Cell cycleâdependent uptake and cytotoxicity of arsenic-based drugs

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Cell cycle–dependent uptake and cytotoxicity of arsenic-based drugs in single leukemia cells Ying Zhou, Haibo Wang, Eric Tse, Hongyan Li, and Hongzhe Sun Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02444 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 7, 2018

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Analytical Chemistry

Cell cycle–dependent uptake and cytotoxicity of arsenic-based drugs in single leukemia cells Ying Zhou1, Haibo Wang1, Eric Tse2, Hongyan Li1, Hongzhe Sun1,* 1

Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, P. R. China; 2Department of Medcine, Queen Mary Hospital, The University of Hong Kong, Hong Kong, P.R. China * Corresponding Author: Hongzhe Sun. Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, P. R. China. E-mail: [email protected]

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 ICP-MS. 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 G1 phase (50.2% - 46.9%) and minimum value was observed in S phase (27.6%-24.6%); whereas xCT, a ZIO-101 transporter, is maximally expressed in G2/M phase (74.8% - 76.1%), and minimally expressed in 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 arsenicbased drugs at single cell levels, may have general implications for precise evaluation of other anticancer drugs by considering cell cycle phase.

Arsenic has long been used as therapeutic agents both in Traditional Chinese Medicine and Western Medicine1. In the 1970s, Chinese researchers found that arsenic trioxide (ATO) exhibited exquisite efficiency for the treatment of acute promyelocytic leukemia (APL)2 through induction of apoptosis, stimulation of differentiation, inhibition of proliferation, inhibition of angiogenesis and so on3. Subsequently, it was approved as a first-line drug for the treatment of APL by the US FDA4. Considering that ATO at clinically achievable levels exhibits reduced antitumor effects for non-APL malignancies due to its systemic toxicities, many organic arsenical compounds, including phenylarsine oxide5, 4-(N-(Sglutathionylacetyl) amino) phenylarsenoxide (GSAO)6 and Sdimethylarsino-thiosuccinic acid (MER1)7, have been developed which show better antitumor efforts in comparison with inorganic arsenicals. ZIO-101 (N-[S-(dimethylarsino)-N-Lgamma-glutamyl-L-cysteinyl]-glycine; S-dimethylarsinoglutathione) is a good representative of organic arsenical compounds, and was well-tolerated in phase I/II clinical trials with both oral and intravenous administration8. It is highly active against a large panel of tumor cell lines including APL, multiple myeloma, B lymphoblastoid and acute lymphocytic leukemia cell lines9,10. Given the success of arsenic-based drugs, further exploration of their anticancer mechanism will help us better understand their therapeutic effects, enabling the development of more effective arsenic drugs.

The cell cycle is a ubiquitous process that leads to cell division and replication11. Generally, the cell cycle could be divided into four distinct phases including G1 (preparing for DNA synthesis), S (DNA synthesis), G2 (preparing for mitosis) and M phase (mitosis). Numerous regulatory proteins are involved in the cell cycle process to direct cells from specific phase to the production of two daughter cells12. It was originally believed that cancer cells are exquisitely sensitive to cell-cycle perturbations due to their rapid replication compared with normal cells13. Most anticancer drugs exert their therapeutic effects through arresting cells at specific cell phase then inducing apoptosis11. Considering the dynamic difference in different cell cycle phase, numbers of cell-cycle-specific chemotherapy drugs14-18 were developed to kill cancer cells in a specific cell phase. Therefore, the combination of chemotherapy and synchronization agents of cell cycle may be an alternative strategy to improve the efficacy of anticancer drugs. Drug uptake is the first step of drug action in cancer cells which is related to cancer burden19. Intracellular active drug contents are also key factors of cell sensitivity and resistance towards certain anticancer drugs. Understanding the cellular uptake of arsenic-based drugs allows the development of new strategies and therapies to increase sensitivity of cancer cells towards arsenicals. During each cell cycle phase, the cell performs different biological functions, which may change the drug absorption rate of a cell20. Considering the importance of

