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Ziegler + Müller In vivo Inhibition of S180 Tumors by the SynergisticEffect of the Chinese Medicinal Herbs Coptis chinensisand Evodia rutaecarpa Xiao-na Wang1, Li-na Xu1, Jin-yong Peng1,2, Ke-xin Liu1, Li-hua Zhang2, Yu-kui Zhang2 1 College of Pharmacy, Dalian Medical University, Dalian, P. R. China2 National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian, P. R. China TNF-α levels of mice treated with ZJW were l" Coptis chinensis Franch 50.54 %, 64.91 % and 1.04 ng/mL, respectively, l" Evodia rutaecarpa (Juss.) The aim of the present paper was to investigate much higher than HL and WZY when singly used.
the synergistic effect and mechanism of anti- Furthermore, the activities of acid phosphatase cancer activity of Zuojinwan (ZJW) comprising and alkaline phosphatase were significantly in- Coptis chinensis Franch (HL) and Evodia rutaecar- creased and the activities of creatine kinase, aldo- pa (Juss.) Benth (WZY) at a ratio of 6 : 1 (w/w). In lase and lactate dehydrogenase were reduced in vivo anticancer activity testing was carried out serum, and the expressions of Bax and wild-type by inhibiting the growth of S180 tumor. Tumor P53 proteins were much higher for the mice growth inhibition, spleen index, lymphocyte pro- treated by ZJW compared with HL and WZY sin- liferation, apoptosis, tumor necrosis factor-α gle-treatment groups. A clear synergistic effect (TNF-α) level, activities of serum tumor markers on the anticancer activity was observed with (TMs), increase in life span (ILS), histopathology ZJW, and the mechanism of antitumor growth and gene expression were tested. The results indi- may be due to an effect on gene expression and cated that ZJW could significantly induce apopto- activities of tumor markers in serum.
February 16, 2009 February 26, 2009 sis of cancer cells. The inhibition ratio, ILS and BibliographyDOI 10.1055/s-0029-1185538Planta Med 2009; 75: 1–6 by TCMs, an action that is different to that pro- Georg Thieme Verlag KG duced by synthetic anticancer agents [5].
Stuttgart · New York · The cost of treatment and prevention of cancer is A well-known TCM, Zuojinwan (ZJW), comprises 100 million dollars per year. Many research de- two traditional Chinese medicinal herbs, Coptis partments focus on the explooration of antitumor chinensis Franch (Huanglian HL in Chinese) and drugs for just this reason.
Evodia rutaecarpa (Juss.) Benth (Wuzhuyu WZY College of Pharmacy Many anticancer drugs from the West, which can in Chinese) mixed in the ratio of 6 : 1 (w/w). It Dalian Medical UniversityNo. 9 West Part of Lvshun South rapidly inhibit the growth of tumors, have been was first recorded in Danxi Xinhuo·Liufa, a famous used, but some serious side effects (e.g., impair- ancient medical manuscript, and is listed in the ment of immune system, hepatotoxicity, nephro- Chinese Pharmacopoeia [6]. Coptis chinensis Peopleʼs Republic of ChinaPhone: + 86 4 11 86 11 04 11 toxicity, cardiotoxicity) [1] are often produced.
" Fig. 1) is a buttercup, and its active con- Another type of side effect, multiple-drug resis- stituents are considered to be alkaloids, primarily tance (MDR), [2] can also occur, which can se- palmatine, berberine, epiberberine and coptisine verely limit the effects of anticancer agents [3].
[7]. It has been used for the treatment of inflam- CorrespondenceDr. Lihua Zhang Traditional Chinese medicines (TCMs) have been mation, diabetes, vomiting and diarrhea, as well National Chromatographic widely used to treat cancers for thousands of as for detoxify cations [8]. Another medicinal Research and Analysis Center years in China because they are highly efficacious plant, Evodia rutaecarpa (Juss.) Benth (l Dalian Institute of ChemicalPhysics and have low toxicities. TCMs can also reverse is a dry ripe fruit, and its main active constituents The Chinese Academy of MDR [4] to enhance the anticancer effect of syn- are evodimaine, rutaecarpine, dehydroevo-dia- thetic drugs.
mine and evocarpine [9]. It has been widely used █street? /if necessary)Dalian 116 023 The actions of TCMs are due to the combined ac- for the treatment of gastric mucosal injury, in- Peopleʼs Republic of China tions of many types of chemical compounds in flammation, and diarrhea [10–12].
