Say No to DMSO: Dimethylsulfoxide Inactivates Cisplatin, Carboplatin, and … · cisplatin stock solution used for culturing CP.5 cells was prepared in PBS. The cisplatin-resistant

Therapeutics, Targets, and Chemical Biology

Say No to DMSO: Dimethylsulfoxide Inactivates Cisplatin,Carboplatin, and Other Platinum Complexes

Matthew D. Hall1, Katherine A. Telma1, Ki-Eun Chang1, Tobie D. Lee1, James P. Madigan1, John R. Lloyd2,Ian S. Goldlust3, James D. Hoeschele4, and Michael M. Gottesman1

AbstractTheplatinumdrugs cisplatin, carboplatin, andoxaliplatin arehighlyutilized in the clinic andasa consequence are

extensively studied in the laboratory setting. In this study, we examined the literature and found a significantnumber of studies (11%–34%) in prominent cancer journals utilizing cisplatin dissolved in DMSO. However,dissolving cisplatin in DMSO for laboratory-based studies results in ligand displacement and changes to thestructure of the complex.We examined the effect of DMSOonplatinumcomplexes, including cisplatin, carboplatin,and oxaliplatin, finding that DMSO reacted with the complexes, inhibited their cytotoxicity and their ability toinitiate cell death. These results render a substantial portion of the literature on cisplatin uninterpretable. Raisingawareness of this significant issue in the cancer biology community is critical, and we make recommendations onappropriate solvation of platinum drugs for research. Cancer Res; 74(14); 3913–22. �2014 AACR.

IntroductionCisplatin (cis-[PtCl2(NH3)2]), carboplatin ([Pt(O,O'-cdbca)

(NH3)2], cbdca¼ cyclobutane-1,1-dicarboxylate), and oxalipla-tin ([Pt(R,R-cyclohexane-1,2-diamine)(O,O'-ethanedioato)]) areused in combination with other agents to treat a range ofmalignancies including testicular, ovarian, head and neck,bladder, esophageal, and small cell lung cancer (1–4). Withthe exception of testicular cancer, acquired or intrinsic resis-tance generally occurs and tumors are not eliminated bytreatment (3–5). Cisplatin exerts its toxicity by DNA bindingand downstream apoptotic signaling, and resistance is con-ferred by reducing apoptotic signaling, upregulating DNAdamage repair mechanisms, altering cell-cycle checkpoints,and disrupting assembly of the cytoskeleton (4, 6). The pleio-tropic mechanisms underlying platinum resistance are welldescribed, but their clinical significance is uncertain (4, 6).These factors have prompted continued experimentation withplatinum drugs to understand their mechanisms of actionalone and in combination with other therapeutics (7), as wellas the synthesis and testing of new platinum complexes withthe prospect of uncovering compoundswith promising activity

(8), some of which have entered clinical trials (e.g., picoplatin,satraplatinand BBR3464; ref. 9).

A critical aspect of the activity of platinum drugs in vitro andin vivo is their interaction with the solvent environment. Forexample, platinum drugs are activated via replacement ofleaving groups with water inside the cell, a process termedaquation (10, 11). For cisplatin, this is the loss of a chlorideligand and its replacement withwater, which is amore reactiveleaving group. For this reason, cisplatin is formulated forclinical use in saline solution with a high chloride concentra-tion (154 mmol/L) to prevent drug aquation before adminis-tration, stabilizing the drug and preventing side reactionsbefore cell entry. A limiting factor for platinum drugs istheir relatively low solubility, and clinical cisplatin is formu-lated at a concentration of 1 mg/mL (3.3 mmol/L). In labora-tory and drug screening settings, stock solutions of organic-based drugs are predominately prepared in the solvent DMSO(O ¼ S(CH3)2), which is viewed as a virtual "universal solvent"able to solubilize most small molecules at high concentrations(up to 100 mmol/L, for example; ref. 12). DMSO contains anucleophilic sulfur, which allows it to coordinate with plati-num complexes, displacing ligands and changing the structureof the complexes (13–16). This renders platinum complexesunstable in DMSO. Massart and colleagues first reported thatDMSO reduced cisplatin's cytotoxicity toward cultured thyr-ocytes (17), andDernel and colleagues reported that a polymer-based drug delivery system limited activity of cisplatin againststage IIb appendicular osteosarcoma in dogs (18). Little infor-mation exists on the effect of DMSO on other platinum drugsand complexes.

Yet, as discussed in this article, cisplatin and other platinumcomplexes are regularly dissolved in DMSO for biologic experi-ments, both in vitro and in vivo in experimental models, andDMSO solutions of cisplatin have been utilized in the clinicalveterinary setting (19). This use may be due to the lack of acomprehensive understanding of the effect of DMSO on the

Authors' Affiliations: 1Laboratory of Cell Biology, Center for CancerResearch, National Cancer Institute; 2AdvancedMass Spectrometry Facil-ity, National Institute of Diabetes & Digestive & Kidney Diseases, NIH,Bethesda; 3Division of Preclinical Innovation, National Institutes of HealthChemical Genomics Center, National Center for Advancing TranslationalSciences, NIH, Rockville, Maryland; and 4Department of Chemistry, East-ern Michigan University, Ypsilanti, Michigan

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Michael M. Gottesman, Laboratory of Cell Biol-ogy,NationalCancer Institute, NIH, 37ConventDrive, Rm. 2108, Bethesda,MD 20892. Phone: 301-496-1921; Fax: 301-402-0450; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-14-0247

�2014 American Association for Cancer Research.

