Sorafenib

Drug-induced reactive oxygen species- mediated inhibitory effect on growth of Trypanosoma evansi in axenic culture system

Rajender Kumar1 & Ruma Rani1 & Saroj Kumar2 & Khushboo Sethi1 & Shikha Jain1 & Kanisht Batra3 & Sanjay Kumar1 & B. N. Tripathi1

Abstract

Trypanosoma evansi, an extracellular haemoflagellate, has a wide range of hosts receptive and susceptible to infection, in which it revealed highly inconsistent clinical effects. Drugs used for the treatment of trypanosomosis have been utilized for more than five decades and have several problems like local and systemic toxicity. In the present investigation, imatinib and sorafenib were selected as drugs as they are reported to have the potential to cause reactive oxygen species (ROS)–mediated effect in cancer cells. Both have also been reported to have potential against T. brucei, T. cruzi and Leishmania donovani. To date, imatinib and sorafenib have not evaluated for their growth inhibitory effect against T. evansi. Imatinib and sorafenib showed significant (p < 0.001) inhibition on parasite growth and multiplication with IC50 (50% inhibitory concentration) values 6.12 μM and 0.33 μM respectively against T. evansi. Both the drug molecules demonstrated for the generation of ROS in T. evansi and were found up to 65% increased level of ROS as compared with negative control in the axenic culture system. Furthermore, different concentrations of imatinib and sorafenib were found non-toxic on horse peripheral blood mononuclear cells and Vero cell lines. Also, in conclusion, our results demonstrated that imatinib- and sorafenib-induced generation of ROS contributed inhibitory effect on the growth of Trypanosoma evansi in an axenic culture system. Keywords Trypanosomaevansi .Surra .Trypanosomosis .ROSmeasurementassay .Growthinhibitionassay .Invitroresazurin cytotoxicity assay Introduction Trypanosoma evansi causes trypanosomosis, commonly known as “Surra”, one of the most important haemoprotozoan diseases affecting livestock productivity and agricultural activity. Tabanids and stomoxes are mechanical transmitters of T. evansi and have the widest host range among trypanosomes (Mihok et al. 1995; Desquesnes et al. 2009). It is believed to be that T. evansi originate from Trypanosoma brucei brucei, but exhibit differences on subcellular levels (Carnes et al. 2015). The disease is widely distributed in tropical and subtropical countries, and its impact varies in different hosts from regiontoregion. In India, it is endemic inequines inNorth and North-western regions of the country and had direct and indirect economic impact on livestock productivity including mortality losses, reproductive losses, reduction in milk yield and draught power (Kumar et al. 2013; Kumar et al. 2017). Though this contagion is mainly restricted to animals including livestock, pet and wild animal species, some recent reports specify their ability for zoonosis also (Shegokar et al. 2006; Van Vinh Chau et al. 2016). Therefore, there is an urgent need for effective control strategies for trypanosomosis in livestock species. Chemotherapeutic treatment against T. evansi contagion is currently reliant on four drugs namely quinapyramine methyl sulphate/chloride, diminazene aceturate, isometamedium chloride and cymelarsan, in which first three have been available for more than half a century and latter one has been used for more than two decades (Desquesnes et al. 2013). However, the currently existing chemotherapeutic agents are not suitable due to their drug resistance, toxic effects and administration schedules (Kumar et al. 2016). Therefore, there is a need to search a non-toxic, economically viable and high therapeutic potential drug compound, which could affect the metabolic pathways essential for survival of parasite and also act as an alternative to currently available above-mentioned drugs, for combating animal trypanosomosis. In the present study, imatinib and sorafenib were selected to evaluate for their therapeutic potential against Trypanosoma evansi. Imatinib (Fig. 1A) is a tyrosine kinase inhibitor targeted cancer drug (biological therapy) and is also known by its brand name Glivec (Pardanani and Tefferi 2004; Patel et al. 2008). Along with neoplasias, imatinib has also