Research Article Zuo Jin Wan, a Traditional Chinese Herbal...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 957078 13 pageshttpdxdoiorg1011552013957078

Research ArticleZuo Jin Wan a Traditional Chinese Herbal FormulaReverses P-gp-Mediated MDR In Vitro and In Vivo

Hua Sui12 Xuan Liu12 Bao-Hui Jin1 Shu-Fang Pan2 Li-Hong Zhou12

Nikitin Alexander Yu3 Jie Wu4 Jian-Feng Cai5 Zhong-Ze Fan2 Hui-Rong Zhu1 and Qi Li12

1 Department of Medical Oncology Shuguang Hospital Shanghai University of Traditional Chinese Medicine Shanghai 201203 China2 Interventional Cancer Institute of Integrative Medicine amp Putuo Hospital Shanghai University ofTraditional Chinese Medicine Shanghai 200062 China

3Department of Biomedical Sciences Cornell University Ithaca NY 14850 USA4Department of Molecular Oncology H Lee Moffitt Cancer Center and Research Institute Tampa FL 33612 USA5Department of Chemistry University of South Florida Tampa FL 33620 USA

Correspondence should be addressed to Hui-Rong Zhu zhu huirong126com and Qi Li lzwfhotmailcom

Received 16 December 2012 Accepted 29 January 2013

Academic Editor Pradeep Visen

Copyright copy 2013 Hua Sui et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Zuo Jin Wan (ZJW) a typical traditional Chinese medicine (TCM) formula has been identified to have anticancer activity inrecent studies In this study we determined the underlying mechanism of ZJW in the reversal effect of multidrug resistance oncolorectal cancer in vitro and in vivo Our results showed that ZJW significantly enhanced the sensitivity of chemotherapeuticdrugs in HCT116L-OHP SGC7901DDP and BelFuMDR cells Moreover combination of chemotherapy with ZJW could reversethe drug resistance of HCT116L-OHP cells increase the sensitivity of HCT116L-OHP cells to L-OHP DDP 5-Fu and MMC invitro and inhibit the tumor growth in the colorectal MDR cancer xenograftmodel ICP-MS results showed that ZJW could increasethe concentration of chemotherapeutic drugs in HCT116L-OHP cells in a dose-dependent manner Furthermore we showed thatZJW could reverse drug resistance of colorectal cancer cells by decreasing P-gp level in vitro and in vivo which has been representedas one of the major mechanisms that contribute to the MDR phenotype Our study has provided the first direct evidence that ZJWplays an important role in reversing multidrug resistance of human colorectal cancer and may be considered as a useful target forcancer therapy

1 Introduction

Multidrug resistance (MDR) a phenomenon that has beenrecognized for several decades remains one of the primaryobstacles to successful cancer therapy [1] It is defined asresistance to one drug accompanied by resistance to otherdrugs that may have different structures and mechanisms ofaction The overexpression of ATP-binding cassette (ABC)transporters on themembrane of cancer cells which generateenergy from ATP hydrolysis to actively extrude a varietyof compounds across the membrane is one of the bestcharacterized mechanisms for this phenomenon [2] As faras we know the ABC transporter-subfamily B member 1(ABCB1MDR1P-glycoprotein P-gp) subfamily C member-12 (ABCC-12 MRP-12) subfamily G member 2 (ABCG2

BCRP) and lung resistance-related protein (LRP) play amajor role in producing MDR in tumor cells

P-glycoprotein (P-gp) a membrane-associated proteintypically found on the plasma membrane has been studiedfor comparatively long time (35 years) and many facts aregathered in this field It has been unequivocally proventhat the lower intracellular drug accumulation and MDRphenotype are associated with the elevated expression ofP-gp which is a large transmembrane glycoprotein withmolecular weight approximately 170 kD [3] Functioning as aldquobarrierrdquo P-gp transport acts are by reduction in intracellularchemical concentration via active efflux against concentra-tion gradients at the plasma membrane

BCRP is known to be located in the canalicular mem-brane of hepatocytes and the apical membrane of kidney

2 Evidence-Based Complementary and Alternative Medicine

proximal tubule cells and has the potential to increasebiliary and urinary elimination of xenobiotics that are BCRPsubstrates [4]

The MRP family is now composed of 9 related ABCtransporters that are able to transport structurally diverselipophilic anions and function as drug efflux pumps [5]MRP1 is a major active transporter of glutathione glucuro-nate and sulfate conjugates and a resistance factor for anthra-cyclines epipodophyllotoxins vinca alkaloids and camp-tothecins [6] MRP-2 (ABCC2) a ubiquitously expressedATP-binding cassette transporter has previously been impli-cated in cellular efflux of cisplatin [7] Interestingly clinicalevidence exists indicating that common ABCC2 polymor-phisms may affect disposition or efficacy of drugs which areknown ABCC2 substrates [8]

Lung resistance protein (LRP) is a member of the vaultproteins involved in MDR LRP has been shown to shuttleanthracyclines out of the nucleus [9] Some conclusive datashowed that the expressions of LRP in patients with gastriccancer without prior chemotherapy are high indicating thatinnate drug resistance may exist in gastric cancer [10]

Traditional Chinesemedicines (TCMs) have been used asmedicines or health supplements in China over thousands ofyears Traditional Chinese prescriptions and formulae whichare based on TCM principles have been also identified tobe an effective anti-cancer drugs in cancer patients suchas breast carcinoma [11] gastric cancer [12] and colorectalcancer [13] Compared with chemotherapeutic drugs whichwere defined as the major drug for a long time traditionalChinese prescriptions and formulae are a therapy to effec-tively control cancer progression improve quality of life andprolong survival times

Zuo Jin Wan (ZJW) a typical TCM formula consists ofthe Rhizoma Coptidis and Fructus Evodiae in the ratio of6 1 (ww) Coptis chinensis Franch the main component ofRhizoma Coptidis has recently been reported to have theeffect of reversing MDR [14 15] Fructus Evodiae has beendemonstrated to induce some cancer cell apoptoses such ashuman melanoma A375-S2 cells [16] human hepatocellularcarcinoma SMMC-7721 cells [17] and Human colorectalcarcinoma COLO-205 cells [18] In addition previous studieshave shown that ZJW can lessen the degree of treatingabdominal pain acid regurgitation nausea and so on andenhance the immune function of patients in the gastric ulcertherapy [19]

Although ZJW herbal formula had been found to havean anti-cancer effect the underlying mechanisms remainunknown In this study our objective was to elucidatethe effect and the molecular mechanism of Chinese herbsformula ZJW herbal formula in human cancer cells both invitro and in vivo

2 Materials and Methods

21 Preparation of the Extracts for ZJW ZJWwas formulatedby Rhizoma Coptidis and Evodia (in a ratio of 6 1) Allthe herbs were purchased from Putuo Hospital herb storeThe mixture (70 g) was extracted twice for 1 h each time byrefluxing in ethanol (1 8 vv)The filtrates were concentrated

and dried in vacuum at 60∘C The concentrated extract wasthen dried by lyophilization to obtain the ZJW extract at ayield of dried powder of 244The extract was stored at 4∘Cand its preparations were standardized regulated and qualitycontrolled according to the guidelines defined by ChineseState Food and Drug Administration (SFDA)

22 Cell Culture and Reagents The human colorectal can-cer HCT116 parental cell gastric cancer SGC7901 cell andhepatic carcinoma Bel7402 parental cell were purchased fromthe Shanghai Cell Collection (Shanghai China) HCT116L-OHP MDR cell lines were established and maintained inour laboratory SGC7901DDP MDR cell line and BelFuMDR cell lines were obtained from Keygen Biotech CoLtd (Nanjing China) The cells were grown in RPMI 1640medium supplemented with 10 (vv) heat-inactivated fetalcalf serum 2mM glutamine 100 unitsmL penicillin and100 120583gmL streptomycin (Invitrogen Carlsbad CA USA)at 37∘C in a 5 CO

2humidified atmosphere HCT116L-

OHP cells were routinely maintained in a medium con-taining 5000 ngmL Oxaliplatin (L-OHP) SGC7901DDPcells were routinely maintained in a medium containing1000 ngmL Diamminedichloroplatinum (DDP) and BelFucells were routinely maintained in a medium containing2000 ngL Mitomycin (MMC) and L-OHP were purchasedfrom Shenzhen Main Luck Pharmaceuticals Co Ltd (Shen-zhen China) DDPwas purchased fromQilu PharmaceuticalCo Ltd (Shandong China) and 5-Fu was purchased fromShanghai XudongHaipu Pharmaceutical Co Ltd (ShanghaiChina) Monoclonal antibodies against ABCB1 ABCC-12BCRP and LRP and dehydrogenase (GAPDH) antibodieswere the products of Cell Signaling Technology (BeverlyMAUSA)

23 Animals and Xenograft Model Male athymic nude mice(NCr-nu) were purchased from Sino-British SIPPRBK labAnimal Co Ltd (Shanghai China license no SCXK 2003-0002) and maintained under specific pathogen-free condi-tions for the studies All animal protocols were approvedby the Institutional Animal Use and Care Committee Allthe experiments and animal care were approved by ShanghaiMedical Experimental Animal Care Commission and inaccordance with the Provision and General Recommen-dation of Chinese Experimental Animals AdministrationLegislation The mice used in these experiments were 8- to12-week old

