Celecoxibantagonizesperifosine'santicanceractivity ......2and15d-PGJ 2...

Celecoxib antagonizes perifosine's anticancer activityinvolving a cyclooxygenase-2-dependent mechanism

Heath A. Elrod, Ping Yue, Fadlo R. Khuri,and Shi-Yong Sun

Department of Hematology and Medical Oncology,Winship Cancer Institute, Emory University Schoolof Medicine, Atlanta, Georgia

AbstractPerifosine is an orally bioavailable alkylphospholipid cur-rently being tested in phase II clinical trials as a potentialanticancer drug. In this study, we reveal a novel mecha-nism underlying the anticancer activity of perifosine thatinvolves the induction of cyclooxygenase 2 (COX-2) in hu-man cancer cells. Perifosine induced apoptosis and/or cellcycle arrest in several lung and head and neck cancer celllines. However, the combination of perifosine with lowconcentrations of celecoxib rendered cells less sensitiveto perifosine both in cell culture systems and in lung can-cer xenograft models. Subsequently, we examined the ef-fects of perifosine on COX-2 expression and activity in aset of lung and head and neck cancer cell lines, and foundthat perifosine rapidly and potently increased COX-2 le-vels and activity, the degrees of which correlated to theabilities of perifosine to inhibit the growth of cancer cells.We also detected increased COX-2 levels in lung cancerxenografts treated with perifosine. Moreover, blockageof COX-2 induction by both antisense and small interferingRNA approaches decreased cell sensitivity to perifosine.Collectively, these data indicate that the activation ofCOX-2 contributes to the anticancer activity of perifosine,including apoptosis induction and growth arrest. Thesedata are clinically relevant as they suggest that the com-

bination of perifosine and COX-2 inhibitors such ascelecoxib, may produce a potential drug contradiction.[Mol Cancer Ther 2009;8(9):2575–85]

IntroductionAlkylphospholipids represent a class of antitumor agentsthat can induce apoptosis in tumors cells while sparing nor-mal cells (1). These agents act at the cell membrane and dis-rupt membrane phospholipid metabolism (1). Miltefosine,the first alkalphospholipid to be tested in clinical trials,has been approved for topical treatment of cutaneous lym-phomas and cutaneous metastases from breast cancer (2).Perifosine, in which the choline moiety of miltefosine is re-placed by a cyclic aliphatic piperidy residue, has greateroral bioavailability with limited gastrointestinal side effectsand better activity in preclinical models when comparedwith miltefosine. Perifosine effectively inhibited the growthof a variety of human tumor cell lines, including melanoma,lung, prostate, colon, and breast cancer cells and suppressedthe growth of mammary carcinomas induced by 7,12-dimethylbenz(a)anthracene in Sprague-Dawley rats (3).In addition, perifosine when combined with other can-cer therapeutic agents exhibited enhanced apoptosis-inducing and anticancer activity (4–7). Perifosine is currentlybeing tested in phase II clinical trials and has shown single-agent partial responses in certain types of cancer such asrenal cell carcinoma (8).Howperifosine exerts its antitumor effect is not completely

understood. Perifosine has been shown to inhibit the plasmamembrane localization of Akt andAkt phosphorylation lead-ing to antiproliferative effects in tumor cells (9). Likewise,perifosine causes cell cycle arrest in tumor cells by inducingp21WAF (10). Our recent study has shown that perifosine in-duces apoptosis in human lung cancer cells involving activa-tion of the extrinsic death receptor–mediated apoptoticpathway (11).The cyclooxygenase-2 (COX-2) inhibitor celecoxib is a

marketed drug for use in humans for management of arthri-tis and acute pain as well as in patients with familial adeno-matous polyposis for reduction of adenomatous colorectalpolyps (12). In addition, celecoxib is currently being testedpreclinically and clinically for its chemopreventive and ther-apeutic efficacy against a broad spectrum of epithelial ma-lignancies, including lung cancers, either as a single agent orin combination with other agents (13, 14). In an effort toidentify perifosine-based combination regimens, we unex-pectedly found that celecoxib diminished the growth inhib-itory effects of perifosine in human non–small cell lungcancer (NSCLC) and head and neck cancer cell lines.COX-2 catalyzes the conversion of arachadonic acid to

prostaglandins (PG). COX-2 expression is generally low orabsent in most tissues, but its expression is inducible upon

Received5/4/09; revised6/18/09; accepted6/22/09; publishedOnlineFirst9/15/09.

Grant support: The Georgia Cancer Coalition Distinguished Cancer Scholaraward (S.-Y. Sun), Department of Defense grants W81XWH-04-1-0142-VITAL (S.-Y. Sun for Project 4), National Cancer Institute SPORE P50 grantCA128613-01 (S.-Y. Sun for Project 2), and American Cancer SocietyPostdoctoral Fellowship PF-07-033-01-CCG (H.A. Elrod).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

H.A. Elrod is a recipient of an American Cancer Society Fellowship. F.R.Khuri and S-Y. Sun are Georgia Cancer Coalition Distinguished CancerScholars.

Request for reprints: Shi-Yong Sun, Department of Hematology andMedical Oncology, Winship Cancer Institute, Emory University School ofMedicine, 1365-C Clifton Road NE, C3088, Atlanta, GA 30322.Phone: 404-778-2170; Fax: 404-778-5520. E-mail: [email protected]

Copyright © 2009 American Association for Cancer Research.

doi:10.1158/1535-7163.MCT-09-0390

Mol Cancer Ther 2009;8(9). September 2009

2575

Research. on December 24, 2020. © 2009 American Association for Cancermct.aacrjournals.org Downloaded from

Published OnlineFirst September 15, 2009; DOI: 10.1158/1535-7163.MCT-09-0390

http://mct.aacrjournals.org/

stimulation by such molecules as cytokines and growth fac-tors (15). COX-2 is usually undetectable in most normal tis-sues, but is overexpressed in a variety of premalignant andmalignant tissues (16). In general, COX-2 overexpression isassociated with resistance to apoptosis, angiogenesis, tumor-igenesis, and poor prognosis (15, 16), all of which are the sci-entific rationale for COX-2 as a preventive or therapeutictarget of cancer. However, COX-2 has also been shown tosuppress tumorigenesis in one model of skin cancer (17), tobe associated with good prognosis in some studies (18–20),and to induce cell cycle arrest (21) and apoptosis (22–27).Interestingly, another member of the alkylphospholipid

family, edelfosine, up-regulates the expression of COX-2which contributes to apoptotic death induced by edelfosine(28). Therefore, we investigated why celecoxib antagonizedthe anticancer effects of perifosine by understanding the roleof COX-2 in perifosine-mediated anticancer activity, includ-ing cell cycle arrest and induction of apoptosis. Our resultsshow that perifosine induces COX-2 expression, which con-tributes to perifosine-induced apoptosis and cell cycle ar-rest. Therefore, our study reveals a novel mechanismunderlying the anticancer activity of perifosine that involvesCOX-2 activation.

