Synchrotron X‐Ray Fluorescence Nanoprobe Reveals Target Sites for Organo‐Osmium Complex in Human Ovarian Cancer Cells

Abstract A variety of transition metal complexes exhibit anticancer activity, but their target sites in cells need to be identified and mechanisms of action elucidated. Here, it was found that the sub‐cellular distribution of [Os(η6‐p‐cym)(Azpy‐NMe2)I]+ (p‐cym=p‐cymene, Azpy‐NMe2=2‐(p‐[dimethylamino]phenylazo)pyridine) (1), a promising drug candidate, can be mapped in human ovarian cancer cells at pharmacological concentrations using a synchrotron X‐ray fluorescence nanoprobe (SXRFN). SXRFN data for Os, Zn, Ca, and P, as well as TEM and ICP analysis of mitochondrial fractions suggest localization of Os in mitochondria and not in the nucleus, accompanied by mobilization of Ca from the endoplasmic reticulum, a signaling event for cell death. These data are consistent with the ability of 1 to induce rapid bursts of reactive oxygen species and especially superoxide formed in the first step of O2 reduction in mitochondria. Such metabolic targeting differs from the action of Pt drugs, offering promise for combatting Pt resistance, which is a current clinical problem.


S3
nm thick silicon nitride windows; Silson Ltd) were deposited on 24-well plates and irradiated with UV light for 20 min. Then, A2780 ovarian carcinoma cells were seeded (1 x 10 5 cells/well) and left to attach for 24 h in RPMI1640 medium. Samples were then treated with IC50 or 1 µM of 1. After 24 h, cells were washed twice with PBS and fixed in methanol at 253 K for 3 min and 30 s exactly, or with 2% PFA at 277 K for 20 min, and then rinsed 3x in PBS, followed by a short wash in ultrapure water (1-2 s).
The excess liquid was blotted using pin filter paper and the samples were allowed to dry at room temperature in a clean and dry environment.

Preparation of XRF and TEM samples (sections). A2780 ovarian carcinoma cells
were seeded in 100 mm petri dishes (5x10 6 cells/well) and left to attach for 24 h in RPMI1640 medium. Samples were then treated with 1 µM 1. After 24 h, cells were washed twice with PBS, and fixed for 20 min with 2% glutaraldehyde in sodium cacodylate buffer at pH 7.6 (Agar Scientific) for 1 h, with regular shaking. Then, cells were washed 3x with PBS, and transferred to Falcon tubes using a scrapper prior to centrifugation. Finally, cells were dehydrated with graded levels of ethanol (20-100% ethanol), and then infiltrated with 100% propylene oxide for 1 h followed by a 1:1 mixture of propylene oxide and EPON resin for 6 h. This was replaced with several changes of 100% resin over 18 h before curing for 24 h at 333 K. Blocks were trimmed and sectioned on a Leica Ultracut E ultramicrotome (Leica Microsystems). Sections 500 nm-(for XRF) or 100 nm-(TEM) thick were collected on appropriate sample holders for XRF (5x5 mm wide, 200 µm thick silicon frames with 1.5x1.5 mm-wide and 500 nm-thick silicon nitride windows; Silson Ltd) or TEM (400 mesh Cu grids; sections additionally stained with 2% uranyl acetate), and then imaged according to requirements.
TEM Imaging: Sections prepared for TEM were imaged on Jeol 2010F using a Gatan Ultrascan 4000 camera.
Image Analysis: Images were opened and processed using FIJI ImageJ package 4 with the EDFread plugin. Colocalisation and covariance were calculated selecting the whole cells as ROI and using the Coloc2 plugin.
Metal accumulation in isolated mitochondria. The accumulation of Os from complex 1 in the mitochondrial fraction of A2780 ovarian cells was investigated.
Briefly, 20 x 10 6 cells were seeded on a P145 cell culture dish. After 24 h of pre-S4 incubation in drug-free medium at 310 K, the complex was added to give final concentrations equal to IC50 (0.16 µM) and 1 µM, and a further 24 h of drug exposure was allowed. After this time, cells were washed treated with trypsin/EDTA, and cell pellets were collected. The isolation of mitochondria was carried out using the Mitochondrial isolation kit for profiling cultured cells from Sigma Aldrich (MITOISO2), according to the supplier's instructions. Isolated mitochondrial pellets were digested overnight in concentrated nitric acid (73%) at 353 K. The resulting solutions were diluted with a solution containing thiourea and ascorbic acid 2 to a final concentration of 5% v/v HNO3 (final concentration 10 mM thiourea, 0.1g/L ascorbic acid) and the amount of Os taken up by the cells was determined by ICP-MS as described above.
These experiments did not include any cell recovery time in drug-free medium; they were carried out in triplicate and the standard deviations were calculated. The amount of Os determined was normalised against the protein content of each sample determined using the Bradford assay.
Cell cycle analysis. Cells were seeded in a 6-well plate using 1 x 10 6 cells per well.
They were pre-incubated in drug-free medium at 310 K for 24 h, after which complex 1 was added at concentrations equal to IC50 (0.16 µM) and 1 µM. After 24 h of drug exposure, supernatants were removed by suction and cells were washed with PBS.
Finally, cells were harvested using trypsin/EDTA and fixed for 2 h using cold ethanol.