Ardisiphenol D, a resorcinol derivative identified from Ardisia brevicaulis, exerts antitumor effect through inducing apoptosis in human non-small-cell lung cancer A549 cells

Abstract

Context: The in vitro and in vivo antitumor activities of ardisiphenol D, a natural product isolated from the roots of Ardisa brevicaulis Diels (Myrsinaceae), have been studied.

Objective: Previously, we have isolated and identified some chemical constituents from this plant. Furthermore, these compounds showed significant inhibition of the proliferation of human pancreatic PANC-1, human lung A549, human gastrointestinal carcinoma SGC 7901, human breast MCF-7, and human prostate PC-3 cancer cells. In the present paper, a major resorcinol derivative called ardisiphenol D was further studied for its antitumor mechanism.

Materials and methods: MTT assay was used to detect the proliferation of A549 cancer cells. Apoptosis induced by ardisiphenol D was observed by Hoechst 33258 fluorescence staining. Caspase-3 enzyme activity was measured by a commercial caspase-3 enzyme activity detection kit. Protein expression of bax, bcl-2, and caspase-3 was tested by Western blots. In vivo antitumor activity of ardisiphenol D was evaluated by determination of A549 tumor growth in nude mice.

Results: Ardisiphenol D significantly inhibited the proliferation of A549 cells with an IC₅₀ of 0.997 μM with a 48 h treatment. Hoechst 33258 fluorescence staining results indicated the apoptosis of A549 cells induced by 3.125 μM of ardisiphenol D. About 0.39 and 0.78 μM of ardisiphenol D also potently increased the caspase-3 enzyme activity in 24 h. Furthermore, 0.39–3.125 μM of ardisiphenol D induced the activation of caspase-3 protein and the up-regulation of the ratio of bax/bcl-2 protein expression in A549 cells. After i.p. injection, ardisiphenol D (5 mg/kg) also strongly suppressed the A549 tumor growth in nude mice.

Discussion and conclusion: Ardisiphenol D induced apoptosis of A549 cells via activation of caspase-3 and up-regulation of the ratio of bax/bcl-2 protein expression. Ardisiphenol D also strongly suppressed the A549 tumor growth in nude mice and exerted antitumor activity in vivo.

• Antitumor activity
• bax
• bcl-2
• caspase-3

Introduction

Ardisia species (Myrsinaceae) have been used as ornamental plants, food, and medicines. Recent studies demonstrated that species of Ardisia are a rich source of structurally diverse natural products (Kobayashi & De Mejia, 2005). Phytochemical studies of Ardisia species resulted in the identification of constituents with various interesting biological activities, such as ardisiacrispins A and B with antitumor activity from Ardisia crenata Sims and Ardisia crispa (Thunb.) A. DC. (Jansakul et al., 1987), polysaccharide with in vitro antiviral activity against coxsackie B3 virus from Ardisia chinensis Benth (Su et al., 2006), ardipusillosides I and II with immuno-stimulatory and anticancer properties from Ardisia pusilla A. DC. (Zhang et al., 1993), bergenin and norbergenin from Ardisia japonica with weak anti-HIV activity (Piacente et al., 1996), alk(en)ylphenols with antioxidative and cytotoxic activities from the fruits of Ardisia colorata (Sumino et al., 2001), and quinones from Ardisia crispa showing antimetastatic and integrin receptor-binding antagonistic activities (Kang et al., 2001). Previously, we reported the isolation and structure identification of several constituents from the genus Ardisia which is commonly distributed in South and Southwest of China (Bao et al., 2010; Liu et al., 2009, 2010). In our previous studies, these isolated compounds were shown to significantly inhibit the proliferation of certain cancer cell lines, such as human pancreatic PANC-1, human lung A549, human gastrointestinal carcinoma SGC 7901, human breast MCF-7, and human prostate PC-3 cancer cells (Chen et al., 2011). Ardisiphenol D (2-methoxy-4-hydroxy-6-tridecyl-benzene-1-O-acetate) is a major potent active resorcinol derivative among all the constituents. In the present article, we describe ardisiphenol D as a natural product with potent inhibitory activity against A549 cancer cell growth both in vitro and in vivo.

