cyclin a2 and cdk2 as novel targets of aspirin and ... · cell cycle and senescence cyclin a2 and...

Cell Cycle and Senescence

Cyclin A2 and CDK2 as Novel Targets of Aspirinand Salicylic Acid: A Potential Role in CancerPreventionRakesh Dachineni, Guoqiang Ai, D. Ramesh Kumar, Satya S. Sadhu,Hemachand Tummala, and G. Jayarama Bhat

Abstract

Data emerging from the past 10 years have consolidated therationale for investigating the use of aspirin as a chemopreventiveagent; however, themechanisms leading to its anticancer effects arestill being elucidated. We hypothesized that aspirin's chemo-preventive actionsmay involve cell-cycle regulation throughmod-ulation of the levels or activity of cyclin A2/cyclin-dependentkinase-2 (CDK2). In this study, HT-29 and other diverse panel ofcancer cells were used to demonstrate that both aspirin and itsprimary metabolite, salicylic acid, decreased cyclin A2 (CCNA2)andCDK2 protein andmRNA levels. The downregulatory effect ofeither drugson cyclinA2 levelswas preventedbypretreatmentwithlactacystin, an inhibitor of proteasomes, suggesting the involve-ment of 26Sproteasomes. In-vitro kinase assays showed that lysatesfrom cells treated with salicylic acid had lower levels of CDK2activity. Importantly, three independent experiments revealed thatsalicylic acid directly binds to CDK2. First, inclusion of salicylic

acid in na€�ve cell lysates, or in recombinant CDK2 preparations,increased the ability of the anti-CDK2 antibody to immunopre-cipitate CDK2, suggesting that salicylic acid may directly bind andalter its conformation. Second, in 8-anilino-1-naphthalene-sulfo-nate (ANS)-CDK2 fluorescence assays, preincubation of CDK2with salicylic acid dose-dependently quenched the fluorescencedue to ANS. Third, computational analysis using molecular dock-ing studies identified Asp145 and Lys33 as the potential sites ofsalicylic acid interactions with CDK2. These results demonstratethat aspirin and salicylic acid downregulate cyclin A2/CDK2 pro-teins in multiple cancer cell lines, suggesting a novel target andmechanism of action in chemoprevention.

Implications: Biochemical and structural studies indicate that theantiproliferative actions of aspirin are mediated through cyclinA2/CDK2. Mol Cancer Res; 14(3); 241–52. �2015 AACR.

IntroductionEvidence from epidemiologic studies has demonstrated a sig-

nificant correlation between regular aspirin use and reducedcancer incidence and mortality. This inverse correlation is nowestablished for the cancers of the colon (1–3), breast, prostate,lung, and skin (4–7). Animal and human studies also support arole for aspirin in chemoprevention. Aspirin suppresses aberrantcrypt foci formation in colorectal cancer patients (8). Its use afterthe colorectal and breast cancer diagnosis was associated with adecreased risk and increased patient survival (9, 10). The evidencethat aspirin prevents cancer is compelling; however, the under-lying mechanisms leading to its anticancer effect is enigmatic as

numerous protein targets and pathways have been suggested.Which of these primarily contributes to its anticancer effects isnot clear; however, one widely accepted mechanism is the inhi-bition of COX-1 and COX-2 by acetylation leading to decreasedprostaglandin synthesis (2). Interestingly, several COX-indepen-dent pathways have been proposed (2, 11). We and others havedemonstrated that aspirin acetylates multiple cellular proteins,which further gives it the potential to affect simultaneouslymultiple cellular pathways (12–17). Additional mechanismsdescribed include inhibition of NF-kB (18), inhibition of Wnt/b-catenin pathway (19), downregulation of Sp transcriptionfactors (20), and inhibition of mTOR signaling (21) amongothers. Thus, aspirin affects multiple pathways rather than onesingle target; thus, the broadly-specific nature of its actionmay bethe key to its chemopreventive properties.

Aspirin is mainly absorbed intact in the gastrointestinal tractand later hydrolyzed to acetate and salicylate ions in the plasma,liver, and within the cell. The plasma concentration of salicylicacid obtained from the hydrolysis of analgesic and anti-inflam-matory doses of aspirin can vary from0.5 to 2.5mmol/L (11), andits half-life is approximately 4.5 hours. Some studies showed thatsimilar to aspirin, salicylic acid also exhibits potent antiprolifera-tive and antitumor activity in vitro and in vivo (20, 22, 23). Thus,the contribution of the salicylic acid to aspirin's anticancer effectscannot be discounted.

Cyclins control the progression of cells through the cell cycleby physically interacting and activating cyclin-dependent kinase

Department of Pharmaceutical Sciences and Translational CancerResearch Center, South Dakota State University College of Pharmacy,Brookings, South Dakota.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Current address for D. Ramesh Kumar: Aquatic Animal Health Division, CIBA,Chennai, India.

CorrespondingAuthor:G. JayaramaBhat, SouthDakota StateUniversity Collegeof Pharmacy, 1 Administration Lane, Box 2202C, Brookings, 57007. Phone: 605-688-4090; Fax: 605-688-5993; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-15-0360

�2015 American Association for Cancer Research.

MolecularCancerResearch

www.aacrjournals.org 241

on August 19, 2019. © 2016 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst December 18, 2015; DOI: 10.1158/1541-7786.MCR-15-0360

http://mcr.aacrjournals.org/

(CDK) enzymes (24). The cell cycle is regulated by multiplecyclins such as A, B, D, and E; CDKs such as 1, 2, 4, and 6; andCDK inhibitors such as p16, p21, and p27. Several isoforms alsoexist for cyclin familymembers; for example, humans contain twodistinct types of cyclin A: cyclin A1, the embryonic specific form;and cyclin A2, the somatic form. Cyclin A2 can activate twodifferentCDKs:CDK1andCDK2 (25). Its levels are lowduring theG1 phase, increase at the onset of S phase, and remain high duringG2 and early mitosis. By associating with CDK2 during the Sphase, it regulates DNA synthesis through phosphorylation ofproteins involved in DNA replication. Cyclin A2 is also importantduring the G2 to M phase transition (26). During early mitosis, itassociates with CDK1 and drives chromosome condensation andnuclear envelope breakdown (27). It is degraded during prome-taphase throughubiquitination by the anaphase-promoting com-plex/cyclosome (APC/C; ref. 28).

In this article, we focused our study on cyclin A2 and its bindingpartner CDK2 because, first, they regulate DNA synthesis duringthe S phase; second, both proteins are deregulated or upregulatedin breast, liver, and lung cancers (29–33). In addition, there hasbeen significant interest in targeting cell cycle through inhibitionof CDK2 activity as a strategy to treat cancer (34–36). Becauseaspirin is known to inhibit cell proliferation, we hypothesizedthat its anticancer effects may involve downregulation of cyclinA2/CDK2 proteins or their mRNA levels or both. Our goal in thisresearch paper was to study the effect of aspirin and salicylic acidon cyclin A2/CDK2 in multiple cancer cell lines representingcancers of various tissues, such as colon, breast, lung, skin,prostate, and ovary, which would also establish the universalityof the observation. Here, we report that cyclin A2 and CDK2 arenovel targets of aspirin and salicylic acid, as both drugs causedtheir downregulation in a concentration-dependent fashion in thehuman colon cancer HT-29 and also in 10 other cancer cell lines.Aspirin- and salicylic acid–mediated decrease in cyclin A2 proteinlevels required a lactacystin-sensitive protease. Both drugs causeda decrease in exogenously expressed DDK-tagged cyclin A2 levels.Moreover, cells treated with aspirin and salicylic acid had reducedamounts of cyclin A2 and CDK2 mRNA levels. The decrease incyclin A2/CDK2 protein levels was associated with a decrease inCDK2 kinase activity. Through anti-CDK2 antibody immunopre-cipitations, molecular docking studies, and CDK2-ANS (8-ani-lino-1-naphthalene sulfonate) fluorescence assay, we show thatsalicylic acid binds and interacts with Asp145 and Lys33 in theCDK2 protein. Our results show that aspirin and salicylic acidregulate cyclin A2 gene expression at the transcriptional/posttran-scriptional and posttranslational levels. We suggest that down-regulation of the cyclin A2/CDK2 mRNA and protein levels mayrepresent one important mechanism by which aspirin exerts itsanticancer effect via the formation of salicylic acid.

Materials and MethodsMaterialsCell lines. HCT 116, HT-29, SW480 (human colon cancer cells);SK-MEL-28 and SK-MEL-5 (human skin melanoma cells), MDA-MB-231 and MCF7 (human Breast cancer cells); NCI-H226(human lung cancer cells); OVCAR-3 (human ovarian cancercells); PC-3 (human prostate cancer cells); and B16-F10 (mousemelanoma cells) were purchased from the American Type CultureCollection (ATCC). Authentication of cell lines was done by theATCC through their DNA-STR profile.

