Morphological and biochemical alterations activated by antitumor clerodane diterpenes

• Casearia sylvestris leaves have oxygenated tricyclic bioactive clerodane diterpenes.
• Fraction and compounds from leaves were cytotoxic on human and mice tumor cells.
• Cas F was 53.4-fold more selective against leukemia cells.
• Cas F and Cas X triggered typical biochemical and morphological changes of apoptosis.
• Cas X-treated leukemia cells exhibited cell cycle arrest in G0/G1 phase.

Casearia sylvestris Swartz (Salicaceae) is a plant commonly widespread in the Americas. It has oxygenated tricyclic bioactive clerodane diterpenes with antimicrobial, antiulcer, larvicidal, chemopreventive, anti-inflammatory, antioxidant and antiproliferative properties. Due to this requirement for the developing of new anticancer drugs, it was initially evaluated the cytotoxic activity of a fraction with Casearins (FC) and its clerodane diterpenes Casearin B (Cas B), D (Cas D), X (Cas X) and Caseargrewiin F (Cas F) isolated from C.sylvestris leaves against 7 tumor cell lines, Sarcoma 180 cells (S180) and on normal peripheral blood mononuclear cells (PBMC). All substances tested showed cytotoxic potential. Cas F and X were the most active compounds. Cell death analyzes with Cas F (0.5 and 1 μM) and Cas X (0.7 and 1.5 μM) using the HL-60 leukemia line as experimental model showed DNA synthesis and membrane integrity reduction, DNA fragmentation and mitochondrial depolarization, specially after 24 h exposure, cell cycle arrest in G0/G1 phase caused by Cas X, activation of the initiator -8/-9 and effector -3/-7 caspases and phosphatidylserine externalization, all biochemical features of apoptosis corroborated by chromatinic condensation, karyorrhexis, cytoplasmic vacuolation and rarefaction and cellular shrinkage, morphological findings specially observed after 12 and 24 h of incubation. Therefore, Cas X and F were the most functional molecules with more pronounced lethal and discriminating effects on tumor cells and antiproliferative action predominantly mediated by apoptosis, highlighting clerodane dipertenes as promising lead antineoplastic compounds.