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Nutrition and Cancer, 62(8), 1025–1035Copyright C© 2010, Taylor & Francis Group, LLCISSN: 0163-5581 print / 1532-7914 onlineDOI: 10.1080/01635581.2010.492087
The Flavonoid Quercetin Transiently Inhibits the Activityof Taxol and Nocodazole Through InterferenceWith the Cell Cycle
Temesgen Samuel, Khalda Fadlalla, Timothy Turner,and Teshome E. YehualaeshetTuskegee University, Tuskegee, Alabama, USA
Quercetin is a flavonoid with anticancer properties. In thisstudy, we examined the effects of quercetin on cell cycle, viability,and proliferation of cancer cells, either singly or in combinationwith the microtubule-targeting drugs taxol and nocodazole. Al-though quercetin induced cell death in a dose-dependent manner,12.5–50 µM quercetin inhibited the activity of both taxol and noco-dazole to induce G2/M arrest in various cell lines. Quercetin alsopartially restored drug-induced loss in viability of treated cellsfor up to 72 h. This antagonism of microtubule-targeting drugswas accompanied by a delay in cell cycle progression and inhi-bition of the buildup of cyclin-B1 at the microtubule organizingcenter of treated cells. However, quercetin did not inhibit the mi-crotubule targeting of taxol or nocodazole. Despite the short-termprotection of cells by quercetin, colony formation and clonogenicityof HCT116 cells were still suppressed by quercetin or quercetin-taxol combination. The status of cell adherence to growth ma-trix was critical in determining the sensitivity of HCT116 cells toquercetin. We conclude that although long-term exposure of can-cer cells to quercetin may prevent cell proliferation and survival,the interference of quercetin with cell cycle progression dimin-ishes the efficacy of microtubule-targeting drugs to arrest cellsat G2/M.
INTRODUCTIONConsumption of foods of plant origin, especially fruits, veg-
etables, and whole grains, is associated with a reduced risk ofdifferent types of cancer, including those of the lung, oral cavity,esophagus, stomach, prostate, and colon (1–4). Dietary com-pounds are being intensively studied for their chemopreventive,chemotherapeutic, or adjuvant potential in cancer management.Dietary polyphenolic compounds, in particular, have attracted
Submitted 17 June 2009; accepted in final form 23 February 2010.Address correspondence to Temesgen Samuel, Pathobiology De-
partment, Tuskegee University, College of Veterinary Medicine, Nurs-ing and Allied Health; and the Tuskegee University Center for CancerResearch, Tuskegee, AL 36088. Phone: 334-724-4547. Fax: 334-724-4110. E-mail: [email protected]
much attention because of their abundance and due to well-documented bioactivity that includes their antioxidant effects.
Quercetin is one of the most abundant dietary flavonoids.Quercetin and its derivatives constitute about 99% of theflavonoids in apple peel (5), and it is also one of the ma-jor constituents in foods consumed in the United States (6,7).Numerous in vitro and animal model studies using quercetinalone or quercetin in combination with other bioactive com-pounds have shown the anticancer activities of the compound(8–15).
Although much is known about the bioactivities and the ma-jor signaling pathways modulated by quercetin (see ref in (16)),less is known about the potentials of the compound as a com-plementary supplement once the cancer has established itselfand therapy has been implemented to treat the cancer. The ben-efits and dangers of the concomitant use of antioxidants andchemotherapeutic agents has been controversial (17). This hasespecially been true for therapeutic agents that induce oxidativestress as the mechanism of action, as antioxidants may also re-duce the side effects of chemotherapeutic agents. A definitiverecommendation is still lacking as to whether or when antiox-idants should at all be used in the course of chemotherapy orradiation therapy (17–20). Drug–diet interactions among an-tioxidants and classes of drugs that act through nonoxidativemechanisms is not well known.
We examined the effect of the cotreatment of cancer cells withthe flavonoid quercetin and 2 antimicrotubule drugs, namely,taxol and nocodazole. We analyzed cells treated with singleagents or a drug-flavonoid combination. We hypothesized anadditive or a synergistic effect with this drug-flavonoid com-bination, but unexpectedly, quercetin protected cells from theactivity of these antimicrotubule drugs and sustained the via-bility of the cells. However, prolonged exposure of the cells tothe highest protective dose of quercetin was still able to pre-vent cell proliferation. Thus, we identify bimodal activity of theflavonoid quercetin, a short-term activity that is cytoprotectiveagainst chemotherapeutic drugs and a long-term activity that isinhibitory to cancer cell growth.