Reagents. Aspirin, salicylic acid, trypsin-EDTA solution were pur-chased from Sigma; SuperSignal West Pico ChemiluminescentSubstrate and protease inhibitor tablets from Thermo Scientific;lactacystin, Immobilon membranes, and H1 Histones from EMDMillipore; FuGENE HD Transfection Reagent from Promega;protein G agarose, Halt Phosphatase Inhibitor Cocktail, GeneJETGel Extraction Kit, and Mlu I and EcoR I restriction enzymes fromLife technologies; TRIzol reagent from Ambion; [g-32P] ATPand [a-32P] dCTP from MP Biochemical; Random Primer DNAlabeling Kit from Clontech; Zeta probe blotting membranes fromBio-Rad; and all other chemicals were obtained from ThermoFischer Scientific.

Recombinant proteins, plasmidDNA, and antibodies. Recombinanthuman CDK2 was obtained from Prospec; Myc-DDK-tagged(C-terminus) human cyclin A2 (CCNA2) and anti-DDK antibodywere obtained from OriGene; anti-cyclin A2 and anti–b-actinantibodies from Cell Signaling Technology; anti-cyclin A2 anti-body was from Abcam, anti-CDK2 antibody from EMDMilliporeor Santa Cruz Biotechnologies; and goat anti-rabbit and goatanti-mouse antibodies were obtained from Bio-Rad.

MethodsCell culture. The cells were grown in an appropriate ATCC-recommended medium containing 10% FBS for 24 to 48 hoursbefore adding aspirin or salicylic acid for indicated times.

Cell proliferation. Approximately 100,000 cells were seeded per100 mm plates containing 10% FBS and grown overnight. Drugswere added at various concentrations and incubated for 48 hours.The floating cells (if any) were collected from the conditionedmedia, pooled with the trypsinized adhered cells, and counted intheNexcelomCellometer Auto T4 cell counter. The viability of thecells was determined by trypan blue staining.

Total cell lysate preparation, immunoprecipitation, and Westernblotting. Cells were treated with aspirin or salicylic acid atdifferent concentrations for the indicated time and washedwith PBS. Cells were scraped in lysis buffer (10 mmol/L Tris-Cl, pH 7.4, 150 mmol/L NaCl, 15% glycerol, 1% Triton X-100with protease inhibitors). Samples containing 50 mg of proteinswere separated by an 8% or 10% PAGE and immunoblottedwith respective antibodies. For immunoprecipitations, 500 mgof the total proteins isolated from cells, or 300 ng of therecombinant CDK2 protein, were diluted to 1 mL of lysisbuffer, immunoprecipitated with anti-CDK2 antibodies over-night at 4�C, the immune complex was captured by adding 35mL of protein G agarose for 3 hours. The immunocomplexeswere washed 3 times with PBS and dissolved in SDS-samplebuffer. The samples were analyzed on a SDS-PAGE, immuno-blotted with either anti-cyclin A2 antibody or anti-CDK2 anti-body. Immunoreactive bands were detected using chemilumi-nescence reagents. The intensities of bands were determinedusing NIH ImageJ software.

Expression of recombinant DDK-tagged proteins. Cells were seededon a 100 mm plate at 50% confluency overnight and transfectedwith 3 mg of recombinant cyclin A2 or CDK2 plasmids usingFuGENE HD Transfection Reagent (Promega, Inc.). The cellswere incubated for 24 to 48 hours to allow for the expressionof recombinant proteins. Cell lysates were prepared and

Dachineni et al.

Mol Cancer Res; 14(3) March 2016 Molecular Cancer Research242




immunoblotted with either anti-DDK antibodies, or anti-cyclinA2 or anti-CDK2 antibodies.

Northern blot analysis. The pCMV6-Entry vector carrying full-length cyclin A2 and CDK2 plasmid DNA was digested with MluIand EcoRI to release the cDNA insert and the DNA purified. TotalRNA was extracted from cells untreated or treated cells with drugsusing TRIzol reagent, andNorthernblot analysiswas carriedout aspreviously described (14). The blots were washed with 0.1� SSCbuffer containing 0.1% SDS for 1 hour at 65�C, dried and exposedto X-ray film.

In-vitroCDKassay.TheCDKassaywas performed according to thepreviously publishedmethod (37). In brief, 500 mg of the proteinfrom cell lysates was diluted with 1 mL of the lysis buffer andimmunoprecipitated using anti-CDK2 antibody followed by theaddition of protein G agarose as described above. After washing 3times with lysis buffer, the immunocomplexes were washed twicewith lysis buffer containing no Triton X-100 and once with kinasebuffer (40 mmol/L Tris-HCl, pH 8.0, 5 mmol/L MgCl2, and 5%glycerol). The final pellet was suspended in kinase buffer contain-ing 20 mmol/L ATP, 2mci of [g-32P] ATP, 5mg ofH1Histone, 0.5�phosphatase inhibitor, and incubated for 30minutes at 37�C. Thereaction was stopped by the addition of SDS-sample buffer andloaded on a 10% SDS-PAGE, gel stained with Coomassie blue,and dried and exposed to X-ray film.

Molecular docking studies. An in-silico approach was adopted toidentify potential target inhibitors through molecular dockingstudies. In an attempt to understand the ligand–protein interac-tions in terms of binding affinity, aspirin and salicylic acid weresubjected to docking with CDK2 using AutoDockVina. The small-molecule topology generator Dundee PRODRG2 server (38) wasused for ligand optimization. The crystallographic three-dimen-sional structures of selected target proteins (PDB ID: 1FIN (2.30Å)were retrieved from the Protein Data Bank (PDB) http://www.pdb.org. The human cyclin A2 (PDB ID: 1FIN B chain), CDK2(PDB ID: 1AQ1), and cyclin A2/CDK2 complex (PDB ID: 1FINA, B chain) protein molecule was selected for energy minimi-zation using Gromacs 3.3.1 package with the GROMOS96 forcefield (39). These molecules were used as the receptor for virtualsmall molecule docking with the ligand aspirin and salicylicacid using AutoDockVina. Python molecular viewer with Auto-Dock Tools was used for conversion to pdbqt format, requiredby AutoDockVina.

CDK2/ANS fluorescence assay. The CDK2/ANS assay is based onthe fluorescence emitted from the interaction of ANS within theallosteric pocket of CDK2 (40). For the assays, the previouslyrecommended concentrations of ANS and CDK2 at 50 mmol/Land 1.6 mmol/L (0.05 mg/mL), respectively, were used. Commer-cially obtained recombinant CDK2 protein was mixed with ANSin a total volume of 50 mL in a 96-well plate, and the fluorescencewas measured at excitation and emission wavelengths of 405 and460 nmusing a SpectramaxM2 spectrophotometer. Alternatively,recombinant CDK2 was first preincubated with salicylic acid atdifferent concentrations before the addition of ANS, and then thefluorescence was measured.

Statistical analysis. All experiments were repeated 3 to 6 timesindependently of each other. A one-way ANOVA followed by

Tukey range tests was adopted to compare group differences tocontrol, and significance was defined as P < 0.05.

ResultsAspirin and salicylic acid decrease cell proliferation in HT-29,SK-MEL-28, and MDA-MB-231 cells

Previous studies have shown that treatment of cells with aspirinand salicylic acid, at concentrations ranging from 2.5 mmol/L to10mmol/L, induced a profound reduction in cell proliferation inHT-29 and other cancer cells (20, 41, 42), but a comparison atlower concentrations was not reported. In this experiment, wechose HT-29 (colon), SK-MEL-28 (skin), and MDA-MB-231(breast) cancer cells to evaluate the effect of aspirin and salicylicacid on proliferation rate at the concentrations ranging from 0.25to 2.5 mmol/L. Cells were treated separately with both drugs for48 hours, trypsinized and counted. We observed that aspirin andsalicylic acid progressively reduced the cell number particularlyfrom0.5mmol/L to 2.5mmol/L (Supplementary Fig. S1). The cellviability was unaffected at all concentrations of the drugs tested.These results show that both drugs are effective in reducing the cellproliferation rate upon exposure for 48 hours, without affectingthe viability.

Effect of aspirin and salicylic acid on cell-cycle–regulatoryproteins

We hypothesized that aspirin and salicylic acid may exert theirantiproliferative effects through modulation of cell-cycle regu-latory proteins. Therefore, we sought to determine whether thesedrugs would affect the levels of cyclins A, B, D, and E; CDKs 1, 2,4, and 6; and CDK inhibitors p16, p21, and p27. To address this,HT-29 cells were left untreated or treated with aspirin or salicylicacid at various concentrations for 24 hours. Cell lysates wereprepared and immunoblotted with various anti-cyclin, anti-CDK, and anti-CDK inhibitor antibodies. We observed that bothaspirin and salicylic acid down regulated the levels of cyclins A2,B1, and D3 and CDKs 1, 2, 4, and 6. Interestingly, both drugsupregulated the levels of cyclin E1 as well as CDK inhibitors, p27and p21. The levels of p16were not detected in these experimentspossibly reflecting the lower expression. The data on the effect ofaspirin and salicylic acid on cyclin A2 in HT-29 cells are shownin Fig. 1A and B. The results obtained on CDK2 are discussedelsewhere in Fig. 4 (see below). The data on cyclins B1, E1, andD3 are shown in Supplementary Fig. S2; CDKs 1, 4, and 6 inSupplementary Fig. S3; and CDK inhibitors 21 and p27 inSupplementary Fig. S4. It is clear from these results that aspirinexposure toHT-29 cells causes differential regulation of cell-cycleregulatory proteins. Among these identified protein targets, wefocused mainly on cyclin A2 and CDK2 in the present studybecause (a) they play an important role in the regulation of DNAsynthesis during cell-cycle progression and (b) they are deregu-lated or upregulated in several cancers such as breast, liver, andlung (29–33). We hypothesized that aspirin and salicylic acidmay primarily target cyclin A2/CDK2 to cause the cell-cyclearrest, which has been previously reported by other investigators(20, 41, 42).

