Steroid Biochem Mol Biol. 2002;  82 315-22
  CrossrefPubMedSearch in Google Scholar
• 13 Ma J, Fu N Y, Pang D B, Wu W Y, Xu A L. Apoptosis induced by isoliquiritigenin in human gastric cancer MGC-803 cells.  Planta Medica. 2001;  67 754-7
  Thieme ConnectPubMedSearch in Google Scholar
• 14 Somasundaram S, Edmund N A, Moore D T, Small G W, Shi Y Y, Orlowski R Z. Dietary curcumin inhibits chemotherapy-induced apoptosis in models of human breast cancer.  Cancer Res. 2002;  62 3868-75
  PubMedSearch in Google Scholar
• 15 Jiang S, Song M J, Shin E C, Lee M O, Kim S J, Park J H. Apoptosis in human hepatoma cell lines by chemotherapeutic drugs via Fas-dependent and Fas-independent pathways.  Hepatology. 1999;  29 101-10
  CrossrefPubMedSearch in Google Scholar
• 16 Iwashita K, Kobori M, Yamaki K, Tsushida T. Flavonoids inhibit cell growth and induce apoptosis in B16 melanoma 4A5 cells.  Biosci Biotechnol Biochem. 2000;  64 1813-20
  CrossrefPubMedSearch in Google Scholar

Professor Chun-Ching Lin

Graduate Institute of Natural Products

College of Pharmacy

Kaohsiung Medical University

100 Shih-Chuan 1st Road

Kaohsiung 807

Taiwan

ROC

Phone: +886-7-3121101 ext. 2122

Fax: +886-7-3135215

Email: aalin@ms24.hinet.net