Research Article Reversal of Multidrug Resistance by the...

Research ArticleReversal of Multidrug Resistance by the Chinese MedicineYiqi Jianpi Huaji Decoction and the Mechanism of Action inHuman Gastric Cancer SGC7901VCR Cells

Wei-Bing Li1 Yang Li2 Chen Yu1 and Yong-Ming He1

1Department of Integrated Traditional and Western Medicine Jiangsu Cancer Hospital Baizi Ting No 42 NanjingJiangsu 210000 China2Department of Radiation Oncology Cancer Center Sun Yat-sen University Guangzhou Guangdong 510006 China

Correspondence should be addressed to Wei-Bing Li 41186709qqcom

Received 25 November 2014 Revised 6 January 2015 Accepted 6 January 2015

Academic Editor Oliver Micke

Copyright copy 2015 Wei-Bing Li et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Yiqi Jianpi Huaji Decoction (YJHD) a traditional Chinese medicinal formula composed of twelve ingredients has recentlybeen reported to have a good clinical curative effect The purpose of the present study was to evaluate the effects of YJHD onSGC7901VCR gastric cancer cells and to elucidate the possible mechanism of action First the effects of a low dose of YJHD incombination with chemotherapeutic agents on SGC7901VCR cells were assessed using the CCK-8 assay and flow cytometry andthe effects of YJHD on genes and proteins involved in drug resistance (MDR1 MRP TUBB3 STMN1 and TS) were evaluatedFurthermore transfection of SGC7901VCR cells with siRNAs targeting these genes inhibited their expression and the efficacy ofvincristine against the cells was dramatically improved in vitro when these genes were silencedThese results demonstrate that low-dose YJHD inhibited cell proliferation induced apoptosis reversed MDR and increased sensitivity to chemotherapeutic agents invitro by downregulating P-gp MRP TUBB3 and STMN1 expression MDR can be reversed by siRNAs targeting genes involved inMDR and this strategy for cancer treatment should be evaluated in future studies

1 Introduction

Stomach cancer was the cause of 738000 deaths in 2008corresponding to approximately 97 of the total number ofcancer-related deaths [1] and the incidence and mortalityrates for stomach cancer are significantly higher in Chinathan in the rest of the world [2]Themajor factors responsiblefor the increased mortality and morbidity associated withgastric cancer are the progression of the disease and failureof chemotherapy caused by multidrug resistance (MDR) [3]via the overexpression of the MDR1 and MRP genes [4] Themajor cause of MDR in tumor cells is the overexpressionof a membrane-bound protein P-glycoprotein (P-gp) andother members of the adenosine triphosphate (ATP) bindingcassette (ABC) transporter superfamily [5] which translo-cate a substrate from the intracellular compartment to theextracellular compartment leading to a reduced intracellularconcentration of the substrate and resistance to antineoplastic

drugs [6] The overexpression of P-gp and MRP is associatedwith a poor prognosis In normal gastric tissue P-gp andMRP mRNAs are either not present or are only slightly ele-vated [7] whereas P-gp andMRPoverexpression is associatedwith MDR in gastric cancer [8ndash10]

However several other mechanisms are also involved inthe development ofMDR in tumor cells including alterationsin drug targets (eg TUBB3 STMN1 and TS) the activationof detoxifying systems the interruption of signaling path-ways and alterations in regulators involved in cell cycle con-trol [6] and the above genes exhibit chemotherapy-relatedeffects A number of studies have confirmed that a highTS expression level contributes to the resistance to 5-Fu anda poor clinical outcome [11] Several studies using drugsthat target microtubules have also found that 120573-tubulin III(TUBB3) and stathmin 1 (STMN1) are directly related to theefficacy of chemotherapy [6] Additionally elevated TUBB3expression is related to vincristine resistance and a short

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 390812 11 pageshttpdxdoiorg1011552015390812

2 Evidence-Based Complementary and Alternative Medicine

survival time [7 8] and STMN1 affects the formation of themitotic spindle promoting microtubule depolymerization orpreventing microtubule polymerization [8]

Herbal medicines are an important source of novel agentswith pharmaceutical potential [9] Modern pharmacologicalexperiments have led to the use of herbal medicines for com-plementary and alternative therapy and this approach hasbecome increasingly popular particularly in cancer patientswho exhibit resistance after several chemotherapy treatments[10 12] Considering the narrow therapeutic window ofchemotherapeutic agents synergistic or additive interactionsmay improve the outcome of a therapy and reverse long-termchemotherapy drug resistance As a result of the variationin adjuvant components each formula can have a differentname and the precisemechanisms of action of these formulaealso remain to be addressed in studies investigating their clin-ical efficacy One such formula Yiqi Jianpi Huaji Decoction(YJHD) is currently under study in our laboratory YJHDis a prescribed Chinese complex that contains the followingtwelve ingredients Codonopsis pilosula Astragalus mem-branaceus Atractylodes macrocephala koidz Radix angelicasinensis Radix paeoniae alba Tangerine peel Pinellia tuberZedoary Rhizoma sparganii Salvia chinensis licorice andOldenlandia diffusa YJHD shows good effect in clinical effi-cacy and three components (Salvia chinensis Zedoary andOldenlandia diffusa) exhibit potential anticancer activity andrestore the sensitivity of MCF-7ADR and A549Taxol cellsmodulating the MDR phenotype and the function of P-gpin vitro [13 14] Oldenlandia diffusa extracts exert antiprolif-erative effects and induce apoptosis in human breast cancercells [15] Additionally the individual use of other YJHDcomponents has been reported in China and was found tostrengthen the immune system improve appetite and actas antiangiogenesis and as an anticonvulsant [16] Howeverthe potential role of this formula in cancer therapy has notyet been clearly addressed by modern science To furtherelucidate mechanisms that may be involved in the interactionbetween YJHD and chemotherapeutic agents we also investi-gated apoptosis levels and the expression of chemotherapeu-tic agent resistance-related genes in gastric cancer cells aftertreatment with YJHD and chemotherapeutic agents singlyand in combination

RNAi is a fundamental cellular mechanism for silencinggene expression and can be used for the development of newdrugs [17] Accordingly this study focused on the down-regulation of MRP MDR1 TUBB3 and STMN1 expressionby siRNA After transfection of siRNAs silencing at theprotein level was demonstrated by western blot analysisFurthermore we also examined the in vitro effects of siRNA-mediated MRP MDR1 TUBB3 and STMN1 suppression onthe chemotherapeutic sensitivity of human SGC7901VCRcell lines

2 Materials and Methods

21 Preparation of YJHD Extract All of the crude com-ponents including 15 g Codonopsis pilosula 15 g Astra-galus membranaceus 10 g Atractylodes macrocephala koidz10 g Radix angelica sinensis 10 g Radix paeoniae alba 6 g