HCT116L-OHP cells were grown in culture and thendetached by trypsinization washed and resuspended inHanks (HBSS) 02mL HBSS with cells (10 times 106) were sub-cutaneously injected into the athymic nude mice to initiatetumor growth When the tumors were allowed to reach anaverage size of 100mm3 the mice were randomized into 6groups (119899 = 8 per group) Mice in group 1 were administeredwith distilled water daily that served as vehicle control Micein groups 2ndash5 were given oxaliplatin as an intraperitonealinjection every two days and the injection dosage (5mgkg)was according to half of the maximum tolerated dose (MTD)of oxaliplatin as previously described [20] Mice in groups3 4 and 5 received intragastric administration of ZJW at

Evidence-Based Complementary and Alternative Medicine 3

the doses of 10275mgkg 2055mgkg and 4110mgkg Inthe clinical practice of the Chinese herbal medicine ZJWis usually prescribed at a daily dose of 10000mg of herbalmaterials When this human dose was converted into ananimal dose (a person of 60 kg and a conversion factor of1233 between human and mouse) it was equivalent to themiddle dose (2055mgkg) used in this study Mice in group 6only received intragastric administration of ZJW at the dosesof 10275mgkg which used as excluding evodiamine toxicitygroup

The body weight of the animals and the two perpendicu-lar diameters (119860 and 119861) were recorded every 3 days and thetumor volume (119881) was estimated according to the followingformula [21] 119881 = 1205876 times [(119860 + 119861)2]3 The curve of tumorgrowth was drawn according to tumor volume and time ofimplantation 6 mice were sacrificed in each group on 28thday after treatment the other 6 mice in the same group wereobserved for the survival time Tumor tissues were excisedfrom the mice and their weight was measured The time ofsurvival for each group and overall significance were plottedon a Kaplan-Meier survival curve also using GraphPadPrism

24 Immunohistology Analysis Was Carried Out Using Paraf-fin Section Paraffin section was incubated in a blockingsolution (10 donkey serum +5 nonfat dry milk +4 BSA+01 Triton X-100) for 10min and then hydrated sectionswere incubated at 4∘C overnight with anti-P-gp antibodyAfter washing with phosphate-buffered solution (PBS) thesections were incubated with diluted (1 200) biotinylatedsecondary antibody for 30min Subsequently the slides werewashed again in PBS and incubated for 30min with the pre-formed avidin-horseradish peroxidasemacromolecular com-plex Development of peroxidase reaction was achieved byincubation in 001 33-diaminobenzidine tetrahydrochlo-ride (DAB) in PBS containing 001 hydrogen peroxide forapproximately 5min at room temperature Sectionswere thenwashed thoroughly in tap water counterstained in haema-toxylin dehydrated in absolute alcohol cleared in xylene andmounted in synthetic resin for microscopic examination

25 Cell Viability Assays Cell proliferation was determinedusing the cell counting kit-8 (CCK-8) Briefly the cells wereseeded in 96-well plates at 1 times 104 cellswell When thecells reached 60 confluence the medium was removedand replaced with fresh medium containing varying concen-trations of ZJW or its admixture with antitumor drug (L-OHP DDP 5-FU and MMC) and incubated for 48 hoursThe CCK-8 assay was performed after 48 hours After 4hours of incubation with culture medium containing theCCK-8 reagent the absorbance was read at 450 nm usinga microplate enzyme-linked immunosorbent assay reader(Labsystems Dragon Wellscan) Relatively inhibitory rate ofcell growth was calculated according to the formula listedabove 119877 = (1198602 minus 1198601)1198602 times 100 and 119875 = 11986011198602 times 100in which 119877 was relatively inhibitory rate and 119875 was relativelyproliferation ratio of cell growth 1198601 was mean absorbancevalue of transfected cells and 1198602 was mean absorbance valueof untransfected control cells without any drug treatment

All experiments were done with 5 wells per experiment andrepeated at least three times

26 Apoptosis Assay In Vitro The cells were seeded in 6-wellplates (4 times 105well) 12 h later three dose concentrations ofZJW (obtained from the result of ZJW IC

10in CCK-8 assay)

were added Flow cytometry was used to detect apoptosis bydetermining the relative amount of AnnexinV-FITC-positivePI-negative cells as previously described Unstained cellscells stained with Annexin V-FITC alone and cells stainedwith propidium iodide alone were used as controls Singlystained cells were used to adjust electronic compensation onFL1 and FL2 channels

27 ICP-MS Analysis The cells were harvested immediatelyafter the end of drug exposure (L-OHP) washed twice withPBS digestion with 025 trypsin centrifuged at 12000timesgfor 15min at 4∘C and fragmentized for the sample processingUltrafiltrate samples (100mL) were diluted using a GilsonASPECXLi programmed to deliver 18mL of iridium internalstandard (0005mgmL in 1 nitric acid) Samples were thenmixed thoroughly prior to ICP-MS analysis Intracellular L-OHP accumulation was determined by ultrasensitive mul-ticollector inductively coupled plasma mass spectrometry(ICP-MS) as previously described

28 Western Blot Analysis Whole cell lysate for SDS-PAGEand Western blot analysis for P-gp BCRP MRP12 and LRPexpression were prepared as previously reported [1] Thelysate was incubated on ice in immunoprecipitation assaybuffer for 2 h before being homogenized using a mortar andpestle The homogenized sample was centrifuged and thesupernatant was collected and stored at minus80∘C Equal loadingwas confirmed with GAPDH (01 120583gmL) Densitometricanalysis was done using the Scion Imaging software (ScionCorporation) with GAPDH being a control for each sample

29 MDR1 Promoter Activity by Vector Transient Transfectionand Dual Luciferase Assay The cells (2 times 104 cells) wereseeded in each well of 96-well culture plates in 100 120583L RPMI-1640 containing 10 fetal bovine serum and incubated at37∘C for 24 h in a 5 CO

2-humidified atmosphere until

the cells reached 90ndash95 confluence at the time of trans-fection MDR1 promoter recombinant vector pGL3-Basic-MDR1 promoter (08 120583gwell) was mixed with a controlvector (10 ngwell) pRL-SV40 in 25120583L RPMI-1640 withoutfetal bovine serum and antibiotics The solution was mixedwith 05120583L Lipofectamine 2000 reagent diluted in 25 120583LRPMI-1640 without fetal bovine serum and antibiotics andincubated at room temperature for 20min two vectors in50120583L solutions were cotransfected into each group cells afterthe cells were washed twice with RPMI-1640 without fetalbovine serum and antibiotics The cells were incubated at37∘C for 12 h in a 5 CO

2humidified atmosphere After

transfection with plasmids the medium was replaced with100 120583L fresh RPMI-1640 without fetal bovine serum

After incubation overnight the cells were washed with100 120583L PBS and lysed by adding 20 120583L lysis buffer (ShanghaiLairsquoan Biotech Co Ltd Shanghai China) After incubation


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Figure 1 HPLC chromatographs of Rhizoma Coptidis and Evodia

for 15min at room temperature on a rocking bed (200 rpm)the lysate was centrifuged at 15000 g for 5min at 4∘C andthe supernatant was harvested and analyzed using a dual-luciferase assay kit (Shanghai Lairsquoan Biotech Co Ltd Shang-hai China) as previously reported [1]

210 Statistical Analysis All experimental data were expres-sed as mean plusmn standard of at least three independent exper-iments performed in duplicate The statistical analysis wasperformed with the Studentrsquos 119905-test or covariance analysis forstatistical significance Statistical significance was set at a 119875-value of less than 005 All analyses were carried out usingSPSS130

3 Results

31 Quantitative Analysis of Various Active Compounds in theExtracts of ZJW To ensure the quality and stability of ZJWsolution we used high-performance liquid chromatography(HPLC) to identify the components and confirm the finalconcentration of this solution The resultant sample wasanalyzed on an Agilent 1100 HPLC system (Santa Clara CAUSA) using a C-18 column (250mm times 46mm 5120583m) Themobile phase consisted of acetonitrile and water containing

005 formic acid All analyses were performed at a col-umn temperature of 30∘C with mobile phase consisting ofCH3CN ddH

2O (85 15 vv) at a flow-rate of 1mLmin with

an injection volume of 10 120583L The detection wave lengthwas set to monitor at 225 nm As shown in Figure 1(a)quantitative results of jatrorrhizine palmatine and berberinein RhizomaCoptidis were 38 64 and 571 respectivelyAs shown in Figure 1(c) quantitative results of evodiamineand rutaecarpine in Evodia were 229 and 318 respec-tively Various components in ZJW extracts were determinedby referring to the calibration curve established by therunning standard at varying concentrations under the sameconditions (Figures 1(b) and 1(d))

32 Noncytotoxic Dose of ZJW Previous work from this labhas shown that ZJW is a potent drug with the effect of anti-cancer in solid tumor cells such as human colon carcinomaand pancreatic cancer cells [22] In order to avoid the possi-bility that inhibition of cell proliferation is due to cytotoxicitywe first examined the cytotoxic effect of ZJW on differentcancer cell lines including colorectal cancer cell (HCT116L-OHP) gastric cancer cell carcinoma (SGC7901DDP) andhepatic carcinoma cell (BelFu) According to the permittedstandardization of the noncytotoxic dose in our study data


showed that the dosage of IC10

in HCT116L-OHP cell was50120583gmL 54120583gmL in SGC7901DDP cells and 60 120583gmLin BelFu cells (Figure 2(a)) Less than these dosages cellsurvival was not found to be significantly different fromuntreated cells (100 survival) Therefore for all cell prolif-eration experiments the cells were treated with ZJW in theconcentration range of 50120583gmL which is the lowest dosageof the IC