CancerResearch

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Published OnlineFirst May 8, 2014; DOI: 10.1158/0008-5472.CAN-14-0247

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activity of platinum complexes in the cancer biology commu-nity. Irrespective of the reason, the implications for publishedstudies on cisplatin's mechanism using DMSO solutions areprofound.

We sought to examine the range of solvent systems utilizedfor platinumdrugs in peer-reviewed research, and the effects ofDMSO on platinum drug activity. A number of journals wereassessed to gauge the solvent types used in studies of cisplatin.We then assessed the impact of DMSO versus clinical formula-tions of a number of platinum complexes on the cytotoxicity,cellular recognition of DNA damage, and cell death signaling.Mass spectrometry was used to directly assess the interactionof DMSO and clinical formulations with each platinumcomplex.

Materials and MethodsLiterature review

Five journals that regularly publish studies on small-mole-cule therapeutics and theirmechanismwere examined:CancerResearch (http://cancerres.aacrjournals.org),Molecular CancerTherapeutics (http://mct.aacrjournals.org),Molecular Pharma-cology (http://molpharm.aspetjournals.org), Journal of Phar-macology and Experimental Therapeutics (JPET; http://jpet.aspetjournals.org), and the Public Library of Science (PLoS;http://www.plos.org) journals. In each case, the word "cisplat-in" was entered as a search term on the respective journalwebsite search engine, restricted to the term appearing in thetitle or abstract of articles. Manuscripts were then individuallyreviewed to identify and assess only those papers reporting invitro data. These papers were then assessed for the solvent orsolution used for dissolving cisplatin, and these were deter-mined and recorded; similarly, it was noted if the solventsystem was not disclosed. In the majority of cases, if thesolvent used was not mentioned in the Materials and Methodssection, it was not explicitly disclosed anywhere in the man-uscript in a manner that allowed unambiguous determinationof the experimental strategy used—this was recorded as "Notreported." Thirty-five manuscripts were assessed for eachjournal, with the exception of the JPET, where only 28 relevantarticles were identified.

MaterialsCisplatin, carboplatin, DMSO, dimethylformamide (DMF),

dimethylacetamide (DMA), formamide, acetamide, and cre-mophor EL were purchased from Sigma-Aldrich. Oxaliplatinwas purchased from LC Laboratories. Satraplatin was pur-chased from Sequoia Research Products. [PtCl2(en)] and trans-platin were purchased from Alpha Aesar. Clinical formulationsof cisplatin, carboplatin, and oxaliplatin were kindly providedby Dr. Tito Fojo, Clinical Center, National Cancer Institute(Bethesda, MD). Compounds were assessed in various solventsystems as described below.

Cell lines and cell cultureThis study used theDLD1 human colorectal carcinoma cells,

parental human cervical carcinoma cell line KB-3-1 (a sublineof HeLa), and its cisplatin-resistant subline KB-CP.5. KB-CP.5cells were originally selected in a single step in 0.5 mg cisplatin/

mL (1.6 mmol/L) in our laboratory, as described previously (20,21). KB lineswere originally generated in the laboratory ofM.M.Gottesman. DLD-1 cells were provided by the National CancerInstitute (part of the NCI-60 collection). All cell lines werethawed immediately before experimentation, and cell lines arecharacterized by NCI using short tandem repeat profiling. Thecisplatin stock solution used for culturing CP.5 cells wasprepared in PBS. The cisplatin-resistant cells were main-tained in the presence of cisplatin, which was removed fromgrowth medium 3 days before all experiments. All cell lineswere grown as monolayer cultures at 37�C in 5% CO2, usingeither DMEM (KB cells) or RPMI (DLD1 cells) with 4.5 g/Lglucose (both from Invitrogen), supplemented with L-gluta-mine, penicillin, streptomycin, and 10% FBS (BioWhittaker).Resistance of CP.5 cells to cisplatin was confirmed on aregular (at least monthly) basis, using cell viability assays asdescribed herein.

Cytotoxicity and cell growthCell survival was measured by the MTT (Invitrogen) assay.

