Velocidad de propagación

Human cell culture experiments were carried out under the guidance of Dr. Isolda Romero-Canelón with assistance from Dr. Anthony Knight, Mrs. Ji-Inn Song, Mrs. Sukhbinder Heer and Mrs. Hannah E. Bridgewater at the School of Life Sciences and Department of Engineering, University of Warwick.8

A2780, SK-OV-3 and SW-626 human ovarian carcinomas, A2780cis cisplatin- resistant human ovarian carcinoma, A549 human lung carcinoma, HCT116 human colorectal carcinoma, HCT116-p21-/- P21-knockout colorectal carcinoma, HCT116- p53-/- P53-knockout colorectal carcinoma, HEPG2 human hepatocellular carcinoma, OE19 human esophageal carcinoma, MCF7 human breast adenocarcinoma, MRC5 human fetal fibroblasts, and PC3 human prostate carcinoma cells were purchased from the European Collection of Cell Cultures (ECACC). HOF human ovarian fibroblasts were purchased from ScienCell. Mycoplasma-free status was confirmed every 6 months by Public Health England. Cell culture media (RPMI-1640, DMEM, McCoy 5A), phosphate buffered saline, L-glutamine, penicillin / streptomycin, fetal bovine serum and trypsin / EDTA were purchased from PAA Laboratories and kindly prepared by technicians at the School of Life Sciences (University of Warwick, UK). RPMI-1640 media (without phenol red) and phosphate buffered saline were purchased from Lonza. Fibroblast medium was purchased from ScienCell Research Laboratories.

Cell maintenance: Defrosting

Ampoule(s) were rapidly defrosted in a water bath (310 K). Cells were transferred to falcon tubes and centrifuged (5 min, 1000 rpm, 295 K) to remove supernatant freezing medium. Pellets were re-suspended in media (5 mL) and transferred to a T25 flask.

Cell maintenance: Passaging

A2780, MRC5 and PC3 cells were grown in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% fetal bovine serum, 1% glutamine (2 mM) and 1% penicillin / streptomycin. A549, HEPG2 and MCF7 cells were grown in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1% glutamine (2 mM) and 1% penicillin / streptomycin. HCT116 cells were grown in McCoy’s Modified 5A medium supplemented with 10% fetal bovine serum, 1% glutamine (2 mM) and 1% penicillin / streptomycin. HOF cells were grown in fibroblast growth medium supplemented with 5% fetal bovine serum, 1% penicillin / streptomycin and 1% fibroblast growth factor serum.

Cells were grown as adherent monolayers in 75 cm2 _{culture flasks (surface-treated,}

fitted with filters) at 310 K in a 5% CO2 humidified atmosphere until 80-90%

confluence was achieved.

Upon reaching 80-90% confluence, cells were washed with PBS (5 mL). 0.25% trypsin / EDTA (2 mL) was added, and the flask was placed in an incubator (310 K) for 3-5 min. Once cells were observed to be detaching from the bottom of the flask, the solution was diluted with the corresponding medium to obtain a single cell suspension. A suitable volume (typically 1:4) was transferred into a T75 (75 cm2_{) cell}

culture flask containing fresh media and placed in an incubator (310 K).

When passaging HOF fibroblasts, trypsin / EDTA activity was quenched with foetal bovine serum (FBS: 5 ml, 5 min). The supernatant containing trypsin was removed after centrifugation (5 min, 1000 rpm, 295 K) and the pellet re-suspended in (supplemented) fibroblast growth medium. Primary cell lines (MRC5 and HOF) were not used after passage 5 due to cells entering senescence.

Sulforhodamine B (SRB) colorimetric assay

The sulforhodamine B (SRB) assay can be used to determine cell density based on the protein concentration per sample.9 _{Sulforhodamine B (Figure 2.2) electrostatically}

binds basic amino acid residues present in viable cells under mild acidic conditions. Release under mild basic conditions is quantitative, and dye absorbance is linear with protein concentration.9

An alternative assay, the MTT assay (MTT = 3-(4,5)-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide), quantifies the number of viable cells by reducing colourless MTT into a dark blue (formazan) species, requiring metabolic processes in live cells.9, 10 Similarly to the SRB assay, the production of formazan is linear with the number of cells.10 _{Because of the metabolic dependence, the MTT assay can}

distinguish between live and dead cells,10 unlike the SRB assay.9 Reduction of MTT may however be influenced by certain reagents, and requires optimisation for some cell lines due to the metabolic dependence.9 Conversely, the SRB assay has been found to provide better linearity and sensitivity, and is compatible with a range of cell lines, including those investigated in this thesis.11

