Reagents and characterization. High quality chemicals and reagents were purchased from standard sources such as Sigma-Aldrich (St. Louis, MO, USA) and VWR International (Radnor, PA, USA). Deionized water (18.2 Ω) used for making solutions was obtained from Milli-Q Direct Ultrapure Water System from Millipore (Billerica, MA, USA). All intermediates were characterized by proton nuclear magnetic resonance (1H NMR) and mass spectrometry (MS) analysis, and the purity of compounds was analyzed by high-performance liquid chromatography (HPLC). 1H NMR data were collected on a Bruker 400MHz spectrometer (Bruker, Billerica, MA) using standard parameters, while chemical shifts are reported in ppm (δ) in reference to residual non-deuterated solvent. Electrospray ionization (ESI) MS analysis was performed on new compounds with an LTQ Orbitrap Elite system (ThermoFisher Scientific, Waltham, MA, USA) at the Mass Spectrometry and Biomarker Discovery Core facility of the Cedars-Sinai Medical Center, Los Angeles, CA.
Synthesis of the DZ-CIS conjugate. Detailed protocol for DZ-CIS synthesis has been reported [24]. In brief, CIS was oxidized with hydrogen peroxide to form cis, cis, trans-diaminedichloro-dihydroxyplatinum (IV) (oxoplatin) according to the reported method [25]. DZ (500 mg, 0.71 mmol) was added to a suspension of the oxoplatin compound (350 mg, 1.05 mmol) in dimethyl sulfoxide (DMSO, 20 mL), followed by 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (204 mg, 1.06 mmol) and dimethylaminopyridine (20 mg, 0.16 mmol) and the mixture was stirred for 20 hours at room temperature. The product was purified with C18 reversed-phase (RP) silica chromatography and eluted with methanol-water to afford DZ-CIS as a dark green solid.
Cell culture. Human ccRCC cell lines of Caki-1, 786-O, ACHN, and SN12C were obtained from American Type Culture Collection (ATCC, Manassas, VA). Mouse Renca cells were provided by Dr. Kim Kyung of the Department of Surgery of the Cedars-Sinai Medical Center. Human primary renal epithelial cell culture was obtained from ex vivo culture of a ccRCC surgical tumor specimen with approval by the Institutional Review Board (IRB No. Pro00031870). Primary cells at passages 4 and 5 were used. Cells were cultured in RPMI 1640 medium (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA), 100 IU/ml penicillin, and 100 µg/ml streptomycin (ThermoFisher Scientific) at 37°C in a humidified incubator supplemented with 5% CO2.
Cell proliferation assay. Cells (5×103/well) of quadruplet wells in 96-well plates (USA Scientific, Irvine, CA) were exposed to CIS (Selleck Chemicals, Houston, TX) or DZ-CIS for 24 hours, with the final concentration of solvent DMSO (Sigma-Aldrich) never exceeding 1%. Cells were then stained with 10% 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma-Aldrich) for 4 hours and decolorized by adding 100 µl of acidic 2-propanol. The extinction of supernatant was read at an absorbance maximum of 595 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA). For drug synergism testing, human cancer cells were exposed to exponentially increasing concentrations (0 µM to 64 µM) of everolimus or temsirolimus (Selleck Chemicals, Houston, TX) plus 4 µM DZ-CIS for 24 hours. The coefficient of drug interaction (CDI) was calculated based on the Combination Index Theorem by Chou T.C. and Talalay P, using a formula CDI = AB/(A + B) where CDI < 1 indicates synergism, CDI = 1 indicates additivity, and CDI > 1 indicates antagonism [26].
Apoptosis assay and western blotting. For the caspase activity assay, cells treated with DZ-CIS for 24 hours were measured for caspase 3/7 enzymatic activities by the Caspase-Glo® 3/7 Assay System (Promega, Madison, WI) with the recommended protocol by the manufacturer. Luminescence intensity was acquired using a LUMIstar Omega microplate luminometer (BMG Labtech, BioTek, Winooski, VT). For western blot analysis, our previously reported protocol was used [27]. Antibodies to poly ADP-ribose polymerase (PARP), caspase 3, and caspase 9 were from Cell Signaling Technology (Danvers, MA). Antibodies to β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX). Horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology) were used.
Fluorescence microscopy. Cells cultured in chamber slides (Nalge Nunc International, Rochester, NY) were stained with 4 µM DZ-CIS. After removing the staining medium, the cells were washed in phosphate buffered saline (PBS) 3 times. The slide was then counterstained with Hoechst 33342 (1 µg/ml, ThermoFisher Scientific) for 10 minutes, fixed in 4% paraformaldehyde (Sigma-Aldrich), and subjected to analysis of NIR fluorescence dye uptake with an Eclipse Ti-E confocal microscope (Nikon Corporation, Tokyo).
Tumor implant study. Animal studies were conducted in accordance with the protocol approved by the Institutional Animal Care and Use Committee (IACUC). Male 4- to 6-week-old NCr nude (Foxn1Nu/Foxn1Nu) mice (n = 10) and BALB/cJ mice (n = 15) purchased from the Jackson Laboratory (Bar Harbor, ME) were subjected to subcutaneous (s.c) tumor inoculation, for which 5 × 106 ACHN cells or 2 × 105 Renca cells in 50 µl of 50% Matrigel© (Corning, Corning, NY) were injected to each flank, producing 2 inoculations per mouse. The mice were then randomized into three groups and were treated intraperitoneally (i.p) twice a week with 10 mg/kg of DZ-CIS in 100 µl of PBS consisting of 5% DMSO and 15% PEG-40 (Sigma-Aldrich). Tumor growth was monitored by measuring the tumor dimensions with a caliper, and tumor volume was calculated using the formula: tumor volume = (length × width2)/2. The endpoint of the study was set as statistically significant tumor suppression in the treatment group. At the end of the studies s.c tumors, livers and kidneys were excised, fixed in 4% paraformaldehyde, and embedded in paraffin for histopathological analyses.
NIR fluorescence tumor imaging. Animals were subjected to imaging 72 hours after the final DZ-CIS i.p injection, with an IVIS Lumina XR Imaging System (PerkinElmer, Waltham, MA) equipped with fluorescent filter sets (excitation/emission, 745/820 nm), with automatic background fluorescence subtraction.
Immunohistochemical analysis (IHC). IHC analysis was performed as we previously reported [28]. Primary antibodies against cleaved caspase 3 (Cell Signaling Technology), PECAM-1 (CD31), Ki-67, and the M30 epitope of soluble caspase-cleaved keratin 18 were used (Sigma-Aldrich). Image acquisition was performed using a digital camera (Nikon Corporation).
Statistical analysis. All results were presented as mean ± standard error of mean (SEM) obtained from at least three independent tests. The normality of distribution was assessed using the Kolmogorov-Smirnov test. Parametric data were compared using Student's t-test. Nonparametric data were compared using Mann-Withney U test and SPSS software version 15 (IBM Corporation, Armonk, NY). The half maximal inhibitory concentration (IC50) values were calculated using a nonlinear regression method after normalizing the acquired values using GraphPad Prism version 5 Software (GraphPad Software, La Jolla, CA).