The phytochemical p-hydroxycinnamic acid suppresses the growth and stimulates the death in human liver cancer HepG2 cells

Abstract

Hepatocellular carcinoma (HCC) is one of the most prevalent malignant diseases and causes a third of cancer-related death. The prognosis and effective treatment of advanced HCC remains poor in spite of the development of novel therapeutic strategies. In the present study, we investigate anticancer effects of the botanical molecule p-hydroxycinnamic acid (HCA) in the HepG2 liver cancer model in vitro. Culturing with HCA (10–1000 nM) suppressed colony formation and growth of HepG2 cells. Mechanistically, culturing with HCA decreased levels of Ras, PI3K, Akt, MAPK, NF-κB p65 and β-catenin, which are linked to processes of cell signaling and transcription, and increased levels of retinoblastoma and regucalcin, which are suppressors for carcinogenesis. These alterations may lead to the suppression of cell growth. Furthermore, culturing with HCA (10–1000 nM) stimulated cell death due to increased caspase-3 levels. Interestingly, the effects of HCA on the growth and death of HepG2 cells were inhibited by culturing with CH223191, an antagonist of aryl hydrocarbon receptor (AHR), suggesting that the flavonoid effects are, at least partly, mediated by activation of AHR signaling. Notably, HCA blocked stimulatory effects of Bay K 8644, an agonist of L-type calcium channel, on the growth of HepG2 cells. Thus, our study demonstrates that HCA suppresses the growth and stimulates the death of human liver cancer HepG2 cells in vitro. The botanical molecule HCA may therefore be a useful tool in the treatment of HCC, providing a novel strategy for the therapy of human liver cancers.

Introduction

Primary liver cancer hepatocellular carcinoma (HCC) is one of the most prevalent malignant diseases and causes a third of cancer-related death [1]. HCC originates on a background of cirrhosis from a chronic and diffuse hepatic disease, which results from continuous liver injury and regeneration [2,3]. The majority of HCC cases are also related to chronic viral infections, including hepatitis B virus or hepatitis C virus, which induces malignant transformation [4,5]. Hepatocarcinogenesis is a multistep process initiated by external stimuli that leads to genetic changes in hepatocytes or stem cells, resulting in proliferation, inhibition of apoptosis, dysplasia and neoplasia. The prognosis of advanced HCC remains poor in spite of the development of novel therapeutic strategies. Knowledge of the oncogenic processes and signaling pathways, which regulate tumor cell proliferation, differentiation, angiogenesis, invasion and metastasis, may lead to the identification of new potential therapeutic targets [6,7]. One effective therapeutic tool for advanced HCC, which can slightly improve patient survival, is the use of multi-kinase inhibitors [8,9] and the microRNAs, a novel class of noncoding small RNAs [10]. However, alternative therapeutic approaches are needed.

The botanical molecule p-hydroxycinnamic acid (HCA), which is an intermediate-metabolic substance in plants and fruits, is synthesized from tyrosine. The anticancer effects of HCA are poorly understood, although there is growing evidence that HCA plays a functional role in health and diseases. We previously showed that HCA has a specific anabolic effect on bone metabolism in vitro and in vivo, among botanical factor cinnamic acid-related compounds, including cinnamic acid, HCA, ferulic acid, caffeic acid and 3,4-dimethoxycinnamic acid [11–16]. HCA suppresses osteoclastogenesis and stimulates osteoblastogenesis by inhibiting nuclear factor kappa B (NF-κB) signaling in vitro [14]. Furthermore, HCA suppresses adipogenesis by regulating extracellular signal-regulated kinase (ERK) and mitogen-activated protein kinase (MAPK) in bone marrow cells in vitro [17]. Notably, the oral intake of HCA has been shown to prevent bone loss in an animal model for postmenopausal osteoporosis [15] and hyperglycemia in an animal model for type 1 diabetes in vivo [16]. Thus, HCA may be significant as a pharmacologic tool with few side effects.

