Subcellular Partitioning of Arsenic Trioxide Revealed by Label-Free Imaging

Abstract

Subcellular partitioning of therapeutic agents is highly relevant to their interactions with target molecules and drug efficacy, but studying subcellular partitioning is an enormous challenge. Here, we describe the application of nanoscale secondary ion mass spectrometry (NanoSIMS) analysis to define the subcellular pharmacokinetics of a cytotoxic chemotherapy drug, arsenic trioxide (ATO). We reasoned that defining the partitioning of ATO would yield valuable insights into the mechanisms underlying ATO efficacy. NanoSIMS imaging made it possible to define the intracellular fate of ATO in a label-free mannerand with high resolution and high sensitivity. Our studies of ATO-treated cells revealed that arsenic accumulates in the nucleolus. After prolonged ATO exposure, ∼40 nm arsenic- and sulfur-rich protein aggregates appeared in the cell nucleolus, nucleus, and membrane-free compartments in the cytoplasm, and our studies suggested that the partitioning of nanoscale aggregates could be relevant to cell survival. All-trans retinoic acid increased intracellular ATO levels and accelerated the nanoscale aggregate formation in the nucleolus. This study yielded fresh insights into the subcellular pharmacokinetics of an important cancer therapeutic agent and the potential impact of drug partitioning and pharmacokinetics on drug activity.

Tool to Resolve Distortions in Elemental and Isotopic Imaging

“…In practice, the ratio of different ion channels or composites of multiple secondary ions is often used to visualize and quantify “signals of interest”. For instance, they can determine the distribution and local enrichment of stable isotope-labeled molecules or biological therapeutics. ,,,,, We can also create composite images that display signals from molecules of interest along with signals that reveal structural or morphological information. ,,,,, These studies necessitate higher levels of image quality in single-channel images. To assess improvements with the NanoSIMS Stabilizer in these scenarios, we compared the results of channel ratio and channel-merging in two previously acquired samples (Figures c–d and S7).…”

“…The NanoSIMS instrument bombard samples with a primary ion beam (e.g., 133 Cs + ), and images are created, pixel by pixel, from secondary ions (e.g., 2 H – , 13 C – , 79 Br – , and 127 I – ) . The ability to visualize isotopic and elemental distributionsand the ability to quantify the signalshas been a boon for cell biology, microbiology, materials science, and geochemistry. − Correlating NanoSIMS images with fluorescence microscopy, electron microscopy, and atomic force microscopy has been useful for studies of metabolism, drug transport, and microbial interactions. Combining NanoSIMS imaging with other imaging modalities has made it possible to correlate chemical information with high-resolution ultrastructural morphology. , …”

Atomic spectrometry update: review of advances in elemental speciation

“…The application of nanoscale SIMS analysis to define the subcellular pharmacokinetics of the cytotoxic chemotherapy drug As trioxide has been reported. 58 The aim of the study was to gain insights into the mechanisms underlying ATO efficacy. It was found that ATO-treated cells revealed As accumulated in the nucleolus.…”

“…LC-MS/MS but are rarely quantied and this latter aspect has been addressed in a comprehensive paper from researchers in Pau. 81 In this work coconut water was spiked with Fe enriched with either 57 Fe or 58 Fe which was allowed to equilibrate with the naturally occurring citrate and malate ligands present in the sample for 12 hours. Subsequently, the spiked sample was diluted and separated on a HILIC column (150 × 2.1 mm, 2.6 mm Ø) with a gradient elution using 5 mmol L −1 ammonium acetate in water and 5 mmol L −1 ammonium acetate in 95% acetonitrile, each at a pH of 5.5, owing at 0.5 mL min −1 for 23 minutes.…”