Oxali(IV)Fluors: Fluorescence Responsive Oxaliplatin(IV) Complexes Identify a Hypoxia-Dependent Reduction in Cancer Cells

Platinum(IV) anticancer agents have demonstrated the potential to overcome the limitations associated with the widely used Pt(II) chemotherapeutics, cisplatin, carboplatin, and oxaliplatin. In order to identify therapeutic scenarios where this type of chemotherapy can be applied, an improved understanding on the intracellular reduction of Pt(IV) complexes is needed. Here, we report the synthesis of two fluorescence responsive oxaliplatin(IV)(OxPt) complexes, OxaliRes and OxaliNap. Sodium ascorbate (NaAsc) was shown to reduce each OxPt(IV) complex resulting in increases in their respective fluorescence emission intensities at 585 and 545 nm. The incubation of each OxPt(IV) complex with a colorectal cancer cell line resulted in minimal changes to the respective fluorescence emission intensities. In contrast, the treatment of these cells with NaAsc showed a dose-dependent increase in fluorescence emission intensity. With this knowledge in hand, we tested the reducing potential of tumor hypoxia, where an oxygen-dependent bioreduction was observed for each OxPt(IV) complex with <0.1% O2 providing the greatest fluorescence signal. Clonogenic cell survival assays correlated with these observations demonstrating significant differences in toxicity between hypoxia (<0.1% O2) and normoxia (21% O2). To the best of our knowledge, this is the first report showing carbamate-functionalized OxPt(IV) complexes as potential hypoxia-activated prodrugs.


Materials and Chemicals
All chemicals and reagents were purchased commercially and were of analytical grade. Absorption spectra were obtained on a Jasco V-770 spectrophotometer and fluorescence spectra were obtained on a PerkinElmer LS55 Luminescence spectrometer using quartz cuvettes of 1 cm path length. Column chromatography was carried out using Merck® silica gel 60 under a positive pressure of nitrogen. Eluent ratios are reported by volume percentages. NMR spectra were recorded on a Bruker AVIII 400, Bruker NEO 600, Bruker AVII 500 (with cryoprobe) and Bruker AVIII 500 spectrometers. Chemical shifts are reported as δ values in ppm. Mass spectra were performed using Waters Micromass LCT and Bruker microTOF spectrometers.

Cell Culture
FLO-1 (Prof. Ricky Sharma, UCL), and HCT116 (Prof. Bert Vogelstein, Johns Hopkins Medicine) were cultured at 37°C, 5% CO2 in a humidified incubator. Cells were grown in DMEM medium supplemented with 10% FBS. All cell lines were routinely mycoplasma tested using a HEK-Blue™ detection kit (Invivogen) and found to be negative. Inhibitors/compounds used were Ascorbate (Sigma Aldrich).

Hypoxia exposure
Hypoxia treatments at <0.1% O2 were carried out in a Bactron II Chamber (Shel Laboratory). Oxygen concentrations were periodically validated using anaerobic oxygen indicator strips (ThermoFisher). Hypoxia treatments at 0.5-4% O2 were carried out in a M35 variable atmosphere workstation (Don Whitley Scientific).

Flow Cytometry
Cells were seeded onto glass 6 cm 2 dishes. Cells were treated with 10 μM of probe. Cells were scraped in 1 mL of 1x PBS into a 1.5 mL tube and fixed inside the hypoxia chamber with 4% PFA for 10 minutes. Samples were washed three times with 1 x PBS. Samples were run on a CytoFLEX (Beckman Coulter) and data analyzed using FlowJo software. Filter sets used for OxaliNap were FITC (ex 488 nm / em 525/40 nm) and for OxaliRes PC5.5 (ex 561 nm / em 690/50 nm). S4

Fluorescent Microscopy
Cells were seeded onto autoclaved cover slips (Menzel-Glaser) before treatment. Cells were fixed inside the hypoxia chamber in 4% PFA (w/v paraformaldehyde in PBS) for 10 minutes. Cells were mounted onto microscopy slides (Menzel Glaser) with ProLong™ Gold Antifade Mountant with DAPI to visual the nucleus (Invitrogen™). Cells were visualized with an LSM780 confocal microscope (Carl Zeiss Microscopy Ltd) at 63x magnification. At least 100 cells were counted per condition for quantification. Excitation and emission wavelengths used were: DAPI 370/470 nm, OxaliRes 571/584 nm and OxaliNap 491/516 nm.

