Reagents
RPMI-1640 and fetal calf serum (FCS) were purchased from GIBCO; In Situ Cell Death Detection Kit (TUNEL Kit) from Roche Applied Science, USA; Miltefosine from Zentaris, GmbH and Ethanol from Merck. Caspase -3 Assay Kit, Gallic acid, Sodium Dodecyl Sulphate (SDS), MTT, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide, 2'-7'-Dichlorodihydrofluorescein diacetate (H2DCFDA), JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimidazolylcarbocyanine iodide), Acridine Orange, Ethidium Bromide, Propidium Iodide (PI) and RNase A were purchased from Sigma (St. Louis, MO, USA). All other reagents used were of the highest analytical grade possible.
In vitro culture of parasites
Promastigotes of the WHO reference strain Leishmania donovani Dd8 (MHOM/IN/80/Dd8) was kindly provided byDr. Patole (NCCS, India). They were cultured in RPMI-1640 medium supplemented with 10%heat-inactivated fetal calf serum at 23°C. For all experiments carried out, promastigotes in the latelogarithmic phase of growth were used.
In vitro cytotoxicity measurement
Late log-phase promastigotes (5 × 105 per well) were resuspended in fresh RPMI medium and incubated in 96-well culture plates with increasing concentrations of Gallic Acid (0, 0.01, 0.05, 0.1, 1 and 2 mM) or Miltefosine (0, 0.01, 0.025, 0.06, 0.125 and 0.25 mM) for 24 h and promastigote viability was evaluated by the quantitative colorimetric MTT assay (Pai et al. 1997). Briefly, after drug treatment, MTT (0.4 mg/ml) was added to each well and plates were incubated for 4 h at 23°C. The purple formazan crystals were solubilized in 10% SDS (W/V) and absorbance was measured in a microplate reader at 550 nm. The concentration of drug that decreases cell growth by 90% (IC90) was calculated by the formula given:
% Inhibition = 100 – [Absorbance of Drug treated cells / Absorbance of Untreated cells].
The results were expressed as the means of three independent experiments and their standard errors.
Cell size analysis
Promastigotes were treated with the antileishmanial drugs for 24 h, washed with 1×PBS and fixed in 2% (V/V) formaldehyde. Control included promastigotes without any drug treatment. The difference in the cell size of 1 mM Gallic acid or 0.2 mM Miltefosine treated and untreated cells was analyzed on a Partec Flow Cytometer (Sysmex Partec, GmbH, Münster, Germany) where, a forward scatter Vs side scatter was plotted for 20,000 cells (Paris et al. 2004).
Analysis of cellular morphology
The morphology of promastigotes after Gallic acid or Miltefosine treatment for 24 h was determined under a phase contract microscope (Carl Zeiss, Axioskop 40, 100X/ 1, 30 Oil immersion/ 0, 17 Plan - NEOFLUAR with an ocular W-PI 10X/ 23) and images were captured using AxioCam MRC camera and analyzed by AxioVision LE Rel. 4.2 software.
Measurement of Reactive Oxygen Species (ROS)
ROS generation in Leishmania promastigotes was measured by fluorescent probe H2DCFDA. 1 × 106 Promastigotes were incubated with or without 1 mM Gallic acid or 0.2 mM Miltefosine for 0-6 h. After each time point, live cells were washed and then probed with H2DCFDA (100 μM) for 30 min at 23°C. Intracellular ROS was measured fluorometrically (Dutta et al. 2007) on Chameleon fluorescence plate reader at excitation 485 nm and emission 535 nm and analyzed by Micro Win software.
In situ detection of DNA fragmentation by terminal deoxynucleotidyltransferase (TdT)-mediated dUTP end labeling (TUNEL)
DNA fragmentation with the untreated and drug treated promastigotes was analyzed in situ by TUNEL (Roche Applied Science, Indianapolis, IN, USA) assay, according to the manufacturer’s instructions with some modifications. Briefly, after 24 h of 0.2 mM Miltefosine or 1 mM Gallic acid treatment, promastigotes were fixed in 4% formaldehyde solution for at least 30 min, washed and permeabilized by a mixture of 0.1% Sodium citrate and 0.2% Triton X-100 in PBS for 10 min. Permeabilized cells were washed and fragmented DNA was tagged with TUNEL Reaction Mixture for 1 h. At least 20,000 cells were washed and acquired on a Partec flow cytometer (Sysmex Partec, GmbH, Münster, Germany) by a blue laser at an excitation of 488 nm and emission detection at 530 nm. The data was analyzed by FloMax software.
