Antiproliferation and apoptosis induction of paeonol in HepG2 cells

Abstract

AIM: To investigate the antiproliferative effect of paeonol (Pae) used alone or in combination with chemotherapeutic agents [cisplatin (CDDP), doxorubicin (DOX) and 5-fluorouracil (5-FU)] on human hepatoma cell line HepG₂ and the possible mechanisms.

METHODS: The cytotoxic effect of drugs on HepG₂ cells was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetra-zolium bromide (MTT) assay. Morphologic changes were observed by acridine orange (AO) fluorescence staining. Cell cycle and apoptosis rate were detected by flow cytometry (FCM). Drug-drug interactions were analyzed by the coefficient of drug interaction (CDI).

RESULTS: Pae (7.81-250 mg/L) had an inhibitory effect on the proliferation of HepG₂ cells in a dose-dependent manner, with the IC₅₀ value of (104.77 ± 7.28) mg/L. AO fluorescence staining and FCM assays showed that Pae induced apoptosis and arrested cell cycle at S phase in HepG₂ cells. Further, different extent synergisms were observed when Pae (15.63, 31.25, 62.5 mg/L) was combined with CDDP (0.31-2.5 mg/L), DOX (0.16-1.25 mg/L), or 5-FU (12.5-100 mg/L) at appropriate concentrations. The IC₅₀ value of the three drugs decreased dramatically when combined with Pae (p < 0.01). Of the three different combinations, the sensitivity of cells to drugs was considerably different.

CONCLUSION: Pae had a significant growth-inhibitory effect on the human hepatoma cell line HepG₂, which may be related to apoptosis induction and cell cycle arrest. It also can enhance the cytotoxicity of chemotherapeutic agents on HepG₂ cells, and the S phase arrest induced by Pae may be one of the mechanisms of these interactions.

Key Words: Paeonol, Hepatocellular carcinoma, Apopto-sis, Cell cycle, Cisplatin, Doxorubicin, 5-fluorouracil, Synergistic effect

INTRODUCTION

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer-related death worldwide[1]. Eighty percent of the burden is borne by countries in Asia and Sub-Saharan Africa[2]. Although recent advances in management with a multidisciplinary approach results in improved local and regional disease control, the 5-year survival rate is still less than 10%[3]. Thus it is imperative to develop more effective and low-toxic chemotherapy agents.

Chinese herbal medicines are now attracting great attention in the world, which also show promising effects in treatment of cancers, including HCC[4]. Paeonol (Pae, 2-hydroxy-4-methoxyacetophenone, Figure 1), is a natural product extracted from the root of Paeonia Suffruticosa Andrew[5]. In our previous study, the antineoplastic activity of Pae has been demonstrated both in various cell lines[6] and in animal models[7,8]. The present study was designed to investigate the antiproliferative effect of Pae used alone or in combination with chemotherapeutic drugs [cisplatin (CDDP), doxorubicin (DOX) and 5-fluorouracil (5-FU)] on human hepatoma cell line HepG₂ and the possible mechanisms.

Figure 1 Structure of Pae (2-hydroxy-4-metho-xyacetophenone).

MATERIALS AND METHODS

Cells and culture conditions

Human hepatocellular carcinoma cell line HepG₂ was purchased from Shanghai Institute of Hepatocarcinoma. HepG₂ cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and incubated at 37°C in a humidified atmosphere with 5% CO₂.

Drugs and reagents

Pae Injection was purchased from the First Pharmaceutical Factory of Shanghai, China (Cat. No. 990402, 10 mg/2 mL); CDDP Injection was purchased from Nanjing Pharmaceutical Co. Ltd, China (Cat. No. 20050602, 20 mg/20 mL); DOX was provided by Wanle Pharmaceutical Inc., Shenzhen, China (Cat. No. 0407E1, 10 mg/ampoule); 5-FU Injection was supplied by Shanghai Haipu Pharmaceutical Factory, China (Cat. No. 031109, 0.25 g/10 mL); DMEM was purchased from GIBCO BRL, Life Technologies Inc. (New York, USA); 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetra-zolium bromide (MTT) and acridine orange (AO) were from Sigma Co., USA. DNA-Prep-Reagents Kit was provided by Beckman Coulter Co. USA(Cat. No. 760279K).