been reported to have activity against various parasites such as Schistosoma mansoni (Beckmann et al. 2014; Buro et al. 2014), Filarial spp. (O’Connell et al. 2015, 2017) and Leishmania amazonensis (Wetzel et al. 2012). Moreover, imatinib has also been tested against the other strains of trypanosomes, T. brucei and T. cruzi (Behera et al. 2014; Simões-Silva et al. 2019). Similarly, the second drug sorafenib (Fig. 1B) was reported as an oral multikinase inhibitor and implicated in tumeriogenesis and tumour progression (Wilhelm et al. 2008; Patt et al. 2017). It has also been reported active against Leishmania donovani (Sanderson et al. 2014), and T. brucei (Guyett et al. 2016). Moreover, both the drugs, imatinib and sorafenib, in combined form have been showed a promising antimalarial activity with different strains of Plasmodium falciparum (Pathak et al. 2015). Both the drugs are protein kinase inhibitors and also available for anticancer activity commercially. Both have also been reported for induction of reactive oxygen species (ROS)–induced apoptosis in a cancer cell (Chang et al. 2011; Lange et al. 2016; Teppo et al. 2017). ROS are also produced by the cells in response against an infection, when infected by pathogens. Some drugs may also produce more level of ROS intracellularly and work on the similar mechanism as some antiprotozoal drugs killing parasites (Fonseca-Silva et al. 2013; Bombaça et al. 2019). This property of a drug is important so as to produce ROS which cause the demolition of cellular macromolecular components and leads to death of parasites. Sorafenib has been reported to induce a dosedependent generation of ROS in human HCC cell lines in vitro as well as in vivo (Coriat et al. 2012; Wan et al. 2013). Moreover, report also published on induction of apoptosis via oxidative stress due to more ROS generation by imatinib in gastric cancer cells (Kim et al. 2019). We also suggest repurposing of these drugs against T. evansi whether they may inhibit the growth of parasite in vitro and can also subside the process of new drug development. However, to date, no reports have been published on the anti-trypanosomal activity of selected drugs against T. evansi. Therefore, the aim of the present study is to investigate the growth inhibitory effect of imatinib and sorafenib against T. evansi. Furthermore, both were also evaluated for generation of ROS and their in vitro cytotoxicity. Materials and methods Propagation of T. evansi in culture medium In the present study, one cryostabillate of T. evansi isolate (T. ev-India-NRCE-Horse1, Hisar), maintained in Parasitology Laboratory of ICAR-National Research Centre on Equines (NRCE), Hisar, India, was used. Swiss albino male mice were used for the propagation of parasite for which each mouse was inoculated intra-peritoneally with 1 × 105 parasites. The mice showed high parasitaemia (1 × 108 trypanosomes/ml) on the 5th day post-infection; then from infected mouse blood, trypanosomes were purified as reported by Kumar et al. (2015). Prior approval was taken for animal experimentation in the present study from Institutional Animal Ethics Committee of ICAR-NRCE, Hisar. Trypanosomes were cultivated in HMI-9 medium modified slightly as described by Hirumi and Hirumi (1989). Briefly, Iscove’s Modified Dulbecco’s medium (IMDM) supplemented with 100 μM bathocuproic acid, 1 mM sodium pyruvate, 100 μM hypoxanthine, 16 μM thymidine, 2 μM mercaptoethanol, 1.94 μM L-cysteine, 60 μM HEPES, 4 mM L-glutamine, 0.4% BSA, 1% antibiotics and 0.001% holotransferrin, and 20% FBS. All chemicals used for preparation of HMI-9 medium were procured from Sigma-Aldrich Co. Then, the parasites were adapted and maintained in axenic culture condition for in vitro drug efficacy study. In vitro growth inhibition assay Before the start of experiment, parasite culture was initiated with a cell density of 105 cells/ml and maintained. After stabilization of axenic culture, 500 μl of the parasite culture was dispensed in each well of culture plate for experimental drug evaluation. Different concentrations of imatinib and sorafenib (Sigma-Aldrich Co.) in DMSO were tested against T. evansi parasite in triplicate. At regular interval of time (24 h, 48 h and72 h), the cell density was counted on a