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Synergistic effect of paeonol and cisplatin on oesophageal cancer cell lines
Available online at www.sciencedirect.com
Digestive and Liver Disease 40 (2008) 531–539
Alimentary Tract
Synergistic effect of paeonol and cisplatin on oesophageal cancer cell lines夽 X.-A. Wan a , G.-P. Sun a,∗ , H. Wang b , S.-P. Xu a , Z.-G. Wang a , S.-H. Liu a b
a Department of Oncology, The First Affiliated Hospital of Anhui Medical University, Ji Xi Road 210, Hefei 230022, PR China Department of Oncology, The Affiliated Provincial Hospital of Anhui Medical University, Lu Jiang Rood 17, Hefei 230001, PR China
Received 13 October 2007; accepted 30 January 2008 Available online 12 March 2008
Abstract Background/aim. Paeonol, a phenolic component from the root bark of Paeonia moutan, has shown great promise in antitumour activities in our previous studies. The present study was designed to investigate whether paeonol has synergistic effect with cisplatin on the growth-inhibitory of human oesophageal cancer cell lines and the possible mechanism. Methods. Cell viabililty was measured by MTT assay. Drug–drug interactions were analysed by the coefficient of drug interaction. Apoptosis was detected by acridine orange fluorescence staining and flow cytometry assay. Bcl-2, Bax and caspase-3 expression was assayed by immunohistochemical staining. Results. A synergistic inhibitory effect on viability of the two cell lines was observed after combination of paeonol with various concentrations of cisplatin. Further study showed the combination induced greater apoptosis than the groups treated with paeonol or cisplatin alone. The expression of Bcl-2 was decreased and that of Bax was increased in treatment groups, especially in the combination group, with the ratio of Bcl-2/Bax decreased correspondingly. And the combination also resulted in greater activation of caspase-3 than did either agent alone. Conclusions. Paeonol, in combination with cisplatin, had a significantly synergistic growth-inhibitory effect on oesophageal cell line, which may be related to apoptosis induction. © 2008 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. Keywords: Cisplatin; Drug interaction; Human oesophageal cell line; Paeonol
1. Introduction Oesophageal cancer (OEC) is one of the most common and malignant tumours [1,2]. The most frequent histological type is squamous cell carcinoma, with the proportion more than 95%. However, the proportion of adenocarcinomas reaches almost 50% in Western Europe and the U.S.A. Despite recent advances in surgical technique and treatment strategies for OEC, the 5-year survival of postoperation is only 20–30% 夽 Grant support: This work was supported by grants from the Natural Science Foundation of China (No. 30772537), the Natural Science Foundation of Anhui Province (No. 050430901), the Program of Science and Technology of Anhui Province (No. 07010302184), the Key Project of the Natural Science Foundation of the Department of Education of Anhui Province (No. KJ2007A032). ∗ Corresponding author. Tel.: +86 551 2922354; fax: +86 551 5161208. E-mail address: [email protected] (G.-P. Sun).
[3–5]. Chemotherapy is one of the commonly used strategies in OEC treatment, especially for unresectable patients [6]. Conventional chemotherapeutic drugs in OEC treatment such as cisplatin (CDDP) often have severe side effects that limit their efficacy. Combination therapy is a common practice in the treatment of cancers, which can achieve therapeutic effects greater than those provided by a single and can reduce the side effects and resistance to drugs. Natural products are potential sources of novel anticancer drugs over the decades. Paeonol (Pae, 2-hydroxy-4methoxyacetophenone), a major active component isolated from a Chinese herbal medicine and prepared from the root bark of the plant Paeonia Suffruticosa Andrew [7], possesses extensive pharmacological activities such as antioxidation, antiinflammation, and immunoregulation [8]. In our previous study, the antineoplastic activity of Pae has been demonstrated in different tumour cell lines [9–12]. In the present
1590-8658/$30 © 2008 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dld.2008.01.012
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study, oesophageal adenocarcinoma cell line and squamous cell carcinoma cell line were used to investigate the growthinhibitory effect of Pae alone or combined with CDDP on human oesophageal cancer cell lines and the possible mechanism, in order to develop an effective therapy for OEC.
[12–14]. CDI is calculated as follows: CDI = AB/(A × B). AB is the ratio of the two-drug combination group to the control group in OD490 ; A or B is the ratio of the single drug group to the control group in OD490 . Therefore, CDI < 1 indicates synergism, CDI < 0.7 indicates a significantly synergistic effect, CDI = 1 indicates additivity, and CDI > 1 indicates antagonism.
2. Materials and methods
2.5. AO staining and flow cytometry (FCM) assay
2.1. Culture of oesophageal cell lines
Apoptotic cells were examined morphologically by AO staining and by using FCM after staining with propidium iodide (PI). For morphological analysis, briefly, cells incubated with drugs for 24 h were dyed with 0.1 mg/mL AO and observed under fluorescence microscope. For FCM analysis, cells were collected and fixed with 70% ethanol for 4 h, washed twice with PBS, and then labeled with 500 L PI staining buffer in the dark for 30 min. Fluorescent signals of PI were detected by an EPICS XL-MCL model Coulter counter. At least 10,000 cells were counted.
