retracted: tumor necrosis factor enhances sn38-mediated apoptosis in mesothelioma cells : the role...

Tumor Necrosis Factor Enhances SN38-MediatedApoptosis in Mesothelioma CellsThe Role of Nuclear Factor-�B Pathway Activation

Patrizia Russo, Ph.D.1

Alessia Catassi, Ph.D.2

Davide Malacarne, Ph.D.1

Stefano Margaritora, M.D., Ph.D.2

Alfredo Cesario, M.D., Ph.D.3,4

Luigi Festi, M.D., Ph.D.5

Antonino Mule, M.D., Ph.D.5

Luigi Ferri, M.D., Ph.D.4

Pierluigi Granone, M.D., Ph.D.2

1 Laboratory of Translational Research B(LungCancer), Department of Integrated Medical Oncol-ogy, National Cancer Institute, Genoa, Italy.

2 Department of Surgical Science, Division of Gen-eral Thoracic Surgery, Catholic University, Rome,Italy.

3 Department of Biology (DIBIO), University ofGenova, Genoa, Italy.

4 Pulmonary Rehabilitation, IRCCS San Raffaele,Rome, Italy.

5 Division of Thoracic Surgery, “Insubria” Univer-sity, Varese, Italy.

6 Department of Pathology, Catholic University,Rome, Italy.

Supported in part by TENDER (Grant 2000/S 118-076796: “Induction of conformational changes inp53 mutants and modulation of sensitivity to se-lective anticancer drugs”) awarded by the Euro-pean Community, Ispra, Italy (2003).

The authors thank Dr. Francesco Pezzella (TumourPathology Group, Nuffield Department of ClinicalLaboratory Science, University of Oxford, Oxford,UK) for his useful discussions.

Address for reprints: Patrizia Russo, Ph.D., Laboratoryof Translational Research B(Lung Cancer), Depart-ment of Integrated Medical Oncology, National Can-cer Institute, Genoa, Italy, Largo Rosanna Benzi 10,I-16132 Genoa, Italy; Fax: (011) 39 0105600217;E-mail: [email protected]

Received March 3, 2004; revision received No-vember 2, 2004; accepted December 3, 2004.

BACKGROUND. Despite the best and most aggressive, often integrated, standard

therapeutic approaches for mesothelioma, overall survival remains very poor. The

actual failure points out clearly the need for the development of novel therapy. One

of the promising paths of experimentation is artificial induction of apoptosis. A

therapeutic strategy that relies on the down-regulation of BCL-XL inhibition nu-

clear factor �B (NF-�B) with a combination of SN38 and tumor necrosis factor

(TNF) was studied in human mesothelioma cell lines (MSTO-221H, IST-MES1,

IST-MES2, MPP89, H28, H513, H2052, and H290).

METHODS AND RESULTS. Cell proliferation (clonogenic assay) was inhibited

strongly by the combination of TNF and SN38. Examining the persistence of the

NF-�B complexes using an electrophoretic mobility-shift assay, it appeared that

they still were present at 24 hours in TNF-treated cells. In SN38-treated cells,

NF-�B complexes persisted for 6 hours. In cells that were treated with combined

SN38 and TNF, NF-�B complexes disappeared quickly and became undetectable at

6 hours. In flow cytometry analysis, only cells that were treated with combined

SN38 and TNF demonstrated significant cellular accumulation in the sub-G0–G1

phase, suggesting a specific induction of apoptosis. Morphologic examination

(4,6-diamidino-2-phenylindole staining and electron microscopy) and internu-

cleosomal DNA fragmentation (gel ladder) confirmed rigorously the induction of

apoptosis.

CONCLUSIONS. Because of NF-�B inhibition with the combination of SN38 and

TNF, the expression of BCL-XL (both the protein [Western blot analysis] and the

mRNA [reverse transcriptase-polymerase chain reaction analysis]) was down-reg-

ulated, cytochrome c was released into the cytoplasm, caspase 3 was activated

(Western blot analysis), and, consequently, apoptosis was triggered. The authors

hope that the results of the current study may contribute to the design and

implementation of a novel therapeutic approach that improves patients’ responses

to treatment for mesothelioma. Cancer 2005;103:1503–18.

© 2005 American Cancer Society.

KEYWORDS: malignant pleural mesothelioma, tumor necrosis factor, camptothecin,apoptosis, nuclear factor-�B, survival, BCL-XL, caspase 3, DNA damage, cell cycle.

Malignant pleural mesothelioma (MPM) is an uncommon, locallyinvasive, and rapidly fatal malignancy with an incidence that

continues to increasing in the Western world. It is linked to asbestosexposure and genetic susceptibility.1 Despite the best and most ag-gressive, often integrated, standard therapeutic approaches, overallsurvival remains very poor.2 The failure rate points clearly to the needfor the development of novel therapy for patients with MPM. In thissetting, one of the promising paths of experimentation is artificial

1503

© 2005 American Cancer SocietyDOI 10.1002/cncr.20924Published online 7 February 2005 in Wiley InterScience (www.interscience.wiley.com).

TRACTEDften inteten int

rvival remainsvival remains

evelopment of novel thvelopment of novel

artificial inductionartificial induction of apof

wn-regulation of BCL-XL inwn-regulation of BCL-XL

ination of SN38 and tumonation of SN38 and tumo

sothelioma cell lines (MSTsothelioma cell lines (MS

H2052, and H290).H2052, and H290).

Cell proliferation (clonogCell proliferation (clono

nation of TNF and SN38. Enation of TNF and S

using an electrophoretic msing an electrophoretic m

present at 24 hours in TNsent at 24 hours in T

plexes persisted for 6 hourpersisted for 6 h

d TNF, NF-d TNF �B complexes dB complexes d�

ours. In flow cytometry anIn flow cytometry an

SN38 and TNF demonstratTNF demonstra

phase, suggesting a spphase, suggesting a sp

(4,6-diamidino-2-ph4,6-diamidino-2-

cleosomal DNAcleosomal DNA

apoptosis.apoptosis.

CONCLUSCONCLUTNF,NF

m

RETRle,e,

bria” Univer-ria” Univer-

, Catholic University,Catholic U

by TENDER (Grant 2000/S 118-by TENDER (Grant 2000/S 118-ction of conformational changesction of conformational changes

s and modulation of sensitivitys and modulation of sensitivityncer drugs”) awarded byer drugs”) awarded by

y, Ispra, Italy (2003)spra, Italy (2003

Francranc

TR

induction of apoptosis. To our knowledge, however,the mechanism and pathway of induction of apoptosisin MPM is known only partially.3,4 In this malignancy,which is extremely rich in peculiarities, p53 mutationsare uncommon.3–5 This certainty may suggest thatalterations in the expression of this gene are unlikelyto be responsible for either the creation of an MPMphenotype or MPM insensitivity to apoptotic induc-tion itself. In simian virus 40 (SV40)-positive MPM,this occurrence may be explained by an inactivation ofthe p53 function coming from the viral TAG.6 Ho-mozygous deletions of the INK4a/ARF locus, however,have been shown to be the predominant events thatoccur at a frequency of � 70% in this malignancy.7–9

This deletion results in the loss of p14ARF, in the in-crease of MDM2, and in the functional inactivation ofp53, thereby diminishing the p53 response to geno-toxic stress.10,11 It was reported previously that thep14ARF transfection resulted in apoptotic cell deaththrough the induction of p14ARF overexpression.12

Conversely, it is interesting to note that others foundthat high BCL-XL expression is relatively uniform forthis tumor, in which BCL-2 expression usually israre.13,14 BCL-X, as a result of alternative splicing, en-codes two death regulators that exhibit opposing ac-tivities. BCL-XL exerts a protective effect, whereas theshorter form, BCL-XS, is proapoptotic.15 The NationalCancer Institute Anticancer Drug Screen (NCI-ACDS)study16 reported a strong negative correlation betweenbasal BCL-XL protein and mRNA levels and drug sen-sitivity. Conversely, BAX and BCL2 showed no corre-lation at all with drug sensitivities. These observationssuggest that BCL-XL may play a unique role in generalresistance to cytotoxic agents (70,000 drugs were con-sidered in the study by Amundson et al.16). The broadnature of the association between endogenous BCL-XL expression and protection from chemotoxicity in-dicates that this gene is an extremely important gen-eral determinant of cell death and suggests a p53-independent mechanism of BCL-XL action as a majorcomponent of chemoresistance in tumor cells.