Tangerine peel 10 g Pinellia tuber 10 g Rhizoma sparganii 10 gZedoary 30 g Salvia chinensis 5 g licorice and 30 g Olden-landia diffusa were purchased from the Jiangsu ProvincialHospital of Traditional Chinese Medicine (Nanjing JiangsuProvince China) First a mixture of YJHDwas homogenizedwith a Waring blender then soaked in 10 L (10-fold of theplant) double-distilled water for 2 h and boiled for 1 h Thecooled decoction was filtered through two layers of cottongauze The filtrates obtained from three cycles of the proce-dure were combined and the filtered solution was concen-trated into a residue in a vacuum evaporator and distilled intoa liquid The yield of the aqueous extract was 21 gmL basedon the original amounts of the herbal ingredients

The extract was reconstituted with sterile distilled waterto prepare working solutions with final concentrations of 64 3 2 1 05 and 025mgmL for the treatment of cancercells The quality control of the YJHD preparation includingthe verification of the correct plant components origin ofproduction and growth harvesting and processing was per-formed according to the guidelines defined by Nanjing HerbPharmaceutics Ltd (Nanjing Jiangsu Province China)

22 Cell Lines and Cell Culture The human gastric cell lineSGC7901 was kindly provided by the center laboratory ofJiangsu Cancer Hospital Nanjing China The human MDRgastric cancer cell line SGC7901VCRwas purchased from theShanghai Institutes for Biological Sciences Chinese Academyof Sciences (Shanghai China) The cells were propagated inRPMI 1640 medium (GIBCO BRL CA USA) supplementedwith 10 bovine serum penicillin (100UmL) and strepto-mycin (100 120583gmL) at 37∘C in a water-saturated atmospherewith 5 CO

2 For the SGC7901VCR cell line the medium

contained an additional 1 120583gmL vincristine (Shenzhen MainLuck Pharmaceuticals)

23 Drugs 5-Fluorouracil (5-Fu) was supplied by JiangsuHengrui Medicine Company (Jiangsu China) Vincristinesulfate (VCR) was purchased from Shenzhen Main LuckPharmaceuticals (Shenzhen Guangdong China) The dilu-tions of all of the reagents were prepared fresh before eachexperiment An Annexin V-FITC flow cytometry detectionkit was purchased from Nanjing KeyGEN Biotech Co Ltd(Nanjing Jiangsu China)

24 Analysis of Cell Viability The CCK8 assay was used tomeasure cell viability Exponentially growing SGC7901VCRcells (100 120583L 5 times 104 cellsmL) were seeded into 96-well plates(Corning Incorporated Corning NY USA) After 24 h thecells were cultured in an RPMI-1640medium containing 10FBS and then treated with VCR or 5-Fu at concentrationsranging between 40 and 0625 120583gmL as well as YJHD atconcentrations between 6 and 05mgmL Then we exam-ined the combined effect of a low-dose YJHD and VCRor 5-Fu The treatments were repeated three times at eachconcentration The plates were then incubated in a humid-ified incubator (Sanyo Osaka Japan) at 37∘C and 5 CO

2

for 48 h Subsequently 2 h prior tomeasuring the absorbance10 120583L of CCK-8 solution was added to each well The opticaldensity was measured at an absorbance of 450 nm using a

Evidence-Based Complementary and Alternative Medicine 3

microplate reader (BioTek InstrumentsWinooski VTUSA)The rate of inhibition of cellular proliferation was calculatedusing the following formula cellular proliferation inhibitionrate = (1 minus mean A

450of experimental groupmean A

450of

control group) times 100 We chose to evaluate the chemother-apy drugs with the highest fold reversal levels

25 Flow Cytometry The effects of the evaluated concen-trations of YJHD and chemotherapeutic drugs on apoptosiswere determined by flow cytometry The cells were seeded at3 times 105 cellswell in six-well plates and incubated overnightA low dose of YJHD (05mgmL) was added in combinationwith 0625120583gmLndash25120583gmL of 5-Fu and the plates wereincubated for 48 h Dimethylsulfoxide-treated cells (01)served as the control The cells were collected and washedtwice with 4∘C PBS The cells were then added to 500 120583L ofbinding buffer and 5120583L of Annexin VFITC and 5 120583L of pro-pidium iodide (PI) were then mixed The experiments wereperformed in triplicate

26 Cell Cycle Analysis For the cell cycle analysis SGC7901VCR cells were cultured in 6-well plates (at least 30 times105 cellswell) in an RPMI-1640 medium containing 10FBS and 100UmL of penicillin and streptomycin for 24 hand then treated with a low dose of YJHD (05mgmL)and 0625 120583gmL of 5-Fu for 48 h The cells were washedcollected and fixed using 70 ethanol Following that stepthe cells were treated with TrisHCl buffer (pH 74) containing1 RNase A (KeyGen Biotech Co Ltd) and stained with PIFlow cytometry (FACSCalibur Becton-Dickinson) was per-formed to determine the distribution of cells with varyingDNA contents Multicycle DNA content data were analyzedusing cell cycle analysis software (KeyGen Biotech Co Ltd)

27 RNA Extraction and Real-Time Reverse Transcriptase-PCR Total RNA was extracted using Trizol (Invitrogen)according to the manufacturerrsquos instructions For mRNAanalysis real-time PCR was performed using the PowerSYBR green PCR master mix (Applied Biosystems) withan ABI 7500 series PCR machine (Applied Biosystems)and the data were normalized to GAPDH expression andthen further normalized to the negative control unlessotherwise indicated Custom primers for TUBB3 STMN1TS MDR1 and MRP were synthesized by Ruian Biotech(GAPDH forward primer 51015840-CCATGGAGAAGGCTG-GGG-31015840 and reverse primer 51015840-CAAAGTTGTCATGGA-TGACC-31015840 TUBB3 forward primer 51015840-GCCTGACAATTT-CATCTTT-31015840 and reverse primer 51015840-TCACACTCCTTCCGCACCA-31015840 STMN1 forward primer 51015840-AGAATACAC-TGCCTGTCGCTT G-31015840 and reverse primer 51015840-AGGCAC-GCTTCTCCAGTT-31015840 TS forward primer 51015840-ACCTGA-ATCACAATC GAGCCA-31015840 and reverse primer 51015840-TTGGATGCGGATTGTACCCT-31015840 MDR1 forward primer 51015840-CACGTCAGCCTTGGA CACAGA-31015840 and reverse primer51015840-CAATGACTCCATCATCGAAACCAG-31015840MRP forwardprimer 51015840-TCTCTCCCGACATGACCGAGG-31015840 and reverseprimer 51015840-CCAGGAATATGCCCCGACTTC-31015840)

28Western Blot Analysis Theproteins in the SGC7901VCRcell culture supernatant were analyzed directly for STMN1TUBB3MDR1MRP andTS detection and cell extracts wereprepared in a suitable lysis buffer In brief each sample wasmixed with loading buffer separated by electrophoresis ata constant voltage and electrotransferred onto PVDF mem-branes (Millipore Billerica MA USA)Themembranes wereincubated with a primary antibody at 4∘C overnight washedthree times with TBS-T buffer and incubated with a sec-ondary antibody conjugated to horseradish peroxidase (CellSignaling Technology CST Beverly MA USA) for 1 h Afterthree washes the blots were visualized using the enhancedchemiluminescence method (Millipore) Rabbit monoclonalantibodies against STMN1 TUBB3 MDR1 MRP and TSwere purchased from Abcam (Abcam Cambridge UK)