Human oesophageal adenocarcinoma cells SEG-1 was kindly provided by Dr. Liping Ma, the central Laboratory of the people’s hospital of Peking university. Human oesophageal squamous cell carcinoma cell line Eca-109 was purchased from Shanghai cell bank, Chinese Academy of Sciences. Two cell lines were both cultured in RPMI-1640 medium (Gibco, New York, USA) supplemented with 10% fetal bovine serum (FBS) and incubated at 37 ◦ C in humid atmosphere with 5% CO2 .
2.6. Immunocytochemistry 2.2. Drugs and chemicals Pae Injection was purchased from First Pharmaceutical Factory of Shanghai, China (Cat. No. 990402, 10 mg/2 mL); CDDP Injection was purchased from Nanjing Pharmaceutical Factory Co. Ltd. (Cat. No. 20050602, 1 mg/1 mL); MTT and acridine orange (AO) were from Sigma Co. (St. Louis, MO, USA); DNA-Prep-Reagents Kit was bought from Beckman Coulter Co. (Miami, FL, USA. Cat. No. 760279K); Rabbit polyclonal antibodies against human Bcl-2, Bax and caspase3 were all purchased from Lab Vision Corporation (Fremont, California, USA) and streptavidin-biotin-peroxidase (S-P) reagents kit was obtained from Fuzhou Maxim Biotech, Ltd. (Fuzhou, Fujian, China).
Immunohistochemical staining for Bcl-2, Bax and caspase-3 was measured using the standard S-P method. The detailed manipulation was conducted according to the manufacturer’s instructions. As negative control, PBS was used instead of primary antibody and other steps were followed in the same way. The immunocytochemical results were quantitatively analysed by biological image analysis system which consists of Nikon ECLIPSE 80i biology microscope, Nikon Digital Camera DXM 1200F, ACT-1 version 2.63 software (Japan), and JEOA 801D morphological biological image analysis software version 6.0 (Jie Da Technologies. Inc., China). The average absorbance value was analysed by 5 randomly selected optical fields by microscopy (×200).
2.3. In vitro cytotoxicity assay 2.7. Statistical analysis Cells were cultured in 96-well plates at a density of 1–5 × 103 cells/well overnight. Then cells were treated with various concentrations of Pae or CDDP alone or their combination. After 44 h of drug exposure, MTT solution (5 mg/mL) was added to the plates for another 4 h. The formazine was solved in 150 L/well DMSO and absorbance was detected at 490 nm using ELx800 Strip reader (Bio-Tek, USA). The percentage of cytotoxicity was calculated as follows: Cytotoxicity (%) = (1 − OD490 of experimental well)/OD490 of control well. The median inhibitory concentration (IC50 ) was expressed as the drug concentration with which cell growth was inhibited by 50%.
Biostatistical analyses were done using SPSS 11.5 software package. All results were expressed as mean ± S.D. One-way analysis of variance (ANOVA) was used for evaluating the differences between groups. Spearman correlation coefficient was used to analyse continuous independent and dependent variables. A level of P < 0.05 was accepted as statistically significant.
3. Results
2.4. Analysis of in vitro drug interaction
3.1. Inhibitory effect of Pae on oesophageal cancer cell proliferation
The coefficient of drug interaction (CDI) was used to analyse the synergistically inhibitory effect of drug combination
Cells incubated with various concentrations of Pae for 48 h showed a dose-dependent reduction of cell viability.
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Fig. 1. The synergistic effect and CDI of Pae and CDDP on the proliferation of SEG-1 and Eca-109 cells. (A) SEG-1 cells treated with Pae and CDDP for 48 h. (B) CDI values of the combination groups in SEG-1 cells. (C) Eca-109 cells treated with Pae and CDDP for 48 h. (D) CDI values of the combination groups in Eca-109 cells. Data are presented as mean ± S.E. (error bar) of triplicate cultures. * P < 0.05, ** P < 0.01, vs. CDDP alone.
