Evaluation of the antitumor and antiangiogenic potential of two new analogues of thalidomide

PM Costa1,3, PMP Ferreira2, AS Meira3, FWA Barros3, VS Bolzani4, ACL Leite5, SMT Cavalcanti5, MO Moraes3, C Pessoa3. 1State University of Ceara, Morphological Sciences 60740 903, Brazil, 2Federal University of Piauí, Biological Sciences 64600 000, Brazil, 3Federal University of Ceara, Physiology and Pharmacology 60430 270, Brazil, 4State University of São Paulo, Organic Chemistry 50740 520, Brazil, 5Federal University of Pernambuco, Pharmacy 50740 520, Brazil

Introduction: Phthalimides are analogues of thalidomide that have been researched for its antiangiogenic and immunomodulatory properties. Objective: To assess the in vitro cytotoxic and antiangiogenic activities in vivo of thalidomide and two analogues SC-10: 1-(1,3-dioxo-3a,4-dihydro-1H-isoindol-2(3H-7H-7aH)-yl)urea and SC-11: 1-(1,3-dioxo-3a,4-dihydro-1H-isoindol-2(3H-7H-7aH)-yl)thiourea. Methods: The in vitro studies were performed with Sarcoma 180-treated and -untreated cells harvested from the intraperitoneal cavity under aseptic conditions after 24 hours exposure (50 and 100µg/mL). Sarcoma cells were evaluated for cellular membrane integrity and internucleosomal DNA fragmentation by flow cytometry (Guava EasyCyte Mine). The 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). The antitumor effect of thalidomide and two analogues was analyzed in male mice swiss (25g) transplanted with the Sarcoma 180 tumor and treated intraperitoneally (50mg/kg/7 days). Antiangiogenic activity was evaluated by the quantification of microvascular density (DMV) with immunostaining of intratumor endothelial cells with anti-CD-31. The digital images captured (200x) with windows® bitmap format (512 x 384 pixels) were analyzed by Morphometric Analysis System (SAMM), a software developed specifically for this purpose. Data were compared by one-way analysis of variance (ANOVA) followed by the Dunnets test (P < 0.01). Results: The cytometry analyzes of Sarcoma-treated cells showed that the analogues (SC-10 and SC-11) and the both concentrations tested (50 and 100 μg/mL) were capable to reduce cell membrane integrity (68.3 ± 0.5, 68.0 ± 1.7 and 69.6 ± 0.9, 46.1 ± 1.6 %) and to cause dose-dependent internucleosomal DNA fragmentation (24,9 ± 2,5, 48.4 ± 1.2 and 42.0 ± 1.6, 58.4 ± 1.1%, respectively) when compared to the negative control (86.1 ± 1.5 and 8.7 ± 0.5%) (P < 0.01), indicating the presence of cells with late stage apoptotic characteristics or in the process of secondary necrosis. In a similar way, Dox caused membrane disruption (58.4 ± 0.2 %) and DNA fragmentation (50.6 ± 2.0 %) (P < 0.01). The inhibition of the tumor growth was only significant in mice treated with thalidomide (53.5%) and the analogues SC-10 and SC-11 (67.9% and 67.4%, respectively) (P < 0.01). The immunohistochemistry analyzes of intratumor endothelial cells showed reduction of DMV in the groups treated with analogues SC-10 and SC-11 (2.7 ± 0.1 and 4.1 ± 1.5%) (P < 0.01), though thalidomide did not present antiangiogenic action (5.4 ± 0.5, P > 0.05), when compared to the negative control (7.8 ± 0.6). Conclusion: These results suggest antitumor activity of the two phthalimides analogues, probably triggered by apoptotic pathways and antiangiogenic action.