Abstract: Leishmania major is causative agent of cutaneous leishmaniasis. Leishmaniasis is most public health problem in developing countries. Cantharidin is vesicant found in the Meloidae beetles hemolymph. Protein phosphatase 1 and 2A is the primary target of cantharidin. Cantharidin induce apoptosis in variant cancer cells. In this study, the effect of various concentrations of cantharidin on the Leishmania major viability was investigated in vitro by flow cytometry and MTT assay. Cantharidin also was applied as ointment by three doses (0.05, 0.5 and 0.1%) on leishmaniasis lesion in BALB/c for 4 weeks, quantity of parasite assessed by Real-Time PCR. MTT test results indicated that 0.5 μg mL-1 and 50 μg mL-1 cantharidin had 14.26 and 49.86% cytotoxicity in leishmania promastigotes after 72 h, respectively. But, flow cytometry data showed 14.29% cytotoxicity (as 13.12% apoptosis, 1.12% late apoptosis and 0.05% necrosis) and 68.51% cytotoxicity (as 5.28% apoptosis, 63.23% late apoptosis and 0% necrosis) in promastigotes due to 0.5 and 50 μg mL-1 cantharidin after 72 h, respectively. Mean of parasite load in control, 0.05, 0.1 and 0.5% cantharidin-treated groups were 85000, 2400, 2200 and 250 parasite/μL, respectively. It can be concluded that cantharidin induces apoptosis in the Leishmania major promastigotes and infected macrophages in the time and dose-dependent manner; also it can decrease parasite load in infected mice.
INTRODUCTION
Leishmania major is causative agent of Cutaneous Leishmaniasis (CL). Leishmaniasis is most health problem in developing countries. There is 12 million people infected with incidence of 1.5 to 2 million new case of CL, annually. There is no effective vaccine. Chemotherapy by antimony components, sodium stibogluconate and meglumine antimoniate is the most way to treatment. Resistance and relapse have been reported (Hepburn, 2000; Simranjeet et al., 2009).
Cantharidin is vesicant, principally found in Meloidae beetles hemolymph. Robiquet (1810) first isolated it from Lytta vesicaoria (Bonness et al., 2006). Cantharidin (2,6-Dimethyl-4,10-dioxatricyclo-decane-3, 5-dione) belongs to terpenoids and soluble in organic solvents. Chinese used it as traditional medicine about 2000 years ago (Moed et al., 2001; Deponte, 2008). About 100 years ago, blister beetles used to cure wart and cutaneous lesions (Moed et al., 2001). Hakim Jorjani described cantharidin to cure wart, hair loss, rabidity and black nails (Yousefi, 2004).
Cantharidin is protein phosphatase 1 and 2A (PP1 and 2A)inhibitor, PP1 and 2A are primary target of cantharidin (Bonness et al., 2006; Li and Casida, 1992; Greenway et al., 1995).
Apoptosis is programmed cell death, involved morphological changes (blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation) and biochemical changes (externalization of phosphatidyl serine in the plasma membrane, oxidative stress, release of proteins from mitochondria e.g., cytochrome c, endonuclease G, apoptosis-inducing factor, activity of proteases e.g., caspases) (Deponte, 2008; Shemarova, 2010). Cantharidin can induce apoptosis by increasing caspase 3, 8 and 9 caspase and block caspase inhibitors simultaneously (Wang et al., 2007; Huan et al., 2006; Sagawa et al., 2008). Sometimes cantharidin induces apoptosis through p53 pathway (Efferth et al., 2005; Huh et al., 2004; Kok et al., 2005; Huh et al., 2003). Several studies show that cantharidin induces apoptosis in various cancer cells (Wang et al., 2007; Sagawa et al., 2008; Fan et al., 2004; Rauh et al., 2007). Apoptosis occur in Leishmania by caspase analogs named metacaspase (Shaha, 2006; Barcinski and DosReis, 1999; Wanderley and Barcinski, 2010; Debrabant et al., 2003; Nguewa et al., 2004; Kosec et al., 2006). Leishmania inhibit macrophages from apoptosis. Macrophage has critical role in host immune response and cytokines release. L. donovani and L. major inhibit apoptosis of macrophages by remove macrophage colony-stimulating factor (M-CSF). L. major also prevent macrophage caspase 3 activation by inhibiting cytochrome c release from mitochondria (Moore and Matlashewski, 1994; Akarid et al., 2004).Therefore, activation of apoptosis is one of the most mechanisms to eliminate intracellular infections (Donovan et al., 2009; Valdes-Reyes et al., 2009). Anti-Leishmania drugs induce apoptosis in Leishmania. Miltefosine induces apoptosis in L. donovani through increase cytochrome c releasing followed by mitochondrial membrane permeability decrease (Verma et al., 2007).
