OBJECTIVE: This study investigates the chemical constituents, anti-proliferative, and apoptotic properties of C. nutans root extracts.
MATERIALS AND METHODS: The roots were subjected to solvent extraction using methanol and ethyl acetate. The anti-proliferative effects of root extracts were tested at the concentrations of 10 to 50 μg/mL on MCF-7 and HeLa by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay for 72 h. Morphological changes were observed under light microscope. Pro-apoptotic effects of root extracts were examined using flow cytometric analysis and RT-PCR. The chemical compositions of root extracts were detected using GC-MS.
RESULTS: The proliferation of MCF-7 cells was inhibited with the IC50 values of 35 and 30 μg/mL, respectively, for methanol and ethyl acetate root extracts. The average inhibition of HeLa cells was ∼25%. Induction of apoptosis in MCF-7 was supported by chromatin condensation, down-regulation of BCL2 and unaltered expression of BAX. However, only ethyl acetate extract caused the loss of mitochondrial membrane potential. GC-MS analysis revealed the roots extracts were rich with terpenoids and phytosterols.
DISCUSSION AND CONCLUSIONS: The results demonstrated that root extracts promote apoptosis by suppressing BCL2 via mitochondria-dependent or independent manner. The identified compounds might work solely or cooperatively in regulating apoptosis. However, further studies are required to address this.
Method: In this study, the potential targets of miR-145 were identified bio-informatically using different target prediction tools. The identified target genes were validated in vitro by dual luciferase assay. Wound healing and soft agar colony assay assessed cell proliferation and migration. miR-145 expression level was measured quantitatively by RT-PCR at different stages of breast tumor. Western blot was used to verify the role of miR-145 in EMT transition using key marker proteins.
Result: Wound healing and soft agar colony assays, using miR-145 over-expressing stably transfected MCF7 cells, unraveled its role as a pro-proliferation candidate in cancerous cells. The association between miR-145 over-expression and differential methylation patterns in representative target genes (DR5, BCL2, TP53, RNF8, TIP60, CHK2, and DCR2) supported the inference drawn. These in vitro observations were validated in a representative set of nodal positive tumors of stage 3 and 4 depicting higher miR-145 expression as compared to early stages. Further, the role of miR-145 in epithelial-mesenchymal (EMT) transition found support through the observation of two key markers, Vimentin and ALDL, where a positive correlation with Vimentin protein and a negative correlation with ALDL mRNA expression were observed.
Conclusion: Our results demonstrate miR-145 as a pro-cancerous candidate, evident from the phenotypes of aggressive cellular proliferation, epithelial to mesenchymal transition, hypermethylation of CpG sites in DDR and apoptotic genes and upregulation of miR-145 in later stages of tumor tissues.