Materials and methods

Reagents and chemicals

RPMI 1640 medium and fetal bovine serum were purchased from Invitrogen Co. (Carlsbad, CA) Penicillin–streptomycin stock solution, 0.25% trypsin solution, and Bradford protein assay kit were products of Yantai Science & Biotechnology Co., Ltd. (Shandong, China). Methyl thiazolyl tetrazolium (MTT), Hoechst 33258, and cyclophosphamide (cytoxan, CTX) were purchased from Sigma-Aldrich (St. Gallen, Switzerland). Caspase-3 enzyme activity detection kit Annexin V-FITC Apoptosis Detection KitAnnexin V-FITC Apoptosis Detection Kit was product of Beyotime Institute of Biotechnology (Shanghai, China). 3-[(4-Methylphenyl)sulfonyl]-(2 E)-propenenitrile (BAY-11-7082) and all primary antibodies for Western blot experiment (bax, bcl-2, caspase-3, and β-actin) were products of Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Plant material

The root of Ardisia brevicaulis Diels was collected in Hunan Province, PR China, in October 2007, and identified by Dr. Manyuan Wang, School of Traditional Chinese Medicine, Capital University of Medical Sciences. A voucher specimen (LHW-2007-1201) was deposited at the School of Chemical Biology and Pharmaceutical Sciences, Capital University of Medical Sciences, PR China.

Extraction, isolation and identification of ardisiphenol D

The dried roots of A. brevicaulis (1.5 kg) were ground and macerated with methanol (5 l, three-times, 100% v/v) at room temperature. The extract solution was evaporated under reduced pressure to yield the methanol extract. The dried methanol extract (120 g) was suspended in 1000 ml of water and then partitioned with ethyl acetate (EtOAc, 1000 ml × 3) to yield EtOAc extract (ABE, 48 g). Part of the EtOAc extract (20 g) was then subjected to a silica gel column chromatography (hexane/EtOAc 1:0–0:1, v/v; CHCl₃/MeOH 8:2, 7:3, v/v) to afford 20 subfractions (ABE1–ABE20). The second fraction (ABE2, eluted by hexane/EtOAc 50:1, 0.15 g) was subjected to Sephadex LH-20 column chromatography (Pharmacia Biotech AB, Stockholm, Sweden) (eluted with CHCl₃/MeOH, 1:1, v/v) to afford three subfractions (ABE2-1–ABE2-3). Ardisiphenol D (15 mg, retention time 18.6 min) was further purified by preparative reverse phase HPLC (MeOH/H₂O, 95:5, v/v) from ABE2-2. Ardisiphenol D (2.5 g) was also obtained from ABE3 (hexane/EtOAc 20:1, 3.2 g) by recrystallization in methanol (Figure 1).

Figure 1. Isolation procedure and chemical structure of ardisiphenol D.

Ardisiphenol D: needles (in methanol), for which the molecular formula is C₂₂H₃₆O₄. Positive ESI-MS: 382 [M + NH₄]⁺; 365 [M + H]⁺. ¹H NMR δ 6.24 (1 H, d, 2.7 Hz, phenyl), 6.18 (1 H, d, 2.7 Hz, phenyl), 2.29 (3 H, s, –OCOCH₃), 3.71 (3 H, s, –OCH₃), 0.86 (3 H, t, 6.9 Hz, alkyl), 1.23–1.49 (18 H, m, alkyl), 2.38 (2 H, t, 7.2 Hz, alkyl). ¹³C NMR δ 131.6, 153.8, 98.1, 151.7, 107.5, 136.5 (phenyl), 20.5 (–OCOCH₃), 169.8 (–OCOCH₃). Ardisiphenol D was dissolved in pure DMSO (cell culture level) at the concentration of 50 mg/ml and stored at −20 °C until use.

Cell culture

Human lung A549 cancer cells were cultured in RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum, 1% penicillin–streptomycin stock solution in a humidified incubator with 5% CO₂, and 95% air at 37 °C. The medium was routinely changed every 2 d. The cells were passaged by trypsinization until they attained confluence.

MTT assay for measuring cell proliferation

Cell viability was evaluated using the MTT assay protocol. A549 cancer cells were seeded in a 96-well plate at the density of 1 × 10⁵ cells/ml and cultured for 24 h. The cells were then treated with ardisiphenol D at different concentrations (100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.79, 0.39, 0.19, 0.1, and 0.05 μM) for 48 h or 72 h. The control group received an equal amount of DMSO, which resulted in a final concentration of 0.2% DMSO in the medium. Cisplatin, a commonly used antitumor clinical drug, was used as the positive control. The mitochondrial-dependent reduction of MTT to formazan was used to measure cell respiration as an indicator of cell viability. Briefly, after the treatments, an MTT solution (final concentration is 200 μg/ml) was added and the cells were incubated for another 4 h at 37 °C. After removing the supernatant, 100 μl of DMSO was added to dissolve the formazan. The absorbance of each group was measured by using a microplate reader at a wavelength of 570 nm, using 630 nm as a reference wavelength. The absorbance values were deducted by the absorbance of blank which is made up with sample without the MTT dye, and then used to calculate the survival rates. The untreated control group was considered as 100% of viable cells. The survival rates were calculated by following formula:

Hoechst 33258 staining and morphological observation of apoptotic cells

A549 cells were seeded in a 6-well plate at a density of 2 × 10⁵ cells/ml for 24 h. After treated with indicated concentrations of ardisiphenol D (1.56 and 3.12 μM) for 48 h, the cells were fixed with 2% formaldehyde in PBS for 10 min, and then washed with PBS for three times. The cells were then incubated with 20 μg/ml of Hoechst 33258 on ice for 20 min. After washed with PBS, the cells were observed under a fluorescence microscope and the cell morphology was observed and photographed. The cells exhibiting condensed chromatin were scored as apoptotic cells.

Measurement of the enzymatic activity of caspase-3

The enzymatic activity of caspase-3 was measured by using a caspase enzyme activity detection kit. A549 cells were seeded in 60 mm dishes at the density of 4 × 10⁵ cells/ml for 24 h. After treatment with indicated concentrations of ardisiphenol D (0.39 and 0.78 μM) for 1, 2, 4, 8, and 24 h, the cells were centrifuged at 600 g for 5 min, the supernatant was removed and the cells were washed with PBS and then lysed in a lysis buffer for 15 min on ice. The total protein concentration of the cell lysate was determined by the Bradford method. Cell lysates (25 μl) were incubated with a caspase-3 colorimetric substrate (Ac-DEVD-pNA) for 4 h at 37 °C. The absorbance was measured by a universal microplate reader (at 405 nm) subsequently, and the activity of caspase-3 was calculated according to the manufacturer’s instructions.

Western blot

A549 cells were treated with indicated concentrations of ardisiphenol D (0.39, 0.78, 1.56, and 3.12 μM) for 48 h, and then lysed immediately by sonication in PBS containing 1% phenylmethanesulfonyl fluoride (PMSF). The lysate was centrifuged at 12 000 rpm for 5 min, and the supernatant was collected and the total protein concentration was assayed with Bradford reagent (Sigma-Aldrich, St. Gallen, Switzerland). Equal amounts of protein (50 µg) were separated by 12% SDS-PAGE and then electrotransferred onto PVDF membranes. The membranes were blocked with 5% skimmed milk solution in TBS-T at room temperature for 2 h. After blocking, the membranes were incubated with an appropriate dilution of specific primary antibodies (bax, bcl-2, caspase-3, and β-actin) overnight. The membranes were washed four-times and then incubated with a 1:1000 dilution of horseradish peroxidase conjugated secondary antibody for 2 h at room temperature. The membranes were washed and then the blots were developed using an enhanced chemiluminescence kit. Images were collected and the bands corresponding to bax, bcl-2, procaspase-3, caspase-3, and β-actin protein were quantitated by densitometric analysis using DigDoc100 program (Alpha Ease FC software, Alpha Innotech, San Leandro, CA). Data of bax, bcl-2, and caspase-3 were normalized on the basis of β-actin level. Densitometric analysis of bax, bcl-2, and caspase-3 protein expression represents the mean from three separate experiments.

Tumorigenesis in nude mice

Five-week-old male Balb/c nude mice were obtained from SLAC Laboratory animal [Shanghai, China, animal care and use committee (ACUC) approval number SCXK(Hu)2007-0005] and 1.5 × 10⁷ human lung cancer A549 cells in 0.2 ml RPMI 1640 were injected s.c. into the right side of the back of the animals. Two weeks later, 36 mice bearing visual and uniform tumors around 5 mm in diameter were randomly divided into three treatment groups (20 mg/kg CTX group, 7.5 mg/kg BAY group, and 5 mg/kg ardisiphenol D group) and a vehicle control group. Because ardisiphenol D is poorly soluble in water, it was first dissolved in DMSO at 10 mg/ml and kept frozen until use. Just before administration, the stock solution of ardisiphenol D was diluted in saline to the indicated final concentrations. About 0.2 ml of either ardisiphenol D solution or vehicle was injected i.p. every other day until the end of the experiment. CTX solution and BAY solution were injected i.p. every day until 24 d. The tumor size and body weight were measured every other day and the tumor volume was calculated using the following formula: where L is the length of the tumor and W is its width.

Statistical analysis

All data were analyzed by SPSS 13.0 software (SPSS Inc., Chicago, IL); results are expressed as means ± SD. Statistical comparison was conducted using a post hoc test after ANOVA. The differences were considered to be significant when p < 0.05.