The main pathway involved in inducible resis-tance is the activation of nuclear factor �B (NF-�B)within tumors in response to chemotherapy or to tu-mor necrosis factor (TNF).17–19 NF-�B suppression ofapoptosis appears to be a transcriptional event, be-cause it activates expression of TRAF-1 and TRAF-2and of c-IAP1 and c-IAP2 to block caspase 8 activa-tion.19,20 It is interesting to note that other antiapop-totic genes that are activated transcriptionally byNF-�B include BCL-XL.21

Different studies have described the inducible an-tiapoptotic function of the transcription factor NF-�B

as a principle mechanism by which cancer cells areprotected from undergoing programmed cell deathafter exposure to TNF, � irradiation, and antineoplas-tic drugs, including SN38.19,22–25 Activation of the tran-scription factor NF-�B by extracellular signals involvesits release from the NF-�B inhibitor � protein (I�B-�)in the cytoplasm and subsequent nuclear transloca-tion.19 NF-�B modulates the expression of many genesthat control cell survival.19 Although some of the targetgene products are protective and others induce celldeath, the primary role of NF-�B is to promote cellsurvival, because massive apoptosis of liver cells hasbeen observed in embryonic NF-�B knockout mice.26

Paradoxically, NF-�B can be activated by anticanceragents, including topoisomerase I and II inhibitors.19

It is accepted generally that camptothecin (CPT) in-duces NF-�B activation during the process of apopto-sis in different mammalian cells, such as HeLa cells,colon carcinoma cells, and lung carcinoma cells.Huang et al.27 have elucidated a series of nuclearevents induced by CPT that converge with cytoplasmicsignaling events responsible for the activation of NF-�B, which can provide an antiapoptotic function, andshowed that inhibition of NF-�B augments CPT-in-duced apoptosis. Similar results were obtained by Cu-sack et al.,23,24 who showed enhanced chemosensitiv-ity to CPT inhibiting NF-�B activation throughadenovirus-mediated transfer of the superrepressorI�B-� or utilizing the proteasome inhibitor PS-341. Tounderstand better the response of tumor cells to CPTand to identify potential targets for adjuvant therapy,Carson et al.28 examined the global changes in mRNAabundance in HeLa cells after CPT treatment usingAffymetrix U133A Gene Chips, which include all an-notated human genes (22,283 probe sets). This phar-macogenomic approach led those authors to identifytwo pathways that are CPT-induced: 1) the epidermalgrowth factor receptor; and 2) NF-�B-regulated anti-apoptotic factors. In that study, the authors concludedthat inhibitors of these respective pathways enhancedthe cytotoxicity of CPT superadditively, suggestingtheir potential as targets for adjuvant therapy withCPT. We previously showed that TNF increased CPTsensitivity through the inhibition of NF-�B activation(shortening of its duration) in a human ovarian carci-noma cell line.25

We used this therapeutic approach (shortening ofNF-�B persistence) in human MPM; thus, TNF wasadministered in combination with SN38 (the activemetabolite of irinotecan) in 8 human MPM cell lines(MSTO-221H, IST-MES1, IST-MES2, MPP89, H28,H513, H2052, and H290). The actions of TNF on 1)SN38-induced NF-�B activation, 2) the level of apo-

1504 CANCER April 1, 2005 / Volume 103 / Number 7

TEDsis o

F-F-��B knoB kno��

e activated bye activated byomerase I and II inhmerase I and II i

lly that camptothecin (ly that camptothecination during the procesation during the proces

mammalian cells, suchmammalian cells, suchnoma cells, and lungnoma cells, and lun

t al.t al.2727

RETRACTac-

as thetheNationalnal

(NCI-ACDS)(NCI-ACDS)elation betweenelation between

vels and drug sen-vels and drugCL2 showed no corre-CL2 showed no corre-

vities. These observationvities. These observatioplay a unique role in gelay a unique role in ge

agents (70,000 drugs wagents (70,000 drugs wy by Amundson et al.by Amundson et al.1616))

ssociation between endociation between endn and protection fromn and protection from

at this gene is an extreat this gene is an extreerminant of cell deaterminant of cell dea

ent mechanism ont mechanismof chemoref chemo

pathwpath

A

have elucidatedhave elucidateds induced by CPT that coinduced by CPT that c

naling events responsibnaling events respon�B, which can provideB, which can provide�

showed that inhibishowed that induced apoptosisduced apoptossack et al.,ck et al.,23,223,2

ity to CPity toadenovadenovI�BB�

ptosis, 3) the transcription of BCL-XL, and 4) the ac-tivation of apoptotic effectors (caspases) were evalu-ated.

MATERIALS AND METHODSCell Culture and DrugNCI-H28, H513, and H290 mesothelioma cells were akind gift from Dr. J. D. Minna (Hamon Center forTherapeutic Oncology Research, Dallas, TX). H2052(CRL-5915) and MSTO-211H (CRL-2081) mesotheli-oma cells; renal carcinoma A498, ACHN, and CAKI-1cells; prostate carcinoma PC-3 cells; and lymphoidHL60 and K562 cells were obtained from AmericanType Culture Collection (Manassas, VA). IST-MES1,IST-MES2, and MPP89 mesothelioma cells were kindlydonated by Dr. S. Ferrini (National Institute for Re-search on Cancer, Genoa, Italy). All cells were culturedin RPMI-1640 complete medium containing 10% fetalcalf serum. Cell counts were determined using aCoulter Counter with channelyzer attachment tomonitor cell size (Coulter Electronics, Hialeah, FL).Cell membrane integrity was determined with aTrypan blue-dye exclusion assay.

SN38, which is the active metabolite of the topo-isomerase I (Top I) inhibitor irinotecan, was kindlyprovided by Dr. J. Patrick McGovren (Pharmacia Up-john Inc., Kalamazoo, MI). Recombinant human TNF(rHuTNF) was obtained from KNOLL-BASF (Ludwig-shafen, Germany). A stock solution of rHuTNF thatcontained 0.1 mg/mL of protein was stored at � 80 °C.Specific activity was 8.74 � 106 U/mg protein (1000U/mL � 1.38 ng/mL; 48-hour L929 bioassay withoutActinomycin D, as determined in the KNOLL-BASFlaboratory). The caspase 3 inhibitor, z-DEVD-fmk, wasobtained from Calbiochem (San Diego, CA). The pro-teasome inhibitor dipeptide boronic acid analog PS-34123 was provided by Millennium Pharmaceuticals(Cambridge, MA).

Cytotoxicity Assay and Morphologic AssessmentDrug-induced cytotoxicity was determined using astandard clonogenic assay (drug exposure for 1 hour),as described previously.25 Cell colonies were countedafter 10 days.

The 50% inhibitory concentration (IC50) valueswere extrapolated from the dose-response curves, asestimated by performing a simple linear regression: Y� log (fa/fu) versus X � log (drug concentration), inwhich fa is the fraction affected by the dose calculatedas a ratio (treated cells to untreated cells), and fu is theunaffected fraction (i.e., 1-fa).29,30

Morphologic assessment was determined eitherby staining cells with 4,6-diamidino-2-phenylindole

(DAPI) or by transmission electron microscopy (TEM)analysis. Briefly, 1) 105 cells were seeded on a slide 24hours before treatment; after treatment, slides werewashed twice in phosphate buffered saline (PBS), fixedfor 5 minutes at room temperature in 95% ethanol, airdried, and then dipped for 5 minutes in 0.1 �gDAPI/mL in a methanol solution. One thousand cellswere scored for each slide. (2) Untreated and treatedMSTO-211H cells (attached and suspension cells, re-spectively) were fixed for 2 hours at 4 °C in 2.5%glutaraldehyde and 0.5% tannic acid in 0.1 M cacody-late buffer, pH 7.4, and analyzed by JFE Enterprises(Brookeville, MD) at a magnification of � 2000.

Preparation of Mitochondria-Free Cytosolic Extracts andWhole Cell ExtractsCytosolic extracts were prepared as described previ-ously.31 In brief, MSTO-211H cells were harvested bygently scraping and were incubated in a buffer contain-ing 220 nM mannitol and 60 mM sucrose on ice for 30minutes. Then, cells were broken in a Dounce homoge-nizer by 70 gentle strokes of a type B pestle. The homog-enates were centrifuged at � 16,000 g for 15 minutes,and the mitochondria-free supernatant fluids were fro-zen at � 70 °C until further analysis. Both extracts of thepellets and whole cell extracts were obtained by dissolv-ing in lysis buffer, followed by repetitive vortexing, andfreeze thawing. After centrifugation at � 16,000 g, thesupernatant fluids were stored at � 70 °C. To control forsimilar protein loading or contamination of cytosolicextracts with mitochondria, the immunoblots (seeabove) were stripped and reprobed with antiactin anti-bodies (diluted 1:3,000) or anticytochrome c oxidase an-tibodies (diluted 1:300; COX Vb, Boehringer, Mannheim,Germany).

