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The Anti-Tumor Effects of Alkaloids from the Seeds of Strychnos Nux-Vomica

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The Anti-Tumor Effects of Alkaloids from the Seeds of Strychnos Nux-Vomica Journal of Ethnopharmacology 106 (2006) 179–186 The anti-tumor effects of alkaloids from the seeds of Strychnos nux-vomica on HepG2 cells and its possible mechanism Xu-Kun Deng a,1,WuYinb,1, Wei-Dong Li a, Fang-Zhou Yin a, Xiao-Yu Lu a, Xiao-Chun Zhang a, Zi-Chun Hua b, Bao-Chang Cai a,∗ a College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210029, China b State Key Lab of Pharmaceutic Biotechnology, Nanjing University, Nanjing 210093, China Received 6 April 2005; received in revised form 12 December 2005; accepted 15 December 2005 Available online 26 January 2006 Abstract To screen the anti-tumor effects of the four alkaloids: brucine, strychnine, brucine N-oxide and isostrychnine from the seed of Strychnos nux- vomica, MTT assay was used to examine the growth inhibitory effects of these alkaloids on human hepatoma cell line (HepG2). Brucine, strychnine and isostrychnine revealed significant inhibitory effects against HepG2 cell proliferation, whereas brucine N-oxide didn’t have such an effect. In addition, brucine caused HepG2 cell shrinkage, membrane blebbing, apoptotic body formation, all of which are typical characteristics of apoptotic programmed cell death. The results of flow cytometric analysis demonstrated that brucine caused dose-dependent apoptosis of HepG2 cells through cell cycle arrest at G0/G1 phase, thus preventing cells entering S or G2/M phase. Immunoblot results revealed that brucine significantly decreased the protein expression level of cyclooxygenase-2, whereas increased the expression caspase-3 as well as the caspase-3-like protease activity in HepG2 cells, suggesting the involvement of cyclooxygenase-2 and caspase-3 in the pro-apoptotic effects exerted by brucine. Therefore, this paper indicate that the major alkaloids present in the seed of Strychnos nux-vomica are effective against HepG2 cells proliferation, among which brucine proceed HepG2 cells death via apoptosis, probably through the participation of caspase-3 and cyclooxygenase-2. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Strychnos nux-vomica; Brucine; HepG2; Apoptosis; Cyclooxygenase-2; Caspase-3 1. Introduction tablet in liver cancer therapy. In addition to its anti-tumor effects, nux vomica is also claimed to have an ability of improving cir- Hepatocellular carcinoma is one of the most common malig- culatory system and relieving pains in patients with rheumatic nant tumors worldwide. In last decades, surgical resection, the disorders (Guizhi, 1996). However, the mechanism underly- commonly used technique is frequently challenged in view of ing the anti-tumor property of the nux vomica remains largely metastasis and other pathological changes. Therefore, the devel- unknown. opment of new agents for hepatocellular cancer is important to Alkaloids are the main bioactive chemicals in nux vomica reduce the mortality caused by this disease. (Bisset and Phillipson, 1971), responsible for the pharmacolog- Nux vomica, the dried seed of Strychnos nux-vomica L. ical and toxic effects exerted by nux vomica to a great extent. In (Loganiaceae), has been effectively used in Chinese folk our previous study, 16 alkaloids have been separated and iden- medicine for the treatment of liver cancer and associated patho- tified from the crude nux vomica, among which strychnine and logical abnormalities for a long history. As a major ingredient, it brucine take up 80% (Cai et al., 1994). Based on the principles has been frequently prescribed in some classic herbal remedies of Chinese medicine, nux vomica is commonly prescribed in such as “Ping-xiao” capsule, “Ci-dan” capsule, “Ma-qian-zi” clinical practice after sand processing, the purpose of which is not only to clean its fine hairs which causes throat irritation (Wu et al., 1994), but also reduce its toxic side effects because after processing, the intrinsic alkaloids such as brucine and strychnine ∗ Corresponding author. Tel.: +86 25 86798281; fax: +86 25 86798281. E-mail address: [email protected] (B.-C. Cai). transform into their isoforms or nitrogen oxidative derivatives 1 These authors contribute equally to this work. with less toxicity (Fig. 1)(Cai et al., 1990, 1993). However, the 0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.12.021 180 X.