Results

Inhibitory effect of ardisiphenol D on the proliferation of A549 cancer cells

The in vitro cytotoxic activity of ardisiphenol D on human lung cancer A549 cell lines was evaluated by the MTT assay. As shown in Figure 2(A), ardisiphenol D significantly inhibited the proliferation of A549 cells and showed good dose dependency. Cisplatin was used as a positive control drug. The IC₅₀ values of ardisiphenol D and cisplatin are shown in Table 1. To determine whether the loss of cell viability is relative to apoptosis, the nuclear morphology of A549 cells was monitored by Hoechst 33258 staining. As illustrated in Figure 2(B), the untreated cells were stained roundly and uniformly, but the cells treated with different concentrations of ardisiphenol D obviously appeared apoptotic features. These results indicated that ardisiphenol D could induce the apoptosis of A549 cells.

Figure 2. (A), Effect of ardisiphenol D on the survival of A549 cells. Cells were seeded at a density of 2 × 10⁴ per well in 96-well plates and incubated for 24 h. After treatment with graded concentrations of ardisiphenol D, the cell survival rate was measured by the MTT method, as described in Materials and methods section. (B) Ardisiphenol D induced apoptosis cell death. A549 cells were cultured in the presence or absence of ardisiphenol D. After incubation, the cells were analyzed by the Hoechst 33258 staining method.

Table 1. IC₅₀ values of ardisiphenol D and positive drug cisplatin on A549 cell proliferation.

	IC50 (μM)
Ardisiphenol D	0.997 ± 0.05
Cisplatin	18.74 ± 1.16

Ardisiphenol D activates caspase-3 enzyme in A549 cells

Caspase plays an important role in the execution of apoptosis. There are two well-established pathways of caspase activation for propagating death signals (Daniel, 2000; Sun et al., 1999). Nevertheless, both pathways result in the activation of the major downstream effector caspase-3 that cleaves various cellular targets and leads to cell death (Stroh & Schulze-Osthoff, 1998). Based on this sense, A549 cells were treated with ardisiphenol D and determined for the activity of caspase-3 enzyme. The proteolytic activity of caspase-3 was measured in terms of its ability to cleave Ac-DEVD-pNA, which is a specific substrate for caspase-3 and can be measured at 405 nm. As shown in Figure 3, the level of caspase-3 enzymatic activity after being treated with ardisiphenol D at indicated period of time was detected to be significantly increased compared with the control group. One unit means the amount of enzyme that will cleave 1.0 mM of the colorimetric substrate Ac-DEVD-pNA per hour at 37 °C under saturated substrate concentration.

Figure 3. Effect of ardisiphenol D on the enzymatic activity of caspase-3. A549 cells were treated with 0, 0.39, and 0.78 μM of ardisiphenol D for 1, 2, 4, 8, and 24 h. The activity of caspase-3 in A549 cells was measured by using a caspase enzyme activity assay kit. Values are expressed as mean ± SE for three independent experiments, in which each measurement was performed in triplicate. **p < 0.01 versus untreated control group.

Effect of ardisiphenol D on the expression of bax, bcl-2, and caspase-3 proteins in A549 cells

In order to explain the mechanism of ardisiphenol D inducing apoptosis, the protein levels of bax, and bcl-2, together with caspase-3, were investigated by Western blots. As shown in Figure 4, the treatment of ardisiphenol D notably induced up-regulation of bax and down-regulation of bcl-2. Furthermore, the protein level of procaspase-3 was significantly decreased when treated with ardisiphenol D. The cleavage of caspase-3 protein can be observed clearly. These results suggested that ardisiphenol D induced apoptosis of A549 cells by activating caspase-3, up-regulation of bax, and down-regulation of bcl-2.

Figure 4. Effect of ardisiphenol D on the expression of bax, bcl-2, and caspase-3. A549 cells were treated with 0 (lane 1), 0.39 (lane 2), 0.78 (lane 3), 1.56 (lane 4), and 3.125 μM (lane 5) of ardisiphenol D for 48 h. (A) The expression of bax, bcl-2, and caspase-3 protein was assessed by Western blot analysis. (B) and (C) Densitometric analysis of protein expressions represented the mean from three separate experiments. Data were normalized on the basis of β-actin levels. **p < 0.01 versus untreated control group.