29 Transfection with siRNAs Cells were seeded in 6-wellplates and after 24 h of growth were then transfectedwith theindicated siRNAs (MDR1 MRP STMN1 and TUBB3) usingLipofectamine RNAiMAX Reagent according to the manu-facturerrsquos protocol Silencingwas examined at 24 h after trans-fection by western blotting The siRNA-transfected cells (5 times104 SGC7901VCR cellsmL)were plated in 96-well plates andtreated with various concentrations of the test agents After48 hr the IC

50values for vincristine in these cells were deter-

mined by the CCK-8 assay The siRNAs were synthesizedby RiboBio (Guangzhou) The target sequences were as fol-lows MDR1 siRNA 51015840-AA GACATGACCAGGTATGCCT-31015840 MRP siRNA 51015840-CGAUGAAGACCAAGAC GUAUU-31015840 TUBB3 siRNA 51015840-UCUCUUCAGGCCUGACAAUTT-31015840 STMN1 siRNA 51015840-CAGTGGATTGTGTAGAGTGTA-31015840A negative control 51015840-UUCUCCGAACGUGUCACGUTT-31015840 was also obtained from Dharmacon

210 Statistical Analysis All of the data are expressed asthe mean plusmn SD The results were analyzed using 119905-tests andanalyses of variance with the SPSS 130 software Values of119875 lt 005 were considered statistically significant

3 Results

31 Inhibition of SGC7901VCR and SGC7901 Cell Prolif-eration by YJHD SGC7901VCR and SGC7901 cells weretreated with 0ndash6mgmL YJHD and the results demonstratethe dose- and time-dependent inhibition of proliferation byYJHD (Figure 1(a)) inducing a greater than 90 reduction inthe number of cells at the highest concentration (6mgmL)To evaluate whether YJHD increases the sensitivity ofchemotherapeutic agents in anMDRgastric cancer cell line invitro we evaluated a nontoxic dose of YJHD combined withvarious concentrations of VCR and 5-Fu The concentrationsof the chemotherapeutic agents were selected according totheir peak plasma concentrations in vivo 40 20 10 5 25125 and 0625 120583gmL The concentrations of YJHD were6 4 2 1 and 05mgmL The effects of the low dose ofYJHD in combination with the chemotherapeutic agents oncell proliferation were measured using the CCK-8 assay Asshown in Figures 1(b)-1(c) a concentration of 05mgmL of


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YJHD increased the sensitivity of SGC7901VCR cells to thechemotherapeutic agents and to some extent reversedMDRThe chemosensitivity of MDR cells to the chemotherapydrugs was measured by comparing IC

50values expressed

as RF The IC50

values of YJHD were 271 plusmn 013 and086 plusmn 0046mgmL respectively for the SGC7901 andSGC7901VCR cell lines (Table 1) At less than or equal to05mgmL YJHDdid not cause any cytotoxicity to SGC7901VCR cells Therefore 05mgmL was chosen as the workingconcentration in the subsequent experiments As expected

the SGC7901VCR cells weremore resistant to VCRThe IC50

values for 5-Fu and VCR in SGC7901 cells were 063 plusmn 0073and 084 plusmn 008 120583gmL whereas the IC

50values for 5-Fu

and VCR were 4378 plusmn 012 and 1701 plusmn 032 120583gmL forthe SGC7901VCR cells (Table 1) YJHD dose dependentlyincreased the sensitivity of SGC7901VCR cells to 5-Fu andVCR (Figures 1(b) and 1(c))When administered at a nontoxicdose YJHD shifted the IC

50values for 5-Fu and VCR to

1462 plusmn 0085 and 729 plusmn 024120583gmL with RF values of 299 plusmn010-fold and 233 plusmn 010-fold respectively (Tables 2 and 3)


Table 1 IC50 values (120583gmL) of anticancer drugs in gastric carci-noma cells

Cell line 5-Fu (120583gmL) VCR (120583gmL) YJHD (mgmL)SGC7901 063 plusmn 0073 084 plusmn 008 271 plusmn 013SGC7901VCR 4378 plusmn 012 1701 plusmn 032 086 plusmn 0046RI 694 2025Reversal factors (RFs) were calculated according to the following equationRFs = IC50 antitumor drug aloneIC50 antitumor drug + modulator

Table 2 Magnitude of the reversal of multiple drug resistance by alow dose of YJHD

Cell line 5-Fu (120583gmL) 5-Fu + 05MYJHD(120583gmL)

SGC7901VCR 4378 plusmn 012 1462 plusmn 0085RF (5-Fu IC505-Fu + YJHDIC50)

299 plusmn 010


Cell line VCR (120583gmL) +05MYJHD(120583gmL)

SGC7901VCR 1701 plusmn 032 729 plusmn 024RF (VCR IC50VCR + YJHD IC50) 233 plusmn 010

However treatmentwithYJHDdid not significantly affect thedrug sensitivity of SGC7901 cells

32 Apoptosis in SGC7901VCR Cells Induced by YJHD inCombination with Chemotherapeutic Drugs Apoptosis wasmeasured using flow cytometry The apoptosis ratios in thecontrol group were 739 plusmn 125 after a 48 h treatment with05mgmL YJHD The apoptosis ratios for the YJHD and 5-Fu groups were significantly increased compared to the 5-Fu single-drug group (119875 lt 005) These results indicate adose-dependent effect on apoptosis in SGC7901VCR cells(119875 lt 005 Figure 2) confirming the apoptotic and inhibitoryeffects of a low dose of YJHD when administered in com-bination with 5-Fu on the growth of MDR gastric cancerSGC7901VCR cells

33 Enhancement of 5-Fu-Induced S-Phase Arrest in SGC7901VCR Cells by YJHD In general agents that induce cyto-toxicity by inhibiting DNA synthesis (eg 5-Fu) arrest thecell cycle at S phase [18] As expected at concentrationsequal to 0625120583gmL 5-Fu-induced S-phase arrest Althougha low dose of YJHD (05mgmL) did not affect the cellcycle when administered by itself treatment with YJHD incombination with 5-Fu induced a significant S-phase arrestin up to 5062 plusmn 081 of the cells (Figure 3) These resultssuggest that YJHD enhanced 5-Fu-induced S-phase arrestwhich may be attributed to its effect on drug resistance

34 Influence of YJHD on the Expression of mRNA and ProteinEncoding Chemotherapeutic Agent Resistance-Related GenesTo investigate the mechanisms underlying the synergistic

Table 4 Effect of siRNAs on the sensitivity of SGC7901VCR tumorcells to chemotherapeutic drugs (vincristine) after transfection

Drug IC50 (120583gmL)

Vincristine STMN1 TUBB3 MDR1 MRPsiRNA siRNA siRNA siRNA

1701 plusmn 032 713 plusmn 115lowast 505 plusmn 029lowast 177 plusmn 038lowast 269 plusmn 029lowast

Cells were plated in 96-well multiwell plates After 24 hr of transfection thecells were exposed to various concentrations of cytotoxic drugs for 2 daysAfter CCK-8 was added the absorbance in individual wells was determinedat 450 nm and the IC50 was calculatedThe results represent the mean plusmn SD of three separate experimentslowast

