Leptin is a peptide hormone, which has a central role in the regulation of body weight; it also exerts many potentially atherogenic effects. Ferulic acid ethyl ester (FAEE) has been approved for antioxidant properties. The aim of this study was to investigate whether FAEE can inhibit the atherogenic effects of leptin and the possible molecular mechanism of its action. Both of cell proliferation and migration were measured when the aortic smooth muscle cell (A10 cell) treated with leptin and/or FAEE. Phosphorylated p44/42MAPK, cell cycle-regulatory protein (for example, cyclin D1, p21, p27), beta-catenin and matrix metalloproteinase-9 (MMP-9) proteins levels were also measured. Results demonstrated that leptin (10, 100 ng ml-1) significantly increased the proliferation of cells and the phosphorylation of p44/42MAPK in A10 cells. The proliferative effect of leptin was significantly reduced by the pretreatment of U0126 (0.5 muM), a MEK inhibitor, in A10 cells. Meanwhile, leptin significantly increased the protein expression of cyclin D1, p21, beta-catenin and decreased the expression of p27 in A10 cells. In addition, leptin (10 ng ml-1) significantly increased the migration of A10 cells and the expression of MMP-9 protein. Above effects of leptin were significantly reduced by the pretreatment of FAEE (1 and 10 muM) in A10 cells. In conclusion, FAEE exerts multiple effects on leptin-induced cell proliferation and migration, including the inhibition of p44/42MAPK phosphorylation, cell cycle-regulatory proteins and MMP-9, thereby suggesting that FAEE may be a possible therapeutic approach to the inhibition of obese vascular disease.
Animals ; Antioxidants/*pharmacology ; Aorta/cytology/*drug effects ; Caffeic Acids/*pharmacology ; Cell Line ; Cell Movement/*drug effects ; Cell Proliferation/*drug effects ; Leptin/*metabolism ; Matrix Metalloproteinase 9/metabolism ; Muscle, Smooth, Vascular/cytology/drug effects ; Myocytes, Smooth Muscle/cytology/*drug effects ; Rats ; beta Catenin/metabolism

BACKGROUND AND OBJECTIVES: Zehneria japonica belongs to the Cucurbitaceae family which is one of the most important plant families. It is commonly known as "Pipinong-gubat," widely distributed in Central Luzon regions and in areas along streams and clearings at low and medium altitudes in the Philippines. This study aimed to evaluate the potential cytotoxic and angiosuppressive properties of Zehneria japonica (Thunb. ex Murray) S.K. Chen (Cucurbitaceae) leaf extracts.

METHODOLOGY: The Z japonica semi-crude extracts were obtained by sequential extraction using hexane, ethyl acetate, and n-butanol. A modified duck egg chorioallantoic membrane (CAM) assay was aided by AngioQuant, a digital imaging software used to evaluate angiogenic activity. Inhibition of angiogenesis was evaluated by percent increase or decrease in mean length of blood vessels, mean size of blood vessels, and total number of blood vessel junctions. Moreover, the cytotoxic effects of the extracts were determined through MTT Assay. Osteosarcoma (U20s) and hepatocellular carcinoma (HepG2) cells were used as cancer representatives while human umbilical vein endothelial cells (HUVEC) were used as the normal cell control.

RESULTS: Analysis with AngioQuant revealed that treatment of the duck egg CAM with Z. japonica semi-crude extracts suppressed angiogenesis with ICso values of 1,810.00 ug/mL, 192.50 ug/ml, and 147.70 ug/mL for hexane, ethyl acetate, and n-butanol, respectively, with Celecoxib (20 ug/mL) as the positive control. For MTT assay, Z. japonica extracts exhibited strong cytotoxic effect against U2Os with an ICso values of 19.65 ug/mL, 9.89 ug/ml, and 31.04 ve/mL for the hexane, ethyl acetate, and n-butanol extracts, and no cytotoxic effects against HepG2 with IC50 values of 770.90 ug/mL, 130.10 ug/mL and 231.60 ug/mL for the hexane, ethyl acetate, and n-butanol extracts. Doxorubicin (0.544 ug/mL) was used as the positive control. The extracts also inhibited the growth of the normal cells, with IC50 values of 69.46 ng/mL, 42.23 ug/mL and 63.44 ug/mL for the hexane, ethyl acetate, and n-butanol extracts. There were no mortality and toxic symptoms observed for 14 days after the administration of the crude butanolic extract of Z. japonica in six female Sprague Dawley rats.

CONCLUSION: Z. japonica crude leaf extracts exhibited angio-suppressive activity through CAM assay. In MTT assay, the extracts exhibited strong cytotoxicity in U20s (IC50 S20 ug/mL), no cytotoxic effect in HepG2 (ICso >100 ug/mL) cells, and mild cytotoxic effect in HUVEC (IC50 40-60 ug/mL). Phytochemical screening through TLC revealed that the extracts contain alkaloids, anthrones, flavonoids, and sterols. 

Plant ; Cucurbitaceae