The inhibitory rate of Pae (7.81, 15.63, 31.25, 62.5, 125, 250 mg/L) on SEG-1 cells was 4.56 ± 1.14%, 10.08 ± 1.58%, 18.35 ± 4.34%, 63.45 ± 3.28%, 75.63 ± 2.54%, 85.15 ± 1.11% (r = 0.961, P < 0.01) and that on Eca-109 cells was 4.55 ± 0.52%, 8.39 ± 3.43%, 12.55 ± 2.08%, 17.48 ± 1.11%, 53.06 ± 11.78%, 95.04 ± 0.23% (r = 0.889, P < 0.05). However, the sensitivity of the two cell lines to Pae was considerably different. SEG-1 cell line was more sensitive to Pae, when compared the IC50 . The IC50 of Pae on SEG-1 and Eca-109 cells were 57.73 and 124.77 mg/L, respectively. 3.2. Synergistic cytotoxicity of Pae combined with CDDP To investigate the synergistic inhibitory effects of Pae and CDDP, three doses of Pae (7.81, 15.63 and 31.25 mg/L) were used to combine with different concentrations of CDDP mixed at a fixed ratio (1:1, v/v). The results showed that Pae increased the cytotoxicity of CDDP on SEG-1 (Fig. 1A) and Eca-109 cells (Fig. 1C). CDI was used to evaluate the nature of the interaction. Pae and CDDP had synergistic effect on SEG-1 cells (Fig. 1B), which was stronger when CDDP in low concentration (0.078 mg/L)
(P < 0.01). Similarly, in Eca-109 cells (Fig. 1D), CDI values are all less than 1, which indicated that Pae and CDDP had synergistic effect on Eca-109 cells at desired concentrations. 3.3. Apoptosis induction of Pae combined with CDDP Fig. 2 showed that AO permeated through all cells and made the nuclei appear green. The cells treated with Pae and/or CDDP showed typically apoptotic changes, including reduction in cell volume, chromatin condensation, deformed and fragmented nuclei, especially in the combination groups. In addition, the induction of apoptosis by treatment groups was also evident from the FCM assay. Compared to Pae or CDDP group, the sub-G1 peak was dramatically increased in the combination group (Fig. 2). 3.4. Effect of Pae and/or CDDP on the expression of Bcl-2 and Bax The standard positive Bcl-2 and Bax expressions were stained brown or yellow mainly in cytoplasm or membrane. After treatment for 24 h, the expression of Bcl-2 decreased (Figs. 3A and 4A), and in contrast, there was a
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Fig. 2. Apoptosis of SEG-1 and Eca-109 cells cells treated with Pae and CDDP. Morphological changes of cells treated with drugs for 24 h were detected by AO staining (×200). The percentage of apoptotic cells were determined by FCM assay. (a) Control group. (b) Pae 31.25 mg/L. (c) CDDP 1.25 mg/L. (d) Pae (31.25 mg/L) plus CDDP (1.25 mg/L).
significant increase of Bax expression (Figs. 3B and 4B), especially in the combination groups. The results were quantitatively analysed by biological image analysis system and the ratio of Bcl-2/Bax decreased correspondingly (Figs. 3C and 4C).
3.5. Effect of Pae and/or CDDP on the expression of caspase-3 To determine whether caspase-3 plays a role in Pae and/or CDDP mediated apoptosis of SEG-1 and Eca-109 cells,
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Fig. 3. Effect of Pae and/or CDDP on the expression of Bcl-2 (Panel A) and Bax (Panel B) in SEG-1 cells. (a) Untreated cells. (b) Pae 31.25 mg/L. (c) CDDP 1.25 mg/L. (d) Pae (31.25 mg/L) plus CDDP (1.25 mg/L). S-P, ×200. (Panel C) Quantitative analysis of Bcl-2 and Bax expression by biological image analysis system. Data are presented as mean ± S.D. (error bar). * P < 0.05 vs. CDDP alone.
we assessed the activated caspase-3 protein level of the two cell lines before and after treatment with Pae and/or CDDP using S-P method. The standard positive caspase-3 expressions were stained brown or yellow mainly in cyto-
plasm or nucleus. As shown in Fig. 5, the expression of activated caspase-3 increased especially in the combination group after the two cell lines were exposed to drugs for 24 h.
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Fig. 4. Effect of Pae and/or CDDP on the expression of Bcl-2 (Panel A) and Bax (Panel B) in Eca-109 cells. (a) Untreated cells. (b) Pae 31.25 mg/L. (c) CDDP 1.25 mg/L. (d) Pae (31.25 mg/L) plus CDDP (1.25 mg/L). S-P, ×200. (Panel C) Quantitative analysis of Bcl-2 and Bax expression by biological image analysis system. Data are presented as mean ± S.D. (error bar). * P < 0.05 vs. CDDP alone.
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Fig. 5. Effect of Pae and/or CDDP on the expression of caspase-3 (Panel A) in SEG-1 and Eca-109 cells. (a) Untreated cells. (b) Pae 31.25 mg/L. (c) CDDP 1.25 mg/L. (d) Pae (31.25 mg/L) plus CDDP (1.25 mg/L). S-P, ×200. (Panel B) Quantitative analysis of caspase-3 expression by biological image analysis system. Data are presented as mean ± S.D. (error bar).* P < 0.01, vs. control, # P < 0.01, vs. CDDP alone.