119875 lt 005 compared with control

interaction between a low dose of YJHD and 5-Fu or VCRwe hypothesized that YJHD might affect the expressionof chemotherapeutic agent resistance-related genes that isMDR1 MRP TS STMN1 and TUBB3 by influencing theirsensitivity to 5-Fu and VCR We first found that the geneexpression of MDR1 and MRP in SGC7901VCR cells ishigher than that in SGC7901 cells After incubating the cellswith YJHD at their respective IC

40values for 48 h the levels

of mRNA expression of these genes in SGC7901VCR cellswere assessed by real-time PCR As shown in Figure 3 thelevels of MDR1 MRP TUBB3 and STMN1 were significantlydownregulated by YJHD treatment though YJHD did notdownregulate the expression of TS Similar results wereobtained by western blotting indicating that the expressionlevels of P-gp MRP TUBB3 and STMN1 were stronglypositive in SGC7901VCR cells for 48 hThe expression levelswere slightly positive in cells treated with 05mgmL ofYJHD for 48 h (Figure 4) with very little effect on TS Theseresults demonstrate that YJHD decreased the expression ofP-gp MRP TUBB3 and STMN1 in SGC7901VCR cells andincreased the antitumor effects of chemotherapeutic drugs byinhibiting their expression

The stable knockdown of oncogenic STMN1 TUBB3MRP and MDR1 by siRNA significantly reversed the drugresistance of SGC7901VCR cells in vitro

Thus to investigate the possible effects of knockdownof these genes in vitro all four were downregulated in theSGC7901VCR cell line (Figure 5) All cells were evaluated bycomparison with IC

50values using a CCK-8 assay revealing

that the proliferation of STMN1- MDR1- MRP- TUBB3-knockdown cells was significantly diminished comparedwiththe negative-control cells Moreover these knockdown cellsexhibited significantly higher sensitivity to vincristine thanthe control cells (119875 lt 001 Table 4)

4 Discussion

Although chemotherapy is an important treatment modalityfor gastric cancer because of MDR its effectiveness is signifi-cantly reduced as metastasis and recurrence ratios increaseThus the reversal of MDR and increase in the sensitiv-ity to chemotherapeutic agents are very important aspectsof tumor therapy YJHD is a traditional Chinese medicinethat is an extract comprising 12 species of medicinal plantsSeveral components have recently been reported to exert


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Figure 2 (a) Flow cytometric analysis of apoptosis in SGC7901VCR cells treated with VCR or 5-Fu with or without 05mgmL YJHD for48 h Analysis of the apoptosis ratio lowast119875 lt 005 versus control (b) Bar graph of the apoptosis rate lowast119875 lt 005 versus control


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Figure 4 (a) MDR1 and MRP gene expression in SGC7901 and SGC7901VCR cells lowastlowastlowast119875 lt 001 versus control (b) YJHD suppressed themRNA expression of chemotherapeutic agent resistance-related genes in SGC7901VCR cells Fold changes relative gene expression aftertreatment with YJHD at 05mgmL for 48 h compared to control lowast119875 lt 005 compared with control (c) Western blot analysis of STMN1 TSTUBB3 MRP and P-gp in control cells and cells treated with YJHD at 05mgmL 1mgmL and 2mgmL (d) Bar graph of the densitometricvalues of the bands corresponding toMRP P-gp STMN1 TS and TUBB3 normalized relative to 120573-actin lowast119875 lt 005 versus control lowastlowast119875 lt 001versus control


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Figure 5 STMN1 MDR1 TUBB3 and MRP expression in stably transfected cells The transfected cells were identified by western blotting120573-actin was used as an internal control

antiproliferative effects on several cancer cell lines in vivoand in vitro [14ndash16] including lung cancer breast cancerand colon cancer cells with several components reducingresistance in breast cancer cell lines [14] Our previous studiesconfirmed that YJHD has an antiproliferative effect on theSGC7901 human gastric cancer cell line in vitro [19] Inthe present study we explored the effects of YJHD in anMDR gastric cancer cell line and investigated the underlyingmechanism of actionThe results of the CCK-8 assay and flowcytometry demonstrated that YJHD dose dependently inhib-ited proliferation and induced apoptosis in SGC7901VCRcells (119875 lt 005) According to the peak plasma concentrationsof chemotherapeutic agents in vivo we observed inhibitoryeffects of the combination of a low dose of YJHD with 5-Fuand VCR on cell proliferation showing that low-dose YJHDincreased the inhibitory effects

The influence of YJHD on the expression of intracellularcyclins can cause cell cycle arrest and induce apoptosisin SGC7901 cells [19] However our experimental resultsindicate that a low dose of YJHD does not affect the cellcycle Our results also suggested that 5-Fu dose dependentlyinduces cell cycle arrest at S phase in SGC7901VCR celllines Moreover a low concentration of YJHD (05mgmL)

enhanced the sensitivity of SGC7901VCR cells to 5-Fu (cellcycle arrest at S phase)

Furthermore because several drug-metabolizing geneshave been shown to be prognostic markers for gastric cancertherapy we examined the additional influence of YJHD ontheir mRNA and protein expression

Microtubules are composed of tubulin dimers and inter-act with a variety of microtubule-associated proteins [20]Studies in lung cancer have shown that high expressionlevels of brain-specific TUBB3 are associated with vincristineresistance in a preclinical setting which has been furtherconfirmed in patients with advanced NSCLC [21] STMN1has been shown to regulate the dynamics of the microtubulesthat compose themitotic spindle and STMN1 overexpressionleads to the resistance to antimicrotubule agents [22] Adecreased sensitivity of breast cancer cells to Vinca alka-loids and Taxol in response to the overexpression of themicrotubule-associated protein STMN1 was reported by Alliet al [22] and a high TS expression level was reported to beassociated with resistance to 5-Fu therapy and a poor clinicaloutcome [23] A meta-analysis [24] demonstrated that theTS expression level should be considered one of the mostimportant markers of the 5-Fu response A previous study


demonstrated that P-gp is overexpressed inH pylori positivepatients particularly in patients who do not respond toeradication therapy [5] and a causal link has been reportedbetweenMRP and P-gp activity whichmay have implicationsfor MDR in tumors in which MRP is overexpressed Ourdata also indicate that YJHD downregulated the expressionofMRP TUBB3 STMN1MDR1mRNA and protein thoughthe expression of TS was not significantly affected In thisstudy we found that the capacity of the Chinese medicine toreverse resistance to fluorouracil is stronger than that of vin-cristine Accordingly we suggest that the drug-induced resis-tance mechanism of the latter is more complex with othermechanisms of resistance in addition to an increase in theexpression of four genes

We then used RNAi technology to knock down theexpression of these four genes and found that the sensitivity tochemotherapy drugs by multidrug-resistant cells was notablyincreased confirming our results