10in the three cell lines

33 Effect of ZJW on the Sensitivity to ChemotherapeuticDrug in Human Cancer Cell To determine the best effect ofZJW on the sensitivity to chemotherapeutic drug in humancancer cell lines all cell lines were treated for ZJW withtheir own induced chemotherapeutic drug and then analyzedby CCK-8 assay with IC

50of L-OHP DDP and 5-Fu As

shown in Figure 2(b) ZJW displayed significant cytotoxicityin HCT116L-OHP cell with an IC

50value from 15748 plusmn

1673 120583gmL to 7140 plusmn 648 120583gmLwhile the IC50against the

parental cells was 1984 plusmn 142 120583gmL Notably the resistanceeffect of ZJW in SGC7901DDP cells and BelFu cells wasless than that of HCT116L-OHP cell Therefore our resultssuggested that ZJW owned the best effect on synergistictherapy in human colorectal cancer

34 Increasing Effect of ZJW on the Sensitivity to Chemothera-peutic Drug in HCT116L-OHP Cell In order to confirm theeffects of ZJW on proliferation in HCT-116L-OHP cells wetested the inhibition in MDR cells after cells treatment with3 different kinds of chemotherapeutic drugs As the resultsuggested HCT-116L-OHP cells were more resistant to L-OHP DDP 5-Fu andMMC respectively in a dose dependentmanner (Figure 2(c)) Therefore these data further suggestthat ZJW was responsible for increasing the sensitivity tochemotherapy to reverse MDR in HCT-116L-OHP cells

35 ZJW-Induced Apoptosis without Affecting Cell Cycle Dis-tribution of HCT-116L-OHP Cells To further investigate themechanisms underlying the inhibiting proliferation effect ofZJW Annexin V and propidium iodide (PI) double stainingwas performed to see the change of cell apoptotic inducedby L-OHP after treatment with ZJWThe results showed thatZJW did not influence the HCT-116L-OHP cell distributionin normal (both annexin V and PI negative) and earlyapoptosis (annexin V positive and PI negative) but signif-icantly increase the late apoptotic or necrotic populations(both annexin V and PI positive) (Figure 3(a)) To furtherexamine whether the effect of ZJWon cell apoptosis occurredin different time cell apoptosis induced by L-OHP wasdetermined at ZJW treatment for 24 h 48 h and 72 h Asshown in Figure 3(b) exposure to ZJW also increased L-OHP-induced cell apoptosis in a time-dependent mannerHowever the cell cycle analyses obtained from the Flowcytometric analysis showed that there was no change in anyphase arrest in response to treatment with ZJW comparedwith control group (Figure 3(c)) All of these observationssuggest that ZJW did not alter cell cycle in HCT-116L-OHPcells These data suggest that the reversal effect of ZJW wasmost likely obtained by direct induced apoptosis as opposedto later the cell cycle

36 Effect of ZJW on Intracellular L-OHP Accumulation Todetermine whether ZJW causes an increase on the intra-cellular chemotherapeutic drug accumulation in colorectalcancer cell we evaluated the effect of ZJW on concentrationof L-OHP in HCT-116L-OHP by ICP-MS assay As shownin Figure 4 the intracellular L-OHP accumulation in HCT-116L-OHP cells was significantly lower than it was in HCT-116 cells After 48 h treatment of ZJW with different dosageintracellular L-OHP accumulation in HCT-116L-OHP cellswas increased in a concentration-dependent manner As thedata suggested ZJW at a concentration of 25120583gmL had a lit-tle effect of increasing chemotherapeutic drug accumulationbut significantly increased intracellular L-OHP accumulationat the concentration of 100 120583gmL These results showed thatZJW significantly increased chemotherapeutic drug accumu-lation in a dose-dependent manner in HCT-116L-OHP cells

37 Effect of ZJW on the Level of P-gp BCRPMRP12 and LRPIn Vitro To further investigate the mechanisms underlyingthe resistant effect of ZJW on colorectal cancer MDR cellswe determined the effect of ZJW on the levels of the four cellmembrane-bound ATP binding cassette (ABC) transportersin HCT-116L-OHP cells which is P-gp BCRP MRP2 andLRP As shown in Figure 5(a) the incubation of cell with ZJW(25 50 and 100 120583gmL) for 48 h did not significantly alterthe level of MRP2 and LRP however the level of P-gp wassignificantly decreased at a concentration-dependentmannerin HCT116L-OHP cell In addition Western blot analysesresults also indicated that there was no expression of MRP1and BCRP in the MDR cells (data not shown) This suggeststhat the MDR reversal effect of ZJW in HCT116L-OHP cellsmay be dependent of the inhibition on the level of P-gp

38 Effect of ZJW on the Activity of MDR1 Promoter In VitroTo further ascertain whether the effect of ZJW on MDR1P-gp expression occurred at the level of transcription theactivity of MDR1mRNAwas determined by the recombinantvector pGL3-MDR1-promoter which has been transfectedinto HCT116L-OHP cells We observed a downregulationin the expression of MDR1 promoter in HCT116L-OHPcells treatmed with ZJW at 24 h 48 h and 72 h In additionour data clearly show that ZJW could inhibit the activity ofMDR1 promoter in a dose-dependent manner (Figure 5(b))Considered together our findings imply that the reversaleffect of ZJW on MDR1P-gp mediated MDR in HCT116L-OHP cells is by influencing the transcription and translationfromMDR1gene to P-gp expression

39 ZJW Reverses MDR in Nude Mice Xenograft ModelTo explore whether ZJW reverses cancer MDR in vivo weemployed a colorectal MDR cancer xenograft model Thetumor volume and body weight were observed in the sixgroups respectively There was no significant difference intumor size between animals treated with saline or ZJWindicating the anti-cancer effect of ZJW is not obvious in vivoHowever the combination of L-OHP and ZJW produceda significant inhibition of tumor growth compared withanimals treated with saline or L-OHP alone (119875 lt 005)As shown in Figure 6(a) ZJW helped L-OHP inhibit the


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Concentrations of ZJW (120583gmL)

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Concentrations of 5-Fu (120583gmL) Concentrations of MMC (120583gmL)

Concentrations of L-OHP (120583gmL) Concentrations of DDP (120583gmL)

(c)

Figure 2 Effect of ZJW on the sensitivity to chemotherapeutic drug in human cancer cells (a) The cells were treated with variousconcentrations of ZJW and analysis by CCK-8 analyses (b) Antiproliferative IC

50

values of L-OHP DDP and 5-Fu on HCT116L-OHPSGC7901DDP and BelFu cells were analyzed by CCK-8 analyses Data are presented as mean plusmn SD of triplicate experiments lowast119875 lt005

lowastlowast

119875 lt 001119875 lt 005 and

119875 lt 001 versus control (c) CCK-8 assay was used to detect cell inhibition of L-OHP DDP 5-Fuand MMC in HCT116L-OHP cells treated with ZJW at 50 120583gmL for 48 h


Annexin-V FITC100101102103104



046 237 664

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Figure 3 Effect of ZJWon apoptosis and cell cycle inHCT116L-OHP cells (a) Flow cytometry analysis of apoptosis withAnnexinV-FITCPIbinding to HCT116L-OHP cells Viable cells (Annexin VminusPIminus) are located in the lower left apoptotic cells (Annexin V+PIminus) in the lowerright postapoptotic secondary necrotic cells (Annexin V+PI+) in the upper right and primary necrotic cells (Annexin VminusPI+) in the upperleft quadrants respectively Numbers in each quadrant are percentages of cells L-OHP group means HCT116L-OHP cells exposure of L-OHP for 24 h L-OHP + ZJW group means HCT116L-OHP cells exposure of L-OHP and ZJW (50120583gmL) for 12 h and HCT116L-OHP cellswithout any treatment as the control (b) Flow cytometry analysis of apoptosis with Annexin V-FITCPI binding to HCT116L-OHP cellsafter treatment with ZJW at 24 h 36 h and 48 h (c) Flow cytometry analysis of cell cycle distribution in three groups which described as (a)


mice tumor volume at a concentration-dependent mannerFurthermore we also measured the difference in survivaltimes among these groups The control group began to die at52 days and all succumbed to disease by 55 days (Figure 6(b))In comparison the first mouse from the H-ZJW + L-OHPgroup died in 74 days and the last two in the group diedafter 77 days The results suggest a significant increase in thesurvival time (119875 lt 001) with synergy effect of ZJW althoughthere were no animals that ultimately survived the disease

310 ZJW Reverses P-gp-Mediated MDR In Vivo To confirmwhether the synergy effect of ZJW on xenograft tumorgrowth was mediated by decreasing the level of P-gp weinvestigated the expression level of P-gp in xenograft tumorof mice treated with combination of L-OHP and ZJW byimmunohistology analysis As shown in Figure 7(a) the levelof P-gp is decreased in xenograft tumor of mice treated withZJW and L-OHP compared with that in control group