Neubauer haemocytometer. Parasite cultures without any drug molecule and cultures treated with DMSO (1%) were taken as experimental negative control and solvent control respectively. Moreover, quinapyramine methyl sulphate (QPS, TriquinS™, Vetoquinol India Animal Health Pvt. Ltd) was also used as the standard drug against T. evansi at concentration of 10 μg/ml (20 μM) for comparative evaluation with tested compounds. The parasite cultures treated with different concentrations of both the drugs were incubated in CO2 incubator (37 °C, 5% CO2) for a period of 72 h. The IC50 values for each drug molecule were calculated at 24 h using Graph Pad Prism software version 7.02. Intracellular ROS measurement assay The intracellular ROS level was measured by using 2′,7′dichlorofluorescin diacetate (DCF-DA) assay (SigmaAldrich). The reagent is a non-fluorescent probe with an ability of cell permeability and, upon oxidation with available reactive oxygen species, turns to extremely fluorescent compound 2′,7′-dichlorofluorescin. Therefore, this reagent employed for the evaluation of intracellular ROS and the fluorescence generated is directly proportional to the amount of ROS available intracellularly (Chen et al. 2010; Fonseca-Silva et al. 2011). Parasites (2 × 105 cells/ml) were cultured for 24 h in the presence of sorafenib and imatinib at its IC50 and IC100 value as calculated for T. evansi. The working stock in culture medium (20 μM) of DCF-DA (1 mM stock in DMSO) was added to the parasite culture and incubated for 30 min at 37 °C in a 5% CO2 incubator. The fluorescence intensity was detected using a plate spectrofluorometer (SpectraMax i3X Multimode Microplate Reader-Molecular Devices) at 480 and 520 nm for excitation and emission, respectively. Parasite culture treated with hydrogen peroxide (H2O2) solution and non-treated parasite culture was taken as positive control and negative control in the present experiment. Moreover, the fluorescence intensity was quantified at different time intervals such as at 0 h, 2 h, 4 h, 6 h and 24 h to evaluate the kinetics of ROS production. Percent ROS levels were measured in drug-treated and untreated trypanosomes according to the formula given below: In vitro cytotoxicity assay In vitro cytotoxicity effect in terms of percentage was determined for different concentrations of both the drugs on peripheral blood mononuclear cells (PBMCs) and Vero cell lines. Initial drug was chosen based on the IC50 value of respective drug, and then further 1×, 5× and 10× concentrations were taken to evaluate the cytotoxic effect. In vitro cytotoxicity assay on PBMCs Cytotoxicity assay using resazurin dye was done on PBMCs in 96-well culture plate. Density gradient method was used for isolation of PBMCs from fresh horse blood (Fuss et al. 2009). Diluted blood in isotonic phosphate buffer saline (PBS) pH.7.2 was added to histopaque-1077, lymphocyte separation media (LSM) (Sigma-Aldrich) in 1:2 ratio and centrifuged at 500g for 30 min at room temperature. At the interface of two liquids, a clear white ring was formed and it was transferred aseptically to another tube and further washing of cells was done with an equal volume of PBS. The pellet was resuspended in 1 ml of RPMI complete medium (containing 10% FBS, 1 ml penicillin 100 IU/ml and streptomycin 100 μg/ml solution, 1 ml 7% sodium bicarbonate solution). The live cells were counted in cell suspension using haemocytometer by equal volume of cell suspension and trypan blue stain. PBMCs (100 μl) at a density of 1 × 105 cells were cultured in a 96-well plate. For each drug, triplicate wells were treated with 100 μl of different concentration of imatinib (6.0 μM, 30 μM, 60 μM) and sorafenib (0.35 μM, 1.75 μM, 3.5 μM) and incubated for 72 h at 37 °C with 5% CO2 with the addition of concanavalin A (10 μg/ml) as it is a lymphocyte-stimulating agent. QPS (10 μg/ml, 50 μg/ml, 100 μg/ml) and DMSO (1%, 5%,10%) were also included in the experiment to serve as experimental reference control and solvent control respectively. After 72 h of treatment with drug, 25 μl of resazurin was added to all the wells. The reduction of blue (resazurin) to pink colour (resofurin) directly corresponds to the reduction in cytotoxicity. After 6 h of the dye addition, the pinkcoloured