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. 2013 Jul 10;65(9):1329–1336. doi: 10.1111/jphp.12100

Cellular distribution studies of the nitric oxide-generating antineoplastic prodrug O2-(2,4-dinitrophenyl)1-((4-ethoxycarbonyl)piperazin-1-yl)diazen-1-ium-1,2-diolate formulated in Pluronic P123 micelles

Imit Kaur 1, Moises Terrazas 2, Ken M Kosak 2, Steven E Kern 1, Kenneth M Boucher 3, Paul J Shami 1,2,✉
PMCID: PMC4009949  NIHMSID: NIHMS572109  PMID: 23927471

Abstract

Objective

Nitric oxide (NO) possesses antitumour activity. It induces differentiation and apoptosis in acute myeloid leukaemia (AML) cells. The NO prodrug O2-(2,4-dinitrophenyl)1-((4-ethoxycarbonyl)piperazin-1-yl)diazen-1-ium-1,2-diolate, or JS-K, has potent antileukaemic activity. JS-K is also active in vitro and in vivo against multiple myeloma, prostate cancer, non-small-cell lung cancer, glioma and liver cancer. Using the Pluronic P123 polymer, we have developed a micelle formulation for JS-K to increase its solubility and stability. The goal of the current study was to investigate the cellular distribution of JS-K in AML cells.

Methods

We investigated the intracellular distribution of JS-K (free drug) and JS-K formulated in P123 micelles (P123/JS-K) using HL-60 AML cells. We also studied the S-glutathionylating effects of JS-K on proteins in the cytoplasmic and nuclear cellular fractions.

Key findings

Both free JS-K and P123/JS-K accumulate primarily in the nucleus. Both free JS-K and P123/JS-K induced S-glutathionylation of nuclear proteins, although the effect produced was more pronounced with P123/JS-K. Minimal S-glutathionylation of cytoplasmic proteins was observed.

Conclusions

We conclude that a micelle formulation of JS-K increases its accumulation in the nucleus. Post-translational protein modification through S-glutathionylation may contribute to JS-K's antileukaemic properties.

Keywords: JS-K, leukaemia, nitric oxide, Pluronic, poloxamer

Introduction

Nitric oxide (NO) is a naturally occurring biological molecule with multiple functions in the vascular, neurological and immune systems.[1] It has direct cytotoxic effects on tumour cells.[2] It can lead to apoptosis by targeting various cellular sites and bringing about post-translational modifications of proteins through mechanisms such as S-nitrosylation, S-glutathionylation and adenosine diphosphate ribosylation.[3] NO has also been found to trigger apoptosis by inducing endoplasmic reticulum (ER) stress.[4] We have previously shown that NO induces differentiation in acute myeloid leukaemia (AML) cells.[5] Also, diazeniumdiolates generate NO spontaneously and induce apoptosis in AML cells.[6] However, these compounds cannot be used clinically for the treatment of malignant diseases because of the pleiotropic effects of NO and particularly NO-induced vasodilatation through activation of the soluble guanylate cyclase/cyclic guanosine monophosphate pathway.[1] An alternative strategy relies on the use of prodrugs that target NO to the malignant cells. Arylated diazeniumdiolates react with glutathione (GSH) to release NO. The reaction is catalysed by GSH S-transferases (GSTs).[7] This drug design exploits upregulation of GST in malignant cells.[8] Our initial screen of arylated diazeniumdiolates revealed that O2-(2,4-dinitrophenyl)1-((4-ethoxycarbonyl)piperazin-1-yl)diazen-1-ium-1,2-diolate or JS-K (Figure 1) has potent antileukaemic activity in vitro and in vivo.[7] JS-K inhibits the growth of HL-60 myeloid leukaemia cells with a 50% growth inhibitory concentration of 0.25–0.5 μm.[7] In in vivo murine models, JS-K was also found to be effective against prostate cancer,[7] hepatoma,[9] multiple myeloma,[10] non-small-cell lung cancer[11] and glioma.[12] JS-K also possesses anti-angiogenic activity both in vitro and in vivo.[13] JS-K was shown to be selectively toxic towards tumour cells. It did not affect the growth of normal human peripheral blood mononuclear cells[10] or normal mammary epithelial cells.[14] Thus, JS-K is a promising candidate for development as an antineoplastic agent with a broad spectrum of activity.

