The natural triterpene maslinic acid induces apoptosis in HT29 colon cancer cells by a JNK-p53-dependent mechanism - PubMed (original) (raw)
The natural triterpene maslinic acid induces apoptosis in HT29 colon cancer cells by a JNK-p53-dependent mechanism
Fernando J Reyes-Zurita et al. BMC Cancer. 2011.
Abstract
Background: Maslinic acid, a pentacyclic triterpene found in the protective wax-like coating of the leaves and fruit of Olea europaea L., is a promising agent for the prevention of colon cancer. We have shown elsewhere that maslinic acid inhibits cell proliferation to a significant extent and activates mitochondrial apoptosis in colon cancer cells. In our latest work we have investigated further this compound's apoptotic molecular mechanism.
Methods: We used HT29 adenocarcinoma cells. Changes genotoxicity were analyzed by single-cell gel electrophoresis (comet assay). The cell cycle was determined by flow cytometry. Finally, changes in protein expression were examined by western blotting. Student's t-test was used for statistical comparison.
Results: HT29 cells treated with maslinic acid showed significant increases in genotoxicity and cell-cycle arrest during the G0/G1 phase after 72 hours' treatment and an apoptotic sub-G0/G1 peak after 96 hours. Nevertheless, the molecular mechanism for this cytotoxic effect of maslinic acid has never been properly explored. We show here that the anti-tumoral activity of maslinic acid might proceed via p53-mediated apoptosis by acting upon the main signaling components that lead to an increase in p53 activity and the induction of the rest of the factors that participate in the apoptotic pathway. We found that in HT29 cells maslinic acid activated the expression of c-Jun NH2-terminal kinase (JNK), thus inducing p53. Treatment of tumor cells with maslinic acid also resulted in an increase in the expression of Bid and Bax, repression of Bcl-2, release of cytochrome-c and an increase in the expression of caspases -9, -3, and -7. Moreover, maslinic acid produced belated caspase-8 activity, thus amplifying the initial mitochondrial apoptotic signaling.
Conclusion: All these results suggest that maslinic acid induces apoptosis in human HT29 colon-cancer cells through the JNK-Bid-mediated mitochondrial apoptotic pathway via the activation of p53. Thus we propose a plausible sequential molecular mechanism for the expression of the different proteins responsible for the intrinsic mitochondrial apoptotic pathway. Further studies with other cell lines will be needed to confirm the general nature of these findings.
Figures
Figure 1
(Panel A) Comet assay images of HT29 cells treated with maslinic acid after 24, 48 and 72 h at concentrations of IC50 and IC80. An increase in the tail moment can be seen after 48 hours' treatment at IC50. (Panel B) Tail-moment values observed in HT29 cells treated with maslinic acid at concentrations of IC50 and IC80 after 24, 48 and 72 h. Percentages compared to untreated control cells are also shown. The values represent means ± SD of four separate experiments. (Panel C) Histograms of cell cycle of HT29 cells after 72 hours' treatment with maslinic acid. Diagram of forward scatter (FS) versus side scatter (SS) of HT29 cells. It can be seen that the central population corresponding to cells in the G0/G1 cell cycle phase was increased. (Panel D) Histograms of cell cycle of HT29 cells after 96 hours' treatment with maslinic acid. A hypodiploid peak can be seen in the sub-G0/G1 region. Diagram of FS versus SS of HT29 cells. Sub-G0/G1 populations are seen to appear at both IC50 and IC80. Values represent means of three separate experiments. Significances are represented as (*) P < 0.05, and (**) P < 0.01.
Figure 2
Western blottings of the levels of JNK (panel A), p53 (panel B) and Bid (panel C) proteins. HT29 cells were treated with maslinic acid at concentrations of IC50 and IC80 for 12, 24, 48 and 72 h. The levels of protein expression are expressed as arbitrary intensity units of each band compared to arbitrary intensity units of actin. The variations in relative percentages of the expression of proteins for each time and concentration are also shown. The values represent means ± SD of at least three separate experiments.
Figure 3
Western blottings of the levels of Bax (panel A), Bcl-2 (panel B) and cytochrome-c (panel C) proteins. HT29 cells were treated with maslinic acid at concentrations of IC50 and IC80 for 48 and 72 h. The levels of protein expression are expressed as arbitrary intensity units of each band compared to arbitrary intensity units of actin. The variations in relative percentages of the expression of Bax, Bcl-2 and cytochrome-c for each time and concentration are also shown. The values represent means ± SD of at least three separate experiments.
Figure 4
Western blottings of the levels of pro- and caspase-9 (panel A), pro- and caspase-3 (panel B), pro- and caspase-7 (panel C), and pro- and caspase-8 (panel D) proteins. HT29 cells were treated with maslinic acid at concentrations of IC50 and IC80 for 48 and 72 h. The levels of protein expression are expressed as arbitrary intensity units of each band compared to arbitrary intensity units of actin. The variations in relative percentages of the expression of proteins for each time and concentration are also represented in this figure. The values represent means ± SD of at least three independent experiments.
Figure 5
Schematic representation of the plausible molecular mechanism proposed for the induction of apoptosis by maslinic acid in HT29 colon-cancer cells. This molecular mechanism is regulated via the induction of JNK and p53, resulting in mitochondrial disruption, the release of cytochrome-c and finally the activation of a cascade of caspases.
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