resorufin was formed in the metabolically active cells. Absorbance of samples was recorded using spectrophotometer at two wavelengths of 570 nm and 650 nm. OD value at 650 nm was deducted from OD value at 570 nm because 650 nm OD value was the normal basic OD value of plate. In vitro cytotoxicity assay on Vero cell lines Vero cell line was taken from the National Centre of Veterinary Type Cultures (NCVTC), NRCE, Hisar, India, and maintained in Eagle’s minimum essential medium (EMEM). For determination of cytotoxic activity of selected drugs, 100 μl of cell suspension (1 × 105/ml) was placed into each well and incubated for 24 h (Manuja et al. 2016). After 24 h incubation, plate was washed and treated with different concentrations of drugs (as mentioned in section In vitro cytotoxicity assay on PBMCs) for 72 h. Thereafter, 25 μl of resazurin was added to all the wells and percentage of cytotoxicity was calculated with reference to the control as mentioned above in Eq. 2. Statistical analysis Analysisofexperimental data was performed using Graph pad prism version 7.02 software to know anti-parasitic efficacy (IC50) of the tested drug molecules against T. evansi. The experimental data were statistically analysed using two-way ANOVA followed by Tukey’s multiple comparison test. Results are presented as mean ± standard deviation (SD). Differences were considered significant at p < 0.05. Results In vitro evaluation of imatinib and sorafenib based on growth inhibition efficacy against T. evansi Different concentrations of sorafenib and imatinib were per se evaluated for their effect on T. evansi for successive 3 days at 24-h, 48-h and 72-h interval. There was a significant (p < 0.0001) increase in parasite count after 24 h, 48 h and 72 h as compared with parasite count at 0 h in negative control wells. Imatinib concentrations from 2 to 15 μM did not show any significant effect on parasite concentration, while 20 μM and 25 μM showed significant (p < 0.001) or 100% parasite inhibition as compared with negative control at 24 h. At 48 h, only 15 μM, 20 μM and 25 μM of imatinib exhibited significant (p < 0.001) parasite inhibition. At 72 h, imatinib concentration (2 μM, 4 μM and 6 μM) did not show a significant decrease in parasite concentration as compared with negative control, while other higher concentrations of imatinib 8 to 25 μM showed significant (p < 0.001) decrease in parasite population (Fig. 2A). The IC50 value of imatinib against T. evansi was calculated at 24 h and found at 6.1 μM, while the complete growth inhibition was found at 20 μM concentration. Figure 2B depicts effect of different concentrations of sorafenib on growth of T. evansi. Sorafenib concentrations from 0.5 to 2.5 μM did not show any significant effect on parasite concentration, while 5 μM, 7.5 μM and 10 μM showed significant inhibitory effect p < 0.05, p < 0.001 and p < 0.001 respectively as compared with negative control at 24 h. At 48 h, 0.5 μM concentration of sorafenib did not exhibit any significant inhibitory effect, while other concentrations exhibited significant inhibitory effect on parasite count as compared with negative control. At 72 h, all concentrations of sorafenib from 0.5 μM to 10 μM showed high significance effect (p < 0.001) on parasite count as compared with the negative control group. The IC50 value of sorafenib for T. evansi was found to be 0.33 μM, and complete growth inhibition was found at 7.5 μM concentration at 24 h. QPS (10 μg/ml or 20 μM) was used as a reference drug in the present investigation, which exhibited significant (p < 0.001) inhibition of parasite at 24 h, 48 h and 72 h as compared with normal parasite control. Moreover, vehicle treated control (DMSO, 1%) showed significant increase in parasite count as compared with parasite count at 0 h and showed no inhibitory effect on T. evansi growth. Measurement of ROS generation Intracellular ROS levels were measured by using the cellpermeable dye DCFDA (Fonseca-Silva et al. 2011; Ferreira et al. 2018). The levels of ROS in drug-treated wells were increased with time from 0 to 24 h (Fig. 3). Both, sorafenib and imatinib, also showed dose-dependent generation of percent ROS as compared with the ROS produced in control cells during the experiment. The well