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4. Discussion The results in the present study demonstrated that Pae possessed growth inhibition to SEG-1 and Eca-109 cells in a dose-dependent manner. However, the IC50 of Pae on SEG-1 and Eca-109 cells were 57.73 and 124.77 mg/L, respectively, which indicated that Pae was more sensitive to SEG-1. There was a synergistic interaction between Pae and CDDP in the two cell lines. The cytotoxity of the combination group was significantly higher than that treated with Pae or CDDP alone in appropriate concentrations. Our results are in accordance with the findings of JI Chun-yan et al. who demonstrated that Pae in low concentration had synergetic effect with 5-FU, MMC and CDDP in inhibiting the proliferation of human colorectal cancer cell line HT-29 [15]. Similar results were observed in the HepG2 and SMMC-7721 cells [12]. Although the exact mechanism of the cytotoxicity of Pae against tumour cells is not entirely clear, apoptosis induction has been proposed for the growth inhibition of Pae [10–12,16]. Apoptosis is a physiological process in controlling cell number and proliferation that helps in maintaining the homeostasis of multicellular organisms [17]. The hypothesis that failure to undergo apoptosis contributes to the development of resistance to anticancer agents has been the subject of extensive research [17,18]. Therefore, agents that facilitate apoptosis should improve therapeutic efficacy. In our study, the cells treated with drugs showed typical morphological changes of apoptosis and sub-G1 population accumulation, which was more prominent in the combination group. For example, in SEG-1 cells, the apoptosis rate of combination group reached to 21.6% (compared to Pae group 7.82% and CDDP group 9.84%). The Bcl-2 family proteins are key regulators of mitochondria pathway which is an important mechanism of apoptosis [19] and its effects are more dependent on the balance between antiapoptotic Bcl-2 and proapoptotic Bax [20]. In response to apoptotic stimuli such as DNA damage, Bcl-2 family members regulate mitochondrial release of cytochrome c to cytosol, where it subsequently forms a complex with Apaf-1 and caspase-9, leading to the activation of the caspase-3, which subsequently activates the rest of the caspase cascade and leads to apoptosis [20–24]. So we examined the expression of Bcl-2 protein family and caspase-3. In the present study, treatment with Pae and/or CDDP decreased the expression of Bcl-2 and increased the expression of Bax, especially in the combination group, which followed by the activation of caspase-3. The up-regulation of Bax expression and the reduction of Bcl-2 expression in the treated cell lines led to a decrease in the ratio of Bcl-2/Bax, which may be responsible for the drug-induced apoptotic processes. And the synergistic apoptosis-induction effect of the combination might occur through the mitochondria-dependent pathway and associated with caspase-3 activation. In conclusion, the results obtained in the present study indicate that Pae in combination with CDDP results in greater growth inhibition and apoptosis induction than did either
agent alone on the two cell lines, which may be related with down-regulation of Bcl-2, up-regulation of Bax, activation of caspase-3 and this combination might be useful for OEC treatment.
Practice points • Conventional chemotherapeutic drugs in OEC treatment such as cisplatin often have severe side effects that limit their efficacy. • Combination therapy with multiple drugs or multiple modalities is a common practice in the treatment of cancers, which can achieve therapeutic effects greater than those provided by a single and can reduce the side effects and resistance to drugs. • Natural products are potential sources of novel anticancer drugs. It has been reported that paeonol has anti-neoplastic activity in vitro and in vivo. Paeonol also has synergistic effect with traditional therapeutic agents on human hepatoma cell lines. • Paeonol may merit further investigation as a potential therapeutic agent of OEC.
Research agenda • Investigate whether paeonol has synergistic effect with cisplatin on the growth-inhibitory of human oesophageal cancer cell lines. • Search in basic molecular mechanisms involved in the synergistic effect. • Aim to apply this combination against OEC, clinically.
Conflicts of interest statement There is no conflict of interest.
Acknowledgements We thank Dr. Wei Wei for the experiment equipment support, institute of Clinical Pharmacology of Anhui Medical University. We are grateful to Dr. Zhimin Zhai and Qing Li for FACS analysis, the Central Laboratory of the Provincial Hospital of Anhui. We are also indebted to Dahai Huang for image analysis, the Central Laboratory of morphology of Anhui Medical University.
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References [1] Pisani P, Parkin DM, Bray F, Ferlay J. Estimates of the world wide mortality from 25 cancers in 1990. Int J Cancer 1999;83:18–29. [2] Parkin DM. Global cancer statistics in the year 2000. Lancet Oncol 2001;2:533–43. [3] Kollarova H, Machova L, Horakova D, Janoutova G, Janout V. Epidemiology of esophageal cancer—an overview article. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2007;151:17–20. [4] Orringer MB, Marshall B, Lannettoni MD. Transhiatal esophagectomy: clinical experience and refinements. Ann Surg 1999;230:392–400. [5] Hagen JA, Demeester SR, Peters JH, Chandrasoma P, DeMeester TR. Curative resection for esophageal adenocarcinoma: analysis of 100 enbloc esophagectomies. Ann Surg 2001;234:520–30. [6] Hwang JJ. Role of chemotherapy in the treatment of gastroesophageal cancers. Oncology 2007;21:579–86. [7] Riley CM, Ren TC. Simple method for the determination of paeonol in human and rabbit plasma by high performance liquid chromatography using solid-phase extraction and ultraviolet detection. J Chromatogr 1989;489:427–32. [8] Sun YC, Shen YX, Sun GP. Advances in the studies of major pharmacological activity of paeonol. Chin Tradit Patent Med 2004;26:579– 82. [9] Sun GP, Wang H, Shen YX, Xu SY. Inhibitory effects of paeonol on the proliferation on four tumor cell lines. Anhui Med Pharm J 2004;8:85–7. [10] Liu CQ, Tan SY, Ji CY, Luo HS, Yu JP. The effects of paeonol on inhibiting the proliferation of human colorectal cancer cell line HT-29 and its molecule mechanism. Chin Pharmacol Bull 2005;21:1251–4. [11] Xu SP, Sun GP, Shen YX, Wei W, Peng WR, Wang H. Antiproliferation and apoptosis induction of paeonol in HepG2 cells. World J Gastroenterol 2007;13:250–6.