The aim of present work was investigating the effect of cantharidin in inducing apoptosis in L. major.
MATERIALS AND METHODS
In vitro study: The present work is designed by an experimental study. The control groups included of Leishmania major promastigotes, macrophages and infected macrophages with Leishmania major and experimental groups included Leishmania promastigotes, macrophages and infected macrophages with L. major treated by various doses of cantharidin.
Cantharidin preparation: Cantharidin was purchased from Sigma (Germany). It dissolved in dimethyl sulfoxide (DMSO) and stored as 20% stock solution. Various concentrations 0.5, 1, 2, 5, 10, 20 and 50 μg mL-1 prepared by dissolving stock solution in the RPMI 1640 medium, then stored at 4°C.
Parasite culture: L. major (MRHO/IR/75/ER) was cultured in the RPMI 1640 medium (GibCo) with 10% heat-inactivated fetal bovine serum (FBS) (GibCo) and incubated at 21°C.
Peritoneal macrophages culture and infecting macrophages with Leishmania major: Resident peritoneal macrophages were harvested through injecting and aspirating phosphate buffered saline (PBS) to peritoneal cavity of BALB/c mice. Macrophages were cultured in the RPMI 1640 supplemented with 10% heat-inactivated FBS and 100 U mL-1 penicillin, 100 μg mL-1 streptomycin, then incubated at 37°C with 5% CO2 (Tanaka et al., 2007). Promastigotes in stationary phase was added to macrophage (10:1) in 24-well plates. After 6 h, supernatant was discharged to remove dead cells and non-entered promastigotes and fresh medium added to wells. Plate incubated at 37°C with 5% CO2.
MTT assay to promastigotes, macrophage and infected macrophages viability: By hemocytometer chamber (Neubauer chamber) 106 promastigotes in log phase were counted and cultured in RPMI 1640 medium with 10% FBS in 96 well plate (Nunc, Denmark).Cantharidin with final concentrations 0.5, 1, 2, 5, 10, 20 and 50 μg mL-1 was added to wells as triplicate. Plate incubated at 21°C. Plate was investigated by MTT [3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] (sigma, Germany) after 24, 48 and 72 h.MTT was dissolved in Phosphate Buffer Saline (PBS) by 5 mg mL-1 and was filtered by 0.2 μm filter. Then 20 μL of prepared MTT was added to each well (0.5 mg mL-1) and plate was incubated 3-5 h at 21°C in dark. Plate was centrifuged in 2500 rpm, 10 min, supernatant discharged and 100 μL DMSO added to wells. After 10 min Optical Density (OD) of plate was read by ELISA reader at 540 nm. Cytotoxicity percentage calculate by [1-(AT-AB)/(AC-AB)]x100. AB is OD of blank well, AC is OD of control and AT is OD of treated cells.
Flow cytometry: Annexin V-FITC Apoptosis Detection Kit (BioVision; USA) was used to flow cytometry assay. Leishmania, macrophage and infected macrophages were cultured in the 24-well plates with cantharidin (0.5, 1, 2, 5, 10, 20 and 50 μg mL-1 for Leishmania and 5, 20 and 50 μg mL-1 for macrophages and infected macrophages) as mentioned above. Promastigotes were collected after 24, 48 and 72 h, but macrophages and infected macrophages harvested by adding 0.1% trypsin and 0.2% EDTA as described (Macey, 2007). Samples centrifuged in 3000 rpm 5 min, supernatant was discharged and 500 μL binding buffer, 5 μL annexin and 5 μL Propidium Iodide (PI) added to residue. Samples were incubated at room temperature and dark for 5 min. Then the samples were analyzed with FACSCalibur flow cytometer (Becton Dickinson) and Cell Quest software.
In vivo study: Ointments doses (0.05, 0.1 and 0.5%) were made by dissolving cantharidin in eucerin as ointment base. Thirty BALB/c female mice (6-8 week) were infected by injection 2x106 mL-1 promastigotes in base of tail and divided them into 5 groups as control, eucerin, 0.05, 0.1 and 0.5% cantharidin ointment groups. Ointment applied daily once for 4 weeks.
