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Effects of camptothecin derivatives and topoisomerase dual inhibitors on Trypanosoma cruzi growth and ultrastructure
- Otto Kischlat Lacombe†1,
- Aline Araujo Zuma†1,
- Camila Cristina da Silva1,
- Wanderley de Souza1, 2, 3 and
- Maria Cristina M Motta1Email author
© Lacombe et al.; licensee BioMed Central Ltd. 2014
Received: 10 January 2014
Accepted: 22 May 2014
Published: 10 June 2014
Trypanosoma cruzi is the etiological agent of Chagas’ disease that is an endemic disease in Latin America and affects about 8 million people. This parasite belongs to the Trypanosomatidae family which contains a single mitochondrion with an enlarged region, named kinetoplast that harbors the mitochondrial DNA (kDNA). The kinetoplast and the nucleus present a great variety of essential enzymes involved in DNA replication and topology, including DNA topoisomerases. Such enzymes are considered to be promising molecular targets for cancer treatment and for antiparasitic chemotherapy. In this work, the proliferation and ultrastructure of T. cruzi epimastigotes were evaluated after treatment with eukaryotic topoisomerase I inhibitors, such as topotecan and irinotecan, as well as with dual inhibitors (compounds that block eukaryotic topoisomerase I and topoisomerase II activities), such as baicalein, luteolin and evodiamine. Previous studies have shown that such inhibitors were able to block the growth of tumor cells, however most of them have never been tested on trypanosomatids.
Considering the effects of topoisomerase I inhibitors, our results showed that topotecan decreased cell proliferation and caused unpacking of nuclear heterochromatin, however none of these alterations were observed after treatment with irinotecan. The dual inhibitors baicalein and evodiamine decreased cell growth; however the nuclear and kinetoplast ultrastructures were not affected.
Taken together, our data showed that camptothecin is more efficient than its derivatives in decreasing T. cruzi proliferation. Furthermore, we conclude that drugs pertaining to a certain class of topoisomerase inhibitors may present different efficiencies as chemotherapeutical agents.
KeywordsCell proliferation Kinetoplast Nucleus Topoisomerase inhibitors Trypanosomatid protozoa Ultrastructure
The Trypanosomatidae family comprises protozoa of medical and veterinary importance. This group includes species that are the etiological agents of numerous human diseases, such as Chagas’ disease (caused by Trypanosoma cruzi), African sleeping sickness (caused by Trypanosoma brucei), and leishmaniasis (caused by Leishmania spp). Chagas’ disease was discovered in 1909 and nowadays affects about 8 million people in Latin America and new cases are being reported in non-endemic areas due to emigrations .
T. cruzi is a flagellated protozoan and like other trypanosomatids presents a single mitochondrion with an enlarged region, termed kinetoplast, which contains the mitochondrial DNA (kDNA). T. cruzi also has a single spherical nucleus presenting a condensed heterochromatin next to the nuclear envelope and around the nucleolus [2–6]. Since the nucleus and the kinetoplast are cellular compartments that contain DNA, their structural organization depends on enzymes such as topoisomerases, that play a key role during replication, transcription, recombination and repair [7–9].
DNA topoisomerases are classified into type I and type II. Type I attaches to DNA and breaks one strand of the double helix that can rotate around its own axis to revert supercoiling. On the other hand, type II binds to a DNA double strand and makes a gate allowing a second DNA double helix pass .
Several topoisomerase inhibitors have been developed based on different types of these enzymes that have been considered as potent targets in chemotherapeutic studies, especially with tumor cells. Topo I inhibitors, such as camptothecin, form a ternary complex, since they can trap the enzyme and DNA together [11–14]. Topo II inhibitors, such as mitoxantrone and etoposide, bind to the enzyme preventing the re-ligation of the DNA double strand. Furthermore, some inhibitors share characteristics of the two groups described above and target both topo I and topo II, thereby being called dual inhibitors .
Many topoisomerase inhibitors are natural products extracted from plants, such as camptothecin, isolated from Camptotheca accuminata, and several alkaloids, such as evodiamine, isolated from Evodia rutaecarpa. Topotecan and irinotecan are camptothecin derivatives that have been used for ovarian and colorectal cancer treatments, respectively. These inhibitors target topo I and bind to DNA, forming a cleavable complex. The collision between this ternary complex and a replication fork generates DNA double-strand breaks, which may be related to the S-phase cytotoxicity, the G2/M cell cycle arrest and DNA damage that activates repair proteins .