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MATERIALS AND METHODS
Cell Culture and TreatmentsThe human colorectal cancer HCT116 cell lines (wild type
and p53 null) were generous gifts from Bert Vogelstein (JohnsHopkins). The cells were maintained in McCoy’s medium(Lonza, Walkersville, MD) supplemented with 10% fetal bovineserum (FBS) and penicillin/streptomycin. Prostate cancer PPC1cells (gift from John C. Reed, Burnham Institute) were grown inRPMI medium (Invitrogen, Carlsbad, CA), supplemented with10% FBS and penicillin/streptomycin. RKO colorectal cancercells [American Type Culture Collection (ATCC), Manassas,VA] were maintained in similarly supplemented Dulbecco’smodified eagle medium (DMEM). MCF7 cells were kindly pro-vided by Leslie Wilson (University of California, Santa Barbara)and were maintained in DMEM. For most experimental treat-ments, cells were seeded in 96-well, 6-well, or 6-cm dishesat approximate densities of 103, 104, or 105 cells per well, re-spectively. For experiments requiring longer than 48 h, the cellnumbers for the entire experimental setup were reduced by half.All cell cultures were incubated at 37◦C and 5% CO2 in a humid-ified incubator. Cells were synchronized by the double thymi-dine block method. Exponentially growing cells were treatedovernight with 2 mM thymidine in growth medium. The nextmorning, culture medium was removed, and the monolayer waswashed 3 times with plain growth medium to remove thymidine.The cells were allowed to grow in complete medium for 8 h andwere treated again overnight with 2 mM thymidine. The syn-chronized monolayer cells were washed again and released intocomplete growth medium. Cell cycle was analyzed at differenttime points after the release.
ReagentsQuercetin (Q0125), nocodazole, and taxol were purchased
from Sigma (St. Louis, MO). A stock solution of 50 mMquercetin was prepared in DMSO, aliquoted in single use por-tions, and stored at –20◦C. Unused portions of any thawedaliquots were discarded. Nocodazole and taxol were also dis-solved in DMSO as 5 mM and 10 mM stock solutions. Work-ing dilutions of the stock were prepared in culture medium.Polyclonal antibodies to cyclin-B1 (AHF0062) and CDK1(AHZ0112) were purchased from Invitrogen. Monoclonal an-tibody to α-tubulin (clone DM1A) was purchased from Sigma(St. Louis, MO).
Colony Formation and Clonogenic AssaysColony formation assay was performed by seeding approxi-
mately 500 cells per well of a 12-well dish. Depending on thecell types or experimental designs, cells were allowed to adherefor up to 18 h and then treated with quercetin, or they were di-rectly seeded in culture medium containing quercetin. Culturemedium was changed every 48 h until discreet colonies werevisible to the naked eye, after which they were stained with10% crystal violet in methanol, washed, and air dried. Clono-genic assay was performed as described (21). The number of
cells in colonies was counted microscopically (×200 magnifi-cation), whereas the number of established clonal colonies wascounted using a stereo microscope. Due to their small sizes,HCT116 cells were allowed to grow up to about 130 cells percolony before staining with crystal violet.
Flow CytometryCells were harvested and prepared for flow cytometry as de-
scribed, with some modifications (21). Cells were harvested bytrypsinization using 0.25% trypsin EDTA. Prior to trypsiniza-tion, floating or loose cells were harvested by gentle manualrocking of the culture dishes and transferring the culture mediumcontaining the cells into centrifuge tubes. Trypsinized and loosecells were then combined and centrifuged. Pellets were resus-pended in 300 µl phosphate buffered saline (PBS), fixed by theaddition of 700 µl 100% ethanol while vortexing, and stored at–20◦C for at least overnight. Fixed cells were centrifuged andstained in FACS staining solution (320 mg/ml RNase A, 0.4mg/ml propidium iodide) in PBS without calcium and magne-sium for 30 min at 37◦C. Stained cells were filtered through a 70microns pore sized filter and analyzed by flow cytometry (FAC-Scalibur Beckton Dickinson, BD Biosciences, San Jose, CA,and C6 Accuri flow cytometers, Acuri Cytometers, Inc., AnnArbor, MI). Data were analyzed, and histograms were preparedusing CellQuest and CFlow software.
MTT/MTS AssaysMTT reagent was obtained from ATCC, whereas the MTS
assay was performed using CellTiter 96 AQueous One Solutioncell proliferation assay kit from Promega (Madison, WI). Theassays were performed on cells seeded in triplicates in 96-wellplates, according to the manufacturer’s instructions. Absorbancewas recorded at 570 nm (MTT) or 490 nm (MTS) using Syn-ergy HT multimode plate reader or PowerWave XS2 (BioTek,Winooski, VT). To account for absorbance of quercetin at490 nm, during each MTT or MTS experiment, separate wellswere set where quercetin was diluted in culture medium with-out cells. The average A490 readings from wells containingquercetin in culture medium were subtracted from the readingsof treated cells. To calculate MTT viability index, absorbancereadings from DMSO treated control wells were set at 100%,and the relative A490 was calculated as a percentage of thecontrol.
BrdU Incorporation Enzyme-Linked ImmunoabsorbentAssay (ELISA)
BrdU incorporation was analyzed by Cell Proliferation Bio-Trak ELISA (GE Healthcare Life Sciences, Piscataway, NJ) ac-cording to the manufacturer’s instructions. For this assay, about5 × 103 cells were seeded per well of 24-well plates. After theywere allowed to adhere for about 12 h, cells were serum de-prived for about 24 h by culturing them in serum-free medium.Cells were then released into serum containing culture mediumand after 3 h treated with quercetin, taxol, or quercetin and
INTERACTION OF QUERCETIN WITH TAXOL AND NOCODAZOLE 1027
taxol. Five hours after the treatment, BrdU labeling reagent wasadded to the culture medium to label those cells synthesizingDNA. Cells were labeled overnight, fixed the next morning, andprocessed for BrdU ELISA as recommended. Absorbance read-ings were taken at 405 nm using PowerWave XS2 plate reader(BioTek).