In vitro cytotoxicity assay

HepG₂ cells were seeded in 96-well plates at a density of 1-5 × 10³ cells/well in 100 μL DMEM containing 10% FBS overnight. Nonadherent cells were removed by gentle washing. Then cells were treated with various concentrations of the drugs. After 44 h of drug exposure, 20 μL MTT solution (5 g/L) was added to each well for another 4 h at 37°C. The formazine was solved in 150 μL/well dimethyl sulfoxide (DMSO) and the absorbance was detected at 490 nm using ELx800 Strip reader (Bio-Tek, USA). The percentage of cytotoxicity was calculated as follows: Cytotoxicity (%) = (1-A₄₉₀ of experimental well)/ A₄₉₀ of control well. The median inhibitory concentration (IC₅₀) (defined as the drug concentration at which cell growth was inhibited by 50%) was assessed from the dose-response curves.

Analysis of in vitro drug interaction

The coefficient of drug interaction (CDI) was used to analyze the synergistically inhibitory effect of drug combinations[9]. CDI is calculated as follows: CDI = AB/ (A × B). According to the absorbance of each group, AB is the ratio of the combination groups to control group; A or B is the ratio of the single agent groups to control group. Thus CDI value less than, equal to or greater than 1 indicates that the drugs are synergistic, additive or antagonistic, respectively. CDI less than 0.7 indicates that the drugs are significantly synergistic.

AO fluorescence staining

Cells were cultured in 6-well plates containing cover slips overnight. After incubation with Pae for 24 h, the cover slips were washed twice with PBS, fixed with 95% ethanol for 15 min, acidified with 1% acetic acid for 30 s, dyed with 0.1 g/L AO for 10 min, differentiated with 0.1 mol/L CaCl₂ for 2 min, and then washed with PBS 3 times. The cover slips were sealed and observed under a fluorescence microscope (OLYMPUS, Japan).

Flow cytometry assay

Cells were cultured in 6-well plates and allowed to grow to 75%-80% confluency. Nonadherent cells were removed by gentle washing, and the media were removed and replaced with fresh medium containing Pae at the desired concentrations. After exposure to drugs for 24 h, cells were collected and centrifuged at 1500 r/min in a 15 mL tube for 10 min. The cells were washed twice with PBS and resuspended in 50 μL fixing buffer at a room temperature for 20 s, then 500 μL propidium iodide (PI) staining buffer was added in the dark at room temperature for 30 min (according to the procedure program of the DNA-Prep Coulter reagents kit). A minimum of 1 × 10⁵ cells for each group was analyzed using an EPICS XL-MCL model Coulter counter. Cell cycle distribution was analyzed using Mcycle software.

Statistical analysis

Biostatistical analyses were done using the SPSS 11.5 software package. All experiments were repeated at least three times. Results of multiple experiments are given as the mean ± SE. Non-parametric Kruskal-Wallis test was used to detect differences among the different experimental groups. Mann-Whitney U test was subsequently used for statistical evaluation in two-group comparisons. Pearson correlation coefficients were used for continuous independent and dependent variables. A level of P < 0.05 was accepted as statistically significant.

RESULTS

Effect of pae on the proliferation of HepG₂ cells

We first examined the effect of Pae on the proliferation of HepG₂ cells. As shown in Figure 2, a dramatic dose-dependent reduction of cell viability was seen in cells incubated with Pae at concentrations of 7.81-250 mg/L for 48 h. The r value of dose-effect curves was 0.959 (P < 0.01) and the IC₅₀ value of Pae was (104.77 ± 7.28) mg/L (P < 0.01).

