First, we studied the cell death mechanism. Apoptosis and induction of caspase activity were checked by Western blotting analysis showing clea vage of PARP. The experiments were done at a concen tration equal to the cytotoxicity IC50 selleck chemical value of G28UCM and anti HER drugs in AU565 cells. Co treatment of AU565 cells with G28UCM plus trastuzumab during 24 h induced a marked increase in the levels of the PARP cleavage product compared to 24 h single agent treatment. The apoptotic effect Inhibitors,Modulators,Libraries of the combined regimes was validated by flow cytometry using the Annexin V Alexa Fluor 488 staining. Similar results in PARP cleavage were obtained when AU565 cells were co treated with G28UCM plus lapatinib during 12 hours or plus erlotinib during 24 hours.
Therefore, we sought to compare the effects of combined treatments versus single drug treatments Inhibitors,Modulators,Libraries on HER2, AKT, and ERK1 2 activation. The phosphorylated form of HER2 was noticeably decreased after 24 h exposure to G28UCM plus trastuzumab, and p AKT protein decreased after 48 h of co treatment with G28UCM and trastuzumab. Co incubation of cells with G28UCM and lapatinib was significantly corre lated Inhibitors,Modulators,Libraries with a decreased level of the phosphorylated form of HER2 and p ERK1 2, which occurred as soon as 12 h after treatment compared to 12 h cell treat ment with either G28UCM or lapatinib alone. Co exposure of G28UCM plus erlotinib induced a decrease of p HER2 and p AKT after 24 hours. During all time course co treatment experiments no sig nificant change either in the total level of the correspond ing proteins or in FASN levels was detected.
As we expected, under the same culture conditions, co treatment of AU565 cells with G28UCM plus cetuximab did not induce apoptosis Inhibitors,Modulators,Libraries and did not block HER2 phosphorylation or its downstream related signal transduction pathways ERK1 2 and PI3K AKT. Effect of G28UCM on cells resistant to trastuzumab or lapatinib The vast majority of HER2 positive advanced breast can cer patients develop resistance to trastuzumab based therapies within the first year of treatment. Conse quently, identification of novel agents that inhibit the growth of trastuzumab resistant cells tumours is critical to improving the survival of metastatic HER2 breast cancer. For this purpose, we extended our study to examine the anti cancer effect of G28UCM Inhibitors,Modulators,Libraries on HER2 breast selleck chem MG132 cancer cells that were continuously exposed in culture medium supplemented with trastuzu mab or lapatinib over a period of at least six months. Trastuzumab resistant or lapatinib resistant cells were developed in our laboratory as described in the Materi als and methods section.