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[12] Xu SP, Sun GP, Shen YX, Peng WR, Wang H, Wei W. Synergistic effect of combining paeonol and cisplatin on apoptotic induction of human hepatoma cell lines. Acta Pharmacol Sin 2007;28:869–78. [13] Cao SS, Zhen YS. Potentiation of antimetabolite antitumor activity in vivo by dipyridamole and amphotericin B. Cancer Chemother Pharmacol 1989;24:181–6. [14] Liao ZY, Zhang SH, Zhen YS. Synergistic effects of geldanamycin and antitumor drugs. Yao Xue Xue Bao 2001;36:569–75. [15] Ji CY, Tan SY, Liu CQ. Inhibitory effect of paeonol on the proliferation of human colorectal cancer cell line HT-29 and its synergistic effect with chemotherapy agents. Chin J Clin Gastroenterol 2005;17:122–4. [16] Sun GP, Wang H, Shen YX, Zhai ZM, Wei W, Xu SY. Study on effects of paeonol in inhibiting growth of K562 and inducing its apoptosis. Chin Pharmacol Bull 2004;20:550–2. [17] Trump BF, Berezesky IK, Chang SH, Phelps PC. The pathways of cell death: oncosis, apoptosis, and necrosis. Toxicol Pathol 1997;25:82–8. [18] Gottesman MM. Mechanisms of cancer drug resistance. Annu Rev Med 2002;53:615–27. [19] Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science 1998;281:1322–6. [20] Reed JC. Bcl-2 family proteins: regulators of apoptosis and chemoresistance in hematologic malignancies. Semin Hematol 1997;34:9–19. [21] Reed JC. Bcl-2 and the regulation of programmed cell death. J Cell Biol 1994;124:1–6. [22] Scorrano L, Korsmeyer S. Mechanisms of cytochrome c release by proapoptotic BCL-2 family members. Biochem Biophys Res Commun 2003;304:437–44. [23] Green DR, Evan GI. A matter of life and death. Cancer Cell 2002;1:19–30. [24] Mehmet H. Apoptosis: caspase find a new place to hide. Nature 2002;403:29–30.
Digestive and Liver Disease 40 (2008) 531–539
Alimentary Tract
Synergistic effect of paeonol and cisplatin on oesophageal cancer cell lines夽 X.-A. Wan a , G.-P. Sun a,∗ , H. Wang b , S.-P. Xu a , Z.-G. Wang a , S.-H. Liu a b
a Department of Oncology, The First Affiliated Hospital of Anhui Medical University, Ji Xi Road 210, Hefei 230022, PR China Department of Oncology, The Affiliated Provincial Hospital of Anhui Medical University, Lu Jiang Rood 17, Hefei 230001, PR China
Received 13 October 2007; accepted 30 January 2008 Available online 12 March 2008
Abstract Background/aim. Paeonol, a phenolic component from the root bark of Paeonia moutan, has shown great promise in antitumour activities in our previous studies. The present study was designed to investigate whether paeonol has synergistic effect with cisplatin on the growth-inhibitory of human oesophageal cancer cell lines and the possible mechanism. Methods. Cell viabililty was measured by MTT assay. Drug–drug interactions were analysed by the coefficient of drug interaction. Apoptosis was detected by acridine orange fluorescence staining and flow cytometry assay. Bcl-2, Bax and caspase-3 expression was assayed by immunohistochemical staining. Results. A synergistic inhibitory effect on viability of the two cell lines was observed after combination of paeonol with various concentrations of cisplatin. Further study showed the combination induced greater apoptosis than the groups treated with paeonol or cisplatin alone. The expression of Bcl-2 was decreased and that of Bax was increased in treatment groups, especially in the combination group, with the ratio of Bcl-2/Bax decreased correspondingly. And the combination also resulted in greater activation of caspase-3 than did either agent alone. Conclusions. Paeonol, in combination with cisplatin, had a significantly synergistic growth-inhibitory effect on oesophageal cell line, which may be related to apoptosis induction. © 2008 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. Keywords: Cisplatin; Drug interaction; Human oesophageal cell line; Paeonol
1. Introduction Oesophageal cancer (OEC) is one of the most common and malignant tumours [1,2]. The most frequent histological type is squamous cell carcinoma, with the proportion more than 95%. However, the proportion of adenocarcinomas reaches almost 50% in Western Europe and the U.S.A. Despite recent advances in surgical technique and treatment strategies for OEC, the 5-year survival of postoperation is only 20–30% 夽 Grant support: This work was supported by grants from the Natural Science Foundation of China (No. 30772537), the Natural Science Foundation of Anhui Province (No. 050430901), the Program of Science and Technology of Anhui Province (No. 07010302184), the Key Project of the Natural Science Foundation of the Department of Education of Anhui Province (No. KJ2007A032). ∗ Corresponding author. Tel.: +86 551 2922354; fax: +86 551 5161208. E-mail address: [email protected] (G.-P. Sun).