Figure 2 Dose-dependent cytotoxicity of Pae in HepG₂ cells. Data are presented as mean ± SE (error bar) of triplicate experiments.

Effects of Pae on apoptosis in HepG₂ cells

Morphological evidence of apoptosis was demonstrated by AO fluorescence staining. AO could be seen in all cells and the nuclei appeared green and chromatin was stained yellow (Figure 3A). Cells treated with Pae showed typically apoptotic changes, such as chromatin condensation, membrane blebing, deformed and fragmented nuclei.

Figure 3 Effect of Pae on apoptosis in HepG₂ cells. A: Morphological changes of HepG₂ cells treated with Pae 62.5 mg/L (× 320); B: Flow cytometry analysis of HepG₂ cells treated with Pae for 24 h. (a). Control; (b).Pae 31.25 mg/L; (c).Pae 62.5 mg/L; (d).Pae 125 mg/L.

FCM assay was performed to analyze apoptosis in HepG₂ cells treated with various concentrations of Pae for 24 h. It was found that the sub-G₁ peak appeared before G₁ phase, which represents apoptotic cell population (Figure 3B), in a dose- and time-dependent manner (Figure 4).

Figure 4 Effect of Pae on cell cycle in HepG₂ cells. The distribution of cells in the sub-G₁, G₀/G₁, S, and G₂/M phases of the cell cycle were calculated and plotted. (A): Dose-dependent curve of cell cycle distribution induced by Pae. (B): Time-dependent curve of cell cycle distribution induced by Pae 31.25 mg/L. Each point represents triplicate experiments. Bars ± SE.

Effects of Pae on cell cycle in HepG₂ cells

Mcycle software was used to analyze the kinetic changes of cell cycle distribution. In untreated HepG₂ controls, cells were present in G₀/G₁ (71.79% ± 2.76%), S (20.31% ± 0.58%), and G₂/M (7.16% ± 0.57%) phases. For HepG₂ cells exposed to various concentrations of Pae, the S-phase fraction increased while G₀/G₁ fraction decreased in a dose-dependent manner (Figure 4A). And the percentages of cells in S phase increased to 24.98% ± 1.63%, 26.54% ± 1.53%, 31.72% ± 4.85% after 24, 48, and 72 h, respectively, when compared with untreated control cells, which was accompanied by a concomitant decrease of cells in the G₀/G₁ phase of the cell cycle (Figure 4B). It indicated that Pae might arrest the cell cycle at the S phase, and this blockage of cell cycle may prevent cells from entering M phase.

Pae enhancing the cytotoxicity of chemotherapeutic drugs on HepG₂ cells

Growth-inhibition assays were performed to investigate whether Pae can enhance the antiproliferative effects of chemotherapeutic agents on HepG₂ cells. Three doses of Pae (15.63, 31.25 and 62.5 mg/L) were combined with different concentrations of CDDP, DOX, and 5-FU, respectively. For each experiment, a dose-response curve of each single chemotherapeutic agent and its combination with Pae was drawn, which showed that Pae increased the cytotoxicity of CDDP, DOX, and 5-FU on HepG₂ cells. The IC₅₀ value of the three drugs decreased dramatically at different extents when combined with Pae. For example, in the presence of 15.63, 31.25 and 62.5 mg/L Pae, the IC₅₀ of CDDP reduced from 0.591 ± 0.053 mg/L to 0.366 ± 0.011, 0.161 ± 0.018, 0.007 ± 0.002 mg/L, respectively. That of DOX reduced from 0.489 ± 0.124 mg/L to 0.175 ± 0.043, 0.037 ± 0.012, 0.032 ± 0.005 mg/L. And that of 5-FU reduced from 310.783 ± 13.094 mg/L to 161.759 ± 9.507, 8.646 ± 2.331, 5.021 ± 0.962 mg/L, respectively (P < 0.01, Figure 5A-C).