Aspirin and salicylic acid decrease cyclin A2 levels in multiplecell lines

We compared the ability of aspirin and salicylic acid atthree different concentrations (0.5, 1.5, and 2.5 mmol/L) to

Aspirin and Salicylic Acid Downregulate Cyclin A2/CDK2

www.aacrjournals.org Mol Cancer Res; 14(3) March 2016 243




downregulate cyclin A2 levels in 11 different cancer cell linesrepresenting human colon (HCT 116, HT-29, and SW480);breast (MDA-MB-231 and MCF7); skin (SK-MEL-28 and SK-MEL-5); lung (NCI-H226); prostate (PC-3); ovary (OVCAR-3),and also in mouse skin melanoma (B16-F10) cells. Figure 1Cand D respectively demonstrates the comparison of the down-regulatory effect of aspirin and salicylic acid on cyclin A2 levelsin these cell lines. It is clear from Fig. 1C and D that the decreasein cyclin A2 was greater at higher concentrations of the drugs,although the sensitivity of cells toward drug treatment differed.Most dramatic downregulation was observed in HCT 116,MCF7, SK-MEL-5, and OVCAR-3 cells at all drug concentrationstested.

Lactacystin completely prevents aspirin and salicylic acid–mediated downregulation of cyclin A2 levels

Cyclin A2 naturally undergoes degradation via ubiquitin-proteasomal pathway (28). During prometaphase, it is ubiqui-tinated by the APC/C, and this tags cyclin A2 for degradation bythe 26S proteasomes (28). To determine a role for the protea-somal pathway, we tested the ability of lactacystin, a 26Sproteasomal inhibitor (43), to prevent aspirin and salicylicacid–mediated decrease in cyclin A2 levels. Cells were leftuntreated or first treated with lactacystin (10 mmol/L) for 1hour, then aspirin or salicylic acid (2.5 mmol/L) was added for24 hours. Cell lysates were prepared and immunoblotted withthe anti-cyclin A2 antibody. Figure 2A demonstrates that lacta-cystin pretreatment completely prevented the degradation ofcyclin A2 caused by aspirin and salicylic acid. Quantification ofthese bands showed that aspirin and salicylic acid decreased thecyclin A2 levels by 47% and 52%, respectively (Fig. 2B).Lactacystin treatment alone stabilized the cyclin A2 proteinlevels; it was 2.3-fold higher compared with untreated control.However, in the presence of lactacystin, both drugs failed tocause the downregulation of the cyclin A2. This suggests that26S proteasomes are involved in aspirin and salicylic acid–mediated downregulatory effects.

Aspirin and salicylic acid decrease exogenously expressed,DDK-tagged, cyclin A2 protein levels

To investigate whether aspirin and salicylic acid could decreasethe exogenously expressed DDK-tagged cyclin A2 protein levels,HT-29 cells were left untransfected or transfected with DDK-tagged full-length cyclin A2 cDNA cloned in the pCMV6 vector.After transfections, cells were incubated for 24 hours to allow forthe expression DDK-tagged cyclin A2. Following this, cells wereeither left untreatedor treatedwithdrugs at a concentrations of 2.5mmol/L for 24 hours. Cell lysates were prepared and immuno-blotted with anti-DDK or anti-cyclin A2 antibodies. Figure 2Cdemonstrates that anti-DDK antibody detected the expression ofthe DDK-tagged cyclin A2 protein in transfected cells (lane 4).Interestingly, both drugs decreased the DDK-tagged cyclin A2(lanes 5 and 6). When the samples were immunoblotted withanti-cyclin A2 antibody, the levels of exogenously expressed,DDK-tagged cyclin A2, as well as the endogenous cyclin A2protein, were decreased following aspirin or salicylic acid treat-ment (Fig. 2D). Reprobing the blot of Fig. 2C with b-actinantibody showed equal amounts of the protein in all lanes. Thus,both aspirin and salicylic acid caused a decrease in the endoge-nous as well as exogenous cyclin A2 protein levels.

Aspirin and salicylic acid decrease cyclin A2 mRNA levelsTo investigate if aspirin and salicylic acid regulate cyclin A2

expression at the transcriptional/posttranscriptional level, wemeasured cyclin A2 mRNA levels in HT-29 cells treated withdrugs following 24-hour treatment. Cells were left untreated ortreated with aspirin or salicylic acid at different concentrations for24 hours; total RNA was prepared and analyzed for cyclin A2mRNA in Northern blots. Figure 3A and C demonstrates that inuntreated control cells (lane 1), abundant cyclin A2 mRNA wasdetected; both aspirin and salicylic acid respectively caused asignificant decrease in cyclin A2 mRNA levels at 1.5 mmol/L and2.5 mmol/L concentrations. Figure 3B and D shows the ethidiumbromide–stained pattern of the ribosomal 28S and 18S RNA,representing the blot in Fig. 3A and C, which shows equal RNA

D

C

‡‡

‡

‡

‡

‡

‡

‡

‡

†

‡

‡

‡

†

0%

20%

HCT-116

HT-29SK M

el-28

MDA MB-23

1SW48

0MCF-7

NCI H22

6

PC-3SK M

el 5

OVCAR-3B16

F10

HCT-116

HT-29SK M

el-28

MDA MB-23

1SW48

0MCF-7

NCI H22

6

PC-3SK M

el 5

OVCAR-3B16

F10

40%

60%

80%

100%

120%Aspirin

cont 0.5 mmol/L 1.5 mmol/L 2.5 mmol/L

‡

‡

‡

‡

‡‡

‡

‡‡

‡

‡

‡

‡

‡

‡

‡

Perc

enta

ge o

f con

trol

‡

‡‡ ‡

‡

0%

20%

40%

60%

80%

100%

120%Salicylic acid

cont 0.5 mmol/L 1.5 mmol/L 2.5 mmol/L

‡

‡

‡

‡

‡

‡‡

‡

‡

‡‡

‡

‡

‡

‡

‡‡

†

‡

‡‡

‡

‡‡

‡

‡Perc

enta

ge o

f con

trol

A Aspirin (mmol/L)

B

Probe: anti-cyclin A2 antibody

Probe: anti-β-actin antibody

Con 0.25

0.5

1.0

1.5

2.0

2.5

Con 0.25

0.5

1.0

1.5

2.0

2.5

Salicylic acid (mmol/L)



Figure 1.Aspirin and salicylic aciddownregulate cyclin A2 protein levelsin multiple cell lines. A and B, theeffect of aspirin and salicylic acid oncyclin A2 protein levels in HT-29 cells.C and D, comparison of the effect ofaspirin and salicylic acid in multiplecancer cell lines. For C and D, theintensity of bands in various Westernblotswas quantified and expressed aspercentage of control. z, P < 0.001; †,P < 0.01.

Dachineni et al.





loading in all lanes. These results show that the observed decreasein cyclin A2 protein levels following treatment with drugs is atleast in part due to decreased presence of cyclin A2 mRNA levels.

Aspirin and salicylic acid downregulate CDK2 protein andmRNA levels in HT-29 cells

We then determined whether exposure of cells to aspirin andsalicylic acidmodulates CDK2 protein andmRNA levels. For this,cells were left untreated or treated with aspirin or salicylic acidat various concentrations, and total lysates or mRNAs wereprepared and analyzed by Western blots and Northern blots,respectively. Figure 4A and B demonstrates that aspirin andsalicylic acid decreased the CDK2 protein levels at all concentra-

tions tested. Figure 4C demonstrates that salicylic acid caused areduction in CDK2mRNA levels. Similar results were obtained inSK-MEL-28 and other cancer cell lines (data not shown).

Salicylic acid decreases CDK2 activity in HT-29 and SK-MEL-28cells

It is clear from the results described above that both aspirin andsalicylic acid decrease cellular cyclin A2 (Fig. 1) and CDK2 (Fig.4A) levels. To investigate if this is associated with a correspondingreduction in the CDK2 activity, we carried out an in-vitro kinaseassay to measure the CDK2 activity using anti-CDK2 immuno-precipitates isolated from cells treated with salicylic acid.Samples representing Fig. 4A (control, 0.5 and 1.5 mmol/L) were


Probe: anti-β-actin

A

B

Probe: anti-DDK tagged antibody



C

D

DDK tagged Cyclin A2

EndogenousC

on

Asp

2.5

mm

ol/L

Sal 2

.5 m

mol

/L

Lac

Lac

+ A

sp

Lac

+ Sa

l

Con

Asp

Sal

Con

Asp

Sal

Untransfected Transfected

Exogenous

1 2 3 4 5 6

1 2 3 4 5 6

1 2 3 4 5 6

100%

53% 48%

233%226%

292%

0%

50%

cont

ASP 2.5 m

MSAL 2

.5 mM

Lacta

cysti

neASP +

Lac

SAL + La

c

100%

150%

200%

250%

300%

350%% Cyclin A2

Perc

enta

ge o

f con

trol

Figure 2.Downregulation of cyclin A2 byaspirin and salicylic acid is mediatedby 26S proteasomal pathway. A,effect of lactacystin on the ability ofaspirin and salicylic acid to decreasecyclin A2 protein levels in HT-29cells. B, the quantification of thebands in blot A. C and D, aspirin andsalicylic acid downregulateexogenously expressed DDK-tagged cyclin A2 protein andimmunoblots probed withanti–DDK-tagged antibody andanti-cyclin A2 antibody. Positions ofthe exogenous and endogenouscyclin A2 were shown by arrows(see the text for details).