Materials and MethodsReagents

Perifosine (Fig. 1A) was supplied by Keryx Biopharma-ceuticals, Inc. with a chemical purity of >99%. This agentwas dissolved in PBS and stored at −20°C. Celecoxib waspurchased from LKT Laboratories. The other COX-2 inhibi-tors SC-58125 and DuP697 were purchased from CaymanChemical. The celecoxib derivative 2,5-dimethyl-celecoxib(DMC), which lacks COX-2–inhibitory activity (29), wasprovided by Dr. Axel H. Schönthal (University of SouthernCalifornia, Los Angeles, CA). These agents were dissolvedin DMSO and stored at −20°C. Stock solutions were dilutedto the appropriate concentrations with growth medium im-mediately before use.Cell Lines and Cell Culture

The human NSCLC cell lines used in this study and theirculture conditions were described previously (30). The H157stable cell line that expresses antisense COX-2 (H157-AS)and its matched vector control line (H157-V) were describedpreviously (31) and provided by Dr. S. M. Dubinett (Univer-sity of California, Los Angeles, CA). The head and neck can-cer cell line 686Ln and its derived cell lines M4c, M4d, andM4e, which have high metastatic potential, were providedby Dr. Z. G. Chen in our institute (32).Cell Viability Assay

Cells were cultured in 96-well cell culture plates and trea-ted the next day with the agents indicated. Viable cell num-ber was estimated using the sulforhodamine B (SRB) assay,as previously described (30).Western Blot Analysis

Preparation of whole-cell protein lysates and Westernblot analysis were described previously (33, 34). Mouseanti–caspase-3 monoclonal antibody was purchased from

Figure 1. Perifosine (A) inhibits the growth of human NSCLC cells(B) through induction of apoptosis (C) and G2-M arrest (D). A, the chem-ical structure of perifosine. B, the indicated cell lines were treated withincreased concentrations of perifosine (1–20 μmol/L) for 3 d. Cell numberswere then estimated using the SRB assay. IC50 values were calculatedfrom a dose-response curve. C and D, the indicated cell lines were treatedwith 10 μmol/L perifosine for 48 h. The cells were trypsinized, harvested,and subjected to flow cytometric analysis for measuring apoptosis usingAnnexin V staining (C) and for analyzing cell cycle using propidium iodidestaining (D). Columns, means of triplicate determinations; bars, ± SD.

Celecoxib Antagonizes Perifosine's Anticancer Activity


2576




Imgenex. Rabbit anti–caspase-8, anti–caspase-9, and anti–poly(ADP-ribose) polymerase (PARP) polyclonal anti-bodies were purchased from Cell Signaling Technology.Rabbit anti–COX-2 polyclonal antibody was purchasedfrom Oxford Biomedical. Rabbit anti-G3PDH polyclonalantibody was purchased from Trevigen. Rabbit anti–β-actinpolyclonal antibody was purchased from Sigma. Secondaryantibodies goat anti-mouse and goat anti-rabbit-horseradishperoxidase conjugates were purchased from Bio-Rad.Apoptosis Assays

Apoptosis was detected either by analysis of caspase ac-tivation using Western blot analysis as described above orby Annexin V staining using Annexin V-PE apoptosis detec-tion kit (BD Bioscience) following the manufacturer's in-structions and analyzed by flow cytometry using theFACScan (Becton Dickinson).Cell Cycle Assay

Control cells or cells treated with the indicated agents for24 or 48 h were harvested, washed with cold PBS, and fixedin 70% ethanol. DNA was stained by incubating cells withPBS containing 50 μg/mL propidium iodide and 15 μg/mLRNase A for 20 min at room temperature. Fluorescence wasmeasured using the FACScan and analyzed using CellQuestsoftware (BD Biosciences).ELISA for Detection of PGE2 and 15d-PGJ2Cells were cultured in 60-mm dishes in culture medium

supplemented with 5% fetal bovine serum and treated withperifosine alone, celecoxib plus perifosine, or celecoxibalone. After a 16-h treatment, medium was collected andmeasured for the levels of PGE2 using the ELISA kit fromCayman Chemical and for the levels of 15d-PGJ2 using theELISA kit from Assay Designs following the manufacturer'sinstructions. Because treatment with perifosine induces sig-nificant cell death, prostaglandin levels were normalizedbased on protein concentration for each sample after treatment.Silencing of COX-2 Expression Using small interfering

RNA (siRNA)

Silencing of COX-2 was achieved by transfecting siRNAusing RNAifect transfection reagent (Qiagen) following themanufacturer's instructions. Control Stealth siRNA oligonu-c leot ides target ing the sequence 5 ′ -GAATTCCT-CAATTCCCTCTCGCATT-3′ and COX-2 Stealth siRNAoligonucleotides targeting the sequence 5′-AAAGACTGG-TATTTCATCTGCCTGC-3′ were purchased from Invitro-gen. Cells were plated in 24-well plates and the next daytransfected with siRNAs. Twenty-four hours later the cellswere re-plated and the next day treated with perifosine asindicated. Gene silencing effects were evaluated by Westernblot analysis as described above.Lung Cancer Xenografts and Treatments