To our knowledge the present study is the first todemonstrate the effects of YJHD on an MDR human gas-tric cancer cell line YJHD inhibited proliferation inducedapoptosis circumvented MDR and increased sensitivity tochemotherapeutic agents in the P-gp-overexpressing gastriccancer cell line SGC7901VCR The effects of YJHD maybe attributed to the downregulation of the expression ofMDR1P-gp MRP TUBB3 and STMN1 thus weakening thelevel of P-gp-mediated MDR and increasing the sensitivity ofSGC7901VCR cells to chemotherapy

The present study suggests that the use of chemothera-peutic agents in combinationwithYJHDas a chemosensitizeror adjuvant may be helpful for the treatment of gastriccancer We also found that chemotherapy together with RNAinterference appears to exert a promising therapeutic effectAlthough clinical application needs to be evaluated thesefindings provide an intriguing possibility for future cancertherapy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Wei-Bing Li and Yang Li contributed equally to this work

Acknowledgments

This project was funded by the State Administration ofTraditional Chinese Medicine of the Peoplersquo Republic ofChina the National Traditional Chinese Medicine ClinicalResearch Base Construction of Special Topic Research noJDZX2012090 and Jiangsu Province Hospital of TCM

References

[1] J Ferlay H R Shin F Bray et al GLOBOCAN 2008 CancerIncidence and Mortality Worldwide IARC Cancer Base No 10Lyon France International Agency for Research on CancerLyon France 2010

[2] X Sun R Mu Y Zhou et al ldquo1990ndash1992 mortality of stomachcancer in Chinardquo Chinese Journal of Oncology vol 24 no 1 pp4ndash8 2002

[3] S Labialle L Gayet E Marthinet D Rigal and L G BaggettoldquoTranscriptional regulators of the humanmultidrug resistance 1gene recent viewsrdquo Biochemical Pharmacology vol 64 no 5-6pp 943ndash948 2002

[4] C Mignogna S Staibano V Altieri et al ldquoPrognostic signifi-cance of multidrug-resistance protein (MDR-1) in renal cancercell carcinomas a five year follow-up analysisrdquo BMC Cancervol 6 article 293 2006

[5] X-Q Yu C C Xue G Wang and S-F Zhou ldquoMultidrugresistance associated proteins as determining factors of phar-macokinetics and pharmacodynamics of drugsrdquo Current DrugMetabolism vol 8 no 8 pp 787ndash802 2007

[6] E K Fetisova A V Avetisyan D S Izyumov M V Korotet-skaya B V Chernyak and V P Skulachev ldquoMitochondria-targeted antioxidant SkQR1 selectively protectsMDR (Pgp 170)-negative cells against oxidative stressrdquo FEBS Letters vol 584 no3 pp 562ndash566 2010

[7] G Nardone A Rocco D Vaira et al ldquoExpression of COX-2mPGE-synthase

1

MDR-1 (P-gp) and Bcl-x119871

a molecular path-way of H pylori-related gastric carcinogenesisrdquo The Journal ofPathology vol 202 no 3 pp 305ndash312 2004

[8] Y Zhang X Qu X Hu et al ldquoReversal of P-glycoprotein-mediated multi-drug resistance by the E3 ubiquitin ligase Cbl-b in human gastric adenocarcinoma cellsrdquo The Journal ofPathology vol 218 no 2 pp 248ndash255 2009

[9] H-W Xu L Xu J-HHao C-Y Qin andH Liu ldquoExpression ofP-glycoprotein and multidrug resistance-associated protein isassociated with multidrug resistance in gastric cancerrdquo TheJournal of International Medical Research vol 38 no 1 pp 34ndash42 2010

[10] W Liu Y-H Yu X-N Ouyang et al ldquoClinical significance of P-glycoprotein and topoisomerase II expression in gastric cancerrdquoThe World Chinese Journal of Digestology vol 18 no 34 pp3640ndash3647 2010

[11] V Vallacchi M Daniotti F Ratti et al ldquoCCN3nephroblastomaoverexpressed matricellular protein regulates integrin expres-sion adhesion and dissemination in melanomardquo CancerResearch vol 68 no 3 pp 715ndash723 2008

[12] M Cervello and G Montalto ldquoCyclooxygenases in hepatocel-lular carcinomardquoWorld Journal of Gastroenterology vol 12 no32 pp 5113ndash5121 2006

[13] L Yang and D D Wei ldquoReversal effects of traditional Chineseherbs on multidrug resistance in cancer cellsrdquo PhytotherapyResearch vol 26 no 4 pp 562ndash567 2012

[14] L Yang D D Wei Z Chen J S Wang and L Y Kong ldquoRever-sal of multidrug resistance in human breast cancer cells byCurcuma wenyujin and Chrysanthemum indicumrdquo Phytomed-icine vol 18 no 8-9 pp 710ndash718 2011

[15] G Gu I Barone L Gelsomino et al ldquoOldenlandia diffusaextracts exert antiproliferative and apoptotic effects on humanbreast cancer cells through ER120572Sp1-mediated p53 activationrdquoJournal of Cellular Physiology vol 227 no 10 pp 3363ndash33722012

[16] J Sun S Wang and Y-H Wei ldquoReproductive toxicity ofRhizoma Sparganii (Sparganium stoloniferum Buch-Ham) inmice mechanisms of anti-angiogenesis and anti-estrogen phar-macologic activitiesrdquo Journal of Ethnopharmacology vol 137 no3 pp 1498ndash1503 2011


[17] D M Dykxhoorn D Palliser and J Lieberman ldquoThe silenttreatment SiRNAs as small molecule drugsrdquoGeneTherapy vol13 no 6 pp 541ndash552 2006

[18] P B Mullan J E Quinn P M Gilmore et al ldquoBRCA1 andGADD45 mediated G2M cell cycle arrest in response toantimicrotubule agentsrdquo Oncogene vol 20 no 43 pp 6123ndash6131 2001

[19] S Peng andG Ting-Jie ldquoJian Pi YangWei compound on humangastric cancer cell line SGC7901 experiment on growth inhi-bition and apoptosisrdquo Jiangsu Journal of Traditional ChineseMedicine vol 45 no 3 pp 70ndash72 2013

[20] P Seve S Isaac O Tredan et al ldquoExpression of class iii 120573-tubulin is predictive of patient outcome in patients with nonmdashsmall cell lung cancer receiving vinorelbine-based chemother-apyrdquo Clinical Cancer Research vol 11 no 15 pp 5481ndash54862005

[21] Y Zhang H Yang J Liu et al ldquoHigh expression levels of classIIIbeta-tubulin in resected non-small cell lung cancer patientsare predictive of improved patient survival after vinorelbine-based adjuvant chemotherapyrdquo Oncology Letters vol 6 no 1pp 220ndash226 2013

[22] E Alli J-M Yang J M Ford and W N Hait ldquoReversal ofstathmin-mediated resistance to paclitaxel and vinblastine inhuman breast carcinoma cellsrdquoMolecular Pharmacology vol 71no 5 pp 1233ndash1240 2007

[23] C Aschele S Lonardi and S Monfardini ldquoThymidylate syn-thase expression as a predictor of clinical response to fluoropy-rimidine-based chemotherapy in advanced colorectal cancerrdquoCancer Treatment Reviews vol 28 no 1 pp 27ndash47 2002