Colony Survival Assay
Cells were seeded at low density in 6-well plates and incubated for 4 h at 37°C to adhere. Cells were incubated at indicated oxygen concentrations for 16 h. Colonies were returned to normoxic conditions, underwent a media change after 3 days and then left to form colonies for 7-10 days. Colonies were stained with 2% crystal violet diluted in 50% methanol and 20% ethanol and counted manually. Plating efficiency was calculated by dividing the number of colonies by the number of cells seeded. Surviving fraction was determined by dividing plating efficiency for treatment by the plating efficiency for the respective control.

MTT Assay
4,000 RKO cells per well were seeded in 96 well plates and allowed to adhere overnight. Cells were treated with the indicated probe and concentration for 3 days. Cells were exposed to hypoxia (<0.1% O2) for 16 hours. This was the same time point used for the clonogenic assays performed in HCT116 cells. Cells were incubated with 0.5 mg/mL MTT reagent (Sigma) in complete media for 3 hours at 37 °C protected from light. MTT was removed and formazan crystals were solubilized with 100 µL of DMSO for 15 minutes at 37 °C protected from light. Absorbance was read at 570 nm (Clariostar, BMG). Data are shown as percentage viability relative to untreated control.   Figure S3. Absorbance spectra of OxaliRes (15 µM) in PBS buffer (pH = 7.40) before (black) and after (red) addition of NaAsc (4 mM). The second measurement was recorded after 60 minutes incubation. Figure S4. Absorption spectra of OxaliNap (15 µM) in PBS buffer (pH = 7.40) before (black) and after (green) addition of NaAsc (4 mM). The second measurement was recorded after 60 minutes incubation.          Scale bar represents 20 µM. HCT116 cells were treated with OxaliRes (10 µM) for the times indicated in hypoxia followed by flow cytometry. HCT116 cells were treated with OxaliNap (10 µM) for the times indicated in hypoxia followed by flow cytometry. In B and C relative fluorescence is displayed as geometric mean intensity (MFI). Error bars represent SD. Significance compared to normoxic control. * p < 0.05, ** p < 0.01, and *** p < 0.001. n=3. Figure S21. HCT116 cells were treated with the indicated concentrations of Resorufin or Naphthalimide (4-NH2Nap) for 3 days. Hypoxic cells were exposed to <0.1% O2 for 16 hours with addition of either Resorufin or Naphthalimide. Cell survival was measured via clonogenic assay. A. Resorufin surviving fraction. B. Naphthalimide surviving fraction. C. Representative images of clonogenic assay. Error bars represent SD. S24 Figure S22. HCT116 cells were treated with the indicated concentrations of Oxaliplatin(OxPt(II)) or OxPt(OH)(OAc)(IV) (1) for 3 days. Hypoxic cells were exposed to <0.1% O2 for 16 hours with addition of either OxPt(II) or 1. Cell survival was measured via clonogenic assay. A. OxPt(II) surviving fraction. B. 1 surviving fraction. C. Representative images of clonogenic assay. Error bars represent SD. * p < 0.05, ** p < 0.01, and *** p < 0.001. n=3. S25 Figure S23. RKO cells were treated with the indicated concentrations of OxaliRes or OxaliNap for 3 days. Hypoxic cells were exposed to <0.1% O2 for 16 hours with addition of either OxaliRes or OxaliNap. Cell viability was measured via MTT assay. A. OxaliRes cell viability. B. OxaliNap cell viability. Data presented are percentage cell viability relative to untreated control. Error bars represent SD. * p < 0.05, ** p < 0.01, and *** p < 0.001. n=3. Figure S24. RKO cells were treated with the indicated concentrations of OxPt(II) or 1 for 3 days. Hypoxic cells were exposed to <0.1% O2 for 16 hours with addition of either OxPt(II) or 1. Cell viability was measured via MTT assay. A. OxPt(II) cell viability. B. 1 cell viability. Data presented are percentage cell viability relative to untreated control. Error bars represent SD. * p < 0.05, ** p < 0.01, and *** p < 0.001. n=3. Figure S25. RKO cells were treated with the indicated concentrations of 4-AmBn for 3 days. Hypoxic cells were exposed to <0.1% O2 for 16 hours with addition of 4-AmBn. Cell viability was measured via MTT assay. Data presented are percentage cell viability relative to untreated control. Error bars represent SD. * p < 0.05, ** p < 0.01, and *** p < 0.001. n=3.

OxPt(OH)(OAc) (1)
The title compound was synthesized following a previously reported literature procedure. 1 In brief, hydrogen peroxide -34.5-36.5 % in water (2 mL) was added to oxaliplatin (0.200 g, 0.503 mmol) in acetic acid (80 mL) and the reaction was stirred overnight at room temperature.