In recent years, HCA has been shown to have an anti-cancer effect in various types of human cancer cells, including bone metastatic breast cancer MDA-MB-231 [18,19], pancreatic MIA PaCa-2 and Pt45P1 cells [20], human colon cancer cells [21–24] and neuroblastoma N2a cells [25]. We found that HCA suppresses the proliferation of MDA-MB-231 and MIA PaCa-2 cells by inhibiting various signaling processes, which are linked to NF-κB, ERK/MAPK, protein kinase C, phosphatidylinositol 3-kinase (PI3K) and nuclear transcription activity, inducing G1 and G2/M phase cell cycle arrest [19,20]. HCA, furthermore, has been shown to stimulate the death in various types of human cancer cells in vitro [19–24]. Interestingly, the in vitro anticancer effects of HCA on MDA-MB-231 [19] and MIA PaCa-2 [20] cells were as effective as that of gemcitabine, a potent cancer drug [26]. Notably, HCA, which has an anabolic effect on bone, has been shown to prevent repressed osteoblastic mineralization and enhanced osteoclastogenesis that is caused by coculturing with bone metastatic MDA-MB-231 cells and bone marrow cells [19]. This finding gives insight into the treatment of cancer bone metastases [18,19]. Thus, HCA may be a useful tool in the therapy of various types of human cancer.

There are few reports concerning the effects of HCA on human liver cancer. The present study, therefore, is undertaken to investigate the anticancer effects of HCA on the HepG2 liver cancer cell model in vitro. HCA was found to suppress colony formation and the growth of HepG2 cells and stimulate the death, leading to a reduction of cancer cell numbers. Therefore, the botanical molecule HCA may be a useful tool in the treatment of HCC, providing a novel strategy for the therapy of various types of human cancers, including liver cancer.

Materials and methods

Reagents

Dulbecco’s modification of Eagle’s medium (DMEM) with 4.5 g/l glucose, L-glutamine and sodium pyruvate and antibiotics (100 μg/ml penicillin and 100 μg/ml streptomycin; P/S) were purchased from Corning (Mediatech, Inc. Manassas, Virginia, USA). Fetal bovine serum (FBS) was from Hyclone (Logan, Utah, USA). HCA (p-coumaric acid), caspase-3 inhibitor (CAS 169332–60-9-Calbiochem), Bay K 8644 and crystal violet were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). 2-Methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH223191) was obtained from Selleckchem Com. (Houston, Texas, USA). All other reagents were purchased from Sigma-Aldrich. Caspase-3 inhibitor was diluted in PBS, CH223191 was dissolved in dimethyl sulfoxide (DMSO), and HCA and other reagents were dissolved in 100% ethanol before use.

Human liver cancer cells

We used human liver cancer HepG2 cells, which were obtained from the American Type Culture Collection (Rockville, Maryland, USA). The HepG2 cell line was derived from a 15-year-old child with primary hepatoblastoma [27]. Although HepG2 cells were not derived from HCC [27], this cell line was reported to be genetically the best model for HCC tumor studies [28]. The cells were cultured in a DMEM containing 10% FBS and 1% P/S.

Colony formation assay

HepG2 cells were seeded into 6-well dishes at a density of 1 × 10³/well and cultured in medium containing 10% FBS and 1% P/S under conditions of 5% CO₂ and 37 °C for 15 days with or without HCA (1000 nM), when visible clones were formed on the plates [29]. After culture, the colonies were washed with PBS and fixed with methanol (0.5 ml per well) for 20 min at room temperature, and then washed three times with PBS. The colonies were then stained with 0.5% crystal violet for 30 min at room temperature. Stained cells were washed five times with PBS. The plates were air-dried for 2 h at room temperature. Colonies containing >50 cells were counted under a microscope (Olympus MTV-3; Olympus Corporation, Tokyo, Japan).

Cell growth assay

HepG2 cells (1 × 10⁵/ml per well) were cultured using a 24-well plate in DMEM containing 10% FBS and 1% P/S for 1, 2, 3 or 5 days in a water-saturated atmosphere containing 5% CO₂ and 95% air at 37 °C in the presence or absence of vehicle (1% ethanol as a final concentration) or HCA (10, 100, 250, 500 or 1000 nM) [30,31]. In separate experiments, HepG2 cells (1 × 10⁵/ml per well) were cultured for 3 days in the presence of either vehicle (1% DMSO or 1% ethanol), CH22319 (1, 10 or 25 μM) or CH22319 (1, 10 or 25 μM) plus HCA (1000 nM). In other experiments, HepG2 cells (1 × 10⁵/ml per well) were cultured for 3 days in the presence of either vehicle (1% ethanol), Bay K 8644 (0.1 or 1 μM), HCA (10, 100 or 1000 nM) or HCA (10, 100 or 1000 nM) plus Bay K 8644 (1 nM). After culture, the cells were detached from the culture dishes by adding a sterile solution (0.1 ml per well) of 0.05% trypsin plus EDTA in Ca²⁺/Mg²⁺-free PBS (Thermo Fisher Scientific, Waltham, Massachusetts, USA) with incubation for 2 min at 37 °C. To each well was then added 0.9 ml of DMEM containing 10% FBS and 1% P/S. The cell number in the cell suspension was counted as described below in the section ‘Cell counting’.