Cell Monolayer ImmunocytochemistryHCT116 cells were seeded in 4-well chamber slides and
allowed to adhere for about 16 h. Then, cells in each wellwere treated with control (DMSO), single agents (quercetin ortaxol or nocodazole), or a combination of quercetin and taxol orquercetin and nocodazole. Approximately 6 h after treatment,the culture medium was removed, and the cells were fixed in 4%formaldehyde for 15 min at room temperature. The fixed cellswere washed with PBS and processed for immunocytochemi-cal staining at the immunohistochemistry lab of the College ofVeterinary Medicine, Nursing and Allied Health (CVMNAH),Tuskegee University. Cyclin-B1 primary antibody (InvitrogenAHF0062) was used at 15 mg/ml concentration. Peroxidaseconjugated secondary antibody (Envision+ Dual Link System,Dako, Carpinteria, CA) and DAB+ Chromogen (Dako) wereused for the detection. Mayer’s Hematoxylin (Lillie’s modifi-cation, Dako) was used as counter stain. Slides were mountedusing Micromount mounting medium (Surgipath, Richmond,IL) and cover slips.
Immunofluorescent Staining and MicroscopyImages of unstained live cells and immunocytochemically
stained cells were taken at ×20 and ×40 magnification objec-tives using Leica or Olympus microscopes fitted with digitalimage capture cameras (Digital Microscopy Lab, CVMNAH).Photographs saved in TIFF format were directly imported toMicrosoft PowerPoint and cropped or adjusted for brightness,contrast, or grayscale conversion. MCF7 cells for immunofluo-rescent staining were grown in 4-well chamber slides. Stainingwas performed as described (22). Confocal images were takenat the Tuskegee University Research Centers at Minority In-stitutions (RCMI) core facility with an Olympus DSU spinningdisk confocal microscope using ×40 dry objective. Images werecaptured using Metamorph Premium software and further pro-cessed in Adobe Photoshop.
ImmunoblottingCell lysates were prepared in NP-40 lysis buffer (20 mM Tris-
Cl pH 7.5, 150 mM NaCl, 10% glycerol, and 0.2% NP-40 plusprotease inhibitor cocktail), and protein concentrations weredetermined using NanoVue spectrophotometer (GE HealthcareLife Sciences, Piscataway, NJ). Samples containing equivalentprotein concentrations were mixed with Laemmli buffer andboiled for 5 min. Proteins were resolved by SDS-PAGE, trans-ferred to PVDF membranes (GE Healthcare Life Sciences),and blocked in 5% nonfat dry milk. Primary antibodies usedwere rabbit anticyclin-B1 (Invitrogen) at 1:200, rabbit anti-CDK1 (Invitrogen) at 1:500, and β-actin (Cell Signaling) at
1:1000. Peroxidase conjugated antirabbit and antimouse IgGsecondary antibodies were purchased from GE Healthcare LifeSciences and used at 1:5000 dilution. Chemiluminescent de-tections were done using LumiSensor Chemiluminescent HRPSubstrate (Genescript, Piscataway, NJ).
RESULTSWe examined the bioactivity of quercetin singly and in com-
bination with two chemotherapeutic drugs known to act viadisruption of the microtubule dynamics, namely, taxol and noco-dazole. Both drugs induce G2/M arrest as phenotype.
Dose-Dependent Induction of Apoptosis by QuercetinTo examine the apoptosis-inducing activity of quercetin, we
exposed human colorectal tumor HCT116 cells to increasingdoses of quercetin and analyzed the cell cycle profile of the cellsat 24 and 48 h posttreatment. As shown in Fig. 1A, quercetin in-duced apoptosis, which was evident as increased sub-G1 (s-G1)population, most significantly by 48 h of exposure, accompa-nied by reduction in the G2/M population. We also examined theproapoptotic effect of quercetin on an adherent PPC1 prostatecancer cell line. PPC1 cells were treated with 0 to 100 µMquercetin in growth medium. As shown in Fig. 1B, by 48 h ofexposure to 25 µM and 50 µM quercetin, the s-G1 populationof PPC1 cells began to increase. The increase in apoptosis wasconcurrent with the reduction in the proportion of cells at G1as well as G2/M phases of the cell cycle. At the dose level of100 µM, over 40% of the cells were in s-G1 state (apoptotic),indicating that higher doses of quercetin are cytotoxic. Similarresults on the cell death inducing potential of quercetin havepreviously been reported (23). From these data, the bioactivityof quercetin appears to be similar in both colorectal and prostatecancer cells, although the latter seemed to be more sensitive tothe flavonoid.
