Say No to DMSO: Dimethylsulfoxide Inactivates Cisplatin, Carboplatin, and Other Platinum Complexes
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Published OnlineFirst May 8, 2014; DOI: 10.1158/0008-5472.CAN-14-0247 Cancer Therapeutics, Targets, and Chemical Biology Research 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 Abstract The platinum drugs cisplatin, carboplatin, and oxaliplatin are highly utilized in the clinic and as a consequence are extensively studied in the laboratory setting. In this study, we examined the literature and found a significant number 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 the structure of the complex. We examined the effect of DMSO on platinum complexes, including cisplatin, carboplatin, and oxaliplatin, finding that DMSO reacted with the complexes, inhibited their cytotoxicity and their ability to initiate cell death. These results render a substantial portion of the literature on cisplatin uninterpretable. Raising awareness of this significant issue in the cancer biology community is critical, and we make recommendations on appropriate solvation of platinum drugs for research. Cancer Res; 74(14); 3913–22. Ó2014 AACR. Introduction (8), some of which have entered clinical trials (e.g., picoplatin, satraplatinand BBR3464; ref. 9). Cisplatin (cis-[PtCl2(NH3)2]), carboplatin ([Pt(O,O'-cdbca) A critical aspect of the activity of platinum drugs in vitro and (NH3)2], cbdca ¼ cyclobutane-1,1-dicarboxylate), and oxalipla- tin ([Pt(R,R-cyclohexane-1,2-diamine)(O,O'-ethanedioato)]) are in vivo is their interaction with the solvent environment. For used in combination with other agents to treat a range of example, platinum drugs are activated via replacement of malignancies including testicular, ovarian, head and neck, leaving groups with water inside the cell, a process termed bladder, esophageal, and small cell lung cancer (1–4). With aquation (10, 11). For cisplatin, this is the loss of a chloride the exception of testicular cancer, acquired or intrinsic resis- ligand and its replacement with water, which is a more reactive tance generally occurs and tumors are not eliminated by leaving group. For this reason, cisplatin is formulated for treatment (3–5). Cisplatin exerts its toxicity by DNA binding clinical use in saline solution with a high chloride concentra- and downstream apoptotic signaling, and resistance is con- tion (154 mmol/L) to prevent drug aquation before adminis- ferred by reducing apoptotic signaling, upregulating DNA tration, stabilizing the drug and preventing side reactions damage repair mechanisms, altering cell-cycle checkpoints, before cell entry. A limiting factor for platinum drugs is and disrupting assembly of the cytoskeleton (4, 6). The pleio- their relatively low solubility, and clinical cisplatin is formu- tropic mechanisms underlying platinum resistance are well lated at a concentration of 1 mg/mL (3.3 mmol/L). In labora- described, but their clinical significance is uncertain (4, 6). tory and drug screening settings, stock solutions of organic- These factors have prompted continued experimentation with based drugs are predominately prepared in the solvent DMSO ¼ platinum drugs to understand their mechanisms of action (O S(CH3)2), which is viewed as a virtual "universal solvent" alone and in combination with other therapeutics (7), as well able to solubilize most small molecules at high concentrations as the synthesis and testing of new platinum complexes with (up to 100 mmol/L, for example; ref. 12). DMSO contains a the prospect of uncovering compounds with promising activity nucleophilic sulfur, which allows it to coordinate with plati- num complexes, displacing ligands and changing the structure of the complexes (13–16). This renders platinum complexes Authors' Affiliations: 1Laboratory of Cell Biology, Center for Cancer unstable in DMSO. Massart and colleagues first reported that 2 Research, National Cancer Institute; Advanced Mass Spectrometry Facil- DMSO reduced cisplatin's cytotoxicity toward cultured thyr- ity, National Institute of Diabetes & Digestive & Kidney Diseases, NIH, Bethesda; 3Division of Preclinical Innovation, National Institutes of Health ocytes (17), and Dernel and colleagues reported that a polymer- Chemical Genomics Center, National Center for Advancing Translational based drug delivery system limited activity of cisplatin against Sciences, NIH, Rockville, Maryland; and 4Department of Chemistry, East- ern Michigan University, Ypsilanti, Michigan stage IIb appendicular osteosarcoma in dogs (18). Little infor- mation exists on the effect of DMSO on other platinum drugs Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). and complexes. Yet, as discussed in this article, cisplatin and other platinum Corresponding Author: Michael M. Gottesman, Laboratory of Cell Biol- ogy, National Cancer Institute, NIH, 37 Convent Drive, Rm. 2108, Bethesda, complexes are regularly dissolved