Cell death assay

HepG2 cells (1 × 10⁵/ml per well) were cultured using a 24-well plate in DMEM containing 10% FBS and 1% P/S for 3 days. On reaching subconfluence, the cells were cultured for an additional 24 h in the presence or absence of either vehicle (1% ethanol as a final concentration) or HCA (10, 100, 250, 500 or 1000 nM) with or without caspase-3 inhibitor (10 μM) [32]. In separate experiments, HepG2 cells (1 × 10⁵/ml per well) were cultured for 3 days, and then the cells reaching subconfluency were cultured for an additional 24 h in the presence or absence of either vehicle (1% ethanol or 1% DMSO), CH223191 (1, 10 or 25 nM) or CH223191 (1, 10 or 25 nM) plus HCA (1000 nM). In other experiments, HepG2 cells (1 × 10⁵/ml per well) were cultured for 3 days, and then the cells reaching subconfluency were additionally cultured for 24 h in the presence or absence of either vehicle (1% ethanol), Bay K 8644 (25 or 100 nM), HCA (100 or 1000 nM) or HCA (100 or 1000 nM) plus Bay K 8644 (25 or 100 nM). After culture, the cells were detached by the addition of a sterile solution (0.1 ml per well) of 0.05% trypsin plus EDTA in Ca²⁺/Mg²⁺-free PBS per well as described above in the section of ‘Cell proliferation assay’, and the cell number was counted as described below in the section ‘Cell counting’.

Cell counting

To detach cells on each well, the culture dishes were incubated for 2 min at 37 °C after the addition of a solution (0.1 ml per well) of 0.05% trypsin plus EDTA in Ca²⁺/Mg²⁺-free PBS, and the cells were detached through pipetting after the addition of DMEM (0.9 ml) containing 10% FBS and 1% P/S [30–33]. Medium containing the suspended cells (0.1 ml) was mixed by the addition of 0.1 ml of 0.5% trypan blue staining solution. The number of viable cells was counted under a microscope (Olympus MTV-3) with a Hemocytometer (Sigma-Aldrich) using a cell counter (Line Seiki H-102P, Tokyo, Japan). For each dish, we took the average of two counts. Cell numbers are shown as number per well.

Western blotting

HepG2 cells were plated in 100 mm dishes at a density of 1 × 10⁶ cells/well in 10 ml of DMEM containing 10% FBS and 1% P/S, the cells were cultured for 3 days in the presence of either vehicle (1% ethanol as a final concentration) or HCA (1000 nM) [33]. After culture, the cells were washed three times with cold PBS and removed from the dish by scraping using the cell lysis buffer (Cell Signaling Technology, Danvers, Massachusetts, USA) with the addition of protease and protein phosphatase inhibitors (Roche Diagnostics, Indianapolis, Indiana, USA). The lysates were then centrifuged at 17000 × g, at 4 °C for 10 min. The protein concentration of the supernatant was determined for Western blotting using the Bio-Rad Protein Assay Dye (Bio-Rad Laboratories, Inc., Hercules, California, USA) with BSA as a standard. The supernatant was stored at −80 °C until used. Samples of 40 µg of supernatant protein per lane were separated by SDS-PAGE (12%, SDS-PAGE) and transferred to nylon membranes for immunoblotting using specific antibodies against various proteins obtained from Cell Signaling Technology, including Ras [Catalog number (cat. no.) 14429 rabbit; dilution 1:1000], PI3K p1100 α (cat. no. 4255, rabbit; dilution 1:1000), Akt (cat. no. 9272, rabbit; dilution 1:1000), MAPK (cat. no. 4695, rabbit; dilution 1:1000), retinoblastoma (Rb; cat. no. 9309, rabbit; dilution 1:1000), p21 (cat. no. 2947, rabbit; dilution 1:1000), caspase-3 (cat. no. 9662, rabbit; dilution 1:1000), cleaved caspase-3 (cat. No. 9661, rabbit, dilution 1:1000), aryl hydrocarbon receptor (AHR; cat. No. 83200, rabbit, dilution 1:1000), β-actin (cat. no. 3700, rabbit; dilution 1:1000) and Santa Cruz Biotechnology, Inc. (Santa Cruz, California, USA), including p53 (cat. no. sc-126, mouse; 1:200), NF- κ B p65 (cat. no. sc-109, rabbit; dilution 1:1000), β-catenin (cat. no. sc-39350, mouse; dilution 1:250) and the cytochrome P450 family 1 subfamily A member 1 (CYP1A1, cat. no. sc-25304, mouse, dilution 1:200). Rabbit anti-regucalcin antibody was obtained from Sigma-Aldrich (cat. no. HPA029102, rabbit, dilution 1:1000). Target proteins were incubated with one of the primary antibodies (1:1,000) overnight at 4 °C, followed by horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc., mouse sc-2005 or rabbit sc-2305; diluted 1:2,000). The immunoreactive blots were visualized with a SuperSignal West Pico Chemiluminescent Substrate detection system (Thermo Scientific, Rockford, Illinois, USA) according to the manufacturer’s instructions. β-actin (diluted 1:2,000; Cell Signaling Biotechnology, #3700, mouse) was used as a loading control. Three blots from independent experiments were scanned on an Epson Perfection 1660 Photo scanner, and bands quantified using Image J software.