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cell cycle in cancer therapy, there is a need to evaluate how the state of cells affects the uptake and cytotoxicity of a drug. Most studies were usually performed on large cell populations, which gave rise to average results for biological events. Since individual cells can differ dramatically in size, protein levels and expressed RNA transcripts, single cell analysis is needed to accurately characterize samples with high cellular heterogeneity21. Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful technique for trace elemental analysis based on counting the number of atoms in a sample22. Owing to multiple advantages including low matrix effect, high sensitivity and wide dynamic range, ICP-MS had been utilized to monitor intracellular element contents at the singlecell level using time-resolved mode23. Our previous report24 has firstly applied this method to track the uptake of ATO at single leukemia cells, providing more precise information on intracellular arsenic contents. To further explore the intrinsic impact of different cell cycle phase on arsenic uptake, timeresolved ICP-MS was introduced to monitor intracellular arsenic contents at single leukemia cells after treatment with two arsenic-based drugs (ATO and ZIO-101) at certain cell cycle phase. It was reported that different transporter systems are involved in the uptake of ATO and ZIO-101. ATO is imported into cells via glucose transporters and aquaglyceroporins25, while ZIO-101 enters the cells via the cysteine/cysteine cellular import system26. To dissect the relationship of drug uptake and expression levels of corresponding transporter proteins, the expression levels of aquaglyceroporin 9 (AQP 9) and xCT, which are key regulators to facilitate ATO and ZIO-101 transport26,27, were detected using flow cytometry. As critical therapeutic efficient indicator, the cytotoxicity of ATO and ZIO-101 at different stages was also evaluated after treatment with ATO and ZIO-101 at certain specific phase, which may provide a new strategy to evaluate the efficacy of arseniccontaining drugs.

EXPERIMENTAL SECTION Materials and Reagents. NB4 and HL60 cells were kindly provided by Dr TSE Wai Choi (Department of Medicine, Li Ka Shing Faculty of Medicine, the University of Hong Kong). AQP 9 antibody with ALEXA FLUOR® 488 conjugate and Rabbit IgG Isotype Control were from Bioss. xCT antibody with FITC conjugate were from Biorbyt. The following chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA): Arsenic(III) trioxide (purity, ≥99.0%); Thymidine; Propidium Iodide; paraformaldehyde; RNase. ZIO-101 was bought from TRC Canada. RPMI-1640 medium, Fetal bovine serum (FBS), Penicillin Streptomycin Sol were purchased from Life Technologies. The deionized water (18.2 MΩ.cm) was used throughout the experiments. Apparatus. A quadrupole-based inductively coupled plasma mass spectrometer (ICP-MS) (Agilent 7700x, Agilent Technologies, CA, USA), equipped with a glass concentric nebulizer and an impact spray chamber. BD FACS Canto II Analyzer. Cell cycle synchronization using a double thymidine block. Two different leukemia cell lines, NB4 and HL60 were used in this work. Cells were cultured in RPMI1640 medium supplemented with 10% FBS and 1% penicillin−streptomycin