in DMSO for biologic experi- MD 20892. Phone: 301-496-1921; Fax: 301-402-0450; E-mail: ments, both in vitro and in vivo in experimental models, and [email protected] DMSO solutions of cisplatin have been utilized in the clinical doi: 10.1158/0008-5472.CAN-14-0247 veterinary setting (19). This use may be due to the lack of a Ó2014 American Association for Cancer Research. comprehensive understanding of the effect of DMSO on the www.aacrjournals.org 3913 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst May 8, 2014; DOI: 10.1158/0008-5472.CAN-14-0247 Hall et al. activity of platinum complexes in the cancer biology commu- mL (1.6 mmol/L) in our laboratory, as described previously (20, nity. Irrespective of the reason, the implications for published 21). KB lines were originally generated in the laboratory of M.M. studies on cisplatin's mechanism using DMSO solutions are Gottesman. DLD-1 cells were provided by the National Cancer profound. Institute (part of the NCI-60 collection). All cell lines were We sought to examine the range of solvent systems utilized thawed immediately before experimentation, and cell lines are for platinum drugs in peer-reviewed research, and the effects of characterized by NCI using short tandem repeat profiling. The DMSO on platinum drug activity. A number of journals were cisplatin stock solution used for culturing CP.5 cells was assessed to gauge the solvent types used in studies of cisplatin. prepared in PBS. The cisplatin-resistant cells were main- We then assessed the impact of DMSO versus clinical formula- tained in the presence of cisplatin, which was removed from tions of a number of platinum complexes on the cytotoxicity, growth medium 3 days before all experiments. All cell lines cellular recognition of DNA damage, and cell death signaling. were grown as monolayer cultures at 37 Cin5%CO2,using Mass spectrometry was used to directly assess the interaction either DMEM (KB cells) or RPMI (DLD1 cells) with 4.5 g/L of DMSO and clinical formulations with each platinum glucose (both from Invitrogen), supplemented with L-gluta- complex. mine, penicillin, streptomycin, and 10% FBS (BioWhittaker). Resistance of CP.5 cells to cisplatin was confirmed on a Materials and Methods regular (at least monthly) basis, using cell viability assays as Literature review described herein. Five journals that regularly publish studies on small-mole- cule therapeutics and their mechanism were examined: Cancer Cytotoxicity and cell growth Research (http://cancerres.aacrjournals.org), Molecular Cancer Cell survival was measured by the MTT (Invitrogen) assay. Therapeutics (http://mct.aacrjournals.org), Molecular Pharma- Cells were seeded at a density of 5,000 cells per well in 96-well fi cology (http://molpharm.aspetjournals.org), Journal of Phar- plates and incubated at 37 C in humidi ed 5% CO2 for 24 macology and Experimental Therapeutics (JPET; http://jpet. hours. Serially diluted (1:3, RPMI or DMEM used as diluent, aspetjournals.org), and the Public Library of Science (PLoS; chloride concentration 120 mmol/L) compound was added fi http://www.plos.org) journals. In each case, the word "cisplat- to give the intended nal concentrations. Solvent tolerance fi in" was entered as a search term on the respective journal testing up to 0.5% under identical conditions con rmed website search engine, restricted to the term appearing in the growth of all cell lines was unaffected. Cells were then incu- title or abstract of articles. Manuscripts were then individually bated an additional 72 hours, and the MTT assay was per- reviewed to identify and assess only those papers reporting in formed according to the manufacturer's instructions (Molec- vitro data. These papers were then assessed for the solvent or ular Probes). Absorbance values were determined at 570 nm on solution used for dissolving cisplatin, and these were deter- a Spectra Max 250 spectrophotometer (Molecular Devices). All mined and recorded; similarly, it was noted if the solvent MTT assays were performed in triplicate. The IC50 values were fi system was not disclosed. In the majority of cases, if the de ned as the drug concentrations required to reduce cellular solvent used was not mentioned in the Materials and Methods proliferation to 50% of the untreated control well. We used section, it was not explicitly disclosed anywhere in the man- Prism 6 (GraphPad Software) software for graphs and statis- Æ fi uscript in a manner that allowed unambiguous determination tics. All data are expressed as mean SD. For bright- eld  5 of the experimental strategy used—this was recorded as "Not imaging, DLD-1 cells were seeded at a density of 3 10 cells fi reported." Thirty-five manuscripts were assessed for each per well in 6-well plates and incubated at 37 C in humidi ed journal, with the exception of the JPET, where only 28 relevant 5% CO2 for 24 hours. Drug was added directly to each well and articles were identified.