Cells were seeded at a density of 5,000 cells per well in 96-wellplates and incubated at 37�C in humidified 5% CO2 for 24hours. Serially diluted (1:3, RPMI or DMEM used as diluent,chloride concentration �120 mmol/L) compound was addedto give the intended final concentrations. Solvent tolerancetesting up to 0.5% under identical conditions confirmedgrowth of all cell lines was unaffected. Cells were then incu-bated an additional 72 hours, and the MTT assay was per-formed according to the manufacturer's instructions (Molec-ular Probes). Absorbance values were determined at 570 nmona Spectra Max 250 spectrophotometer (Molecular Devices). AllMTT assays were performed in triplicate. The IC50 values weredefined as the drug concentrations required to reduce cellularproliferation to 50% of the untreated control well. We usedPrism 6 (GraphPad Software) software for graphs and statis-tics. All data are expressed as mean � SD. For bright-fieldimaging, DLD-1 cells were seeded at a density of 3 � 105 cellsper well in 6-well plates and incubated at 37�C in humidified5% CO2 for 24 hours. Drug was added directly to each well andcells were incubated for another 72 hours. Media were aspi-rated and cells were rinsed with PBS before imaging.

H2AX immunofluorescence stainingFixed DLD-1 cells were stained with Phospho-Histone

H2A.X (Se139; 20E3) Rabbit mAb (Alexa Fluor 488 Conjugate;all components from Cell Signaling Technology) followingthe manufacturer's instructions and as previously described(22). Briefly, cells were seeded at 5� 104 cells per chamber in8-chamber coverslips and incubated for 24 hours. Cells werethen incubated with Pt complexes for 24 hours before beingwashed with PBS, fixed in 4% paraformaldehyde, thenblocked with blocking buffer (PBS/5% normal horseserum/0.3% Trition X-100) for one hour. Diluted antibody(1:50 in PBS/1% BSA/0.3% Triton X-100) was then applied,and cells were incubated overnight at 4 �C. Cells were thenwashed and stained with 40, 6-diamidino-2-phenylindolenuclear DNA stain, and imaged on a Zeiss LSM 710 NLOconfocal microscope.

Hall et al.

Cancer Res; 74(14) July 15, 2014 Cancer Research3914




Preparation of cell lysates, quantification of protein, andWestern blot analysisAfter 48-hour drug treatments, both floating and adherent

DLD-1 colorectal cancer cells were washed with PBS andlysed together in NP-40 lysis buffer [50 mmol/l Tris/HCl, pH8.0, 150 mmol/L NaCl, 1% NP-40, and supplemented withcomplete protease inhibitor cocktail tablets (all from Sigma-Aldrich) and PhosStop phosphatase inhibitor tablets(Roche)] and centrifuged to remove insoluble material. 2XLaemmli sample buffer (Sigma-Aldrich) was added to equiv-alent amounts of cellular lysates (40 mg), which were thenresolved by SDS-PAGE on 4% to12% Bis-Tris gels (Invitro-gen) and transferred onto Immobilon PVDF membrane(Millipore). Membranes were blocked in 5% (w/v) nonfatdried skimmed milk powder in TBS-Tween 20 (TBST; 20mmol/L Tris/HCl, pH 7.6, 137 mmol/L NaCl, and 0.2% Tween20; Sigma-Aldrich; blocking buffer) and probed with appro-priate primary antibodies overnight followed by anti-mouseor anti-rabbit IgG horseradish peroxidase (HRP)-conjugatedsecondary antibody (Cell Signaling Technology) for onehour. Membranes were washed in TBST and incubated ineither Western Blotting Luminol Reagent (Santa Cruz Bio-technology) or Immobilon Western Chemiluminescent HRPSubstrate (Millipore) and the signal developed on HyBlot ESfilm (Denville Scientific). All primary antibodies were fromCell Signaling Technology: PARP (#9542), cleaved caspase-3(#9664), phospho-H2A.X (#9718), and b-actin (#3700).

Mass spectrometryAccurate mass data were obtained on a Waters Premiere

LCT time-of-flight mass spectrometer operated in the positiveion W-mode at 10 K resolution. The high-performance liquidchromatography solvent pumpwas operated at 200mL/minuteand the solvent composition was 50:40:10 water:methanol:acetonitrile. All solvents were LC/MS grade and were pur-chased from Sigma-Aldrich. Samples were flow injected via asample loop and ionized in the electrospray ion source. Theelectrospray capillary was operated at 3.5 kV and at 350 �C. Thedesolvation gas was purified nitrogen. The MS data wereanalyzed using Waters MassLynx 4.1 software.

ResultsSolvent utilization in in vitro studies of cisplatin

Five journals that regularly publish studies on small-mole-cule therapeutics and their mechanism were examined forreports pertaining to the activity and mechanism of cisplatin:Cancer Research, Molecular Cancer Therapeutics, MolecularPharmacology, JPET, and the PLoS journals. Thirty-five papersreporting in vitro data were assessed for each journal (28 forJPET), and the solvent used for dissolving cisplatin in each casewas determined and recorded. Solvent systems utilized weresimple salt buffer, cell culture media, clinical formulation(often indicated as sourced from a research hospital's in-housepharmacy), DMF, DMSO, saline, PBS, and water (Table 1).Where the solvent used was not identified in the Materialsand Methods, this was recorded as "Not reported," with a verysmall number of exceptions where the solvent was categori-cally identified elsewhere in the manuscript. Manuscriptsassessed are listed in Supplementary Tables S1–S5, with thesolvent used assigned for each. Although only in vitro datawereassessed, it should be noted that cisplatin dissolved in DMSOhas also been used for in vivo studies (23).