[3–5]. Chemotherapy is one of the commonly used strategies in OEC treatment, especially for unresectable patients [6]. Conventional chemotherapeutic drugs in OEC treatment such as cisplatin (CDDP) often have severe side effects that limit their efficacy. Combination therapy is a common practice in the treatment of cancers, which can achieve therapeutic effects greater than those provided by a single and can reduce the side effects and resistance to drugs. Natural products are potential sources of novel anticancer drugs over the decades. Paeonol (Pae, 2-hydroxy-4methoxyacetophenone), a major active component isolated from a Chinese herbal medicine and prepared from the root bark of the plant Paeonia Suffruticosa Andrew [7], possesses extensive pharmacological activities such as antioxidation, antiinflammation, and immunoregulation [8]. In our previous study, the antineoplastic activity of Pae has been demonstrated in different tumour cell lines [9–12]. In the present
1590-8658/$30 © 2008 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dld.2008.01.012
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study, oesophageal adenocarcinoma cell line and squamous cell carcinoma cell line were used to investigate the growthinhibitory effect of Pae alone or combined with CDDP on human oesophageal cancer cell lines and the possible mechanism, in order to develop an effective therapy for OEC.
[12–14]. CDI is calculated as follows: CDI = AB/(A × B). AB is the ratio of the two-drug combination group to the control group in OD490 ; A or B is the ratio of the single drug group to the control group in OD490 . Therefore, CDI < 1 indicates synergism, CDI < 0.7 indicates a significantly synergistic effect, CDI = 1 indicates additivity, and CDI > 1 indicates antagonism.
2. Materials and methods
2.5. AO staining and flow cytometry (FCM) assay
2.1. Culture of oesophageal cell lines
Apoptotic cells were examined morphologically by AO staining and by using FCM after staining with propidium iodide (PI). For morphological analysis, briefly, cells incubated with drugs for 24 h were dyed with 0.1 mg/mL AO and observed under fluorescence microscope. For FCM analysis, cells were collected and fixed with 70% ethanol for 4 h, washed twice with PBS, and then labeled with 500 L PI staining buffer in the dark for 30 min. Fluorescent signals of PI were detected by an EPICS XL-MCL model Coulter counter. At least 10,000 cells were counted.
Human oesophageal adenocarcinoma cells SEG-1 was kindly provided by Dr. Liping Ma, the central Laboratory of the people’s hospital of Peking university. Human oesophageal squamous cell carcinoma cell line Eca-109 was purchased from Shanghai cell bank, Chinese Academy of Sciences. Two cell lines were both cultured in RPMI-1640 medium (Gibco, New York, USA) supplemented with 10% fetal bovine serum (FBS) and incubated at 37 ◦ C in humid atmosphere with 5% CO2 .
2.6. Immunocytochemistry 2.2. Drugs and chemicals Pae Injection was purchased from First Pharmaceutical Factory of Shanghai, China (Cat. No. 990402, 10 mg/2 mL); CDDP Injection was purchased from Nanjing Pharmaceutical Factory Co. Ltd. (Cat. No. 20050602, 1 mg/1 mL); MTT and acridine orange (AO) were from Sigma Co. (St. Louis, MO, USA); DNA-Prep-Reagents Kit was bought from Beckman Coulter Co. (Miami, FL, USA. Cat. No. 760279K); Rabbit polyclonal antibodies against human Bcl-2, Bax and caspase3 were all purchased from Lab Vision Corporation (Fremont, California, USA) and streptavidin-biotin-peroxidase (S-P) reagents kit was obtained from Fuzhou Maxim Biotech, Ltd. (Fuzhou, Fujian, China).
Immunohistochemical staining for Bcl-2, Bax and caspase-3 was measured using the standard S-P method. The detailed manipulation was conducted according to the manufacturer’s instructions. As negative control, PBS was used instead of primary antibody and other steps were followed in the same way. The immunocytochemical results were quantitatively analysed by biological image analysis system which consists of Nikon ECLIPSE 80i biology microscope, Nikon Digital Camera DXM 1200F, ACT-1 version 2.63 software (Japan), and JEOA 801D morphological biological image analysis software version 6.0 (Jie Da Technologies. Inc., China). The average absorbance value was analysed by 5 randomly selected optical fields by microscopy (×200).
2.3. In vitro cytotoxicity assay 2.7. Statistical analysis Cells were cultured in 96-well plates at a density of 1–5 × 103 cells/well overnight. Then cells were treated with various concentrations of Pae or CDDP alone or their combination. After 44 h of drug exposure, MTT solution (5 mg/mL) was added to the plates for another 4 h. The formazine was solved in 150 L/well DMSO and absorbance was detected at 490 nm using ELx800 Strip reader (Bio-Tek, USA). The percentage of cytotoxicity was calculated as follows: Cytotoxicity (%) = (1 − OD490 of experimental well)/OD490 of control well. The median inhibitory concentration (IC50 ) was expressed as the drug concentration with which cell growth was inhibited by 50%.
Biostatistical analyses were done using SPSS 11.5 software package. All results were expressed as mean ± S.D. One-way analysis of variance (ANOVA) was used for evaluating the differences between groups. Spearman correlation coefficient was used to analyse continuous independent and dependent variables. A level of P < 0.05 was accepted as statistically significant.