Statistical analysis

Data are presented as the mean ± SD. Statistical significance was evaluated using GraphPad InStat version 3 for Windows XP (GraphPad Software Inc. La Jolla, California, USA). Multiple comparisons were performed using one-way analysis of variance (ANOVA) with Tukey–Kramer multiple comparisons post-test for indicated parametric data. P < 0.05 was considered statistically significant.

Results

p-hydroxycinnamic acid suppresses colony formation and growth of HepG2 cells

We first investigated whether or not culturing with HCA impacts colony formation and growth of HepG2 cells. Cells were cultured in the presence of HCA (1000 nM) for 15 days (Fig. 1a and b). Colony formation of HepG2 cells was suppressed by culturing with HCA. Furthermore, we determined the effect of HCA on the growth of HepG2 cells in vitro. The cells were cultured in the presence of HCA (10, 100, 250, 500 and 1000 nM) for 1 (Fig. 1c), 2 (Fig. 1d), 3 (Fig. 1e) and 5 (Fig. 1f) days. Growth of HepG2 cells was suppressed by culturing with HCA. Thus, HCA was found to have anticancer effects on the HepG2 liver cancer model in vitro. In this study, we used only HepG2 cells as human liver cancer cells which are without virus infection. Further studies remain to be used in other hepatoma cell lines.

Fig. 1.

p-Hydroxycinnamic acid (HCA) suppresses colony formation and growth in human liver cancer HepG2 cells in vitro. HepG2 cells (1 × 10³ cells/2 ml per well in 6-well plates) were cultured in Dulbecco’s modification of Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% P/S for 15 days in the presence or absence of either vehicle (1% ethanol as a final concentration) or HCA (1000 nM). After culture, the colonies were stained with 0.5% crystal violet, and counted. (a) Representative photo. (b) Colonies containing more than 50 cells were counted under a microscope. Data are presented as the mean ± SD obtained from six wells of two replicate plates per data set using different dishes and cell preparations. In separate experiments, HepG2 cells (1 × 10⁵ cells/ml per well in 24-well plates) were cultured in DMEM containing 10% FBS and 1% P/S for 1 (Fig. 1c), 2 (Fig. 1d), 3 (Fig. e), or 5 (Fig. 1f) days in the presence or absence of either vehicle (1% ethanol) or HCA (10, 100, 250, 500 or 1000 nM). After culture, the number of attached cells was counted. Data are presented as the mean ± SD obtained from eight wells of two replicate wells per data set using different dishes and cell preparations. *P < 0.001 versus control (gray bar). 1- way analysis of variance, Tukey–Kramer post-test.

Mechanistically, we investigated whether HCA regulates levels of proteins linked to cell signaling and transcription in HepG2 cells (Fig. 2a and b). Interestingly, culturing with HCA (1000 nM) increased levels of Rb and regucalcin, which are suppressors for carcinogenesis [34]. Furthermore, the levels of Ras, PI3K, Akt, MAPK, NF-κB p65 and β-catenin were decreased by culturing with HCA. These results suggest that culturing with HCA suppresses processes of cell signaling and transcription linked to stimulation of cell growth.

