Gammasecretase Inhibition Results in Both Caspase Dependant and Independent Apoptosis in Breast Cancer Cell Lines.
Authors: Balasubramanian R. , Rashied S. , Filipovic A. , Slade M. , Yague E. , Coombes C. 
Published: December 2010

Abstract
BackgroundNotch signalling is normally involved in lateral inhibition during embryogenesis and is found to be de regulated in many breast cancers. Gamma-secretase is an enzyme complex formed by constitution of Nicastrin, APH1, Presenilin and Pen2 and is involved in the cleavage of notch proteins. We investigated the effects of γ-secretase inhibition and its mechanism of action in breast cancer cell lines.Materials and MethodsGrowth assay was performed using Gammasecretase inhibitor-1(Z-Leu-Leu-Nle-CHO) in MCF-7, MDA-MB-231, T47D and ZR-75 breast cancer cell lines and 226 U19 and MCF10A non tumourigenic epithelial cell lines at concentrations between10nM and 60uM. Expression of Bcl-2, Bcl-XL, P-Bad and XIAP were measured by Western blot and cell cycle analysis was done by FACS in MCF-7 and MDA-MB-231 cells treated with GSI-1 at 0.75μM, 2μM and 5μM for 48 hours. Caspase 3 and 7 activity was measured in MCF-7 and MDA-MB-231 cells treated with GSI-1 at 0.75μM, 2μM and 5μM for 24 and 48 hours using Promega assay kit. MCF-7 cells were also treated with 0.75μM GSI-1 and DMSO in five replicates for 48 hours and gene array was performed using 44K Agilent array. Fold changes of AKR1B10 and Osteopontin genes were confirmed by Real time PCR at 0.75μM of GSI-1 in MCF-7 and they were also measured at concentrations of 2μM and 5μM in MCF-7 and MDA MB 231 cells using Taqman.ResultsThe 4 cancer cell lines MCF-7, MDA-MB231, ZR-75 and T47D showed growth inhibition in a dose dependant manner and were killed at 5uM. However this concentration did not affect the non tumourigenic cell lines. Expression of Bcl-2, Bcl-XL, inhibitor of apoptosis (XIAP) and P-BAD went down with escalating doses of GSI-1 at 48 hours in both MCF-7 and MDA MB 231 cells. Cell cycle analysis confirmed increase in the percentage of cells in G2M arrest leading to apoptosis at 48 hours in the same cell lines. However we observed increase in Caspase 3 and 7 activity only in MDA-MB-231 not in MCF-7 cells. Gene array in MCF-7 cells treated with GSI1 showed 15 and 4 fold changes in the expression of AKR1B10 and Osteopontin. There was also dose dependant increase in the expression these two genes with GSI-1 in MCF-7 but no such change was observed in MDA MB 231 cells.DiscussionThis is the first time we are reporting the effect of GSI-1 and its possible mechanism of action in breast cancer cell lines. Apoptosis appears to be the main mechanism of cell death as evidenced by our Western blots and FACS analysis in MCF-7 and MDA MB 231 cell lines. However, MCF-7 did not show any Caspase 3/7 activity implying other mechanisms may be involved in the induction of apoptosis. Production of reactive oxygen species (ROS) triggers the damage to mitochondrial membrane and release of cytochrome c, Apoptosis inducing Factor and Endonuclease G resulting in the initiation of Caspase independent apoptosis. AKR1B10 and Osteopontin were shown to be highly expressed in conditions resulting in increased production of ROS. Thus inhibition of γ-secretase enzyme results in breast cancer cell death in a dose which was non lethal to non tumourigenic cell lines. Both Caspase dependant and independent apoptosis appear to be involved in the mechanism of cell death.
Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3129.