Quantitative real-time PCR: Briefly, 100 mg of leishmaniasis lesion and spleen of some mice in our control and experimental groups cut off using scalpel. Samples maintained in -80°C freezer until use. RNA of samples was extracted by RNX -plus solution kit (cinnagen) and RT-PCR carried out by First Strand cDNA Synthesis Kit (Roche, Germany)according to the manufacture’s instruction. Real-Time PCR was performed by LightCycler 1.5 system and SYBR green I kit (Roche, Germany). To normalize the parasite load a 224 bp fragment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was amplified with the primer forward: 5’-TCAAGGGTGGTGCGAAGAAG-3’ and reverse: 5’-TCGCCGTGTAGGAGTGGATG-3’ designed previously (Pal et al., 2010). The reactions mixture included a 5 μL sample of DNA, 0.5 μm of each primer; 9 μL of PCR graded H2O and 4 μL of LightCycler® FastStart DNA Master SYBR Green I. The reaction was performed with an initial denaturation step at 95°C for 10 min, followed by 45 cycles of amplification (95°C/10 sec, 61°C/10 sec, 72°C/25 sec), melting curve (95°C/0 sec, 65°C/60 sec, 95°C Ramp Rate = 0.1°C/sec, 0 sec) and cooling (40°C/30 sec).
Standard preparation: PCR product was cloned in PTZ57/T by InsTAclone™ PCR Cloning Kit according to manufacture’s protocol (Fermentas, USA) and then transformed into the Escherichia coli strain TG1. The plasmids were purified by commercial kit (Roche, Germany), linearized and DNA concentrations were determined by UV spectrophotometry. Five 10-fold dilutions of DNA solution (45 ng mL-1 to 4.5 pg mL-1, containing about 183300000-1833 copies of the target/μL) were prepared triplicate to design standard curve. According to standards, samples were quantified.
Data analysis: Each experiment in vitro was performed in triplicate. Result showed as mean and standard deviation values. All statistical analysis of cytotoxicity in control and experimental group were performed using SPSS 16 for Windows. The non-parametric independent groups were compared with Mann-Whitney test.
RESULTS
In vitro Cytotoxic effect of cantharidin on L. major and macrophages by MTT assay: Cytotoxic effect of cantharidin with various doses on the L. major promastigotes, macrophages and macrophages infected with L. major was estimated by MTT assay 24, 48 and 72 h after incubation. Data showed in Table 1 and 2 present cell viability {Cytotoxicity % = 100 - cell viability %}. Cytotoxicity of Leishmania promastigotes due to minimum dose of cantharidin (0.5 μg mL-1) at 24, 48 and 72 h was 8.20, 31.47 and 14.26%, respectively. But cytotoxicity in Leishmania with maximum dose of cantharidin (50 μg mL-1) after 24, 48 and 72 h was 32.08, 55.54 and 49.86%, respectively. Data showed in Table 1 present cell viability % of Leishmania major promastiogotes.
Minimum dose of cantharidin (0.5 μg mL-1) caused 0, 13.83 and 15.23% cytotoxicity in macrophages after 24, 48 and 72 h, respectively. Cytotoxicity in macrophages with maximum dose of cantharidin (50 μg mL-1) after 24, 48 and 72 h was 0, 13.01 and 22.86%, respectively. Data showed in Table 2 present cell viability % of macrophages.
| Table 1: | Percentage of cell viability of promastigotes after treatment with different dose of cantharidin by MTT assay |
| aLeishmania major (L. m) | |
| Table 2: | Percentage of macrophages viability after treatment with different dose of cantharidin by MTT assay |
| Table 3: | Infected macrophages viability after treatment with different dose of cantharidin on macrophages infected with Leishmania major by MTT test |
| aLeishmania major (L. m) | |
Minimum dose of cantharidin (0.5 μg mL-1) caused 0, 5.04 and 2.33% cytotoxicity in infected macrophages after 24, 48 and 72 h, respectively. Cytotoxicity in infected macrophages with maximum dose of cantharidin (50 μg mL-1) after 24, 48 and 72 h was 10.08, 19.95 and 29.97%, respectively. Data showed in Table 3 present cell viability % of infected macrophages.