Inhibition of Microtubule-Acting Drugs by QuercetinBioactive compounds with antioxidant properties have been
suggested to antagonize the activity of chemotherapeutic agentsthat induce oxidative stress (20). However, it is not well knownif flavonoids may enhance or inhibit the activities of otherclasses of anticancer drugs. We investigated the bioactivity ofquercetin in the presence of microtubule-targeting chemothera-peutic drugs. Since quercetin alone induced apoptosis in colonand prostate cancer cells, we hypothesized the cell cycle in-hibitory activity of the antimicrotubule drugs nocodazole andtaxol would be enhanced by cotreatment with quercetin. Totest this, we first examined the effect of combination treat-ment of nocodazole, a microtubule-destabilizing agent, andquercetin on HCT116 colon cancer cells. Adherent wild typeand p53-null HCT116 cells were treated with the carrier alone(DMSO), with single agents (nocodazole 10 µM or quercetin50 µM), or with a combination of both agents. Cell morphologywas examined by microscopy, and cell cycle profile was ana-lyzed by flow cytometry. Whereas HCT116 cells treated with
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FIG. 1. The effect of quercetin (Qctn) on the cell cycle profile of HCT116 colorectal and PPC1 prostate cancer cells. A: HCT116 cells were treated with 10 µMnocodazole (NOC), 100 nM taxol (TAX), or with the indicated concentrations of quercetin for 24 h or 48 h. Cells were harvested and analyzed by flow cytometry.The proportions of cells in each phase of the cell cycle (sub-G1, G1, S, G2/M) for each treatment are indicated in the table. B: PPC1 cells are treated with 0 to100 µM quercetin (as shown) for 24 h. Cells were harvested and analyzed by flow cytometry. Histograms of the cell cycle profiles of the cells are shown on theupper panel. The lower panel shows the proportion of cells in phases of the cell cycle (sub-G1, G1, S, G2/M) for each dose of quercetin. Representative data from2 independent experiments are shown. DMSO, dimethyl sulfoxide.
nocodazole alone were completely rounded as expected, surpris-ingly, cells treated with a combination of quercetin and noco-dazole were morphologically indistinguishable from the controlcells (Fig. 2A–D). HCT116 cells treated with quercetin alonedid not show any major morphological alteration within 24 h.Additionally, flow cytometric analysis showed that whereas 10µM nocodazole induced 70–90% G2/M accumulation of cells,cotreatment with 50 µM quercetin completely abolished theG2/M arrest induced by nocodazole in both wild type and p53-null cells (Fig. 2G–I). Quercetin at 25 µM dose showed moder-ate inhibition of nocodazole activity in wild type cells within 24h. Within this time frame, lower doses of quercetin had neitherinhibitory nor enhancing effects on nocodazole activity (G2/Marrest). Additionally, to assess the inhibitory effect of quercetinon another microtubule-targeting drug, we tested the combi-nation of taxol and quercetin on colon cancer cells. Unlikenocodazole, taxol prevents cell cycle progression by stabiliz-ing the microtubules. We performed similar single (quercetinor taxol) and combination (quercetin and taxol) treatments ofHCT116 cells with the two agents. As with nocodazole, thecells treated with the combination of quercetin and taxol weremorphologically indistinguishable from control DMSO-treated
cells (Fig. 2E, 2F) and displayed cell cycle profile similar to thecontrol cells (not shown). This suggested that quercetin-treatedcells may not have responded to the cell cycle effects of themicrotubule targeting drugs.
To further test that quercetin protected cells from taxol activ-ity, we treated PPC1 prostate cancer cells with taxol or with taxoland quercetin, and examined the cells by flow cytometry. To thisend, we treated the cells overnight with increasing doses of taxol(0–400 nM) with or without cotreatment with 50 µM quercetin.The cell cycle profiles of treated and untreated cells were ana-lyzed by flow cytometry. As with HCT116 cells, cotreatment ofPPC1 prostate cancer cells with quercetin completely abolishedthe prominent G2/M arrest induced by the drug taxol (Fig. 3A,3B).
Since we found that quercetin blocked the cell cycle arrestinduced by nocodazole and taxol, we became interested in ex-amining if the viability of cells treated with the microtubule-acting drugs would be restored by quercetin. To assess this, weperformed MTT assay on singly (quercetin or nocodazole) ordoubly (quercetin and nocodazole) treated cells at 24, 48, and72 h after the treatments. The MTT viability index showed thatquercetin alone in doses above 50 µM reduced the viability of
INTERACTION OF QUERCETIN WITH TAXOL AND NOCODAZOLE 1029
FIG. 2. Quercetin blocks the activity of nocodazole (NOC) and taxol (TAX). HCT116 cells were treated with carrier A: dimethyl sulfoxide (DMSO); B: 50 µMquercetin alone; C: 10 µM nocodazole; D: 10 µM nocodazole plus 50 µM quercetin; E: 100 nM taxol; or F: 100 nM taxol plus 50 µM quercetin. Cells remainedunder treatment for 24 h (A–D) or 16 h (E, F), and phase contrast images were taken at ×200 magnification. G–I: Quercetin inhibits G2/M arrest in HCT116cells. Wild type (G) and p53-null (H) HCT116 cells were treated with DMSO, 50 µM quercetin, 10 µM nocodazole, or the indicated decreasing concentrationsof quercetin in the presence of 10 µM nocodazole as shown. 50 µM quercetin effectively blocked the cell cycle effect of nocodazole on both cell types, whereaslower concentration showed weaker or no inhibition. I: Tabular presentation of the data in G and H.
