As shown in Fig. 1, the chromatographic profile revealed four bufadienolides in R. marina extracts (RMF-1 and RMM-5), namely telocinobufagin (1), marinobufagin (2), bufalin (3) and resibufogenin (4) ( Figs. 1 and 2), whereas in R. guttatus
venom (RGF-6 and RGM-9), only one bufadienolide was identified (marinobufagin – 2). The compounds were identified by comparison of retention times with standards and on the basis of UV and mass spectra. These findings are in agreement with previous data for B. marinus ( Gao et al., 2010). Regarding the biological assessments, the cytotoxicity of R. marina and R. guttatus venom extracts was first evaluated in a variety of tumor cell lines after 72 h exposure using the colorimetric CH5424802 price MTT assay. All extracts of R. marina male/female venoms revealed higher cytotoxic activity,
with IC50 values ranging from 0.01 μg/mL [RMF-1, RMF-3 and RMF-4 (HL-60); RMF-3 and RMF-4 (SF-295) and RMF-3 (HCT-116)] to 0.23 μg/mL (OVCAR-8) ( Table 1). Meanwhile, R. guttatus venom extracts exhibited a lower cytotoxic effect when compared selleck inhibitor to those of R. marina, with their IC50 values being around 2.9–6.6 μg/mL. Second, the cytotoxicity of the extracts was determined against normal cells, using human PBMC for this purpose. Herein, higher IC50 values were found for proliferating leukocytes (0.8, 0.5, 0.4, 0.3, 1.1, 0.8, 16, 13.1 and 13.9 μg/mL for RMF-1, RMF-2, RMF-3, RMF-4, RMM-5, RGF-6, RGF-7, RGF-8 and RGM-9, respectively) ( Table 2). Statistically, there were no differences in the cytotoxicity outcomes between samples obtained from female and male animals belonging to the
same species (p > 0.05). To better understand this potent cytotoxic activity, in vitro cytolytic analyses were performed with PLEK2 human erythrocytes. Interestingly, the most promising extracts obtained from R. marina (RMF-1, RMF-2, RMF-3, RMF-4 and RMM-5) were not able to cause hemolysis even at the highest concentration tested (200 μg/mL) ( Table 2). On the other hand, all R. guttatus venom extracts led to hemolysis, with EC50 values ranging from 20.8 (RGF-8) to 33.7 μg/mL (RGF-6). BrdU incorporation into DNA was measured in HL-60-treated cells with R. marina venom extracts after 24 h exposure. As seen in Fig. 3, all extracts (RMF-1, RMF-2, RMF-3, RMF-4 and RMM-5) decreased BrdU incorporation, showing labeling of 35.4 ± 3.4, 30.7 ± 1.0, 25.1 ± 1.8, 28.0 ± 1.7 and 38.3 ± 2.6% at 0.1 μg/mL and 19.7 ± 1.3, 19.6 ± 1.2, 15.8 ± 1.8, 16.5 ± 0.8 and 29.5 ± 1.6% at 1 μg/mL, respectively, when compared to untreated cells (73.0 ± 3.2%) (p < 0.05). Dox (0.1 and 1 μg/mL) treatment resulted in 22.6 ± 1.9 and 12.7 ± 0.9% BrdU incorporation (p < 0.05). Drug discovery and development have established a respectable armamentarium of useful chemotherapeutic agents as well as a number of important successes in the treatment and management of human cancer.