How do the alkaloids emetine and homoharringtonine kill trypanosomes? An insight into their molecular modes of action

Sonja Krstin , Tamer Mohamed , Xiaojuan Wang , Michael Wink

ABSTRACT

Background: Although Trypanosoma brucei causes deadly sleeping sickness, the number of the registered medications is rather limited. Some plant alkaloids are potent trypanocidal agents.
Purpose: In this study, we wanted to elucidate the molecular modes of trypanocidal activity of the alkaloids emetine and homoharringtonine against Trypanosoma brucei brucei.
Methods: We investigated the activity of both alkaloids regarding growth recovery from alkaloid-induced stress. We measured the inhibition of protein biosynthesis using the Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay kit. Reduction of mitochondrial membrane potential and cell cycle arrest were measured by means of flow cytometry. Additionally, we determined spectrophotometrically the inhibition of the trypanosome specific enzyme trypanothione reductase activity and DNA intercalation.
Results: Both alkaloids prevented that parasites could resume normal growth after pretreatment with the alkaloids. They inhibited protein biosynthesis in a time- and concentration-dependent manner. In contrast to homoharringtonine, emetine is also a DNA intercalator. Homoharringtonine decreased the mitochondrial membrane potential. Both alkaloids caused cell cycle arrest. Both alkaloids failed to affect trypanothione reductase, a crucial component of the redox system of trypanosomes.
Conclusion: We assume that both alkaloids are primarily inhibitors of protein biosynthesis in trypanosomes, with DNA intercalation as an additional mechanism for emetine. This is the first study that elucidates the molecular mode of trypanocidal action of emetine and homoharringtonine.

Keywords: Homoharringtonine, Emetine, Trypanosome, Protein biosynthesis, Cell cycle, Mitochondrial membrane potential

1. Introduction

Trypanosoma brucei is a unicellular parasitic flagellate, which, if left untreated, causes deadly sleeping sickness in humans and animals. The World Health Organization (WHO) marks the disease as neglected, since it is prevailing in the developing areas of the world, affecting mainly Subsaharan Africa among them suramin and pentamidine that have been in clinical use for more than 70 years (http://www.who.int/mediacentre/factsheets/fs259/en/ accessed March 2016).
Some alkaloids are potent trypanocidal secondary plant metabolites. Two alkaloids with reported high anti-parasitic activity are emetine and homoharringtonine (Krstin et al., 2015; Merschjohann et al., 2001; Rosenkranz and Wink, 2008). Emetine (Fig. 1a) is an isoquinoline alkaloid that has been isolated from Psychotria ipecacuanha (Brot.) Stokes (Rubiaceae) and used in folk medicine and phytomedicine as emetic, expectorant and amebicide (Cheong et al., 2011; Wink and Schimmer, 2010). Homoharringtonine (Fig. 1b) is a cephalotaxine derivative that has been isolated from Cephalotaxus harringtonii (Knight ex J.Forbes) K. Koch (Taxaceae) and used in traditional Chinese medicine against helminths and malignancies (Al Ustwani et al., 2014; Efferth et al., 2007; Wink and Schimmer, 2010). US Food and Drug Administration (FDA) approved the use of its derivative omacetaxine mepesuccinate for treatment of patients suffering from chronic myeloid leukemia (Chen and Li, 2014). The biological activity of emetine and homoharringtonine is attributed to their ability to inhibit protein biosynthesis, while emetine was also reported to intercalate DNA (Wink and Schimmer, 2010).
Treatment of sleeping sickness lacks innovation; it was 2009, when a combination of nifurtimox and eflornithine was put on the market (http://www.who.int/mediacentre/factsheets/fs259/en/, accessed March 2016). There is therefore a high demand for new drugs to fight sleeping sickness, especially since some strains have developed resistance against common trypanocidal agents (Wink, 2012). Although emetine and homoharringtonine are active against trypanosomes, information of their trypanocidal mode of action is limited. In order to proceed with clinical studies and consequently put these alkaloids in clinical use for the treatment of trypanosomiasis, we require a sound understanding at a molecular level of the way they are killing trypanosomes.
In this study we aimed to elucidate the powerful trypanocidal activity of the alkaloids emetine and homoharringtonine against Trypanosoma brucei brucei (T. b. brucei). In our previous study (Krstin et al., 2015), where 6 alkaloids were included, emetine and homoharringtonine showed the highest trypanocidal activity. We investigated the underlying mode of action regarding their influence on protein biosynthesis, mitochondrial membrane potential, cell cycle, activity of the trypanosome-specific enzyme Trypanosoma brucei trypanothione reductase and DNA intercalation. This is the first study that clarifies the molecular mode of action of emetine and homoharringtonine in trypanosomes, which should help to infer potential side effects.