Mitochondrial membrane potential determination (ΔΨm)
Difference in the mitochondrial membrane potential in untreated and drug treated promastigotes was detected by JC-1 (Mukherjee et al. 2009). Briefly, after individual drug treatments, promastigotes were washed and incubated with JC-1 (1μM) for 30 min at 37°C. Subsequently, after washing 20,000 cells were acquired on Partec flow cytometer (Sysmex Partec, GmbH, Münster, Germany) and ratio of J-aggregate forms of JC-1 (excitation at 535 nm and emission at 600 nm) against the monomeric form (excitation at 485 nm and emission at 535 nm) was plotted using the FloMax software for analysis.
Acridine Orange/ Ethidium Bromide staining for Apoptosis detection
Treated or untreated promastigotes were washed and suspended in Acridine Orange and Ethidium Bromide (100 μg/ml) (Kern and Kehrer, 2002; Liegler et al., 1995). At least 20,000 cells were acquired on a Partec flow cytometer (Sysmex Partec, GmbH, Münster, Germany) and analyzed by FloMax software. A ratio of green fluorescence (excitation at 485 nm and emission at 535 nm) Vs red fluorescence (excitation at 535 nm and emission at 630 nm) was plotted.
Flow cytometry analysis of cell cycle
After drug treatments, promastigotes were washed and incubated at -20°C overnight in 70% (V/V) ethanol. Cells were washed and resuspended in 100 μg/ml Propidium Iodide (PI) in 1×PBS containing RNase A [0.1 mg/ml] ( Sen et al. 2004; Sen et al. 2007). The fluorescence intensity of PI incorporated in 20,000 promastigotes was analyzed on a FACS Partec flow cytometer (Sysmex Partec, GmbH, Münster, Germany) and FloMax software upon excitation at 535 nm and emission at 630 nm.
Determination of Caspase 3 activity
To determine the role of caspase 3 in Gallic acid induced Leishmania cytotoxicity, Caspase 3 Assay Kit (colorimetric) was used according to the manufacturer’s protocol. Briefly, 5 × 106 Leishmania promastigotes were treated with 1 mM Gallic acid or 0.2 mM Miltefosine for 24 h at 23°C. Promastigotes were washed and lysed in 50 μl of lysis buffer on ice for 20 min. Cell lysates were collected after centrifugation at 600 g for 20 min at 4°C. 5 μl of cell lysate or positive control (5 μg/ml Caspase 3, supplied by the kit) was mixed with 85 μl Assay buffer in a 96-well microtiter plate. 10 μl Caspase 3 substrate (acetyl-Asp-Glu-Val-Asp p-nitroanilide) was added to each well and incubated at 37°C for 2 h. Plate was read at 405 nm in an ELISA plate reader.
Estimation of hemolysis (%) for Gallic acid-induced toxicity by Human Red Blood Cells (RBC) lysis assay
Drugs, their metabolites, or excipients used in formulation can cause toxic hemolysis. Therefore, the FDA recommends testing for hemolytic potential (FDA guidance for Industry). Any drug administered needs to be checked for its cytotoxicity, as high concentration (IC90 1mM) of gallic acid was used against parasites in the present study so RBC hemolysis assay was carried out to evaluate the toxicity of compounds or drugs. The collection of human blood and ex vivo assessment of toxicity of gallic acid by RBC lysis assay was carried out after pre-approval by the Institutional Ethical Committee (IEC) (SPPU/IEC/2019/53 dated: 4/10/2019). Briefly, 5 ml blood was drawn from a healthy donor directly into K2-EDTA-coated Vacutainer tubes to prevent coagulation. This was followed by centrifugation of the blood at 500×g for 5 min and removal of the upper yellowish plasma which was discarded into biohazard waste after bleach treatment. The RBCs were washed with 1×PBS and centrifuged at 500×g for 5 min and this was carried out twice. Human RBC suspension was prepared in 10mM PBS, pH-7.4. RBC suspension was incubated with different concentrations of drug ranging from 1×IC90 up to 32×IC90 for different time intervals such as 1, 3, 6, 12 and 24 h. RBC suspension treated with 2% Triton X-100 was taken as positive control while, RBC suspension containing 1×PBS was negative control. After incubation, cells were centrifuged at 2000 rpm for 5 min and supernatant was taken in microtiter plate. Absorbance was measured at 541 nm for the estimation of hemolysis as a function of hemoglobin absorbance. Percent hemolysis was calculated using the following formula –
Statistical analysis
Data are expressed as mean ±S.E.M. unless mentioned. Comparisons were made between treatments and untreated controls using unpaired Student’s t-test. P-values <0.05 were considered significant.