Stable clones overe pressing AMPK B1 in two ovarian cancer cell lines with relatively lower AMPK B1 level or depleted of AM PK B1 by shRNAi mediated gene silencing in another two ovarian cancer cell lines with relatively higher AMPK B1 e pression were generated. The TT cell proliferation assay demonstrated that enhanced e pression of AMPK B1 significantly inhibited ovarian cancer cell growth by 45 to 50% in A2780cp and SKOV3 stable clones compared with the parental lines and vector controls. Further more, transient upregulation of AMPK B1 elevated pAM PK and mitigated cell proliferation in ovarian cancer cells in a dose dependent manner.

Additionally, we demonstrated that enforced e pression of AMPK B1 e hibited 60 to 70% less foci in A2780cp and SKOV3 stable clones by the focus formation assay, and we demonstrated that the AMPK B1 overe pressed clones of A2780cp and SKOV3 cells showed a 70% to 75% reduction in the number and size of colonies compared with the vector controls by the focus formation assay. Conversely, by depleting en dogenous AMPK B1 in OV2008 and OVCA 433 cells, which highly e press AMPK B1, using the sh B1 shRNA, we demonstrated that cell prolif eration increased 20 25% in all stable clones that overe pressed the sh B1 shRNA. Similarly, the stable AMPK B1 knockdown clones e hibited a 2 3 fold increase in cell growth based on the focus formation assay and a 4 5 fold increase in colony for mation using the anchorage independent growth ability assay. Given that overe pression of AMPK B1 could inhibit ovarian cancer cell growth, we investigated how AMPK B1 affected the cell cycle kinetics of ovarian cancer cells.

We then demonstrated that overe pression of AMPK B1 induced G1 phase arrest in A2780cp and SKOV3 stables clones compared to the controls by a cell cycle analysis using flow cytometry. On the other hand, stable knock down of endogenous AMPK B1 enhanced the G1 phase in OV2008 and OVCA433 cells. In sum, these findings suggest that AMPK B1 plays a sup pressive role in the cell growth and anchorage independent growth capacity of ovarian cancer cells by inducing G1 phase arrest. Loss of AMPK B1 promotes ovarian cancer cell migration and invasion We also studied the functional role of AMPK B1 in ovar ian cancer cell migration and invasion.

Using transwell migration and invasion assays, enhanced AMPK B1 e pression was found Anacetrapib to significantly attenuate the cell mi gration and invasive capacities of SKOV3 stable clones. In contrast, stable depletion of endogenous AMPK B1 in AMPK B1 e pressing OVCA433 cells using the sh B1 shRNA enhanced cell migration and invasion. These results indi cate that down regulation of AMPK B1 enhances the ag gressiveness of ovarian cancer and e plains why its level is progressively decreased in advanced stage and high grade ovarian cancers. AMPK B1 modulates AKT mTOR and JNK pathways Because AMPK B1 is a subunit of the AMPK comple , we further e amined its functional role in AMPK activity.