Figure 1.

Figure 1

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Structure of JS-K.

In preliminary studies, JS-K was found to have challenging pharmacological properties that hinder its clinical development. These include poor solubility in aqueous media and reactivity with blood components resulting in a short half-life. To solve these problems, we have developed a nanoscale formulation of JS-K using Pluronic micelles. Pluronic tri-block copolymers (poloxamers) are amphiphilic polymers composed of ethylene oxide (EO) and propylene oxide (PO) arranged in the sequence EOx-POy-EOx.[15] Depending on the length of the EO and PO blocks, Pluronic polymers form nanoscale micelles at different critical micelle concentrations.[16] These micelles have a hydrophobic core that traps sparingly soluble molecules and a hydrophilic shell that allows solubilization of the latter in aqueous media. Pluronic polymers are excellent candidates to solubilize anticancer agents because they have low toxicity, circumvent the multidrug resistance phenotype, and lead to higher intratumoural drug accumulation through the enhanced permeability and retention effect.[15–18] After screening multiple Pluronic polymer candidates, we have developed a formulation for JS-K using the Pluronic P123. The purpose of the current study was to compare the intracellular distribution of free JS-K as opposed to JS-K formulated in Pluronic P123 micelles (P123/JS-K). Results show that Pluronic P123 micelles increase the penetration of JS-K in HL-60 AML cells. We also observed that JS-K induces S-glutathionylation of nuclear proteins.

Materials and Method

Materials

JS-K was synthesized at Richman Chemical (Lower Gwynedd, PA, USA) as previously described.[19] Pluronic P123 polymers were obtained from BASF (Florham Park, NJ, USA). All other chemicals were from Sigma (St. Louis, MO, USA), unless otherwise noted.

Cells and culture conditions

Human myeloid leukaemia HL-60 cells (ATCC, Rockville, MD, USA) were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. Cells were cultured at 37°C in a 5% CO2 humidified atmosphere. For experiments in different media, cells were suspended in phosphate-buffered saline (PBS) with or without 10% FBS, or RPMI/10% FBS as indicated in the individual experiment. For each experiment, JS-K was added at the time of culture initiation, cells were harvested at the indicated time points, washed in PBS, and assays were conducted.

Pluronic micelle formulation of JS-K

JS-K stocks (50 mm) in dimethyl sulfoxide (DMSO) were mixed with stocks of Pluronic P123 polymers prepared in PBS. Micellization was allowed to occur spontaneously with gentle heating at 55–60°C. The Pluronic stock concentration was 2.25%. The proportion of JS-K to P123 in solution was 1.7% by weight. pH of the Pluronic solution of JS-K was maintained at 6.5. The final concentration of JS-K in the Pluronic solution was 1 mm. For comparison, experiments were conducted with free JS-K. In the latter case, a DMSO stock (50 mm) of JS-K was serially diluted in PBS to a final concentration of JS-K of 1 mm in PBS/60% DMSO. For the cellular uptake and cell distribution studies, the final JS-K concentration in culture with either formulation was 50 μm. This high concentration was used to ensure detection of measurable levels of JS-K within the limitations of our assays. For the S-glutathionylation assays (see later), the final concentration of JS-K in culture using either formulation was 5 μm.

HPLC

HPLC analysis to measure JS-K levels was conducted on a Waters Alliance HPLC system consisting of a 2695 Separation Module, a 2996 Photodiode Array Detector using a Waters XTerra MS C18 chromatography column (4.6 × 150 mm; 3.5 mm particle size), controlled by Empower software. We used an isocratic mobile phase consisting of A = 30% 0.02 m ammonium formate (pH 3), 40% acetonitrile (ACN) and 30% methanol, and D = 100% methanol. Flow rate was kept constant at 1 ml/min for 6 min. During chromatographic separation, JS-K was monitored by absorbance at 300 nm. The method results in a standard detection curve over a linear range of 6.1–49.1 ng with a lower limit of detection of 6.4 ng.

