Enhancement of apoptosis of human hepatocellular carcinoma SMMC-7721 cells through synergy of berberine and evodiamine.
Berberine and evodiamine, two kinds of alkaloids, have been reported to show many activities. In the present paper, inhibitory activities of the two compounds and their mixtures on human hepatocellular carcinoma SMMC-7721 cells were investigated, and the inhibitory rates, apoptosis, cell cycle distribution and tumor necrosis factor-[alpha] (TNF-[alpha]) were all tested and described. The results indicate that the mixtures of the two compounds showed the highest inhibition effect (50.00%) as compared with berberine and evodiamine used individually (20.24% and 16.33%, respectively) over 48h. Through fluorescence microscope and flow cytometry (FCM) analysis, the cell apoptosis and cell cycle distribution of SMMC-7721 induced by the synergy of the two compounds was made evident. Furthermore, the TNF-[alpha] value in the mixture treated group was much higher (p < 0.05) than in the other two groups. Thus, the combined use of berberine and evodiamine could significantly enhance the apoptosis of SMMC-7721 cells, which will be useful to further anti-cancer therapy and research.
[C] 2008 Elsevier GmbH. All rights reserved.
Keywords: Berberine; Evodiamine; Human hepatocellular carcinoma SMMC-7721 cells; Synergy effects
A famous traditional Chinese medicine formula, Zuojinwan, composed of two traditional Chinese medicinal herbs, Coptis chinensis Franch. (Huanglian in Chinese) and Evodiae Fructus (Wuzhuyu in Chinese) at the ratio of 6: 1(w/w), was first recorded in Danxi Xinhuo ... Liufa, a famous ancient medicine treatise, and is also officially listed in the Chinese Pharmacopoeia (China Pharmacopoeia Committee, 2005). Recent pharmacological studies have revealed that this formula is active against inflammation, ulceration and gastric acid (Zhang, 2007). Chemical investigations have shown that the active constituents are alkaloids including palmatine, berberine, epiberberine and coptisine from C. chinensis Franch. (Yang et al., 1998), carboxyevodiamine, dehydroevodiamine, evodiamine and rutaecarpine from E. Fructus (Chuang et al., 1996).
Among them, the isoquinoline alkaloid named berberine (Ber, shown in Fig. 1) has been reported to show anti-inflammatory (Kuo et al., 2004) and antitumor (Fukuda et al., 1999) activities, and is often used for heat-clearing, detoxification and diarrhea (Yu et al., 2000). Recently, there are multiple reports that the compound shows other effects, including ride on CYP450 total content and expression in BCG-induced immune hepatic injury (Wang et al., 2004), and inhibits human bladder tumor cells by suppression of arylamine N-acetyltransferase (NAT) activity (Chung et al., 1999). Another alkaloid, evodiamine (Evo, shown in Fig. 1), is often used for the treatment of abdominal pain, as a vasorelaxant (Chiou et al., 1992), and for gastric emptying (Wu et al., 2002). Pharmacological experiments have revealed that this compound exhibits strong effects on many kinds of cells, including prostate cancer cells (Huang et al., 2005), leukemic T-lymphocytes (Huang et al., 2004), breast cancer NCI/ADR-RES cells (Liao et al., 2005) and rat testicular interstitial cells (Lin et al., 1999).
[FIGURE 1 OMITTED]
Generally, an enhancement or synergy effects are observed when different medicinal plants were used combined in TCM research. For example, the excellent pharmacological action of Zuojinwan was produced by the synergy of C. chinensis Franch and E. Fructus. Thus, synergy research on the combined use of Ber and Evo was critically important for elucidating the principle of our ancient formula.
The aim, therefore, of the present paper was to investigate the synergy effects between Ber and Evo on human hepatoma SMMC-7721 cells, and the mechanism was also discussed. The result indicated that a significant enhancement of anticancer activity was observed when Ber and Evo were used together, as compared with use of the single compound. To the best of our knowledge, no publications have yet appeared on the synergy effects of Ber and Evo in an anti-tumor pharmacology study.