Baicalein, luteolin and evodiamine are topoisomerase dual inhibitors. Baicalein is an alkaloid isolated from Scutellaria baicalensis used in the treatment of hypertension, atherosclerosis, dysentery and inflammatory diseases . Luteolin is a flavonoid, a group of natural compounds with therapeutic properties that causes apoptosis in promastigote forms of L. donovani[17–19]. Evodiamine is an alkaloid extracted from Evodia rutecarpa used as an anticancer, anti-inflammatory and antiobesity agent . This compound was initially classified as a topo I inhibitor, but then it was proposed that evodiamine could also bind to topoisomerase II .
In the present work, we evaluated the effects of the eukaryotic topoisomerase I inhibitors, topotecan and irinotecan, and the eukaryotic dual inhibitors baicalein, luteolin and evodiamine on the epimastigote forms of T. cruzi, considering its proliferation and ultrastructural organization.
Materials and methods
T. cruzi epimastigote forms were grown at 28°C for 24 h in liver infusion tryptose (LIT) medium  supplemented with 10% fetal calf serum.
Topotecan, irinotecan, baicalein, luteolin and evodiamine were purchased from Sigma Aldrich and diluted in dimethyl sulfoxide (DMSO) at 5 mM and 30 mM. The drug was added to the culture medium after 24 h of initial growth, which corresponds to the exponential phase. Drug concentrations were used as follows: 1, 5, 10, 50, 100, 200 and 300 μM. Every 24 h cells were collected and counted in a Neubauer chamber during the 96 h of cultivation. Paired t-tests were applied to the results using 95% confidence interval (GraphPad Prism version 5.00 for windows; GraphPad Software Inc., San Diego, CA).
Cell viability was performed using the MTS/PMS colorimetric method, which is based on dehydrogenase activity and the conversion of MTS into formazan, that indicates the number of metabolically active cells . Parasites were incubated with MTS/PMS solution for 4 h and formaldehyde 0.4% was used as negative control. The percentage of viable protozoa was obtained through a spectrofluorimeter (Molecular Devices Microplate Reader (SpectraMax M2/M2e, Molecular Devices) using a 490 nm wavelength. MTS/PMS is a colorimetric assay, based on dehydrogenase activity and the conversion of MTS into formazan, that indicates the number of metabolically active cells.
Transmission electron microscopy
Protozoa were fixed in 2.5% glutaraldehyde diluted in 0.1 M cacodylate buffer (pH 7.2) for 1 h at room temperature and were washed in the same buffer. Cells were post-fixed for 1 h in 0.1 M cacodylate buffer containing 1% OsO4 and 0.8% potassium ferricyanide. Protozoa were washed in the same buffer and were dehydrated in a graded series of acetone and embedded in Epon (Electron Microscopy Sciences, Hatfield, PA). Ultrathin sections were stained with uranyl acetate and lead citrate and were observed using a Zeiss 900 transmission electron microscope (Zeiss, Oberkochen, Germany).
In terms of T. cruzi ultrastructure, the parasites treated with dual inhibitors did not present alterations when compared to the control cells. These compounds did not lead to modifications in the kDNA topology or in the heterochromatin organization, as was observed after treatment with topo I inhibitors (data not shown for Additional file 3).
Effects of topoisomerase I and dual inhibitors on Trypanosoma cruzi after 72 h of treatment
Eukaryotic Topo I Inhibitor
Unpacking of nuclear heterochromatin and mitochondrial swelling
Eukaryotic Topo I Inhibitor
Eukaryotic Dual Inhibitor
Eukaryotic Dual Inhibitor
Eukaryotic Dual Inhibitor
In the present work, the effects of different topoisomerase inhibitors were evaluated considering T. cruzi proliferation and ultrastructure. Irinotecan and topotecan are derivatives of camptothecin, thus they act by binding to DNA and to topoisomerase I by forming a ternary complex, referred to as a cleavable complex. These compounds interfere with the re-join of the double-strand break, leading to cell cycle blockade, activation of DNA repair and apoptosis .