Cellular uptake and nuclei isolation

HL-60 cells (5 × 106 cells per ml) were cultured in six-well tissue culture plates. Free or P123/JS-K was added to HL-60 cells at final concentrations of 50 μm. At the indicated time points, cells were collected by centrifugation and washed with PBS at pH 6.5. Cells were then lysed in lysis buffer. For whole cell lysates, the lysis buffer consisted of 1% sodium dodecyl sulfate (SDS) formate. The lysis buffer for cytosolic and nuclear extraction consisted of 40 mm sodium citrate, 1% Triton X-100 adjusted to pH 6.5 or lower to increase the stability of JS-K. The lysed samples were then vortexed for few seconds and refrigerated for 30 min at 4°C. Nuclei were then separated from the cytosol by centrifugation for 10 min at 600 g. The efficiency of the nuclear isolation was verified by microscopic inspection of cytospins of the nuclear and cytoplasmic fractions stained with Wright/Giemsa stain. The isolated cellular fractions were then extracted with ACN (60%)/formate (40%) for measurements of JS-K levels by HPLC as described earlier.

Glutathionylation assays

To evaluate protein S-glutathionylation, HL-60 cells (5 × 106 cells/ml) were treated with free JS-K or P123/JS-K at a concentration of 5 μm for 30 min. Cytosolic and nuclear protein isolation was performed using the Qproteome Nuclear Protein Isolation Kit (Qiagen, Valencia, CA, USA) following the manufacturer's protocol with the exception of excluding dithiothreitol (DTT) in order not to confound results of the S-glutathionylation assay. Protein concentration of the samples was determined using the Bradford assay. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 4–12% polyacrylamide gel under non-reducing conditions. Proteins were transferred to polyvinylidene fluoride membranes. After blocking in 5% non-fat dry milk in tris-buffered saline with 0.2% tween, the membrane was incubated overnight at 4°C with anti-GSH monoclonal antibody (1 : 1000, Virogen, Watertown, MA, USA). Blots were developed by incubating the membrane with horseradish peroxidase conjugated secondary antibody (1 : 5000, Jackson Immuno Research Laboratories, INC, Westgrove, PA, USA) and analysed by enhanced chemiluminescence (GE Healthcare, Piscataway, NJ, USA).

Statistical analysis

Two-way analysis of variance was performed using SigmaPlots (Systat Software, Inc., Chicago, IL, USA). Results obtained for free JS-K or P123/JS-K were compared at different time points. Post-hoc analysis was performed using the Holm Sidak test. Differences were considered statistically significant for P-values below 0.05.

Results

Cellular uptake studies

We started by performing exploratory experiments to compare the recovery of JS-K from whole cell extracts when the drug was formulated in P123 micelles (P123/JS-K) as opposed to the unformulated compound (dissolve in PBS/DMSO). Cells were incubated in PBS or RPMI/10% FBS and JS-K added at a concentration of 50 μm for both formulations. After 15, 30 and 60 min, JS-K recovery from whole cell extracts was conducted as detailed in the Methods section. JS-K recovery was higher with the P123 micelles than with the free drug (data not shown for these exploratory experiments). Because with these exploratory experiments, it seemed that the P123 formulation enhanced the cellular penetration of JS-K, we next sought to determine if it affected its intracellular distribution.

We initially conducted the experiments with cells incubated in PBS to avoid the confounding effect of proteins or other elements in full media. For both formulations, recovery of JS-K from nuclei was higher than from the cytosol. However, P123 micelles increased the recovery of JS-K from nuclei. When cells were incubated in PBS, the percent recovery of free JS-K from nuclei at 15, 30, 60 and 120 min was 5.03 ± 2.7, 5.00 ± 3.2, 10.83 ± 6.1 and 0.58 ± 0.2, respectively (Figure 2). Under the same conditions and at the same time points, the percent recovery of P123-formulated JS-K from nuclei was 9.05 ± 4, 20.00 ± 8.1, 21.15 ± 3.6 and 27.73 ± 4.9, respectively (Figure 2). The differences between the two formulations were statistically significant when they were compared (P = < 0.001), but the differences between the different time points of the same formulation were not significant. Also, the differences between the two formulations were statistically significant at 30 and 120 min. On the other hand, percent recovery of free JS-K from cytosol at 15, 30, 60 and 120 min was 1.47 ± 0.5, 0.87 ± 0.5, 1.4 ± 0.5 and 1.74 ± 0.8, respectively (Figure 2). Under the same conditions and at the same time points, the percent recovery of P123-formulated JS-K from cytosol was 2.73 ± 1.5, 0.83 ± 0.2, 1.22 ± 0.4 and 0.95 ± 0.3 (Figure 2). It is notable that with free JS-K, nuclear recovery peaked at 60 min and was negligible afterwards. On the other hand, when JS-K was formulated in P123 micelles, nuclear recovery peaked at 120 min.