Real-time qPCR was also used to investigate the mRNAexpression of MDR1 which is P-gp target gene in xenografttumor treated with ZJW and L-OHP Following the treatmentof ZJW there was a significant decline of the expression ofMDR1 mRNA in ZJW group compared with that in controlgroupMoreover the significant decline ofMDR1mRNAwasobserved in the combination of L-OHP and ZJW than thatin L-OHP alone (Figure 7(b)) Therefore the results in vivofurther confirmed that the anti-MDReffect of ZJWwas partlymediated by decreasing the level of MDR1P-gp

4 Discussion

Over a period of time MDR is a critical problem thatcontinues to hamper the success of modern chemotherapyagainst cancer [23] It is a complicated multifaceted resultand it is mediated by a series of integral membrane proteinsincluding ABCB1P-gp ABCC-12 ABCG2BCRP and LRPTo reverse chemotherapeutic drugs-mediated MDR numer-ous studies have attempted to develop some more effectivechemotherapeutic drugs [24ndash26] However the tolerance ofchemotherapeutic drugs still exists even modern medicinecontinues to develop and create in some studies Moreoveralthough many clinical trials have been conducted for somespecific targets most results have been disappointing andthe toxicity of these modern medicine themselves is oneimportant factor that led to the failure of these studies [27]

Since it has been a critical problem of chemotherapy withpoor effect in the treatment of cancer the development ofanti-MDR agents has become a major focus on overcomingcancer drug resistance Traditional Chinese prescriptions andformulae as a major constituent of several herbal productsrepresent an ideal compound for reversing MDR due to itslow toxicity Previous studies have confirmed that ZJW haspotent anti-cancer and synergistic effects by inhibiting thegrowth of S180 tumor in vivo [28] Recent findings havefound that berberine and coptisine which are the majoractive constituents of Coptis were found to reverse ABCB1-mediated MDR in human MDR cancer cells [14 15]

In order to elucidate its anti-cancer molecular mecha-nisms the present research focused on the effects of ZJW

0

100

200

300

400HCT116

HCT116L-OHP

Intr

acel

lula

r con

cent

ratio

n of

L-O

HP

ZJW (120583gmL)25 50 100

lowast

lowast

lowast

mdash mdash

(ng5times105

cells

)

Figure 4 Effect of ZJW on intracellular L-OHP accumulation Avalidated ICP-MS method was used to detect intracellular L-OHPaccumulation in HCT116 and MDR HCT116L-OHP cells whichwere treated with ZJW at 25120583gmL 50 120583gmL and 100120583gmL for48 h meanwhile HCT116 cells were taken as control groupThe dataare representative of at least three experiments which are presentedas the mean plusmn SD lowast119875 lt 005 ZJW (25120583gmL 50 120583gmL and100120583gmL) versus ZJW (0 120583gmL)

ethanol extracts in reversing MDR In our present studythe indicator components of ZJW extract including Rhi-zoma Coptidis and Fructus Evodiae have been detected byHPLCESI-MS analysis To investigate the anti-MDR effectsof ZJW on human cancer HCT116L-OHP SGC7901DDPand BelFu cells growing exponentially were treated withZJW (0ndash600120583gmL) and the IC

10of ZJW was measured by

CCK-8 In the three MDR cell lines the survival at the IC10

was not found to be significantly different from untreatedcells and it permitted standardization of the noncytotoxicdose These results suggest that concentrations of ZJW below50120583gmL are not toxic to the three MDR cells Thereforefor all cell proliferation experiments the cells were traded byZJW in the concentration range of 0ndash50120583gmL The MDRcells used in cell proliferation experiments at a noncytotoxicdose are similar to those reported previously for U251 cancercells which were treatmented with a noncytotoxic dose [29]Furthermore CCK-8 analyses were performed in HCT116L-OHP SGC7901DDP and BelFu cells to determine their rela-tive ability in increasing concentrations of the chemotherapydrugs L-OHP DDP and 5-Fu Data showed that HCT-116L-OHP cells are much more resistant to death induced by L-OHP after ZJW treatment when compared with the othercells Because colorectal cancer cell displayed the maximumdegree of downregulation in IC

50of chemotherapeutic drugs

we used colorectal cancer for the next experiment assaysIn addition ZJW significantly increased the cellular toxicityof L-OHP DDP 5-Fu and MMC in HCT-116L-OHP cellsand greatly enhanced the inhibition rate of chemotherapeuticagents in a dose-dependent manner


HCT116L-OHPHCT116 25 50 100 ZJW

P-gp

LRP

MRP2

GAPDH

170 kD

95 kD

190 kD

36 kD

mdash

(a)

0

02

04

06

08

1

Fold

chan

ge o

f MD

R1 p

rom

oter

activ

ity

Control

24 h 48 h 72 h

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

ZJW (50 120583gmL)ZJW (25 120583gmL) ZJW (100 120583gmL)

(b)

Figure 5 Inhibition effect of ZJW on the ABCB1 gene and its encoded P-gp in vitro (a) Western blotting assay was carried out to detect thelevel of P-gp LRP and MRP2 in HCT116 cells HCT116L-OHP cells and HCT116L-OHP cells treated with ZJW for at 25 120583gmL 50120583gmLand 100120583gmL for 48 h respectively Western blotting with an antibody to GAPDH was used to ensure equal loading of proteins in eachlane The bolts were photographed and quantitated for each sample the data are from three independent experiments (b) The activity ofMDR1 promoter in HCT116L-OHP cells treatment with ZJW at 24 h 48 h and 72 h was analyzed by dual-luciferase assay kit The resulteddata is firefly luciferaserenilla luciferase from different groups Data are means plusmn standard deviation of values from triplicate experimentslowast

119875 lt 005 lowastlowast119875 lt 001 represents that ZJW treatment group is significantly different from control group at 24 h 48 h and 72 h

In our study we found ZJW had the synergistic effectin chemotherapeutic drugs induced-cell apoptosis in a time-dependent manner as several previous reports also haveshown the synergy effect of traditional Chinese prescriptionsand formulae [30 31] However we did not observe obviousalterations in any cell cycle arrest of HCT-116L-OHP cellsafter treatment with the formula One possible explanationis that there might be interaction between different herbconstituents of this formula which could lead to complicatedeffects Another factor is that ZJW could not alter L-OHP-induced cell cycle arrest since it is considered L-OHP as anoncycle specific antineoplastic agents These findings are inaccordance with some reports [32 33] which show oppo-site roles for some drugs in tumor development and causeapoptosis but not cell cycle alterations

To elaborate the reversal ability of ZJW we investi-gated the effect of ZJW on the accumulation of L-OHP inHCT116L-OHP cells Based on these data by ICP-MS analy-sis we have shown that ZJW remarkably enhanced the intra-cellular accumulation of L-OHP in a dose-dependent man-nerThese results were in accordancewith those of the CCK-8assay which altogether proved that ZJW was able to increasethe sensitivity of MDR cells to chemotherapeutic agents

Other studies showed that targeted agents or inhibitorsmodulators of efflux transporters could inhibit energy-dependent efflux of protein on the cell membrane andcause transport mechanism of membrane protein in humanMDR cancer cells [34] Earlier work from this laboratorydemonstrated a correlative relationship between signal

transduction pathway andP-gp in the colorectalMDRcell [1]Herewe have extended these studies using three humanMDRcell lines and detected the effect of ZJW on the expression ofABCB1P-gp ABCC-12 ABCG2BCRP and LRP We foundthat ABCB1P-gp ABCC-2 and LRP were more upregulatedin HCT116L-OHP than in HCT116 cells Further analysisrevealed that ZJW-reverse MDR was accompanied by thedownregulation of P-gp but not through ABCC-2 andLRP-mediated MDR pathway It suggests that ZJW-reversedMDR may be dependent on the inhibition of ABCB1P-gpOur data support the suggestion by Chao et al about the linkbetween ZJW and TPA-induced AP-1 activities by alteringanchorage-independent cellular growth in a concentration-dependent manner [35] Because it is known now that thereare AP-1 binding sites on the promoters of ABCB1 blockage oftumor promoter-induced AP-1 activation inhibits neoplastictransformation Therefore the reversal effect of ZJW may beregulated through the inhibition of AP-1 mediated P-gp

In light of our in vitro data on the effect of ZJW in humancolorectal MDR cancer cells to chemotherapeutic drugs weexamined the in vivo therapeutic potential of ZJW Indeedanimal experiments results showed that the anticancer effectof ZJW on resistant cancer cells xenograft is better than thatof L-OHP control group In this study we provided evidencethat combination of chemotherapy with herbal medicineformula ZJWprolonged the overall survival time of xenograftmodel And these results demonstrated that ZJW exhibited agood downregulation on the expression of ABCB1P-gp bothin vitro and in vivo


10 12 14 16 18 20 22 24 26 280

200

400

600

800

1000

1200

1400

1600

1800

2000

Time (days)0 2 4 6 8

Tum

or v

olum

e (m

m3)

ControlZJWL-OHP

L-ZJW + L-OHPM-ZJW + L-OHPH-ZJW + L-OHP

lowast

lowast

◼

◼◼

(a)

Control

ZJW

L-OHP

L-ZJW + L-OHP

M-ZJW + L-OHP

H-ZJW + L-OHP

(b)

40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80Survival time (days)

Prob

abili

ty o

f sur

viva

l

0

02

04

06

08

12

1

H-ZJW + L-OHPM-ZJW + L-OHPL-ZJW + L-OHP

L-OHPZJWControl

(c)