2. Material and methods

2.1. Chemicals

Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay kit, MEM medium, methionine-free DMEM medium, non-essential amino acids (NEAA), penicillin, streptomycin and L-glutamine were purchased from Invitrogen, Germany. Cycloheximide (≥93%), fetal bovine serum (FBS), dimethylsulfoxide (DMSO; ≥ 99.9%), HEPES (≥95%), glucose (≥95%), sodium pyruvate, hypoxanthine (98%), thymidine (99-100%), adenosine, bathocuproinedisulfonic acid disodium salt, ethidium bromide, emetine dihydrochloride hydrate, homoharringtonine (≥ 98%), carbonyl cyanide 3-chlorophenylhydrazone (>97%), NADPH, propidium iodide (≥ 94%), rhodamine 123 and β-mercaptoethanol were obtained from Sigma-Aldrich GmbH, Germany. RNAse A was acquired from Applichem GmbH, Germany and Lambda DNA from Thermo Fisher Scientific, Germany.

2.2. Cell culture

We used a T. b. brucei TC 221 derived from stock 427 bloodstream cell line- pathogenic only for animals, which justifies its use in laboratories; the strain as ac uired from rof. eter Overath (Max lan nstitut f r iologie, ingen, ermany). It was maintained in complete Baltz medium (Baltz et al., 1985) and cultivated at 37 °C, 5% CO2 and 95% humidity. All experiments were performed with cells being in their logarithmic growth phase.

2.3. Recovery assay

The assay was executed as previously described with minor modifications (Kessler et al., 2013) . A cell density of 1.3 x 106 cells/ml was exposed to different doses of homoharringtonine (0.01, 0.25 and 1 μM) and emetine (0.02, 0.25 and 2 μM) for short periods of time (30, 120 and 240 min). Cells were then centrifuged at 1800 x g for 5 min at room temperature, washed two times with sterile phosphate-buffered saline (PBS) and transferred to fresh, drug-free Baltz medium at a density of 104 cells/ml. The cell density was determined every 24 h in the improved Neubauer counting chamber. Growth recovery was monitored for 72 h after drug contact.

2.4. Protein biosynthesis inhibition

To investigate the inhibition of global protein biosynthesis in trypanosomes, Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay kit was used. The assay was performed following the kit´s instructions. Briefly, 2 x 106 T. b. brucei cells/ml were incubated with emetine (0.02, 0.06 and 0.12 μM) and homoharringtonine (0.01, 0.25 and 1 μM) for 4, 8 and 24 h. From this section onwards, lower concentrations of emetine ere used than in the recovery assay ecause of emetine’s high toxicity and longer incubation time. After washing, the cells were incubated for 30 min in a methionine-free medium ith 50 μM of L-azidohomoalanine- an amino acid analog of L-methionine containing an azido moiety, which is being incorporated into proteins during protein biosynthesis. Afterwards, the cells were washed, fixed and permeabilized. The cells were then incubated with Click-iT® reaction cocktail for 30 min, at room temperature, protected from light. The cocktail contained AlexaFluor 488 conjugated alkyne that detected (by “click“ reaction) the amino analog in the azido modified protein. The resulting fluorescence was measured at excitation/emission wavelengths of 485/535 nm using a Tecan Safire Infinite F200 microplate reader (Tecan Crailsheim, Germany). The antibiotic cycloheximide, a known inhibitor of protein synthesis, was used as a positive control.

2.5. Mitochondrial membrane potential assay

Decrease in the potential of the mitochondrial mem rane (ΔΨm) as measured using the fluorescent probe Rh123, which accumulates within mitochondria (Divo et al., 1993). Briefly, 2 x 106 T. b. brucei cells/ml were incubated with emetine (0.02, 0.06 and 0.12 μM) and homoharringtonine (0.01, 0.25 and 1 μM) for 4 and 24 h. After ashing, cells ere incu ated ith 10 μg/ml Rh123 (Rhodamine 123) on 37 °C for 15 min to verify changes in ΔΨm. Data acquisition and analysis were performed at excitation/emission wavelengths of 488/530 nm using FACSCaliburTM flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) equipped with CellQuestTM software. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as a positive control. A total of 10000 events were acquired in the region that was previously established as the one that corresponded to the parasites.