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solution (37 °C, 5% CO2). Cells were grown in culture medium to approximately 40% confluency and then Thymidine was added into culture medium at a final concentration of 2 mM and incubated for 14 hrs. The medium was removed and washed with 3×PBS. Fresh cell culture medium was added and incubated for 9 hrs and 2 mM of thymidine blocking solution were added for the second time, incubated for additional 14 hrs. The medium was removed and washed with 3×PBS and fresh culture medium was added. The cells were harvested at different time points for flow cytometry analysis. Cell debris was excluded from analysis by appropriately raising the forward scatter threshold. The cell fractions in sub-G1- (apoptotic cells), G1-, S-, and G2/M-phases were quantified with FlowJo X 10.0.7 software after exclusion of cell debris and doublets. Time-resolved ICP-MS for arsenic measurement. To monitor arsenic uptake, NB4 and HL60 cells were treated with 15 µΜ ATO and ZIO-101 for 1.5 hrs at different time points after release from the double thymidine block. The culturing solution of 3 mL of cell suspension was removed by centrifugation (1000 × g and 5 mins) for 3 times and resuspended in ice-cold phosphate-buffered saline (PBS of 1.9 mM NaH2PO4, 8.1 mM Na2HPO4, and 154 mM NaCl) at pH 7.4. Prior to ICPMS measurement, the pellet was resuspended in PBS to a cell density of 106/mL. The numbers of cells were counted by haemocytometer under an optical microscope. The cell suspension was sprayed into aerosols using microconcentric nebulizer and introduced into the ICP directly for time-resolved ICP-MS measurements. The experimental details of the single cell analysis were described in our previous report28. Briefly, the sample (leukemia cell suspensions) was placed in a 500 µL syringe for injection into the nebulizer. A micro-syringe pump (SPLab01. Baoding Shenchen precision pump Co., Ltd., Hebei, China) was used to precisely control the flow rate at 0.03 mL/min. A microconcentric nebulizer and a single-pass spray chamber were used for aerosol generation. During each integration time (5 ms), at most one cell was allowed to be analyzed after precisely controlling the cell density and sampling rate. Only one isotope was monitored in each measurement. Helium gas mode was applied for 75As detection to minimize polyatomic interference from 40Ar35Cl on 75As detection. Before each experiment, the ICP-MS was tuned using an aqueous multi-element standard solution (10 mg/L each of Li, Y, Co, Ce and Tl) for consistent sensitivity (7Li, 89Y and 205Tl) and minimum levels of doubly-charged ions and oxide species of 140 Ce. The measurement duration was typically 60 s. All measurements were performed in triplicate. Data were analyzed offline using Origin (Origin version 8) and Microsoft Excel (Microsoft Excel 2013). The error bars in all figures represent the standard deviation values for triplicate experiments to demonstrate the replication and reliability of all data which are 1.13 times that of confidence intervals for 95% confidence level29. Flow cytometry analysis of cell cycle with propidium iodide DNA staining. Cells were harvested and washed with PBS for 3 times. And cold 70% ethanol was added dropwise to the cell pellet while vortexing. Cells were fixed overnight at 4°C and washed with PBS for 2 times, then centrifuged at 3500 rpm and the supernatant was discarded. RNase A (100 µg/ml) and propidium iodide (50 µg/ml) were added in PBS solution. The samples were analyzed by flow cytometry in PI/RNaseA solution.


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Analytical Chemistry Analysis of AQP9 and xCT expression by flow cytometry. The cells were harvested and washed and the total numbers of cell were determined. The cells were resuspended to approximately 1×106 cells/mL in PBS and 4% paraformaldehyde were added in PBS, and fixed for 10 mins in a 37°C water bath. The cells were washed twice with PBS containing 0.5% BSA. Resuspend 1×106 cells in 100 µL PBS containing 0.5% BSA and ALEXA FLUOR® 488 conjugated AQP 9 and FITC conjugated xCT antibodies were added and incubated for 1 h at room temperature. The negative control samples were stained with IgG Isotype. The cells were washed twice with PBS containing 0.5% BSA, resuspended in 0.5mL PBS and analyzed on a flow cytometer.

RESULTS AND DISCUSSION Experimental design. The workflow of monitoring arsenic uptake across the cell cycle in single leukemia cells by timeresolved ICP-MS is shown in Scheme 1. The cell culture media generally comprise of cells at different phases of the cell cycle, which simultaneously undergo progression and cell division. To evaluate whether the different states of cells affect the uptake of arsenic-based drugs, the cells were synchronized by a double thymidine block (an inhibitor of DNA synthesis), which could arrest cells in early S phase30. The cells were collected at different time points after being synchronized by the double thymidine to obtain large cell populations at certain phases of the cell cycle, then incubated with arsenic compounds. The arsenic contents in individual leukemia cells were then measured by time-resolved ICP-MS after precisely controlling the sampling rate and integration time as reported previously24. The log values of spike intensity were plotted as a histogram and the maximum of the histograms was taken as the arsenic contents in individual cells. The arsenic contents of synchronized leukemia cells at different phases of the cell cycle were compared.