3. Results
2.4. Analysis of in vitro drug interaction
3.1. Inhibitory effect of Pae on oesophageal cancer cell proliferation
The coefficient of drug interaction (CDI) was used to analyse the synergistically inhibitory effect of drug combination
Cells incubated with various concentrations of Pae for 48 h showed a dose-dependent reduction of cell viability.
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Fig. 1. The synergistic effect and CDI of Pae and CDDP on the proliferation of SEG-1 and Eca-109 cells. (A) SEG-1 cells treated with Pae and CDDP for 48 h. (B) CDI values of the combination groups in SEG-1 cells. (C) Eca-109 cells treated with Pae and CDDP for 48 h. (D) CDI values of the combination groups in Eca-109 cells. Data are presented as mean ± S.E. (error bar) of triplicate cultures. * P < 0.05, ** P < 0.01, vs. CDDP alone.
The inhibitory rate of Pae (7.81, 15.63, 31.25, 62.5, 125, 250 mg/L) on SEG-1 cells was 4.56 ± 1.14%, 10.08 ± 1.58%, 18.35 ± 4.34%, 63.45 ± 3.28%, 75.63 ± 2.54%, 85.15 ± 1.11% (r = 0.961, P < 0.01) and that on Eca-109 cells was 4.55 ± 0.52%, 8.39 ± 3.43%, 12.55 ± 2.08%, 17.48 ± 1.11%, 53.06 ± 11.78%, 95.04 ± 0.23% (r = 0.889, P < 0.05). However, the sensitivity of the two cell lines to Pae was considerably different. SEG-1 cell line was more sensitive to Pae, when compared the IC50 . The IC50 of Pae on SEG-1 and Eca-109 cells were 57.73 and 124.77 mg/L, respectively. 3.2. Synergistic cytotoxicity of Pae combined with CDDP To investigate the synergistic inhibitory effects of Pae and CDDP, three doses of Pae (7.81, 15.63 and 31.25 mg/L) were used to combine with different concentrations of CDDP mixed at a fixed ratio (1:1, v/v). The results showed that Pae increased the cytotoxicity of CDDP on SEG-1 (Fig. 1A) and Eca-109 cells (Fig. 1C). CDI was used to evaluate the nature of the interaction. Pae and CDDP had synergistic effect on SEG-1 cells (Fig. 1B), which was stronger when CDDP in low concentration (0.078 mg/L)
(P < 0.01). Similarly, in Eca-109 cells (Fig. 1D), CDI values are all less than 1, which indicated that Pae and CDDP had synergistic effect on Eca-109 cells at desired concentrations. 3.3. Apoptosis induction of Pae combined with CDDP Fig. 2 showed that AO permeated through all cells and made the nuclei appear green. The cells treated with Pae and/or CDDP showed typically apoptotic changes, including reduction in cell volume, chromatin condensation, deformed and fragmented nuclei, especially in the combination groups. In addition, the induction of apoptosis by treatment groups was also evident from the FCM assay. Compared to Pae or CDDP group, the sub-G1 peak was dramatically increased in the combination group (Fig. 2). 3.4. Effect of Pae and/or CDDP on the expression of Bcl-2 and Bax The standard positive Bcl-2 and Bax expressions were stained brown or yellow mainly in cytoplasm or membrane. After treatment for 24 h, the expression of Bcl-2 decreased (Figs. 3A and 4A), and in contrast, there was a
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Fig. 2. Apoptosis of SEG-1 and Eca-109 cells cells treated with Pae and CDDP. Morphological changes of cells treated with drugs for 24 h were detected by AO staining (×200). The percentage of apoptotic cells were determined by FCM assay. (a) Control group. (b) Pae 31.25 mg/L. (c) CDDP 1.25 mg/L. (d) Pae (31.25 mg/L) plus CDDP (1.25 mg/L).
significant increase of Bax expression (Figs. 3B and 4B), especially in the combination groups. The results were quantitatively analysed by biological image analysis system and the ratio of Bcl-2/Bax decreased correspondingly (Figs. 3C and 4C).
3.5. Effect of Pae and/or CDDP on the expression of caspase-3 To determine whether caspase-3 plays a role in Pae and/or CDDP mediated apoptosis of SEG-1 and Eca-109 cells,
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Fig. 3. Effect of Pae and/or CDDP on the expression of Bcl-2 (Panel A) and Bax (Panel B) in SEG-1 cells. (a) Untreated cells. (b) Pae 31.25 mg/L. (c) CDDP 1.25 mg/L. (d) Pae (31.25 mg/L) plus CDDP (1.25 mg/L). S-P, ×200. (Panel C) Quantitative analysis of Bcl-2 and Bax expression by biological image analysis system. Data are presented as mean ± S.D. (error bar). * P < 0.05 vs. CDDP alone.
we assessed the activated caspase-3 protein level of the two cell lines before and after treatment with Pae and/or CDDP using S-P method. The standard positive caspase-3 expressions were stained brown or yellow mainly in cyto-
plasm or nucleus. As shown in Fig. 5, the expression of activated caspase-3 increased especially in the combination group after the two cell lines were exposed to drugs for 24 h.