2.6. Cell cycle arrest

To determine whether the alkaloids arrest the cell cycle of trypanosomes, emetine (0.02, 0.06 and 0.12 μM) and homoharringtonine (0.01, 0.25 and 1 μM) were incubated with 106 T. b. brucei cells/ for 4 and 24 h at 37 °C. Afterwards, the cells were washed two times with PBS and fixed overnight with 70 % EtOH at –20 °C. The ethanol was then removed; the cells were washed twice with the ice-cold PBS, followed by 30 min incubation at 37 °C with 200 μg/ml RNAse A. Immediately before reading, cells were stained for 30 min with 50 μg/ml of propidium iodide (PI) at room temperature. The analysis of cell growth phases was performed at excitation/emission wavelengths of 488/585 nm using FACSCaliburTM flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) equipped with CellQuestTM software.

2.7. Trypanosoma brucei trypanothione reductase (TbTR) inhibition assay

The recombinant enzyme TbTR and the substrate TS2 were prepared following published procedures (Comini et al., 2009; Persch et al., 2014; Sullivan and Walsh, 1991). Reversible inhibition of TbTR was measured spectrophotometrically as described by (Jockers-Scherubl et al., 1989). The assay contained in a total volume of 1 mL: 40 mM Hepes, 1 mM EDTA; pH 7.5, 100 μM NAD H, 2 U R and 100 μM of each alkaloid or the respective amount of the solvent (DMSO) in the negative controls. The reaction was started by adding 100 μM S2 and the absorption decrease due to NADPH consumption at 340 nm was followed at 25 °C (Hitachi Spectrometer, Model 150-20, Japan). The activity of the enzyme was also validated after 1h incubation of the mixture at the room temperature.

2.8. DNA intercalation

DNA intercalation was determined by measuring the changes in the melting temperature (Tm) in the absence and presence of the alkaloids homoharringtonine or emetine, using DNA from the bacteriophage Lambda. The measurements were conducted using double beam spectrophotometer (Jasco V-630) fitted with a thermostated cell holder according to an already established procedure with some modifications (McCoubrey et al., 1996). Briefly: Drug/DNA complexes were heated at a rate of 0.5 °C/min from 25 to 90 °C. Drug/DNA ratio was 1:3. The absorption was continuously determined at 260 nm per 0.5 °C increase in temperature. Tm was taken as the midpoint of the hyperchromic transition. Ethidium bromide was used as a positive control.

2.9. Statistical analysis

All experiments were carried out three times and the data are expressed as mean ± standard deviation from three independent experiments. Data were analyzed with GraphPad Prism® software (GraphPad Prism® 5.0a, GraphPad Software, Inc., CA, USA). Student´s t-test was used to analyze differences between controls and different treatments in all experiments except the cell cycle arrest, where two way analysis of variance and Bonferroni’s post hoc test ere used. A p-value below 0.05 was considered significant. 3. Results In our previous study (Krstin et al., 2015), we determined the IC50 values of 0.01 and 0.03 μM for the cytotoxicity of homoharringtonine and emetine (using the MTT assay) against T. b. brucei cell line, respectively. Based on these IC50 values, we proceeded with further experiments.

3.1. Antiproliferative effect of emetine and homoharringtonine

The parasites lost their ability to resume normal growth in a concentration- and time- dependent manner after removal of both alkaloids from the cultures, which was most evident after 72 h (Fig. 2). After ending the induced stress from a 4 h emetine treatment (0.02, 0.25 and 2 μM), the effect was apparent already after 48 h. 2 h of incubation with the highest concentration of emetine (2 μM) or homoharringtonine (1 μM) was sufficient to slow down the growth for about 50 % and 30 %, respectively. After 4 h incubation with any concentration of emetine, the cell growth was reduced by about 60, 70 and 80 % after 24, 48 and 72 h, respectively.

3.2. Inhibition of protein biosynthesis

Emetine and homoharringtonine could inhibit protein biosynthesis in vitro in a time- and concentrationdependent manner (Fig. 3). The concentration of 1 μM homoharringtonine could significantly inhibit protein biosynthesis by 10 % after only 4 h (Fig. 3b). In all tested concentrations, both alkaloids significantly inhibited protein biosynthesis after 24 h (Fig. 3a and 3b), with the highest concentrations of homoharringtonine (1 μM) and emetine (0.12 μM) inhibiting the protein biosynthesis by 50 and 20 %, respectively. The inhibition of protein biosynthesis by homoharringtonine is comparable to the activity of the positive control, cycloheximide.

