Calotropis procera extract inhibits prostate cancer through regulation of autophagy.

Current treatment options available for prostate cancer (PCa) patients have many adverse side effects and hence, new alternative therapies need to be explored. Anticancer potential of various phytochemicals derived from Calotropis procera has been studied in many cancers but no study has investigated the effect of leaf extract of C. procera on PCa cells. Hence, we investigated the effect of C. procera leaf extract (CPE) on cellular properties of androgen-independent PC-3 and androgen-sensitive 22Rv1 cells. A hydroalcoholic extract of C. procera was prepared and MTT assay was performed to study the effect of CPE on viability of PCa cells. The effect of CPE on cell division ability, migration capability and reactive oxygen species (ROS) production was studied using colony formation assay, wound-healing assay and 2',7'-dichlorodihydrofluorescein diacetate assay, respectively. Caspase activity assay and LDH assay were performed to study the involvement of apoptosis and necrosis in CPE-mediated cell death. Protein levels of cell cycle, antioxidant, autophagy and apoptosis markers were measured by western blot. The composition of CPE was identified using untargeted LC-MS analysis. Results showed that CPE decreased the viability of both the PCa cells, PC-3 and 22Rv1, in a dose- and time-dependent manner. Also, CPE significantly inhibited the colony-forming ability, migration and endogenous ROS production in both the cell lines. Furthermore, CPE significantly decreased NF-κB protein levels and increased the protein levels of the cell cycle inhibitor p27. A significant increase in expression of autophagy markers was observed in CPE-treated PC-3 cells while autophagy markers were downregulated in 22Rv1 cells after CPE exposure. Hence, it can be concluded that CPE inhibits PCa cell viability possibly by regulating the autophagy pathway and/or altering the ROS levels. Thus, CPE can be explored as a possible alternative therapeutic agent for PCa.

Epigenetic regulation of epithelial to mesenchymal transition: a trophoblast perspective.

Epigenetic changes alter the expression of genes at both pre- and post-transcriptional levels without changing their DNA sequence. Accumulating evidence suggests that such changes can modify cellular behavior and characteristics required during development and in response to various extracellular stimuli. Trophoblast cells develop from the outermost trophectoderm layer of the blastocyst and undergo many phenotypic changes as the placenta develops. One such phenotypic change is differentiation of the epithelial natured cytotrophoblasts into the mesenchymal natured extravillous trophoblasts. The extravillous trophoblasts are primarily responsible for invading into the maternal decidua and thus establishing connection with the maternal spiral arteries. Any dysregulation of this process can have adverse effects on the pregnancy outcome. Hence, tight regulation of this epithelial-mesenchymal transition (EMT) is critical for successful pregnancy. This review summarizes the recent research on the epigenetic regulation of the EMT occurring in the trophoblast cells during placental development. The functional significance of chemical modifications of DNA and histone, which regulate transcription, as well as non-coding RNAs, which control gene expression post-transcriptionally, is discussed in relation to trophoblast biology.

MicroRNA-141-3p and miR-200a-3p regulate insulin-like growth factor 2 during mouse placental development.

Insulin-like growth factor 2 (IGF2) plays a vital role in fetal and placental development throughout gestation. Placental expression of IGF2 decreases substantially in intra-uterine growth restriction (IUGR) and Igf2 null mice develop small placentas. In this report, we examined the role of microRNAs in regulating Igf2 gene expression during mouse placental development. Using bioinformatic analysis, we have identified microRNAs that have conserved binding sites in the 3'-UTR of Igf2. Using luciferase reporter assay, we demonstrated that miR141-3p and miR-200a-3p mimics substantially down regulated relative luciferase activity by binding to 3'-UTR of Igf2, which was reversed by using miR141-3p and miR-200a-3p inhibitors. Furthermore, in a similar assay, use of Igf2 3'-UTR that lacked the binding site for the microRNAs did not have any effect on luceiferase activity. Interestingly, the expression of miR141-3p and miR-200a-3p were inversely and temporally correlated to the expression of IGF2 during mouse placental development. Overexpression of miR141-3p and miR-200a-3p in mouse trophoblast stem cells suppressed endogenous expression of IGF2. Consequently, IGF2 silencing by miR141-3p and miR-200a-3p diminished Akt activation in mouse trophoblast stem cells. Our study provides evidence for regulation of Igf2 by microRNAs and further elucidates the role of miR141-3p and miR-200a-3p in the mouse placental development.