Animal experiments were approved by the InstitutionalAnimal Care and Use Committee of Emory University.Four- to six-week-old (about 20 g of body weight) femaleathymic (nu/nu) mice (Taconic) were housed under patho-gen-free conditions in microisolator cages with laboratorychow and water ad libitum. 5 × 106 H358 cells or H460 cellsin PBS were injected s.c. into the flank region of nude mice.When tumors reached certain size ranges (50–100 mm3), themice were randomized into four groups (n = 6/group) ac-

cording to tumor volumes and body weights for the follow-ing treatments: vehicle control, perifosine in PBS (20 or30 mg/kg/d, oral gavage), celecoxib in DMSO (50 or100 mg/kg/d; oral gavage), and the combination of perifo-sine and celecoxib. Tumor volumes were measured usingcaliper measurements once every 2 d and calculated with theformula V = π(length × width2)/6. After a 5-, 6-, 10-, or 11-dtreatment, the mice were sacrificed with CO2. The tumorswere then removed, weighed, and frozen in liquid nitrogenor fixed with 4% fomaldehyde. Certain portions of tumortissues from each tumor were homogenized in protein lysisbuffer for preparation of whole-cell protein lysates asdescribed previously (30). Western blotting results werequantitated using NIH Image J (NIH).Immunohistochemistry for Detection of COX-2 in

Tumor Tissues

Five-micrometer formaldehyde-fixed, paraffin-embeddedsections from H358 tumor xenografts were dewaxed, hy-drated though graded alcohols, and microwaved in100 mmol/L sodium citrate for 5 min on high power and10 min on low power for antigen retrieval. Immunohisto-chemistry analysis was done following the protocol forthe DakoCytomation EnVision + Dual Link System. Sec-tions were incubated overnight at 4°C with rabbit poly-clonal anti-COX-2 (1:500 dilution; Oxford BiomedicalResearch). COX-2–positive cells were counted using Meta-Morph Imaging Software (Universal Imaging Corporation).Statistical Analysis

The statistical significance of differences between twogroups was analyzed with two-sided unpaired Student's ttests when the variances were equal or with Welch's cor-rected t test when the variances were not equal, by use ofGraphpad InStat 3 software (GraphPad Software). Datawere examined as suggested by the same software to verifythat the assumptions for use of the t tests held. Results wereconsidered to be statistically significant at P < 0.05. All sta-tistical tests were two-sided.

ResultsPerifosine Inhibits the Growth of Human NSCLC Cells

through Induction of Apoptosis and Cell Cycle Arrest

Human NSCLC cell lines exhibited varied sensitivities toperifosine (Fig. 1B). Among these cell lines, H460 and H358were the most sensitive to perifosine, whereas H226 was re-sistant to perifosine. Both A549 and H157 exhibited interme-diate sensitivities to perifosine (Fig. 1B). Detection ofapoptosis and cell cycle alteration revealed that H460 cellsprimarily underwent apoptotic cell death (84.8 ± 1.1% inperifosine-treated cells versus 7.7 ± 1.8% in PBS-treatedcells), whereas H358 cells were very sensitive to G2-M arrestby perifosine (44.8 ± 2.9 versus 21.6 ± 1.5 in PBS-treatedcells) with limited sensitivity to undergo apoptotic celldeath (18.4 ± 2.4% in perifosine-treated cells versus 10.8 ±3.9% in PBS-treated cells). No apoptosis but very weakG2-M arrest (25.8 ± 1.9% in perifosine-treated cells versus22.9 ± 0.9% in control cells) was detected inH226 cells exposedto 10 μmol/L perifosine. A549 andH157 cells underwent both

Molecular Cancer Therapeutics


2577




Figure 2. Celecoxib protects NSCLC cells from perifosine-induced decrease in cell survival (A) and apoptosis (B and C) and antagonizes the anticanceractivity of perifosine in mouse xenograft models (D). A, both H358 and H460 cells were treated with perifosine alone, celecoxib alone, or perifosine pluscelecoxib at the indicated concentrations for 48 h. Cell survival was estimated using the SRB assay. Columns, means of four replicate determinations; bars,± SD. P values at all combination treatments are at least < 0.01 when compared with perifosine alone. B, H460 cells were treated with 10 μmol/Lperifosine in the absence and presence of 10 μmol/L celecoxib for 24 h and analyzed for apoptosis by Annexin V staining. C, H460 cells were treatedwith 5 μmol/L perifosine in the absence or presence of increasing doses of celecoxib (5, 10, 20 μmol/L) for 12 h. The cells were harvested for preparationof whole-cell protein lysates and detection of caspase and PARP cleavage by Western blot analysis. CF, cleaved from. D, the indicated lung cancer xeno-grafts were treated with vehicle, 20 mg/kg perifosine, 50 mg/kg celecoxib, or the combination of celecoxib and perifosine for 9 d (H460) or 30 mg/kgperifosine, 100 mg/kg celecoxib, or the combination for 11 d (H358). Tumor sizes were measured once every 2 d. Tumor growth rates were calculated bycomparing with the initial size of each individual tumor. Each measurement is a mean ± SE (n = 6). *, P < 0.05; **, P < 0.01; and ***, P < 0.001compared with vehicle control. #, P < 0.05 compared with perifosine treatment. PRFS, perifosine; CCB, celecoxib.



2578




Figure 3. Perifosine increases COX-2 expression (A) and prostaglandin production (B) in NSCLC cells and induces COX-2 expression in lung cancerxenografts (C and D). A, the indicated cell lines were treated with the given concentrations of perifosine for 8 h (top) or with 10 μmol/L perifosine for theindicated times (bottom). The cells were subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis. Actin served as aloading control. B, H460 cells were treated with the indicated concentrations of perifosine for 16 h and then subjected to estimation of PGE2 levels and15d-PGJ2 levels in media using ELISA kits according to the manufacturers' instructions. Columns, means of duplicate determinations; bars, ± SD.C, Western blot analysis of COX-2 expression in H358 tumor lysates after 6 d of treatment with vehicle control, 20 mg/kg perifosine, or 40 mg/kgperifosine by oral gavage in nude mice. These data were also quantitated using the NIH ImageJ software (bottom). D, immunohistochemistry stainingof COX-2 expression in H358 tumors after 5 d of treatment with vehicle control or 30 mg/kg perifosine by oral gavage in nude mice. The COX-2–positivecells were scored using the MetaMorph Imaging Software and graphed (right). Left, the representative immunohistochemistry results of COX-2. Theresults in C and D are the means of triplicate tumors (n = 3). Bars, ± SDs.