Figure 5 The synergistically antiproliferative effect of Pae combined with CDDP (A), DOX (B), or 5-FU (C) on HepG₂ cells. The dots represent the concentrations of chemotherapeutic drugs as 0 on the dose-response curves which means treatment with Pae alone. Data are presented as mean ± SE of triplicate experiments. ^aP < 0.05, ^bP < 0.01, vs chemotherapeutic drugs alone. And CDI for the combination treatment of Pae with CDDP (D), DOX (E), or 5-FU (F) on HepG₂ cells.

We analyzed the nature of the interaction between Pae and the three drugs using CDI, which quantitatively measures the interaction of two drugs. As shown in Figure 5D, Pae and CDDP yielded synergistic interactions across a wide concentration range. The synergistic effect was most prominent when 15.63 mg/L Pae was combined with 1.25 mg/L CDDP (CDI < 0.7). While a significant synergistic effect was only obtained when Pae concentration reached 31.25 and 62.5 mg/L in combination with 0.16 mg/L and 0.31 mg/L DOX, respectively. When DOX reached 1.25 mg/L, the interaction was antagonistic (Figure 5E). Pae had a relatively weak activity to enhance the antiproliferative effect of 5-FU in HepG₂ cells. If the concentrations of drugs were too high or too low, the synergistic cytotoxic effects could not be achieved. The combinations of Pae at 31.25 mg/L and 5-FU at 12.5 and 25 mg/L exhibited significantly synergistic activity against HepG₂ cells, while an antagonistic effect was observed at 62.5 mg/L of Pae in combination with 25-100 mg/L of 5-FU (Figure 5F).

DISCUSSION

Currently, a variety of cytotoxic and antiproliferative agents have been tested in HCC treatment, which are used alone, or in combination with other drugs or other treatment modalities[10]. Agents with partial response rates near or above 10% include DOX, CDDP and 5-FU[11-13]. However, high doses of these drugs lead to severe toxicities, which have a negative effect on patients’ survival. The use of less toxic doses in combination with other anti-proliferative agents would be desirable[14-17].

Pae is isolated from the herb Pycnostelma paniculatum (Bunge) K.S., and the root of the plant Paeonia Suffruticosa Andrew[5]. It is a white needle crystal with a relatively low-melting point of 51°C-52°C. The molecular weight of Pae is 166.18 ku and the molecular formula is C₉H₁₀O₃[18] . Pae possesses extensive pharmacological activities such as sedation, hypnosis, antipyresis, analgesic, antioxidation, antiinflammation, and immunoregulation[19]. Additionally, Pae had minimal systemic toxicity (LD₅₀ 3430 mg/kg) when it was orally administrated to mice[20]. In our previous study, the antineoplastic activity of Pae has been demonstrated both in cell lines, such as human erythromyeloid cell line K562, breast cancer gene cell line T6-17, human hepatoma cell line Bel-7404, and cervical cancer cell line Hela[6], and in animal models bearing HepA hepatocarcinoma[7,8]. Ji et al[21] demonstrated that Pae at a low concentration had synergetic effects with 5-FU, MMC and CDDP on inhibiting the proliferation of human colorectal cancer cell line HT-29.