ImmunoblottingSamples were washed twice with PBS, scraped off theplates, and lysed in cell lysis buffer (50 mM Tris-HCl,pH 7.5; 150 mM NaCl; 1% Nonidet P-40; 0.5% sodiumdeoxycholate; and 0.1% sodium dodecyl sulphate[SDS]). Whole cell lysates were boiled, and the proteinconcentration was determined with the Bradford as-say (Bio-Rad Laboratories, Hercules, CA). Equalamounts of protein (20 – 40 �g) were separated bySDS-polyacrylamide gel electrophoresis under reduc-ing conditions in 4 –20% linear gradient polyacryl-amide gels (Ready-Gel; Bio-Rad Laboratories). Afterthe proteins were transferred to an Immobilon-Pmembrane (Millipore Corp., Bedford, MA), the mem-branes were blocked with 5% nonfat milk powder and0.2% Tween 20 in Tris-buffered saline (TBS-T) over-night at 4 °C and then incubated with the primary

TNF in SN38-Induced Apoptosis/Russo et al. 1505

RETRACTEDdly

a Up-Up-man TNFNF

SF (Ludwig-SF (Ludwig-of rHuTNF thatf rHuTNF that

s stored atstored at �� 80 °C.8U/mg protein (1000U/mg protein (1000

ur L929 bioassay withouur L929 bioassay withomined in the KNOLL-Bmined in the KNOLL-

se 3 inhibitor, z-DEVD-fe 3 inhibitor, z-DEVD-fbiochem (San Diego, CAiochem (San Diego, CA

or dipeptide boronic acr dipeptide boronic arovided by Millenniumovided by Millenniu

ge, MA).ge, MA).

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bycation ofation of

a-Free Cytosolic ExtraFree Cytosolic Ex

were prepared as descwere prepared as descf, MSTO-211H cells werf, MSTO-211H cells we

ng and were incubated ing and were incubatedM mannitol and 60 mMM mannitol and 60 mM

es. Then, cells were broks. Then, cells were brozer by 70 gentle strokes ozer by 70 gentle strokes

enates were centrifugednates were centrifugeand the mitochondrand the mitochzen aten at � 70 °C un°Cpellets and whllets and whing in lysising infreezefreezesupup

antibody for 1 hour at room temperature. Membraneswere then washed in TBS-T for 3 5-minute periods.Primary antibodies for p14ARF, p16INK4a, caspase 8,caspase 9, Bcl-2, Bax, BCL-XL, I�B-�, and actin wereobtained from Santa Cruz Biotechnology (Santa Cruz,CA). Polyclonal antibody to caspase 3 and monoclonalantibodies to poly(ADP-ribose) polymerase (PARP)and cytochrome c were obtained from PharMingen(San Diego, CA). Horseradish peroxidase-conjugatedgoat antimouse or donkey antigoat antibodies wereused as secondary antibodies (Santa Cruz Biotechnol-ogy). Proteins were visualized with chemiluminescentluminol reagents (Santa Cruz Biotechnology).

Flow CytometryCells were plated in log phase in T75 flaks (2700 cells/cm2) in complete medium for 24 hours; treated for 1hour with SN38 alone, TNF alone, or combined SN38and TNF; incubated in drug-free medium for addi-tional 24 hours; then counted before flow cytometricanalysis. Samples were prepared for flow cytometryessentially as described previously.25 Briefly, cells werewashed with 1 � PBS, pH 7.4, and then fixed withice-cold 70% ethanol. Samples were washed with 1� PBS and stained with propidium iodide 60 �g/mL

(Sigma Chemical Company, St. Louis, MO) containingRNase 2 �g/mL (Sigma Chemical Company) for 30minutes at 37 °C. Cell cycle analysis was performedusing a Becton Dickinson fluorescence-activated cellanalyzer and Cell Quest software (version 1.2; BectonDickinson Immunocytometry Systems, Mansfield,MA). For each sample, at least 15,000 cells were ana-lyzed, and quantitation of the cell cycle distributionwas performed using ModFit LT software (version1.01; Verity Software House Inc., Topsham, ME).

Determination of DNA Damage by Alkaline ElutionAlkaline elution was performed to assess DNA dam-age by detecting DNA strand breaks as describedpreviously.25,32 Before alkaline elution and drugtreatments, cells were radiolabeled with 0.02�Ci/mL of [14C]thymidine for 24 hours (2 doublingtimes) at 37 °C and then chased in nonradioactivemedium for 4 hours. After drug treatments (1 hour),cells were scraped in Hanks balanced salt solution,counted, and aliquots of cell suspensions wereplaced in drug-containing ice-cold Hanks balancedsalt solution. After alkaline elution, filters were in-cubated at 65 °C with 1 N HCl for 45 minutes; then,0.04 M NaCl were added for an additional 45 min-

TABLE 1Evaluation of the 50% Inhibitory Concentration in Human Mesothelioma Cell Lines and Combination Indexa

Cell line

Drug combinationa

Combination indexb

Sign of theinteraction

SN38 (nM) rHuTNF (U/mL)SN28 (nM) plus rHuTNF(1000 U/mL)

IC50

95% CL(lower/upper)IC50

95% CL(lower/upper) IC50

95% CL(lower/upper)

IC50

(nM)95% CL(lower/upper)

H2052 8.05E-02 5.10E-02/1.27E-01 1.24E � 05 1.36E � 04/1.13E � 06 1.55E-03 1.15E-03/2.08E-03 2.744E-02 1.294E-02/5.570E-02 Strong SYNH290 3.94E-02 2.25E-02/6.90E-02 1.78E � 04 3.33E � 03/8.69E � 04 1.15E-03 5.18E-04/2.54E-03 8.684E-02 3.904E-02/1.736E-01 Strong SYNH513 5.35E-02 2.75E-02/1.04E-01 1.15E � 05 1.60E � 04/8.31E � 05 3.19E-03 1.82E-03/5.60E-03 6.880E-02 3.363E-02/1.589E-01 Strong SYNIST-MES1 4.41E-02 1.48E-02/1.32E-01 6.39E � 04 1.10E � 04/3.72E � 05 3.26E-03 1.87E-03/5.60E-03 9.0070E-02 4.941E-02/1.993E-01 Strong SYNIST-MES2 2.66E-02 1.06E-02/6.69E-02 4.28E � 04 1.03E � 04/1.78E � 05 8.86E-03 3.16E-03/2.48E-02 3.638E-01 1.383E-01/1.175E � 00c SYNMPP89 3.16E-02 1.19E-02/8.14E-02 4.26E � 04 1.04E � 04/1.74E � 05 6.31E-03 3.34E-03/1.19E-02 2.279E-01 9.320E-02/7.354E-01 SYNMSTO-211H 2.48E-02 1.34E-02/4.56E-02 6.41E � 04 8.38E � 03/4.90E � 03 5.79E-04 9.79E-05/3.42E-03 3.934E-02 1.418E-02/9.572E-02 Strong SYNH28 2.52E-02 1.31E-02/4.87E-02 1.06E � 05 8.22E � 03/1.37E � 06 1.83E-03 9.53E-04/3.50E-03 8.253E-02 3.946E-02/1.950E-01 Strong SYN

IC50: 50% inhibitory concentration; rHuTNF: recombinant human tumor necrosis factor; 95% CL: 95% confidence limits; SYN: synergism.a Cells were exposed to different concentrations of SN38 (nM), to different concentrations of rHuTNF (U/mL), or to a combination of SN38 (different concentrations in nM) plus recombinant human tumor necrosis

factor (at a dose of 1000 U/mL) for 1 hour and then were incubated for 10 days in drug-free medium before they were counted. For each combination, the combination index (CI) and its 95% confidence limits were

estimated.b To calculate the CI, we referred to the equation proposed by Chou and Talalay29,30 for 2 mutually nonexclusive drugs CI � (D)1/(Dx)1�(D)2/(Dx)2�((D)1 � (D)2)/((Dx)1 � (Dx)2), where Dx represents the dose of

the drug alone, as extrapolated from linear regression (see Materials and Methods), capable of producing the same effect of the combined drugs (D1 � D2). In this case, we calculate a CI referred to the drug

combination (�, nM SN38 � 1000 U/mL TNF) estimated as capable of inducing a 50% inhibition of the cell growth: CI � (1000 U/mL)/(IC50TNF) � (IC50 SN38c)/(IC50 SN38a), where SN38a and SN38c indicate

the drug tested, respectively, alone and in combination with TNF 1000 U/mL. In outline, a CI near 1.0 indicates additivity, CI � 1.0 indicates antagonism, CI � 1.0 indicates synergism, and CI � 0.03 indicates strong

synergism. Confidence limits for the CIs were estimated performing a parametric bootstrapping using mean, standard deviation, and covariance of the regression coefficient and Y intercept as estimated from the

three dose-response regression analyses (SN38 alone, TNF alone, and SN38 combined with TNF1000 U/mL) conducted for each cell line.c Not statistically significant.