[24] S Popat A Matakidou and R S Houlston ldquoThymidylate-synthase expression and prognosis in colorectal cancer a sys-tematic review andmeta-analysisrdquo Journal of Clinical Oncologyvol 22 no 3 pp 529ndash536 2004

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



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


survival time [7 8] and STMN1 affects the formation of themitotic spindle promoting microtubule depolymerization orpreventing microtubule polymerization [8]

Herbal medicines are an important source of novel agentswith pharmaceutical potential [9] Modern pharmacologicalexperiments have led to the use of herbal medicines for com-plementary and alternative therapy and this approach hasbecome increasingly popular particularly in cancer patientswho exhibit resistance after several chemotherapy treatments[10 12] Considering the narrow therapeutic window ofchemotherapeutic agents synergistic or additive interactionsmay improve the outcome of a therapy and reverse long-termchemotherapy drug resistance As a result of the variationin adjuvant components each formula can have a differentname and the precisemechanisms of action of these formulaealso remain to be addressed in studies investigating their clin-ical efficacy One such formula Yiqi Jianpi Huaji Decoction(YJHD) is currently under study in our laboratory YJHDis a prescribed Chinese complex that contains the followingtwelve ingredients Codonopsis pilosula Astragalus mem-branaceus Atractylodes macrocephala koidz Radix angelicasinensis Radix paeoniae alba Tangerine peel Pinellia tuberZedoary Rhizoma sparganii Salvia chinensis licorice andOldenlandia diffusa YJHD shows good effect in clinical effi-cacy and three components (Salvia chinensis Zedoary andOldenlandia diffusa) exhibit potential anticancer activity andrestore the sensitivity of MCF-7ADR and A549Taxol cellsmodulating the MDR phenotype and the function of P-gpin vitro [13 14] Oldenlandia diffusa extracts exert antiprolif-erative effects and induce apoptosis in human breast cancercells [15] Additionally the individual use of other YJHDcomponents has been reported in China and was found tostrengthen the immune system improve appetite and actas antiangiogenesis and as an anticonvulsant [16] Howeverthe potential role of this formula in cancer therapy has notyet been clearly addressed by modern science To furtherelucidate mechanisms that may be involved in the interactionbetween YJHD and chemotherapeutic agents we also investi-gated apoptosis levels and the expression of chemotherapeu-tic agent resistance-related genes in gastric cancer cells aftertreatment with YJHD and chemotherapeutic agents singlyand in combination

RNAi is a fundamental cellular mechanism for silencinggene expression and can be used for the development of newdrugs [17] Accordingly this study focused on the down-regulation of MRP MDR1 TUBB3 and STMN1 expressionby siRNA After transfection of siRNAs silencing at theprotein level was demonstrated by western blot analysisFurthermore we also examined the in vitro effects of siRNA-mediated MRP MDR1 TUBB3 and STMN1 suppression onthe chemotherapeutic sensitivity of human SGC7901VCRcell lines

2 Materials and Methods

21 Preparation of YJHD Extract All of the crude com-ponents including 15 g Codonopsis pilosula 15 g Astra-galus membranaceus 10 g Atractylodes macrocephala koidz10 g Radix angelica sinensis 10 g Radix paeoniae alba 6 g

Tangerine peel 10 g Pinellia tuber 10 g Rhizoma sparganii 10 gZedoary 30 g Salvia chinensis 5 g licorice and 30 g Olden-landia diffusa were purchased from the Jiangsu ProvincialHospital of Traditional Chinese Medicine (Nanjing JiangsuProvince China) First a mixture of YJHDwas homogenizedwith a Waring blender then soaked in 10 L (10-fold of theplant) double-distilled water for 2 h and boiled for 1 h Thecooled decoction was filtered through two layers of cottongauze The filtrates obtained from three cycles of the proce-dure were combined and the filtered solution was concen-trated into a residue in a vacuum evaporator and distilled intoa liquid The yield of the aqueous extract was 21 gmL basedon the original amounts of the herbal ingredients

The extract was reconstituted with sterile distilled waterto prepare working solutions with final concentrations of 64 3 2 1 05 and 025mgmL for the treatment of cancercells The quality control of the YJHD preparation includingthe verification of the correct plant components origin ofproduction and growth harvesting and processing was per-formed according to the guidelines defined by Nanjing HerbPharmaceutics Ltd (Nanjing Jiangsu Province China)

22 Cell Lines and Cell Culture The human gastric cell lineSGC7901 was kindly provided by the center laboratory ofJiangsu Cancer Hospital Nanjing China The human MDRgastric cancer cell line SGC7901VCRwas purchased from theShanghai Institutes for Biological Sciences Chinese Academyof Sciences (Shanghai China) The cells were propagated inRPMI 1640 medium (GIBCO BRL CA USA) supplementedwith 10 bovine serum penicillin (100UmL) and strepto-mycin (100 120583gmL) at 37∘C in a water-saturated atmospherewith 5 CO

2 For the SGC7901VCR cell line the medium

contained an additional 1 120583gmL vincristine (Shenzhen MainLuck Pharmaceuticals)

23 Drugs 5-Fluorouracil (5-Fu) was supplied by JiangsuHengrui Medicine Company (Jiangsu China) Vincristinesulfate (VCR) was purchased from Shenzhen Main LuckPharmaceuticals (Shenzhen Guangdong China) The dilu-tions of all of the reagents were prepared fresh before eachexperiment An Annexin V-FITC flow cytometry detectionkit was purchased from Nanjing KeyGEN Biotech Co Ltd(Nanjing Jiangsu China)

24 Analysis of Cell Viability The CCK8 assay was used tomeasure cell viability Exponentially growing SGC7901VCRcells (100 120583L 5 times 104 cellsmL) were seeded into 96-well plates(Corning Incorporated Corning NY USA) After 24 h thecells were cultured in an RPMI-1640medium containing 10FBS and then treated with VCR or 5-Fu at concentrationsranging between 40 and 0625 120583gmL as well as YJHD atconcentrations between 6 and 05mgmL Then we exam-ined the combined effect of a low-dose YJHD and VCRor 5-Fu The treatments were repeated three times at eachconcentration The plates were then incubated in a humid-ified incubator (Sanyo Osaka Japan) at 37∘C and 5 CO

2

for 48 h Subsequently 2 h prior tomeasuring the absorbance10 120583L of CCK-8 solution was added to each well The opticaldensity was measured at an absorbance of 450 nm using a




450of










3 Results



0 1 2 3 4 5 60

20

40

60

80

100

SGC7901VCRSGC7901

Inhi

bitio

n ra

te (

)

YJHD (mgmL)

(a)

0 10 20 30 40 500

20

40

60

80

100

SGC7901VCR

SGC7901In

hibi

tion

rate

()

VCR (120583gmL)

SGC7901VCR + YJHD

(b)

0 5 10 15 200

20

40

60

80

100

SGC7901SGC7901VCR

Inhi

bitio

n ra

te (

)

SGC7901VCR + YJHD

5-Fu (120583gmL)

(c)