In the present study, Pae exhibited growth inhibition to HepG₂ cells in a dose-dependent manner, with the IC₅₀ value of 104.77 (± 7.28 mg/L) mg/L. Although the exact mechanism of the cytotoxicity of Pae against HepG₂ cells is not entirely clear, many potential mechanisms have been proposed for the growth inhibition of Pae in cultured cells and animal models. These mechanisms include induction of apoptosis[22-23] and immunoregulation such as promoted lymphocyte proliferation, IL-2 production by splenocytes, and TNF-α production by PMφ from the model mice[7,8]. Apoptosis is a mechanism by which cells undergo death to control cell proliferation or in response to DNA damage. A tumor occurs when the balance of cell proliferation and cell death is broken[24]. Induction of apoptosis is an effective strategy for cancer therapy[25]. In the present study, the cells treated with Pae showed typical characteristics of apoptosis. Similarly, apoptotic peak appeared before G₁ phase after treatment with Pae, which resulted from the internucleosomal degradation of DNA, in a dose- and time-dependent manner. Moreover, the HepG₂ cells exposed to Pae for 24 h showed depletion of the G₀/G₁ fraction and accumulation in S-phase. Accumulation in S-phase has also been reported by Liu et al[22], in which Pae could induce cell cycle disturbance and S phase of the HT-29 cells was increased, while G₀/G₁ and G₂/M phase of the cells were decreased. The S phase arrest and apoptosis induction of Pae on HepG₂ cells might be its main mechanism.

Meanwhile, HepG₂ cells were treated with the combinations of Pae and different chemotherapeutic agents. The results indicated that the growth inhibitory effect of CDDP, DOX, or 5-FU, respectively, was enhanced significantly by Pae at appropriate concentra-tions. Among the three agents examined, CDDP showed the most wide synergistic effect with Pae. The synergistic effect was most prominent (CDI < 0.7 =) when 15.63 mg/L Pae was combined with 1.25 mg/L CDDP. This indicated that the combination of Pae and CDDP at certain concentrations might help reduce nausea, vomiting and serious kidney toxicity of CDDP. Similar results that Pae in combination with anticancer drugs had synergistic effects at lower concentrations and had antagonistic effects at higher concentrations were observed in DOX and 5-FU, but with different sensitivities. The S phase arrest of Pae may be one of the mechanisms related with these interactions. 5-FU belongs to cell cycle specific agents, which acts specifically on cells in S phase[26]. The cytotoxic effects of CDDP and DOX are generally considered to be non-cell-cycle specific[27-28]. Nevertheless, DOX has the most killing effect on S phase cells[28]. CDDP is most specific to G₁ phase cells, while it also has strong effects on cells in S phase[29]. Further studies are needed to investigate the mechanisms of these synergisms, which favor the reasonable application of Pae to HCC treatment.

ACKNOWLEDGMENTS

We thank Dr. Zhimin Zhai and Qing Li for FACS analysis, the Central Laboratory of the Provincial Hospital of Anhui.

COMMENTS

Background

Hepatocellular carcinoma (HCC) is a major contributor to cancer incidence and mortality in the world. No effective treatment is available by now. Therefore, there is a critical need to develop more strategies for chemotherapy of hepatoma.

Research frontiers

Currently, a variety of cytotoxic and antiproliferative agents have been tested in HCC treatment, which are used alone, or in combination with other drugs or with different modalities of treatment. Chinese herbal medicines are now attracting great attention in the world, which also show promising effects in HCC therapy. Paeonol, a natural product extracted from the root of Paeonia Suffruticosa Andrew, has shown antineoplastic activities both in cell lines and in animal models.

Innovations and breakthroughs

This is the first report on the antiproliferation, induction of apoptosis and cell cycle arrest by Pae in HepG₂ cells.

Applications

Pae may be expected to be effective and useful as a new agent in hepatoma chemotherapy.

Peer review

The authors examine the cytotoxic effect of Pae only in HepG₂ cells. It remains unclear whether the effect of Pae in HepG₂ cells can be generalized to other hepatoma cells. The authors should examine the effect of Pae using a panel of hepatoma cell lines; The data in Figure 4 suggested that Pae induces S-phase arrest in HepG₂ cells. However, the molecular basis for S-phase arrest is not clearly shown. The authors should examine whether Pae has an effect in cells arrested at G₁/S phase using pre-treatment of cells such as hydroxyurea. It would be important to examine the expression of p21, p27 CDK1 and cyclinA after treatment with Pae.