Here, we observed that topotecan promoted a moderate effect on cell proliferation, whereas irinotecan did not affect protozoa growth. These results revealed that such inhibitors were not efficient in impairing T. cruzi growth when compared to camptothecin, the precursor compound, which presented IC50 values of 2.08 μM. The typical ultrastructural alterations, such as the unpacking of nuclear heterochromatin, promoted by topoisomerase I inhibitors were observed in cells treated with topotecan; however such modifications were only noticed after using high drug concentrations .
As described previously, topotecan and irinotecan were able to inhibit tumor cell proliferation, and were more effective and less toxic than camptothecin [26, 27]. Such effects have also been reported on T. brucei and on Leishmania infantum promastigotes. In both these trypanosomatid species topotecan presented more efficacy than irinotecan, especially in T. brucei. The IC50 values correspond to 1.23 μM for topotecan and 21.5 μM for irinotecan on T. brucei, whereas values are equivalent to 10.86 μM for topotecan and superior to 200 μM for irinotecan on L. infantum[28, 29]. A previous work also showed that camptothecin was cytotoxic to T. brucei and L. donovani, with IC50 values ranging from 1 to 3 μM .
Baicalein was the most effective compound against T. cruzi proliferation and viability considering all the inhibitors evaluated in this study. The treatment of Leishmania promastigotes with concentrations inferior to 15 μM of baicalein for 24 h was previously reported to reduce parasite growth up to 89% . Furthermore, published data demonstrated that this drug inhibited tumor cell growth in vitro and in vivo and presented low toxicity [16, 32].
Baicalein, evodiamine and luteolin are all classified as dual inhibitors of topoisomerase; but the latter compound did not promote any effect on T. cruzi cell proliferation. However, luteolin inhibits the growth of several cancer cell lines, blocking the cell cycle in the G1 phase . In Leishmania, this inhibitor was also able to induce cell cycle arrest and apoptosis . In this work, evodiamine promoted a slight inhibition of T. cruzi proliferation (IC50 90 μM) when compared to baicalein (IC50 62.83), however this compound has presented efficacy against different cancer cell lines . Dual inhibitors target topoisomerases I and II, thus it was expected that such compounds could present high efficiency in blocking cell proliferation and also promoting ultrastructural changes in the nucleus and kinetoplast; however these effects were not observed in T. cruzi after treatment with these inhibitors.
DNA topoisomerases represent an interesting target for anti-parasitic chemotherapy, since their inhibition interferes with the replicative process, which can lead to parasite death. In this work, we showed that compounds pertaining to the same topoisomerase inhibitor class had different effects on T. cruzi proliferation and ultrastructure. All inhibitors evaluated in this work are efficient for cancer therapy and sometimes blocked trypanosomatid growth, however their effects on T. cruzi proliferation and ultrastructure had never been investigated. Thus, we considered that they could be promissory agents in chemotherapeutic studies against T. cruzi, however these compounds presented considerably high IC50 values. The low effects observed in this parasite can be related to distinct factors such as the differences in human and protozoan topoisomerase domains, affinity for the target enzyme, cell membrane permeability and cell resistance, including mechanisms of drug efflux. Our results reinforce the idea that it is necessary to develop new compounds that may be successfully used in the therapy against neglected diseases.
