The mTOR pathway in estrogen response: A potential for combining the rapamycin derivative RAD001 with the aromatase inhibitor Letrozole (Femara®) in breast carcinoma

Abstract

5620 Fatty acid synthase (FAS) is a 250-270 kd cytosolic multifunctional polypeptide required for the conversion of dietary carbohydrates to fatty acids. While high levels of FAS expression compared to normal tissue have been observed in several cancers, including breast, prostate, colon and lung carcinoma, there is little information regarding gliomas. For the first time, we show high levels of FAS expression associated with glioma cell lines. Western blotting revealed higher FAS expression (4-20 fold increase) in U251, U138, NSB-19, and U373 human glioma cell lines and in C6 rat glioma cells compared to normal rat astrocytes. Cerulenin-mediated inhibition of lipid synthesis was determined using 14C labeled acetic acid. After labeling, total lipids were extracted and counted for 14C by scintillation counting. Endogenous fatty acid synthesis in cerulenin-treated glioma cells decreased by 49%, 49.7% and 51.1% at 2 hours in NSB-19, U251, and C6 cells, respectively. Cell cycle analysis, using flow cytometry, demonstrated that 3 μg/mL of cerulenin resulted in S-phase cell arrest 24h after treatment; the number of G0/G1 and G2/M cells was decreased Further, treatment with cerulenin resulted in dose- and time-dependent decreases in glioma cell viability, assessed using the MTT assay. The mean IC50 values for U251 were 4.5 μg/mL and 2 μg/mL at 24 and 96h, respectively. For NSB-19 glioma cells, that expressed lower levels of FAS protein, the mean IC50 values were 5.5 μg/mL and 2.7 μg/mL at 24 and 96h, respectively. Cerulenin also reduced glioma cell clonogenic survival; incubating NSB-19 cells with 3μg/mL cerulenin reduced clonogenic cell survival 97% compared to controls. Increased apoptotic cell death and activated caspase-3 expression was observed in U251 and NSB-19 cells treated with cerulenin. To test whether FAS inhibition led to selective glioma cell cytotoxicity, primary rat astrocytes that express little FAS were incubated with 0.5-7.5 μg/mL cerulenin for 24h; no decrease in cell viability was observed. These data demonstrate that :(1) human and rat glioma cell lines express high levels of FAS compared with normal brain astrocytes; (2) FAS inhibition is selectively cytotoxic to glioma cells; normal astrocytes are unaffected; and (3) cerulenin mediated cytotoxicity is due in part to apoptotic cell death and cell cycle arrest. These findings suggest that FAS might be a novel target for anti-glioma therapy. (Supported by NIH Grant CA82722)