Differences in cisplatin distribution in sensitive and resistant ovarian cancer cells: a TEM/NanoSIMS study.

Cisplatin is a widely used anti-cancer drug, but its effect is often limited by acquired resistance to the compound during treatment. Here, we use a combination of transmission electron microscopy (TEM) and nanoscale-secondary ion mass spectrometry (NanoSIMS) to reveal differences between cisplatin uptake in human ovarian cancers cells, which are known to be susceptible to acquired resistance to cisplatin. Both cisplatin sensitive and resistant cell lines were studied, revealing markedly less cisplatin in the resistant cell line. In cisplatin sensitive cells, Pt was seen to distribute diffusely in the cells with hotspots in the nucleolus, mitochondria, and autophagosomes. Inductively coupled plasma mass spectrometry (ICP-MS) was used to validate the NanoSIMS results.

The Differential Distribution of RAPTA-T in Non-Invasive and Invasive Breast Cancer Cells Correlates with Its Anti-Invasive and Anti-Metastatic Effects.

Nanoscale secondary ion mass spectrometry (NanoSIMS) combined with transmission electron microscopy (TEM) can be a powerful approach to visualize the exact distribution of drugs at the sub-cellular level. In this work, we exploit this approach to identify the distribution and localisation of the organometallic ruthenium(II)-arene drug Ru(η6-C6H5Me)(pta)Cl2, termed RAPTA-T, in MDA-MB-231 and MCF-7 human breast cancer cells. These cell lines have been chosen because the former cell lines are highly invasive and resistant to most chemotherapeutic agents and the latter ones are very sensitive to hormonal-based therapies. In the MDA-MB-231 cells, RAPTA-T was found to predominantly localise on the cell membrane and to a lesser extent in the nucleolus. These findings are consistent with the previously reported anti-metastatic properties of RAPTA-T and the observation that once internalized RAPTA-T is associated with chromatin. RAPTA-T shows a lack of membrane accumulation on the non-invasive MCF-7 cells, which correlates well with its selective anti-metastatic properties on invasive cell lines.

Biochemical and biophysical characterization of ruthenation of BRCA1 RING protein by RAPTA complexes and its E3 ubiquitin ligase activity.

RAPTA compounds, ([Ru(η6-arene)(PTA)Cl2], PTA = 1,3,5-triaza-7-phosphaadamantane), have been reported to overcome drug resistance in cisplatin resistant cells. However, the exact mechanism of these complexes is still largely unexplored. In this study, the interaction of some RAPTA compounds with the N-terminal fragment of the BRCA1 RING domain protein was investigated. The binding of the RAPTA compounds to the BRCA1 protein resulted in a release of Zn2+ ions in a dose and time dependent manner, as well as thermal alteration of ruthenated-BRCA1 proteins. Electron Transfer Dissociation (ETD) fragmentation mass spectrometry revealed the preferential binding sites of the RAPTA complexes on the BRCA1 zinc finger RING domain at a similar short peptide stretch, Cys24Lys25Phe26Cys27Met28Leu29 and Lys35 (residues 44-49 and 55 on full length BRCA1). Changes in the conformation and binding constants of ruthenium-BRCA1 adducts were established, resulting in inactivation of the RING heterodimer BRCA1/BARD1-mediated E3 ubiquitin ligase function. These findings could provide mechanistic insight into the mode of action of RAPTA complexes for on tested BRCA1 model protein.

Expression proteomics study to determine metallodrug targets and optimal drug combinations.

The emerging technique termed functional identification of target by expression proteomics (FITExP) has been shown to identify the key protein targets of anti-cancer drugs. Here, we use this approach to elucidate the proteins involved in the mechanism of action of two ruthenium(II)-based anti-cancer compounds, RAPTA-T and RAPTA-EA in breast cancer cells, revealing significant differences in the proteins upregulated. RAPTA-T causes upregulation of multiple proteins suggesting a broad mechanism of action involving suppression of both metastasis and tumorigenicity. RAPTA-EA bearing a GST inhibiting ethacrynic acid moiety, causes upregulation of mainly oxidative stress related proteins. The approach used in this work could be applied to the prediction of effective drug combinations to test in cancer chemotherapy clinical trials.

Application of imaging mass spectrometry approaches to facilitate metal-based anticancer drug research.

Mass spectrometry imaging is being increasingly used in metal-based anticancer drug development to study elemental and/or molecular drug distributions in different biological systems. The main analytical tools employed are SIMS (especially nanoSIMS), LA-ICP-MSI and MALDI-MSI as well as a combination of complementary imaging techniques. Main challenges are appropriate sample preparation methods, reliable and validated quantification strategies and a trade-off between sensitivity and spatial resolution. So far, research has mostly focused on the development of analytical methods for imaging with the long term goal to study drug uptake into tumor tissue and toxicity affected organs and to identify cellular targets of metal-based drugs. In this review we cover the technological features of the mass spectrometry imaging methods used and give an overview of the applications in metal-based anticancer drug research as well as some future perspectives.

NanoSIMS analysis of an isotopically labelled organometallic ruthenium(II) drug to probe its distribution and state in vitro.

The in vitro inter- and intra-cellular distribution of an isotopically labelled ruthenium(II)-arene (RAPTA) anti-metastatic compound in human ovarian cancer cells was imaged using nano-scale secondary ion mass spectrometry (NanoSIMS). Ultra-high resolution isotopic images of (13)C, (15)N, and Ru indicate that the phosphine ligand remains coordinated to the ruthenium(II) ion whereas the arene detaches. The complex localizes mainly on the membrane or at the interface between cells which correlates with its anti-metastatic effects.

Lyophilization of cholesterol-free PEGylated liposomes and its impact on drug loading by passive equilibration.

The obstacles in translating liposome formulations into marketable products could be attributed to their physical instabilities upon long-term storage as aqueous dispersions. Lyophilization is the most commonly used technique to improve physical stability of liposomes. The development of stable, lyophilized liposomes is focused primarily on the cholesterol-containing liposomes or pure phosphatidylcholine-based liposomes, with minimal studies on cholesterol-free, pegylated (CF-PEG) liposomes which have emerged as an important class of liposome drug carriers. Hence, it is our interest to investigate the effect of lyophilization on CF-PEG liposomes, and specifically, on drug loading via the passive equilibration method. Three different sugar cryoprotectants were used at two different sugar-to-lipid molar ratios (S/L). Our results demonstrated that CF-PEG liposomes lyophilized with sucrose at S/L=5:1 yielded the best cryoprotective effect, as characterized by size, polydispersity indices, and microscopic examination upon liposome reconstitution. The lyophilized liposomes had low water content of 2.59 ± 0.18%. Of note, lyophilized CF-PEG liposomes exhibited two-fold increase in drug content when carboplatin was loaded via the passive equilibration method, and the in vitro drug release profile of these liposomes were not different from that of the non-lyophilized counterparts. Taken together, we envisioned that a stable, lyophilized empty CF-PEG liposome system could be coupled to hydrophilic drug loading via the passive equilibration method to produce a liposomal drug kit product.