ACTEDED/1.5

E-02/1.993E 02/1.993383E-01/1.175E383E-01/1.175E ��

9.320E-02/7.354E-019.320E-02/7.354E-01-02 1.418E-02/9.572E-0202 1.418E-02/9.572E-02

53E-02 3.946E-02/1.950E-0153E-02 3.946E-02/1.950E-01

8 (different con8 (different concentrations in nM) plus reccentrations in nM) plus rec

ach combination, the combach combination, the combination index (ination index

CI � (D)(D)11/(Dx)1��(D)(D)22/(D/(Dx)2�((D)1 � (D)D)2)/)

e effect of the combined drugs (De effect of the combined drugs (D1 � D )

owth: CIwth: C � (1000 U/mL)/(IC50TNF)NF) �� (IC(IC

ates additivity, CIaddi �

RETRACure. Membranesre. Membranes

5-minute periods.5-minute perip16p16INK4aK4a, caspase 8,, caspase 8,

XL, IL, I��B-B� �, and actin wed actin wez Biotechnology (Santa CBiotechnology (Santa

dy to caspase 3 and mondy to caspase 3 and moy(ADP-ribose) polymer(ADP-ribose) polymer

cc were obtained fromwere obtained fromCA). Horseradish peroxCA). Horseradish per

mouse or donkey antigmouse or donkey antigsecondary antibodiessecondary antibodies

eins were visualizns were visualigents (Santants (San

(SigigR

1.0 indicates antagonisates antagoni

g mean, standard deviation, and covariancandard deviation, and cov

F1000 U/mL) conducted for each cell line.F1000 U/mL) conducted for each cell lin

AC

utes. Radioactivity in all fractions was measuredwith a liquid scintillation analyzer (Packard Instru-ments, Meriden, CT).

Determination of DNA Single-Strand Breaks

DNA single-strand breaks (SSB) were assessed by al-kaline elution under deproteinizing, DNA denaturingconditions. Briefly, after treatment, radiolabeled cellswere harvested at 4 °C, loaded onto polycarbonatefilters (2-�m pore size; Poretics, Livermore, CA), andlysed with SDS buffer (0.1 M glycine; 0.025 M ethyl-enediamine tetraacetic acid [EDTA]; 2% weight/vol-ume SDS; and 0.5 mg/mL proteinase K, pH 10.0). Thelysis solution was washed from filters with 0.02 MEDTA, pH 10, and the DNA was eluted with tetrapro-pylammonium hydroxide-EDTA, pH 12.1, containing0.1% SDS at a flow rate of 0.035 mL per minute into 5fractions at 3-hour intervals.

Gel Mobility-Shift AssayNucleic extracts were prepared according to themethod described by Vikhanskaya et al.31 Briefly, 5� 105 cells were collected, washed in PBS, and pel-leted. Pellets were resuspended in 400 mL of hypo-tonic buffer (20 mM N-2-hydroxyethyl piperazine-N�-2-ethane sulphonate [HEPES], pH 7.9; 10 mM KCl; 0.1mM EDTA; 0.1 mM ethylene guanine tetraacetic acid[EGTA]; 1 mM dithiothreitol [DTT]; and 0.5 mMphynel methyl sulfonyl fluoride [PMSF]). The cellswere allowed to swell on ice for 15 minutes; then, 25

FIGURE 1. (A) Tumor necrosis factor (TNF) receptor

1 (TNF-R1) mRNA expression in human malignant

pleural mesothelioma (MPM) cell lines. Lanes 2–9:

human MPM cell lines; lane 10: without reverse tran-

scriptase (RT�). Glycerol-3-phosphate dehydrogenase

(G3PDH) was used as a positive control (data not

shown). Results are from one of three representative,

independent experiments that were performed in du-

plicate. (B) Expression of p14ARF and p16INK4A in me-

sothelioma cell lines. The C33A cervical carcinoma cell

line was used as a positive control for the expression

of both p14ARF and p16INK4A. Results are from one of

three representative, independent experiments that

were performed in duplicate.

FIGURE 2. DNA single strand breaks induced by different treatment in the

MSTO-211H cell line. (A) Cells were treated for 1 hour and were lysed

immediately or (B)were incubated in drug-free medium for additional time (B).


CTE

RETRu

assessed by al-assessed by al-g, DNA denaturing, DNA denatu

ent, radiolabeled cellsent, radiolabeled cellsded onto polycarbonaded onto polycarbona

oretics, Livermore, CA)retics, Livermore, CA)(0.1 M glycine; 0.025 M(0.1 M glycine; 0.025 M

cetic acid [EDTA]; 2% wetic acid [EDTA]; 2%.5 mg/mL proteinase K,mg/mL proteinase K

n was washed from filn was washed fromH 10, and the DNA was10, and the DNA wasonium hydroxide-EDTonium hydroxide-ED

at a flow rate oft a flow rate of3-hour intehour in

TRAC

�L of an 18% solution of Nonidet NF-40 were added,and the tubes was vortexed vigorously for 10 seconds.The homogenate was centrifuged for 30 seconds in amicrofuge. The nuclear pellet was resuspended in 50�L ice-cold buffer (20 mM HEPES, pH 7.9; 0.4 M NaCl;1 mM EDTA; 1 mM EGTA; 1 mM DTT; and 1 mMPMSF), and the tubes were rocked vigorously at 4 °Cfor 15 minutes. Nucleic extracts were centrifuged for 5

minutes in a microfuge at 4 °C, and the supernatantfluid was frozen as aliquots at � 70 °C. One milligramto 3 mg of each cell treatment were incubated on icefor 30 minutes in 15 mL of buffer containing 10 mMTris, pH 7.5; 50 mM NaCl; 1 mM EDTA; 1 mM DTT; 3Mg poly [dI-dC]; 2 mL pab65 (Santa Cruz Biotechnol-ogy) or nonspecific antibodies; 1I ng of 32[P] end-labeled oligonucleotide; and part of the enhancer se-

TABLE 2Analysis of Cell Cycle Perturbation in Human Mesothelioma Cell Linesa

Cell line ControlSN38IC50 (nM)

SN38(1.0 � 10�3 nM)

rHuTNF(1000 U/mL)

SN38 (1.0 � 10�3 nM)and rHuTNF (1000 U/mL)

MSTO-211HSub-G0/G1 phase 4.5 � 1.2 24.6 � 1.5 4.7 � 1.9 4.1 � 1.7 48.2 � 5.3G0/G1 phase 44.6 � 2.1 30.9 � 2.4 44.1 � 3.2 46.4 � 4.2 38.6 � 2.4S-phase 28.4 � 3.1 44.5 � 1.7 30.5 � 2.6 20.4 � 2.6 9.2 � 2.1G2/M-phase 27.0 � 1.8 24.6 � 1.8 25.4 � 1.4 33.2 � 2.4 57.2 � 2.2

IST-MES1Sub-G0/G1 phase 8.7 � 2.8 34.1 � 2.8 8.2 � 1.6 4.1 � 1.1 54.2 � 7.8G0/G1 phase 62.4 � 3.3 38.7 � 4.4 47.2 � 4.4 47.9 � 5.4 46.4 � 4.4S-phase 22.4 � 1.4 36.8 � 2.8 28.2 � 1.5 23.4 � 1.5 9.4 � 3.6G2/M-phase 15.2 � 3.1 24.5 � 2.6 24.6 � 3.4 28.7 � 4.3 44.2 � 3.2

IST-MES2Sub-G0/G1 phase 5.9 � 3.2 24.2 � 4.8 2.7 � 1.1 5.2 � 1.3 41.3 � 4.7G0/G1 phase 62.6 � 2.8 33.3 � 2.8 54.2 � 4.8 44.9 � 1.4 45.6 � 4.1S-phase 22.8 � 3.4 46.2 � 8.8 27.4 � 3.3 21.7 � 3.6 12.6 � 1.3G2/M-phase 14.6 � 2.2 20.5 � 2.1 18.4 � 1.2 33.4 � 2.2 41.8 � 2.2

