Supporting Information - PNAS · 0.4 0.2 0.0 -0.2 -0.4 0 10 20 30 40 Current mV vs. Ag/AgCl 0 50...

Supporting InformationDhar and Lippard 10.1073/pnas.0912276106SI TextThe complexes cis-[Pt(NH3)2Cl2] (1) and c,c,t-[Pt(NH3)2Cl2(OH)2] (2) were synthesized as described. Distilledwater was purified by passage through a Millipore Milli-Q Biocelwater purification system (18.2 M�) containing a 0.22-�m filter.Anti-cytochrome c (Ab-1) sheep polyclonal antibody was pro-cured from Calbiochem. Alexa Fluor 488-labeled secondaryantibody donkey anti-(sheep IgG) was obtained from Invitrogenfor cytochrome c detection. For AIF detection, we used a rabbitpolyclonal IgG antibody from Santa Cruz Biotechnology, Inc.Alexa Fluor 546-labeled secondary antibody goat anti-(rabbitIgG) was purchased from Invitrogen. The detection of thecisplatin 1,2-d(GpG) intrastrand adduct was carried out using amonoclonal adduct-specific antibody R-C18 which was kindlyprovided by Jurgen Thomale (University of Duisburg-Essen).FITC-labeled secondary antibody rabbit anti-(rat Ig) was ob-tained from Invitrogen. Specific adhesion slides for immunoflu-oresecence were purchased from Squarix Biotechnology. JC-1(5,5�,6,6�-tetrachloro-1,1�,3,3� tetraethylbenzimidazolylcarbo-cyanine iodide) was obtained from Cayman Chemicals. 1H, 13Cand 195Pt NMR spectra were recorded on a Bruker AVANCE-400 NMR spectrometer with a Spectro Spin superconductingmagnet in the Massachusetts Institute of Technology Depart-ment of Chemistry Instrumentation Facility (MIT DCIF).Atomic absorption spectroscopic measurements were taken ona Perkin-Elmer AAnalyst 300 spectrometer. HRMS analysis wascarried out on a Bruker Daltonics APEXIV 4.7 Tesla FourierTransform Ion Cyclotron Resonance mass spectrometer in theMIT DCIF. Fluorescence imaging studies were performed withan Axiovert 200M inverted epifluorescence microscope (Zeiss)equipped with an EM-CCD digital camera C9100 (Hamamatsu).An X-Cite 120 metal-halide lamp (EXFO) was used as the lightsource. The microscope was operated with Volocity software(Improvision).

Electrochemistry. Electrochemical measurements were made at25 °C on a EG&G PAR Model 263 Potentiostat/Galvanostatwith electrochemical analysis software 270 and a three-electrodeset-up comprising a glassy carbon working electrode, platinumwire auxiliary electrode and a Ag/AgCl reference electrode. Theelectrochemical data were uncorrected for junction potentials.KCl was used as a supporting electrolyte.

Cytotoxicity Studies. Cell lines and cell culture. Human testicularcancer NTera-2, human cervical cancer HeLa, human osteosar-coma U2OS, human breast adenocarcinoma MCF-7, and healthynormal fibroblast GM61869 cell lines were obtained from theATCC. Human ovarian carcinoma cell lines A2780 and A2780/CPwere kindly provided by Thomas Hamilton (Fox Chase CancerCenter, Jenkintown, PA). A549 lung carcinoma and normal lungMRC-5 cells were obtained from David E. Root (WhiteheadInstitute for Biomedical Research). Cells were grown at 37 °C in 5%CO2 in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were passed every 3–4 days and restarted fromfrozen stocks upon reaching pass number 20.MTT assay. The cytotoxic behavior of mitaplatin, DCA, andcisplatin was evaluated by using the MTT assay. Solutions of theplatinum complexes were freshly prepared in sterile PBS beforeuse and quantitated by atomic absorption spectroscopy. Cellswere seeded on a 96-well plate in 100 �L of DMEM andincubated for 24 h. The cells were then treated with mitaplatin,

DCA, or cisplatin, separately at varying concentrations andincubated for 72 h at 37 °C. The cells were then treated with 20�L of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT) (5 mg/mL in PBS) and incubated for 5 h. Themedium was removed, the cells were lysed by adding 100 �LDMSO, and the absorbance of the purple formazan was re-corded at 550 nm using a BioTek Synergy HT multidetectionmicroplate plate reader. Each condition was repeated in tripli-cate in three independent experiments for each cell line.