kemia cells at specific stages of the cell cycle. As shown in Figure 1, significant different histograms of the propidium iodide (PI) fluorescence profiles could be observed for asynchronous NB4/HL60 (Figure 1A) and synchronized cells (Figure 1B) collected at different time points after the double thymidine block. The percentages of cells in each cell cycle phase were calculated (Figure 1C). Both NB4 and HL60 cells were synchronized for around 7-10 hrs after release from the double thymidine block. After synchronization, a pronounced enrichment of cells in G1 phase was achieved, i.e., 85% for NB4 cells and 87% for HL60 cells, in comparison with asynchronous cells, 35.3% and 51.4% of cells were in G1 phases for NB4 and HL60 cells respectively. Over 65% of the cells were arrested in G1 phase for both NB4 and HL60 cells after release from the double thymidine block from 0 to 2 hrs. An increase of cell proportions in S phase was observed and maintained for the next 3 hrs. By 6 hrs, most of the cells were in G2/M phase for further 3 hrs. The cells became less synchronized after 11 hrs. The results show that the cells could maintain synchronized and go successively through four phases (G1-S-G2/M) in around 9 hrs after the double thymidine block. Cells at different cell cycle phase were then collected at different time points for further studies of drug uptake.

Scheme 1. Workflow on monitoring the uptake of ATO and ZIO-101 in single leukemia cells across the cell cycle by timeresolved ICP-MS. To further uncover the relationship between the amounts of drug uptake and expression levels of corresponding transporter proteins as well as the cytotoxicity of ATO and ZIO-101 at different stages of the cell cycle, flow cytometry was introduced to monitor the expression levels of two transporter proteins, AQP9 and xCT, which were reported to be the key regulators to facilitate the transport of ATO and ZIO-101 respectively. The apoptosis ratios of cells at certain phases of the cell cycle were also analyzed by flow cytometry to evaluate the cytotoxicity of ATO and ZIO-101 in single leukemia cells. Cell cycle synchronization by a double thymidine block. Cell growth is a complex and dynamic process. Under normal conditions, cell cultures are a mixture of cells at different stages of the cell cycle. To arrest cells in a certain cell cycle phase, cell synchronization is needed. In this work, a double thymidine block synchronization method was utilized to enrich leu-

Figure 1. FACS analysis of synchronized NB4/HL60 cells by a double thymidine block. (A) PI fluorescence profiles of asynchronous NB4 and HL60 cells; (B) PI fluorescence profiles of synchronous NB4 (Left) and HL60 (Right) cells at different time points (0, 1, 2, 3, 4, 5, 6, 7, 10 hrs) after release from the double thymidine block; (C) The proportions of cells in each phase of the cell cycle.

Arsenic uptake in different cell cycle phase. Intracellular accumulation of arsenic is critical for therapeutic effects of As-based drugs. Deeper understanding of mechanism of drug uptake will help us to better evaluate the therapeutic efficiency. The uptake of ATO and ZIO-101 at different cell cycle phases was monitored based on time-resolved ICP-MS. After release from the double thymidine block, both NB4 and HL60 cells



were allowed to grow in cell culture medium. Cells were then treated with 15 µM of ATO or ZIO-101 for 1.5 hrs at 0, 1.5, 3, 4.5, 6 and 7.5 hrs after thymidine treatment, corresponding to G1, G1, S, S, G2/M and G2/M phases respectively. The uptake of arsenic in NB4 and HL60 cells at different cell cycle phase was monitored by time-resolved ICP-MS. The detailed operation conditions were described in a previous report24. Generally, helium collision cell was used to minimize polyatomic interference from 40Ar35Cl for 75As detection. The suspensions of ATO and ZIO-101 treated leukemia cells were injected and sprayed in sequence, at most one cell was introduced into the ICP over the duration of the residence time of ICP upon the sampling rate and integration time being optimized so that only one cell was analyzed at one time. ICP-MS spikes, corresponding to arsenic signals of individual cells, appeared in the mass spectrum. The frequency of the spikes directly reflects the numbers of cells and the intensity of spikes (counts per second) is proportional to the concentration of arsenic within one cell. The typical 75As profiles of ATO/ZIO-101 treated NB4/HL60 cells are shown in Figure 2A, B (upper) and the 75As profiles of ATO/ZIO-101 treated NB4/HL60 cells at different time points corresponding to different cell cycle phase are shown in Figure S1. The spike intensity was plotted as histograms (Figure 2A, B (lower)), showing that the distribution of arsenic spike intensity as log values approximately follows a Gaussian distribution. The spike intensity distribution correlated with the distribution of arsenic contents in single leukemia cells. The maximum of the histograms was taken as the average arsenic contents of the cells.