Figure 2.

Figure 2

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Percent recovery of JS-K from cytoplasmic and nuclear fractions when cells were incubated in phosphate-buffered saline. Values are expressed as percent of initial JS-K added. Cytosolic recovery of JS-K is low as compared with nuclear recovery. At any time point, JS-K recovery from the nucleus was higher when the drug was formulated in P123 Pluronic micelles. Free cytosol = O2-(2,4-dinitrophenyl)1-((4-ethoxycarbonyl)piperazin-1-yl)diazen-1-ium-1,2-diolate recovery from cytosol with phosphate-buffered saline/dimethyl sulfoxide formulation. P123 cytosol = JS-K recovery from cytosol with P123 formulation. Free nuclei = JS-K recovery from nuclei with phosphate-buffered saline/dimethyl sulfoxide formulation. P123 nuclei = JS-K recovery from nuclei with P123 formulation. The plot shows averages and standard errors of the mean of at least two separate experiments for each time point.

As the recovery from the cytosol was negligible when the cells were incubated in PBS, the cytosolic fraction was not tested for JS-K recovery in subsequent studies. To determine the effect of proteins in the culture medium on JS-K recovery, we then measured JS-K levels when the cells were incubated in PBS/10% FBS. When cells were incubated in PBS/10% FBS, the percent recovery of free JS-K from nuclei at 30, 60 and 120 min was 3.55 ± 2.1, 3.15 ± 1.5 and 2.13 ± 1.1, respectively (Figure 3). Under the same conditions and at the same time points, the percent recovery of P123/JS-K from nuclei was 20.74 ± 7.2, 12.70 ± 3.7 and 16.03 ± 3.9, respectively (Figure 3). The differences between the two formulations were statistically significant (P < 0.001), but the differences between the different time points of the same formulation were not significant. When the formulations were compared at different time points, the differences were statistically significant at 30 and 120 min.

Figure 3.

Figure 3

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Percent recovery of JS-K from the nuclear fraction when cells were incubated in phosphate-buffered saline/10% fetal bovine serum. Values are expressed as percent of initial JS-K added. At any time point, JS-K recovery from the nucleus was higher when the drug was formulated in P123 Pluronic micelles. The plot shows averages and standard errors of the mean of at least two separate experiments for each time point.

To determine the effect of full media on JS-K recovery, we repeated the experiments when the cells were incubated in RPMI/10% FBS. When cells were incubated in RPMI/10% FBS, the percent recovery of free JS-K from nuclei at 15, 30, 60 and 120 min was 0.38 ± 0.17, 0.45 ± 0.23, 0.43 ± 0.25 and 0.2 ± 0.11, respectively (Figure 4). Under the same conditions and at the same time points, the percent recovery of P123/JS-K was 2.93 ± 1.65, 4.68 ± 1.62, 9.30 ± 1.58 and 11.90 ± 4.4, respectively (Figure 4). Overall, JS-K recovery from nuclei was lower than with the less complex matrices, but again, recovery was higher with P123/JS-K. The two formulations showed statistically significant differences (P < 0.001), but the differences between the different time points of the same formulation were not significant. When the formulations were compared at different time points, the differences were statistically significant at 60 and 120 min.

Figure 4.

Figure 4

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Percent recovery of JS-K from the nuclear fraction when cells were incubated in RPMI/10% fetal bovine serum. Values are expressed as percent of initial JS-K added. At any time point, JS-K recovery from the nucleus was higher when the drug was formulated in P123 Pluronic micelles. The plot shows averages and standard errors of the mean of three separate experiments for each time point.