Antitumor activity of ardisiphenol D in vivo

To determine whether ardisiphenol D also exerted antitumor activity in vivo, mice were inoculated with 1.5 × 10⁷ A549 cancer cells s.c. on the back and then administered ardisiphenol D, CTX, BAY, or vehicle, as described in the “Materials and methods” section. The body weight of the animals was monitored every other day (Figure 5A) and no significant body weight loss was recognized either in the treated group or in the positive control groups (CTX and BAY groups) versus the vehicle control group at any time during the experimental period. The treatment was initiated from the 15th day by i.p. injection of ardisiphenol D at the dose of 5 mg/kg every other day (or vehicle in the control group) until the 22nd day. CTX was administered by i.p. injection at the dose of 20 mg/kg, and BAY was administered by i.p. injection at the dose of 7.5 mg/kg every day. The tumor size was measured every other day.

Figure 5. Effect of ardisiphenol D on the growth of A549 cells in nude mice. (A) Body weight of mice. Y-axis, body weight (g); X-axis, time (day). (B) The tumor volume in the mice. Y-axis, tumor volume (mm³); X-axis, time (day). (C) Wet weight of the tumor in the mice on the last day of the experiment (n = 9). Y-axis, wet weight of the tumor (g). **p < 0.01 versus untreated control group. (D) Photographs of the tumor after sacrifice on the last day.

As is evident from the tumor growth curve shown in Figure 5(B), the tumor volume increased steadily in the control group. As one positive drug, CTX significant inhibited the increment of tumor volume. Furthermore, the increase was significantly less prominent in the ardisiphenol D treatment group as well as in the BAY treatment group. There was a significant difference in the tumor size between the ardisiphenol D treatment group and the control group (p < 0.01), and the mean wet weight of the tumor was significantly higher in the control group than in the ardisiphenol D treatment group (Figure 5C). These data indicated that ardisiphenol D also exerted antitumor activity in vivo. At the end of the experiment, the mean tumor volume in the ardisiphenol D treatment group was less than half of that in the control group. More importantly, no toxicity was observed in any of the animals at the dose used.

Discussion

Phytochemical studies on the chemical constituents of Ardisia species resulted in the identification of many compounds. These constituents have been reported to show various interesting biological activities, such as antitumor activity (Jansakul et al., 1987), antiviral activity against coxsackie B3 virus (Su et al., 2006), immuno-stimulatory and anticancer properties (Zhang et al., 1993), anti-HIV activity (Piacente et al., 1996), antioxidative and cytotoxic activities (Sumino et al., 2001), and antimetastatic and integrin receptor-binding antagonistic activities (Kang et al., 2001). Recently, Zhu isolated some alkylphenols from the roots of A. brevicaulis and found that these compounds can induce G1 arrest and apoptosis through endoplasmic reticulum stress pathway (Zhu et al., 2012). Although the cytotoxic effects of various constituents isolated from Ardisia species have been reported, but the biological studies on these constituents and their mechanism of action still remain rare.

Our research group has isolated and identified many active constituents from Ardisia species (Bao et al., 2010; Liu et al., 2009, 2010) and found that these compounds have significant cytotoxic activities on the proliferation of many cancer cell lines (Chen et al., 2011) in recent years. In the present study, the cytotoxic effect of ardisiphenol D, the most potent active agent, on the proliferation of human non-small-cell lung cancer A549 cells and its mechanism have been investigated.

Ardisiphenol D significantly inhibited the proliferation of A549 cancer cells in a dose-dependent manner. Caspase plays an important role in the execution of apoptosis. There are two well-established pathways of caspase activation for propagating death signals (Daniel, 2000; Sun et al., 1999). Nevertheless, both pathways result in the activation of the major downstream effector caspase-3 that cleaves various cellular targets and leads to cell death (Stroh & Schulze-Osthoff, 1998). Based on this sense, ardisiphenol D was further investigated for its effect on the activity of caspase-3 enzyme in A549 cells. The results indicated that ardisiphenol D potently induced the activation of caspase-3 enzyme. Furthermore, in order to explain the mechanism of ardisiphenol D-inducing apoptosis, the protein levels of bax and bcl-2, together with caspase-3 were investigated by Western blot. The results suggested that ardisiphenol D activated the apoptosis of A549 cells via activation of caspase-3 enzyme, up-regulation of bax, and down-regulation of bcl-2 protein expression. Further studies in vivo indicated that ardisiphenol D potently inhibited the tumor growth of A549 cells in nude mice as well as positive drugs CTX and BAY. All evidence elucidated the potent antitumor effect of ardisiphenol D, from which we may expect the potential of such resorcinol derivatives to be developed as antitumor agents.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This research work was supported by National Key Basic Research Project of China (2009CB522300), Project of the Ministry of Science and Technology of China (2007DFB31620), and Taishan Scholar Project to Fenghua Fu.