As shown in Table 1, for each journal the majority of papersdid not report the solvent used (26%–50% of papers). Followingthis, a DMSO solution or clinical formulation was the secondmost used solvent. DMSO was the most heavily utilized in-labsolvent. For example, DMSO was used in 34% of CancerResearch and 20% of Molecular Cancer Therapeutics articlesthat were examined.

In the clinic, cisplatin is typically provided as a lyophilizedpowder in a vial containing 50 mg cisplatin, 450 mg NaCl, and500mgmannitol.Whendissolved in 50mLofwater, this resultsin a 1 mg/mL solution (3.3 mmol/L) of cisplatin dissolved in150 mmol/L saline. The saline prevents aquation of the com-plex in solution before administration to the patient (24).Clinical formulation was used for in vitro experiments in11% to 23% of papers. However, in some studies, cisplatin wasdissolved in PBS (chloride concentration 140 mmol/L—phos-phate has been reported to not appreciably alter cisplatinaquation; ref. 25) or saline (chloride concentration 154mmol/L). When these chloride-containing solutions are

Table 1. Reported solvent or formulation of cisplatin used for in vitro studies in selected peer-reviewedscientific journals, stated as percentage of total (with number of reports in parentheses)

FormulationCancerResearch

Molecular CancerTherapeutics PLoS

MolecularPharmacology JPET

Not reported 43 (15) 37 (13) 43 (15) 26 (9) 50 (14)DMSO 34 (12) 20 (7) 17 (6) 11 (4) 14 (4)Water — 3 (1) 11 (4) 11 (4) 7 (2)Buffer — — — 3 (1) —

DMF — 3 (1) 3 (1) 3 (1) —

Cell media — — 3 (1) 6 (2) —

Clinical 14 (5) 23 (8) 17 (6) 14 (5) 11 (3)Saline 3 (1) 11 (4) — 20 (7) 14 (4)PBS 3 (1) 3 (1) 3 (1) 6 (2) 4 (1)

DMSO Inactivates Platinum Drugs

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considered along with clinical formulation, their use waspredominant, accounting for 20% to 40% of reports (CancerResearch, 20%;Molecular Cancer Therapeutics, 37%; PLoS, 20%;Molecular Pharmacology, 40%; JPET, 29%).

Other solvents were used in only a small number of cases. Itis possible that DMF (on one occasion) was used as analternative organic solvent as it is able to solubilize cisplatinand does not contain a sulfur group. Similarly, water cansolubilize cisplatin. However, in the absence of chloride, cis-platin and other complexes with chlorido leaving groupsbecome aquated, producing a mixture of species withincreased reactivity and altered cytotoxicity (26, 27).

In vitro assessment of effect of DMSO on platinumcomplexes

To assess the effect of DMSO on the activity of platinumdrugs and complexes, the activity of the drugs cisplatin,carboplatin, oxaliplatin, and satraplatin, and the experimentalcomplexes transplatin (trans-[PtCl2(NH3)2]) and [PtCl2(en)](structures shown in Fig. 1) were assessed. Satraplatin is astable, lipophilic platinum(IV) complex designed to be orallyavailable (28), and although it entered clinical trials againstadult and pediatric malignancies (most prominently prostatecancer in combination with prednisone), it has not beenapproved for use by the FDA (29). Transplatin is the "inactive"geometric isomer of cisplatin (30), and [PtCl2(en)] is regularlyused in experimental papers, and like cisplatin contains twocis-chlorido leaving groups (8).

The activity of clinical formulations of the drugs cisplatin(3.3 mmol/L in 0.9% saline with 10 mg/mL mannitol), carbo-platin (27 mmol/L in 5% glucose solution; ref. 31), and oxali-platin (12.6 mmol/L in 5% glucose solution; 32) was comparedwith analogous solvent preparations to the clinical formulation(i.e., saline for cisplatin, water for carboplatin, and oxaliplatin),

and solutions of each drug in DMSO (at 20 mmol/L stocksolution). Satraplatin is only available as capsules for oral use,and aqueous solubility is poor. As such, a 4:1 water:DMSOsolution was prepared to enable assessment of the effect ofwater on activity. No clinical formulation exists for transplatinor [PtCl2(en)], but given that both contain chlorido leavinggroups (like cisplatin), both complexeswere dissolved in saline.The responses of DLD-1 and KB-3-1 parental cells, and thecisplatin-resistant subline of KB-3-1, termed KB-CP.5, wereevaluated to examine whether sensitivity to each platinumagent, and cross-resistance in cisplatin-resistant cells, wereaffected byDMSO (Table 2; Fig. 2). TheCP.5 cells demonstratedcross-resistance to all Pt complexes (>10-fold) with the excep-tion of transplatin. The clinical formulations of cisplatin,carboplatin, and oxaliplatin showed equivalent cytotoxicity totheir respective saline/aqueous solutions.