both wild type and p53-null HCT116 cells (Fig. 4A, 4B). How-ever, doses of quercetin as low as 3.13 µM attenuated the activityof nocodazole, whereas nocodazole (10 µM) alone reduced theviability of the treated cells (Fig. 4C, 4D). At 72 h after treat-
ment, the viability index of nocodazole treated HCT116 cellswas about 65%, whereas the viability index of cells treated withnocodazole plus 50 µM quercetin was comparable to that of thecarrier treated control cells. Quercetin at 100 µM dose was less
FIG. 3. Quercetin inhibits the activity of taxol on PPC1 prostate cancer cells. PPC1 cells were treated with A: 0–400 nM taxol as shown or B: a combination of0–400 nM taxol and 50 µM quercetin, and incubated for 12 h. Cells were harvested and analyzed by flow cytometry. The histograms in upper panels show the cellcycle profiles of the cells, and the lower panels (tables) show the numerical proportion of cells in each phase of the cell cycle for each treatment in A and B. Oneof 3 independent experiments is shown. Qctn, quercetin.
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FIG. 4. Quercetin maintains the viability of colorectal cancer cells treated with nocodazole but delays cell cycle progression. A–D: Effect of quercetin orquercetin-nocodazole combination on the viability of HCT116 cells. Wild type (WT) or p53-null HCT116 cells were treated with A,B: quercetin alone or with C,D:combinations of 10 µM nocodazole and increasing doses of quercetin as shown. Cell viability was measured after 24, 48, and 72 h by MTT assay. Cell viability isplotted as MTT index, relative to that of the control dimethyl sulfoxide (DMSO)-treated cells. E: Bromodeoxyuridine (BrdU) uptake in wild type HCT116 cellstreated with DMSO, 100 nM taxol, 50 µM quercetin, or a combination of taxol and quercetin was measured by BrdU incorporation ELISA. Relative BrdU uptakeis shown as a percentage of uptake by the control cells. The difference in BrdU incorporation between taxol, quercetin, and combination treated cells was notsignificant. F: RKO colorectal cancer cells were synchronized by double thymidine (2 mM) block and released into growth medium containing DMSO control(Contr.) or quercetin (Qctn). Aliquots of cells growing asynchronously or at different time points [at release (t0), 2 h, 4 h, or 9 h] after release from the block wereanalyzed by flow cytometry. Cell cycle profiles are shown as histograms in the top panels (E); and the proportion of cells in G1, S, or G2 at the time points areshown in the lower panels (F: tables). Cells exposed to quercetin medium showed considerable delay (underlined values) in cell cycle progression compared tocontrol cells.
INTERACTION OF QUERCETIN WITH TAXOL AND NOCODAZOLE 1031
protective than 50 µM, suggesting the cytotoxicity of quercetinat higher doses.
To assess if the viability of cells treated with quercetin andtaxol was accompanied with cell cycle progression, we per-formed BrdU incorporation assay as an indicator of cellularDNA synthesis and analyzed BrdU incorporation in singly orcombination treated cells. As shown in Fig. 4E, cells treatedwith the combination of taxol and quercetin incorporated BrdUto a degree comparable to singly treated cells. Therefore, itappears that the sustained viability of quercetin-taxol combina-tion treated cells may not necessarily be accompanied by DNAreplication but by steady state maintenance of viability.
To further test the effect of quercetin on cell cycle progres-sion, we synchronized HCT116 cells at G1-S boundary by thedouble thymidine block method and released them into culturemedium containing 50 µM quercetin. Progression of the re-leased cells through the cell cycle was assessed by flow cytome-try of cells harvested at different time points after the release. Wefound that cells released into quercetin medium showed markeddelay in cell cycle. By 9 h after release, most cells in the controlmedium were in G1 phase of the next cell cycle, whereas mostof the cells in quercetin medium were still in S-G2 phase of thefirst cell cycle after the release (Fig. 4F).
Quercetin Does Not Interfere With MicrotubuleTargeting of Taxol and Nocodazole
The inhibition of the activity of taxol and nocodazole byquercetin led us to speculate that quercetin might interfere withthe uptake, intracellular distribution, or microtubule targetingof the two drugs. To rule out this possibility, we examinedthe α-tubulin architecture in MCF7 cells treated with taxol ornocodazole in the presence or absence of quercetin. Similar toHCT116 and PPC1 cells, treatment of MCF7 cells with taxoland nocodazole in the presence of quercetin also resulted inabsence of G2/M arrest of the cells. However, unlike HCT116and PPC1 cells, 50 µM and 25 µM quercetin were cytotoxicto MCF7 cells, whereas 12.5 µM was protective against theG2/M arrest of cells (not shown). Confocal images of cellsimmunostained for α-tubulin showed that in the presence ofquercetin, nocodazole and taxol were still able to destabilizeor stabilize microtubules, respectively (FIG. 5). Because thedrugs target microtubule dynamics in the presence of quercetin,we conclude that the absence of G2/M arrest of combination-treated cells is not due to lack of uptake or increased efflux ofthe antimicrotubule drugs.