Flow cytometry results: A dot plot of PI on the y-axis against annexin V on the x-axis can be used to distinguish viable cells which are negative for both PI and annexin V, apoptotic cells(annexin V-positive but PI-negative) and late apoptotic cells (double-positive for PI uptake and annexin V staining)or necrotic cells (annexin V-negative but PI-positive).
Apoptosis in macrophages caused by 5, 20 and 50 μg mL-1 cantharidin after 48h was 34.90, 34.99 and 21.35%, respectively and necrosis was 7.27, 15.36 and 23.76%, respectively. But, apoptosis in infected macrophages caused by 5, 20 and 50 μg mL-1 cantharidin after 48h was 31.05, 43.50 and 43.42%, respectively and necrosis was 10.08, 11.98 and 17.11%, respectively. Apoptosis and necrosis in control group (macrophages without cantharidin) was 19.06% and 1.97% after 48 h, respectively (Fig. 1).
Apoptosis in L. major promastigotes due to minimum dose of cantharidin (0.5 μg mL-1) and maximum dose of cantharidin (50 μg mL-1) after 72 h was 13.12 and 63.23%respectively, but necrosis was 0.05 and 0%. Apoptosis and necrosis in control group (promastigotes without cantharidin) was 2.44 and 0.29 after 72 h, respectively. Other data of flow cytometry are shown in Fig. 2 and not mentioned in the context.
| Fig. 1(a-e): | Flow cytometry results show the effect of cantharidin with different concentrations on the non-infected macrophages and infected macrophages viability, (a) Control sample, (b) Macrophage with 5 μg mL-1 cantharidin after 48 h, (c) Macrophage with 50 μg mL-1 cantharidin after 48 h, (d) Infected macrophage with 5 μg mL-1 cantharidin after 48 h, (e) Infected macrophage with 50 μg mL-1 cantharidin after 48 h. Regions of quadrate show necrosis in upper lift, late apoptosis in upper right, apoptosis in low right and live cells in low lift |
In vivo Real-time PCR results: Amplification curve of samples was shown in Fig. 3a. Samples with high concentration amplified in low cycle show. Non-detected samples were amplified in more than 38 cycles. Standard curve was shown in Fig. 3b, according to standard curve Parasite load of samples was quantified.
Load of parasite in cutaneous lesion and in spleen in control, 0.05, 0.1 and 0.5% of cantharidin-treated groups is shown in Table 4. Parasites load mean in lesions samples of control and Eucerin-treated groups was 85000; it was 34000 and 136000 in spleen samples, respectively. Parasite load in lesion samples of 0.05% cantharidin-treated group was not detected exception one sample that was 11000; parasite load mean in spleen samples was about 5000.
| Fig. 2(a-h): | Flow cytometry results shown the effect of cantharidin with different concentrations on the promastigotes viability, (a) Control sample, (b) Promastigotes with 0.5 μg mL-1 cantharidin after 72 h, (c) Promastigotes with 1 μg mL-1 cantharidin after 72 h, (d) Promastigotes with 2 μg mL-1 cantharidin after 72 h, (e) Promastigotes with 5 μg mL-1 cantharidin after 72 h, (f) Promastigotes with 10 μg mL-1 cantharidin after 72 h, (g) Promastigotes with 20 μg mL-1 cantharidin after 72 h, (h) Promastigotes with 50 μg mL-1 cantharidin after 72 h. Regions of quadrate show necrosis in upper lift, late apoptosis in upper right, apoptosis in low right and live cells in low lift |
Parasite load in lesion samples of 0.1% cantharidin-treated group was not detected exception one sample that was 10000; parasite load mean in spleen samples was about 4500. In 0.5% cantharidin-treated group lesion and spleen samples parasite was not detected.