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Fig. 4. Effect of Pae and/or CDDP on the expression of Bcl-2 (Panel A) and Bax (Panel B) in Eca-109 cells. (a) Untreated cells. (b) Pae 31.25 mg/L. (c) CDDP 1.25 mg/L. (d) Pae (31.25 mg/L) plus CDDP (1.25 mg/L). S-P, ×200. (Panel C) Quantitative analysis of Bcl-2 and Bax expression by biological image analysis system. Data are presented as mean ± S.D. (error bar). * P < 0.05 vs. CDDP alone.
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Fig. 5. Effect of Pae and/or CDDP on the expression of caspase-3 (Panel A) in SEG-1 and Eca-109 cells. (a) Untreated cells. (b) Pae 31.25 mg/L. (c) CDDP 1.25 mg/L. (d) Pae (31.25 mg/L) plus CDDP (1.25 mg/L). S-P, ×200. (Panel B) Quantitative analysis of caspase-3 expression by biological image analysis system. Data are presented as mean ± S.D. (error bar).* P < 0.01, vs. control, # P < 0.01, vs. CDDP alone.
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4. Discussion The results in the present study demonstrated that Pae possessed growth inhibition to SEG-1 and Eca-109 cells in a dose-dependent manner. However, the IC50 of Pae on SEG-1 and Eca-109 cells were 57.73 and 124.77 mg/L, respectively, which indicated that Pae was more sensitive to SEG-1. There was a synergistic interaction between Pae and CDDP in the two cell lines. The cytotoxity of the combination group was significantly higher than that treated with Pae or CDDP alone in appropriate concentrations. Our results are in accordance with the findings of JI Chun-yan et al. who demonstrated that Pae in low concentration had synergetic effect with 5-FU, MMC and CDDP in inhibiting the proliferation of human colorectal cancer cell line HT-29 [15]. Similar results were observed in the HepG2 and SMMC-7721 cells [12]. Although the exact mechanism of the cytotoxicity of Pae against tumour cells is not entirely clear, apoptosis induction has been proposed for the growth inhibition of Pae [10–12,16]. Apoptosis is a physiological process in controlling cell number and proliferation that helps in maintaining the homeostasis of multicellular organisms [17]. The hypothesis that failure to undergo apoptosis contributes to the development of resistance to anticancer agents has been the subject of extensive research [17,18]. Therefore, agents that facilitate apoptosis should improve therapeutic efficacy. In our study, the cells treated with drugs showed typical morphological changes of apoptosis and sub-G1 population accumulation, which was more prominent in the combination group. For example, in SEG-1 cells, the apoptosis rate of combination group reached to 21.6% (compared to Pae group 7.82% and CDDP group 9.84%). The Bcl-2 family proteins are key regulators of mitochondria pathway which is an important mechanism of apoptosis [19] and its effects are more dependent on the balance between antiapoptotic Bcl-2 and proapoptotic Bax [20]. In response to apoptotic stimuli such as DNA damage, Bcl-2 family members regulate mitochondrial release of cytochrome c to cytosol, where it subsequently forms a complex with Apaf-1 and caspase-9, leading to the activation of the caspase-3, which subsequently activates the rest of the caspase cascade and leads to apoptosis [20–24]. So we examined the expression of Bcl-2 protein family and caspase-3. In the present study, treatment with Pae and/or CDDP decreased the expression of Bcl-2 and increased the expression of Bax, especially in the combination group, which followed by the activation of caspase-3. The up-regulation of Bax expression and the reduction of Bcl-2 expression in the treated cell lines led to a decrease in the ratio of Bcl-2/Bax, which may be responsible for the drug-induced apoptotic processes. And the synergistic apoptosis-induction effect of the combination might occur through the mitochondria-dependent pathway and associated with caspase-3 activation. In conclusion, the results obtained in the present study indicate that Pae in combination with CDDP results in greater growth inhibition and apoptosis induction than did either
agent alone on the two cell lines, which may be related with down-regulation of Bcl-2, up-regulation of Bax, activation of caspase-3 and this combination might be useful for OEC treatment.
Practice points • Conventional chemotherapeutic drugs in OEC treatment such as cisplatin often have severe side effects that limit their efficacy. • Combination therapy with multiple drugs or multiple modalities is a common practice in the treatment of cancers, which can achieve therapeutic effects greater than those provided by a single and can reduce the side effects and resistance to drugs. • Natural products are potential sources of novel anticancer drugs. It has been reported that paeonol has anti-neoplastic activity in vitro and in vivo. Paeonol also has synergistic effect with traditional therapeutic agents on human hepatoma cell lines. • Paeonol may merit further investigation as a potential therapeutic agent of OEC.
Research agenda • Investigate whether paeonol has synergistic effect with cisplatin on the growth-inhibitory of human oesophageal cancer cell lines. • Search in basic molecular mechanisms involved in the synergistic effect. • Aim to apply this combination against OEC, clinically.
Conflicts of interest statement There is no conflict of interest.
Acknowledgements We thank Dr. Wei Wei for the experiment equipment support, institute of Clinical Pharmacology of Anhui Medical University. We are grateful to Dr. Zhimin Zhai and Qing Li for FACS analysis, the Central Laboratory of the Provincial Hospital of Anhui. We are also indebted to Dahai Huang for image analysis, the Central Laboratory of morphology of Anhui Medical University.
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