In vitro antiproliferative activity of coronarin D

Author(s)

Márcia Yumi Okubo

• Document type: Master's Dissertation
• Press: Piracicaba, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba
• Defense date: 2016-04-13

Examining board members

João Ernesto de Carvalho; Luiz Eduardo Nunes Ferreira; Denise Gonçalves Priolli

• Advisor: João Ernesto de Carvalho

Abstract

This study aimed to evaluate the antiproliferative activity of coronarin D in human tumor cell panel consisting of ten cell lines by the sulforhodamine B method. Among these cell lines, the glioblastoma (U251) was one that showed greater selectivity, and so it was selected for studies of cell death and cell cycle control using flow cytometry. After evaluation of cell viability, in which U251 cells were treated with different times and concentrations were determined by the treatment conditions applied in the analysis of flow cytometry. Non-toxic concentrations were selected, which are 2.5 µM; 5 µM and 10 µM and times of 24 and 48 hours for quantitation of DNA in phases G1, S and G2/M using propidium iodide, whereas the assessment of cell death involving exposure of phosphatidylserine residues (labeled by Annexin V- EP), the exposure times of 12 and 24 hours were selected at concentrations of 10, 20 and 40 µM of coronarin D (cell viability 70% to 80%). Then, tests were performed: activation of caspases using fluorophore SR-VAD-FMK, loss of mitochondrial membrane potential indicated by the reduction in fluorescence of rhodamine 123 and release of hydrogen peroxide (H2O2) measured by DCF fluorescence. The results indicated that coronarin D in the concentration of 10 µM, altered cell cycle kinetics with accumulation in the G1 phase after 24 and 48 hours of exposure. Furthermore, signaling was observed for death process through exposure phosphatidylserine in concentrations at 12 and 24 hours of treatment. At concentrations of 20 and 40 µM, after 24 hours of treatment there was caspase activation with a reduction of mitochondrial membrane potential observed in 6, 9 and 12 hours was caused by the release of H2O2, a reactive oxygen species, after 1 hour 30 minutes of treatment. These tests were used to two-way analysis of variance (ANOVA) with significance level of p <0.05. In addition, a preliminary assessment of treatment safety with coronarin D through the micronucleus induction test in cell line of Chinese hamster ovarian (CHO-K1) were evaluated in the absence and presence of enzymes of human metabolism (S9 fraction). A high frequency of micronucleus in the concentration of 10 µM which was reduced in the presence of metabolizing enzymes S9. These results were submitted to analysis of variance of a single (ANOVA) followed by Tukey test (p <0.05). Therefore, it is suggested that the coronarin D was able to induce the arrest of the U251 cells in the checkpoint between G1 and S phase, to lead them to cell death by intrinsic mechanism of apoptosis initiated by a originated stress release H2O2 leads to a reduction of mitochondrial membrane potential and caspase activation, and interact with DNA causing further damage to the genetic material without S9 fraction (AU)

• FAPESP's process: 14/06636-7 - In vitro antiproliferative activity of Coronarin-D
• Grantee: Márcia Yumi Okubo
• Support Opportunities: Scholarships in Brazil - Master