-K. Deng et al. / Journal of Ethnopharmacology 106 (2006) 179–186 Fig. 1. Chemical structures of brucine, brucine N-oxide, strychnine, isostrychnine, major present alkaloids in nux vomica and the transformations between them after hot sand processing. (1) Strychnine transformed into its isoform, isostrychnine after sand processing. (2) Brucine transformed into its nitrogen derivatives, brucine N-oxide after sand processing. alterations in chemical structures also result in different phar- Sigma Chemical Co. (St. Louis, MO, USA); brucine and brucine macological effects possessed by these alkaloids. For example, N-oxide were supplied by Carl Roth GmbH+ Co. (German); brucine, in crude nux vomica, was claimed to be a morphine-like isostrychnine was obtained from nux vomica based on the pre- analgesic substance, whereas its derivatives brucine N-oxide, in viously described method (Cai et al., 1994). In brief, after sand processed nux vomica, act as a more NSAIDs-like com- extraction from nux vomica, isostrychnine was confirmed by pound (Yin et al., 2003a). comparing the physical and chemical properties such as melting In our previous study, the total alkaloid fractions from nux point and thin layer chromatography with standard compounds vomica were shown to remarkably suppress the HeLa and K562 and 1H NMR spectra. Its purity was more than 99%. The seeds cells growth (Cai et al., 1995, 1998), the results of which pro- of Strychnos nux-vomica L. were collected and identified by vided the possibility that the traditional application of nux vom- professor Bao-Chang Cai and De-Kang Wu from Nanjing Uni- ica in the therapy of liver cancer is also due to the direct cytotoxic versity of Chinese Medicine. A voucher sample (Wu 200052) effects on liver tumor cells elicited by these alkaloids. Therefore, was deposited in the College of Pharmacy, Nanjing University in order to test this hypothesis and identify the alkaloid which of Chinese Medicine. All water soluble compounds were dis- is mainly responsible for the anti-tumor effect of nux vomica solved in RPMI 1640 medium (Gibco, USA), whereas insoluble as well as its mechanisms, we planned to screen the cytotoxic chemicals were dissolved into DMSO, the final concentration effects of strychnine, brucine, isostrychnine and brucine N-oxide of DMSO in each sample were less than 0.1%. Adjustment as described above on HepG2 cells, and choose a representative of the solutions to pH 7.2–7.4 was made with HCl (0.5 M) or compound to further investigate its anti-tumor mechanisms. In NaOH (0.5 M) if necessary. All solutions were passed through these four alkaloids, strychnine and brucine were selected to rep- a 0.22 ␮m filter (GVMP 01230, Millipore) and stored at 4 ◦C resent the alkaloids stemmed from crude nux vomica, whereas until use. isostrychnine and brucine N-oxide stood for the alkaloids from processed nux vomica. The results of this investigation might 2.2. Cell culture provide a scientific explanation for the traditional application of this herbal medicine in liver cancer therapy. Human hepatoma cell lines (HepG2) was purchased from cell bank of Shanghai Institute of Cell Biology (Shanghai, 2. Materials and methods China), maintained in RPMI 1640 culture medium plus 10% calf serum and 100 U/ml penicillin, 75 U/ml streptomycin, in ◦ 2.1. Materials a37 C incubator supplied with 95% room air and 5% CO2. After 60–80% confluency, the cells were trypsinized with 0.25% Strychnine, Hoechst 33258, 3-(4,5-dimethylthiazol-2-yl)- trypsin (AMRESCO, dissolved in PBS, pH 7.4), counted and 2,5-diphenylterazolium bromide (MTT) were purchased from placed down at needed density for treatment. X.-K. Deng et al. / Journal of Ethnopharmacology 106 (2006) 179–186 181 2.3. MTT-colorimetric assay confluent cells were lysed and collected, resolved by a 12% SDS- PAGE gel, the protein was then electrophoretically blotted onto Assessment of in vitro neoplastic activity was determined polyvinylidene fluoride (PVDF) membranes. The membranes based on the previously described method (Fatope et al., 1996). were first hybridized with primary antibodies and then with Briefly, the HepG2 cells in exponential growth were placed down horseradish peroxidase-conjugated anti-mouse or anti-rabbit at a final concentration of 6 × 104 cells/ml in 96-well plate. IgG secondary antibody (Sigma, St. Louis, MO, USA). The After 40% confluency, the cells were treated with strychnine, antibody against the human cyclooxygenase-2 was purchased brucine, brucine N-oxide or isostrychnine at varying concen- from Santa Cruz Biotech (Santa Cruz, CA, USA), antibody trations, 100 ␮l MTT (1 mg/ml) was then added at 24, 48, 72 h against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) after treatments. For MTT assay, the supernatant was discarded were purchased from Upstate Biotechnology (Lake Placid, NY, and 200 ␮l DMSO was added, the 96 wells plate was vibrated USA). The immune complexes were detected using chemilumi- on micro-vibrator for additional 10 min, the optical density of nescence (ECL) system (Amersham Pharmacia Biotech, Buck- each well was measured at λ492nm by Enzyme-immunoassay inghamshire, UK). instrument (Nanjing, China). The growth inhibitory ratio of each alkaloid was calculated based on the following formula: (average O.D. of control group − average O.D. of treated group) Inhibitory ratio of growth (%) = × 100 average O.D. of control group 2.4. Apoptosis analysis by Hoechst 33258 staining 2.7. Measurement of caspase-3-like activity The Hoechst 33258 staining was carried out as previously described (Ramonede and Tomas, 2002). Briefly, the HepG2 The activity of caspase-3-like proteases was mea- cells in exponential growth were placed down at a final con- sured as increases in hydrolysis of fluorogenic tetrapeptide centration of 1 × 105/well in 12-well culture plate.
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