2579




G2-M arrest and apoptosis upon perifosine treatment (P < 0.05or 0.01; Fig. 1C and D). Thus, it seems that perifosine inducesapoptosis and/or G2-M arrest leading to inhibition of thegrowth of human NSCLC cells.Celecoxib Reduces the Anticancer Activity of

Perifosine in Cell Culture and In vivoWe were interested in enhancing the anticancer activity of

perifosine. To this end, we tested the effects of perifosine incombination with other cancer therapeutic agents includingcelecoxib. Unexpectedly, the presence of celecoxib at thetested concentrations ranging from 5 to 20 μmol/L signifi-cantly attenuated perifosine-mediated growth-inhibitoryeffects in both H460 and H358 cells (P < 0.01 or 0.001;Fig. 2A). Similarly, other COX-2 inhibitors, includingSC-58125 and DUP697, also significantly protected cellsfrom perifosine-induced growth inhibition. However, thecelecoxib derivative, DMC, which lacks COX-2–inhibitoryactivity, failed to protect cells from perifosine-induced celldeath (Supplementary Fig. S1).Furthermore, we analyzed the effects of perifosine on in-

duction of apoptosis and G2-M arrest in the presence of cel-ecoxib. Perifosine induced 50% of H460 cells to undergoapoptosis in the absence of celecoxib, but only 24% apopto-sis in the presence of celecoxib (Fig. 2B). Perifosine alonecaused cleavage of caspase-8, caspase-9, caspase-3, andPARP. However, the presence of celecoxib abrogated theability of perifosine to cause cleavage of caspases and PARP(Fig. 2C). Thus, these results clearly show that celecoxib pro-tects cells from perifosine-induced apoptosis. Because H358

cells are more sensitive to G2-M arrest when treated withperifosine, we also examined the impact of celecoxib onthe ability of perifosine to induce G2-M arrest in this cellline. We found that perifosine alone induced 70% of cellsin G2-M phase, but only arrested 39% of cells in G2-M phasein the presence of celecoxib (Supplementary Fig. S2). There-fore, we conclude that celecoxib also protects cells fromperifosine-induced cell cycle arrest.We also determined whether celecoxib antagonized the

anticancer efficacy of perifosine in vivo using lung cancer xe-nografts in mice. As presented in Fig. 2D, perifosine alonesignificantly inhibited the growth of both H460 and H358xenografts (P < 0.05). Celecoxib alone at the tested doses(50 or 100 mg/kg) had minimal effects on the growth of ei-ther tumors. When perifosine was combined with celecoxib,perifosine lost its activity to inhibit the growth of lung tu-mors in both xenograft models (P < 0.05). Thus, it is appar-ent that celecoxib also antagonizes the anticancer activity ofperifosine in vivo.Perifosine Increases COX-2 Expression and Activity in

NSCLC Cells and in Lung Cancer Xenografts

Because the addition of celecoxib could abrogate perifosine-induced cell death and growth arrest, we next determinedwhether perifosine modulated COX-2 expression andactivity in NSCLC cells. By Western blot analysis, we de-tected dose-dependent increases in COX-2 expression inH460, H358, A549, and H157 cells, but not in H226 cells(Fig. 3A, top). Of these NSCLC cell lines, H460 and H358cells exhibited the highest degree of COX-2 induction

Figure 4. Celecoxib inhibits perifosine-induced prostaglandin production (A) and COX-2 expression (B) in human NSCLC cells. A, H460 cells wereexposed to 10 μmol/L perifosine in the absence or presence of 10 μmol/L celecoxib or celecoxib alone for 16 h. The media were then collected for mea-surement of PGE2 (top) or 15dPGJ2 (bottom) levels by ELISA kits. Columns, means of duplicate determinations; bars, ± SD. B, the indicated cell lines weretreated with 5 μmol/L perifosine in the absence and presence of increasing concentrations of celecoxib (5, 10, 20 μmol/L) or celecoxib alone (5, 10,20 μmol/L) for 12 h. The cells were subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis for detection of thegiven proteins.



2580




(Fig. 3A) and are the most sensitive to perifosine (Fig. 1B),whereas the H226 cells that did not show COX-2 inductionafter perifosine treatment were the least sensitive to perifo-sine (Fig. 1B), suggesting that COX-2 induction by perifo-sine correlates with sensitivity to perifosine. Importantly,the induction of COX-2 in the H460 and the H358 cellslines occurred as early as 6 hours after perifosine treatment(Fig. 3A, bottom), suggesting that COX-2 induction is an earlyevent in response to perifosine treatment. Perifosine had noeffect on the expression of COX-1 in H460 or H358 cell lines(data not shown). In addition, significantly increased levels ofthe prostaglandins, PGE2 and 15d-PGJ2, were detected incells exposed to perifosine (P < 0.05 or 0.01; Fig. 3B), indicat-ing that perifosine increases COX-2 activity as well. Collec-tively, these results show that perifosine increases COX-2expression and activity in human NSCLC cells.To determine whether perifosine increased COX-2 expres-

sion in vivo, we treated H358 lung cancer xenografts withperifosine or vehicle control and detected COX-2 levels inthese xenografts by both Western blotting (6-day treatment)and immunohistochemistry (5-day treatment). As we ob-served in cell cultures, we detected significant increased le-vels of COX-2 by Western blot analysis (Fig. 3C) andimmunohistochemistry (Fig. 3D) in perifosine-treated xeno-grafts compared with vehicle-treated tumors (P < 0.05).