treated with IC100 dose concentrations showed more ROS production as compared with the well treated with IC50 dose concentration. Sorafenib IC50 dose exhibited 11.2%, 13.7%, 18.3%, 20.7% and 50.4% ROS generation, while sorafenib IC100 dose exhibited 16.4%, 20.8%, 27.6%, 34.5% and 64.1% ROS generation as compared with untreated at 0 h, 2 h, 4 h, 6 h and 24 h respectively. Likewise, imatinib IC50 dose exhibited 9.4%, 12.1%, 17.7%, 21.5% and 48.1% ROS generation, while imatinib IC100 dose exhibited 13.8%, 16.2%, 21.6%, 34.6% and 58.6% ROS generation as compared with untreated at 0 h, 2 h, 4 h, 6 h and 24 h respectively. Moreover, QPS, the commercial drug, also showed a time-dependent ROS generation which was 4.9%, 6.3%, 9.8%, 22.9% and 52.9% as compared with untreated at 0 h, 2 h, 4 h, 6 h and 24 h respectively. In vitro cell cytotoxicity assay on peripheral blood mononuclear cells Cell cytotoxicity was calculated for the imatinib and sorafenib (selected drugs), QPS (positive control) and DMSO (solvent control) at different concentrations such as 1×, 5× and 10× on peripheral blood mononuclear cells (Fig. 4A). All the drug concentrations showed a dose-dependent cell cytotoxicity. Drug molecule imatinib showed less than 10% cytotoxicity at its 1× (6 μM), and 5× (30 μM) concentrations, while its 10× (60 μM) concentrations showed 19.56% cytotoxicity. The second drug molecule sorafenib showed below 3% cytotoxicity at its 1× (0.35 μM), 5× (1.75 μM) and 10× (3.5 μM) concentrations. Standard drug QPS also exhibited a dosedependent cytotoxicity such as 1.28%, 6.08% and 10.97% at its 1×, 5× and 10× drug concentrations, respectively. Vehicle control DMSO (1%) showed below 25% cytotoxicity at 10× (10%) concentration. In vitro cell cytotoxicity assay on Vero cell lines Figure 4B depicts the percent cytotoxicity of different concentrations on Vero cell lines. Drug molecules imatinib showed 3.95% and 8.89% cytotoxicity at its 1× (6 μM) and 5× (30 μM) concentrations respectively, while its 10× (60 μM) concentration showed 42.52% cytotoxicity. The second Fig. 3 Percent ROS generation of different concentrations of sorafenib and imatinib in Tryopanosoma evansi. Each value expressed as increase in percent ROS production as compared with untreated/control parasite. Values shown are the mean ± SD. H2O2 (100 μl) taken as a positive control. SC, sorafenib; IMA, imatinib; QPS, quinapyramine methyl sulphate; H2O2, hydrogen peroxide selected drug molecule sorafenib showed below 5% cytotoxicity at its 1× (0.35 μM), 5× (1.75 μM) and 10× (3.5 μM). Standard drug QPS exhibited less than 10% cytotoxicity at its 1×, 5× and 10× concentrations. Moreover, vehicle control DMSO (1%) showed below 40% cytotoxicity at 10× (10%) concentration. Discussion Imatinib and sorafenib, both have been reported as drug target for protein kinase which regulate different cellular processes like progression of cell cycle, differentiation of cell cycle and transcriptional (Naula et al. 2005; Johnson 2009). At present, identification of potent drug targets is the need to treat trypanosomatid-associated infections in human and livestock. Among various drug targets, protein tyrosine kinases inhibitors are druggable targets in trypanosomatid protozoan parasites and effective kinase inhibitors in one species would likely support similar studies in the other trypanosomatid species (Merritt et al. 2014). Imatinib and sorafenib have been reported as anticancer drugs which have already passed different phase trials of drug development and also available orally (Ranieri et al. 2012; Goswami et al. 2016). Current chemotherapies cannot be administered orally and showed problem regarding drug resistance and cytotoxicity. Therefore, a new therapeutic compound with oral bioavailability and less toxicity is required. Moreover, repurposing of these drugs against trypanosomes also sidelines the immense protocol of new drug development as some reports have already been published (Dichiara et al. 2017; SimõesSilva et al. 2019). Both the drugs have been reported for ROSmediated cell death in cancer cells. Along with anticancer activity, both also have been reported to possess therapeutic potential against different parasites as described in the “Introduction” section. Imatinib and sorafenib