Materials and methods
Complete medium (CM) composed of RPMI-1640 containing 10% (v/v) fetal calf serum (FCS) was kindly provided by GIBCO, Inc. Some reagents including PBS, SDS, 3-(4, 5-dimethyl- thiazol-zyl)-2, 5-diphenyltetrazolium bromide (MTT) and 0.25% trypsin solution were provided by Sigma, St. Louis, MO, USA. The compounds berberine and evodiamine, with purities over 99%, were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).
For this experiment, human hepatocellular carcinoma SMMC-7721 cell lines were obtained from the Tumor Department of Second Affiliate Hospital of Dalian Medical University (Dalian, China), and cultured in RPMI-1640 containing 10% (v/v) FCS at 37[degrees]C in an incubator containing 0.5% [CO.sub.2].
The cultured SMMC-7721 cells were detached by trypsinization, centrifuged at 1000 rpm for 5 min and resuspended in fresh CM at a density of 2 x [10.sup.5] cells [ml.sup.-1]. Then 100[micro]l of the cells were planted onto 96-well flat bottom plates. After incubation in 5% [CO.sub.2]-air mixture at 37 [degrees] C for 12h, the compounds of Ber and Evo at final concentrations of 0.015 and 0.045 [micro]mol [ml.sup.-1], respectively, and their mixtures at the same concentrations were added. Six repeated wells were used for each concentration, and the cancer cell without drugs was used as control. The cells were incubated for another 20, 32, 44, 68 h, and then 10 [micro]l MTT (5 mg [ml.sup.-1]) were added into each well. After another 4 h incubation, 100 [micro]l SDS were added, and the cell was then subjected to MTT cellular viability assay according to literature procedures (Mosmann, 1983).
Cells were plated onto 96-well plates (100 [micro]l well (-1)) at a density of 2 x [10.sup.5] cells [ml.sup.-1] and incubated overnight. Ber (0.015 [micro]mol [ml.sup.-1] final concentration), Evo (0.045 [micro]mol [ml.sup.-1] final concentration) and their mixtures (Ber 0.015 [micro]mol [ml.sup.-1] and Evo 0.045 [micro]mol [ml.sup.-1]) were then added into each well and incubated for 48 h. In addition, blank wells were installed in the experiment. After incubation, cells were stained according to the introduction of the examination agent box (purchased from Kaiji Biological Technology Co. Ltd. Nanjing, China). Images of the cells were then taken using an OLYMPUS fluorescence microscope (Japan).
Flow cytometry (FCM) assay
The incubated SMMC-7721 cells were added into 6-well plates at a density of 1 X [10.sup.6] cells [ml.sup.-1] in the presence or absence of Ber, Evo and the mixtures for 48 h after overnight incubation. Cells were then harvested by trypsinization, washed twice with PBS and centrifuged at 1000 rmp for 5 min. The cells were fixed with 4[degrees]C ethanol (70-75%) for 24 h. The DNA of the cells was stained with PI staining solution in the dark room for 30 min and analyzed using a EPICS XL Flow Cytometry System with Medical MCYCLE software (Coulter, USA).
Tumor necrosis factor-[alpha] (TNF-[alpha]) assay
Cells supernatants of Ber, Evo and the mixture were collected after 48 h incubation. The determination of TNF-[alpha] concentration was made according to the introduction of the Tumor Necrosis Factor Radio-immunoassay kit (TNF-[alpha] RIA Kit, purchased form Dongya Immunity Tech. Inst, China). The data were analyzed using a ZC-2010 y-counter (USTC CHUANG-XIN Co., Ltd, China).
Statistical analysis was performed using the ANOVA method. All data were expressed as means [+ or -] S.D. The different groups were compared using Student's t-test, and considered significant at p < 0.05.
Effect of Ber, Evo and their mixtures on inhibition of cancer cells
In order to investigate the synergy effects of combined use of Ber and Evo, the signal dose of Ber at 0.015, 0.03, 0.045, 0.059 and 0.089 [micro]mol [ml.sup.-1], and their mixtures with different combination were used to treat human hepatocellular carcinoma SMMC-7721 cells at 48 h. According to the inhibition rates from different concentration mixtures of Ber and Evo, the Isobol-curve (Wagner, 2006) was made and shown in Fig. 2. The outcome indicated that the mixtures could significantly increase inhibition rates as compared to the use of Ber and Evo individually, and E comb > E ((d)Ber) + E ((d) Evo), with E representing observed effect, and (d)Ber and (d)Evo representing the dose of agents Ber and Evo.