MPP89Sub-G0/G1 phase 2.3 � 0.8 18.8 � 4.4 5.5 � 2.2 6.8 � 1.4 38.3 � 1.3G0/G1 phase 54.3 � 6.6 39.2 � 1.6 52.1 � 1.7 44.2 � 2.1 39.7 � 5.9S-phase 22.2 � 4.4 36.2 � 7.2 25.7 � 3.3 18.1 � 3.1 8.1 � 1.6G2/M-phase 23.5 � 3.3 24.6 � 3.3 22.2 � 1.7 37.7 � 4.4 45.2 � 2.8

H28Sub-G0/G1 phase 2.5 � 0.8 12.2 � 2.5 4.7 � 1.8 6.8 � 2.5 31.5 � 1.7G0/G1 phase 47.6 � 1.1 31.2 � 3.8 46.4 � 2.7 40.5 � 4.6 32.6 � 2.1S-phase 28.5 � 1.4 40.1 � 2.3 27.4 � 2.5 27.8 � 2.4 12.5 � 1.2G2/M-phase 23.9 � 3.1 28.7 � 2.5 26.2 � 1.7 31.7 � 1.2 54.9 � 5.7



H290Sub-G0/G1 phase 2.2 � 1.0 17.7 � 2.7 4.4 � 1.2 5.2 � 3.1 38.2 � 4.9G0/G1 phase 48.2 � 3.3 37.8 � 2.1 47.3 � 6.6 41.0 � 2.9 38.1 � 2.8S-phase 27.5 � 2.7 40.1 � 1.3 31.4 � 1.8 23.2 � 1.9 19.6 � 22G2/M-phase 24.3 � 1.8 22.1 � 1.5 21.3 � 1.6 35.8 � 3.3 42.3 � 4.4

IC50: 50% inhibitory concentration; rHuTNF: recombinant human tumor necrosis factor.a Cells were exposed to drug for 1 hour, and flow cytometric analysis was performed after an additional 24-hour incubation in drug-free medium. Values shown are the mean � standard error of at least three

independent experiments that were performed in duplicate.


RE

49.49.444.244.2 ��

41.3 �� 4.74.71.4 45.61.4 �� 4.14.1

77 �� 3.6 12.63.6 12.6 �� 133.433.4 �� 2.2 41.82.2 41.8

6.86 � 1.4444.2 � 2.118.118 � 3.137.737.7 � 4.4

7 � 1.8 6.81.8 6.846.4 �� 2.72.727.4 � 2.52.526.22 � 1.77

5.5 4.54.5 �� 1.41.488 �� 2.1 50.32.1 5 � 2.72.7

40.140.1 � 1.3 31.41.3 � 122.122.1

R

� 1.5 18.35 18.

14.814.8 �� 2.52.5.2 33.22 � 2.42.4

� 2.3 40.42.3 � 4.3.323.123.1 �� 3.6 26.43.6 26.4 � 1

2.22.2 � 1.01.048.248.2 �� 3.327.527.5 �� 2.72.724.3 �� 1.81.8

y concentration; rHuTNF: recncentration; rHuTNF: reRdrug for 1 hour, anug for 1 hour

hat were pewere pRR

quence from the human immunodeficiency virus LTRregion [ENH7 from � 115 to � 81: GCTTGCTACAAGG-GACTTTCCGCTGGGGACTTTCC]) was added for an-other 15 minutes at room temperature. DNA-proteincomplexes were separated by electrophoresis through5% native polyacrylamide gels, dried, and visualized.31

Internucleosomal DNA FragmentationInternucleosomal DNA fragmentation was shown by theharvesting of total cellular DNA, as described previous-ly.33 Briefly, adherent cells and detached cells were har-vested separately, washed, and lysed with 50 mmol/LTris, pH 7.5; 10 mmol/L EDTA; 0.5% Triton-X-100; and0.5 mg/mL proteinase K for 2 hours at 50 °C. Sampleswere then extracted twice with phenol/chloroform/isoamyl alcohol and precipitated with ethanol. The pelletwas resuspended in Tris-EDTA and 10 �g/mL ribonu-clease A, and the DNA was separated on a 2% agarosegel.

Reverse Transcriptase-Polymerase Chain ReactionOne microgram of total RNA was reversed transcribedfor 1 hour at 37 °C in 20-�L reactions containing 50

mM Tris, pH 8.3; 75 mM KCl; 3 mM MgCl2; 500 �Meach deoxyadinosine triphosphate (dATP), deox-yguanosine triphosphate (dGTP), deoxycytidinetriphosphate (dCTP), and deoxythymidine triphos-phate (dTTP); 7.5 �g/mL random hexamers (PromegaCorp., Madison, WI); 10 mM DTT; and 200 U Moloneymurine leukemia virus reverse transcriptase (LifeTechnologies, Inc., Bethesda, MD). For the detectionof BCL-XL mRNA, 2 �L of the reverse-transcribed ma-terial was amplified by polymerase chain reaction(PCR) in 50-�L reactions containing 10 mM Tris, pH8.3; 50 mM KCl; 1.5 mM MgCl2; 200 �M each dATP,dGTP, dCTP, and dTTP; 0.2 �M of each primer; and0.25 U Amplitaq DNA polymerase (Applied Biosystems[formerly Perkin-Elmer Applied Biosystems], FosterCity, CA) for a total of 28 cycles consisting of 94 °C for45 seconds, 55 °C for 60 seconds, and 72 °C for 60seconds, with a final extension step at 72 °C for 10minutes. Amplification products were analyzed byelectrophoresis in ethidium bromide-stained agarosegels. Primer sequences were as follows: BCL-XL, 5�-CCCAGAAAGGATACAGCTGG-3� (forward) and 5�-

FIGURE 3. Gel mobility-shift analysis of nu-

clear factor �B (NF-�B) complexes. Results are

from one of three representative, independent

experiments. The position of NF-�B complex is

shown on the left. (A) Evaluation of constitutive

activation of NF-�B in different human cell lines.

The human prostate carcinoma PC-3 cell line

was used as a positive control. (B) Nucleic ex-

tracts from MSTO-211H cells, which were

treated with different drugs for different times,

were incubated with labeled probe containing an

NF-�B site and in indicated lanes with designed

antibodies for 30 minutes. (C) Gel-shift assay

with extracts from MSTO-211H cells that were

treated for a short time (60–15 minutes). (D)

Gel-shift assay with extracts from MSTO-211H

cells was performed at different times after

long-term drug exposure (3–24 hours).


Reosomal DNA Fragmeneosomal DNA Fragmenosomal DNA fragomal DNA frag

total celluotal celherenerenRETRA

mmunodeficiency virus Lmunodeficiency virus5 toto �� 81: GCTTGCTACA1: GCTTGCTAC

GACTTTCC]) was addeGACTTTCC]) was adat room temperature.at room temperature.

re separated by electropeparated by elecolyacrylamide gels, drieolyacrylamide gels, drie

GCGATCCGACTCACCAATAC-3� (reverse); TNF-R1,5�-AGTGCTGTTGCCCCTGGTCAT-3� (sense), 5�-TTTTCAGGTGTCGATTTCCCA-3� (antisense); andglyceraldehyde 3-phosphate dehydrogenase, 5�-GGTCAT CCC TGA GCT GAA CG-3� and 5�-TTC GTT GTCATA CCA CGA ATT G-3�.

Statistical AnalysisBoth parametric statistics and nonparametric statis-tics (the Student t test and the Mann–Whitney U test,respectively; not significant [P � 0.05]) were used. Foreach drug combination, the combination index (CI)(CI � 0.3, strong synergism; CI � 1, additive; CI � 1,

FIGURE 4. Gel mobility-shift analysis

of nuclear factor �B (NF-�B) complexes

in malignant pleural mesothelioma cell

lines. Results are from one of three

representative, independent experi-

ments. (A) Different cell lines were

treated with tumor necrosis factor (TNF)

at a dose of 1000 U/mL, with SN38 at

the 50% inhibitory concentration value,

or with SN38 1.0 � 10� 3 nM plus TNF

at a dose of 1000 U/mL. Electrophoretic

mobility-shift assay (EMSA) analysis

was performed after long-term drug ex-

posure (6 hours). CTRL: control. (B) Dif-

ferent cell lines (MSTO-211H cells and

PC3 cells) were treated as described for

Panel A, and EMSA analysis was per-

formed after long-term drug exposure (6

hours). In some experiments, cells were

pretreated with PS-341 (at a dose of 1

�M for 1 hour followed by drug treat-

ment).