Figure 6 Inhibition effect of ZJW in vivo (a) Tumor volume change was determined every two days during delivery period Values aremean weight of nude mice plusmn SD Statistical difference was analyzed by Studentrsquos 119905-test lowast119875 lt 005 represents that L-OHP (5mgkg) treatmentgroup is significantly different from control group ◼119875 lt 005 represents that L-ZJW (10275mgkg) + L-OHP (5mgkg) treatment group issignificantly different from control group 119875 lt 005 represents thatM-ZJW(2055mgkg) + L-OHP (5mgkg) treatment group is significantlydifferent from control group 998771119875 lt 005 represents that H-ZJW(4110mgkg) + L-OHP (5mgkg) treatment group is significantly differentfrom control group (b) Tumors removed from nude mice and photographed on the 28th day after administration (c) Overall survival ofxenograft model after injection with HCT116L-OHP cells

In conclusion our data strongly imply that ZJW inhibitedP-gp-mediated MDR both in vitro and in vivo First ZJWreversed MDR via increasing the sensitivity of MDR cellsto chemotherapeutic agents Second ZJW reversed MDRthrough down-regulation of P-gp in vitro and in vivo Andthird combination of chemotherapy with ZJWprolonged theoverall survival time of xenograft model and reduced thetumor volumeThus this study has provided a natural potentinhibitor of humanMDR Compared withmodernmedicine

combination of therapeutic principles represented by multi-ple herbs may yield better results in cancer treatment ZJW atraditional Chinese herbal medicine has been demonstratedto have implications for the rational development of novelregimens in human MDR

Authorsrsquo Contribution

H Sui X Liu and B-H Jin contributed equally to this work


Control H-ZJW L-OHP

L-ZJW + L-OHP M-ZJW + L-OHP H-ZJW + L-OHP

(a)

0

02

04

06

08

1

12

Fold

chan

ge

MDR1 mRNAP-gp

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

ControlH-ZJWL-OHP


998779998779

998779998779

998779998779 998779998779

998779998779

998779998779

(b)

Figure 7 Inhibition effect of ZJW on the level of P-gp and the expression of MDR1 mRNA in vivo (a) The xenograft tumor tissue in mice ofall groups was subjected to immunohistology analysis using P-gp antibody the results were magnified 200 in microscope positive cells werebrown staining negative cells were blue staining (b) The xenograft tumor tissues in mice of all groups were subjected to real-time qPCR todetermine the mRNA expression of MDR1 Quantification of P-gp and MDR1 mRNA was performed by assigning a value of 1 to the grouptreated with H-ZJW (4110mgkg) + L-OHP (5mgkg) Statistical difference was analyzed by Studentrsquos 119905-test lowastlowast119875 lt 001 versus control grouplowastlowast

119875 lt 001 versus L-OHP alone group This is a representative result of three repetitive experiments with similar results

Acknowledgments

This work was funded and supported by the National Nat-ural Science Foundation of China (no 81202812) Scienceand Technology Commission of Shanghai Municipality (no10ZR1427400) Program of Shanghai Municipal EducationCommission (nos 09YZ132 2011JW57 12YZ058) and Shang-hai Municipal Health Bureau (nos 2011ZJ030 20114Y0132010QL050B 20114Y001)

References

[1] H Sui S Zhou Y Wang et al ldquoCOX-2 contributes to P-glyco-protein-mediated multidrug resistance via phosphorylation ofc-Jun at Ser6373 in colorectal cancerrdquo Carcinogenesis vol 32no 5 pp 667ndash675 2011

[2] H Sui Z Z Fan and Q Li ldquoSignal transduction pathways andtranscriptional mechanisms of ABCB1Pgp-mediated multipledrug resistance in human cancer cellsrdquo Journal of International


Medical Research vol 40 no 2 pp 426ndash435 2012[3] H Sui L Zhou P Yin et al ldquoJNK signal transduction path-

way regulates MDR1 p-glycoprotein-mediated multi drug re-sistance in colon carcinoma cellsrdquo World Chinese Journal ofDigestology vol 19 no 9 pp 892ndash898 2011

[4] G An F Wu and M E Morris ldquo57-Dimethoxyflavone andmultiple flavonoids in combination alter the ABCG2-mediatedtissue distribution of mitoxantrone in micerdquo PharmaceuticalResearch vol 28 no 5 pp 1090ndash1099 2011

[5] A J Slot S VMolinski and S P Cole ldquoMammalianmultidrug-resistance proteins (MRPs)rdquo Essays in Biochemistry vol 50 no1 pp 179ndash207 2011

[6] S M He R Li J R Kanwar and S F Zhou ldquoStructural andfunctional properties of human multidrug resistance protein 1(MRP1ABCC1)rdquoCurrentMedicinal Chemistry vol 18 no 3 pp439ndash481 2011

[7] K Taniguchi M Wada K Kohno et al ldquoA human canalicularmultispecific organic anion transporter (cMOAT) gene is over-expressed in cisplatin-resistant human cancer cell lines withdecreased drug accumulationrdquo Cancer Research vol 56 no 18pp 4124ndash4129 1996

[8] I Cascorbi and S Haenisch ldquoPharmacogenetics of ATP-bind-ing cassette transporters and clinical implicationsrdquo Methods inMolecular Biology vol 596 pp 95ndash121 2010

[9] A Saglam M Hayran and A H Uner ldquoImmunohistochemicalexpression of multidrug resistance proteins in mature TNK-cell lymphomasrdquo APMIS vol 116 no 9 pp 791ndash800 2008

[10] W Q Hu C W Peng and Y Li ldquoThe expression and signifi-cance of P-glycoprotein lung resistance protein and multidrugresistance-associated protein in gastric cancerrdquo Journal ofExperimental amp Clinical Cancer Research vol 28 p 144 2009

[11] W Li S Chan D Guo M Chung T Leung and P H YuldquoWater extract of Rheum officinale Baill induces apoptosis inhuman lung adenocarcinoma A549 and human breast cancerMCF-7 cell linesrdquo Journal of Ethnopharmacology vol 124 no 2pp 251ndash256 2009

[12] A Rasul B Yu L Yang et al ldquoInduction of mitochondria-med-iated apoptosis in human gastric adenocarcinoma SGC-7901cells by kuraridin and nor-kurarinone isolated from sophoraflavescensrdquo Asian Pacific Journal of Cancer Prevention vol 12no 10 pp 2499ndash2504 2011

[13] Q Li H Sui X Liu J M Qin P H Yin and Z Z Fan ldquoRever-sal effect of Jianpi Jiedu Recioe on JNKSAPK signal transduc-tion pathway-mediated multidrug resistance in human coloncarcinoma cellsrdquo CJTCMP vol 27 no 3 pp 731ndash735 2012

[14] Y DMinM C Yang KH Lee K R Kim S U Choi andK RLee ldquoProtoberberine alkaloids and their reversal activity of P-gp expressed multidrug resistance (MDR) from the rhizome ofCoptis japonica Makinordquo Archives of Pharmacal Research vol29 no 9 pp 757ndash761 2006

[15] C He R Rong J Liu J Wan K Zhou and J X Kang ldquoEffectsof Coptis extract combined with chemotherapeutic agents onROS production multidrug resistance and cell growth in A549human lung cancer cellsrdquo Chinese Medicine vol 7 no 1 article11 2012

[16] J Yang L Wu S Tashino S Onodera and T Ikejima ldquoCriticalroles of reactive oxygen species in mitochondrial permeabilitytransition inmediating evodiamine-induced humanmelanomaA375-S2 cell apoptosisrdquo Free Radical Research vol 41 no 10 pp1099ndash1108 2007

[17] X N Wang X Han L N Xu et al ldquoEnhancement of apoptosisof human hepatocellular carcinoma SMMC-7721 cells through

synergy of berberine and evodiaminerdquo Phytomedicine vol 15no 12 pp 1062ndash1068 2008

[18] Z G Yang A Q Chen and B Liu ldquoAntiproliferation and apop-tosis induced by evodiamine in human colorectal carcinomacells (COLO-205)rdquo Chemistry amp Biodiversity vol 6 no 6 pp924ndash933 2009

[19] F R Zhao H P Mao H Zhang et al ldquoAntagonistic effects oftwo herbs in Zuojin Wan a traditional Chinese medicine for-mula on catecholamine secretion in bovine adrenal medullarycellsrdquo Phytomedicine vol 17 no 8-9 pp 659ndash668 2010

[20] K Kolinsky B Shen Y Zhang et al ldquoIn vivo activity of novelcapecitabine regimens alone and with bevacizumab and oxali-platin in colorectal cancer xenograft modelsrdquoMolecular CancerTherapeutics vol 8 no 1 pp 75ndash82 2009

[21] Y Mi Y Liang H Huang et al ldquoApatinib (YN968D1) reversesmultidrug resistance by inhibiting the efflux function of mul-tiple ATP-binding cassette transportersrdquo Cancer Research vol70 no 20 pp 7981ndash7991 2010

[22] Q F Tang X Liu Y Ge et al ldquoExperimental study on inhibitingproliferation and inducing apoptosis of zuo jin wan alcoholextracts on human gastric cancer cells infected by Helicobacterpylorirdquo Chongqing Medicine vol 41 no 15 pp 1462ndash1464 2012

[23] H Sui L H Zhou X Liu et al ldquoCOX-2 regulate MDR1P-glycoprotein-mediated multidrug resistance in colon carci-noma cellsrdquo China Oncology vol 21 no 4 pp 241ndash246 2011

