pd-184161 and Carcinoma--Hepatocellular

The extracellular signal-regulated (ERK), mitogen-activated protein kinase (p42/p44 MAPK) pathway is up-regulated in hepatocellular carcinoma (HCC). Molecular targeting of this critical mitogenic pathway may have therapeutic potential for the treatment of HCC; however, chemoresistance to long-term therapy may develop. In the present study, we employed small-molecule MAPK kinase (MEK) inhibitors, including U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene] and PD184161 (Neoplasia 8:1-8, 2006), in HepG2 and Hep3B human HCC cell lines to identify potential mechanism(s) of resistance. U0126 dose-dependently suppressed ERK phosphorylation at both 1- and 24-h time points in HepG2 cells, previously shown to be sensitive to growth inhibition by U0126. In contrast, ERK phosphorylation was only decreased at the 1-h time point but not at 24 h in the more resistant Hep3B cells. It is interesting that the lack of prolonged phospho-ERK suppression was associated with MEK hyperphosphorylation in Hep3B cells. Several MEK/ERK pathway intermediates were up-regulated in Hep3B cells; furthermore, transfection of Raf-1 small interfering RNA to suppress MEK/ERK pathway activation sensitized Hep3B cells to U0126. MEK inhibitor resistance was independent of p53 or hepatitis Bx protein status. Finally, we showed that combining two chemically distinct MEK inhibitors enhanced growth inhibition and apoptosis compared with the single agents. Taken together, these results suggest that up-regulated expression or activity of the MEK/ERK pathway contributes to MEK inhibitor resistance in HCC cells. Our findings also provide preclinical evidence suggesting that the status of the MEK/ERK pathway in patients may predict response to MEK/ERK-targeted therapeutics.

Topics: Aniline Compounds; Apoptosis; Benzamides; Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; Humans; Mitogen-Activated Protein Kinase Kinases; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-raf; ras Proteins; RNA, Small Interfering; Signal Transduction; Trans-Activators; Tumor Suppressor Protein p53; Up-Regulation; Viral Regulatory and Accessory Proteins

Hepatocellular cancer (HCC) is a leading cause of cancer-related death worldwide. Historically, doxorubicin (DOX) has been widely used against unresectable HCC with variable response rates.. We hypothesized that DOX combined with mitogen-activated protein kinase kinase-extracellular signal-regulated kinase (MEK-ERK) targeted therapy may provide enhanced anti-cancer effects. Human HCC cell lines (HepG2, Hep3B) were treated with DOX and MEK enzyme inhibitors, U0126 or PD184161, alone or in combination. Growth, apoptosis, and ERK expression/MEK activity were respectively determined by proliferation assay, DNA fragmentation enzyme-linked immunoassay or fluorochrome inhibitor of caspases, and Western blot.. DOX (0.01-1 microM) decreased cell proliferation in Hep3B cells (IC(50) approximately 0.12 microM) at 48 to 72 h; DOX was less effective in HepG2 cells (IC(50) approximately 0.25 microM). At early time points (30 min) after DOX treatment of Hep3B cells, MEK activity was unchanged at low doses and decreased at higher doses; after 24 h, phospho-ERK levels increased at higher doses. Contrarily, in HepG2 cells, DOX caused a sustained, dose-dependent increase in phospho-ERK levels at early and late time points. The MEK inhibitor U0126 decreased phospho-ERK in both HCC lines. In contrast to DOX, HepG2 cells were more sensitive than Hep3B cells to U0126. The combination of DOX with U0126 (or PD184161) resulted in greater inhibition of proliferation in HepG2 but not in Hep3B cells. This effect may be mediated in part by enhanced apoptosis.. The effect of DOX on early and late induction of MEK activity predicts its chemotherapeutic response in HCC. Furthermore, this effect may also determine the utility of MEK inhibitor combination treatment.

Topics: Aniline Compounds; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzamides; Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Doxorubicin; Enzyme Inhibitors; Humans; Liver Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Nitriles; Phosphorylation