In vitro growth inhibition assay

Cells were washed with PBS and detached using 2 mM trypsin / EDTA (2 mL) with incubation for 5 min (310 K). A single cell suspension was obtained, and counted using a haemocytometer. The suspension was diluted to seed a 96-well plate with 5000 cells per well using 150 µL (with the exception of SK-OV3 which were seeded at a density of 10’000 cells per well using 150 µL) which were left to incubate in drug- free media for 48 h (310 K). 100 µM drug stock solutions were prepared (0.5% DMSO, 95% culture medium). Serial dilutions were performed to prepare 6 drug concentrations (typically 0.01-100 µM). Solutions (200 µL) were added to the wells in triplicate, alongside positive (CDDP) and negative control samples. A drug exposure period of 24 h was allowed (310 K). Supernatants were removed, and the wells washed with PBS (100 µL). A recovery period of 72h was allowed in drug-free medium (200 µL, 310 K). Aliquots of stock solutions (ca. 100 µM) were analysed by ICP-OES to determine the exact metal concentration.

The SRB assay was used to determine cell viability. Cold 50% trifluoroacetic acid (TCA, 50 µL) was added to each well for 1 h (277 K). The plate was washed with slow running water and dried. 0.4% SRB dye (50 µL, prepared in 1% acetic acid) was added for 30 mins (298 K). Excess dye was removed with 1% acetic acid, and the plate was dried. 10 mM Tris base solution (200 µL) was added to each well for 1 h (298 K). Absorbance was read with a BioRad iMark microplate reader. Survival percentages were obtained relative to negative control wells. Data was plotted as survival percentage versus the logarithm of drug concentration using OriginPro 2016. IC50

(half-maximal inhibitory) concentrations were determined as the inflection point of the fitted sigmoidal curve. Experiments were performed as duplicates of triplicates

Metal accumulation in cancer cells

Cells were grown as described previously.8 The medium was removed, cells were washed with phosphate-buffered saline (PBS) and detached using 2 mL of trypsin / EDTA (2 mM, 310 K, 5 min). The solution was diluted using the corresponding medium and mixed to achieve a single cell suspension, and cell density determined using a plastic disposable haemocytometer. The suspension was diluted to seed 4 × 106 cells per well in a P100 petri dish (10 mL).

After 24 h of pre-incubation, solutions of the complexes (DMSO not exceeding 0.5%) and negative controls were added in triplicate to give final (equipotent) concentrations equal to the IC50 determined in A2780 (unless stated otherwise). 24 h drug exposure

was allowed. The experiments did not include any recovery time in drug-free media stated otherwise. Cells were incubated at 310 K unless stated otherwise.

The supernatant was removed. Cells were washed with PBS and detached with trypsin- EDTA. The number of cells in each sample was determined using a haemocytometer. Cells were collected by centrifugation (5 min, 1000 rpm), re-suspended in PBS (1 mL) and transferred to glass microwave vials. After further centrifugation (5 min, 1000 rpm), the supernatant was removed by suction to obtain whole cell pellets. Freshly distilled 72% v/v nitric acid (500 µL) was added to the pellets which were digested using a CEM Discovery microwave (453 K, 3 min, 300W, 450 psi). Solutions were diluted using doubly-distilled milliQ water containing thiourea (10 mM) and ascorbic acid (100 mg/L) to achieve a final acid concentration of 3.6% v/v HNO3 (final working

volume = 10 mL). The amount of metal taken up by the cells was determined by ICP- MS. The experiments were all carried out in triplicate and the s.d. were calculated. Final results were reported as ng Os / Ir / Pt × 106 cells.

2.4

References

1. J. Tönnemann, J. Risse, Z. Grote, R. Scopelliti and K. Severin, Eur. J. Inorg. Chem., 2013, 2013, 4558-4562.

2. J. P. C. Coverdale, C. Sanchez-Cano, G. J. Clarkson, R. Soni, M. Wills and P. J. Sadler, Chem. Eur. J., 2015, 21, 8043-8046.

3. M. Liu, X. Mao, C. Ye, H. Huang, J. K. Nicholson and J. C. Lindon, J. Magn. Reson., 1998, 132, 125-129.

4. O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard and H. Puschmann, J. Appl. Crystallogr., 2009, 42, 339-341.

5. G. Sheldrick, Acta Crystallogr., Sect. A: Found. Crystallogr., 2008, 64, 112- 122.

6. C. Venzago, M. Popp, J. Kovac and A. Kunkel, J. Anal. At. Spectrom., 2013,

28, 1125-1129.

7. J. J. Soldevila-Barreda, Doctor of Philosphy, University of Warwick, 2014. 8. I. Romero-Canelón, Doctor of Philosphy, University of Warwick, 2012. 9. V. Vichai and K. Kirtikara, Nat. Protocols, 2006, 1, 1112-1116.

10. T. Mosmann, J. Immunol. Methods, 1983, 65, 55-63.

11. Y. P. Keepers, P. E. Pizao, G. J. Peters, J. van Ark-Otte, B. Winograd and H. M. Pinedo, Eur. J. Cancer, 1991, 27, 897-900.

Chapter 3

Asymmetric catalytic osmium