Target identification of anti-diabetic and anti-obesity flavonoid derivative (Fla-CN).

Fla-CN is a flavonoid derivative with anti-diabetic and anti-obesity effects; however, its biological targets are still unknown. In this study, we developed bifunctional affinity-based probes to identify the direct targets of Fla-CN. When using probe 3, we observed the co-location of probe 3 and mitochondria in both HepG2 and 3T3-L1 cells. The putative target proteomes were obtained using activity-based protein profiling (ABPP) and photo-affinity labelling. Pyruvate carboxylase, mitochondrial malate dehydrogenase, mitochondrial complex I, and F1FO-ATPase were validated as the direct targets of Fla-CN by surface plasmon resonance (SPR) and biochemical assays. It was elucidated that the Tyr651, Gln870 and Lys912 were the key amino acid residues near the binding site of pyruvate carboxylase with Fla-CN. The direct interaction of Fla-CN and the above four targets allowed elucidation of its complicated molecular mechanism, including the activation of adenosine 5-monophosphate (AMP)-activated protein kinase (AMPK), and the inhibition of gluconeogenesis. Further investigation for activation of AMPK in normal and insulin resistance (IR) HepG2 cells, indicated that Fla-CN could target insulin resistance tissues.

Flavonoid derivative (Fla-CN) inhibited adipocyte differentiation via activating AMPK and up-regulating microRNA-27 in 3T3-L1 cells.

Fla-CN (3-O-[(E)-4-(4-cyanophenyl)-2-oxobut-3-en-1-yl] kaempferol) is a semi-synthesized flavonoid derivative of tiliroside which exhibited anti-diabetic effect in vivo. Our previous study revealed the role of Fla-CN in anti-obesity and anti-diabetes in vivo, but the underlying mechanism remained to be addressed. The present study aimed to investigate the mechanism of anti-adipogenesis in vitro. Fla-CN markedly inhibited intracellular lipid accumulation in a dose-dependent manner, and the inhibitory effect was mainly limited to the early stage of adipocyte differentiation in vitro. Further investigations revealed that Fla-CN up-regulated the expression level of miR-27a/b and suppressed its target genes expression including peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα). Furthermore, the phosphorylation of AMP-activated protein kinase (AMPK) was also enhanced by Fla-CN in pre-adipocyte differentiation. These effects were abolished when cells were treated with miR-27a/b inhibitor and AMPK inhibitor Compound C. Additionally, Fla-CN reduced the expressions of adipocyte-specific genes such as sterol regulatory element-binding transcription factor 1c (SREBP-1c), fatty acid synthase (FAS) and adipocyte fatty acid binding protein (aP2). In conclusion, these results suggested a mechanism of Fla-CN for adipocyte differentiation inhibition of 3T3-L1 cells through miR-27a/b induction and AMPK activation.

Synthesis and anti-metastatic effects of pregn-17(20)-en-3-amine derivatives.

Novel pregn-17(20)-en-3-amine derivatives were synthesized and their anti-metastatic effects were evaluated in human breast cancer cells using chemotaxis assay. Compared with positive control LY294002, a PI3K inhibitor, derivatives 5a, 19a, 20a, 19g, 20f, 5c, 12e and 12f exhibited significant inhibitory effects against cancer cell migration induced by chemokine epidermal growth factor (EGF). Especially, the IC50 for compound 20f was as low as 0.03 µM. Preliminary structure-activity relationship studies suggested that most 3ß-substituted derivatives were more effective than those 3α-substituted derivatives, provided there was no substituted group at position C-16. Moreover, the α,ß-unsaturated fragment in ring D might be critical for their anti-metastatic activities. Further investigations on compound 20f revealed inhibitory effects on cell adhesion, migration and invasion of MDA-MB-231 cells. The mechanisms for the anti-metastatic effect of 20f might be through the inhibition of the phosphorylations of PI3K, Akt, PKCζ, and integrin ß1 in a dose-dependent manner. Taken together, the novel steroidal alkaloid derivative 20f could be further explored as an effective anti-metastatic agent for the treatment of human metastatic breast cancer.

Synthesis and anti-metastatic effects of novel chiral ionone alkaloid derivatives.

Novel chiral ionone alkaloid derivatives were synthesized and evaluated their anti-metastatic effects in human MDA-MB-231 breast cancer cells. The chiral center C-6 of derivatives exerted an important role in response to the anti-metastatic activity. Comparing with a positive control of LY294002, compounds 17b and 19a exhibited potent inhibitory effects on the EGF-induced invasion of MDA-MB-231 cells with IC50 values of 0.026 ± 0.003 and 0.016 ± 0.002 µM, respectively. Moreover, compounds 17b and 19a showed inhibitory effects on the expressions of p-PKCζ and p-integrin ß1 in MDA-MB-231 cells in a dose-dependent manner. Thus, compounds 17b and 19a offer potential to be developed as novel anti-metastasis agents.

Antitumor metastasis pregnane alkaloids from Pachysandra terminalis.

Three new pregnane alkaloids, named terminamines H-J (1-3), together with two known alkaloids (4 and 5), were isolated from the ethanol extract of Pachysandra terminalis. The structures of isolated compounds were elucidated by spectroscopic methods, including (1)H and (13)C NMR, 2D NMR, and HR-ESI-MS. Compounds 1, 4, and 5 revealed significant anti-metastasis activities. In addition, compound 1 inhibited the expression of p-PKCζ in MDA-MB-231 cells, and compound 4 inhibited the expressions of p-PKCζ in MDA-MB-231 and A549 cells.