We examined the effect of adiponectin on pro-proliferative and antiapoptotic actions of leptin using BrdU and TUNEL assays. Adiponectin increased apoptosis in a dose-dependent RO4929097 in vitro manner (Fig. 1A). Kinetics of increasing/decreasing doses of adiponectin in combination with leptin showed that 10 μg/mL adiponectin efficiently inhibited the effect of leptin (Supporting Fig. 1). Adiponectin eliminated the anti-apoptotic effect of leptin

(Fig. 1A). Adiponectin treatment significantly increased caspase-3 activity even in the presence of leptin (Supporting Fig. 3). Importantly, adiponectin inhibited proliferation of HCC cells in a dose-dependent manner, in contrast to leptin treatment, which increased proliferation. Combined treatment with adiponectin and leptin also resulted in significant inhibition of leptin-induced proliferation (Fig. 1B). Cancer progression is a multistep process that involves invasion of the basement membrane by tumor cells and migration to points far from a given primary tumor mass leading to metastasis.32 We examined the effect of adiponectin on leptin-induced invasion and migration of

HCC cells. Leptin increased migration of HCC cells, whereas adiponectin inhibited migration in a conventional scratch-migration Silmitasertib datasheet assay. Adiponectin treatment also inhibited migration of cancer cells in the presence of leptin, overcoming its promigratory potential (Fig. 2A). In a quantitative real-time assay using an ECIS-based technique

to follow migration of HCC cells, we found that cells treated with leptin showed increased resistance, whereas adiponectin treatment inhibited cell migration (showing low resistance). Cells cotreated with both adiponectin and leptin displayed a decreased resistance, showing that BCKDHA adiponectin could inhibit leptin-induced migration (Fig. 2B). Next, we performed Matrigel invasion assays to examine the effect of adiponectin on leptin-induced invasion potential of HCC cells. Leptin treatment increased invasion of cancer cells through Matrigel in comparison to untreated cells, whereas adiponectin treatment inhibited invasion of HCC cells. Importantly, adiponectin treatment significantly inhibited leptin-induced invasion of cancer cells (Fig. 3A). In an ECIS-based invasion assay, established human umbilical vein endothelial cell (HUVEC) cell layers were challenged with HCC cells. The drop in the resistance showed direct interactions of the tumor cells with HUVEC cells and extravasation of HCC cells on the substratum. Leptin treatment induced a steeper drop in resistance than no treatment control, demonstrating that leptin increased invasive potential. Adiponectin inhibited invasive potential of HCC even in the presence of leptin (Fig. 3B). These results showed that adiponectin could effectively inhibit leptin-induced increased migration and invasion of HCC cells.