Thus, perifosine also increases COX-2 levels in tumor tis-sues in vivo.Celecoxib Inhibits Perifosine-Induced COX-2

Expression and Activity

To determine whether celecoxib at the tested concentra-tions indeed inhibits perifosine-induced COX-2 activation,we analyzed the effects of perifosine on the production ofPGE2 and 15d-PGJ2 in the presence and absence of celecoxib.As presented in Fig. 4A, perifosine alone increased thelevels of PGE2 and 15d-PGJ2; however, addition of celecoxibsignificantly inhibited the perifosine-induced increase inPGE2 and 15d-PGJ2 (P < 0.05). Moreover, we found thatthe presence of celecoxib also abrogated the ability of peri-fosine to increase COX-2 expression (Fig. 4B). Collectively,these results clearly indicate that celecoxib at the testedconcentration ranges inhibits perifosine-induced COX-2activation.B lockade o f COX-2 I nduc t i on v i a Gene t i c

Manipulations Attenuates the Anticancer Activity of

Perifosine

To robustly show the role of COX-2 induction on the anti-cancer activity of perifosine, we further evaluated the abilityof perifosine to inhibit the growth of NSCLC cells whenCOX-2 induction was blocked using either antisense or genesilencing approaches. Perifosine exerted a dose-dependent

Figure 5. Inhibition of COX-2 induction by expression of antisense COX-2 (A and B) or transfection of COX-2 siRNA (C and D) protects againstperifosine-induced growth inhibition. A, Western blot analysis of COX-2 expression in H157 vector control (H157-V) and H157 antisense COX-2(H157-AS) cells after treatment with the indicated concentrations of perifosine for 8 h. B, the indicated cell lines were treated with the given con-centrations of perifosine for 24 or 48 h. The cells were subjected to estimation of cell survival using the SRB assay. Points, means of four replicatedeterminations; bars, ± SD. **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001 compared with H157-P or H157-V. C and D, H460 cells weretransfected with control siRNA (Ctrl) or COX-2 siRNA using RNAifect transfection reagent for 48 h and then treated with 10 μmol/L perifosine. After24 h, the cells were subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis for the given proteins (C) or toAnnexin V staining and flow cytometry for detection of apoptosis (D). Columns, means of duplicate determinations; bars, ± SD. **, P < 0.01 com-pared with perifosine in control siRNA–transfected cells.



2581




effect on COX-2 induction in vector control H157-V cells;however, this effect was substantially abrogated in H157-AS cells, which express antisense COX-2 gene (Fig. 5A). Ac-cordingly, the H157-AS cells were significantly less sensitiveto perifosine compared with parental (H157-P) or H157-Vcells (P < 0.01 or less; Fig. 5B). Because H157 cells are moresusceptible to undergo G2-M arrest upon perifosine treat-ment (Fig. 1), we also compared the effects of perifosineon G2-M arrest in these cell lines. As expected, the effectof perifosine on G2-M arrest was abrogated in H157-AS cells

compared with H157-P and H157-V cells (SupplementaryFig. S3). In addition, we silenced COX-2 expression inH460 cells through transfection of COX-2 siRNA and thenexamined its impact on the apoptosis-inducing effect ofperifosine. Transfection of COX-2 siRNA successfully inhib-ited perifosine-mediated COX-2 induction (Fig. 5C). Corre-spondingly, we detected much less cleaved PARP (Fig. 5C)and Annexin V–positive cells (Fig. 5D) in COX-2 siRNA–transfected cells than in control siRNA–transfected cells.Specifically, we detected 29.6 ± 0.8% of apoptotic cells in

Figure 6. Effects of perifosine on the growth of human head and neck cancer cells (A) and COX-2 expression (B) and protective effects ofcelecoxib on perifosine-induced decrease in cell survival (C) and apoptosis (D). A, the indicated cell lines were treated with increased concen-trations of perifosine (1–20 μmol/L) for 3 d and cell numbers were estimated using the SRB assay. B, the indicated cell lines were treated withthe given concentrations of perifosine for 8 h (top). The cells were subjected to preparation of whole-cell protein lysates and subsequent West-ern blot analysis. Actin served as a loading control. C, the indicated cell lines were treated with the given concentrations of perifosine alone,celecoxib alone, and their respective combinations for 48 h and then subjected to the SRB assay for estimation of cell survival. Columns, meansof four replicate determinations; bars, ± SD. **, P < 0.01 and ***, P < 0.001 compared with perifosine alone. D, the indicated cell lines weretreated with 5 μmol/L perifosine alone, 10 μmol/L celecoxib alone, or their combinaiton for 48 h (left). The cells were harvested and subjectedto flow cytometric analysis for measuring apoptosis using Annexin V staining (left). In addition, the same treatments were done in M4c cells for24 h to prepare whole-cell protein lysates for detection of the indicated proteins by Western Blot analysis (right). Tubulin served as a loadingcontrol. CF,cleavedform.



2582




control siRNA–transfected cells, but only 15.8 ± 0.2% of ap-optotic cells in COX-2–transfected cells (Fig. 5D), which issignificantly less than that in control siRNA–transfectedcells (P < 0.01). Collectively, these results provide a robustsupport for a critical role of COX-2 activation in mediatingthe anticancer activity of perifosine.Perifosine Also Induces COX-2–Dependent Apoptosis

in Head and Neck Cancer Cells

To determine whether perifosine exerts COX-2–dependentapoptosis and shows antagonism with celecoxib in othertypes of cancer cells, we further examined the effects of peri-fosine on COX-2 expression in 686Ln and its derivative celllines M4c, M4d, and M4e, which exhibited differential re-sponses to perifosine (Fig. 6A). As we found in the NSCLCcell lines, the M4c, M4d, and M4e cell lines which expressedmuch higher levels of COX-2 after perifosine treatmentcompared with the 686Ln cell line (Fig. 6B) were much moresensitive to perifosine than were the 686Ln cells (Fig. 6A).Perifosine was also able to induce COX-2 expression inthe M4e cells as early as 3 hours, showing that COX-2 in-duction is an early event. The induction of COX-2 reachedpeak levels at 6 hours and was maintained up to 24 hours,indicating that COX-2 induction is also a sustained event(Supplementary Fig. S4). We next examined the effects ofperifosine combined with celecoxib on the growth of thesehead and neck cancer cells including examination of apo-ptosis. As presented in Fig. 6C, in the presence of celecoxibat the tested concentrations ranging from 5 to 20 μmol/L,perifosine-mediated growth-inhibitory effects were signifi-cantly attenuated in both M4c and M4e cells (P < 0.01 or0.001). The addition of celecoxib also substantially reducedapoptosis induced by perifosine as was examined byAnnexin V analysis and cleavage of caspase-3 and PARP(Fig. 6D.) Thus, the protection from perifosine-induced celldeath by the addition of celecoxib is also clearly shownin human head and neck cancer cell lines. Moreover, thepresence celecoxib abrogated perifosine-induced COX-2expression (Fig. 6D, right), further supporting an importantrole of COX-2 in perifosine-induced apoptosis.

