RESUMO
BACKGROUND: Elevation of fatty acid synthase (FAS) in human cancers is often associated with increased tumor aggression. The basic genetic mechanisms leading to increased enzyme content in cancer cells were investigated using cell lines derived from human metastatic breast carcinomas (T47D, Zr75 and SKBr3) and normal human breast epithelium (184A1). MATERIALS AND METHODS: Western analysis, Northern blotting, [2-(14)C]malonyl-CoA incorporation assays, nuclear run-off transcription assays, mRNA decay assays, and poly(A) tail assays were used to measure and compare transcription rates of the FAS gene among the four cell lines. RESULTS: By Western analysis, FAS levels in T47D were 2.6 times lower than ZR75 and SKBr3, but 6.7 times greater than non-neoplastic 184A1 cells. FAS mRNA levels and specific activity correlated with protein content. In contrast, relative rates of FAS gene transcription were significantly higher in non-neoplastic 184A1 cells than T47D, ZR75 and SKBr3. Stability of message was investigated to explain this discrepancy. The half-life of FAS mRNA in 184A1 cells was 5.6 h, or 4-5-fold less than ZR75 and SKBr3. Poly(A) tail assays showed that FAS mRNA species from 184A1 cells tended to be longer than those of breast cancer cell lines (500-1500 nt versus 500-800 nt, respectively). CONCLUSION: Breast cancer cell lines contained significantly more FAS enzyme, message and activity than non-neoplastic 184A1 cells. Yet, 184A1 cells exhibited higher rates
Assuntos
Adenocarcinoma/enzimologia , Adenocarcinoma/genética , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Ácido Graxo Sintases/biossíntese , RNA Mensageiro/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Poli A/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transcrição GênicaRESUMO
The current study characterizes the lipogenic enzyme fatty acid synthase (FAS; EC 2.3.1.85) in pediatric tumor cell lines of neural or neural crest origin [medulloblastoma (Daoy), malignant rhabdoid tumor of kidney (SM II), retinoblastoma (Y79), and neuroblastoma (SK-N-SH)]. Constitutive FAS content and activity in these lines were compared to human fibroblast cell line Hs27. Hs27 exhibits low levels of FAS and recapitulates enzyme status in normal human tissues under most physiological conditions. Western analysis detected significantly larger amounts of FAS protein in Y79 and SK-N-SH than Daoy, SM II and Hs27. Incorporation of radiolabeled malonyl-CoA into total cellular lipid revealed that enzyme activity correlated with amount. Increased FAS content and activity in Y79 and SK-N-SH relative to the other cell lines and Hs27, in particular, implied enzyme activation in retinoblastoma and neuroblastoma lineages. The enzyme also showed evidence of hormonal regulation, as dexamethasone induced FAS protein in Daoy and SK-N-SH. However, hormonal induction of FAS protein levels did not correlate with activity levels, which led us to speculate phosphorylation as a means of regulating the enzyme's activity. Finally, the FAS inhibitor cerulenin was investigated for its ability to suppress tumor cell growth. After four days of propagation, short-term treatment of cell lines with drug produced mean IC50s less than 10.5 micrograms/ml (i.e., 5.6 +/- 1.9 for SM II; 9.3 +/- 1.5 for Daoy; 10.2 +/- 0.2 for SK-N-SH; and 10.4 +/- 2.6 for Y79). Annexin V assays revealed that cerulenin initiated apoptosis. The antineoplastic properties of cerulenin documented here are consistent with prior studies showing its cytotoxic effects upon other types of cancer cells and illustrate the potential utility of FAS inhibition as a novel chemotherapeutic approach.
