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Terpenoids as effective compounds for overcoming multidrug resistance in cancer cells
Publication . Reis, Mariana; Ferreira, Maria José Umbelino, 1956-; Lage, Hermann
Circumventing multidrug resistance (MDR) in cancer is a cornerstone to chemotherapy success. Modulation of P-glycoprotein (P-gp) and evaluation of the collateral sensitivity effect are among the most promising approaches to overcome MDR. The main goal of this work was to explore these two approaches in order to find and optimize new MDR reversers derived from Euphorbia species. The phytochemical study of E. piscatoria yielded four new diterpenes, two with the ent-abietane skeleton (1-2) and two macrocyclic lathyranes (3-4). From E. welwitschii four new jatrophanes (19-22) were obtained. Welwitschines A (19) and B (20) present a unique combination of structural features: a 5/8/8 fused-ring system and an 12,15-ether bridge. Several known terpenoids (5-10 and 23) and flavonoids (11-14, 24 and 25) were also isolated from these plants. Moreover, a small library of 27 macrocyclic lathyrane diterpenes (6.1-6.27), targeted for P-gp efflux modulation, was achieved by molecular derivatization of jolkinol D (6), isolated from E. piscatoria. This process was accomplished mainly by using several alkanoyl and aroyl anhydrides/chlorides as acylating agents. The chemical structures of the compounds were deduced from their physical and spectroscopic data (IR, MS, 1D and 2D NMR experiments). Additionally, the stereochemistry of compounds 19 and 20 was confirmed through X-ray crystallography. Piscatoric acid (2) was a key compound to give insights into the biogenetic pathway of ent-abietane lactones. In the same way, the isolation of compounds 19-22 allowed to propose the 12,17-cyclojatrophanes biogenesis. The MDR reversal activity of compounds 6, 6.1-6.27 and 19-23 was evaluated through combination of transport and chemosensitivity assays, using the mouse lymphoma MDR1-transfected cell model. The most relevant structure-activity relationships for derivatives 6.1-6.27 highlighted the importance of the aroyl moiety and its pattern of substitution for P-gp efflux modulation. Most of the derivatives revealed to interact synergistically with doxorubicin, by increasing its cytotoxic activity. The effects on P-gp ATPase were evaluated for the most potent derivative, jolkinoate P (6.15). This compound showed to interact with P-gp, exhibiting a profile related to compounds that inhibit its ATPase activity. In regard to the jatrophanes 19-23, the results indicated that high conformational flexibility of the twelve-membered ring of compounds 21-23 favored P-gp modulation, in contrast to the tetracyclic scaffold of compounds 19 and 20. Epoxiwelwitschene (22) displayed the highest modulatory effect and may act as a competitive modulator, as indicated by the ATPase activity assay. Drug combination experiments also corroborated the anti-MDR potential of these diterpenes due to their synergistic interaction with doxorubicin. The potential collateral sensitivity effect of compounds 1-6, 8, 19-24 and 6.1-6.27 was evaluated against gastric (EPG85-257), pancreatic (EPP85-181) and colon (HT-29) human cancer cells and their drug-selected counterparts, resistant to novantrone (RN) and to daunorubicin (RDB), using a proliferation assay. Compounds 6.11, 6.22, 22 and 23 were able to decrease aproximately 85% of the level of resistance of EPG85-257RDB cells, in comparison to parental cells. Compounds 6.11, 6.20, 22 and 23 also showed promising results in pancreatic cells, being able to reduce the levels of resistance in about 32 - 65%. The mechanisms of cell death were analyzed, using the annexin V/PI and active caspase-3 assays. It was verified that cell death occurred through caspase-dependent apoptosis. The structural optimization of the compounds with the lathyrane scaffold and isolation of the jatrophanes from E. welwitschii, provided the identification of some important structural requirements, leading to a more effective reversion of MDR. Thus, compounds 6.11, 6.15, 6.20, 6.22, 22 and 23 can be proposed as new lead candidates for the development of MDR reversal agents.
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Fundação para a Ciência e a Tecnologia
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SFRH
Número da atribuição
SFRH/BD/72915/2010