DiscussionInduction of apoptosis and cell cycle arrest by perifosine inother types of cancer cells was documented previously (10,35, 36). Perifosine was reported to induce both G1 and G2-Marrest in glioma cells and head and neck squamous carci-noma cells (10, 35). However, we found that all of the testedhuman NSCLC cells that responded to perifosine under-went G2-M arrest albeit with various degrees, indicatingthat perifosine primarily induces G2-M arrest in humanNSCLC cells. Given that perifosine also induces apoptosisin these cell lines, we conclude that perifosine induces bothapoptosis and G2-M arrest, leading to inhibition of thegrowth of human NSCLC cells.Importantly, the current study reveals a novel and unique

mechanism underlying perifosine-mediated anticancer ac-tivity in human NSCLC and head and neck cancer cellswhich is COX-2–dependent. Specifically, we show that peri-

fosine induces apoptosis and/or cell cycle arrest in humancancer cells through induction and activation of COX-2based on the following compelling evidence: First, perifo-sine increases COX-2 expression in both cell cultures andin mouse lung cancer xenografts. The degree of COX-2 in-duction is associated with the potency of perifosine in inhi-biting the growth of cancer cells. Second, the COX-2 specificinhibitor celecoxib as well as other COX-2 inhibitors sup-press perifosine-induced COX-2 up-regulation and activa-tion and protect cells from perifosine-induced apoptosis orG2-M arrest. In addition, celecoxib's derivative, DMC,which lacks a COX-2-inhibitory effect, failed to abrogatethe antitumor effect of perifosine, suggesting that inductionof COX-2 activity is important in mediating apoptosis andcell cycle arrest induced by perifosine. Finally, blockade ofCOX-2 induction using either antisense or siRNA ap-proaches attenuates the ability of perifosine to inducegrowth arrest or apoptosis. To our knowledge, this is thefirst study to show a critical role of COX-2 activation in me-diating the anticancer activity of perifosine.Given that previous studies have shown proapoptotic or

tumor suppressive roles of COX-2 in various model systems(17, 21–25, 28), our current finding on the critical role ofCOX-2 activation in mediating the anticancer activity ofperifosine should not be surprising. The fundamental ques-tions of how COX-2 mediates perifosine-induced apoptosisor cell cycle arrest and how perifosine increases COX-2 ex-pression are of interest for further exploration in the future.15d-PGJ2, one of the prostaglandins generated by COX-2,can act as a PPARγ ligand and is proapoptotic (37). A recentstudy suggests that edelfosine, another alkylphospholipid,induces COX-2–dependent apoptosis through productionof 15d-PGJ2 (28). They showed that edelfosine could inducethe expression and transcriptional activity of PPARγ inH-ras–transformed human breast epithelial cells, suggest-ing that 15d-PGJ2 may be acting through PPARγ to induceapoptosis. In our study, we detected increased levels of15d-PGJ2 in cells exposed to perifosine, but perifosinehad no effect on the transcriptional activity of PPARγ (datanot shown.). Another recent study showed that the chemo-therapeutic agents paclitaxel, cisplatin, and 5-fluorouracilcould induce COX-2 expression in cervical carcinoma cells,and the inhibition of COX-2 by siRNA or the addition ofthe selective COX-2 inhibitor, NS-398, rendered the cells lesssensitive to apoptosis induced by these chemotherapeuticagents (26). They showed that the addition of 15d-PGJ2–induced apoptosis and inhibition of lipocalin-type PGDsynthase, which contributes to prostaglandin synthesis,could prevent the chemotherapeutically induced apoptosis.Therefore, whether the production of prostagladins, specifi-cally, 15d-PGJ2, directly contributes to perifosine-inducedCOX-2–dependent apoptosis in our system needs further in-vestigation. Our unpublished data show that perifosine in-creased the activity of the COX-2 promoter, suggesting thatperifosine likely induces COX-2 expression at the transcrip-tional level. Therefore, a detailed analysis of the COX-2 pro-motermay help us reveal themechanism bywhich perifosineincreases COX-2 expression.



2583




In this study, perifosine-induced caspase-8 activation isclearly inhibited by celecoxib (Fig. 2C). We previouslyshowed that perifosine induces DR5 expression which con-tributes to perifosine-induced apoptosis (11). Moreover, ithas been shown that 15d-PGJ2 increases DR5 expression(38). Thus, it is plausible to speculate a link betweenperifosine-induced COX-2 and DR5 up-regulation. Ourpreliminary data indeed show that celecoxib is able toabrogate perifosine-induced DR5 expression.1 Our ongoingwork in this direction may shed light on the mechanismsunderlying COX-2–induced apoptosis.We did not see complete protection from perifosine-

induced cell killing or arrest with celecoxib, or blockade ofCOX-2 induction using either an antisense or gene knock-down strategy, suggesting that COX-2 is not the only mech-anism mediating the anticancer activity of perifosine inhuman NSCLC cells. It has been documented that perifosineinhibits Akt activity (9, 36). Our own study shows thatperifosine inhibits Akt activity leading to perifosine-inducedapoptosis in human NSCLC cells (11). Thus, further explora-tion of the relationship between COX-2 activation and Aktinhibition is also of interest in the future.Celecoxib is a prescribed drug for treatment of inflamma-

