WY-14643 Regulates CYP1B1 Expression through Peroxisome Proliferator-Activated Receptor α-Mediated Signaling in Human Breast Cancer Cells.

Human cytochrome P450 1B1 (CYP1B1)-mediated biotransformation of endobiotics and xenobiotics plays an important role in the progression of human breast cancer. In this study, we investigated the effects of WY-14643, a peroxisome proliferator-activated receptor α (PPARα) agonist, on CYP1B1 expression and the related mechanism in MCF7 breast cancer cells. We performed quantitative reverse transcription-polymerase chain reaction, transient transfection, and chromatin immunoprecipitation to evaluate the effects of PPARα on peroxisome proliferator response element (PPRE)-mediated transcription. WY-14643 increased the protein and mRNA levels of CYP1B1, as well as promoter activity, in MCF-7 cells. Moreover, WY-14643 plus GW6471, a PPARα antagonist, significantly inhibited the WY-14643-mediated increase in CYP1B1 expression. PPARα knockdown by a small interfering RNA markedly suppressed the induction of CYP1B1 expression by WY-14643, suggesting that WY-14643 induces CYP1B1 expression via a PPARα-dependent mechanism. Bioinformatics analysis identified putative PPREs (-833/-813) within the promoter region of the CYP1B1 gene. Inactivation of these putative PPREs by deletion mutagenesis suppressed the WY-14643-mediated induction of CYP1B1 promoter activation. Furthermore, WY-14643 induced PPARα to assume a form capable of binding specifically to the PPRE-binding site in the CYP1B1 promoter. Our findings suggest that WY-14643 induces the expression of CYP1B1 through activation of PPARα.

Leptin induces CYP1B1 expression in MCF-7 cells through ligand-independent activation of the ERα pathway.

Leptin, a hormone with multiple biological actions, is produced predominantly by adipose tissue. Among its functions, leptin can stimulate tumour cell growth. Oestrogen receptor α (ERα), which plays an essential role in breast cancer development, can be transcriptionally activated in a ligand-independent manner. In this study, we investigated the effect of leptin on CYP1B1 expression and its mechanism in breast cancer cells. Leptin induced CYP1B1 protein, messenger RNA expression and promoter activity in ERα-positive MCF-7 cells but not in ERα-negative MDA-MB-231 cells. Additionally, leptin increased 4-hydroxyoestradiol in MCF-7 cells. Also, ERα knockdown by siRNA significantly blocked the induction of CYP1B1 expression by leptin, indicating that leptin induced CYP1B1 expression via an ERα-dependent mechanism. Transient transfection with CYP1B1 deletion promoter constructs revealed that the oestrogen response element (ERE) plays important role in the up-regulation of CYP1B1 by leptin. Furthermore, leptin stimulated phosphorylation of ERα at serine residues 118 and 167 and increased ERE-luciferase activity, indicating that leptin induced CYP1B1 expression by ERα activation. Finally, we found that leptin activated ERK and Akt signalling pathways, which are upstream kinases related to ERα phosphorylation induced by leptin. Taken together, our results indicate that leptin-induced CYP1B1 expression is mediated by ligand-independent activation of the ERα pathway as a result of the activation of ERK and Akt in MCF-7 cells.

Optogenetic control of mRNA localization and translation in live cells.

Despite efforts to visualize the spatio-temporal dynamics of single messenger RNAs, the ability to precisely control their function has lagged. This study presents an optogenetic approach for manipulating the localization and translation of specific mRNAs by trapping them in clusters. This clustering greatly amplified reporter signals, enabling endogenous RNA-protein interactions to be clearly visualized in single cells. Functionally, this sequestration reduced the ability of mRNAs to access ribosomes, markedly attenuating protein synthesis. A spatio-temporally resolved analysis indicated that sequestration of endogenous ß-actin mRNA attenuated cell motility through the regulation of focal-adhesion dynamics. These results suggest a mechanism highlighting the indispensable role of newly synthesized ß-actin protein for efficient cell migration. This platform may be broadly applicable for use in investigating the spatio-temporal activities of specific mRNAs in various biological processes.

Determination of optimized oxygen partial pressure to maximize the liver regenerative potential of the secretome obtained from adipose-derived stem cells.

BACKGROUND: A hypoxic-preconditioned secretome from stem cells reportedly promotes the functional and regenerative capacity of the liver more effectively than a control secretome. However, the optimum oxygen partial pressure (pO2) in the cell culture system that maximizes the therapeutic potential of the secretome has not yet been determined. METHODS: We first determined the cellular alterations in adipose tissue-derived stem cells (ASCs) cultured under different pO2 (21%, 10%, 5%, and 1%). Subsequently, partially hepatectomized mice were injected with the secretome of ASCs cultured under different pO2, and then sera and liver specimens were obtained for analyses. RESULTS: Of all AML12 cells cultured under different pO2, the AML12 cells cultured under 1% pO2 showed the highest mRNA expression of proliferation-associated markers (IL-6, HGF, and VEGF). In the cell proliferation assay, the AML12 cells cultured with the secretome of 1% pO2 showed the highest cell proliferation, followed by the cells cultured with the secretome of 21%, 10%, and 5% pO2, in that order. When injected into the partially hepatectomized mice, the 1% pO2 secretome most significantly increased the number of Ki67-positive cells, reduced serum levels of proinflammatory mediators (IL-6 and TNF-α), and reduced serum levels of liver transaminases. In addition, analysis of the liver specimens indicated that injection with the 1% pO2 secretome maximized the expression of the intermediate molecules of the PIP3/Akt and IL-6/STAT3 signaling pathways, all of which are known to promote liver regeneration. CONCLUSIONS: The data of this study suggest that the secretome of ASCs cultured under 1% pO2 has the highest liver reparative and regenerative potential of all the secretomes tested here.