S-glutathionylation studies

Protein S-glutathionylation is a well-established mechanism that controls protein function at the post-translational level.[20,21] In that respect, NO has been shown to be a potent S-glutathionylating agent.[3] We therefore sought to determine whether JS-K had the same effect in HL-60 cells. Based on our results showing increased levels of JS-K in the nucleus, we also investigated whether JS-K had a differential S-glutathionylation effect on nuclear as opposed to cytoplasmic proteins. HL-60 cells were treated with JS-K at a concentration of 5 μm for 30 min upon which nuclear and cytoplasmic proteins were isolated for assays of S-glutathionylation. Results showed that free JS-K induced minimal increase in the S-glutathionylation of cytosolic proteins (Figure 5). The effect was similar when JS-K was formulated in P123 micelles (Figure 5). When the soluble fraction of nuclear proteins was analysed, free JS-K did not induce significant S-glutathionylation. However, for the same protein fraction, P123/JS-K induced significant S-glutathionylation (Figure 6). When the insoluble fraction of nuclear proteins was analysed, both free and P123-formulated JS-K induced significant S-glutathionylation. The effect was slightly more pronounced with the P123 formulation (Figure 6).

Figure 5.

Figure 5

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S-glutathionylation of cytosolic proteins by O2-(2,4-dinitrophenyl)1-((4-ethoxycarbonyl)piperazin-1-yl)diazen-1-ium-1,2-diolate. HL-60 cells were treated with 5 μm JS-K and incubated for 30 min. Evaluation of S-glutahionylation of cytosolic proteins was conducted as detailed in the text. JS-K in either formulation causes minimal S-glutathionylation of cytosolic proteins. 30 μg of protein was loaded into each lane of the gel. Lane 1: dimethyl sulfoxide control; Lane 2: HL-60 cells treated with free JS-K; Lane 3: P123 control; Lane 4: HL-60 cells treated with P123/JS-K. Actin was used as the loading control. The blot shown is representative of four separate experiments.

Figure 6.

Figure 6

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S-glutathionylation of nuclear proteins by JS-K. HL-60 cells were treated with 5 μm JS-K and incubated for 30 min. Evaluation of S-glutahionylation of nuclear proteins was conducted, as detailed in the text. JS-K caused S-glutathionylation of nuclear proteins. The effect is more pronounced with P123/JS-K. Among the nuclear proteins, soluble nuclear proteins are S-glutathionylated to a greater extent than insoluble nuclear proteins. Thirty micrograms of protein was loaded into each lane of the gel. Lane 1: No treatment control for soluble nuclear proteins; Lane 2: S-glutahionylation of soluble nuclear proteins caused by free JS-K; Lane 3: P123 control for soluble nuclear proteins; Lane 4: S-glutahionylation of soluble nuclear proteins caused by P123/JS-K; Lane 5: No treatment control for insoluble nuclear proteins; Lane 6: S-glutahionylation of insoluble nuclear proteins caused by free JS-K; Lane 7: P123 control for insoluble nuclear proteins; Lane 8: S-glutahionylation of insoluble nuclear proteins caused by P123/JS-K. Proliferating cell nuclear antigen was used as the loading control for soluble nuclear proteins, and histone 3 protein was used as the loading control for insoluble nuclear proteins. The blot shown is representative of four separate experiments.

Discussion

In the current work, we evaluated the cellular distribution of JS-K in HL-60 leukaemia cells. When free JS-K was added to the leukaemic cells, there was minimal drug recovery from the different cellular compartments. However, higher levels of JS-K were recovered from the nucleus. When JS-K was formulated in P123 micelles, recovery of JS-K was substantially increased. Again, the highest amounts of JS-K were recovered from the nucleus. Several factors could explain these observations. JS-K reacts with GSH to form the secondary products dinitro-phenyl S-GSH and the diazeniumdiolate 4-carbethoxypiperazi-NO, which releases NO. Intracellular GSH levels are found mainly concentrated in the cytoplasm.[22] One could therefore speculate that higher levels of JS-K were recovered from the nucleus because most of the JS-K in the cytoplasm reacted with GSH. However, the fact that JS-K was measured in the nucleus indicates that it does get transported into that compartment either passively or actively.