tion and pain. In the field of oncology, celecoxib is an ap-proved drug in the clinic for the adjuvant treatment offamilial adenomatous polyposis, an inherited syndromethat predisposes individuals to colon cancer. In addition,celecoxib has been tested in various clinical trials for eithercancer chemoprevention or therapy (13, 16). The clinicallyachievable peak plasma concentrations of celecoxib in hu-mans after oral administration of a single dose of 800 mgare approximately 3.2 to 5.6 μmol/L (39). At these concen-tration ranges, celecoxib significantly attenuated thegrowth-inhibitory effects of perifosine. Consistently, cele-coxib significantly diminished the activity of perifosineagainst the growth of lung cancer xenografts in mouse mod-els (Fig. 2). Collectively, these results clearly indicate thatcelecoxib antagonizes the anticancer activity of perifosineboth in vitro and in vivo. As mentioned above, the COX-2inhibitor NS 398 can render cervical carcinoma cells lesssensitive to apoptosis induced by chemotherapeuticagents (26). These data and our current study add to theclinical relevance of COX-2 inhibition by non-steriod anti-inflammatory drugs in patients also being administeredanticancer agents. The immediate clinical impact of ourstudy is that caution should be taken when combiningCOX-2 inhibitors such as celecoxib with perifosine, so asnot to inhibit the efficacy of perifosine. Given that COX-2induction seems to be associated with cell sensitivity toperifosine-induced apoptosis or cell cycle arrest as shownin our study, it may also be important in the clinicalpractice to consider COX-2 induction or activity as apredictive biomarker for perifosine-based cancer therapy.

Disclosure of Potential Conflicts of Interest

The authors report no potential conflicts of interest.

Acknowledgments

We thank Dr. A.H. Schönthal (University of Southern California, LosAngeles, CA) for providing DMC, Dr. S.M. Dubinett (University of Cali-fornia, Los Angeles, CA) for providing antisense COX-2 stable cell lines,and Dr. Z.G. Chen (Emory University, Atlanta, GA) for providing thehead and neck cancer cell lines.

References

1. Ruiter GA, Verheij M, Zerp SF, van Blitterswijk WJ. Alkyl-lysophospholipidsas anticancer agents and enhancers of radiation-induced apoptosis. IntJ Radiat Oncol Biol Phys 2001;49:415–9.

2. Jendrossek V, Handrick R. Membrane targeted anticancer drugs: po-tent inducers of apoptosis and putative radiosensitisers. Curr Med ChemAnticancer Agents 2003;3:343–53.

3. Hilgard P, Klenner T, Stekar J, Nossner G, Kutscher B, Engel J.D-21266, a new heterocyclic alkylphospholipid with antitumour activity.Eur J Cancer 1997;33:442–6.

4. Dasmahapatra GP, Didolkar P, Alley MC, Ghosh S, Sausville EA, RoyKK. In vitro combination treatment with perifosine and UCN-01 demon-strates synergism against prostate (PC-3) and lung (A549) epithelialadenocarcinoma cell lines. Clin Cancer Res 2004;10:5242–52.

5. Rahmani M, Reese E, Dai Y, et al. Coadministration of histone deacety-lase inhibitors and perifosine synergistically induces apoptosis in humanleukemia cells through Akt and ERK1/2 inactivation and the generationof ceramide and reactive oxygen species. Cancer Res 2005;65:2422–32.

6. Nyakern M, Cappellini A, Mantovani I, Martelli AM. Synergistic induc-tion of apoptosis in human leukemia T cells by the Akt inhibitor perifosineand etoposide through activation of intrinsic and Fas-mediated extrinsiccell death pathways. Mol Cancer Ther 2006;5:1559–70.

7. Li X, Luwor R, Lu Y, Liang K, Fan Z. Enhancement of antitumor activityof the anti-EGF receptor monoclonal antibody cetuximab/C225 by perifo-sine in PTEN-deficient cancer cells. Oncogene 2006;25:525–35.

8. Stephenson J, Schreeder M, Waples J, et al. Perifosien (P), active as asingle agent for renal cell carcinoma (RCC), now in phase I trials combinedwith tryosine kinase inhibitors (TKI). J Clin Oncol 2007 ASCO AnnualMeeting Proceedings Part I 2007;25:15622.

9. Kondapaka SB, Singh SS, Dasmahapatra GP, Sausville EA, Roy KK.Perifosine, a novel alkylphospholipid, inhibits protein kinase B activation.Mol Cancer Ther 2003;2:1093–103.

10. Patel V, Lahusen T, Sy T, Sausville EA, Gutkind JS, Senderowicz AM.Perifosine, a novel alkylphospholipid, induces p21(WAF1) expression insquamous carcinoma cells through a p53-independent pathway, leadingto loss in cyclin-dependent kinase activity and cell cycle arrest. CancerRes 2002;62:1401–9.

11. Elrod HA, Lin YD, Yue P, et al. The alkylphospholipid perifosineinduces apoptosis of human lung cancer cells requiring inhibition of Aktand activation of the extrinsic apoptotic pathway. Mol Cancer Ther2007;6:2029–38.

12. Frampton JE, Keating GM. Celecoxib: a review of its use in themanagement of arthritis and acute pain. Drugs 2007;67:2433–72.

13. Koki AT, Masferrer JL. Celecoxib: a specific COX-2 inhibitor withanticancer properties. Cancer Control 2002;9:28–35.

14. Grosch S, Maier TJ, Schiffmann S, Geisslinger G. Cyclooxygenase-2(COX-2)-independent anticarcinogenic effects of selective COX-2 inhibi-tors. J Natl Cancer Inst 2006;98:736–47.

15. Zha S, Yegnasubramanian V, Nelson WG, Isaacs WB, De Marzo AM.Cyclooxygenases in cancer: progress and perspective. Cancer Lett 2004;215:1–20.

16. Dannenberg AJ, Subbaramaiah K. Targeting cyclooxygenase-2 inhuman neoplasia: rationale and promise. Cancer Cell 2003;4:431–6.

17. Bol DK, Rowley RB, Ho CP, et al. Cyclooxygenase-2 overexpression inthe skin of transgenic mice results in suppression of tumor development.Cancer Res 2002;62:2516–21.

18. Yegnasubramanian S, Kowalski J, Gonzalgo ML, et al. Hypermethyla-tion of CpG islands in primary and metastatic human prostate cancer.Cancer Res 2004;64:1975–86.1 Elrod and Sun, unpublished data.