[FIGURE 2 OMITTED]
As shown in Fig. 3, the highest inhibition rate (59.77% of the cancer cell growth was produced by the combined use of Ber at 0.015 [micro]mol [ml.sup.-1] and Evo at 0.045 [micro]mol [ml.sup.-1] at 48 h, which was higher than any other combination tested. Furthermore, the enhanced inhibition was investigated at different incubation times (24m 36, 48 and 72 h), and the results are shown in Fig. 4. A significant, time-dependent enhancement (p < 0.01) of inhibition in tumor cell growth was produced when Ber and Evo used individually at the same concentration. Thus, the concentrations of Ber and Evo were set at 0.015 and 0.045 [micro]mol [ml.sup.-1]. respectively, for subsequent experiments.
[FIGURE 3 OMITTED]
[FIGURE 4 OMITTED]
Effect of Ber, Evo and the mixtures on cancer cells' apoptosis
SMMC-7721 cells were treated with or without Ber, Evo and the mixtures for 48 h, and the results are shown in Fig. 5. As shown in Fig. 5A, the cancer cells emitted green fluorescence and nearly no death or apoptosis was observed. However, some early and late apoptotic cells with orange and densely cardinal red-stained color appeared when the compounds of Ber, Evo and their mixtures were added (Figs. 5B-D). The apoptotic cells of the group treated with the mixtures were also significantly increased under fluorescent microscope by AO, with EB doubly staining (Fig. 5D) as compared with Ber-and Evo-treated groups. In the fluorescent assay, the enhancement effect of induction cancer cell apoptosis was clear.
[FIGURE 5 OMITTED]
Effect of Ber, Evo and the mixtures on the cancer cell cycle
In this experiment, cell viability under treatment with different drugs was detected by FCM. The results shown in Fig 6 indicated that the highest apoptosis rate (58.1%) was produced in the SMMC-7721 cells cultured with the mixtures (Ber 0.015 [micro]mol [ml.sup.-1] and Evo 0.045 [micro]mol [ml.sup.-1]) (Fig. 6D), and the apoptosis rates in those treated with Ber and Evo only reached 21.6% and 13.3% respectively (Figs 6B, C) as evaluated by the appearance of a sub-[G.sub.1] peak. The inclusions indicated that a better enhancement of apoptosis occurred when the two compounds were used together, as compared with the use of single compounds (p < 0.01).
[FIGURE 6 OMITTED]
Effects of Ber, Evo and the mixtures on cytokine production
The TNF-[alpha] levels of the cancer cell's supernatant treated with Ber, Evo and their mixtures were determined, and the outcome is shown in Fig. 7. The concentration of TNF-[alpha] was significantly increased to 1.22 ng [ml.sup.-1] with the mixtures (Ber 0.015 [micro]mol [ml.sup.-1], Evo 0.045 [micro]mol [ml.sup.-1]), higher (p < 0.05) than Ber (0.727 ng [ml.sup.-1]) or Evo (0.657 ng [ml.sup.-1] used singly.
[FIGURE 7 OMITTED]
Ber and Evo, two important ingredients of many medicinal plants, have received considerable attention as individual chemporeventive agents with anti-tumor activities (Mitani et al., 2001), but no papers have yet reported synergy effect research on the anti-cancer activities of Ber and Evo. In this experiment, we have successfully investigated the anti-tumor effects of Ber, Evo and their mixtures against human hepatocellular carcinoma SMMC-7721 cells. The inhibitory rates of the mixtures (50.00%) were found to be superior to those of Ber (20.24%) and Evo (16.33%) used singly at 48 h, which could help to elucidate their principle activities in the treatment of human diseases.