Phone: + 86 4 11 84 37 97 79 the complex mixture. Thus, a synergistic effect Enhanced apoptosis of human hepatocellular car- Fax: + 86 4 11 84 37 97 may be present in the process of cancer treatment cinoma SMMC-7721 cells caused by the synergis- Wang X-n et al. In vivo Inhibition … Planta Med 2009; 75: 1–6

Ziegler + Müller 6.0 mg raw material/mL, respectively. These solutions were usedfor subsequent experiments.
Animals and cell linesKM mice, weighing 20.0 ± 2.0 g, purchased from the LaboratoryAnimal Center of Dalian Medical University [Dalian, China. Qual-ity certificated Number: SCXK (Liao) 2004–0017], were randomlydivided into five groups (n = 14). The animals were housed fiveper plastic cage with wood chip bedding in an animal room witha 12 h light and 12 h dark cycle at room temperature (24 ± 2 °C)and allowed free access to standard laboratory diet (purchasedfrom the Laboratory Animal Center of Dalian Medical University,Dalian, China).
Photographs of the medicinal herbs Coptis chinensis Franch and S180 tumor cells were obtained from the Histology and Embryol- Evodia rutaecarpa (Juss.) Benth.
ogy Department of Dalian Medical University (Dalian, China), and0.2 mL of the cells (2 × 105 cells/mL) were implanted (s. c.) in theright hind groin of the mice on the eighth day.
The animal experiment was approved by the School of Animal tic effect of berberine and evodiamine was noted in our previous Science of Shenyang Agricultural College.
study [13]. The research has been carried out at the cellular leveland in vitro. Berberine originates from HL and evodiamine from Measurement of body weight, tumors and spleens WZY, but they cannot demonstrate all the actions of the medici- After implantation for 24 h, 0.2 mL of HL, WZY and ZJW, sample nal herbs of HL and WZY [14, 15]. It is also not clear if anticancer solutions were orally administered individually for 10 days ac- activity and the synergistic effect will be produced by the com- cording to the body weight of the animal and the references [16, bined use of HL and WZY in vivo, so these factors merit further 17], and the S180 model group was fed 0.2 mL of sodium chloride solution (0.9 %, w/v). The positive drug CTX was dissolved in The objective of the present study was to investigate whether the water to afford a concentration of 20 mg/kg, and was adminis- anticancer effects of the TCM Zuojinwan (ZJW) are due to syner- tered (i. p.) on alternate days for 10 days. Mice were killed on the gistic effects of its components Coptis chinensis Franch (HL) and eleventh day; tumors and spleens were isolated and weighed.
Evodia rutei█ae?carpa (Juss) Benth (WZY) and also to determine The inhibition ratio of tumor growth was calculated according the mechanisms by which the anticancer actions are mediated.
to the following formula: Body weight, tumor growth inhibition, spleen index, lymphocyteproliferation, apoptosis, tumor necrosis factor-α (TNF-α) level, in- inhibition ratio (%) = [(C – T)/C] × 100 crease in life span (ILS), histopathology, activities of serum tumormarkers (TMs), and expression of Bax and P53 genes were tested where C denotes the mean weight of S180 tumor in the model and discussed. Our data indicated that ZJW is more powerful than group, and T is the mean weight of S180 tumor in the experimen- Coptis chinensis Franch or Evodia rutaecarpa (Juss.) Benth used tal groups. The spleen index was also calculated.
singly in the growth inhibition of S180 tumor in vivo, and a syn-ergistic anticancer effect between the two herbs was clearly present. This is the first report of the synergistic effect of antitu- Mice were inoculated with 0.2 mL of a suspension of S180 cells.
mor actions due to the combined use of HL and WZY.