3.3. Decrease of mitochondrial membrane potential

We observed a marked decrease in total Rh123 fluorescence intensity under the influence of homoharringtonine, indicating significant mitochondrial depolarization in the cells treated for 24 h with 0.25 and 1 μM of the al aloid compared ith the control group (Fig. 4c). On the other hand, treatment with 0.02, 0.06 and 0.12 μM of emetine showed no reduction in ΔΨm (Fig. 4b,d). After only 4 h of incubation, both alkaloids failed to affect the mitochondrial membrane potential (Fig. 4a,b).

3.4. Cell cycle arrest

Both alkaloids arrested the cell cycle of trypanosomes, however in a different manner. Homoharringtonine arrested the cell cycle in G0/G1 phase after only 4 h, when we measured a significant increase of cell number in G0/G1 and decrease in G2 phase (Fig. 5a). After 24 h, the effect switched and the number of cells in the G0/G1 phase was reduced (Fig. 5b). On the other hand, emetine arrested the cell cycle in G2/M phase only after 24 h, while after only 4 h of incubation emetine showed the same trend like homoharringtonine, although the trend was not statistically significant (Fig. 5).

3.5. TbTR inhibition assay

High concentration of alkaloids failed to affect the activity of Trypanosoma brucei trypanothione reductase, regardless of the incubation time (data not shown). Emetine could inhibit the activity of the enzyme after an 1 h incubation at room temperature, but only by 11 %; at an elevated concentration of 100 µM, this activity is probably not relevant for cytotoxicity.

3.6. DNA intercalation

Apparently, emetine can intercalate DNA, since it increased the melting temperature (Tm) of DNA by 24 °C at a concentration of 50 μg/ml (90 μM) compared with the pure DNA, which served as negative control (Fig. 6) confirming earlier results from our laboratory (Wink et al., 1998). The positive control ethidium bromide intercalated the DNA in the strongest fashion, increasing the Tm by 33 °C at a concentration of 10 μg/ml (25 μM). However, homoharringtonine failed to intercalate DNA, regardless of the concentration used in the assay.