FIGURE 5. Inhibitor protein I�B� regulation in malignant pleural mesothelioma cell lines. Cells were untreated (CTRL) or were treated with tumor necrosis factor

(TNF) at a dose of 1000 U/mL, with SN38 at the 50% inhibitory concentration value, or with SN38 1.0 � 10� 3 nM plus TNF at a dose of 1000 U/mL and were

analyzed after 6 hours of drug exposure. Cell lysates were prepared and assessed by Western blot analysis for I�B� protein. Results are from one of three

representative, independent experiments.


RETRACTEDth tumo

dose of 1000 U/dose of 1000 U/

he 50% inhibitory concehe 50% inhibitory con

or with SN38 1.0with SN3 � 1010� 3

at a dose of 1000 U/mL.at a dose of 1000 U/m

mobility-shift assamobility-shift assa

was performedwas performed

posure (6posure (6

ferentferen

PP

antagonism) and its 95% confidence limits were esti-mated.

RESULTSCell CytotoxicityMPM cells were exposed to rHuTNF or to SN38 for 1hour and then plated for 10 additional days in drug-freemedium. The IC50 was reached only after exposure totremendously high concentrations of TNF (� 25,000U/mL) (Table 1). TNF resistance was not correlated withthe absence of specific TNF receptors (TNF-R1s); there-fore, overall, human MPM cell lines (MSTO-221H, IST-MES1, IST-MES2, MPP89, H28, H513, H2052, and H290)showed TNF-R1 mRNA (Fig. 1A). Conversely, MPM cellswere very sensitive to SN38 (Table 1).

All MPM cell lines were examined for p14ARF/p16INK4a protein expression. In MSTO-211H, IST-MES1, H28, H513, H2052, and H290 MPM cell lines,endogenous p14ARF and p16INK4A expression was ab-sent (the C33A cervical carcinoma cell line was used asa positive control for p14ARF/p16INK4a expression).Only IST-MES2 and MPP89 cells expressed the p14ARF

and p16INK4a proteins (Fig. 1B). Data obtained in H28,H513, H2052, and MSTO-211H cells were in agree-ment with previous reports.8 Consequently, the sensi-tivity to SN38 or to TNF (Table 1) was independent ofp14ARF/p16INK4a protein expression.

When MPM cell lines were incubated simulta-neously with different concentrations of SN38 andwith a nontoxic concentration of TNF (1000 U/mL), astrong synergism was observed. Table 1 shows the IC50

values obtained for SN38 or TNF as single agents andfor the combination SN38 and TNF. Table 1 reports

also the combination index and the confident limit at95%.

Different drug schedules were chosen for all ofthe following experiments: 1) rHuTNF, 1000 U/mL(in all experiments); 2) SN38 at the concentrationsneeded to reach the IC50 value (as reported in Table1; equiactive); 3) SN38 at the nontoxic concentrationof 1.0 � 10� 3 nM (the survival fraction was � 95% inoverall cell lines; equimolar); and 4) SN38 plusrHuTNF, 1000 U/mL. The exposure time was 1 hour.After drug removal, cells were allowed to grow for anadditional 3–24 hours in drug-free medium.

Determination of DNA Single Strand BreaksTNF administered simultaneously, as reported bydifferent authors33 and in our previous articles,25,32

increased the level of DNA-SSB (under-deproitein-izing condition) induced by CPT. In MSTO-211Hcells, SN38 (1-hour treatment at the IC50) inducedDNA breaks, as shown in Figure 2. The combinationof SN38 and TNF, as expected, increased stronglythe fraction of eluted DNA from filters (Fig. 2A). Onehour after drug removal, DNA eluted slower; and,after 3 hours, its kinetics were superimposable withthe kinetics of the control (Fig. 2B).

Cell Cycle AnalysisTable 2 shows that TNF alone induced a small butstatistically significant (P � 0.002 Student t test) G2-Marrest; whereas SN38, at the IC50 value, induced astrong S arrest and a small fraction of sub-G0-G1 pop-ulation. SN38 at nontoxic concentrations did not af-fect the composition of the cell cycle. The combina-

TABLE 3Percentage of Apoptotic Cellsa

Cell line ControlSN38 IC50

(nM)SN38(1.0 � 10�3 nM)

rHuTNF(1000 U/mL)

SN38 (1.0 � 10�3 nM)and rHuTNF (1000 U/mL)

MSTO-211H 5.2 � 0.8b 26.6 � 1.8c 2.4 � 0.4b 4.8 � 1.6b 44.5 � 4.6c

IST-MES1 7.4 � 1.6b 38.1 � 5.5c 7.5 � 0.6b 8.3 � 2.2b 58.6 � 4.7c

IST-MES2 4.2 � 0.4b 21.8 � 2.5c 4.6 � 2.2b 5.3 � 1.3b 48.4 � 2.6c

MPP89 3.4 � 1.8b 22.5 � 4.6c 5.5 � 1.7b 3.4 � 0.4b 35.7 � 3.3c

H28 2.2 � 0.6b 11.9 � 1.4 3.4 � 0.8b 5.8 � 2.6b 34.8 � 2.9c

H513 3.9 � 2.1b 18.4 � 2.3d 4.6 � 2.4b 6.6 � 1.4b 34.6 � 2.8c

H2052 9.8 � 2.6b 19.9 � 2.7d 5.8 � 1.3b 7.7 � 1.6b 29.8 � 3.7c

H290 1.7 � 0.3b 24.6 � 1.2c 3.2 � 0.8b 4.1 � 0.3b 41.4 � 4.4c

IC50: 50% inhibitory concentration; rHuTNF: recombinant human tumor necrosis factor.a Mesothelioma cell lines were exposed to drug for 1 hour, and 4,6-diamidino-2-phenylindole (DAP1) staining was performed after an additional 24-hour incubation in drug-free medium. One thousand cells were

scored for each DAP1-stained slide. Values shown are the mean � standard error of two independent experiments that were performed at least in duplicate.b Not statistically significant (P � 0.05; Student t test).c P � 0.002 (Student t test).d P � 0.002 (Student t test).


RETRACTEDfor 1or 1

drug-freereeexposure toexposure to

TNF (TNF (�� 25,0000not correlated withnot correlated

ptors (TNF-R1s); there-tors (TNF-R1s); there-l lines (MSTO-221H, ISTl lines (MSTO-221H, IS

H28, H513, H2052, and HH28, H513, H2052, and H(Fig. 1A). Conversely, M(Fig. 1A). Conversely, M

to SN38 (Table 1).o SN38 (Table 1).ell lines were examinel lines were examine

tein expression. In Mtein expression. In8, H513, H2052, and H8, H513, H2052, and H

nous p14nous p14ARFARF and p16and p16ININ

C33A cervical carc3A cervical carcontrol forntrol f

2 andand

he combination index acombination index a%.%.

Different drug schg schthe following expehe following(in all experimein all experimneeded to reeded to re1; equiact1; equiof 1.0of 1.0ovee

D4

ion in drug-free medium. Oneon in drug-free medium. On

e.

TED

tion of SN38 (1.0 � 10� 3 nM) plus TNF (1000 U/mL)(Table 2) induced G2-M arrest as well as TNF alone, adepletion of the S-phase, and a significant fraction ofthe sub-G0-G1 cell population. This fraction washigher than that induced by SN38 alone at the IC50

value.

NF-�B ActivationOverall, MPM cell lines did not have constitutive, ac-tivated NF-�B complexes (Figs. 3, 4), similar to somecontrol cells (renal carcinoma A498, ACHN, andCAKI-1 cells and human lymphoid HL60 and K562cells) (Fig. 3A). The human prostate carcinoma PC3cell line (Fig. 3A) represented a positive control (highlevel of constitutive activated NF-�B complexes).34

The kinetics of formation and persistence ofNF-�B complexes were studied initially by electro-phoretic mobility-shift assay in MSTO-211H cells.rHuTNF at 1000 U/mL, according to our previousresults,20,24 induced NF-�B complexes after a shorttreatment; they were evident after 30 minutes ofincubation (Fig. 3B,C). SN38 at the IC50 value didnot induce NF-�B complexes until 3 hours (Fig. 3C).The combination of SN38 and rHuTNF inducedNF-�B complexes with a pattern of activity similarto TNF that was reduced in SN38-treated cells(Fig. 3B).