Fig. 2.

p-Hydroxycinnamic acid (HCA) regulates the levels of various proteins related to cell signaling and transcription processes in human liver cancer HepG2 cells in vitro. HepG2 cells (1 × 10⁶ cells/10 ml of medium per dish) were cultured in Dulbecco’s modification of Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% P/S for 3 days in the presence of either vehicle (1% ethanol) or HCA (1000 nM). After culture, the cells were removed from the dish with a cell scraper in cell lysis buffer containing protease inhibitors. Forty micrograms of supernatant protein per lane were separated by SDS-PAGE and transferred to nylon membranes for Western blotting using antibodies against various proteins. (a) Representative data are presented. (b) Immunoblot band intensity was quantitated and is presented as fold of control (without Bay K 8644). Data are presented as the mean ± SD obtained from four dishes per data set using different cell preparations. *P < 0.001 versus control (white bar). Student’s t-test.

p-hydroxycinnamic acid stimulates the death of HepG2 cells

We next investigated whether or not HCA stimulates the death of HepG2 cells. Culturing with HCA (10, 100, 250, 500 and 1000 nM) decreased the number of HepG2 cells attached on dishes (Fig. 3a), indicating that the flavonoid stimulates cell death. The induction of cell death by HCA (100 and 1000 nM) was prevented by culturing with caspase-3 inhibitor (10 μM) (Fig. 3b). This result suggests that the suppressive effects of HCA on cell growth are, at least partly, dependent on stimulated death of HepG2 cells. Furthermore, we determined whether HCA regulates levels of caspase-3 and cleaved caspase-3, which participate in inducing apoptosis and nuclear DNA fragmentaion [35] (Fig. 3c and d). These levels were increased by culturing with HCA (1000 nM), indicating that caspase-3 is activated.

Fig. 3.

p-Hydroxycinnamic acid (HCA) stimulates the death of human liver cancer HepG2 cells in vitro. HepG2 cells (1 × 10⁵ cells/ml per well in 24-well plates) were cultured in Dulbecco’s modification of Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% P/S for 3 days and the cells reaching subconfluence were additionally cultured for 24 h in the presence of vehicle (1% ethanol), HCA (10, 100, 250, 500 or 1000 nM) without caspase-3 inhibitor (a) or HCA (100 or 1000 nM) with caspase-3 inhibitor (10 µM) (b). After culture, the number of attached cells counted. Data are presented as the mean ± SD obtained from eight wells of two replicate wells per data set using different dishes and cell preparations. In separate experiments, HepG2 cells (1 × 10⁶ cells/10 ml of medium per dish) were cultured in DMEM containing 10% FBS and 1% P/S for 3 days in the presence of either vehicle (1% ethanol) or HCA (1000 nM). After culture, the cells were removed from the dish with a cell scraper in cell lysis buffer containing protease inhibitors. Forty micrograms of supernatant protein per lane were separated by SDS-PAGE and transferred to nylon membranes for Western blotting using antibodies against various proteins. (c) Representative data are presented. (d) Immunoblot band intensity was quantitated and is presented as fold of control (without Bay K 8644). Data are presented as the mean ± SD obtained from four dishes per data set using different cell preparations. *P < 0.001 versus control (gray bar). 1- way analysis of variance, Tukey–Kramer post-test.

Involvement of aryl hydrocarbon receptor in p-hydroxycinnamic acid effects on HepG2 cells

The AHR is transcriptionally active in the form of a heterodimer with the AHR nuclear translocator, which then binds to the xenobiotic responsive element [36,37]. CH223191 is an antagonist for activation of AHR signaling by binding to its receptor [38]. We therefore determined whether the effects of HCA on HepG2 cells are attenuated by culturing with CH223191. Culturing with CH223191 (1, 10 and 25 μM) did not have significant effects on the proliferation and death of HepG2 cells in vitro (Fig. 4). Interestingly, the effects of HCA (1000 nM) on the proliferation and death of HepG2 cells were abolished in the presence of CH223191 (1, 10 and 25 μM). Culturing with HCA did not attenuate the levels of AHR and CYP1A1 in HepG2 cells (Fig. 4c and d). These results suggest that the effects of HCA on cell growth and death are, at least partly, dependent on AHR signaling in HepG2 cells in vitro.

Fig. 4.