| Fig. 3(a-b): | (a) Amplification curve of samples by Light Cycler software and (b) Standard curve with efficiency 1.89 |
DISCUSSION
MTT results indicate that cantharidin with 0.5 and 50 μg mL-1 has 14.26 and 49.86% cytotoxicity in the promastigotes after 72 h. Cytotoxicity in control group was 0% by MTT assay. Flow cytometry results showed 14.29% cytotoxicity (as 13.12% apoptosis, 1.12% late apoptosis and 0.05% necrosis) and 68.51% cytotoxicity (as 5.28% apoptosis, 63.23% late apoptosis and 0% necrosis) in promastigotes due to 0.5 and 50 μg mL-1 cantharidin after 72 h respectively. Cytotoxicity in control group was 4.34% (as 2.44% apoptosis, 1.61% late apoptosis and 0.29% necrosis) by flow cytometry. Cytotoxicity of macrophages caused by 5 and 50 μg mL-1 cantharidin in MTT assay was 10.57 and 13.01% after 48 h, respectively. Cytotoxicity in control group was 0% by MTT assay. Flow cytometry results showed 43.78% (as 34.90% apoptosis, 1.61% late apoptosis and 7.27% necrosis) and 49.34% (as 21.35% apoptosis, 4.23% late apoptosis and 23.76% necrosis) cytotoxicity of macrophages caused by 5 μg mL-1 and 50 μg mL-1 cantharidin after 48 h respectively. Cytotoxicity in control group was 23.15% (as 19.06% apoptosis, 2.12% late apoptosis and 1.97% necrosis) by flow cytometry. Harvesting the macrophages using trypsin may increase apoptosis rate. By MTT assay, cytotoxicity of infected macrophages caused by 5 μg mL-1 and 50 μg mL-1 cantharidin was 3.56 and 19.95% after 48 h, respectively. Cytotoxicity in control group was 0% by MTT assay.
| Table 4: | Parasite load of samples quantified by Real-Time PCR |
| aNot detected | |
Flow cytometry results showed 44.44% (as 31.95% apoptosis, 3.31% late apoptosis and 10.08% necrosis) and 61.81% (as 43.42% apoptosis, 1.27 late apoptosis and 17.11% necrosis) cytotoxicity in infected macrophages caused by 5 and 50 μg mL-1 cantharidin after 48 h, respectively. Cytotoxicity in control group was 23.15% (as 19.06% apoptosis, 2.12% late apoptosis and 1.97% necrosis) by flow cytometry. Data of Real-Time PCR indicated decreasing parasite load in cantharidin-treated group. High parasite load in spleen of some sample indicated visceral leishmaniasis in these cases.
MTT results show that cytotoxicity at 72 h was less than it at 48 h. MTT test can assess only total cytotoxicity but can’t distinguish necrosis from apoptosis. Flow cytometry is an accurate test with ability to determine apoptosis, late apoptosis and necrosis.
Macrophages remove Leishmania guyanensis through apoptotic process include reactive oxygen intermediated (Sousa-Franco et al., 2006). It was found that size of lesions in treated groups was larger than it in control group. Cantharidin induces neutrophil inflammation by mediators including IL-8, TNF-α, IL-1β and C3a (Day et al., 2001). Neutrophils secrete variety cytokines that activate T cells. Moreover, neutrophils damage tissues via cytokine, leukotrienes and prostaglandins secretion (Wright et al., 2010).
It was concluded that cantharidin induces apoptosis in promastigotes in dose- and time-depended manner. Apoptosis in macrophages just induces by low concentrations (<10 μg mL-1) of cantharidin, high concentration (>10 μg mL-1) of cantharidin cause necrosis.
So far, there is no study about cantharidin to induce Leishmania apoptosis. In one study, cantharidin with concentrations of 0.5, 1, 2, 5, 10, 20 and 50 μg mL-1 inhibited the growth of Leishmania major promastigotes after 24 h and the resultant inhibition levels were 39.22, 41.95, 49.88, 54.78, 58.01, 68.30 and 80.04%, respectively. After 72 h, the mean number of amastigotes per macrophage in the culture using 2 μg mL-1 of cantharidin, (the 50% inhibitory concentration dose (IC50)), was 1.2 while it was 2.7 in the control group. This results obtained through direct counting by Neubauer chamber with a light microscope (Ghaffarifar, 2010).
It isn’t known how cantharidin induces apoptosis in Leishmania. Expression of PS on the outer layer of cell membrane of promastigotes is sign of apoptosis. Amastigotes through PS on outer layer of membrane don’t undergo apoptosis, but they use PS to enter into macrophages (Wanderley and Barcinski 2010).
CONCLUSION
Cantharidin with apoptosis inducer ability can be considered as drug to cutaneous leishmaniasis. It needs more investigation in vivo to determine cantharidin as cutaneous leishmaniasis drug.
ACKNOWLEDGMENTS
This study was performed as a PhD thesis and supported by the Tarbiat Modares University. The project was authorized by the Ethic Committee of Tarbiat Modares University. The authors declare no conflict of interest.