The method Isobol-curve was made according to Berenbaum (1989). The method included two equations. The first was E comb > E ([d.sub.Ber]) + ([d.sub.Evo]), to indicate that 'a' total effect of a combination is greater than expected from the sum of the effects of single components'. The second equation was E comb > E ([d.sub.Ber]) and E([d.sub.Evo]), to describe that synergy was deemed present 'if the effect of combination was greater than that of each of the individual agents' (Williamson, 2001; Wagner, 2006). The Isobol method is essential to understand the interaction effect of two pure substances.
The Acridine Orangen: Ethidium Bromide (AO: EB) assay (Xiong et al., 1997) is an advanced method to detect the cells apotosis. In AO: EB double staining, EB cannot pass through the cell membrane of normal cells, but AO can do so and emits green fluorescence (Briggs and Jones, 2005). In the apoptosis cells, karyon is crimpled, chromatin is agglomerated, shrunk and broken, and then the adoptotic body is shaped and stained orange. Dead cells are stained densely cardinal red by EB. Thus, the normal, apoptosis and dead cells are distinguished by fluorescence and modality. We observed that many densely red apoptotic cells and cell fragments were found in the mixtures group, but the Ber and Evo groups had a smaller number, while the control group showed many green-yellow normal cells.
Flow cytometry (FCM), a powerful technique for the rapid characterization of cell populations, has been used widely to study prokaryotic cells in different environments (Garcia-Ochoa et al., 1998). Cells can be detected on the basis of their light-scattering properties in the forward (FALS) and the right (RALS) angle direction and, more importantly, staining with fluorescent molecules allowed us to probe the intracellular environment even with living cells. We evaluated the anticancer activity of the synergy of Ber and Evo in human hepatoma SMMC-7721 cells by FCM. The results indicated that the mixtures could more strongly enhance apoptosis cells than Ber and Evo used singly, and the apoptosis rate reached 58.1% higher than those of Ber (21.6%) and Evo (13.3%), respectively. FCM has evolved as a leading tool due to its growing role in bioprocess development and monitoring (Hewitt et al., 1999, 2000).
Tumor necrosis factor-[alpha] (TNF-[alpha]) (Camussi et al., 1991), one of the cytokines released from activated macrophage and mediate by TNR-RI, combined with the receptor of tumor cell membrane, kills tumor cells specifically. It is well known for its effects on inflammation (Parant, 1988) and arthritis (Shen et al., 2007). On the other hand, TNF-[alpha] can inhibit proliferation of tumor cells (Malik and Balkwill, 1988; Zhang et al., 2006; Mizokami et al., 2000) through cytotomicity, and regulate immunomodulation. In our study, the TNF-[alpha] level of the mixtures (1.22 ng [ml.sup.-1] was significantly higher than those of Ber (0.7267 ng [ml.sup.-1]) and Evo (0.6567 ng [ml.sup.-1]) used singly.
The present study was performed to examine the antitumor effect on human hepatocellular carcinoma SMMC-7721 cells produced through the synergy effect of Ber and Evo, two major TCM constituents. The mechanisms of gene and protein level for the enhanced anticancer activity that is part of the synergy effect of Ber and Evo requires clarification through further investigation.
This research was supported by the Excellent Young Scientists Fund (No. 200J23JHo24) of the Science and Technology Foundation of Dalian, China, and the China Post-Doctoral Foundation (No. 20070420207).
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X.-N. Wang, X. Han, L.-N. Xu, L.-H. Yin, Y.-W. Xu, Y. Qi, J.-Y. Peng *
College of Pharmacy, Dalian Medical University, No. 9 the West Part of Lvshun South Road, Dalian 116044, China
* Corresponding author. Tel.fax: 86411 8611 0411.
E-mail address: email@example.com (J.-Y. Peng).
0944-7113/$-see front matter [C] 2008 Elsevier GmbH. All rights reserved. doi: 10.1016/j.phymed.2008.05.002
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|Author:||Wang, X.-N.; Han, X.; Xu, L.-N.; Yin, L.-H.; Xu, Y.-W.; Qi, Y.; Peng, J.-Y.|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Article Type:||Clinical report|
|Date:||Dec 1, 2008|
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