28S

18S

A CB D

28S

18S

Con

1.5

2.5

0.5

Asp (mmol/L) Sal (mmol/L)

Con

1.5

2.5

2.5

Con

1.5

Asp (mmol/L)

Con

Sal (mmol/L)

1.5

2.5

0.5

Cyc

lin A

2

Cyc

lin A

2

4,000

3,000

2,000

1,500

1,000

500

Figure 3.Aspirin (A) and salicylic acid (C) decrease cyclin A2 mRNA levels in a concentration dependent fashion. B and D, the ethidium bromide–stained ribosomal RNApattern of blots in A and C (see the text for details).






immunoprecipitated with anti-CDK2 antibodies, and the immu-nocomplexes were subjected to an in-vitro kinase assays usingradiolabeled [g-32P] ATP andH1histones as substrates. Figure 4Eand F demonstrates that CDK2 kinase activity is significantlyreduced in cells treated with salicylic acid as measured by theability of CDK2 to phosphorylate H1 histones. The amount ofhistones was similar in all lanes (Fig. 4E and F, bottom).

Inclusion of salicylic acid during immunoprecipitationenhances the ability of anti-CDK2 antibody toimmunoprecipitate CDK2 in HT-29 na€�ve total cell lysates

It has been shown that cyclin A2 binds to CDK2 to form aheterodimer and regulates CDK2 activity. The cell culture (in vivo)experiments described in Figs. 1 and 4A respectively demonstratethat exposure of cells to aspirin/salicylic acid downregulates cyclinA2 and CDK2 protein levels. In order to gain insight into themechanisms of downregulation, we hypothesized that salicylicacid may directly bind to CDK2 causing a conformation change.Such a scenario is also supported by reports in literature, whichsuggest that salicylic acid indeed interacts with cellular proteins(18, 44, 45). We further hypothesized that salicylic acid–boundCDK2may still associate with cyclin A2 to form a triad of CDK2/salicylic acid/cyclin A2 complex, and the formation of this un-natural complexmay lead to degradation of these proteins by the26S proteasomes. To address this, we prepared lysates from HT-29 cells that were not treated with aspirin or salicylic acid (na€�vecell lysates). We preincubated these na€�ve cell lysates with dif-ferent concentration of salicylic acid and then tested the ability ofanti-CDK2 antibodies to bind and immunoprecipitate CDK2

protein. We reasoned that salicylic acid–induced changes inCDK2 conformation may affect the ability of anti-CDK2 anti-body to bind (due to changes in the accessibility of the epitope)and immunoprecipitate CDK2. Therefore, measuring the levelsof CDK2 and cyclin A2 (cyclin A2 naturally associates withCDK2) in the anti-CDK2 antibody immunoprecipitates wouldsuggest how salicylic acid affects CDK2 protein recognition byanti-CDK2 antibody. This approach is described in Supplemen-tary Fig. S5 (flow chart).

The association of cyclin A2with CDK2was determined by firstimmunoprecipitating the samples with the anti-CDK2 antibody(mouse monoclonal), followed by reprobing the blots with anti-cyclin A2 antibody (rabbit monoclonal). This approach was usedto avoid detection of immunoglobulin heavy chain (Ig-H) in anti-cyclin A2 immunoblots of the anti-CDK2 immunoprecipitates, asIg-H and cyclin A2 have a similar molecular weight of 54 to 56kDa. The untreated control cell lysate was divided into fouraliquots, each containing 500 mg of protein in a volume of 1 mLimmunoprecipitation buffer. One aliquot was left untreated, andto the other three aliquots, salicylic acid was added at differentconcentrations (0.5, 1.5, and 2.5 mmol/L) for 1 hour at roomtemperature. Preincubation of lysates with salicylic acid wasperformed to allow for the potential binding (if any) of salicylicacid to CDK2. The CDK2 protein was immunoprecipitated byadding monoclonal anti-CDK2 antibody, and immunocom-plexes were immunoblotted with rabbit anti-cyclin A2 antibody(see Supplementary Fig. S5). Consistent with the literature, anti-CDK2 antibody immunoprecipitates from untreated controllysates (no incubation with salicylic acid) contained cyclin A2

Probe: anti-CDK2 antibody


A

B

C

Con

0.5

1.5

2.5

0.5

1.5

2.5

Asp (mmol/L) Sal (mmol/L)

Con

1.5

2.5

Sal (mmol/L)

28S

18S

CDK2

100%

66% 68%60%

81%

62%

28%

0%

20%

40%

60%

80%

100%

120%

SAL 2.5SAL 1.5SAL 0.5ASP 2.5 ASP 1.5ASP 0.5cont

Dose (mmol/L)

% CDK2 decrease HT-29

Perc

enta

ge o

f con

trol

HT-29 Sk-Mel-28

Sal 0

.5

Con

Sal 0

.5

Con

Coomassie-stained H1 Histones

E

0.710.61

HT-29

Sal 1

. 5

Con

Sal 1

.5

Con

Sk-Mel-28F

0.310.51

Coomassie-stained H1 Histones

D

Figure 4.Aspirin/salicylic acid downregulate CDK2 protein/mRNA levels and activity. A, aspirin and salicylic acid downregulate CDK2 protein in HT-29 cells. B, quantificationof the band in blot A, expressed as percentage control. C, Northern blot analysis of CDK2 in response to salicylic acid treatment in HT-29 cells. D, ethidiumbromide–stained ribosomal RNA pattern of blot C. E and F, CDK2 activity at two different concentrations (0.5mmol/L and 1.5mmol/L) in HT-29 and SK-MEL-28 cells;numbers on the blot represent intensities expressed as percentage of control. The bottom plot shows the Coomassie blue–stained histones followingelectrophoresis (see the text for details).

Dachineni et al.





protein, suggesting that it naturally associateswithCDK2 (Fig. 5A,lane 1). We observed that, with increasing salicylic acid concen-tration (preincubated samples), greater amount of cyclin A2was detected in the anti-CDK2 immunoprecipitates (Fig. 5A, lanes2–4). Reprobing the blot in Fig. 5A with anti-CDK2 antibodyshowed that, in samples preincubated with salicylic acid, greateramount of CDK2 protein (33 kDa) was also immunoprecipitatedby anti-CDK2 antibodies (Fig. 5B). There was no significantchange in the pH of the immunoprecipitation buffer before andafter the addition of salicylic acid; therefore, increased CDK2immunoprecipitation does not appear to be due to changes inthe buffer pH. It was also not due to a nonspecific adsorption toprotein G agarose (data not shown). The Ig-H and light-chain(Ig-L) levels remained the same in Fig. 5B, confirming equalamount of anti-CDK2 antibody addition to the immunoprecip-itation reactions. These results provided the first clues on theability of salicylic acid to bind to CDK2 and possibly alter itsconformation.

Intrigued with this observation, we repeated the immuno-precipitations described in Fig. 5A and B several times and inlysates isolated from multiple cell lines (data not shown) toensure reproducibility. We next determined whether theCDK2 present in the immunoprecipitated samples of Fig. 5Ais catalytically active. For this, the experiment was performedsimilar to the one described in Fig. 5A, wherein three differentconcentrations of salicylic acid were added to the na€�ve celllysates for 1 hour before immunoprecipitation with the anti-CDK2 antibody. Following immunoprecipitation, the immu-nocomplexes were subjected to an in-vitro kinase assay using

radiolabeled [g-32P] ATP and H1 histone as a substrate, asdescribed for Fig. 4E. Figure 5C shows a correlation between thepresence of increasing concentration of salicylic acid duringimmunoprecipitation reactions and increased phosphorylationof H1 histones in the in-vitro kinase assay. H1 histone phos-phorylation progressively increased when salicylic acid wasincluded in the immunoprecipitation reactions at 0.5, 1.5, and2.5 mmol/L (lanes 2, 3, and 4). The bottom panel in Fig. 5Cshows that all lanes contained similar amounts of H1 histones.These results suggest that the increased amount of H1 phos-phorylation observed in kinase assay in Fig. 5C probablyreflects the greater amount of CDK2/cyclin A2 protein presentin the anti-CDK2 immunoprecipitates, and that CDK2 in theimmunoprecipitate is catalytically active.