might have therapeutic potential against T. evansi as they have also been reported to possess potential against other species of trypanosome, T. cruzi and T. brucei (Behera et al. 2014; Guyett et al. 2016). The major goal of this investigation is to check the in vitro parasite growth inhibitory activity against T. evansi and the capacity of intracellular generation of ROS in parasite followed by cytotoxicity profile of selected drugs in axenic culture. In in vitro growth inhibition efficacy assay, both the drug molecules were tested for inhibition of trypanosome growth at different concentrations at 24 h, 48 h and 72 h and were found to be significant (p < 0.001) inhibitor of trypanosome growth. In the present study, imatinib and sorafenib showed IC50 at 6.11 μM and 0.33 μM drug concentration, respectively, against T. evansi. Behera et al. (2014) evaluated effect of imatinib against T. brucei (50% growth inhibitory concentration, GC50 > 10 μM) and had not observed any trypanocide effect on T. brucei. Recently, Simões-Silva et al. (2019) reported moderate activity of imatinib (EC50 = 24.8 ± 7.4 μM) in comparison to benznidazole (EC50 = 4.1 ± 1.3 μm), and out of 14 imatinib derivatives, one compound (3,2-difluoro-2phenylacetamide) showed EC50 = 7.4 μm, being 5-fold more active than imatinib and equally potent as benznidazole against T. cruzi. Moreover, sorafenib had also reported to possess antitrypanosomal potential against T. brucei (IC50 = 1.7 μM) (Guyett et al. 2016). In our investigation, the value of IC50 of the drug molecules, imatinib as well as sorafenib against T. evansi, was comparatively lower as reported by researcher earlier against other trypanosomes species. Furthermore, complete inhibition of parasite (100% inhibition of parasite replication and static effect) by imatinib was observed in range of 15–20 μM, while sorafenib showed 100% parasite inhibition in the range of 5–10 μM. From this growth inhibitory assay, it was concluded that both the drug molecules have growth inhibitory potential against T. evansi and sorafenib showed more efficacy against T. evansi as compared with imatinib.
Inmammalian cells,enhancement incellularROS level has been proposed to be responsible for dysfunction of mitochondria and leads to cell death (Bolisetty and Jaimes 2013). The mitochondria of T. evansi showed an inimitable structure in comparison to mammalian mitochondria that makes this organelle a drug target for researchers. Here, we determine for the first time that sorafenib and imatinib exert its inhibitory effect on T. evansi by generating ROS that might have effect on mitochondrial functions of parasite. To investigate the effect of drugs toward mitochondrial dysfunction, ROS level was measured using DCF-DA assay. The ROS level in treated wells was increased with time from 0 to 24 h. Different drug concentrations evaluated for ROS, i.e. sorafenib IC50 dose, sorafenib IC100 dose, imatinib IC50 dose, imatinib IC100 dose and QPS, showed a time-dependent effect on ROS production, and it was 45 to 65 times higher ROS as compared with normal untreated parasite culture at 24 h. Moreover, all the drug concentrations also exhibited comparable percent ROS production as QPS, a reference drug.
Different concentrations (1×, 5× and 10×) of imatinib and sorafenib wereevaluated for their cytotoxiceffect onVero cell lines and horse PBMCs. From the experimental evaluation, it was observed that imatinib showed up to 20% and 40% cytotoxicity at its 10× (60 μM) concentration on PBMCs and Vero cell lines respectively, although sorafenib showed up to 5% cytotoxicity at its 10× (3.5 μM) concentration on both the cell lines. Therefore, it was concluded that sorafenib is safer as compared with imatinib.
In this work, imatinib and sorafenib showed significant inhibitory effect with low IC50 value as compared with our standard reference drug QPS. Furthermore, both the drugs exhibited a dose-dependent and time-dependent increased ROS generation. Our investigation demonstrates, for the first time, growthinhibitioneffect and ROS productionbyimatinib and sorafenib treatment on T. evansi and suggests that this might be the basic mechanism of inhibition of cell growth in T. evansi.

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