Taxol/nocodazole and Quercetin Combination TreatmentPrevents Accumulation of Cyclin-B1 at the MicrotubuleOrganizing Center (MTOC)
As shown above, cells treated with quercetin and taxol orquercetin and nocodazole did not accumulate at the G2/M phaseof the cell cycle. Since mitotic entry is regulated mainly bythe cell cycle dependent subcellular dynamics and stability ofcyclin-B1 and its partner CDK1 through the MTOC (24), we
FIG. 5. Quercetin does not interfere with microtubule targeting of taxol andnocodazole. MCF7 cells were treated for 16 h (overnight) with carrier [dimethylsulfoxide (DMSO)], quercetin (Qctn; 10 µM), taxol (TAX; 50 nM), nocodazole(NOC; 10 µM) or combinations of taxol and quercetin (TAX + Qctn), ornocodazole and quercetin (NOC + Qctn) as shown. Cells were then fixedand immunofluorescently stained for tubulin (upper row). 4’,6-diamidino-2-phenylindole (DAPI) was used as a counterstain for nuclei (middle row). Mergedimages (tubulin and DAPI) are shown in the bottom row. Confocal images weretaken using a ×40 dry objective.
examined the localization of these proteins in HCT116 cellstreated singly with quercetin or taxol or nocodazole or by acombination of quercetin and taxol or quercetin and nocodazolefor 8 h. Monolayers of HCT116 cells grown in chamber slideswere immunohistochemically stained using an antibody againstcyclin-B1. Interestingly, combination-treated cells showed weakto no detectable accumulation of cyclin-B1 at the MTOC in con-trast to those cells treated with either the drugs or quercetin alone(Fig. 6A). This indicates that the lack of cell cycle arrest by taxoland nocodazole in the presence of quercetin is accompanied bythe absence of proper mobilization of cyclin-B1–CDK complexto the MTOC to initiate mitosis. However, since the cells did notaccumulate in S-phase, combination treated cells could also beblocked at other phases of the cell cycle. Indeed, as shown above(Fig. 4E), cells treated with quercetin alone or quercetin-taxolcombination did not incorporate BrdU more than taxol treatedcells, suggesting quercetin treatment may have stalled the pro-gression of the cell cycle also before the S-phase. The decreasein the levels of cyclin-B1 in combination-treated cells was alsoconfirmed by immunoblotting. Whereas taxol-treated cells ac-cumulated cyclin-B1 as expected, taxol-quercetin treated cellshad markedly low levels of cyclin-B1 (Figs. 6B).
Quercetin Inhibits Colony Formation of Both Wild Typeand p53-Null Colorectal Tumor Cells
It is estimated that more than 50% of human cancers carryp53 protein mutations, almost all of which have been cataloged(25,26). As p53 is also a key protein regulating the apoptoticand cell cycle signaling, we became interested to examine if theantiproliferative activity of quercetin would be dependent on thep53 status of colon cancer cells.
To address this, we exposed wild type and the isogenic p53-null human colorectal tumor HCT116 cells to varying con-centrations of quercetin and examined growth of the cells by
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FIG. 6. Treatment of HCT116 cells with a combination of quercetin and taxol disrupts the localization of cyclin-B1 at the MTOC. A: HCT116 wild typecells grown in chamber slides were exposed to dimethyl sulfoxide (DMSO), 50 µM quercetin (Qctn), 100 nM taxol (TAX), or 50µM quercetin and 100 nMtaxol combination (TAX + Qctn). After 8 h of treatment, cell monolayers were stained with anticyclin-B1 antibody by immunocytochemistry. Arrows indicatethe localization of cyclin-B1 at the MTOC. B: HCT116 cells grown in 6-cm diameter dishes were treated with DMSO, 50 µM quercetin, 100 nM taxol, or acombination of 50 µM quercetin and 100 nM taxol for 8 h. Cell lysates were prepared as described in Materials and Methods. Cyclin-B1, CDK1, and β-actinproteins were detected by immunoblotting. ∗, indicates a non-specific band.
colony formation assay. Both wild type and p53-null cells wereseeded in the presence of 0–100 µM concentrations of quercetinunder two different conditions. In one instance, the cells wereallowed to adhere for overnight before adding quercetin; andunder the second instance, the dissociated cells were seeded inthe presence of quercetin. Growth medium was replaced at 72-hintervals with a fresh supplementation of quercetin at the sameconcentration as the initial dose.
As shown in Fig. 7A, 7B, long-term exposure to quercetin(50 µM or more) inhibited colony formation in both p53 positiveand negative cells at a comparable dose, which suggests thatthe long-term cell proliferation inhibitory effect of quercetinprobably does not require cellular p53. Moreover, the same doseof quercetin (50 µM) that abrogated the G2/M arrest by taxol andnocodazole also inhibited colony formation by HCT116 cells.Additionally, we observed that both wild type and p53-null cellswere more sensitive to the activity of quercetin when the cellswere seeded in the presence of the flavonoid. Whereas 50 µMquercetin was needed to inhibit colony formation of adherentHCT116 cells, 12.5 µM quercetin was sufficient to achieve aneven stronger inhibition of colony formation of both wild typeand mutant cells when they were treated before they adhered tothe culture dishes.