Inclusion of salicylic acid during in-vitro kinase assay does notaffect the CDK2 activity

The experiments described in Fig. 5C did not contain anysalicylic acid added during the kinase assay; therefore, it was ofinterest to determine if inclusion of salicylic acid during kinaseassay reaction would affect the H1 histone phosphorylation. Forthis, na€�ve cell lysates were immunoprecipitated with anti-CDK2antibody (without preincubation with salicylic acid), and immu-noprecipitates were subjected to an in-vitro kinase assays in theabsence or presence of different concentrations of salicylic acid(0.5, 1.5, and2.5mmol/L). Figure 5Ddemonstrates that inclusionof salicylic acid during the kinase assay had no effect on the abilityof CDK2 to phosphorylate H1 histones at all concentrationstested.

IP: Anti-cdk-2 antibodyProbe: anti-cyclin A2 antibody

Ig-H

Ig-L

Sal (mmol/L)A

B

C

H1 HistonesD

E

IP: Anti-CDK2 antibody Probe: anti-cdk-2 antibod

Cdk-236 kDa

1.5

2.5

0.5

Con

Cyclin A2

Sal (mmol/L)

1.5

2.5

0.5

Con

Sal (mmol/L)

1.5

2.5

0.5

Con

H1 Histones

Sal (mmol/L)

1.5

2.5

0.5

Con

Ig- H

Ig-L

Cdk-236 kDa

1.5

2.5

0.5

Con

Sal (mmol/L)

IP: Anti-CDK2 antibody Probe: anti-cdk-2 antibody

1 2 3 4

1 2 3 4

1 2 3 4

Figure 5.The inclusion of salicylic acid increases the ability of anti-CDK2 antibody to immunoprecipitate CDK2 from na€�ve cell lysates and recombinant CDK2 protein.A, anti-cyclin A2 antibody (cat. number ab32386; Abcam) immunoblots of anti-CDK2 immunoprecipitates (cat. number-05-596; EMD Millipore), showing thepresence of increased levels of cyclin A2 with increased concentration of salicylic acid. In this blot, the area that has cyclin A2 bands is only shown. B, anti-CDK2antibody immunoblots of the anti-CDK2 immunoprecipitates show the presence of increased levels of CDK2 with increased concentrations of salicylicacid. C, the results of the in-vitro kinase assay performed on anti-CDK2 immunoprecipitates in an experiment as in A. For the experiment in C, salicylic acid waspreincubated with lysate before immunoprecipitation, but not included in the kinase assay. D, results of in-vitro kinase assay performed on anti-CDK2immunoprecipitates. For the experiments in D, lysates were not preincubated with salicylic acid before immunoprecipitation, but was included during thekinase assay. E, for anti-CDK2 immunoblot of anti-CDK2 immunoprecipitate of recombinant CDK2, immunoprecipitation was carried out in the presence ofincreasing concentration of salicylic acid.






Salicylic acid increases the ability of anti-CDK2 antibodies tobind to the purified recombinant CDK2 protein

In order to determine if salicylic acid can increase the abilityof the anti-CDK2 antibody to recognize/bind directly toCDK2, the experiments performed in Fig. 5A were repeatedexcept that commercially obtained purified CDK2 was usedinstead of total cell lysates. Three hundred ng of the recom-binant CDK2 protein was mixed in 1 mL of immunoprecip-itation buffer and incubated in the absence or presence ofsalicylic acid at different concentrations (0.5, 1.5, and 2.5mmol/L) for 1 hour at room temperature. Following this, theanti-CDK2 antibody was added overnight, and antigen–anti-body complexes were collected and immunoblotted with theanti-CDK2 antibody. As shown in Fig. 5E, salicylic acid dose-dependently increased the ability of anti-CDK2 antibody tobind and immunoprecipitate recombinant CDK2. The amountof the Ig-H chain and Ig-L chains was equal in all lanes. Theincreased CDK2 immunoprecipitation observed was not dueto a change in the pH of the immunoprecipitation reaction asaddition of 0.5 mmol/L salicylic acid, for example, to 1 mL ofthe immunoprecipitation buffer caused only a marginaldecrease in the pH (7.4 vs. 7.18). The isoelectric pH of theunmodified CDK2 is 8.8, and therefore, the increased immu-noprecipitation of CDK2 observed in Fig. 5E in the presence ofsalicylic acid is not due to nonspecific protein precipitationrelated to the isoelectric point.

Preincubation of salicylic acid with CDK2 decreasesfluorescence due to ANS

ANS is an extrinsic fluorophore demonstrated to interact withCDK2 at an allosteric site, leading to a change in the conformationand also increase in fluorescence (40, 46). Based on the resultsobtained in the immunoprecipitation experiments (Fig. 5B andE),wehypothesized that salicylic acidmayphysically interactwithCDK2, causing a conformational change, and thiswould affect thebinding of ANS to CDK2 leading to decreased fluorescence. Toaddress this, ANS (50 mmol/L) was added to recombinant CDK2(1.6 mmol/L) or CDK2 (1.6 mmol/L), which was preincubatedwith salicylic acid at different concentrations, and thefluorescencewas measured. Figure 6A demonstrates that preincubation ofCDK2 with salicylic acid dose-dependently quenched the fluo-rescence due to ANS. This suggests that salicylic acid is likely tobind to CDK2 protein, supporting the results obtained in immu-noprecipitation reactions (Fig. 5A, B, and E).

Molecular docking studies show potential interactions ofsalicylic acid with CDK2 and cyclin A2

Molecular docking is used to predict binding modes and freeenergy calculations between the ligand and the receptor (47). Weused AutoDockVina to understand the interactions between aspi-rin/salicylic acid with CDK2/cyclin A2. The binding-free energyand hydrogen bond lengths were determined to check the abilityof aspirin and salicylic acid to dock separately with CDK2,

0%

20%

40%

60%

80%

100%

120%

0 0.5 1 1.5Salicylic acid (mmol/L)

ANS-CDK2 assayA

%DF

Anti-CDK2Antibody

C

ATPSALSAL ATP ATP

ANS

ANS

Native CDK2Salicylic acid bound CDK2

ANS bound CDK2

SAL ANS

Salicylic acid_ CDK2

OO

O

H

R

Asp145

O

NR

H N

Lys33

HH

H

Phe80

B

CDK2 CDK2 CDK2

Figure 6.A, effect of preincubation of salicylic acid with CDK2 on fluorescence due to ANS. CDK2 (1.6 mmol/L) was incubated with ANS (50 mmol/L) alone or with salicylic acidat different concentrations, and fluorescence was measured as described in the text. Salicylic acid–mediated decrease in fluorescence was compared withfluorescence due toANS/CDK2. Thedecrease influorescencewas expressed as a percentage of control.B,molecular docking studies showing interactions of salicylicacid with CDK2. C, a model showing potential salicylic acid binding to CDK2. We predict that salicylic acid binds to an allosteric site on CDK2, similar to a sitedescribed for ANSbinding toCDK2. Binding of salicylic acid toCDK2 changes the conformation and increases the ability of anti-CDK2 antibody to immunoprecipitateCDK2 due to a better exposure of the epitope. Binding of salicylic acid to CDK2 would also quench the fluorescence due to ANS. We predict that potentialallosteric inhibitors could be developed by screening new salicylic acid derivatives with allosteric binding potential and inhibition of CDK2 activity. The binding sitesfor ATP, ANS and SAL are indicated.

Dachineni et al.





cyclin A2, or CDK2/cyclin A2 complex. The results of the dockingstudies are shown in Table 1 and Supplementary Fig. S6A–S6E.The free-binding energy values for the interactions between aspi-rin or salicylic acid with CDK2 were similar (�5.8 Kcal/mol). Theenergy value was much greater when salicylic acid interacted withcyclin A2 monomer (�6.8 Kcal/mol), or with cyclin A2/CDK2complex (�6.1Kcal/mol), as comparedwith aspirin's interactionswith cyclin A2 monomer (�6.2 Kcal/mol), or with the complex(�5.2 Kcal/mol). Because negative energy values indicate a morefavorable binding of ligands with receptor molecules, our datasuggest that salicylic acid has a better binding affinity to cyclin A2than aspirin. Among the potential interactions shown in Table 1(also see Supplementary Fig. S6), salicylic acid interaction withCDK2 through Asp 145 and Lys 33 is a very significant one (Fig.6B), as it corroborates the results obtained in the immunopre-cipitation experiments (Fig. 5A, B, and E) and ANS-CDK2 fluo-rescence assay (Fig. 6A), which independently suggest that sal-icylic acid binds to CDK2.

DiscussionAspirin has attracted considerable attention as a potential drug

in the chemoprevention of epithelial cancers. However, there is anextensive debate regarding the molecular pathways by which itexerts its anticancer effects. Aspirin contains acetyl and salicylategroups, both of which have their own targets and are believed tocontribute to its chemopreventive actions. Studies from ourlaboratory (12–14, 17) and others (15, 16) showed that, besidesCOX, it can acetylate numerous other proteins. Although theidentification of the aspirin-mediated acetylation targets hasrecently gained momentum (15, 16) after our initial reports(12, 14), identification of direct binding targets for salicylic acidhas been underexplored (13). Till date, salicylic acid has beenshown to bind directly and interact with three cellular proteins inhuman cells: IkB kinase (IKK) b, a component of the NF-kBcomplex (18), AMP-activated protein kinase (44), and HighMobility Group Box1 proteins (45). We hypothesized that sal-icylic acid being a small molecule with hydroxyl (–OH) andcarboxyl (–COOH) functional groups potentially could directlybind/interact with additional cellular proteins and affect theirfunctions.