50values expressed

as RF The IC50




50values for 5-Fu










299 plusmn 010





















4 Discussion



100

101

102

103

104

AVPI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104



171

186

232

756

128

1448

686

37

668

4071

82

643

(a)

0

20

40

60

80

Apop

tosis

rate

()

05

mg

mL

YJH

D

0625

120583g

mL5

-Fu

05

mg

mL

YJH

D=0625

120583g

mL5

-Fu

25120583

gm

L5

-Fu

Con

trol

lowast

lowast

lowast

lowast

05

mg

mL

YJH

D+25120583

gm

L5

-Fu

(b)



Control 05mgmL YJHD


1000

800

400

600

200

0

0 30 60 90 120 150

Num

ber

Channels (FL2-A)

400

600

200

0

0 30 60 90 120 150

Num

ber

Channels (FL2-A)

Channels (FL2-A)

160

120

80

40

0

0 20 40 60 80 100 120

320

240

160

80

0

0 30 60 90 120 150

Num

ber

Num

ber

Channels (PE-A)

0625120583gmL 5-Fu

ApoptosisDip S

ApoptosisDip S

ApoptosisDip S

ApoptosisDip S

Dip G2

Dip G1

Dip G2

Dip G1

Dip G2

Dip G1

Dip G2

Dip G1

(a)

0 50 100

Control

S

Con

cent

ratio

n

()

05mgmL YJHD

0625120583gmL 5-Fu

YJHD + 5-Fu

G2M

G1

(b)


0

G1

G2



MDR1 MRP0

5

10

15

20

25

SGC7901SGC7901VCR

Relat

ive e

xpre

ssio

n of

mRN

A

lowastlowastlowast

lowastlowastlowast

(a)


05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

05mgmL YJHD


(b)

STMN1

TS

TUBB3

ControlYJHDYJHD



P-gp

MRP

(c)

MDR100

05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

STMN1 TS TUBB300

05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

P-gp



lowastlowast


lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

(d)



Control

Control


MRP

ControlSGC7901VCR

TUBB3


STMN1


120573-actin

120573-actin

P-gp

120573-actin

120573-actin
















Acknowledgments


References








1


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















450of










3 Results



0 1 2 3 4 5 60

20

40

60

80

100

SGC7901VCRSGC7901

Inhi

bitio

n ra

te (

)

YJHD (mgmL)

(a)

0 10 20 30 40 500

20

40

60

80

100

SGC7901VCR

SGC7901In

hibi

tion

rate

()

VCR (120583gmL)

SGC7901VCR + YJHD

(b)

0 5 10 15 200

20

40

60

80

100

SGC7901SGC7901VCR

Inhi

bitio

n ra

te (

)

SGC7901VCR + YJHD

5-Fu (120583gmL)

(c)



50values expressed

as RF The IC50




50values for 5-Fu










299 plusmn 010





















4 Discussion



100

101

102

103

104

AVPI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104



171

186

232

756

128

1448

686

37

668

4071

82

643

(a)

0

20

40

60

80

Apop

tosis

rate

()

05

mg

mL

YJH

D

0625

120583g

mL5

-Fu

05

mg

mL

YJH

D=0625

120583g

mL5

-Fu

25120583

gm

L5

-Fu

Con

trol

lowast

lowast

lowast

lowast

05

mg

mL

YJH

D+25120583

gm

L5

-Fu

(b)



Control 05mgmL YJHD


1000

800

400

600

200

0

0 30 60 90 120 150

Num

ber

Channels (FL2-A)

400

600

200

0

0 30 60 90 120 150

Num

ber

Channels (FL2-A)

Channels (FL2-A)

160

120

80

40

0

0 20 40 60 80 100 120

320

240

160

80

0

0 30 60 90 120 150

Num

ber

Num

ber

Channels (PE-A)

0625120583gmL 5-Fu

ApoptosisDip S

ApoptosisDip S

ApoptosisDip S

ApoptosisDip S

Dip G2

Dip G1

Dip G2

Dip G1

Dip G2

Dip G1

Dip G2

Dip G1

(a)

0 50 100

Control

S

Con

cent

ratio

n

()

05mgmL YJHD

0625120583gmL 5-Fu

YJHD + 5-Fu

G2M

G1

(b)


0

G1

G2



MDR1 MRP0

5

10

15

20

25

SGC7901SGC7901VCR

Relat

ive e

xpre

ssio

n of

mRN

A

lowastlowastlowast

lowastlowastlowast

(a)


05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

05mgmL YJHD


(b)

STMN1

TS

TUBB3

ControlYJHDYJHD



P-gp

MRP

(c)

MDR100

05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

STMN1 TS TUBB300

05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

P-gp



lowastlowast


lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

(d)



Control

Control


MRP

ControlSGC7901VCR

TUBB3


STMN1


120573-actin

120573-actin

P-gp

120573-actin

120573-actin
















Acknowledgments


References








1


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 1 2 3 4 5 60

20

40

60

80

100

SGC7901VCRSGC7901

Inhi

bitio

n ra

te (

)

YJHD (mgmL)

(a)

0 10 20 30 40 500

20

40

60

80

100

SGC7901VCR

SGC7901In

hibi

tion

rate

()

VCR (120583gmL)

SGC7901VCR + YJHD

(b)

0 5 10 15 200

20

40

60

80

100

SGC7901SGC7901VCR

Inhi

bitio

n ra

te (

)

SGC7901VCR + YJHD

5-Fu (120583gmL)

(c)



50values expressed

as RF The IC50




50values for 5-Fu










299 plusmn 010





















4 Discussion



100

101

102

103

104

AVPI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104



171

186

232

756

128

1448

686

37

668

4071

82

643

(a)

0

20

40

60

80

Apop

tosis

rate

()

05

mg

mL

YJH

D

0625

120583g

mL5

-Fu

05

mg

mL

YJH

D=0625

120583g

mL5

-Fu

25120583

gm

L5

-Fu

Con

trol

lowast

lowast

lowast

lowast

05

mg

mL

YJH

D+25120583

gm

L5

-Fu

(b)



Control 05mgmL YJHD


1000

800

400

600

200

0

0 30 60 90 120 150

Num

ber

Channels (FL2-A)

400

600

200

0

0 30 60 90 120 150

Num

ber

Channels (FL2-A)

Channels (FL2-A)

160

120

80

40

0

0 20 40 60 80 100 120

320

240

160

80

0

0 30 60 90 120 150

Num

ber

Num

ber

Channels (PE-A)

0625120583gmL 5-Fu

ApoptosisDip S

ApoptosisDip S

ApoptosisDip S

ApoptosisDip S

Dip G2

Dip G1

Dip G2

Dip G1

Dip G2

Dip G1

Dip G2

Dip G1

(a)

0 50 100

Control

S

Con

cent

ratio

n

()

05mgmL YJHD

0625120583gmL 5-Fu

YJHD + 5-Fu

G2M

G1

(b)


0

G1

G2



MDR1 MRP0

5

10

15

20

25

SGC7901SGC7901VCR

Relat

ive e

xpre

ssio

n of

mRN

A

lowastlowastlowast

lowastlowastlowast

(a)


05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

05mgmL YJHD


(b)

