J Pharm Pharmacogn Res 3(suppl. 1): S97, 2015
Proceedings of the 4th International Symposium on Pharmacology of Natural Products FAPRONATURA 2015 September 21st-25th, 2015; Cuban Society of Pharmacology. Topes de Collantes, Sancti Spiritus, Cuba.
PPP-05: PARTICIPATION OF CYSTEINE PROTEINASE CMS2MS3 IN ANTIMETASTATIC ACTIVITY OF FRACTION FROM Vasconcellea cundinamarcensis’ LATEX: EFFECTS ON ADHESION AND CELL DEATH
Dittz D1, Tatsumi GC1, Salas CE2, Lopes MTP1.
Introduction: Our research group has demonstrated that proteolytic fractions from Vasconcellea cundinamarcensis’ latex (P1G10 and CMS2) have antitumor/antimetastatic activity on murine melanoma. We aim to identify a possible protease from CMS2 and its cellular mechanisms that contribute to the effects described previously. Methods: For determination of IC-50, B16F10 cells were exposed to proteases (0.1-500 ug/mL) for 72hs. The cellular viability was assessed by resazurin metabolization, quantified at 570/600 nm. The effect on cell adhesion was determined by exposing B16F10 to proteases (1-50 ug/mL) for 2-24hs. Finally, cells that remaining adhered was quantified by resazurin metabolization. In B16F10 cells, exposed to CMS2MS3 (10 ug/mL) by 2-24hs were evaluated the α5β1 integrin levels (flow cytometer), number of focal adhesion by vinculin measurement (immunohistochemistry), sub-diploid content (flow cytometer), activation of casp3, casp9 and BAX (Western Blot) and calcium transient (confocal microscopy). Results: Among the five analyzed proteases, CMS2MS3 showed the lowest IC-50 (7.81 ug/mL). All proteases promoted loss of adhesion in B16F10, especially CMS2MS3 at 10-50 ug/mL. The level of α5β1 integrin was reduced by CMS2MS3 10 ug/mL in all analyzed time. The number of vinculin/cell reduced 65-85% from 2hs exposure to CMS2MS3. After 2-24hs of exposure to CMS2MS3, sub-diploid DNA increased only at a concentration of 50 ug/mL. When exposed to 10 ug/mL of CMS2MS3, a reduction in intracellular levels of casp3 and casp9 was observed from 2 h of exposure and an increase in BAX levels after 24 h. A rapid increase in nuclear calcium was observed after when B16F10 were exposure to CMS2MS3 10 ug/mL for 10 s. Conclusions: Among the five proteases from V. cundinamarcensis’ latex, CMS2MS3 showed the best cytotoxic effect and ability to reduce the adhesion, mediated by α5β1 integrin in B16F10 cells. The cell death, by apoptosis, occurs after loss of adhesion, an event known as Anoikis. Financial Support: CNPq, FAPEMIG and CAPES.
Citation Format: Dittz D, Tatsumi GC, Salas CE, Lopes MTP (2015) Participation of cysteine proteinase CMS2MS3 in antimetastatic activity of fraction from Vasconcellea cundinamarcensis’ latex: effects on adhesion and cell death. [Abstract]. In: Proceedings of the FAPRONATURA 2015; 2015 Sep 21-25; Topes de Collantes, Sancti Spiritus: CSF. J Pharm Pharmacogn Res 3(Suppl. 1): S97. Abstract nr PPP-05.