Fluorescence Imaging. Cell fixing solution. Paraformaldehyde (4.0 g)and NaOH (0.4 g) were dissolved in 100 mL distilled water. Tothis solution was added NaH2PO4 (1.68 g) and the pH wasadjusted to be between 7.5 and 8.0 by adding NaOH.Fluorescence sample mounting media. For sample mounting, a solu-tion containing 20 mM Tris (pH 8.0), 0.5% N-propyl gallate, and70% glycerol was used.Detection of cisplatin 1,2-d(GpG) intrastrand adducts. Detection of theplatinum 1,2-d(GpG) adducts was carried out by following aprocedure recently reported by us using an antibody specific to thisadduct (3). Briefly, NTera-2 cells were seeded in a six-well plateusing DMEM and incubated overnight at 37 °C. Mitaplatin wasadded to a final concentration of 20 �M and incubated at 37 °C.After 12 h, cells were trypsinized, washed with PBS, resuspendedin HAES-sterile-PBS at a density of 1 � 106 per mL and placed ontoa precoated slide (ImmunoSelect, Squarix) and air dried. Cell fixingwas carried out at �20 °C in methanol for 45 min. Nuclear DNAwas denatured by alkali (70 mM NaOH, 140 mM NaCl, and 40%methanol, vol/vol) treatment for 5 min at 0 °C, and cellular proteinswere removed by a proteolytic procedure involving two steps. Thecells were first digested with pepsin at 37 °C for 10 min and thenwith proteinase K at 37 °C for 5 min. After blocking with milk (1%in PBS; 30 min; 25 °C) the slides were incubated with anti-(Pt-DNA) Mabs (R-C18 0.1 mg/mL in milk) (4) overnight at 4 °C. Afterwashing with PBS, immunostaining was performed by incubationwith FITC-labeled goat anti-(rat Ig) antibody at 37 °C for 1 h. Thenuclei of the cells were stained by using Hoechst (H33258) (250�g/L) and mounted using the mounting solution for imaging.Determination of platinum concentrations from cell extracts. A549 andMRC5 cells were grown in DMEM to �95% confluence in 175-cm2

flasks. These cells were then treated either with 10 �M cisplatin ormitaplatin and subsequently incubated for 24 h at 37 °C. Cells werewashed with PBS three times and released by trypsinization intoPBS. Solutions containing cells were then centrifuged at 800 � g for10 min and the cell pellet obtained was resuspended in 100 �L ofice-cold lysis buffer (1.0 mM DTT, 1.0 mM PMSF, 10 mM KCl, and10 mM MgCl2, pH 7.5) for 15 min. This process was repeated andfinally the pellets were resuspended in 40 �L of ice-cold lysis buffer.The cell membranes were lysed by 10 strokes of a 28-gauge syringe.The resulting suspension was centrifuged at 11,000 � g for 20 minand the cytosolic fraction of the cells was collected as supernatant.The pellet was resuspended in 40 �L of extraction buffer (1.0 mMDTT, 1.0 mM PMSF, 1.5 mM MgCl2, 0.2 M EDTA, 0.42 M NaCl,and 25% glycerol, pH 7.9) and lysed with 10 strokes of a 28-gaugesyringe. The lysate was shaken at 1,000 rpm for 45 min at 4 °C andthen centrifuged at 20,000 � g for 10 min at 4 °C. The supernatantwas collected as the nuclear fraction. Platinum concentrations in allof the fractions were determined by AAS. The protein concentra-tion in each fraction was determined by using bicinchoninic acid(BCA) assay. Total platinum concentrations were expressed asnanograms of Pt per microgram protein.