Figure 2. 75As profiles and the corresponding distribution diagrams of 75As spike intensities of NB4 and HL60 cells treated by ATO (A, B) and ZIO-101(C, D) at 0 h after release from the double thymidine block in time-resolved ICP-MS.

The 75As spike intensities were further analyzed to calculate the arsenic contents in single leukemia cells. The intracellular arsenic concentrations were normalized to the maximum value

of each group. The relative intracellular arsenic concentrations are shown in Figure 3. The intracellular arsenic contents at different cell cycle phases were found to be different after treatment of both NB4 and HL60 cells with either ATO or ZIO-101. Treatment of cells with ATO resulted in most arsenic absorbed in G2/M phase, followed by G1 phase. The least arsenic contents were found in S phase, which is almost 50% lower than that in G2/M phase (Figure 3A). While treatment of cells with ZIO-101 led to similar intracellular arsenic levels when most cells were in G1 and G2/M phase. However, the arsenic contents decreased by 30-40% when the cells moved into the S phase (Figure 3B). Compared with ATO, the variation of ZIO-101 uptake in different cell cycle was less apparent (NB4-G2/M:S=2.5:1 (7.5 h: 3h) for ATO and 1.6 :1 (6 h: 4.5 h) for ZIO-101), suggesting that ATO and ZIO-101 share different transporter in leukemia cells. Subsequently we compared the intracellular arsenic contents in single NB4 (Figure 3C) and HL60 (Figure 3D) cells after treatment with ATO and ZIO-101. It was found that around 4-5-fold higher arsenic contents in ZIO-101-treated NB4 cells than ATO-treated NB4 cells and 7-11-fold higher arsenic contents in ZIO-101-treated HL60 cells than ATO-treated HL60 cells, implying that the higher therapeutic efficiency of ZIO-101 than ATO might be partly caused by the improved intracellular uptake of arsenic in cancer cells. Treatment of ATO led to much higher intracellular arsenic contents in single NB4 than those in HL60 cells (Fig. 3E). In contrast, a comparable arsenic level in single NB4 and HL60 cells was noted after ZIO-101 treatment (Fig. 3F), suggesting the different sensitivity of different cancer cells towards ATO and ZIO-101.

Figure 3. Relative intracellular arsenic contents in single NB4 and HL60 cells after treatment with ATO (A) and ZIO-101 (B) at different cell cycle phase for 1.5 hrs; Relative intracellular arsenic contents after treatment with ATO and ZIO-101 in single NB4 (C) and HL60 (D) cells. Comparison of ATO (E) and ZIO-101 (F) accumulation in different cell lines. All data were normalized to the maximum value of each group for different purposes.