We would like to thank to Rachel Rachid for technical assistance. This work was supported by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
- Rassi JRA, Rassi A, Resende MJ: American Trypanosomiasis (Chagas Disease). Infect Dis Clin North Am. 2012, 26: 275-291. 10.1016/j.idc.2012.03.002.View ArticlePubMedGoogle Scholar
- Ogbadoyi E, Ersfeld K, Robinson D, Sherwin T, Gull K: Architecture of the Trypanosoma brucei nucleus during interphase and mitosis. Chromosoma. 2000, 108: 501-513. 10.1007/s004120050402.View ArticlePubMedGoogle Scholar
- Elias MCQB, Faria M, Mortara RA, Motta MCM, De Souza W, Thiry M, Schenkman S: Chromosome localization changes in the Trypanosoma cruzi nucleus. Eukaryot Cell. 2002, 1: 944-953. 10.1128/EC.1.6.944-953.2002.View ArticlePubMedPubMed CentralGoogle Scholar
- De Souza W: Basic cell biology of Trypanosoma cruzi. Curr Pharm Des. 2002, 8: 269-285. 10.2174/1381612023396276.View ArticlePubMedGoogle Scholar
- Motta MCM, De Souza W, Thiry M: Immunocytochemical detection of DNA and RNA in endosymbiont-bearing trypanosomatids. FEMS Microbiol Letters. 2013, 221: 17-23.View ArticleGoogle Scholar
- Jensen RE, Englund PT: Network News: The replication of kinetoplast DNA. Annu Rev Microbiol. 2012, 66: 473-491. 10.1146/annurev-micro-092611-150057.View ArticlePubMedGoogle Scholar
- Cortes F, Pastor N, Mateos S, Dominguez I: Roles of DNA topoisomerases in chromosome segregation and mitosis. Mutat Res. 2013, 543: 59-66.View ArticleGoogle Scholar
- Obado SO, Bot C, Nilsson D, Andersson B, Kelly JM: Repetitive DNA is associated with centromeric domains in Trypanosoma brucei but not Trypanosoma cruzi. Genome Biol. 2007, 8: R37-10.1186/gb-2007-8-3-r37.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang JC: Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev Mol Cell Biol. 2012, 3: 430-440.View ArticleGoogle Scholar
- Champoux JJ: DNA Topoisomerases: structure, function, and mechanism. Annu Rev Biochem. 2001, 70: 369-413. 10.1146/annurev.biochem.70.1.369.View ArticlePubMedGoogle Scholar
- Hsiang YH, Lihou MG, Liu LF: Arrest of replication forks by drug-stabilized topoisomerase I-DNA cleavable complexes as a mechanism of cell killing by camptothecin. Cancer Res. 1989, 49: 5077-5082.PubMedGoogle Scholar
- D’Arpa P, Beardmore C, Liu LF: Involvement of nucleic acid synthesis in cell killing mechanisms of topoisomerase poisons. Cancer Res. 1990, 50: 6919-6924.PubMedGoogle Scholar
- Tsao YP, D'Arpa P, Liu LF: The involvement of active DNA synthesis in camptothecin-induced G2 arrest: altered regulation of p34cdc2/cyclin B. Cancer Res. 1992, 52: 1823-1829.PubMedGoogle Scholar
- Teicher BA: Next generation topoisomerase I inhibitors: rationale and biomarker strategies. Biochem Pharmacol. 2008, 75: 1626-1271.View ArticleGoogle Scholar
- Motta MCM: Kinetoplast as a potential chemotherapy target of trypanosomatids. Curr Pharm Des. 2008, 14: 847-854. 10.2174/138161208784041051.View ArticlePubMedGoogle Scholar
- Li-Weber M: New therapeutic aspects of flavones: The anticancer properties of Scutellaria and its main active constituents Wogonin, Baicalein and Baicalin. Cancer Treat Rev. 2009, 35: 57-68. 10.1016/j.ctrv.2008.09.005.View ArticlePubMedGoogle Scholar
- Havsteen B: Flavonoids, a class of natural products of high pharmacological potency. Biochem Parmacol. 1983, 32: 1141-1148. 10.1016/0006-2952(83)90262-9.View ArticleGoogle Scholar
- Mittra B, Saha A, Chowdhury AR, Pal C, Mandal S, Ukhopadahyay S, Bandyopadahyay S, Majumder HK: Luteolin, an abundant dietary component is a potent anti-leishmanial agent that acts by inducing topoisomerase II-mediated kinetoplast DNA Cleavage Leading to Apoptosis. Mol Med. 2000, 6: 527-541. 10.1007/s0089400060527.View ArticlePubMedPubMed CentralGoogle Scholar