Dhar and Lippard www.pnas.org/cgi/content/short/0912276106 1 of 10

1. Dhara SC (1970) A rapid method for the synthesis of cis-[Pt(NH3)2Cl2]. Indian J Chem8:193–194.

2. Hall MD, et al. (2003) The cellular distribution and oxidation state of platinum(II) andplatinum(IV) antitumor complexes in cancer cells. J Biol Inorg Chem 8:726–732.

3. Dhar S, Liu Z, Thomale J, Dai H, Lippard SJ (2008) Targeted single-wall carbon nano-tube-mediated Pt(IV) prodrug delivery using folate as a homing device. J Am Chem Soc130:11467–11476.

4. Liedert B, Pluim D, Schellens J, Thomale J (2006) Adduct-specific monoclonal antibodiesfor the measurement of cisplatin-induced DNA lesions in individual cell nuclei. NucleicAcids Res 34:e47.

0.4 0.2 0.0 -0.2 -0.4

mV vs. Ag/AgCl

0 50 100 150 200 250 300

Scan rate/mVs-1

Fig. S1. Redox behavior of mitaplatin (1) in phosphate buffer at pH 7.4. Cyclic voltammograms of 1 in phosphate buffer-0.1 M KCl of pH 7.4 with varied scanrates (Upper). Plot of reduction peak potential maxima of 1 at pH 7.4 as a function of scan rate (Lower).

400 200 0 -200 -400 -600

mV vs. Ag/AgCl

0 50 100 150 200 250 300

Scan rate/mVs-1

Fig. S2. Redox behavior of mitaplatin (1) in phosphate buffer at pH 6.0. Cyclic voltammograms of 1 in phosphate buffer-0.1 M KCl of pH 6.0 with varied scanrates (Upper). Plot of reduction peak potential maxima of 1 at pH 6.0 as a function of scan rate (Lower).

0 10 20 30 40 500

100 MRC-5

[Pt] or 2[DCA] (µΜ)

Cisplatin, IC50

= 9.5 µM Mitaplatin, IC

50 = 18.3 µM

DCA, IC50

> 100 µM

0 10 20 30 40 50 600

100 MCF-7

[Pt] or 2[DCA] (µM)

Cisplatin, IC50

= 13 µM Mitaplatin, IC

50 = 18 µM

DCA, IC50

> 50 µM

0 20 40 60 80 100

120A549

[Pt] (µM) or 2[DCA] (µM)

Cisplatin, IC50 = 12 µM Mitaplatin, IC50 = 14 µM DCA, IC50 > 200 µM

0.00 0.04 0.08 0.12 0.16 0.20 0.24

Mitaplatin, IC50 = 0.051 µM Cisplatin, IC50 = 0.043 µM DCA, IC50 > 0.4 µM

NTera-2%

0 10 20 30 40 50

Cisplatin, IC50 = 3.9 µM Mitaplatin, IC50 = 6.4 µM DCA, IC50 > 100 µM

[Pt] or 2[DCA] (µM)0 1 2 3 4 5 6

Mitaplatin, IC50 = 2.0 µM Cisplatin, IC50 = 1.2 µM DCA, IC50 > 8 µM

0 10 20 30 40 500

100 MRC-5

[Pt] or 2[DCA] (µΜ)

Cisplatin, IC50

= 9.5 µM Mitaplatin, IC

50 = 18.3 µM

DCA, IC50

> 100 µM

0 10 20 30 40 50 600

100 MCF-7

Cisplatin, IC50

= 13 µM Mitaplatin, IC

50 = 18 µM

DCA, IC50

> 50 µM

0 20 40 60 80 100

120A549

[Pt] (µM) or 2[DCA] (µM)