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Analytical Chemistry Variation of cell size across the cell cycle. Cell proliferation is tightly regulated by several checkpoints and layers of control mechanisms. Cellular processes can vary substantially during each phase. It is widely accepted that cell division is associated with changes in cell size. It is possible that the cell size affects the cell cycle-dependent uptake of As-based drugs. To evaluate whether the size variation of cells at different phases was accountable for divergence of cellular uptake of arsenic-based drugs, the size of NB4 and HL60 cells throughout the cell cycle was investigated by comparing forward scattering (FSC) profiles, which has long been recognized as an indicator of cell size31. The Forward/side scattering distribution of NB4 and HL60 appeared as evident cluster at different positions (Figure S2A, B). Quantitative analysis of the median FSC values (Figure S2E, F) based on the FSC histograms of NB4 and HL60 cells at certain stages (Figure S2C, D) showed that cell size increased with the cell cycle progression, and the largest cells were found in G2/M phase and the smallest cells in G1 phase for both NB4 and HL60 although the difference was not very evident (the highest FSC median values were 1.2 and 1.3-fold of the lowest values for NB4 and HL60 cells, respectively). The larger size of NB4 and HL60 cells in G2/M phase may be partly contribute to a higher arsenic accumulation for both ATO and ZIO-101. Expression levels of ATO and ZIO-101 transporters in different cell cycle phase. Transitions through the cell cycle phases are regulated by several control mechanisms related to quantitative changes of different proteins in cells at different states of division and growth. Drug uptake is governed by a complex system. A variety of proteins may affect the rate and amounts of uptake of a drug. Drug delivery often depends on specific transporters, which are major determinants governing drug resistance and accumulation. ATO was reported to be imported into cells via glucose transporters and aquaglyceroporins25, while ZIO-101 enters the cells via the cystine /cysteine cellular import system26. It was reported that aquaglyceroporin 9 (AQP9) is a key regulator to facilitate arsenic transport in yeast, Xenopus and mammalian cells27, and the major cystine/cysteine cellular importer32, xCT, may be a marker of sensitivity of cells towards ZIO-10126. To evaluate whether difference in the amounts of arsenic uptake in different cell cycle phase was due to the different expression levels of transporter proteins, we examined the expression levels of AQP9 and xCT in NB4 cells at different cell cycle phase by flow cytometry. Cells were collected at an interval of 1.5 hrs after release from the double thymidine block. Six cell fractions, which represent cell populations in different cell cycle phase, were stained with AQP9 and xCT antibody. The expression profiles of AQP9 and xCT in different cell cycle phase are shown in Figure 4A and C. The relative expression levels of AQP9 and xCT indicated by positive fluorescence ratios are shown in Figure 4B, D. For AQP9 proteins, the positive fluorescence ratios exhibited fluctuations through the whole cell cycle phase, and maximal values were observed when most cells were in G1 phase (50.2% at 0 h and 46.9% at 1.5 hrs) and minimum in S phase (27.6% at 3 hrs and 24.6% at 4.5 hrs). For xCT proteins, despite of a less evident fluctuation compared with AQP9, difference in the expression levels was also observed amongst different cell cycle phase. The expression levels of xCT increased with the cell cycle progression with higher xCT contents found in G2/M phase (74.8% at 6 hrs and 76.1% at 7.5 hrs) than those in G1 phase (55.4% at 0 h and 59.8% at 1.5 hrs), whereas cells in S-phase

showed an intermediate expression level of xCT. The discrepancy of arsenic uptake in different cell cycle phase was not exactly consistent with the expression level of corresponding transporters, indicating that ATO and ZIO-101 might utilize multiple importers rather than only one transporter protein.

Figure 4. Expression profiles of AQP9 (A) xCT (C) and corresponding histogram profiles of positive ratios of AQP9 (B) xCT (D) in NB4 cells collected at 0, 1.5, 3, 4.5, 6, 7.5 hrs after release from the double thymidine block.

Cytotoxicity of arsenic-based drugs on NB4 cells in different cell cycle phase. Given that both ATO and ZIO-101 exert their therapeutic functions mainly through inducing apoptosis of cancer cells3, the ratios of cell apoptosis may therefore serve as an effective indicator to evaluate the cytotoxicity and therapeutic efficiency of ATO and ZIO-101. It is reported that extensive degradation of DNA occurred during cell apoptosis, leading to lower DNA contents compared with the normal cells33. In the DNA histogram of apoptotic cells (Figure S3, Fig. 5), a peak before the G1 peak appeared, which is called sub G1 cells. The percentage of sub G1 cell could be used to evaluate the extent of cell apoptosis. To optimize the time required to induce observable cell apoptosis by ATO and ZIO-101, NB4 cells were treated with 2 µM of ATO or ZIO-101 for 2, 4, 6, 8 and 10 hrs, stained with propidium iodide (PI), then subjected to flow cytometry analysis. The PI fluorescence profiles of NB4 cells are shown in Figure S2. The apoptosis cell ratios were calculated from the Sub-G1 cell populations. As shown in Figure 6 (A), a timedependent increase in the percentages of cells at Sub-G1 phase was observed upon treatment of cells with ATO and ZIO-101, and at 6 hrs, ca. 9.88 % and 17.7% of cells were in Sub-G1 phase after treatment with ATO and ZIO-101 respectively. We thus selected this time period for the treatment of NB4 cells to evaluate cytotoxicity of ATO and ZIO-101.



CONCLUSION

Figure 5. Histograms of DNA contents in NB4 cells in different cell cycle phase after treatment with 2 µM ATO (upper) and ZIO101 (lower) for 6 hrs.