[24] H YangM Hua H Liu et al ldquoAn epirubicin-conjugated nano-carrier with MRI function to overcome lethal multidrug-resist-ant bladder cancerrdquo Biomaterials vol 33 no 15 pp 3919ndash39302012

[25] F Wang Y J Mi X G Chen et al ldquoAxitinib targeted cancerstemlike cells to enhance efficacy of chemotherapeutic drugs viainhibiting the drug transport function of ABCG2rdquo MolecularMedicine vol 18 no 1 pp 887ndash898 2012

[26] K J Liu J H He X D Su et al ldquoSaracatinib (AZD0530) is apotent modulator of ABCB1-mediated multidrug resistance invitro and in vivordquo Journal of Cancer vol 132 no 1 pp 224ndash2352013

[27] H Sui B H Jin P H Yin Z Z Fan and Q Li ldquoReversal effectof Jianpi Jiedu Recioe on multidrug resistance in human coloncarcinoma cellsrdquo Chinese Journal of Experimental TraditionalMedical Formulae vol 18 no 9 pp 181ndash185 2012

[28] XWang L Xu J Peng K Liu L Zhang and Y Zhang ldquoIn vivoinhibition of s180 tumors by the synergistic effect of the chinesemedicinal herbs coptis chinensis and evodia rutaecarpardquo PlantaMedica vol 75 no 11 pp 1215ndash1220 2009

[29] L J Ostruszka andD S Shewach ldquoThe role of cell cycle progres-sion in radiosensitization by 2101584021015840-difluoro-21015840-deoxycytidinerdquoCancer Research vol 60 no 21 pp 6080ndash6088 2000

[30] J Gao T He Y Li and YWang ldquoA traditional chinesemedicineformulation consisting of Rhizoma corydalis and Rhizoma cur-cumae exerts synergistic anti-tumor activityrdquoOncology Reportsvol 22 no 5 pp 1077ndash1083 2009

[31] Y Wang Z Yang and X Zhao ldquoHonokiol induces paraptosisand apoptosis and exhibits schedule-dependent synergy incombinationwith imatinib in human leukemia cellsrdquoToxicologyMechanisms and Methods vol 20 no 5 pp 234ndash241 2010

[32] Z Chen M Ingouff V Sundaresan and F Berger ldquoChromatinassembly factor 1 regulates the cell cycle but not cell fate duringmale gametogenesis in Arabidopsis thalianardquoDevelopment vol135 no 1 pp 65ndash73 2008


[33] V S Romanov M V Abramova S B Svetlikova et al ldquop21Waf1is required for cellular senescence but not for cell cycle arrestinduced by the HDAC inhibitor sodium butyraterdquo Cell Cyclevol 9 no 19 pp 3945ndash3955 2010

[34] P Mistry A J Stewart W Dangerfield et al ldquoIn vitro and invivo reversal of P-glycoprotein-mediated multidrug resistanceby a novel potent modulator XR9576rdquo Cancer Research vol 61no 2 pp 749ndash758 2001

[35] D Chao L Lin S Kao et al ldquoInhibitory effects of Zuo-Jin-Wan and its alkaloidal ingredients on activator protein 1nuclear factor-120581B and cellular transformation in HepG2 cellsrdquoFitoterapia vol 82 no 4 pp 749ndash758 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



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OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


proximal tubule cells and has the potential to increasebiliary and urinary elimination of xenobiotics that are BCRPsubstrates [4]

The MRP family is now composed of 9 related ABCtransporters that are able to transport structurally diverselipophilic anions and function as drug efflux pumps [5]MRP1 is a major active transporter of glutathione glucuro-nate and sulfate conjugates and a resistance factor for anthra-cyclines epipodophyllotoxins vinca alkaloids and camp-tothecins [6] MRP-2 (ABCC2) a ubiquitously expressedATP-binding cassette transporter has previously been impli-cated in cellular efflux of cisplatin [7] Interestingly clinicalevidence exists indicating that common ABCC2 polymor-phisms may affect disposition or efficacy of drugs which areknown ABCC2 substrates [8]

Lung resistance protein (LRP) is a member of the vaultproteins involved in MDR LRP has been shown to shuttleanthracyclines out of the nucleus [9] Some conclusive datashowed that the expressions of LRP in patients with gastriccancer without prior chemotherapy are high indicating thatinnate drug resistance may exist in gastric cancer [10]

Traditional Chinesemedicines (TCMs) have been used asmedicines or health supplements in China over thousands ofyears Traditional Chinese prescriptions and formulae whichare based on TCM principles have been also identified tobe an effective anti-cancer drugs in cancer patients suchas breast carcinoma [11] gastric cancer [12] and colorectalcancer [13] Compared with chemotherapeutic drugs whichwere defined as the major drug for a long time traditionalChinese prescriptions and formulae are a therapy to effec-tively control cancer progression improve quality of life andprolong survival times

Zuo Jin Wan (ZJW) a typical TCM formula consists ofthe Rhizoma Coptidis and Fructus Evodiae in the ratio of6 1 (ww) Coptis chinensis Franch the main component ofRhizoma Coptidis has recently been reported to have theeffect of reversing MDR [14 15] Fructus Evodiae has beendemonstrated to induce some cancer cell apoptoses such ashuman melanoma A375-S2 cells [16] human hepatocellularcarcinoma SMMC-7721 cells [17] and Human colorectalcarcinoma COLO-205 cells [18] In addition previous studieshave shown that ZJW can lessen the degree of treatingabdominal pain acid regurgitation nausea and so on andenhance the immune function of patients in the gastric ulcertherapy [19]

Although ZJW herbal formula had been found to havean anti-cancer effect the underlying mechanisms remainunknown In this study our objective was to elucidatethe effect and the molecular mechanism of Chinese herbsformula ZJW herbal formula in human cancer cells both invitro and in vivo

2 Materials and Methods

21 Preparation of the Extracts for ZJW ZJWwas formulatedby Rhizoma Coptidis and Evodia (in a ratio of 6 1) Allthe herbs were purchased from Putuo Hospital herb storeThe mixture (70 g) was extracted twice for 1 h each time byrefluxing in ethanol (1 8 vv)The filtrates were concentrated

and dried in vacuum at 60∘C The concentrated extract wasthen dried by lyophilization to obtain the ZJW extract at ayield of dried powder of 244The extract was stored at 4∘Cand its preparations were standardized regulated and qualitycontrolled according to the guidelines defined by ChineseState Food and Drug Administration (SFDA)

22 Cell Culture and Reagents The human colorectal can-cer HCT116 parental cell gastric cancer SGC7901 cell andhepatic carcinoma Bel7402 parental cell were purchased fromthe Shanghai Cell Collection (Shanghai China) HCT116L-OHP MDR cell lines were established and maintained inour laboratory SGC7901DDP MDR cell line and BelFuMDR cell lines were obtained from Keygen Biotech CoLtd (Nanjing China) The cells were grown in RPMI 1640medium supplemented with 10 (vv) heat-inactivated fetalcalf serum 2mM glutamine 100 unitsmL penicillin and100 120583gmL streptomycin (Invitrogen Carlsbad CA USA)at 37∘C in a 5 CO

2humidified atmosphere HCT116L-

OHP cells were routinely maintained in a medium con-taining 5000 ngmL Oxaliplatin (L-OHP) SGC7901DDPcells were routinely maintained in a medium containing1000 ngmL Diamminedichloroplatinum (DDP) and BelFucells were routinely maintained in a medium containing2000 ngL Mitomycin (MMC) and L-OHP were purchasedfrom Shenzhen Main Luck Pharmaceuticals Co Ltd (Shen-zhen China) DDPwas purchased fromQilu PharmaceuticalCo Ltd (Shandong China) and 5-Fu was purchased fromShanghai XudongHaipu Pharmaceutical Co Ltd (ShanghaiChina) Monoclonal antibodies against ABCB1 ABCC-12BCRP and LRP and dehydrogenase (GAPDH) antibodieswere the products of Cell Signaling Technology (BeverlyMAUSA)

23 Animals and Xenograft Model Male athymic nude mice(NCr-nu) were purchased from Sino-British SIPPRBK labAnimal Co Ltd (Shanghai China license no SCXK 2003-0002) and maintained under specific pathogen-free condi-tions for the studies All animal protocols were approvedby the Institutional Animal Use and Care Committee Allthe experiments and animal care were approved by ShanghaiMedical Experimental Animal Care Commission and inaccordance with the Provision and General Recommen-dation of Chinese Experimental Animals AdministrationLegislation The mice used in these experiments were 8- to12-week old

HCT116L-OHP cells were grown in culture and thendetached by trypsinization washed and resuspended inHanks (HBSS) 02mL HBSS with cells (10 times 106) were sub-cutaneously injected into the athymic nude mice to initiatetumor growth When the tumors were allowed to reach anaverage size of 100mm3 the mice were randomized into 6groups (119899 = 8 per group) Mice in group 1 were administeredwith distilled water daily that served as vehicle control Micein groups 2ndash5 were given oxaliplatin as an intraperitonealinjection every two days and the injection dosage (5mgkg)was according to half of the maximum tolerated dose (MTD)of oxaliplatin as previously described [20] Mice in groups3 4 and 5 received intragastric administration of ZJW at








10in CCK-8 assay)











0 2 4 6 8 10 12(min)

0

50

100

150

200

(mAU

)