DMSO had a significant effect on the cytotoxicity of allcomplexes with monodentate (singly coordinated) ligands,diminishing the cytotoxicity of cisplatin, carboplatin, and[PtCl2(en)], and to a lesser effect, transplatin. These effectscould be easily observed by bright-field microscopy (for cis-platin, carboplatin, and oxaliplatin; Fig. 2A–C), and by dose–response cell killing (Fig. 2D–F). The strongest effects were oncisplatin, with a greater than 40-fold loss of cytotoxicity againstboth DLD-1 and KB-3-1 cells (Table 2), and [PtCl2(en)], with agreater than 30-fold loss of cytotoxicity, both complexes con-tain the "'classic" cis-dichlorido leaving group arrangement. Forexample, cisplatin dissolved in saline demonstrated an IC50 of3.8� 0.6mmol/L, whereas its IC50 when dissolved inDMSOwas178 � 9.2 mmol/L. Carboplatin demonstrated reduced cyto-toxicity compared with cisplatin (34.2 � 3.4 mmol/L againstKB-3-1 cells), consistent with its diminished reactivity (32), butDMSO reduced its cytotoxicity further (3- to 10-fold, 243� 11.2mmol/L against KB-3-1 cells). In contrast, DMSO seemed to

Figure 1. Structures of the platinumcomplexes used in this study.

Hall et al.





have a slight potentiating effect on oxaliplatin, with bothKB-3-1 (aqueous ¼ 3.0 � 0.5 mmol/L, DMSO ¼ 0.7 � 0.3mmol/L) and DLD-1 (aqueous ¼ 3.9 � 0.6 mmol/L, DMSO ¼1.6 � 0.2 mmol/L) cells being slightly more sensitive to theDMSO-formulated compound. DMSO had no effect on satra-

platin cytotoxicity (e.g., IC50 for the clinical formulation againstDLD-1 ¼ 1.5 � 0.2 mmol/L, in DMSO ¼ 1.6 � 0.2 mmol/L).

AlthoughDMSOdisrupted the activity of cisplatin, cisplatin-resistant cells retained cross-resistance toward the DMSO-formulated drug. CP.5 cells demonstrated 10-fold resistance to

Table 2. Cytotoxicity (IC50, mmol/L) of platinum complexes against parental (DLD-1 and KB-3-1) andcisplatin-resistant (KB-CP.5) cell lines

IC50 (mmol/L)

DLD-1 KB 3-1 KB CP.5

CisplatinClinical 3.3 � 0.5 1.1 � 1.0 33.2 � 8.5Saline 3.8 � 0.6 1.9 � 0.4 34.3 � 1.6DMSO 178 � 9.2 48.1 � 3.7 376 � 283Saline þ DMSO (384 mmol/L) 3.4 � 0.3 1.1 � 0.2 ND

CarboplatinClinical 68.8 � 22.6 41.4 � 10.8 460.7 � 54.4Aqueous 78.4 � 5.1 34.2 � 3.4 1,014 � 10.4DMSO 573 � 38.0 243 � 11.2 118 �74.3

OxaliplatinClinical 1.5 � 0.2 1.14 � 0.05 NDAqueous 3.9 � 0.6 3.0 � 0.5 74.3 � 34.1DMSO 1.6 � 0.2 0.7 � 0.3 84.0 � 72.7

SatraplatinWater: DMSO (4:1) 3.8 � 0.4 2.4 � 0.3 37.3 � 3.2DMSO 2.9 � 0.6 2.5 � 0.3 8.6 � 1.6

TransplatinSaline 335 � 165 >500 716 � 287DMSO 587 � 31.8 433 � 30.6 134 � 80.3

[PtCl2(en)]Saline 2.7 � 1.9 11.4 � 1.5 569 � 113DMSO 87.1 � 36.3 135 � 7.1 256 � 92.2

Figure 2. Comparison of the effect of DMSO on the biologic activity of cisplatin, carboplatin, and oxaliplatin. Bright-field images (A–C), and dose–responsecurves (D–F) of DLD-1 cells exposed to platinum drugs (cisplatin, carboplatin, and oxaliplatin) in different solvents (water/saline, clinical formulation, DMSO).






cisplatin (CP.5 IC50¼ 34.3� 1.6 mmol/L vs. KB 3-1 IC50¼ 1.9�1.6 mmol/L), and cross-resistance to aqueous carboplatin (32-fold), oxaliplatin (25-fold), and satraplatin (4-fold). CP.5 cellsdemonstrated 8-fold resistance to cisplatin formulated inDMSO, and cross-resistance to oxaliplatin and satraplatin wasmaintained. Both carboplatin and transplatin dissolved inDMSO seem to have greater potency against CP.5 cells thanwhen formulated in aqueous media (water and saline, respec-tively). By way of example, the IC50 of transplatin dissolved inDMSO was very high (433 � 30.6 mmol/L) but lower againstCP.5 cells (134 � 80.3 mmol/L). This collateral sensitivitysuggests that reaction with DMSO produces a complex (videinfra) with greater activity than cisplatin. The poor cytotoxicityand in vivo activity of transplatin are generally ascribed to itsgreater reactivity, but it is known that trans Pt complexes withbulkier ligands have reduced reactivity, andwith this improvedstability comes biologic activity (33).