Involvement of aryl hydrocarbon receptor in HCA effects on human liver cancer HepG2 cells. (a) HepG2 cells (1 × 10⁵ cells/ml per well in 24-well plates) were cultured in Dulbecco’s modification of Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% P/S for 3 days in the presence of either vehicle [1% dimethyl sulfoxide (DMSO)], CH223191 (1, 10 or 100 μM), HCA (1000 nM) or HCA (1000 nM) plus CH223191 (1, 10 or 100 μM). (b) HepG2 cells (1 × 10⁵ cells/ml per well in 24-well plates) were cultured in DMEM containing 10% FBS and 1% P/S for 3 days, and then the cells reaching subconfluence were additionally cultured for 24 h in the presence of either vehicle (1% ethanol and/or 1% DMSO), CH223191 (1, 10 or 100 μM), HCA (1000 nM) or HCA (1000 nM) plus CH223191 (1, 10 or 100 μM). After culture, the number of attached cells was counted. Data are presented as the mean ± SD obtained from eight wells of two replicate wells per data set using different dishes and cell preparations. In separate experiments, HepG2 cells (1 × 10⁶ cells/10 ml of medium per dish) were cultured in DMEM containing 10% FBS and 1% P/S for 3 days in the presence of either vehicle (1% ethanol) or HCA (1000 nM). After culture, the cells were removed from the dish with a cell scraper in cell lysis buffer containing protease inhibitors. Forty micrograms of supernatant protein per lane were separated by SDS-PAGE and transferred to nylon membranes for Western blotting using antibodies against the proteins indicated. (c) Representative data are presented. (d) Immunoblot band intensity was quantitated and is presented as fold of control (without Bay K 8644). Data are presented as the mean ± SD obtained from four dishes per data set using different cell preparations. *P < 0.001 versus control (gray bar). ^#P < 0.001 versus HCA alone.1-way analysis of variance, Tukey–Kramer post-test.

p-hydroxycinnamic acid suppresses the calcium channel agonist-promoted growth of HepG2 cells

Activation of calcium signaling in cells is involved in the development of carcinogenesis [39,40]. Bay K 8644 is an agonist of l-type calcium channel and stimulates calcium entry into the cells [41]. We investigated whether the effects of Bay K 8644 on the growth and death of HepG2 cells are attenuated by culturing with HCA in vitro. Culturing with Bay K 8644 (0.1 and 1 nM) promoted the growth of HepG2 cells (Fig. 5a). Stimulatory effects of Bay K 8644 (1 nM) on cell growth were suppressed by culturing with HCA (10, 100 and 1000 nM). Meanwhile, culturing with Bay K 8644 (25 and 100 nM) stimulated the death of HepG2 cells (Fig. 5b). This effect of Bay K 8644 was not potentiated in the presence of HCA (100 and 1000 nM), which stimulates cell death (Fig. 5d). Thus, HCA suppressed cell growth, which is promoted by activation of calcium signaling with Bay K 8644 in HepG2 cells in vitro.

Fig. 5.

p-Hydroxycinnamic acid (HCA) suppresses the calcium channel agonist Bay K 8644-promoted growth of human liver cancer HepG2 cells in vitro. (a) HepG2 cells (1 × 10⁵ cells/ml per well in 24-well plates) were cultured in Dulbecco’s modification of Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% P/S for 3 days in the presence of either vehicle (1% ethanol), Bay K 8644 (0.1 or 1 nM), HCA (10, 100 or 1000 nM) or HCA (10, 100 or 1000 nM) plus Bay K 8644 (1 nM). (b) HepG2 cells (1 × 10⁵ cells/ml per well in 24-well plates) were cultured in DMEM containing 10% FBS and 1% P/S for 3 days, and then the cells reaching subconflunce were additionally cultured for 24 h in the presence of either vehicle (1% ethanol), Bay K 8644 (25 or 100 nM), HCA (100 or 1000 nM), Bay K 8644 (25 or 100 nM) plus HCA (100 or 1000 nM). After culture, the number of attached cells was counted. Data are presented as the mean ± SD obtained from eight wells of two replicate wells per data set using different dishes and cell preparations. *P < 0.001 versus control (gray bar). ^#P < 0.001 versus Bay K 8644 alone. 1-way analysis of variance, Tukey–Kramer post-test.