In the present study, we report several novel observationsincluding a mechanism by which aspirin and salicylic acid mayexert their anticancer effects in epithelial cell types. We report theidentification of cyclin A2/CDK2 as novel targets of aspirin andsalicylic acid in multiple cancer cell lines. We demonstratedthat both drugs decrease cyclin A2 and CDK2 proteins as wellas their mRNA, in a concentration-dependent fashion. The

downregulatory effect of both drugs on cyclin A2 protein wassensitive to pretreatment with lactacystin, suggesting that 26Sproteasomal enzymes are involved. It is to be noted that cyclin A2protein naturally undergoes degradation mediated by 26S pro-teasomal pathway (28), and our observation, therefore, is con-sistent with the known pathway of cyclin A2 degradation. Thedecrease in cyclin A2/CDK2 levels in aspirin/salicylic acid treatedcells was associated with a corresponding decrease in CDK2activity, which suggests that the cellular CDK2 activity is likelyto be reduced upon drug exposure.

Our findings show that aspirin and salicylic acid regulate cyclinA2 expression at two levels, transcriptional/posttranscriptionaland posttranslational levels. At the posttranslational level, alactacystin-sensitive cysteine protease activated in response toaspirin or salicylic acid within the cells may cause the directdegradation of cyclin A2 protein. Alternatively, the observeddecrease in the levels of cyclin A2 mRNAs in aspirin and salicylictreated cells may be also due to the degradation of a transcriptionfactor (TF; mediated by a lactacystin-sensitive protease) involvedin cyclin A2 gene transcription. In this context, it is important tonote that several TFs, such as c-Myc (48), CREB (cyclic AMPresponse element binding protein), and CREM (cyclic AMPresponse element modulators; ref. 49), have been implicated inthe transcription of cyclin A2 gene; and which of these TFs areaffected by these two drugs requires additional study. In fact, in arecent study, we reported the ability of aspirin and salicylic acid todownregulate c-Myc protein and mRNA in cancer cells (50).Therefore, it is likely that the decreased levels of cyclin A2 mRNAor CDK2 mRNAs observed in aspirin- and salicylic acid–treatedcells are not a nonspecific effect due to a general cell-cycle arrest,but most likely due to the downregulations of TFs.

Results obtained from three independent experiments stronglysuggest that salicylic acid interacts with CDK2 possibly at anallosteric site leading to a change in its conformation. First cluesfor the binding of salicylic acid to CDK2 came from immuno-precipitation studies. We observed the increased ability of anti-CDK2 antibody to immunoprecipitate CDK2 protein in na€�ve celllysates when they were preincubated with salicylic acid (Fig. 5Aand B). The inclusion of salicylic acid also dose-dependentlyenhanced the ability of anti-CDK2 antibodies to immunoprecip-itate purified recombinant CDK2 (Fig. 5E). Second, ourmoleculardocking studies suggest that salicylic acid potentially interactswith Asp145 and Lys 33 in CDK2 (Table 1 and Fig. 6B), both ofwhich havebeenpreviously identified as being present in its activesite (51, 52). Third, preincubation of CDK2 with salicylic aciddose-dependently quenched the fluorescence due to ANS (Fig.6A). Interaction of ANS with CDK2 is well characterized andoccurs at an allosteric pocket near the ATP-binding site, leading to

Table 1. Molecular docking studies on aspirin and salicylic acid with CDK2, cyclin A2, and the CDK2/cyclin A2 complex

Protein LigandBinding affinityKcal/mol Amino acids

Bondlength (Å) Note

CDK2 Aspirin �5.8 LYS33 3.2 Interacts with –COOHSalicylic acid �5.8 ASP145, LYS33 2.4, 2.6 Interacts with –OH and –COOH

Cyclin A2 Aspirin �6.2 LYS194 3.2 Interacts with –COOHSalicylic acid �6.8 ASN237, ASP240, LYS194 2.1, 2.1, 3.1 Interacts with –OH and –COOH

CDK2/cyclinA2 complex

Aspirin �5.2 LYS194 (B chain) 3.2 Interacts with –COOH to cyclin A2 onlySalicylic acid �6.1 ASN237, ASP240, LYS194 (B chain) 2.0, 2.4, 2.6 Interacts with –OH and –COOH to

cyclin A2 only

NOTE: Free energy binding values and hydrogen bond lengths for the interaction of salicylic acid and aspirinwith CDK2, cyclin A2, and CDK2/cyclin A2 complex (seetext for details).






a large conformational change in CDK2 (46), and it has beenshown to interact with Asp145 and Lys33 (40, 46). It is interestingto note that both ANS and salicylic acid share common aminoacid residues Asp 145 and Lys 33, for interactions with CDK2;therefore, it is not surprising that preincubation of salicylic acidwith CDK2 quenched the fluorescence due to ANS. It has beenshown that CDK2 displays significant conformational flexibilityand accommodates the binding of highly diverse small moleculeligands (40). We predict that, similar to ANS, binding of salicylicacid to CDK2 occurs at an allosteric site causing a conformationalchange, and this would explain greater recognition and immu-noprecipitation of CDK2 protein by anti-CDK2 antibody (Fig. 5BandE; also see Fig. 6C). Further confirmationofAsp145 andLys33as salicylic acid–binding sites on CDK2 requires mutational andprotein crystallization studies.

Endogenous cyclin A2 within cells can exist in the monomericor dimeric state, bound to CDK2. Our molecular docking studies(Table 1) suggest that in addition to CDK2, aspirin and salicylicacid can potentially interact with cyclin A2 monomeric forms atspecific amino acid residues (Supplementary Fig. S6B and S6C).However, if cyclin A2/CDK2 dimer has already been formed, itappears that aspirin and salicylic acid can interact only with cyclinA2, but not with CDK2 (Supplementary Fig. S6D and S6E). Thestandard hydrogen bond length between donor and the acceptoratoms is in the order of 2.6 to 3.5 Å, with optimum at 2.8 Å (53).Based on the hydrogen bond length and the associated negativefree energy, salicylic acid showed stronger interaction with bind-ing pockets in CDK2 monomer, cyclin A2 monomer, or withcyclin A2 in the cyclin A2/CDK2 heterodimer than aspirin. Addi-tional studies involving biochemical and protein crystallizationare required to confirm the direct binding of aspirin and salicylicacid with cyclin A2.

The in-vitro experiment described in Fig. 5B shows that pre-incubation of na€�ve cell lysates with salicylic acid increased theability of anti-CDK2antibody to bind and immunoprecipitate theCDK2 protein. These anti-CDK2 immunoprecipitates from sal-icylic acid–preincubated lysates also contained greater levels ofcyclin A2, as cyclin A2 is a natural binding partner with CDK2(coprecipitation; Fig. 5A), and showed increased CDK2 activity asmeasured byH1histone phosphorylation assay (Fig. 5C). In thesekinase experiments, salicylic acid was not included during thekinase reaction. Therefore, the increased CDK2 activity observedin anti-CDK2 immunoprecipitates from salicylic acid–preincu-bated samples (Fig. 5C) reflects the greater amounts of cyclin A2/CDK2protein levels; and thus, it is not due a stabilization effect ofsalicylic acid. Interestingly, the inclusion of increasing amounts ofsalicylic acid during the in-vitro kinase assay performed on theanti-CDK2 immunoprecipitates from na€�ve cell lysates had noeffect on H1 histone phosphorylation (Fig. 5D). Taken together,these results suggest that binding of salicylic acid to CDK2 mostlikely changes the conformation leading to increased CDK2immunoprecipitation. Efficient phosphorylation of H1 histonesin the presence of salicylic acid (Fig. 5D) also suggests that theATP-binding site in CDK2 is unaffected due to interactions withsalicylic acid in the cyclin A2/CDK2 complex.

Although our in vitro experiments (Figs. 5A and B and 6A) andthe molecular docking studies (Fig. 6B; Table 1) suggest thatsalicylic acid binds to CDK2, it is not clear howwithin the cellularmilieu, binding of salicylic acid to CDK2 causes subsequentdegradation of cyclin A2 and CDK2 proteins. It is certain thatproteasomal pathway is involved (Fig. 2). Inside the cellular

milieu, the triad of CDK2/salicylic acid/cyclin A2 complex maybe recognized by proteasomal enzymes as an un-natural complex,leading to degradation of both cyclin A2 andCDK2. Alternatively,the triad of CDK2/salicylic acid/cyclin A2 complex, although stillcatalytically active, may have an altered substrate specificity. Forexample, salicylic acid–bound CDK2 with an altered conforma-tion and substrate specificity may phosphorylate and activateunique targets/proteasomal enzymes specific for the degradationof cyclin A2/CDK2. This view is supported by reports in literaturethat conformational changes in flexible parts of the protein havebeen indeed shown to alter substrate specificity (54). Investiga-tions into the pathways leading to degradation of cyclin A2/CDK2proteins following salicylic acid occupancy represent an impor-tant extension of this study.