They were treated with HL, WZY, and ZJW, individually with thedoses of the samples described above after incubation for 24 h.
After 10 days, drugs were stopped and the survival time of each Materials and Methods animal was recorded. ILS was calculated according to following Plant materialCyclophosphamide (CTX) with a purity of 99 % was purchased ILS (%) = (A/B − 1) × 100 from Jiangsu Hengrui Medicine Company Limited (Jiangsu, Chi-na). HL and WZY were purchased from Yunnan Qiancaoyuan where A is the mean survival time of the mice in the test groups, Pharmaceuticals Limited (Yunnan, China). The voucher speci- and B is the mean survival time of the animals in the model mens (DLMU-HL0701 and DLMU-WZY0701), identified by Dr.
Yunpeng Diao, College of Pharmacy, Dalian Medical University,are deposited in the Herbarium of the School of Pharmacy, Dalian Lymphocyte proliferation assay Medical University, Dalian, China. 21 g ZJW (HL 18 g + 3 g WZY), Mice spleens were extirpated on a very clean bench. Under germ- 18 g HL and 3 g WZY were individually weighed and extracted free conditions, spleens were cut into pieces and sieved through a by reflux with 210, 180 and 30 mL water (solvent-sample = 10 : 1, 200-gauge mesh. 2.0 mL of cell-suspension were used and 1.0 mL v/w) for 2 h in a round-bottomed flask (500 mL) equipped with a of a separated medium of lymphocyte cells was slowly added.
reflux condenser, respectively. The extraction solvent was fil- The mixture was centrifuged for 20 min. The suspension was tered and evaporated to dryness under reduced pressure at 50 ° washed twice with phosphate-buffered saline (PBS). Spleen cells C. Light-yellow powders were obtained and redissolved in water were resuspended in RPMI1640 containing 10% bovine serum al- to afford the sample solutions at the concentrations of 42, 36 and bumin (Giboc, USA) and seeded into 96-well flat-bottomed plates(about 1 × 105 cells/mL) in concanavalin A (ConA; 10 µg/mL). Cells Wang X-n et al. In vivo Inhibition … Planta Med 2009; 75: 1–6 Ziegler + Müller Effect of ILS and inhibition in S180-bearing mice treated with ZJW, HL and WZY (means ± S. D.).
a ILS (%) = (A/B − 1) × 100, in which A is the mean survival time of the S180-bearingmice in test groups; B is the mean survival time of the S180-bearing mice in model group. b Inhibition (%) = [(C – T)/C] × 100, in which C is the mean body weight of S180-bearing mice in model group, and T denotes the average body weight of the S180- bearing mice in experimental group. The ILS of the mice treated with ZJW was signifi-cantly higher than HL and WZY single-use, *p < 0.05. Inhibition of tumor growth inZJW treated group was significantly higher than that in the HL-, WZY‑and CTX- treatedgroups, ** p < 0.001 Effect of drug treatment on body weight, spleen index, tumor weight, and survival time in S180-transplanted tumor mice.
were incubated in 5% CO2/air mixture at 37 °C for 44 h. Ten mi-croliters of lium bromide (MTT; Sigma, USA) were added into each well. After another incubation of 4 h, 100 µL of sodium dodecyl sulfate (SDS) S180 tumor tissue was fixed with 10 % buffered neutral formalin, were added. Cells were subjected to MTT cellular viability assay and non-stained sections (4 µm) were cut from the tissue. After according to a method described elsewhere [18], and values at blocking endogenous peroxidases, incubations were carried out the detection wavelength of 570 nm were recorded.
using primary antibodies of Bax and P53 (Santa Cruz Biotechnol-ogy, USA) at 4 °C overnight, and further enhanced by non-biotiny- lated secondary antibodies for 30 min at 37 °C (Zhongshan Glo- S180 tumor cells were obtained by the following method. S180 denbrighdge Biotechnology Company Limited, China). Samples tumors were carefully removed from mice and ground in trypsin were stained with 3,3′-diaminobenzidine (DAB) and counter- at 37 °C. Tumors were cut into pieces through 200-gauge mesh stained with hematoxylin [19]. Positive rates of Bax and P53 ex- and the suspension was collected. Tumor cells were washed with pressions were calculated according to the criteria of an immuno- PBS and centrifuged at 800 rpm for 10 min. The cell suspension histochemistry reaction [20]. Integrated optical density (IOD) (95 µL) was removed and 5 µL acridine orange : ethidium bromide was analyzed by IPP 6.0 software.