STMN1

TS

TUBB3

ControlYJHDYJHD



P-gp

MRP

(c)

MDR100

05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

STMN1 TS TUBB300

05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

P-gp



lowastlowast


lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

(d)



Control

Control


MRP

ControlSGC7901VCR

TUBB3


STMN1


120573-actin

120573-actin

P-gp

120573-actin

120573-actin
















Acknowledgments


References








1


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















299 plusmn 010





















4 Discussion



100

101

102

103

104

AVPI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104



171

186

232

756

128

1448

686

37

668

4071

82

643

(a)

0

20

40

60

80

Apop

tosis

rate

()

05

mg

mL

YJH

D

0625

120583g

mL5

-Fu

05

mg

mL

YJH

D=0625

120583g

mL5

-Fu

25120583

gm

L5

-Fu

Con

trol

lowast

lowast

lowast

lowast

05

mg

mL

YJH

D+25120583

gm

L5

-Fu

(b)



Control 05mgmL YJHD


1000

800

400

600

200

0

0 30 60 90 120 150

Num

ber

Channels (FL2-A)

400

600

200

0

0 30 60 90 120 150

Num

ber

Channels (FL2-A)

Channels (FL2-A)

160

120

80

40

0

0 20 40 60 80 100 120

320

240

160

80

0

0 30 60 90 120 150

Num

ber

Num

ber

Channels (PE-A)

0625120583gmL 5-Fu

ApoptosisDip S

ApoptosisDip S

ApoptosisDip S

ApoptosisDip S

Dip G2

Dip G1

Dip G2

Dip G1

Dip G2

Dip G1

Dip G2

Dip G1

(a)

0 50 100

Control

S

Con

cent

ratio

n

()

05mgmL YJHD

0625120583gmL 5-Fu

YJHD + 5-Fu

G2M

G1

(b)


0

G1

G2



MDR1 MRP0

5

10

15

20

25

SGC7901SGC7901VCR

Relat

ive e

xpre

ssio

n of

mRN

A

lowastlowastlowast

lowastlowastlowast

(a)


05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

05mgmL YJHD


(b)

STMN1

TS

TUBB3

ControlYJHDYJHD



P-gp

MRP

(c)

MDR100

05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

STMN1 TS TUBB300

05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

P-gp



lowastlowast


lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

(d)



Control

Control


MRP

ControlSGC7901VCR

TUBB3


STMN1


120573-actin

120573-actin

P-gp

120573-actin

120573-actin
















Acknowledgments


References








1


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

101

102

103

104

AVPI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104

100

101

102

103

104

AV

PI

100 101 102 103 104



171

186

232

756

128

1448

686

37

668

4071

82

643

(a)

0

20

40

60

80

Apop

tosis

rate

()

05

mg

mL

YJH

D

0625

120583g

mL5

-Fu

05

mg

mL

YJH

D=0625

120583g

mL5

-Fu

25120583

gm

L5

-Fu

Con

trol

lowast

lowast

lowast

lowast

05

mg

mL

YJH

D+25120583

gm

L5

-Fu

(b)



Control 05mgmL YJHD


1000

800

400

600

200

0

0 30 60 90 120 150

Num

ber

Channels (FL2-A)

400

600

200

0

0 30 60 90 120 150

Num

ber

Channels (FL2-A)

Channels (FL2-A)

160

120

80

40

0

0 20 40 60 80 100 120

320

240

160

80

0

0 30 60 90 120 150

Num

ber

Num

ber

Channels (PE-A)

0625120583gmL 5-Fu

ApoptosisDip S

ApoptosisDip S

ApoptosisDip S

ApoptosisDip S

Dip G2

Dip G1

Dip G2

Dip G1

Dip G2

Dip G1

Dip G2

Dip G1

(a)

0 50 100

Control

S

Con

cent

ratio

n

()

05mgmL YJHD

0625120583gmL 5-Fu

YJHD + 5-Fu

G2M

G1

(b)


0

G1

G2



MDR1 MRP0

5

10

15

20

25

SGC7901SGC7901VCR

Relat

ive e

xpre

ssio

n of

mRN

A

lowastlowastlowast

lowastlowastlowast

(a)


05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

05mgmL YJHD


(b)

STMN1

TS

TUBB3

ControlYJHDYJHD



P-gp

MRP

(c)

MDR100

05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

STMN1 TS TUBB300

05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

P-gp



lowastlowast


lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

(d)



Control

Control


MRP

ControlSGC7901VCR

TUBB3


STMN1


120573-actin

120573-actin

P-gp

120573-actin

120573-actin
















Acknowledgments


References








1


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control 05mgmL YJHD


1000

800

400

600

200

0

0 30 60 90 120 150

Num

ber

Channels (FL2-A)

400

600

200

0

0 30 60 90 120 150

Num

ber

Channels (FL2-A)

Channels (FL2-A)

160

120

80

40

0

0 20 40 60 80 100 120

320

240

160

80

0

0 30 60 90 120 150

Num

ber

Num

ber

Channels (PE-A)

0625120583gmL 5-Fu

ApoptosisDip S

ApoptosisDip S

ApoptosisDip S

ApoptosisDip S

Dip G2

Dip G1

Dip G2

Dip G1

Dip G2

Dip G1

Dip G2

Dip G1

(a)

0 50 100

Control

S

Con

cent

ratio

n

()

05mgmL YJHD

0625120583gmL 5-Fu

YJHD + 5-Fu

G2M

G1

(b)


0

G1

G2



MDR1 MRP0

5

10

15

20

25

SGC7901SGC7901VCR

Relat

ive e

xpre

ssio

n of

mRN

A

lowastlowastlowast

lowastlowastlowast

(a)


05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

05mgmL YJHD


(b)

STMN1

TS

TUBB3

ControlYJHDYJHD



P-gp

MRP

(c)

MDR100

05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

STMN1 TS TUBB300

05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

P-gp



lowastlowast


lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

(d)



Control

Control


MRP

ControlSGC7901VCR

TUBB3


STMN1


120573-actin

120573-actin

P-gp

120573-actin

120573-actin
















Acknowledgments


References








1


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MDR1 MRP0

5

10

15

20

25

SGC7901SGC7901VCR

Relat

ive e

xpre

ssio

n of

mRN

A

lowastlowastlowast

lowastlowastlowast

(a)


05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

05mgmL YJHD


(b)

STMN1

TS

TUBB3

ControlYJHDYJHD



P-gp

MRP

(c)

MDR100

05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

STMN1 TS TUBB300

05

10

15

Control

Relat

ive i

nten

sity

(fold

ove

r con

trol)

P-gp



lowastlowast


lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

(d)



Control

Control


MRP

ControlSGC7901VCR

TUBB3


STMN1


120573-actin

120573-actin

P-gp

120573-actin

120573-actin
















Acknowledgments


References








1


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control

Control


MRP

ControlSGC7901VCR

TUBB3


STMN1


120573-actin

120573-actin

P-gp

120573-actin

120573-actin
















Acknowledgments


References








1


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























Acknowledgments


References








1


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Reversal of Multidrug Resistance by the...

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Transcript of Research Article Reversal of Multidrug Resistance by the...