The generation of NF-�B complexes also wasanalyzed in MSTO-211H cells that were treated for24 hours. NF-�B complexes remained for 12 hoursin TNF-treated cells (Fig. 3D), whereas SN38-treatedcells (IC50 value) were present for 12 hours (Fig. 3D)and disappeared after 24 hours (Fig. 3D) In SN38plus TNF-treated cells (Fig. 3D), NF-�B complexes,which were visible weakly after 3 hours, disappearedquickly and became undetectable at 6 hours.

The persistence of NF-�B complexes was evalu-ated in all MPM cell lines that were treated for 6hours with SN-38 at the IC50 value, with TNF 1000U/mL, or with combined SN38 and TNF (Fig. 4A). Inall cell lines, the NF-�B complexes induced by thecombination disappeared completely after 6 hours(Fig. 4A).

To assess the specific effects of the combination ofTNF and SN38 on shortening the persistence of NF-�Bcomplexes, the activity of PS-341 was evaluated inMSTO-211H cells (Fig. 4B). It is known that PS-341enhances SN38 chemosensitivity through the inhibi-tion of NF-�B activation induced by SN38.23 Treat-ment with PS-341 alone (1 �M for 1 hour) did notresult in activation of NF-�B (Fig. 4B). PS-341 was ableto inhibit the constitutive activation of NF-�B in PC3cells, as reported previously.35 In MSTO-211H cells,

pretreatment with PS-341 prior to drug administrationincompletely inhibited the activation of NF-�B in-duced by exposure to SN38 or to TNF.

Normally, NF-�B resides in the cytoplasm as aninactivated form in a complex with I�B-�. I�B-�

FIGURE 6. Detection of apoptosis in mesothelioma MSTO-211H cells by (A)

transmission electron microscopy analysis (results are from one of two repre-

sentative, independent experiments) and (B) detection of DNA fragmentation by

agarose gel electrophoresis. Cells were exposed to the drug for 1 hour and

were analyzed after additional 24-hour incubation in drug-free medium. Un-

treated cells (CTRL) or cells that were treated with SN38 1.0 � 10� 3 nM, with

SN38 at a concentration needed to reach the 50% inhibitory concentration

value (SN38 IC50), with tumor necrosis factor (TNF) at a dose of 1000 U/mL, or

with SN38 1.0 � 10� 3 nM plus TNF at a dose of 1000 U/mL.


RETRAClarcellsells

es also wases also waswere treated forere treated for

ined for 12 hoursned for 12 howhereas SN38-treatedwhereas SN38-treated

nt for 12 hours (Fig. 3Dnt for 12 hours (Fig. 3D4 hours (Fig. 3D) In Shours (Fig. 3D) In

s (Fig. 3D), NF-s (Fig. 3D), NF-�B comweakly after 3 hours, dweakly after 3 hours, d

came undetectable at 6me undetectable atistence of NF-istence of NF �B compcom

REl MPM cell lines thatl MPM cell lines thawith SN-38 at the ICwith SN-38 at the IC

with combined Swith combineds, the NF-the NF

disappsap

modulates the function or activity of NF-�B throughits proteolytic degradation in response to differentextracellular stimuli, such as TNF.19. This processresults in the translocation of NF-�B to the nucle-us.19 Overall, MPM cell lines showed I�B-� proteindegradation (Fig. 5) after TNF treatment or SN38treatment, whereas the combination treatment up-regulated I�B-� protein expression 6 hours aftertreatment (Fig. 5).

Induction of ApoptosisNuclei of treated and untreated cells were observedfor typical morphologic apoptotic changes afterDAPI staining (Table 3). The combination of SN38and TNF induced a severe induction of apoptosis,

whereas SN38 alone at the IC50 concentration wasmoderately active in inducing apoptosis. rHuTNF (1000U/mL) and nontoxic concentrations of SN38 were com-pletely unable to trigger apoptosis (Table 3).

TEM examination definitely confirmed the dra-matic induction of apoptosis by the appearance oftypical apoptotic bodies when the mesotheliomacell line MSTO-211H was treated with SN38 in com-bination with rHuTNF (% of positive cells, 48.0� 1.2; P � 0.002; Student t test) (Fig. 6A). Apoptosiswas induced in a small population of control cells(1.5 � 0.5), cells treated with a nontoxic concentra-tion of SN38 (5.1 � 0.3; P � 0.05 not statisticallysignificant), and cells that were exposed to 1000U/mL rHuTNF (� 1%; P � 0.1; not statistically sig-

FIGURE 7. (A) Blots illustrate expres-

sion of the Bcl-2 and BCL-XL proteins in

human mesothelioma cell lines: human

lymphoid K-562 and HL-60 cell lines,

the renal carcinoma A-469 cell line, the

ACHM and CAKI-1 cell lines, the prostate

carcinoma PC3 cell lines, and positive

and negative (HL-60) controls for BCL-

XL expression. (B) Human mesothelioma

cell lines were exposed to SN38 1.0

� 10� 3

nM, to tumor necrosis factor

(TNF) at a dose of 1000 U/mL, or to

SN38 1.0 � 10� 3 nM plus TNF at a

dose of 1000 U/mL for 1 hour and then

incubated for an additional 24 hours in

drug-free medium. Cell lysates were

prepared and assessed by Western blot

analysis for the Bcl-2 and Bcl-XL pro-

teins. (C) In some experiments, MSTO-

211H cells were treated with SN38 or

PS-341 1 �M for 1 hour alone or with a

combination of PS-341 plus SN38. Cells

were exposed to PS-341 1 hour prior to

SN38. Results are from one of three

representative, independent experi-

ments that were performed in duplicate.


RETRtes the function or actes the function or acolytic degradatioytic degradatio

r stimuli, sstimulitransrans

r too

f threef three

ent experi-nt experi-

med in duplicate.d in duplicate.

nificant) (Fig. 6A). No necrotic cells were seen in anysample.

Gel-ladder analysis confirmed the induction ofsevere DNA fragmentation related to the induction ofapoptosis (approximately 180 base pairs) only in cellsthat were treated with the combination of SN38 andTNF both in MSTO-211H cells (Fig. 6B) and in all MPMcells (data not shown).

BCL-XL Protein Expression and Cytochrome c ReleaseOverall, the MPM cell lines overexpressed BCL-XLproteins and mRNA (Figs. 7–9) relative to positivecontrols (K-562, A498, ACHN, CAKI-1, and PC3 cells)and negative controls (HL-60 cells) (Fig. 7A).16 In alllines, the combination treatment down-regulatedBCL-XL protein expression (Figs. 7B, 8) and mRNAexpression (Fig. 9) in MSTO-211H cells.

Time-course experiments showed that BCL-XLproteins were present after 3 hours of treatment,started to disappear 6 hours after treatment, and were

undetectable after 12 hours (Fig. 8). Release of cyto-chrome c to cytoplasm was analyzed on Westernblot analysis in mitochondrial and cytoplasmic frac-tions of MSTO-211H cells. Only when cells weretreated with the combination of SN38 and TNF wascytochrome c released from the mitochondria to thecytoplasm (Fig. 9A).

Caspase ActivitiesTo determine the requirement of caspase activitiesduring combined SN38 and TNF-induced apoptosis,caspase activity was evaluated in MSTO-211H cells(Fig. 10). The treatment of cells with the combina-tion caused activation of caspase 8, as indicated bya reduction in the intensity of the proenzyme. Theactivation of caspase 9 by the combination was ev-idenced by the reduction in the intensity of theproenzyme. Activated caspase 3 was detected aftercombination treatment as a double band represent-ing a p19 proteolytic fragment and the active sub-unit p17, respectively. Therefore, the combinationof SN38 and TNF caused a marked increase in thecleavage products of PARP. Caspase activation dur-ing the course of combined SN38 and TNF-inducedapoptosis also was confirmed by using a caspase3-specific inhibitor, z-DEVD-fmk. This caspase in-hibitor was able to prevent the activation ofcaspases and the induction of apoptosis (% of apo-ptotic cells after DAPI staining, 8.7% � 1.2%), asexpected. Therefore, these data suggest that thecombination of SN38 and TNF blocking NF-�B ac-tivation induces dramatic induction of apoptosis,and apoptosis is mediated by BCL-XL down-regulation and consequential caspase 3 activation.