842

7A

rea

1035

25

107

00A

rea

1059

98

118

66

Jatro

rrhi

zine

Palm

atin

e

Berb

erin

e


(a)

0 2 4 6 8 10 12 14(min)

0

100

200

300

400

500

(mAU

)

593

36

324

673

7

780

8

882

28

970 9

303

950

7 100

51

107

22

125

7

136

49

Jatro

rrhi

zine

Palm

atin

e

Berberine

799

68

417

115

04


(b)

177

21

891

243

254

338

386

04

016

410

54

447

468

44

931

620

76

401

668

16

707

610

36

617

086

783

18

112

850

3

040

60

664

886

110

123

107

24 114

2811

808 12

482

130

25

138

71

145

2

155

4816

868

177

80

0 2 4 6 8 10 12 14 16 18(min)

0

100

200

300

400

500

600

700

(mAU

)

800

Evod

iam

ine

Ruta

ecar

pine


(c)

344

8

464

7

138

61

179

48

0 2 4 6 8 10 12 14 16 18(min)

0

10050

150200250300

(mAU

)

Evod

iam

ine

Ruta

ecar

pine


(d)




3 Results























50

60

70

80

90

100

Cel

l sur

viva

l (

)

0 10 20 30 40 50 60 70 80 90



(a)

0123456789

10

L-OHP DDP 5-Fu



lowastlowast

lowastlowast

lowast

Fold

chan

ge o

f IC50

toch

emot

hera

peut

ic d

rugs

(b)

0 16 24 32 40 48 56 640

20

40

60

80

100

Cel

l inh

ibiti

on (

)


8 0 8 12 160

20

40

60

80

100

4

Cel

l inh

ibiti

on (

)


0 60 120 180 2400

20

40

60

80

100

Cel

l inh

ibiti

on (

)

0 40 80 120 1600

20

40

60

80

100C

ell i

nhib

ition

()



(c)


50


lowastlowast

119875 lt 001119875 lt 005 and






046 237 664


102 774 1138

(a)



1102103104


1102103104

889 1822 3212

195 2122 1142

24 h 36 h 48 h

(b)



Dip G2Dip S


Dip G2Dip S


Dip G2Dip S

0 50 100 1500

60

120

180

240

0

80

160

240

320

0

80

160

240

320

Num

ber

Num

ber

Num

ber


(c)






4 Discussion




0

100

200

300

400HCT116

HCT116L-OHP

Intr

acel

lula

r con

cent

ratio

n of

L-O

HP

ZJW (120583gmL)25 50 100

lowast

lowast

lowast

mdash mdash

(ng5times105

cells

)










P-gp

LRP

MRP2

GAPDH

170 kD

95 kD

190 kD

36 kD

mdash

(a)

0

02

04

06

08

1

Fold

chan

ge o

f MD

R1 p

rom

oter

activ

ity

Control

24 h 48 h 72 h

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


(b)









10 12 14 16 18 20 22 24 26 280

200

400

600

800

1000

1200

1400

1600

1800

2000


Tum

or v

olum

e (m

m3)

ControlZJWL-OHP


lowast

lowast

◼

◼◼

(a)

Control

ZJW

L-OHP

L-ZJW + L-OHP

M-ZJW + L-OHP

H-ZJW + L-OHP

(b)


Prob

abili

ty o

f sur

viva

l

0

02

04

06

08

12

1


L-OHPZJWControl

(c)







Control H-ZJW L-OHP


(a)

0

02

04

06

08

1

12

Fold

chan

ge

MDR1 mRNAP-gp

lowastlowast

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

ControlH-ZJWL-OHP


998779998779

998779998779

998779998779 998779998779

998779998779

998779998779

(b)



Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























10in CCK-8 assay)











0 2 4 6 8 10 12(min)

0

50

100

150

200

(mAU

)

842

7A

rea

1035

25

107

00A

rea

1059

98

118

66

Jatro

rrhi

zine

Palm

atin

e

Berb

erin

e


(a)

0 2 4 6 8 10 12 14(min)

0

100

200

300

400

500

(mAU

)

593

36

324

673

7

780

8

882

28

970 9

303

950

7 100

51

107

22

125

7

136

49

Jatro

rrhi

zine

Palm

atin

e

Berberine

799

68

417

115

04


(b)

177

21

891

243

254

338

386

04

016

410

54

447

468

44

931

620

76

401

668

16

707

610

36

617

086

783

18

112

850

3

040

60

664

886

110

123

107

24 114

2811

808 12

482

130

25

138

71

145

2

155

4816

868

177

80

0 2 4 6 8 10 12 14 16 18(min)

0

100

200

300

400

500

600

700

(mAU

)

800

Evod

iam

ine

Ruta

ecar

pine


(c)

344

8

464

7

138

61

179

48

0 2 4 6 8 10 12 14 16 18(min)

0

10050

150200250300

(mAU

)

Evod

iam

ine

Ruta

ecar

pine


(d)




3 Results























50

60

70

80

90

100

Cel

l sur

viva

l (

)

0 10 20 30 40 50 60 70 80 90



(a)

0123456789

10

L-OHP DDP 5-Fu



lowastlowast

lowastlowast

lowast

Fold

chan

ge o

f IC50

toch

emot

hera

peut

ic d

rugs

(b)

0 16 24 32 40 48 56 640

20

40

60

80

100

Cel

l inh

ibiti

on (

)


8 0 8 12 160

20

40

60

80

100

4

Cel

l inh

ibiti

on (

)


0 60 120 180 2400

20

40

60

80

100

Cel

l inh

ibiti

on (

)

0 40 80 120 1600

20

40

60

80

100C

ell i

nhib

ition

()



(c)


50


lowastlowast

119875 lt 001119875 lt 005 and






046 237 664


102 774 1138

(a)



1102103104


1102103104

889 1822 3212

195 2122 1142

24 h 36 h 48 h

(b)



Dip G2Dip S


Dip G2Dip S


Dip G2Dip S

0 50 100 1500

60

120

180

240

0

80

160

240

320

0

80

160

240

320

Num

ber

Num

ber

Num

ber


(c)






4 Discussion




0

100

200

300

400HCT116

HCT116L-OHP

Intr

acel

lula

r con

cent

ratio

n of

L-O

HP

ZJW (120583gmL)25 50 100

lowast

lowast

lowast

mdash mdash

(ng5times105

cells

)










P-gp

LRP

MRP2

GAPDH

170 kD

95 kD

190 kD

36 kD

mdash

(a)

0

02

04

06

08

1

Fold

chan

ge o

f MD

R1 p

rom

oter

activ

ity

Control

24 h 48 h 72 h

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


(b)









10 12 14 16 18 20 22 24 26 280

200

400

600

800

1000

1200

1400

1600

1800

2000


Tum

or v

olum

e (m

m3)

ControlZJWL-OHP


lowast

lowast

◼

◼◼

(a)

Control

ZJW

L-OHP

L-ZJW + L-OHP

M-ZJW + L-OHP

H-ZJW + L-OHP

(b)


Prob

abili

ty o

f sur

viva

l

0

02

04

06

08

12

1


L-OHPZJWControl

(c)







Control H-ZJW L-OHP


(a)

0

02

04

06

08

1

12

Fold

chan

ge

MDR1 mRNAP-gp

lowastlowast

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

ControlH-ZJWL-OHP


998779998779

998779998779

998779998779 998779998779

998779998779

998779998779

(b)



Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 2 4 6 8 10 12(min)

0

50

100

150

200

(mAU

)

842

7A

rea

1035

25

107

00A

rea

1059

98

118

66

Jatro

rrhi

zine

Palm

atin

e

Berb

erin

e


(a)

0 2 4 6 8 10 12 14(min)

0

100

200

300

400

500

(mAU

)

593

36

324

673

7

780

8

882

28

970 9

303

950

7 100

51

107

22

125

7

136

49

Jatro

rrhi

zine

Palm

atin

e

Berberine

799

68

417

115

04


(b)

177

21

891

243

254

338

386

04

016

410

54

447

468

44

931

620

76

401

668

16

707

610

36

617

086

783

18

112

850

3

040

60

664

886

110

123

107

24 114

2811

808 12

482

130

25

138

71

145

2

155

4816

868

177

80

0 2 4 6 8 10 12 14 16 18(min)

0

100

200

300

400

500

600

700

(mAU

)

800

Evod

iam

ine

Ruta

ecar

pine


(c)

344

8

464

7

138

61

179

48

0 2 4 6 8 10 12 14 16 18(min)

0

10050

150200250300

(mAU

)

Evod

iam

ine

Ruta

ecar

pine


(d)




3 Results























50

60

70

80

90

100

Cel

l sur

viva

l (

)

0 10 20 30 40 50 60 70 80 90



(a)

0123456789

10

L-OHP DDP 5-Fu



lowastlowast

lowastlowast

lowast

Fold

chan

ge o

f IC50

toch

emot

hera

peut

ic d

rugs

(b)

0 16 24 32 40 48 56 640

20

40

60

80

100

Cel

l inh

ibiti

on (

)


8 0 8 12 160

20

40

60

80

100

4

Cel

l inh

ibiti

on (

)


0 60 120 180 2400

20

40

60

80

100

Cel

l inh

ibiti

on (

)

0 40 80 120 1600

20

40

60

80

100C

ell i

nhib

ition

()



(c)


50


lowastlowast

119875 lt 001119875 lt 005 and






046 237 664


102 774 1138

(a)