(AO : EB; 100 µg/mL:100 µg/mL) mixed liquor were added. A fluo-rescence microscope (B × 51 TF Olympus, Tokyo, Japan) was used Statistical analysis to image the cells after a 15-min reaction in a darkened room.
Statistical analysis was done using the statistical softwareSPSS11.5. Data were expressed as mean ± S. D. Groups were com- pared using Studentʼs rank sum and ANOVA methods. P < 0.05 Serum from transplanted S180 tumor mice on the eleventh day was considered significant.
was collected. The TNF-α concentration was detected using aTNF-α radioimmunoassay (RIA) kit purchased from Dongya Im-munity Technology Institute (China) according to manufacturerʼs instructions. Data were analyzed by a ZC-2010 γ-counter (USTC Chuangxin Company Limited, China).
Inhibition values of the drugs and the ILS of S180 tumor-trans-planted mice are shown in l " Table 1. A significantly enhanced in- Histopathological and morphological observations hibition of tumor growth was produced by ZJW (50.54 %), which Spleens and tumors of mice treated with ZJW, HL and WZY were was much higher than those by HL (25.87 %) and WZY (15.53 %) obtained and put into 10% buffered neutral formalin. They were used singly (p < 0.001). The ILS of mice treated with ZJW, HL and cut into small pieces and stained with hematoxylin and eosin WZY were 64.91 %, 26.31 % and 21.93 %, respectively. The efficient (HE) for histopathological and morphological analyses.
inhibition and prolonged ILS of S180-bearing mice were ob-served when HL and WZY were used together. ZJW could signifi- cantly enlarge the spleen index and body weight compared with Activities of acid phosphatase (ACP), alkaline phosphatase (AKP), the mice treated with CTX (l " Fig. 2).
creatine kinase (CK), fructose 1,6-diphosphate aldolase (ALD) and The immunological function of S180 tumor-transplanted mice lactate dehydrogenase (LDH) in serum of the transplanted S180 was increased in groups treated with HL, WZY and ZJW (l tumor mice were detected using kits purchased from the Najing and higher than that seen in the CTX-treated group. Compared Jiancheng Bioengineering Institute (China) according to the man- with HL and WZY, the ZJW-treated groups produced the best ac- tions (p < 0.001), indicating that an obvious synergistic effect wasproduced when HL and WZY were used together to influencelymphocyte proliferation in S180 tumor-transplanted mice.
Wang X-n et al. In vivo Inhibition … Planta Med 2009; 75: 1–6

Ziegler + Müller The normal cells emitted a green fluorescence (l " Fig. 4 A). The or- ange apoptotic cells of the group treated with ZJW (l treatment on lympho- were significantly increased compared with HL (l " Fig. 4 D) and cyte proliferation in " Fig. 4 E) as shown by AO : EB staining.
S180-transplanted tu- The effects of ZJW, HL and WZY on TNF-α levels in the serum of S180 tumor-transplanted mice were determined by RIA(l " Fig. 5). The TNF-α concentration was 1.04 ng/mL in the serum of S180-bearing mice treated with ZJW, which was significantlyhigher than those of the HL (0.66 ng/mL)- and WZY (0.56 ng/mL)-treated groups (p < 0.001).
The histopathology of S180 tumors was analyzed. Tumor cells ofthe S180 model group were not only significantly enlarged, butalso had many irregular karyons and polykaryocytes. The mor-phology of tumor cells treated with ZJW was more regular, andkaryon pyknosis was more significant than in the CTX-treatedgroup. HL and WZY had weaker effects on the necrosis of tumorcells than ZJW.