2584




19. Nakopoulou L, Mylona E, Papadaki I, et al. Overexpression ofcyclooxygenase-2 is associated with a favorable prognostic phenotypein breast carcinoma. Pathobiology 2005;72:241–9.

20. O'Kane SL, Cawkwell L, Campbell A, Lind MJ. Cyclooxygenase-2 ex-pression predicts survival in malignant pleural mesothelioma. Eur J Cancer2005;41:1645–8.

21. Trifan OC, Smith RM, Thompson BD, Hla T. Overexpression ofcyclooxygenase-2 induces cell cycle arrest. Evidence for a prostaglandin-independent mechanism. J Biol Chem 1999;274:34141–7.

22. Xu Z, Choudhary S, Voznesensky O, et al. Overexpression of COX-2in human osteosarcoma cells decreases proliferation and increases apo-ptosis. Cancer Res 2006;66:6657–64.

23. Tang HY, Shih A, Cao HJ, Davis FB, Davis PJ, Lin HY. Resveratrol-induced cyclooxygenase-2 facilitates p53-dependent apoptosis in hu-man breast cancer cells. Mol Cancer Ther 2006;5:2034–42.

24. Hinz B, Ramer R, Eichele K, Weinzierl U, Brune K. Up-regulation ofcyclooxygenase-2 expression is involved in R(+)-methanandamide-induced apoptotic death of human neuroglioma cells. Mol Pharmacol2004;66:1643–51.

25. Davaille J, Gallois C, Habib A, et al. Antiproliferative properties ofsphingosine 1-phosphate in human hepatic myofibroblasts. A cyclooxy-genase-2 mediated pathway. J Biol Chem 2000;275:34628–33.

26. Eichele K, Ramer R, Hinz B. Decisive role of cyclooxygenase-2 andlipocalin-type prostaglandin D synthase in chemotherapeutics-induced ap-optosis of human cervical carcinoma cells. Oncogene 2008;27:3032–44.

27. Lin HY, Tang HY, Keating T, et al. Resveratrol is pro-apoptotic and thy-roid hormone is anti-apoptotic in glioma cells: both actions are integrin andERK mediated. Carcinogenesis 2008;29:62–9.

28. Na HK, Inoue H, Surh YJ. ET-18-O-CH3-induced apoptosis is causallylinked to COX-2 upregulation in H-ras transformed human breast epithelialcells. FEBS Lett 2005;579:6279–87.

29. Kardosh A, Soriano N, Liu YT, et al. Multitarget inhibition of drug-resistant multiple myeloma cell lines by dimethyl-celecoxib (DMC), anon-COX-2 inhibitory analog of celecoxib. Blood 2005;106:4330–8.

30. Sun SY, Yue P, Dawson MI, et al. Differential effects of synthetic nu-clear retinoid receptor-selective retinoids on the growth of human non-small cell lung carcinoma cells. Cancer Res 1997;57:4931–9.

31. Krysan K, Merchant FH, Zhu L, et al. COX-2-dependent stabilization ofsurvivin in non-small cell lung cancer. FASEB J 2004;18:206–8.

32. Zhang X, Su L, Pirani AA, et al. Understanding metastatic SCCHNcells from unique genotypes to phenotypes with the aid of an animal modeland DNA microarray analysis. Clin Exp Metastasis 2006;23:209–22.

33. Liu X, Yue P, Zhou Z, Khuri FR, Sun SY. Death receptor regulation andcelecoxib-induced apoptosis in human lung cancer cells. J Natl Cancer Inst2004;96:1769–80.

34. Sun SY, Yue P, Wu GS, et al. Mechanisms of apoptosis induced by thesynthetic retinoid CD437 in human non-small cell lung carcinoma cells.Oncogene 1999;18:2357–65.

35. Momota H, Nerio E, Holland EC. Perifosine inhibits multiple signalingpathways in glial progenitors and cooperates with temozolomide to arrestcell proliferation in gliomas in vivo. Cancer Res 2005;65:7429–35.

36. Hideshima T, Catley L, Yasui H, et al. Perifosine, an oral bioactivenovel alkylphospholipid, inhibits Akt and induces in vitro and in vivo cyto-toxicity in human multiple myeloma cells. Blood 2006;107:4053–62.

37. Na HK, Surh YJ. Induction of cyclooxygenase-2 in Ras-transformedhuman mammary epithelial cells undergoing apoptosis. Ann N Y AcadSci 2002;973:153–60.

38. Straus DS, Glass CK. Cyclopentenone prostaglandins: new insightson biological activities and cellular targets. Med Res Rev 2001;21:185–210.

39. Niederberger E, Tegeder I, Vetter G, et al. Celecoxib loses its anti-inflammatory efficacy at high doses through activation of NF-κB. FASEBJ 2001;15:1622–4.



2585




2009;8:2575-2585. Published OnlineFirst September 15, 2009.Mol Cancer Ther Heath A. Elrod, Ping Yue, Fadlo R. Khuri, et al. involving a cyclooxygenase-2-dependent mechanismCelecoxib antagonizes perifosine's anticancer activity

Updated version

10.1158/1535-7163.MCT-09-0390doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2009/09/16/1535-7163.MCT-09-0390.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/8/9/2575.full#ref-list-1

This article cites 39 articles, 17 of which you can access for free at:

Citing articles

http://mct.aacrjournals.org/content/8/9/2575.full#related-urls

This article has been cited by 3 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] at

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

Permissions

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

.http://mct.aacrjournals.org/content/8/9/2575To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-09-0390

http://mct.aacrjournals.org/content/suppl/2009/09/16/1535-7163.MCT-09-0390.DC1

http://mct.aacrjournals.org/content/8/9/2575.full#ref-list-1

http://mct.aacrjournals.org/content/8/9/2575.full#related-urls

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

mailto:[email protected]

http://mct.aacrjournals.org/content/8/9/2575


Celecoxibantagonizesperifosine'santicanceractivity ......2and15d-PGJ 2...

Documents

Transcript of Celecoxibantagonizesperifosine'santicanceractivity ......2and15d-PGJ 2...