To evaluate whether ATO and ZIO-101 exert different cytotoxicity on cells in different cell cycle phase, NB4 cells at 0, 3, 6 hrs after release from the double thymidine block were treated with 2 µM ATO or ZIO-101 for 6 hrs, and stained with PI for flow cytometry assay. As shown in Figure 5, the Sub G1 peaks were observed obviously on DNA content histograms, indicative of cell apoptosis stimulated by ATO and ZIO-101. The percentages of Sub G1 cells in different cell fractions were calculated and are shown in Figure 6 (B). Different cytotoxicity was observed for both ATO and ZIO-101 on NB4 cells in different cell cycle phase. Supplementation of ATO to cells at 0 h and 3 hrs after release from the double thymidine block for 6 hrs, which corresponds to G1+S and S+G2/M phase respectively, led to apoptosis cell ratios of 7.4% and 14.2% respectively. Similarly, the apoptosis cell ratios of 15.6% and 23.7% were found upon supplementation of ZIO-101 to NB4 cells at G1+S phase and S+G2/M phase respectively. While Supplementation of ATO and ZIO-101 to the synchronize cells at 6 hrs after release from the double thymidine block (G2/M + Unsynchronized cells) resulted in moderate apoptosis cells ratios of 12.1% and 19.2% respectively. Based on this data, we conclude that the therapeutic efficiency of arsenic-based drugs was affected by cell cycle, suggesting that precise evaluation of efficacy of arsenic-based drugs should consider the position of cells in the cell cycle.

By using the double thymidine block to synchronize the cells together with time-resolved ICP-MS, we demonstrate that uptake of two arsenic-based drugs (ATO and ZIO-101) in single NB4 and HL60 cells is cell cycle dependent. The maximal intracellular arsenic contents were found in G2/M phase and the minimum in S phase for both NB4 and HL60 cells, and such variation is less apparent for ZIO-101 than ATO. However, this cannot be solely explained by the expression levels of two transporter proteins AQP9 and xCT, which varied across the cell cycle with AQP9 mostly expressed in G1 phase and xCT in G2/M phase. We therefore suggest that ATO and ZIO-101 may be transported into the cells via multiple importers. Moreover, we show that the cytotoxicity of ATO and ZIO-101 is affected by cell cycle, and the highest cytotoxicity was found in S+G2/M phase and the lowest in G1+S phase, with almost two-fold difference in terms of apoptosis cell populations. To the best of our knowledge, this is the first report to investigate the uptake and cytotoxicity of anticancer drugs across different cell stages at single cell levels. Our studies implicate that cell cycle might need to be taken into account to precisely evaluate the uptake and cytotoxicity of anticancer drugs.

AUTHOR INFORMATION Corresponding Author *Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, P. R. China. E-mail: [email protected].

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest

ACKNOWLEDGMENT This work was supported by the Research Grants Council of Hong Kong (703913P and 17304614P) and the University of Hong Kong (for an emerging strategic research theme (e-SRT) on Integrative Biology and for a scholarship to YZ). We thank Profs. P.J Sadler and Francis Lévi (University of Warwick) for stimulating discussion.

SUPPORTING INFORMATION

Figure 6. The percentages of apoptosis NB4 cells induced by ATO and ZIO-101 as indicated by percentage of sub-diploid DNA-content. (A) Dependent changes of apoptosis cell ratios for different treatment time; (B) Variations of apoptosis cell ratios in NB4 cells at different cell cycle phase after treatment with 2 µM ATO and ZIO-101 for 6 hrs.

Additional information on the 75As profiles of NB4 and HL60 cells treated with 15 µM ATO and ZIO-101 at different time points after release from double thymidine block in time-resolved ICP-MS (Figure S1); Forward/side scattering distribution of NB4 and HL60 cells at different cell cycle phase and the histograms of FSC-A and median FSC values of NB4 and HL60 cells at different cell cycle phase (Figure S2); The Histograms of DNA contents in NB4 cell populations after treatment with 2 µM ATO and ZIO-101 for different time periods (Figure S3) were supplied as supporting information.

REFERENCES (1) Emadi, A.; Gore, S. D. Arsenic trioxide - an old drug rediscovered. Blood Rev. 2010, 24, 191-199.


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Cell cycleâdependent uptake and cytotoxicity of arsenic-based drugs

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Cell cycleâdependent uptake and cytotoxicity of arsenic-based drugs