To examine if quercetin provided long-term survival advan-tage to cancer cells exposed to antimicrotubule drugs, we per-formed clonogenicity assays on wild type HCT116 cells treatedwith only quercetin or a combination of quercetin and taxol. Thenumbers of clonal colonies formed and the number of cells percolony were compared. As shown in Fig. 7C and 7D, quercetindoses (25 µM and 50 µM) that interfered with taxol and nocoda-zole still inhibited the clonogenicity of HCT116 cells. Moreover,the number of cells per colony was lower in cells treated with
12.5 µM or higher quercetin, compared to control cells, suggest-ing that quercetin may have interfered with cell cycle progres-sion and therefore limited the rate of cell proliferation or sur-vival. When we tested the clonogenicity of HCT116 cells treatedwith 25 µM quercetin and taxol (0.6–5 nM) combinations, weobserved that quercetin provided no clonogenicity advantageto cells. On the contrary, the combination of quercetin withtaxol consistently suppressed the clonogenic survival of treatedcells and sensitized the cells to lower doses of taxol, which didnot inhibit clonogenic survival. Cells treated with 1.25 nM and0.6 nM taxol retained clonogenicity, whereas combination of25 µM quercetin with the same doses of taxol markedly inhib-ited clonogenic survival of the cells (Fig. 7E).
DISCUSSIONWe have found that quercetin, a ubiquitous flavonoid abun-
dantly available in green vegetables and fruits, has pleiotropic ef-fects on cancer cell survival as a single agent and when combinedwith conventional chemotherapeutic drugs that target the micro-tubules. Although we initially predicted that quercetin wouldenhance the activity of taxol or nocodazole, we unexpectedlyfound that quercetin antagonized the G2/M arrest induced byboth drugs. We also found that even in the presence of quercetin,the uptake of nocodazole or taxol was not inhibited, as shownby the distinctive effects of the drugs on the microtubules. Theantagonistic activity of quercetin on taxol and nocodazole wasaccompanied by the absence of recruitment of cyclin-B1 to theMTOC in combination-treated cells. Cyclin-B1 and CDK1 arepartner proteins crucial for mitotic entry (23). At the end of theS phase, cyclin-B1 protein level is elevated, cyclin-B1–CDKcomplexes are formed, and the CDK component is activated.
INTERACTION OF QUERCETIN WITH TAXOL AND NOCODAZOLE 1033
FIG. 7. Continued exposure of HCT116 cells to quercetin inhibits colony formation. A: Wild type (wt) and p53-null HCT116 cells were seeded in 12-well cellculture dishes and allowed to adhere to the plate for about 16 h. Adherent cells were treated with the indicated concentrations of quercetin and colony formation wasexamined over 8 days as described in Materials and Methods. B: Wild type and p53-null HCT116 cells were seeded in 12-well cell culture dishes in the presenceof the indicated concentrations of quercetin (Qctn) in culture medium. Colony formation was examined as described. C–E: Quercetin does not provide lastingclonogenicity and survival advantage to HCT116 cells. Clonogenicity of HCT116 cells exposed to 6.25 µM to 100 µM quercetin was examined by clonogenicityassay (21). The colonies that formed after the treatments, and the number of cells per colony for each treatment, are shown in C and D, respectively, relative tothe numbers from control cells. Doses of quercetin that antagonized taxol or nocodazole still inhibited clonogenic survival of the cells. E: Clonogenic survivalof HCT116 cells treated with quercetin (25 µM) or quercetin in combination with taxol (0.6 nM to 5 nM). Clonogenicity of the cells is shown as the number ofcolonies that formed relative to the control dimethyl sulfoxide (DMSO) treatment. Quercetin in combination with taxol provided no clonogenic advantage; on thecontrary, combination treated cells had the poorest clonogenic survival.
Activated cyclin-B1–CDK complex phosphorylates substrateproteins, including those at the MTOC, to drive cells into mito-sis. We propose that quercetin’s interference with the cell cycleprogression inhibits the activity of the two microtubule-actingdrugs to arrest cells at G2/M.
Although we found that quercetin interfered with the mitoticarrest induced by microtubule-targeting drugs, we did not findevidence to suggest that the cells continue to synthesize DNAand proliferate when combination treated. Indeed, quercetin byitself inhibited the long-term growth and survival of cells at thesame concentrations that interfered with antimicrotubule drugs.Although our in vitro observations are limited, our data suggestthat the continued presence of quercetin in the cellular environ-ment may attenuate the activity of microtubule acting agentsin the short run. Since the viability of cells in the presence ofmicrotubule disrupting drugs was maintained even by low con-centration of quercetin (3.13 µM or higher in our study), thecoadministration of quercetin during treatment with antimicro-tubule agents such as paclitaxel may diminish drug activity. Invivo studies need to be performed to elucidate the relevance ofthis interference. However, our clonogenic assays suggest thatlong-term administration of high doses of quercetin alone or
even low doses of quercetin in combination with taxol may notpromote the clonogenic survival of colorectal cancer cells.