Cisplatin, IC50 = 12 µM Mitaplatin, IC50 = 14 µM DCA, IC50 > 200 µM

0.00 0.04 0.08 0.12 0.16 0.20 0.24

Mitaplatin, IC50 = 0.051 µM Cisplatin, IC50 = 0.043 µM DCA, IC50 > 0.4 µM

NTera-2%

0 10 20 30 40 50

Cisplatin, IC50 = 3.9 µM Mitaplatin, IC50 = 6.4 µM DCA, IC50 > 100 µM

[Pt] or 2[DCA] (µM)0 1 2 3 4 5 6

Mitaplatin, IC50 = 2.0 µM Cisplatin, IC50 = 1.2 µM DCA, IC50 > 8 µM

Fig. S3. Cell killing ability of mitaplatin. The cytotoxicity of mitaplatin, cisplatin, and DCA against NTera-2, U2OS, HeLa, A549, and MCF-7 cancer cells and MRC-5normal cells as determined by the MTT assay.

DIC J-monomer J-aggregates Merge

Fig. S4. Changes in the mitochondrial membrane potential as revealed by the JC-1 assay. Mitaplatin attacks the mitochondria of human NTera-2 cancer cellsbut has no effect on healthy normal fibroblasts. A control with cisplatin similarly revealed no effect on mitochondrial function.

25 µm

25 µm 25 µm 25 µm

25 µm 25 µm

Control

Mitaplatin

Nuclei Pt-GG Merge Fig. S5. Visualization of Pt-1,2-d(GpG) intrastrand cross-links. Immunofluorescence of untreated NTera-2 cells (Upper). Pt-GG adducts were observed in thenuclear DNA of NTera-2 cells after treatment with mitaplatin for 12 h (Lower). Nuclei were stained with Hoechst (blue) and Pt-1,2-d(GpG) adducts in DNA werevisualized using Mab R-C18 (green).

0 10 20 30 40 50 60

[Pt] (µM)0 10 20 30 40 50 60

[Pt] (µM)

(A) A2780 (B) A2780/CP70

0 10 20 30 40 50 60

[Pt] (µM)0 10 20 30 40 50 60

[Pt] (µM)

(A) A2780 (B) A2780/CP70

Fig. S6. Effect of mitaplatin on cisplatin-resistant cells. Cytotoxicity profiles of mitaplatin (F), cisplatin (■), and c,c,t-[Pt(NH3)2Cl2(O2CCH3)2] (Œ) with cisplatinsensitive A2780 cells (A) and cisplatin-resistant A2780/CP70 cells (B) after 72 h as determined by the MTT assay.

Table S1. IC50 values of c,c,t-[Pt(NH3)2Cl2(O2CCH3)2] for differentcancer cells

Cell Line* IC50 (�M)

NTera-2 11.0HeLa 17.0U2OS �10.0A2780 14.2A2780/CP70 23.0

*NTera-2, human testicular cancer; HeLa, human cervical cancer; U2OS, hu-man osteosarcoma; A2780, human ovarian carcinoma; A2780/CP70, cisplatin-resistant human ovarian carcinoma.

Table S2. Platinum concentrations in cellular compartments after treatment with cisplatin or mitaplatin

MRC-5 A549

Cytosol (�g/mg protein) Nuclear (�g/mg protein) Cytosol (�g/mg protein) Nuclear (�g/mg protein)

Cisplatin 1.63 � 0.64 2.21 � 0.66 1.05 � 0.39 0.59 � 0.067Mitaplatin 0.147 � 0.03 4.0 � 0.81 4.9 � 1.2 1.54 � 0.54

Supporting Information - PNAS · 0.4 0.2 0.0 -0.2 -0.4 0 10 20 30 40 Current mV vs. Ag/AgCl 0 50...

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