To assess the effect of DMSO on the DNA damage and cellkilling produced by Pt drugs (9), we examined phosphorylationof the histone H2A family member H2A.X. H2A.X becomesphosphorylated at serine 139 (gH2AX) in response to double-stranded breaks elicited by DNA damage, recruiting repairfactors to sites of damage, and is also phosphorylated as aresult of apoptosis induced by DNA damage (34). In this way,H2A.X has been shown to be phosphorylated in cells exposed tocisplatin, carboplatin, and oxaliplatin (35, 36). Cells weretreated with cisplatin, carboplatin, and oxaliplatin formulatedin aqueous solution (saline orwater) orDMSO, and gH2A.X fociwere evaluated by immunofluorescence microscopy after 24hours (Fig. 3A). Cells treated with cisplatin (5 mmol/L) in salineor carboplatin (5 mmol/L) in water clearly elicited a DNAdamage response, whereas cells treated with the compoundsdissolved in DMSO resulted in a striking diminution of gH2A.Xstaining. Despite being approximately equitoxic with cisplatin,oxaliplatin resulted only in low-level staining. Satraplatin inwater and DMSO showed equivalent gH2A.X staining, whereastransplatin and [PtCl2(en)] dissolved in DMSO elicited weakergH2A.X staining than their saline-dissolved comparison (notshown).

Immunoblot analysis of gH2A.X supported immunofluores-cence microscopy staining (Fig. 3B). To directly study whetherDMSO affected cell death, we incubated DLD-1 cells withcisplatin (5 mmol/L), carboplatin (50 mmol/L), and oxaliplatin(5 mmol/L) in either DMSO or water, and whole-cell lysateswere collected after 48 hours for Western analysis (Fig. 3B).Induction of cleaved PARP and cleaved caspase-3 was causedby cisplatin, carboplatin, and oxaliplatin, consistent with theirrole in platinum-mediated apoptosis (37). These events wereprevented by formulation of the three drugs in DMSO.

Modification of platinum complexes by DMSO and othersolvents

To assess the nature of the interactions between cisplatinand DMSO, we compared electrospray ionizationmass spectrain positive mode of all six complexes in aqueous and DMSOsolutions (Supplementary Table S7, only peaks related toDMSO adducts are shown). Previous chemical analysis hasdemonstrated that cisplatin produces multiple species upon

dissolution in DMSO, by replacement of a Cl�withDMSO (m/z¼ 343), and replacement of a Cl� and an NH3 ligand by twoDMSO molecules (m/z ¼ 404; refs. 38, 39). We also observed ahigher molecular weight species (m/z ¼ 665.9) correspondingto a bridged form of DMSO-substituted cisplatin, mNH2-[Pt(NH3)(Cl)(DMSO)]2

þ (Supplementary Table S7) not previouslyreported. These are shown schematically in SupplementaryFig. S3. Carboplatin, transplatin, and [PtCl2(en)] producedreaction products with DMSO, all involving replacement of aCl� ligand (Supplementary Table S7). Carboplatin and oxali-platin also produced self-association multimers, as previouslyreported (32). Mass spectra of satraplatin in DMSO did notproduce any peaks corresponding to interaction with DMSO,consistent with the inertness of platinum(IV) complexes (40).Oxaliplatin did not produce any observable peaks correspond-ing to reaction with DMSO.

The effect of DMSO on aqueous cisplatinThe evidence thus far indicates that most Pt complexes

(including cisplatin and carboplatin) are deactivated upondissolution in DMSO stock solutions, and aqueous-basedsolutions are essential for biologic studies. However, cisplatinis usually used in combination in the clinic, and synergy of Ptdrugs with experimental and established therapeutics areregularly assessed (41). As organic-based therapeutics areusually dissolved in DMSO, we wondered whether cisplatinin aqueous solution or growthmediawould be deactivated by asmall component of DMSO if it is introduced into the samesolution.

We first examined whether DMSO would inhibit cisplatin'scytotoxicity. To examine this, cisplatin dissolved in saline wasdiluted into growthmedium, followed by 30mL/mLDMSO (3%,effective concentration 384 mmol/L in 154 mmol/L saline),followed by serial dilution and dosing to cells. DMSO did nothave any effect on cisplatin's cytoxicity on KB-3-1 or DLD-1cells (e.g., DLD-1 cisplatin saline IC50 ¼ 3.3 � 0.5 mmol/L,cisplatin saline and DMSO IC50 ¼ 3.4 � 0.3 mmol/L; Table 2).Despite the lack of biologic effect, mass spectrometry of salinesolutions of cisplatin and cisplatin in the presence of 3%DMSOrevealed a peak corresponding to the replacement of onechloride ligand with DMSO (Table 3). These data suggest thatDMSO introduced into combination studies with cisplatindoes not affect its activity. However, researchers should beaware that DMSO does interact with cisplatin even in diluteenvironments.

