Cytotoxic action of a fraction rich in casearins on tumor cell lines

FWA Barros1, PMP Ferreira2, AG Santos3, VS Bolzani4, AJ Cavalheiro4, GCG Militão5, AS Meira1, PM Costa1, MO Moraes1, C Pessoa1. 1Federal University of Cear á , Department of Physiology and Pharmacology, 60.431-970, Brazil, 2Federal University of Piau í , Department of Biological Sciences, 64.600-000, Brazil, 3State University of S ã o Paulo, Faculty of Pharmaceutical Sciences, 14.801-902, Brazil, 4State University of S ã o Paulo, Chemistry Institute, 14.800-900, Brazil, 5Federal University of Pernambuco, Department of Physiology and Pharmacology, 50.670-901, Brazil

Introduction: Casearia sylvestris Swartz (Salicaceae) is used in folk Brazilian medicine as cicatrizant, topical anesthesic and to treat snakebites. It presents antileishmanial, trypanocidal, larvicidal, anti-inflammatory and antioxidant activities. Objective: To assess the in vitro cytotoxic activity of a Fraction rich in Casearinas (FC) isolated from Casearia sylvestris leaves on cancer cell lines. Methods: Initially, the chromatographic profile of FC was performed by liquid quantitative chromatography of high efficiency using UV at 230 nm. Secondly, the cytotoxicity of FC was evaluated by MTT assay on tumor leukemia (MOLT-4, HL-60, K-562, CEM), breast (Hs578-T, MDA/MB-231, MX-1), prostate (PC-3, DU-145), colon (HCT-8), nervous system (SF-295) and melanoma (MDA/MB-435, B16/F10) lines after 72h compound exposure (0.019-25 μg/mL). Then, HL-60-treated cells were evaluated for cellular membrane integrity, internucleosomal DNA fragmentation and mitochondrial depolarization by flow cytometry (Guava EasyCyte Mine) after 24h exposure. Cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin, at 37°C with 5% CO2. Negative control was incubated with the vehicle used to dilute the substance (DMSO 1.6%). Doxorubicin (Dox, 0.3 µg/mL) was used as positive control. Data were compared by one-way analysis of variance (ANOVA) followed by the Newman-Keuls test (P < 0.01). Results: The quantification of diterpene clerodanes based on a calibration curve for caseargrewiin F (correlation coefficient: R2 = 0.99; calibration curve: Y = 4.107X + 536575), revealed that the FC is represented by 56.5% (mg/g) of diterpene clerodanes. The compounds caseargrewiin F and casearin X were the main molecules found (9.9 and 14.2% respectively). FC showed potent cytotoxicity in vitro, with IC50 values lower than 0.5µg/mL against all cancer lines tested. Dox was also active on cell lines, with IC50 values ranging from 0.002 (MX-1) to 0.48 µg/mL (MDA/MB-435). Cytometry analyzes in HL-60 cells confirmed these cytotoxicity, since the both concentrations tested (0.4 e 0.8 μg/mL) were capable to reduce cell membrane integrity (93.6 ± 0.7 and 80.4 ± 1.3 %) and to cause dose-dependent internucleosomal DNA fragmentation (13.7 ± 1.5 and 67.8 ± 4.6 %) and mitochondrial depolarization (7.1 ± 0.5 and 12.3 ± 0.9%) when compared to the negative control (96.1 ± 0.8, 0.9 ± 0.1 and 1.1 ± 0.1 %), respectively (P < 0.01). In a similar way, Dox caused membrane disruption (92. 6 ± 0.7 %), DNA fragmentation (43.6 ± 4.3 %) and mitochondrial potential alterations (22.7 ± 1.9 %) (P < 0.01). Conclusion: The FC revealed potent cytotoxic activity on different neoplasic cell lines and its mechanism is probably triggered by apoptotic pathway(s).