Aspirin's ability to inhibit cell proliferation or induce cell-cyclearrest (G0/G1) has been documented in the literature in manycancer cell lines (41, 42, 55, 56). In our study, aspirin and salicylicacid down regulated cyclin A2/CDK2 in 11 different cancer cellsrepresenting the cancers of various epithelial tissues (colon, lung,prostate, ovary, and skin), which suggests that this is a universalphenomenon and applicable to most cancer cells. Our studiesperformed in HT-29 cells shows that exposure of cells to aspirinand salicylic acid caused downregulation of cyclins B1 and D3;CDKs 1, 4, and 6; and upregulation of CDK inhibitors p21 andp27 (Supplementary Figs. 2, 3 and4).Downregulation ofmanyofthe important cyclins and CDKs, and upregulation of CDKinhibitors, would tip the balance strongly toward cell-cycle arrestand will explain the documented ability of aspirin and salicylicacid to cause cell-cycle arrest in literature.

In many cancers, CDK2 activity is deregulated (32), and cyclinA2 is overexpressed (29–31, 33). Therefore, attention is increas-ingly being focused on cell cycle as a potential target for thera-peutic intervention (35, 57, 58). In this context, our finding thataspirin and salicylic acid downregulate cyclin A2/CDK2 proteinand mRNAs in multiple cancer cell lines should initiate newthinking and research on these age-old drugs in cancer treatment.The answer for an effective drug for chemoprevention may lie inrevisiting salicylic acid, an ancient drug known for over twomillennia in plants for its therapeutic properties. The observationthat salicylic acid binds to CDK2 at an allosteric site can beexploited to develop novel anticancer drugs, for example, deri-vatives of salicylic acid can be screened for inhibition of CDK2activity or disruption of the cyclin A2/CDK2 complex. Salicylicacid is abundantly present in many plants where it has beenshown to protect the cells from infection through induction of celldeath (59). It will also be interesting to determine if salicylic acid–induced cell death in infected leaves involves downregulation ofcyclin A2/CDK2 or other related proteins.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: R. Dachineni, G. Ai, H. Tummala, G.J. BhatDevelopment of methodology: R. Dachineni, G. Ai, G.J. BhatAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Dachineni, S.S. Sadhu, G.J. BhatAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Dachineni, G. Ai, D.R. Kumar, G.J. BhatWriting, review, and/or revision of the manuscript: R. Dachineni, H. Tum-mala, G.J. Bhat

Dachineni et al.





Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G.J. BhatStudy supervision: D.R. Kumar, G.J. Bhat

AcknowledgmentsThe support from the Translational Cancer Research Seed Grant, funded as

2010 Research Initiative Center by the State of South Dakota, Faculty ExcellenceFund from South Dakota State University (SDSU), Department of Pharmaceu-tical Sciences, SDSU, and from NIH (5RO3CA133061-02) to G.J. Bhat isgratefully acknowledged. The authors acknowledge Drs. Jane Endicott andMartin Noble of Newcastle University, UK, for the suggestions on the ANS-CDK2assay. They also thankDr.Mohit Tyagi, ChowdhuryAbdullah, Yang Yang,

Mohamed Ismail, SDSU, and Dr. Lloyd Alfonso, D'Youville School College ofPharmacy, Buffalo, NY, for helpful discussions. They thank Dr. KalkunteSrivenugopal of Texas Tech University Health Sciences Center, Amarillo, TX,for suggestions on CDK2 assays.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 25, 2015; revised December 1, 2015; accepted December 3,2015; published OnlineFirst December 18, 2015.

References1. Kaiser J. Will an aspirin a day keep cancer away? Science 2012;337:1471–3.2. ThunMJ, Jacobs EJ, Patrono C. The role of aspirin in cancer prevention. Nat

Rev Clin Oncol 2012;9:259–67.3. Chan AT, Arber N, Burn J, ChiaWK, Elwood P, HullMA, et al. Aspirin in the

chemoprevention of colorectal neoplasia: an overview. Cancer Prev Res2012;5:164–78.

4. Rothwell PM, Price JF, Fowkes FGR, Zanchetti A, Roncaglioni MC,Tognoni G, et al. Short-term effects of daily aspirin on cancer incidence,mortality, and non-vascular death: analysis of the time course of risksand benefits in 51 randomised controlled trials. The Lancet 2012;379:1602–12.

5. Sahasrabuddhe VV, Gunja MZ, Graubard BI, Trabert B, Schwartz LM, ParkY, et al. Nonsteroidal anti-inflammatory drug use, chronic liver disease, andhepatocellular carcinoma. J Natl Cancer Inst 2012;104:1808–14.

6. Gamba CA, Swetter SM, Stefanick ML, Kubo J, Desai M, Spaunhurst KM,et al. Aspirin is associated with lower melanoma risk among postmeno-pausal Caucasian women: the Women's Health Initiative. Cancer 2013;119:1562–9.

7. Veitonmaki T, Tammela TL, Auvinen A, Murtola TJ. Use of aspirin, but notother non-steroidal anti-inflammatory drugs is associated with decreasedprostate cancer risk at the population level. Eur J Cancer 2013;49:938–45.

8. Shpitz B, Klein E, Buklan G, Neufeld D, Nissan A, Freund HR, et al.Suppressive effect of aspirin on aberrant crypt foci in patients with colo-rectal cancer. Gut 2003;52:1598–601.

9. Chan AT, Ogino S, Fuchs CS, Aspirin use and survival after diagnosis ofcolorectal cancer. JAMA 2009;302:649–58.

10. Fraser D, Sullivan FM, Thompson AM, McCowan C. Aspirin use andsurvival after the diagnosis of breast cancer: a population-based cohortstudy. Br J Cancer 2014;111:623–7.

11. DovizioM, BrunoA, Tacconelli S, Patrignani P.Mode of action of aspirin asa chemopreventive agent. Recent Results Cancer Res 2013;191:39–65.

12. Marimuthu S, Chivukula RS, Alfonso LF,MoridaniM,Hagen FK, Bhat GJ,et al. Aspirin acetylates multiple cellular proteins in HCT-116colon cancer cells: Identification of novel targets. Int J Oncol 2011;39:1273–83.

13. Alfonso L, Ai G, Spitale RC, Bhat GJ.Molecular targets of aspirin and cancerprevention. Br J Cancer 2014;111:61–7.

14. Alfonso LF, Srivenugopal KS, Arumugam TV, Abbruscato TJ, Weidanz JA,Bhat GJ, et al. Aspirin inhibits camptothecin-induced p21CIP1 levels andpotentiates apoptosis in human breast cancer cells. Int J Oncol 2009;34:597–608.

15. Bateman LA, Zaro BW, Miller SM, Pratt MR. An alkyne–aspirin chemicalreporter for the detection of aspirin-dependent protein modification inliving cells. J Am Chem Soc 2013;135:14568–73.

16. Wang J, Zhang CJ, Zhang J, He Y, Lee YM, Chen S, et al. Mapping sites ofaspirin-induced acetylations in live cells by quantitative acid-cleavableactivity-based protein profiling (QA-ABPP). Sci Rep 2015;5.

17. AiG,Dachineni R,KumarDR,MarimuthuS, AlfonsoLF, BhatGJ, et al. Aspirinacetylates wild type and mutant p53 in colon cancer cells: identification ofaspirin acetylated sites on recombinant p53. Tumor Biol 2015:1–10.

18. Kopp E, Ghosh S. Inhibition of NF-kappa B by sodium salicylate andaspirin. Science 1994;265:956–9.

19. Bos CL, Kodach LL, van den Brink GR, Diks SH, van SantenMM, Richel DJ,et al. Effect of aspirin on theWnt/b-cateninpathway ismediated via proteinphosphatase 2A. Oncogene 2006;25:6447–56.

20. Pathi S, Jutooru I, Chadalapaka G, Nair V, Lee SO, Safe S, et al. Aspirininhibits colon cancer cell and tumor growth and downregulates specificityprotein (Sp) transcription factors. PLoS One 2012;7:e48208.

21. Din FV, Valanciute A, Houde VP, Zibrova D, Green KA, Sakamoto K, et al.Aspirin inhibits mTOR signaling, activates AMP-activated protein kinase,and induces autophagy in colorectal cancer cells. Gastroenterology 2012;142:1504–15.

22. LawBK,Waltner-LawME, Entingh AJ, Chytil A, AakreME,Nørgaard P, et al.Salicylate-induced growth arrest is associated with inhibition of p70s6kand down-regulation of c-myc, cyclin D1, cyclin A, and proliferating cellnuclear antigen. J Biol Chem 2000;275:38261–7.

23. Borthwick GM, Johnson AS, Partington M, Burn J, Wilson R, Arthur HM,et al. Therapeutic levels of aspirin and salicylate directly inhibit a model ofangiogenesis through a Cox-independent mechanism. FASEB J 2006;20:2009–16.

24. Deshpande A, Sicinski P, Hinds PW. Cyclins and cdks in development andcancer: a perspective. Oncogene 2005;24:2909–15.