Current thought on the bioactivity of quercetin and otherflavonoids is that these compounds act by scavenging free rad-icals induced by endogenous and exogenous pro-oxidants (27).These pro-oxidant agents include DNA damaging chemothera-peutic drugs and irradiation. However, recent studies have sug-gested that polyphenolic compounds and antioxidants may an-tagonize diverse groups of chemotherapeutic drugs. Liu et al.(28) showed that dietary flavonoids, especially quercetin, inhibitbortezomib-induced apoptosis in malignant B-cell lines and pri-mary chronic lymphocytic leukemia (CLL) cells by direct asso-ciation with bortezomib. The authors (28) also found that the in-hibitory effect of quercetin was abolished by boric acid, therebyrestoring the apoptotic effect of bortezomib on CLL cells. Simi-larly, Golden et al. (29) found that green tea polyphenols blockedthe activities of bortezomib and other boronic acid-based pro-teasome inhibitors through direct interference. Our data addstaxol and nocodazole to the list of drugs potentially antagonizedby quercetin.
It is not clear, however, if the antioxidant propertiesof flavonoids explain all of such antidrug bioactivity. For
1034 T. SAMUEL ET AL.
example, a recent study on vitamin C—another antioxidant di-etary compound—showed that vitamin C significantly atten-uated the activity of diverse classes of chemotherapeutic com-pounds such as doxorubicin, cisplatin, vincristine, methotrexate,and imatinib, independent of its antioxidant potential (30). Thechemotherapeutic compounds used in the study and found to beinhibited by vitamin C are known to target cellular DNA, thecytoskeleton, or diverse cell signaling mechanisms.
These results and our data suggest that compounds such asquercetin, other polyphenols, and vitamin C may have hith-erto unknown bioactivities that may be independent of theirantioxidant properties. Competitive interference of polyphe-nols with bortezomib for proteasome inhibition has been doc-umented (28,29), but mechanisms of antagonism of polyphe-nols against other drugs remain unknown. In the cases of taxoland nocodazole, the effects of quercetin do not appear to stemfrom the inhibition of uptake of the drugs. Also, unlike borte-zomib, the two drugs are not known to directly target the pro-teasome, excluding the possibility of competitive proteasomalinhibition. Therefore, it is possible that the cell cycle inhibitoryeffects of quercetin and the resulting lack of cycling cellsmay explain the antagonistic effect of quercetin on taxol andnocodazole.
We also observed that the bioactivity of quercetin varieswith the adherence status of the treated cells. In colony forma-tion assay, nonadherent colon carcinoma cells were inhibitedby a dose of quercetin fourfold less than that required for theadherent cells. This observation, together with lack of a ma-jor difference between p53 wild type and p53-null HCT116cells, suggests that the adherence status rather than the p53status renders tumor cells more sensitive to the bioactivity ofquercetin. Moreover, the observation that adherent cell lines arealso more sensitive to quercetin before they attach to surfacessuggests that the mechanisms and pathways that support cellattachment may confer a degree of resistance to the growthinhibitory effects of quercetin. This in turn may imply thatcells may be more sensitive to the actions of the flavonoidquercetin if they are detached from their anchor, as it may occurduring metastasis. However, this possible mechanism of ac-tion can’t explain the cancer preventive activities of flavonoidssuch as quercetin because metastatic events occur during laterstages of oncogenesis. The chemopreventive mechanisms ofdietary levels of quercetin and other flavonoids remain to beelucidated.
In conclusion, quercetin appears to have a bimodal bioactivityin which it may provide a short-term transient survival benefitto cells exposed to taxol and nocodazole, but it has a long-termanticell proliferative effect. The antiproliferative effects appearto be strong especially when the cells have lost their attachmentto the growth matrix. Although quercetin attenuated the cellcycle effects of taxol and nocodazole in the short term, weobserved diminished survival and clonogenicity of cancer cellsexposed to combinations of quercetin and taxol, which suggestsno long lasting antagonistic effects. Further studies are needed
to examine the in vivo effects of coadministration of quercetinor other flavonoids with microtubule-acting drugs.
ACKNOWLEDGMENTSWe thank Tsegaye Habtemariam, Cesar Fermin, and Fred-
erick Tippett for support through HRSA/COE D34HP00001-22-00; Sibyl Bowie for editorial assistance; John Williams fortechnical assistance at the Tuskegee University RCMI imagingcore facility; John Heath, Clayton Yates, Starlette Sharp, and Pa-tricia Adams for various technical support and advice. We thankBert Vogelstein for HCT116 cells, John Reed for PPC1 cells,and Leslie Wilson for MCF7 cells. We acknowledge the researchtraining support by the MSM/TU/UABCC Cancer Partnershipto T. Samuel. This work was supported by NIH/NCI/NIGMSgrant 1SC2CA138178 and U54 CA 118623. The authors haveno conflict of interest to disclose.
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