RESUMO
Backgorund: Fruits and seed extracts of Annona montana have significant cytotoxic potential in several cancer cells. This study evaluates the effect of A. montana leaves hexane extract on several signaling cascades and gene expression in metastatic breast cancer cells upon insulin-like growth factor-1 (IGF-1) stimulation. Methods: MTT assay was performed to determine the proliferation of cancer cells. Propidium iodide staining and flow cytometry analysis of Annexin V binding was utilized to measure the progression of the cell cycle and the induction of apoptosis. Protein expression and phosphorylation were determined by western blotting analysis to examine the underlying cellular mechanism triggered upon treatment with A. montana leaves hexane extract. Results: A. montana leaves hexane (sub-fraction V) blocked the constitutive stimulation of the PI3K/mTOR signaling pathways. This inhibitory effect was associated with apoptosis induction as evidenced by the positivity with Annexin V and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNNEL) staining, activation of caspase-3, and cleavage of PPAR. It also limited the expression of various downstream genes that regulate proliferation, survival, metastasis, and angiogenesis (i.e., cyclin D1, survivin, COX-2, and VEGF). It increased the expression of p53 and p21. Interestingly, we also observed that this extract blocked the activation of AKT and ERK without affecting the phosphorylation of the IGF-1 receptor and activation of Ras upon IGF-1 stimulation. Conclusion: Our study indicates that A. montana leaves (sub-fraction V) extract exhibits a selective anti-proliferative and proapoptotic effect on the metastatic MDA-MB-231 breast cancer cells through the involvement of PI3K/AKT/mTOR/S6K1 pathways.
RESUMO
The Annona genus is a member of Annonaceae, one of the largest families of plants across tropical and subtropical regions. This family has been used in several ethnomedicinal practices to treat a multitude of human diseases. However, the molecular mechanism underlying its effect on the lipid droplet formation and on the expression of adipogenic markers of this plant remain to be investigated. In this study, we examined whether the extracts from the aerial part of Annona montana affect in vitro differentiation of preadipocytes. For our investigations, both mouse embryo fibroblast 3T3-L1 and normal human primary subcutaneous preadipocytes were incubated with Annona montana extracts (-and its subfractions-) and then analyzed on preadipocyte differentiation, lipid content, lipid droplet size and number, the expression of adipogenic-specific transcriptional factors, as well as cell survival. From our examinations, we found the Annona montana ethyl acetate extract to exhibit a potent inhibitory effect on adipogenesis, without affecting cell survival, in a dose-dependent manner. Such inhibitory effects included a significant decrease in the accumulation of lipid content by both a dramatic reduction of size and number of lipid droplets. This extract strongly attenuated the expression of PPARγ and HMGB2. It also inhibited the expression of CEBPα, FAS, and Akt without influencing Erk1/2 activities. Our findings suggest that specifically, the Annona montana ethyl acetate extract has a prominent inhibitory effect in cellular pathways of adipocyte differentiation by modulating specific gene expression, which is known to perform a pivotal role during adipogenesis.