4. Discussion

The present study helps to understand the molecular modes of trypanocidal action of the alkaloids homoharringtonine and emetine. We found that both alkaloids are primarily inhibitors of nascent protein biosynthesis. Emetine is additionally able to intercalate DNA (thus leading to mutations and strand breaks), meaning it has two main targets and modes of action in trypanosomes.
Proteins are essential cell constituents. Besides promoting almost every chemical reaction in the cell, they also represent building blocks in many cell structures, like cytoskeleton and cell membrane. Proteins play a vital role in the cell movement, traffic of substances in and out of cells and communication between cells (Alberts et al., 2008). Both alkaloids were able to inhibit the protein synthesis in trypanosomes, although homoharringtonine was faster and more powerful by showing its inhibition ability after only 4 h. After 24 h of incubation, homoharringtonine inhibited the protein biosynthesis significantly, similar to the antibiotic cycloheximide. Our data agree with the literature that emetine and homoharringtonine inhibit the synthesis of proteins in human HeLa cells and in vitro (Orozco et al., 1985; Tscherne and Pestka, 1975). Emetine has been used in folk medicine and phytomedicine as an amebicide, and its mode of action against Entamoeba histolytica is proposed to be the inhibition of protein biosynthesis (Orozco et al., 1985).
Considering that trypanosomes have only one mitochondrial organelle- in contrast to human cells that have numerous- the proper function of this organelle is important for the survival of the parasite. Keeping the potential of the mitochondrial membrane stable is necessary for the proper function of the organelle as well as the survival of the parasite (Fidalgo and Gille, 2011). Concentrations of 0.25 and 1 μM of homoharringtonine reduced the mitochondrial membrane by approximately 20 and 40 %, respectively. We observed this effect only after 24 h, which we interpreted as a consequence of targeting other cellprocesses primarily. Beranova et al. (2013) showed that homoharringtonine induces apoptosis via multiple mechanisms in human colorectal cells. Decrease in mitochondrial membrane potential is thought to be one of the markers of cell suicide (Fidalgo and Gille, 2011; Ly et al., 2003). The decrease of the mitochondrial membrane potential could indicate apoptosis, however it is probably a secondary effect generated by inhibition of the protein biosynthesis. A previous study from our laboratory shows that although emetine caused DNA fragmentation, it failed to decrease the mitochondrial membrane potential in trypanosomes (Rosenkranz and Wink, 2008).
The trypanosome cell cycle is, like in any other eukaryotic cell, tightly regulated through several checkpoints: G0/G1, S, G2 and M. They have several different CRK (cyclin related kinase) and cyclins that coordinate the accurate transition through all stages of the cell cycle (Hammarton, 2007; Zuma et al., 2014). Both alkaloids arrested the trypanosomes in a concentration dependent manner after 4 h incubation in G0/G1 phase of the cell cycle. Although emetine-induced arrest did not reach significance, we observed the same trend as with homoharringtonine. Protein biosynthesis is mostly occurring during the G1 phase of the cell cycle (Baaske and Heinstein, 1977). The study by Xu (1981) showed that homoharringtonine can arrest the cell cycle of murine cells in G1 phase. We found that homoharringtonine significantly inhibited the protein biosynthesis after 4 h, while emetine did not, which means that homoharringtonine was able to reduce the amount of proteins and probably lead to a secondary effect of arresting the cell cycle. On the other hand, after 24 h incubation, the alkaloids seemed to arrest the cell cycle in G2/M phase, where less protein biosynthesis is taking place than in G1 phase (Baaske and Heinstein, 1977). It could be that alkaloids first arrested the cell cycle in the G1 phase, but the cells managed to bypass the checkpoint and continue with the cell cycle. Then, after 24 h the cell cycle is arrested at the G2 phase. Both alkaloids are inhibiting protein biosynthesis and it is typical for protein synthesis inhibitors to block both G1 and G2 phase (Baaske and Heinstein, 1977; Blagosklonny, 2004; Zhou et al., 1995).
We hypothesized, that due to their high toxicity against trypanosomes, these alkaloids might affect trypanothione reductase, a crucial enzyme in the survival of trypanosomes and uniquely present in the order Trypanosomatidae. However, both alkaloids failed to influence the activity of TbTR, ruling out that the trypanothione system is a target.
DNA intercalators have the ability to intercalate between the stacks of base pairs of DNA, consequently stabilizing the double helix, which can prevent replication and cause strand breaks, deletions and frameshift mutations, ultimately killing a cell (Wink, 2012). We confirmed previous research that emetine is a
DNA intercalator, while homoharringtonine failed to intercalate DNA in our experiment (Wink et al., 1998). Taking our results into account, emetine is killing trypanosomes at least via two mechanisms, by having two molecular targets, DNA intercalation and protein synthesis inhibition, while homoharringtonine acts predominantly via the inhibition of protein biosynthesis. This could be the reason why emetine is killing the trypanosomes faster than homoharringtonine, the phenomenon we observed in the antiproliferative assay. After only 4 hours of incubation, trypanosomes could recover faster from the effect of homoharringtonine than emetine. On the other hand, homoharringtonine is a much stronger inhibitor of protein biosynthesis, which could be the explanation why it exhibited an anti-parasitic effect 3 times stronger than emetine in our previous study.
When searching for new trypanocidal drugs, it is essential to find not only drugs that are trypanocidal, but also the ones that will have fewer side effects than the common trypanocidal drugs. Both alkaloids are reported to have a cytotoxic activity (Akinboye et al., 2015; Baaske and Heinstein, 1977; Moller et al., 2006). Wang et al. (2016) and Merschjohann et al. (2001) showed that the IC50 values after 48 h of incubation of homoharringtonine and emetine with human cancer cells were 9 and 97 nM, respectively. Both alkaloids (emetine and homoharringtonine) very likely present a potential source of toxicity when used in humans. However, we propose that future in vivo studies should not be disregarded because of their toxicity, since we are referring to a life-threatening disease, such as sleeping sickness, where a higher degree of side effects could be justified. The second stage of the disease, when the parasite crosses the blood brain barrier is the most dangerous one, when death can occur. Unlike suramin, homoharringtonine is capable of crossing the blood brain barrier (Savaraj et al., 1987), which would be advantageous in this context. Damlaj et al. (2016) have concluded that when using omacetaxine mepesuccinate (a derivative of homoharringtonine used in the therapy of leukemia) the side effects like cardiac toxicity could be efficiently avoided by applying an adequate dosage. However, the cytotoxicity towards human cells of homoharringtonine and emetine should not be underestimated and therefore in order to estimate the actual toxicities of these two alkaloids, animal studies should be carried out at concentrations which can eradicate the parasitic infection, and the side effects should be evaluated at these concentrations. Because the alkaloids have a different IC50 value we used different concentrations in our assays. We can nevertheless draw the conclusion that both alkaloids are primarily inhibitors of protein biosynthesis in T. b. brucei parasites, with DNA intercalation being an additional mechanism for emetine. Animal experiments are now necessary to demonstrate the efficacy and explore safety risks of both alkaloids.

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