Cell cycle-dependent uptake and cytotoxicity of arsenic-based drugs in single Leukemia cells

Abstract

Arsenic has long been used as therapeutic agents. Understanding the mechanism of action of arsenic-based drugs enables more effective arsenic drugs to be developed. Cell cycle has been known to play a critical role for cell division and growth. Herein, we establish a methodology to evaluate the uptake of two arsenic-based drugs (ATO and ZIO-101) across the cell cycle in single leukemia cells, i.e., NB4 and HL60, using a double thymidine block combined with time-resolved inductively coupled plasma mass spectrometry (ICPMS). We show that cells absorb maximal amounts of both ATO and ZIO-101 in G2/M phase and minimum in S phase, and such variation is less apparent for ZIO-101 than ATO (NB4-G2/M:S = 2.5:1 for ATO and 1.6:1 for ZIO-101). We subsequently demonstrate that AQP9, an ATO transporter, is highly expressed in the G1 phase (50.2-46.9%) and minimum value was observed in the S phase (27.6-24.6%); whereas xCT, a ZIO-101 transporter, is maximally expressed in the G2/M phase (74.8-76.1%) and minimally expressed in the G1 phases (55.4-59.8%). Different expression levels of AQP9 and xCT are only partially accountable for the observed differences in arsenic uptake across cell cycle, indicative of the presence of other importers for both ATO and ZIO-101. Furthermore, we show that the cytotoxicity of ATO and ZIO-101 on NB4 cells is also cell cycle dependent, with the highest cytotoxicity at S + G2/M phase and the lowest at G1 + S phase. Our studies provide the first evidence on cell cycle dependent uptake and cytotoxicity of arsenic-based drugs at single cell levels, may have general implications for precise evaluation of other anticancer drugs by considering cell cycle phase.