The effects on the spleen in S180-bearing mice were also investi-gated. The white pulp of the spleen was markedly more de-creased in the CTX-treated group than in the S180 model group.
The white pulp in ZJW-treated group was conspicuously in-creased compared with that in the CTX group, which was alsolarger than those in HL and WZY singly-used groups.
S180 tumor-transplanted animals were treated with or withoutZJW, HL and WZY, and the activities of the tumor markers in se-rum were detected. Activities of ACP and AKP were 126.72 U/100 mL and 67.27 U/100 mL after treatment with ZJW, respec- Effect on cell apoptosis. A S180 model 10×; B CTX 10×; C ZJW tively, which were much higher than after HL (78.05 U/100 mL 10×; D HL 10×; E WZY 10×.
and 40.97 U/100 mL) and WZY (67.66 U/100 mL and 31.92 U/100 mL) singly used (p < 0.001) (l " Fig. 6). Activities of CK (20.65 U/mL), ALD (319.13 U/L) and LDH (1029.04 U/L) in serumof ZJW-treated mice were considerably reduced compared with HL (26.79 U/mL, 439.93 U/L and 1715.45 U/L) and WZY (29.45 U/ α in S180-bearing mice.
mL, 513.28 U/L and 2758.42 U/L) used singly.
To explore the relationship between the antitumor action and thelevel of protein expression, Bax and wild-type P53 protein ex-pression were analyzed by immunohistochemistry (l Fig. 8). A mean score for the IOD of staining was determined,and the percentage of positive cases was detected for each groupusing an individual score of > 0 as a positive result. According toresults in l " Table 2, Bax protein appears to be predominantly lo- calized within the cytoplasm of the tumour cells, and the expres-sion was significantly higher in the ZJW-treated group (meanIOD, 932.66 ± 180.72: positive rate, 90 %) than in the HL-, WZY-and CTX-treated groups (mean IOD, 272.52 ± 73.43, 186.35 ±55.50 and 213.46 ± 64.04; positive rate, 50 %, 32 % and 42.86 %, re-spectively). Expression of wild-type P53 protein was the highest(main IOD, 28.03 ± 91.78; positive rate, 86.67 %) compared withHL-, WZY- and CTX- (main IOD, 219.40 ± 43.58, 268.09 ± 26.38and 367.52 ± 87.70; positive rate, 32%, 35% and 40 %, respective-ly) treated groups.
Discussion!ZJW could produce significant antitumor activity compared with Actions of ACP, AKP, CK, ALD and LDH in the serum of mice in S180 HL and WZY used singly. ZJW could not only inhibit the S180 tu- model and in those treated with ZJW, HL and WZY.
mor growth, but also significantly enhance the ILS of tumor-bear-ing mice.
ConA, a lectin protein originally extracted from the jackbean Can-avalia ensiformis, has been utilized in lymphokine production, and glycoprotein purification [21]. In a study by Das et al. [21], mitogenic assays, characterization of normal and malignant cells, the lymphocyte proliferation in HL-, WZY- and ZJW-treated ani- Wang X-n et al. In vivo Inhibition … Planta Med 2009; 75: 1–6

Ziegler + Müller Bax gene immunoreactivity in the cytoplasm of S180-transplanted P53 gene immunoreactivity in karyon of S180-transplanted tu- tumor. Bax staining for S180 model (A), CTX (B), ZJW (C), HL (D) and WZY mors. P53 staining for S180 control (A), CTX (B), ZJW (C), HL (D) and WZY (E). Magnification × 400.
(E). Magnification × 400.
Group (nbax, np53) Expression of Bax and P53 proteins in S180-bearing micetreated with S180 model (20, 20) (mean ± S. D.).
