ERK is phosphorylated and activated by MEK in re sponse to growth factor stimulation, and then activated ERK phosphorylates and activates Volasertib order nuclear targets to up regulate immediate early genes. Therefore, we deter mined the expression levels of p ERK1 2 in AsPC 1 and Capan 2 cells. Intriguingly, the phosphorylation status of ERK2 was much higher than that of ERK1 in AsPC 1 cells, and this phenomenon was completely converse in Capan 2 cells. This observation suggests distinct roles of ERK1 and ERK2 in the regulation of cell behavior in AsPC 1 and Capan 2 cells. To test whether PHB is required for the ERK pathway, we validated a siRNA against PHB in AsPC 1 and Panc 1 cells by quantitative real time PCR. The re sults showed that siPHB reduced the PHB mRNA level by about 80% compared with that using control siRNA.

Furthermore, we checked the phosphorylation status of ERK1 2 in siPHB transfected Inhibitors,Modulators,Libraries AsPC 1 and Panc 1 cells. As expected, stimulation of AsPC 1 cells with epidermal growth factor caused an increase of ERK1 2 phosphorylation, whereas silencing of PHB expression strongly suppressed the EGF induced phosphorylation of ERK. This finding suggested specific involvement of PHB in the RAS RAF ERK pathway. Moreover, a similar result was obtained in Panc 1 cells, indicating general inhibition of ERK activation by PHB depletion. Inhibitors,Modulators,Libraries Thus, these results clearly indicate that PHB is required for EGF induced ERK1 2 activation in pancreatic cancer cells. RocA disrupts the ERK pathway by targeting the CRAF PHB interaction in AsPC 1 cells The oncogenic RAS ERK pathway is a key node for cellular proliferation signals and has been the focus of substantial drug discovery efforts in many cancers.

A previous study has indicated that RocA suppresses the ERK pathway in leukemic cells. To confirm that the anti tumor effect of RocA is indeed caused by suppression of the ERK Inhibitors,Modulators,Libraries pathway, we examined the effect of RocA on ERK activity in AsPC 1 cells. The results showed significant dose dependent inhibition of the phos phorylation status of ERK1 2. Importantly, RocA showed Inhibitors,Modulators,Libraries very strong time dependent suppression of ERK1 2 activities. PHB was previously shown to be required for mem brane association and activation of CRAF. Therefore, we examined whether RocA affects PHB Inhibitors,Modulators,Libraries CRAF mem brane association in AsPC 1 cells.

To this end, cell membrane and cytosol fractions were prepared from AsPC 1 cells treated with Roc A or DMSO to analyze the localization of PHB and CRAF. Immunoblot analysis showed significant reduction of CRAF, particularly phos phorylated selleck chem CRAF, in the membrane fraction after RocA treatment. Notably, RocA also sig nificantly reduced the levels of PHB in the membrane fraction, indicating that binding of RocA to PHB may also interfere with PHB membrane association. However, RocA did not influence membrane localization of RAS.