When formulated in P123 micelles, JS-K was recovered at higher levels than when the drug was formulated in PBS/DMSO. It is to be noted that for these assays, we used high concentrations of JS-K (50 μm) to be able to measure drug levels using the assay we developed. Previous work has shown that the solubility limit of JS-K in aqueous solutions was around 10 μm.[7] Consequently, a possible reason for which there was greater cellular recovery of JS-K with the P123 formulation is that by increasing the solubility of JS-K in the culture media, P123 allowed more drug to reach the cells. However, P123 micelles enhance recovery of JS-K in particular from the nucleus. This may be due to the fact that the micelles temporarily shield JS-K and prevent it from reacting with cytoplasmic GSH and proteins in the cytoplasm. Furthermore, P123 micelles by themselves may enhance the penetration of JS-K into the nucleus. Indeed, Rapoport et al. studied the effect of Pluronic P105 on the cellular distribution of the lipophilic drug Ruboxyl. They showed that Ruboxyl accumulates in the nucleus at P105 concentrations as high as 1%.[23] It is therefore likely that a similar effect occurs when JS-K is formulated with P123 micelles.

The recovery of JS-K from the different cellular compartments diminished when the cells were incubated with complex media such as PBS/10% FBS and RPMI/10% FBS. This could be due to the fact that the stability of JS-K diminishes as it is exposed to proteins and other components of the media. P123 micelles yield to increased recovery of JS-K from the cells as compared with free JS-K in these media. Again, this is likely due to ‘protection’ of JS-K by the micelles.

From a mechanistic standpoint, we show that JS-K induces S-glutathionylation of mainly nuclear proteins. Such a post-translational modification can have important implications. An oxidative cellular environment is linked with cell death, whereas a reducing environment promotes cell survival.[24] Depletion of cellular GSH pool has been associated with apoptosis.[22] Nuclear GSH depletion also triggers apoptosis.[25] The transcription factor nuclear factor-κB (NF-κB) promotes AML survival.[24]  S-glutathionylation of this protein can prevent its nuclear translocation, thus promoting apoptosis. NO inhibits directly the DNA binding activity of NF-κB.[26] Whether, as an NO donor, JS-K affects AML cell survival through S-glutathionylation of NF-κB remains to be proven. We found that in HL-60 cells, S-glutathionylation is less marked on cytoplasmic proteins. This may be due to increased levels of GSH in the cytoplasm with resultant competition for reaction with JS-K.

As demonstrated earlier, P123 micelles increase the stability of JS-K. Micelles also enhance nuclear translocation of JS-K with observable post-translational modification of nuclear proteins through S-glutathionylation. Post-translational modifications such as S-glutathionylation and S-nitrosylation can have important consequences. These modifications can enhance or repress the activity of proteins and cause apoptosis.[3] They can also cause activation of the unfolded protein response because of accumulation of misfolded S-glutathionylated or S-nitrosylated proteins, and result in ER stress.[27,28] If ER stress is strong and prolonged, apoptosis ensues.[29] Induction of ER stress could therefore contribute to JS-K's cytotoxic activity.

S-glutathionylation of nuclear proteins by JS-K raises the possibility that the drug may be exerting some of its effect through a mechanism involving post-translational modification of these proteins. These proteins likely include transcription factors (soluble nuclear proteins) and histones (insoluble nuclear proteins). The latter finding raises the possibility that JS-K may affect leukaemia cell growth through an epigenetic mechanism as has recently been reported for NO.[30] Townsend et al. have shown that the JS-K analogue O2-{2,4-dinitro-5-[4-(N-methylamino)benzyloxy]phenyl}1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA-NO) is a potent S-glutathionylating agent. They further demonstrated that one of the principal proteins that are S-glutathionylated by PABA-NO is protein disulfide isomerase.[28] Identification of proteins that are S-glutathionylated by JS-K will require further investigation.

Conclusions

We show here that JS-K likely affects leukaemic cell viability by multiple mechanisms, including post-translational modification of nuclear proteins. While the drug by itself accumulates in different cellular compartments, a Pluronic micelle formulation affects its cellular distribution and leads to enhanced accumulation in the nucleus. JS-K-induced protein S-glutathionylation raises the possibility that it exerts part of its effect through the post-translational modification of key proteins that are yet to be identified. JS-K shows promise for medicinal applications as an antineoplastic agent.

Declarations

Conflict of interest

Paul Shami is Scientific Founder, Chief Medical Officer and Chairman of the Board of Directors of JSK Therapeutics, Inc.

Funding

This work was supported by grant RO1 CA129611 from the National Cancer Institute.

Authorship Contributions

Research design: Kaur, Kosak, Kern and Shami

Conducted experiments: Kaur and Terrazas

Data analysis: Kaur, Kosak, Kern, Boucher and Shami

Wrote the manuscript: Kaur and Shami

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