- Das A, Dasgupta A, Sengupta T, Majumder HK: Topoisomerases of kinetoplastid parasites as potential chemotherapeutic targets. Trends Parasitol. 2004, 20: 381-386. 10.1016/j.pt.2004.06.005.View ArticlePubMedGoogle Scholar
- Chan ALF, Chang WS, Chen LM, Lee CM, Chen CE, Lin CM, Hwang JL: Evodiamine stabilizes topoisomerase I-DNA cleavable complex to inhibit topoisomerase I activity. Molecules. 2009, 14: 1342-1352. 10.3390/molecules14041342.View ArticlePubMedGoogle Scholar
- Pan X, Hartley JM, Hartley JA, White KN, Wang Z, Bligh SWA: Evodiamine, a dual catalytic inhibitor of type I and II topoisomerases, exhibits enhanced inhibition against camptothecin resistant cells. Phytomedicine. 2012, 7: 618-624.View ArticleGoogle Scholar
- Camargo EP: Growth and differentiation in Trypanosoma cruzi. I. Origin of metacyclic trypanosomes in liquid media. Rev Inst Med Trop. 1964, 6: 93-100.Google Scholar
- Henriques C, Moreira TLB, Maia-Brigagão C, Henriques-Pons A, Carvalho TMU, De Souza W: Tetrazoluim salt based methods for high-throughput evaluation of anti-parasite chemotherapy. Analytical Methods. 2011, 3: 2148-2155. 10.1039/c1ay05219e.View ArticleGoogle Scholar
- Pommier Y: Topoisomerase I, inhibitors: camptothecins and beyond. Nat Rev Cancer. 2006, 6: 789-802. 10.1038/nrc1977.View ArticlePubMedGoogle Scholar
- Zuma AA, Cavalcanti DP, Maia MC, De Souza W, Motta MCM: Effect of topoisomerase inhibitors and DNA-binding drugs on the cell proliferation and ultrastructure of Trypanosoma cruzi. Int J Antimicrob Agents. 2011, 37: 449-456. 10.1016/j.ijantimicag.2010.11.031.View ArticlePubMedGoogle Scholar
- Cohen DP, Adams DJ, Flowers JL, Wall ME, Wani MC, Manikumar G, Colvin OM, Silber R: Pre-clinical evaluation of SN-38 and novel camptothecin analogs against human chronic B-cell lymphocytic leukemia lymphocytes. Leuk Res. 1999, 23: 1061-1070. 10.1016/S0145-2126(99)00133-2.View ArticlePubMedGoogle Scholar
- Bailly C: Homocamptothecins: potent topoisomerase I inhibitors and promising anticancer drugs. Crit Rev Oncol Hematol. 2002, 45: 91-108.View ArticleGoogle Scholar
- Deterding A, Dungey FA, Thompson K, Steverding D: Anti-trypanosomal activities of DNA topoisomerase inhibitors. Acta Tropica. 2005, 93: 311-316. 10.1016/j.actatropica.2005.01.005.View ArticlePubMedGoogle Scholar
- Prada CF, Alvarez-Velilla R, Balaña-Fouce R, Prieto C, Calvo-Alvarez E, Escudero-Martinez JM, Requena JM, Ordonez C, Desideri A, Perez-Pertejo Y, Reguera RM: Gimatecan and other camptothecin derivatives poison Leishmania DNA-topoisomerase IB leading to a strong leishmanicidal effect. Biochem Pharmacol. 2013, 10: 1433-1440.View ArticleGoogle Scholar
- Bodley AL, Shapiro TA: Molecular and cytotoxic effects of camptothecin, a topoisomerase I inhibitor, on trypanosomes and Leishmania. Proc Natl Acad Sci U S A. 1995, 9: 3726-3730.View ArticleGoogle Scholar
- BoseDasgupta S, Das BB, Sengupta S, Ganguly A, Roy A, Dey S, Tripathi G, Dinda B, Majumder HK: The caspase-independent algorithm of programmed cell death in Leishmania induced bybaicalein: the role of LdEndoG, LdFEN-1 and LdTatD as a DNA ‘degradesome’. Cell Death Differ. 2008, 10: 1629-1640.View ArticleGoogle Scholar
- Liu JJ, Huang TS, Cheng WF, Lu FJ: Baicalein and baicalin are potent inhibitors of angiogenesis: inhibition of endothelial cell proliferation, migration and differentiation. Int J Cancer. 2003, 106: 559-565. 10.1002/ijc.11267.View ArticlePubMedGoogle Scholar
- Lin Y, Shi R, Wang X, Shen H: Luteolin, a flavonoid with potentials for cancer prevention and therapy. Curr Cancer Drug Targets. 2008, 7: 634-646.View ArticleGoogle Scholar
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