Assessment of alternative solvent systems for platinumcomplexes

One limitation of aqueous formulations of cisplatin is itsmaximal solubility of just more than 3 mmol/L, whereasDMSO solutions can be prepared of more than 10 mmol/L.We examined whether other organic solvents had a similareffect on cisplatin. Stock solutions of cisplatin were preparedin amenable solvents: DMF, DMA, formamide, acetamide,cremophor EL (all 10 mmol/L stock solutions), and water(3.3 mmol/L; Table 3). Cremophor EL and acetonitrile didnot fully dissolve cisplatin, but were tested as fine ultrasoni-cated suspensions. None of the solvents profoundly affected

Hall et al.





cisplatin's cytotoxicity against DLD-1 cells, and although notstatistically significant, DMF resulted in the greatest decreasein cytotoxicity of cisplatin (IC50 ¼ 5.0 � 1.3 mmol/L comparedwith 3.8 � 0.6 mmol/L for cisplatin in saline).However, each organic solvent alone (at a high concentration

of 3% inmedia, consistentwith the lowest dilution of 10mmol/Lcisplatin in media required to perform a dose–response) dem-onstrated cytotoxicity toward DLD-1 cells (Table 3). The excep-tion was acetonitrile, with the limitation that acetonitrile wasone of the two solvents unable to fully dissolve cisplatin. Themost toxic solvent was cremophor EL, a polyethoxylated castoroil used as the excipient for intravenous administration of

poorly solubledrugs suchaspaclitaxel (42). Although3% solventis a high concentration, the IC50 concentration for cisplatin(�1–3 mmol/L) in our toxicity assays occurs at a drug dilutionthat is in the presence of 0.2% to 0.05% solvent, a concentrationat which no toxicity toward DLD-1 cells was observed.

DiscussionWe have demonstrated here the profound effects of DMSO

on platinum drugs and complexes that contain monodentateligands. Drug cytotoxicity is diminished, as evidenced by cellgrowth andDNA damage response. The replacement of ligandson the platinum complexes could be observed by mass

5 μmol/L, saline 5 μmol/L, water50 μmol/L, water

5 μmol/L, DMSO 5 μmol/L, DMSO50 μmol/L, DMSO

Aqu

eous

Carboplatin OxaliplatinCisplatin

DM

SO

A

B

Total PARP

Cleaved PARP

β-Actin

Cleaved caspase-3

Control

Cisplatin

(DMSO)

Cisplatin

(salin

e)

Oxalip

latin (D

MSO)

Oxalip

latin (w

ater)

Carboplatin (D

MSO)

Carboplatin (w

ater)

Phospho-H2A.X

Figure 3. Comparison of the effectof DMSO on cisplatin, carboplatin,and oxaliplatin-mediated DNAdamage and cell death. A, cellularDNA damage recognition causedby platinum drugs observed viaconfocal images (�63) of DLD-1cells exposed to platinum drugs asindicated and stained with anantibody that recognizesphosphorylated (g ) H2A.X.B, immunoblot analysis of proteinlysate from DLD-1 cells treated asdescribed for A and probed forexpression of total PARP, cleavedPARP, cleaved caspase-3, andphosphor (g ) H2A.X, with b-actinprobed as a control for proteinloading.






spectrometry. This study was prompted by a recognition of thehigh rate of use of DMSO with cisplatin in the literature, andpartly by limitations encountered in developing a high-throughput screen using cisplatin—drugs are transferred bypins and surface tension has been optimized to transfer a fixedvolume of DMSO. Using an alternative solvent would not bepossiblewithout significant optimization. A significant numberof reports in allfive journals examinedutilizedDMSO-dissolvedcisplatin, raising questions about how to interpret the results ofa large number of previous studies. Our findings also reinforcethe current focus on the challenge of reproducing scientificliterature (43).

These findings extend to most platinum complexes. Thou-sands of platinum complexes (and other metal-based com-plexes) have been synthesized and tested in vitro in an effort toidentify complexes with greater potency than cisplatin, adifferent mechanism of action from cisplatin, and/or an abilityto overcome cross-resistance in cisplatin-resistant cells (8, 6).Given the general lack of information about how to formulateexperimental agents and the likelihood that complexes willdissolve in DMSO, most compounds for testing against celllines are also regularly dissolved in DMSO. For example, of the20 most recent papers reporting new chemical entities basedon platinum in the Journal ofMedicinal Chemistry, seven (35%)used DMF to dissolve complexes for biologic testing, five (25%)did not state the solvent used, and three (15%) used DMSO(Supplementary Table S6). Alternative solvents such as DMFhave been reported not to cause cytotoxicity below 0.5% (44),and solutions of cisplatin dissolved in DMF retain activity (45).However, althoughDMF is recognized as cytotoxic toward cells(46), there seems to be no comprehensive assessment of DMF'sutility as a drug (or platinum complex) solvent. It is criticalthen, that an understanding of drug interaction with solvent,and solubility in solvent, be ascertained for experimentalmetal-based therapeutics.