25. Pagano M, Pepperkok R, Verde V, Ansorge W, Draetta G, et al. Cyclin A isrequired at two points in the human cell cycle. EMBO J 1992;11:961.

26. Bendris N, Lemmers B, Blanchard JM, Arsic N. Cyclin A2 mutagenesisanalysis: a new insight into CDK activation and cellular localizationrequirements. PLoS One 2011;6:e22879.

27. Gong D, Pomerening JR, Myers JW, Gustavsson C, Jones JT, et al. Cyclin A2regulates nuclear-envelope breakdown and the nuclear accumulation ofcyclin B1. Curr Biol 2007;17:85–91.

28. Peters JM. The anaphase promoting complex/cyclosome: a machinedesigned to destroy. Nat Rev Mol Cell Biol 2006;7:644–56.

29. Bukholm IR, BukholmG,Nesland JM,Over-expression of cyclin a is highlyassociated with early relapse and reduced survival in patients with primarybreast carcinomas. Int J Cancer 2001;93:283–7.

30. VolmM,Koom~A R,Cyclin A is associatedwith anunfavourable outcome inpatients with non-small-cell lung carcinomas. Brit J Cancer 1997;75:1774.

31. OhashiR,GaoC,MiyazakiM,Hamazaki K, Tsuji T, InoueY, et al. Enhancedexpression of cyclin E and cyclin A in human hepatocellular carcinomas.Anticancer Res 2000;21:657–662.

32. Malumbres M, Barbacid M. Cell cycle kinases in cancer. Curr Opin GenetDev 2007;17:60–5.

33. Kanai M, Shiozawa T, Xin L, Nikaido T, Fujii S. Immunohistochemicaldetection of sex steroid receptors, cyclins, and cyclin-dependent kinases inthe normal and neoplastic squamous epithelia of the uterine cervix. Cancer1998;82:1709–19.

34. Anscombe E,Meschini E,Mora-Vidal R,MartinMP, StauntonD,GeitmannM, et al. Identification and characterization of an irreversible inhibitor ofCDK2. Chem Biol 2015;22:1159–64.

35. ZalzaliH,Nasr B,HarajlyM,BasmaH,GhamloushF,Ghayad S, et al. CDK2transcriptional repression is an essential effector in p53-dependent cellularsenescence—implications for therapeutic intervention. Mol Cancer Res2015;13:29–40.

36. Wang X, Chen X, Han W, Ruan A, Chen L, Wang R, et al. miR-200c targetsCDK2 and suppresses tumorigenesis in renal cell carcinoma. Mol CancerRes 2015:13:1567–77.

37. Choi KS, Eom YW, Kang Y, HaMJ, Rhee H, Yoon J-W, et al. Cdc2 and Cdk2kinase activated by transforming growth factor-b1 trigger apoptosisthrough the phosphorylation of retinoblastoma protein in FaO hepatomacells. J Biol Chem 1999;274:31775–83.






38. KomanderD, Kular GS, Sch€uttelkopf AW,DeakM, Prakash KR, Bain J, et al.Interactions of LY333531 and other bisindolyl maleimide inhibitors withPDK1. Structure 2004;12:215–26.

39. Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJ,et al. GROMACS: fast, flexible, and free. J Comput Chem 2005;26:1701–18.

40. Martin MP, Alam R, Betzi S, Ingles DJ, Zhu JY, Sch€onbrunn E, et al. A novelapproach to the discovery of small-molecule ligands of CDK2. Chembio-chem 2012;13:2128–36.

41. Goel A, Chang DK, Ricciardiello L, Gasche C, Boland CR. A novel mech-anism for aspirin-mediated growth inhibition of human colon cancer cells.Clin Cancer Res 2003;9:383–90.

42. Luciani MG, Campregher C, Gasche C. Aspirin blocks proliferation incolon cells by inducing a G1 arrest and apoptosis through activation of thecheckpoint kinase ATM. Carcinogenesis 2007;28:2207–17.

43. Fenteany G, Schreiber SL. Lactacystin, proteasome function, and cell fate.J Biol Chem 1998;273:8545–8.

44. Hawley SA, Fullerton MD, Ross FA, Schertzer JD, Chevtzoff C, Walker KJ,et al. The ancient drug salicylate directly activates AMP-activated proteinkinase. Science 2012;336:918–22.

45. Choi HW, Tian M, Song F, Venereau E, Preti A, Park SW, et al. Aspirin'sactive metabolite salicylic acid targets high mobility group box 1 tomodulate inflammatory responses. Mol Med 2015;21:526.

46. Betzi S, Alam R, Martin M, Lubbers DJ, Han H, Jakkaraj SR, et al. Discoveryof a potential allosteric ligand binding site in CDK2. ACS Chem Biol 2011;6:492–501.

47. Bikadi Z, Hazai E. Application of the PM6 semi-empirical method tomodeling proteins enhances docking accuracy of AutoDock. J Cheminfor-matics 2009;1:15.

48. Zeller KI, Jegga AG, Aronow BJ, O'Donnell KA, Dang CV. An integrateddatabase of genes responsive to the Myc oncogenic transcription factor:Identification of direct genomic targets. Genome Biol 2003;4:R69.

49. Desdouets C, Matesic G, Molina CA, Foulkes NS, Sassone-Corsi P, BrechotC, et al. Cell cycle regulation of cyclin A gene expression by the cyclic AMP-responsive transcription factors CREB and CREM. Mol Cell Biol 1995;15:3301–9.

50. Ai G, Dachineni R,Muley P, TummalaH, Bhat GJ. Aspirin and salicylic aciddecrease c-Myc expression in cancer cells: a potential role in chemopre-vention. Tumor Biol 2015:1–2.

51. Welburn JP, Tucker JA, Johnson T, Lindert L,MorganM,Willis A, et al. Howtyrosine 15 phosphorylation inhibits the activity of cyclin-dependentkinase 2-cyclin A. J Biol Chem 2007;282:3173–81.

52. Lees E. Cyclin dependent kinase regulation. Curr Opin Cell Biol 1995;7:773–80.

53. Overington J, Johnson MS, Sali A, Blundell TL. Tertiary structural con-straints on protein evolutionary diversity: templates, key residues andstructure prediction. Proc Biol Sci 1990;241:132–45.

54. Yu X, Cojocaru V, Wade RC. Conformational diversity and ligand tunnels ofmammalian cytochromeP450s. BiotechnolAppl Biochem2013;60:134–45.

55. Qiao L,Hanif R, Sphicas E, Shiff SJ, Rigas B. Effect of aspirin on induction ofapoptosis in HT-29 human colon adenocarcinoma cells. Biochem Phar-macol 1998;55:53–64.

56. Shiff SJ, Koutsos MI, Qiao L, Rigas B, et al. Nonsteroidal antiinflammatorydrugs inhibit the proliferation of colon adenocarcinoma cells: effects oncell cycle and apoptosis. Exp Cell Res 1996;222:179–88.

57. Davies TG, Bentley J, Arris CE, Boyle FT, Curtin NJ, Endicott JA, et al.Structure-based design of a potent purine-based cyclin-dependent kinaseinhibitor. Nat Struct Biol 2002;9:745–9.

58. Deng Y, Shipps GW, Zhao L, Siddiqui MA, Popovici-Muller J, Curran PJ,et al. Modulating the interaction between CDK2 and cyclin A with aquinoline-based inhibitor. Bioorg Med Chem Lett 2014;24:199–203.

59. Alvarez ME. Salicylic acid in the machinery of hypersensitive cell deathand disease resistance. In programmed cell death in higher plants. 2000,p. 185–98. Springer Netherlands.


Dachineni et al.




2016;14:241-252. Published OnlineFirst December 18, 2015.Mol Cancer Res Rakesh Dachineni, Guoqiang Ai, D. Ramesh Kumar, et al. A Potential Role in Cancer PreventionCyclin A2 and CDK2 as Novel Targets of Aspirin and Salicylic Acid:

Updated version

10.1158/1541-7786.MCR-15-0360doi:

Access the most recent version of this article at:

Material

Supplementary

http://mcr.aacrjournals.org/content/suppl/2016/01/09/1541-7786.MCR-15-0360.DC1

Access the most recent supplemental material at:

Cited articles

http://mcr.aacrjournals.org/content/14/3/241.full#ref-list-1

This article cites 55 articles, 13 of which you can access for free at:

Citing articles

http://mcr.aacrjournals.org/content/14/3/241.full#related-urls

This article has been cited by 2 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mcr.aacrjournals.org/content/14/3/241To request permission to re-use all or part of this article, use this link



http://mcr.aacrjournals.org/lookup/doi/10.1158/1541-7786.MCR-15-0360

http://mcr.aacrjournals.org/content/suppl/2016/01/09/1541-7786.MCR-15-0360.DC1

http://mcr.aacrjournals.org/content/14/3/241.full#ref-list-1

http://mcr.aacrjournals.org/content/14/3/241.full#related-urls

http://mcr.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mcr.aacrjournals.org/content/14/3/241


cyclin a2 and cdk2 as novel targets of aspirin and ... · cell cycle and senescence cyclin a2 and...

Documents