Positive rates of Bax and P53 proteins in ZJW group were significantly higher than in HL- and WZY-treated groups, ** p < 0.001. Bax proteinexpression in HL group was higher than in WZY group, * p < 0.05 mals was investigated, and the synergistic effect of HL and WZY tosis and acidification of cytolymph [25]. AKP can participate in clearly enhanced lymphocyte proliferation.
the information transfer of cells. Activities of ACP and AKP were The AO : EB assay [22] is used to evaluate the apoptosis of tumor increased significantly, which indicated that ZJW could increase cells. Not only can normal cells be detected, but also early apop- the activities of ACP and AKP to regulate the DNA expression, totic cells, late apoptotic cells and necrotic cells can be distin- phosphorylation and dephosphorylation of related proteins to guished by this method. Early apoptotic cells (densely green/yel- inhibit the growth of tumor cells. ALD is a lyase in glycolysis, low-stained or displaying green-yellow fragments), late apop- and its activity is increased significantly in serum when cancer totic cells (densely cardinal red-stained or displaying cardinal is present. The endocellular enzyme CK [26] can enter the blood red fragments) and cardinal red swollen necrotic cells can be ob- when cells are damaged in cancer. LDH [27], an important oxido- served. Significant apoptosis was induced by ZJW, and apoptotic reductase in glycolysis, can catalyze the reversible production of cells detected by AO : EB staining were markedly increased com- lactic acid. Activities of ALD, CK and LDH were reduced in the se- pared with the other groups.
rum of ZJW-treated mice, which would benefit the inhibition of TNF-α is a cell factor released from activated macrophages that tumor growth.
can be combined with the receptors of tumor cell membranes to The P53 gene family contains wild-type (wtp 53) and mutant- kill tumor cells specifically without injuring healthy cells. It can type (mut p53) genes. The wtp 53 genes are more likely to sup- regulate many genes associated with inflammation, infection press tumor cell expression than the mut P53 genes [28]. Wtp53 and malignancy. TNF-α can inhibit the proliferation of tumor genes are tumor suppressors, and are expressed in karyon genes cells [23] by cytotoxicity and regulate immunomodulation. TNF- when introduced into cells [29]. Expression of high levels of wild- α is an important factor in the detection and study of tumors. The type (but not mutant) P53 can induce cell cycle arrest or apopto- TNF-α concentration in the serum of mice treated by ZJW was sis. Wtp53 protein was expressed at the highest level in ZJW- much higher than in the HL- and WZY-treated groups, and more treated mice compared with the HL and WZY counterparts. P53 tumor cells were killed by ZJW compared with the two herbs used protein also reached a higher level in CTX compared with the control group. Bax, a pro-apoptotic member of the Bcl-2 protein Histopathological and morphological analyses revealed that ZJW family, is responsible for apoptotic cell death. It can induce could significantly increase antitumor activity, and promote im- growth arrest, apoptosis and cell senescence. HL could promote mune function by regulation of the white pulp of the spleen, higher Bax protein expression than WZY, and highest expression which was markedly higher than in the HL- and WZY-treated of the protein was produced in the mice treated with ZJW. Ex- pressions of Bax and wild-type P53 proteins could be signifi- Abnormal genes are expressed after protein synthesis within the cantly increased by ZJW compared with HL and WZY used singly, cell and are changed due to cancer. Tumor markers are displayed and there was a synergistic effect in the expression of the two in the form of enzymes in blood, and have an important role in proteins when HL and WZY were used together.
tumor determination [24]. ACP activity directly affects the apop- Wang X-n et al. In vivo Inhibition … Planta Med 2009; 75: 1–6 Ziegler + Müller The anticancer activity produced by ZJW was much better than nents on neutrophils and microglial cells. Eur J Pharmacol 2007; 555: those of HL and WZY singly used, and an obvious synergistic ef- 12 Yu LL, Liao JF, Chen CF. Anti-diarrheal effect of water extract of Evodiae fect was also shown by the combination use of the two herbs.
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BMC Molecular Biology Research articleHin-mediated DNA knotting and recombining promote replicon dysfunction and mutationRichard W Deibler†1,2,3, Jennifer K Mann†2,4, De Witt L Sumners4 and Lynn Zechiedrich*1,2,4 Address: 1Interdepartmental Program in Cell and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030-3411 USA, 2Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030-3411 USA, 3Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115 USA and 4Department of Mathematics, Florida State University, Tallahassee, Florida 32306-4510 USA

Safe design of custom lithium batteries

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