The problem of cisplatin deactivation by DMSO was firstreported over 20 years ago, and the underlying chemistry hadbeen generally well defined (13, 16). Jones and colleaguesdemonstrated that coadministration of cisplatin with DMSOin Sprague–Dawley rats reduced nephrotoxicity (47), andMassart and colleagues observed that DMSO inhibited cisplat-in cytotoxicity toward cultured thyrocytes while investigating

whether DMSO could act as an antidote for cisplatin-inducedtoxicities, based on previously reported protective effects ofDMSO (17). This work was initiated in the context of the desireto utilize known thiols as reactive "protective agents" to temperthe side effects of cisplatin (48). Fischer and colleagues laterdemonstrated that although the neurotoxic side effects ofcisplatin are tempered by DMSO, loss of cisplatin's cytotoxicityoffset any potential benefit (38).

It is not clear whether the cytotoxicity observed in DMSO-formulated cisplatin is due to the mixture of chemical entitiespresent in solution, or the unreacted portion of cisplatinremaining in equilibrium. Given varying cross-reaction insolutions utilized in laboratories, it would be expected to bedifficult to replicate results with saline, PBS, or clinical for-mulations of platinum complexes. For example, a crystallo-graphic study of cisplatin and carboplatin binding to histidinesof hen egg-white lysozyme found one platinum bound to His15when cisplatin was dissolved in aqueous medium. Whendissolved in DMSO, two platinum atoms bound to His15,leading to the conclusion that DMSO could facilitate plati-num-protein binding (49). Uribe and colleagues reportedincreased sensory hair cell death in zebrafish cotreated withcisplatin andDMSObut not other solvents, whichwas ascribedto the cell permeabilizing effects of DMSO, and warned thatDMSO could produce false-positive effects in other drugscreens due to DMSO's biologic activity (50).

The effects of DMSOonplatinumdrug activity in vitro can beprofound.We believe the practice of dissolving platinum drugsin DMSO must cease, and if the solvent is to be utilized, newplatinum agents must demonstrate a lack of interaction withDMSO. For experimentation, cisplatin should be prepared in asaline-based solvent (3 mmol/L), and carboplatin (27 mmol/L)and oxaliplatin (12.6mmol/L) inwater.When available, clinicalformulation can substitute. It is the hope of the authors thatthis report will raise awareness of this significant issue in thecancer biology community, introduce caution when interpret-ing results of published studies utilizing DMSO, inform futureexperimental design, and guide editorial guidelines for mech-anistic and experimental therapeutic research.

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

Table 3. Cytotoxicity (IC50, mmol/L) of cisplatin, dissolved or suspended in solvents, against DLD-1 cells

Cisplatin IC50 (mmol/L) Stock (mmol/L) Soluble 3% solvent viability (%)

Control 100Clinical 3.3 � 0.5 3.3 Y —

Saline 3.8 � 0.6 3.3 Y 96 � 10DMSO 177.6 � 9.2 20 Y 90 � 5DMF 5.0 � 1.3 10 Y 79 � 5DMA 3.5 � 0.8 10 Y 24 � 3Formamide 3.7 � 1.8 10 Y 57 � 2Cremophor EL 2.5 � 0.2 10 N 0.7 � 0.1Acetonitrile 3.4 � 0.3 10 N 103 � 6Water 4.0 � 0.5 3.3 Y 106 � 7

Hall et al.





Authors' ContributionsConception and design: M.D. Hall, M.M. GottesmanDevelopment of methodology: M.D. Hall, K.A. TelmaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.D. Hall, K.-E. Chang, T.D. Lee, J.P. Madigan, J.R.Lloyd, I.S. GoldlustAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.D. Hall, K.A. Telma, T.D. Lee, J.P. MadiganWriting, review, and/or revisionof themanuscript:M.D.Hall, K.-E. Chang, I.S. Goldlust, J.D. Hoeschele, M.M. GottesmanAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M.D. HallStudy supervision: M.D. Hall

AcknowledgmentsThe authors thank George Leiman for editorial assistance and Dr. Craig

Thomas for helpful insights.

Grant SupportThis work was supported by the Intramural Research Program of the NIH,

National Cancer Institute. K.-E. Chang is an NIH Medical Research ScholarsProgram scholar, a public-private partnership supported jointly by the NIH andgenerous contributions to the Foundation for the NIH from Pfizer Inc, The DorisDuke Charitable Foundation, The Alexandria Real Estate Equities, Inc., Mr. andMrs. Joel S. Marcus, and the Howard Hughes Medical Institute, as well as otherprivate donors.

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 to indicate thisfact.

Received February 21, 2014; revised April 16, 2014; accepted April 16, 2014;published OnlineFirst May 8, 2014.

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Hall et al.




2014;74:3913-3922. Published OnlineFirst May 8, 2014.Cancer Res Matthew D. Hall, Katherine A. Telma, Ki-Eun Chang, et al. Carboplatin, and Other Platinum ComplexesSay No to DMSO: Dimethylsulfoxide Inactivates Cisplatin,

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Say No to DMSO: Dimethylsulfoxide Inactivates Cisplatin, Carboplatin, and … · cisplatin stock solution used for culturing CP.5 cells was prepared in PBS. The cisplatin-resistant