Discussion

HCC is one of the most prevalent malignant diseases and causes a third of cancer-related death. There remains no effective treatment for HCC. Our study demonstrates that the botanical molecule HCA has anticancer effects in the HepG2 cells human liver cancer cell model in vitro. Culturing with HCA of comparatively lower concentrations (10–1000 nM) was found to suppress colony formation and growth of HepG2 cells and stimulate cell death, leading to reduction of liver cancer cells. The botanical molecule HCA may therefore be a useful tool in treatment of HCC with few side effects.

Mechanistically, we demonstrate that HCA regulates levels of proteins linked to cell signaling and transcription in HepG2 cells. Interestingly, culturing with HCA increased levels of Rb and regucalcin, which are suppressors for carcinogenesis [34], and decreased the levels of Ras, PI3K, Akt, MAPK, NF-κB p65 and β-catenin, which are involved cell signaling related to cell growth [42,43]. Suppressive effects of HCA on the growth of HepG2 cells may be based on repression of cell signaling and transcription processes linked to promotion of cell growth. Moreover, increased Rb and regucalcin, which are tumor suppressor, may partly contribute to the anti-cancer effects of HCA on liver cancer cells.

We did not have the experiments of HCA effects on cell cycles in HepG2 cells. Our previous finding demonstrated that the suppressive effects of HCA on cell proliferation of human breast cancer MDA-MB-231 cells [19] and human pancreatic cancer MIA PaCa-2 cells [20] were not potentiated in the presence of cell cycle inhibitors, including butyrate, roscovitine or sulforaphane, suggesting that HCA induces G1 and G2/M phase cell cycle arrest. We speculate that HCA may induce cell cycle arrest in HepG2 cells, although this remains to study.

HCA was also found to stimulate the death of HepG2 cells. Culturing with HCA decreased the number of HepG2 cells attached on dishes and activated caspase-3, indicating that HCA stimulates cell death by apoptosis. Mechanistically, stimulatory effects of HCA on cell death were prevented by culturing with caspase-3 inhibitor, and the flavonoid increased levels of caspase-3 and cleaved caspase-3, which participate in inducing nuclear DNA fragmentation [35,44]. This result suggests that the suppressive effects of HCA on cell growth may be, at least partly, dependent on stimulated death of HepG2 cells.

The AHR binds to the AHR nuclear translocator in the cytoplasm of various types of cells, including liver cancer cells, and then binds to the xenobiotic responsive element on various genes [35,36]. AHR was initially discovered via its ligand, the polychlorinated hydrocarbon, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) [36,37]. We previously showed that TCDD treatment repressed the proliferation and promotes the death of HepG2 cells [45] and human colorectal cancer RKO cells [46] in vitro, which might be, at least partly, implicated in AHR signaling. We therefore investigated an involvement of AHR signaling in the HCA effects on the growth and death of HepG2 cells. Interestingly, the effects of HCA on the growth and death of HepG2 cells were blocked by culturing with CH223191, a specifi antagonist of AHR [38]. Our finding suggests that the effects of HCA on the proliferation and death of HepG2 cells may be, at least partly, related to the activation of AHR signaling.

Furthermore, we determined whether or not HCA suppresses growth of HepG2 cells. Activation of calcium signaling in cells is involved in the promotion of cell proliferation and leads to development of carcinogenesis [39–41]. Bay K 8644 is an agonist of L-type calcium channels, which stimulate calcium entry into cells [41]. Interestingly, culturing with HCA suppressed Bay K 8644-promoted growth of HepG2 cells, suggesting that the flavonoid represses promotion of cell growth linked to activation of calcium signaling. Suppressive effects of HCA on the proliferation of HepG2 cells may therefore be, at least partly, a result of inhibition of calcium signaling. Thus, HCA was shown to have repressive effects on enhanced growth of liver cancer cells.

In conclusion, our study demonstrates that the botanical molecule HCA suppresses the growth and stimulates the death of HepG2 human liver cancer model cells in vitro, leading to reduction of cancer cell numbers. HCA may thereby repress hepatocarcinogenesis, although further studies remain to be done to test this hypothesis using animal models. HCA very few side effects. Therefore, HCA may be significant new therapeutic in the treatment of HCC. Furthermore, HCA has been demonstrated to have anticancer effects on bone metastatic human breast cancer MDA-MB-231 cells [19], human pancreatic cancer MIA PaCa-2 cells [20] and human colorectal cancer cells [21–25] in vitro. This supports the further investigation of the botanical molecule HCA as a therapeutic for various types of human cancers.