DISCUSSIONIn the current study, we showed that TNF sensitizesSN38-induced cytotoxicity of human mesotheliomacancer cell lines (MSTO-221H, IST-MES1, IST-MES2,MPP89, H28, H513, H2052, and H290). In addition, wedemonstrated that the major induction of massiveapoptosis induced by TNF in combination with SN38is related mainly to NF-�B time shortening in me-sothelioma cells; as a result, the expression of BCL-XLis down-regulated, cytochrome c is released into thecytoplasm, caspase 3 is activated, and apoptosis istriggered. The partial inhibition of NF-�B complexesinduced by SN38 with PS-341 in MSTO-211 cells wasnot enough to down-regulate the constitutive BCL-XLexpression significantly (Fig. 7C). Therefore, our ob-servations indicate that a short persistence of NF-�Bcomplexes and BCL-XL down-regulation both are re-

FIGURE 8. Expression of BCL-XL and BCL-XS mRNA in the human mesothe-

lioma cell line MSTO-211H was evaluated by reverse transcriptase-polymerase

chain reaction (RT-PCR) analysis. Cells were exposed to drug for 1 hour, and

cell lysates were prepared and assessed by RT-PCR after an additional 24-hour

incubation in drug-free medium. The K-562 and HL-60 human lymphoid cell

lines were used as positive and negative controls (CTRL), respectively. Results

are from one of three representative, independent experiments that were

performed in duplicate.


CTEDin MS

ells with thells with thcaspase 8, as indcaspase 8, as in

ensity of the proenzynsity of the proene 9 by the combinatioe 9 by the combinati

reduction in the intereduction in the inteActivated caspase 3 wactivated caspase 3 wa

on treatment as a doubon treatment as a doub19 proteolytic fragmen9 proteolytic fragmen

RETRACTnecrotic cells were seenecrotic cells were see

analysis confirmed thenalysis confirmed thefragmentation related toragmentation related

(approximately 180 bas(approximately 180 bare treated with the core treated with the co

in MSTO-211H cen MSTO-211H cot shown).t shown

p17, respectively. Thep17, respectively. ThSN38 and TNF causeSN38 and TNF caus

cleavage products ofcleavage products ofing the course ofng the courseapoptosis alsopoptosis als3-specific in3-specific inhibitorhibitocaspaaspapt

rase

our, and, and

ional 24-hourhour

man lymphoid cellman lymphoid cell

), respectively. Results, respectively. Results

nt experiments that weret experiments that

lated to the ability of the cell to undergo apoptosis. Itis interesting to note that elevated basal expression ofBCL-XL protein was not correlated with constitutiveactivation of NF-�B. This observation is true not onlyfor MPM cell lines but also for lymphoid and renalcarcinoma cell lines.

The critical event in the initiation of apoptosis ismitochondrial dysfunction, which is regulated byBCL-2 family proteins. BCL-2 family proteins consistsof 3 subfamilies: the proapoptotic and antiapoptoticproteins, including Bax (proapoptotic) and BCL-2 (an-tiapoptotic), and the BH-3 subfamily. The antiapop-totic BCL-2 subfamily includes BCL-XL. The switchingon and off of apoptosis is determined by the ratio ofproapoptotic protein to antiapoptotic protein.15

In the NCI-ACDS study,16 a strong negative corre-lation was reported between basal BCL-XL protein andmRNA levels and drug sensitivity, suggesting thatBCL-XL may play a unique role in general resistance tocytotoxic agents (70,000 drugs considered). In view of

these data, it is not surprising that MPM would bestrongly resistant to drugs. Our observations that thecombination of SN38 and TNF down-regulated theexpression of both BCL-XL proteins and mRNA maybe crucial in opening new possibilities for MPM ther-apy. Moreover, ARF is not involved in drug sensitivityin MPM.

The results of the current study demonstrated thatTNF administered simultaneously with SN38 reducesthe time of persistence of NF-�B complexes, causing adramatic induction of apoptosis through the down-regulation of BCL-XL; consequently, a strong, syner-gistic, cytotoxic effect is achieved. CPT derivativessuch as irinotecan and topotecan are approved by theFood and Drug Administration in the U.S., and severalanalogues currently are in various stages of clinicalevaluation (i.e., 9-aminocamptotecin and rubite-can).36 The North Central Cancer Treatment Groupdesigned a Phase II trial to assess the efficacy andtoxicity of topotecan in patients suffering from unre-

FIGURE 9. Human mesothelioma cell

lines were exposed to a combination of

SN38 1.0 � 10� 3 nM and tumor ne-

crosis factor (TNF) at a dose of 1000

U/mL for 1 hour and then incubated for

additional time (3–12 hours) in drug-

free medium. Cell lysates were pre-

pared and assessed by Western blot

analysis for Bcl-Xl protein. Results are

from one of three representative, inde-

pendent experiments that were per-

formed in duplicate. CTRL: control;

G3PDH: glycerol-3-phosphate dehydro-

genase.


RETRhe cell to undergo apophe cell to undergo apopte that elevated basal exe that elevated basal ex

was not correlated witwas not correlated wiNF-NF-��B. This observatioB. This observat�

cell lines but also forcell lines but also forma cell lines.ma cell lines.

ritical event in thtical event in tial dysfuncl dysfu

proteinotein

sectable MPM. Topotecan administration was toler-ated reasonably well; however, the response rate wasinsufficient to warrant an additional study.37 Con-versely, combination studies may be more promis-ing.38 The combination of irinotecan with cisplatin hasdefinite activity against MPM and is tolerated well.38

The Top I inhibitor irinotecan must be con-verted into its considerably more active metaboliteSN-38 (which has 1000 times the potency of theparent compound) by the endogenous enzyme car-boxyl esterase, which is found in the plasma, theintestinal mucosa, and the liver.39 The distributionof irinotecan and its active metabolite SN-38 in thepleural fluid, after intravenous administration, wasevaluated in patients with different histologic sub-types of MPM. Irinotecan appeared in the pleuralfluid 1 hour after intravenous infusion, and the levelpeaked after 6 hours. SN-38 also was observed in thepleural fluid 1 hour after infusion, and a concentra-tion equivalent to that in plasma was achieved after4 hours.37

Most patients with earlier stage MPM present withpleural effusions, which allow chemotherapeuticagents to be administered intrapleurally. This thera-peutic approach has the pharmacologic advantage ofexposing pleural tumors to greater concentrations ofthe agents compared with the concentrations avail-able using the intravenous route. It has been foundthat TNF is tolerated better and is more active when itis infused locally (i.e., intraperitoneally).40,41 In-trapleural administration of TNF in MPM patients is awell tolerated regimen; however, regrettably, it also isassociated with an inconsistent and rather moderateimpact on the production of pleural fluid.42 In allpatients with advanced disease that obliterates thepleural space, the intrapleural administration of drugscan prove difficult. Moreover, MPM easily can metas-tasize despite its pronounced tendency to remain lo-calized in the pleural cavity. Intrapleural therapy maybe limited to patients who have earlier stage diseaseand to those patients with small-volume disease for

Š

FIGURE 10. Cytochrome c release from the mitochondrial intermembrane space

to cytosol (A) and requirement of caspase activities during SN38 and tumor

necrosis factor (TNF)-induced apoptosis (B). MSTO-211H cells were treated with

different drug for 1 hour; cell lysates were prepared after an additional 24-hour

incubation in drug-free medium. The control actin (pellet) and cytochrome c

oxidase (COX Vb) in the pellet and cytosol are shown in Panel A. Results are

from one of three representative, independent experiments that were per-

formed in duplicate. CTRL: control; PARP: poly(ADP-ribose) polymerase.


ACTED9 T

etaboliteetabolitenous administrnous administ

with different histolowith different histootecan appeared in thotecan appeared in

intravenous infusion,intravenous infusion,hours. SN-38 also washours. SN-38 also was

d 1 hour after infusiond 1 hour after infusionuivalent to that in plasmuivalent to that in plas

urs.rs 3737

Most patients with eaMost patients with epleural effusions, wpleural effusions, wagents to be admiagents to be apeutic approaceutic approaexposing plexposing plthe agenthe ageable ubleth

whom adequate direct diffusion into the tumor still ispossible.

We showed previously that weekly intraperitonealadministration of mitoxantrone (at a dose of 6 mg/m2)and TNF (at a dose of 200 mg/m2) is a feasible regimenwith acceptable toxicity.41 Therefore, the regimen ofcombined TNF and irinotecan (SN38 is its active me-tabolite) is expected to have acceptable toxicity. How-ever, studies currently are ongoing in mice to evaluatethe toxicity of this regimen.

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RETRACTEDcites: a

12.2.WH, Stoter G, EggWH, Stoter G, Eg

n of tumour necrosis faof tumour necrosists with mesothelioma: cytos with mesothelioma:

hase protein response.ase protein response EurEu3.

retracted: tumor necrosis factor enhances sn38-mediated apoptosis in mesothelioma cells : the role...

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