1102103104


1102103104

889 1822 3212

195 2122 1142

24 h 36 h 48 h

(b)



Dip G2Dip S


Dip G2Dip S


Dip G2Dip S

0 50 100 1500

60

120

180

240

0

80

160

240

320

0

80

160

240

320

Num

ber

Num

ber

Num

ber


(c)






4 Discussion




0

100

200

300

400HCT116

HCT116L-OHP

Intr

acel

lula

r con

cent

ratio

n of

L-O

HP

ZJW (120583gmL)25 50 100

lowast

lowast

lowast

mdash mdash

(ng5times105

cells

)










P-gp

LRP

MRP2

GAPDH

170 kD

95 kD

190 kD

36 kD

mdash

(a)

0

02

04

06

08

1

Fold

chan

ge o

f MD

R1 p

rom

oter

activ

ity

Control

24 h 48 h 72 h

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


(b)









10 12 14 16 18 20 22 24 26 280

200

400

600

800

1000

1200

1400

1600

1800

2000


Tum

or v

olum

e (m

m3)

ControlZJWL-OHP


lowast

lowast

◼

◼◼

(a)

Control

ZJW

L-OHP

L-ZJW + L-OHP

M-ZJW + L-OHP

H-ZJW + L-OHP

(b)


Prob

abili

ty o

f sur

viva

l

0

02

04

06

08

12

1


L-OHPZJWControl

(c)







Control H-ZJW L-OHP


(a)

0

02

04

06

08

1

12

Fold

chan

ge

MDR1 mRNAP-gp

lowastlowast

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

ControlH-ZJWL-OHP


998779998779

998779998779

998779998779 998779998779

998779998779

998779998779

(b)



Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































50

60

70

80

90

100

Cel

l sur

viva

l (

)

0 10 20 30 40 50 60 70 80 90



(a)

0123456789

10

L-OHP DDP 5-Fu



lowastlowast

lowastlowast

lowast

Fold

chan

ge o

f IC50

toch

emot

hera

peut

ic d

rugs

(b)

0 16 24 32 40 48 56 640

20

40

60

80

100

Cel

l inh

ibiti

on (

)


8 0 8 12 160

20

40

60

80

100

4

Cel

l inh

ibiti

on (

)


0 60 120 180 2400

20

40

60

80

100

Cel

l inh

ibiti

on (

)

0 40 80 120 1600

20

40

60

80

100C

ell i

nhib

ition

()



(c)


50


lowastlowast

119875 lt 001119875 lt 005 and






046 237 664


102 774 1138

(a)



1102103104


1102103104

889 1822 3212

195 2122 1142

24 h 36 h 48 h

(b)



Dip G2Dip S


Dip G2Dip S


Dip G2Dip S

0 50 100 1500

60

120

180

240

0

80

160

240

320

0

80

160

240

320

Num

ber

Num

ber

Num

ber


(c)






4 Discussion




0

100

200

300

400HCT116

HCT116L-OHP

Intr

acel

lula

r con

cent

ratio

n of

L-O

HP

ZJW (120583gmL)25 50 100

lowast

lowast

lowast

mdash mdash

(ng5times105

cells

)










P-gp

LRP

MRP2

GAPDH

170 kD

95 kD

190 kD

36 kD

mdash

(a)

0

02

04

06

08

1

Fold

chan

ge o

f MD

R1 p

rom

oter

activ

ity

Control

24 h 48 h 72 h

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


(b)









10 12 14 16 18 20 22 24 26 280

200

400

600

800

1000

1200

1400

1600

1800

2000


Tum

or v

olum

e (m

m3)

ControlZJWL-OHP


lowast

lowast

◼

◼◼

(a)

Control

ZJW

L-OHP

L-ZJW + L-OHP

M-ZJW + L-OHP

H-ZJW + L-OHP

(b)


Prob

abili

ty o

f sur

viva

l

0

02

04

06

08

12

1


L-OHPZJWControl

(c)







Control H-ZJW L-OHP


(a)

0

02

04

06

08

1

12

Fold

chan

ge

MDR1 mRNAP-gp

lowastlowast

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

ControlH-ZJWL-OHP


998779998779

998779998779

998779998779 998779998779

998779998779

998779998779

(b)



Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















046 237 664


102 774 1138

(a)



1102103104


1102103104

889 1822 3212

195 2122 1142

24 h 36 h 48 h

(b)



Dip G2Dip S


Dip G2Dip S


Dip G2Dip S

0 50 100 1500

60

120

180

240

0

80

160

240

320

0

80

160

240

320

Num

ber

Num

ber

Num

ber


(c)






4 Discussion




0

100

200

300

400HCT116

HCT116L-OHP

Intr

acel

lula

r con

cent

ratio

n of

L-O

HP

ZJW (120583gmL)25 50 100

lowast

lowast

lowast

mdash mdash

(ng5times105

cells

)










P-gp

LRP

MRP2

GAPDH

170 kD

95 kD

190 kD

36 kD

mdash

(a)

0

02

04

06

08

1

Fold

chan

ge o

f MD

R1 p

rom

oter

activ

ity

Control

24 h 48 h 72 h

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


(b)









10 12 14 16 18 20 22 24 26 280

200

400

600

800

1000

1200

1400

1600

1800

2000


Tum

or v

olum

e (m

m3)

ControlZJWL-OHP


lowast

lowast

◼

◼◼

(a)

Control

ZJW

L-OHP

L-ZJW + L-OHP

M-ZJW + L-OHP

H-ZJW + L-OHP

(b)


Prob

abili

ty o

f sur

viva

l

0

02

04

06

08

12

1


L-OHPZJWControl

(c)







Control H-ZJW L-OHP


(a)

0

02

04

06

08

1

12

Fold

chan

ge

MDR1 mRNAP-gp

lowastlowast

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

ControlH-ZJWL-OHP


998779998779

998779998779

998779998779 998779998779

998779998779

998779998779

(b)



Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















4 Discussion




0

100

200

300

400HCT116

HCT116L-OHP

Intr

acel

lula

r con

cent

ratio

n of

L-O

HP

ZJW (120583gmL)25 50 100

lowast

lowast

lowast

mdash mdash

(ng5times105

cells

)










P-gp

LRP

MRP2

GAPDH

170 kD

95 kD

190 kD

36 kD

mdash

(a)

0

02

04

06

08

1

Fold

chan

ge o

f MD

R1 p

rom

oter

activ

ity

Control

24 h 48 h 72 h

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


(b)









10 12 14 16 18 20 22 24 26 280

200

400

600

800

1000

1200

1400

1600

1800

2000


Tum

or v

olum

e (m

m3)

ControlZJWL-OHP


lowast

lowast

◼

◼◼

(a)

Control

ZJW

L-OHP

L-ZJW + L-OHP

M-ZJW + L-OHP

H-ZJW + L-OHP

(b)


Prob

abili

ty o

f sur

viva

l

0

02

04

06

08

12

1


L-OHPZJWControl

(c)







Control H-ZJW L-OHP


(a)

0

02

04

06

08

1

12

Fold

chan

ge

MDR1 mRNAP-gp

lowastlowast

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

ControlH-ZJWL-OHP


998779998779

998779998779

998779998779 998779998779

998779998779

998779998779

(b)



Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















P-gp

LRP

MRP2

GAPDH

170 kD

95 kD

190 kD

36 kD

mdash

(a)

0

02

04

06

08

1

Fold

chan

ge o

f MD

R1 p

rom

oter

activ

ity

Control

24 h 48 h 72 h

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


(b)









10 12 14 16 18 20 22 24 26 280

200

400

600

800

1000

1200

1400

1600

1800

2000


Tum

or v

olum

e (m

m3)

ControlZJWL-OHP


lowast

lowast

◼

◼◼

(a)

Control

ZJW

L-OHP

L-ZJW + L-OHP

M-ZJW + L-OHP

H-ZJW + L-OHP

(b)


Prob

abili

ty o

f sur

viva

l

0

02

04

06

08

12

1


L-OHPZJWControl

(c)







Control H-ZJW L-OHP


(a)

0

02

04

06

08

1

12

Fold

chan

ge

MDR1 mRNAP-gp

lowastlowast

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

ControlH-ZJWL-OHP


998779998779

998779998779

998779998779 998779998779

998779998779

998779998779

(b)



Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















10 12 14 16 18 20 22 24 26 280

200

400

600

800

1000

1200

1400

1600

1800

2000


Tum

or v

olum

e (m

m3)

ControlZJWL-OHP


lowast

lowast

◼

◼◼

(a)

Control

ZJW

L-OHP

L-ZJW + L-OHP

M-ZJW + L-OHP

H-ZJW + L-OHP

(b)


Prob

abili

ty o

f sur

viva

l

0

02

04

06

08

12

1


L-OHPZJWControl

(c)







Control H-ZJW L-OHP


(a)

0

02

04

06

08

1

12

Fold

chan

ge

MDR1 mRNAP-gp

lowastlowast

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

ControlH-ZJWL-OHP


998779998779

998779998779

998779998779 998779998779

998779998779

998779998779

(b)



Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control H-ZJW L-OHP


(a)

0

02

04

06

08

1

12

Fold

chan

ge

MDR1 mRNAP-gp

lowastlowast

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

ControlH-ZJWL-OHP


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Acknowledgments


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Research Article Zuo Jin Wan, a Traditional Chinese Herbal...

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