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Inhibition of farnesyl-protein-transferase in neuroblastoma cells by alpha-hydroxyfarnesylphosphonate
R Girgert1, A Hohnecker, J Wittrock
1Department of Pediatric Surgery, University of Tübingen, Germany.
Abstract:
Oncogenic Ras is responsible for malignant transformation in a variety of tumors. Farnesylation of Ras by farnesyl-protein-transferase (FPTase) is necessary for membrane localization of Ras-proteins, a prerequisite for its biological activity. Although mutations in ras genes are rare in neuroblastoma inactivation of Ras by inhibition of the FPTase is of interest in neuroblastoma. In this tumor, amplification of N-myc is frequently observed and expression of N-myc is induced via Ras signaling. Farnesyl-protein-transferase of neuroblastoma cells is inhibited by alpha-hydroxyfarnesylphosphonate. In homogenates of the cell line SK-N-AS an ID50 = 6.5 microM is estimated, in SK-N-SH the ID50 is 3.4 microM. The consequences of the inhibition of FPTase on the membrane localization was examined by immunoblots. Western blots of membrane proteins analysed with H-ras and N-ras specific antibodies revealed that H-ras protein is more sensitive to the inhibition of FPTase than N-ras protein. After culturing neuroblastoma cells for 24 hrs in the presence of 20 microM alpha-hydroxyfarnesylphosphonate H-ras protein completely dissappeared from the membrane fraction whereas N-ras protein was only affected by 50%. K-ras was not detectable on Western blots of three neuroblastoma cell lines. The experiments showed that FPTase inhibitors are effective in neuroblastoma cells but for complete inactivation of N-ras stronger conditions are required than for H-ras.
Insights
Farnesyl-protein-transferase (FPTase) inhibitors show promise for neuroblastoma treatment by disrupting Ras-protein membrane localization. H-ras is more sensitive than N-ras to FPTase inhibition, requiring stronger conditions for complete N-ras inactivation.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Oncogenic Ras proteins drive malignant transformation in various cancers.
- Ras farnesylation by farnesyl-protein-transferase (FPTase) is crucial for its membrane localization and biological activity.
- While Ras mutations are uncommon in neuroblastoma, Ras signaling impacts N-myc amplification, a frequent event in this tumor.
Purpose of the Study:
- To investigate the efficacy of farnesyl-protein-transferase (FPTase) inhibition in neuroblastoma.
- To assess the impact of FPTase inhibition on Ras-protein membrane localization in neuroblastoma cell lines.
- To compare the sensitivity of H-ras and N-ras to FPTase inhibition.
Main Methods:
- Utilized alpha-hydroxyfarnesylphosphonate to inhibit FPTase in neuroblastoma cell lines (SK-N-AS, SK-N-SH).
- Determined the inhibitory concentration 50 (ID50) for FPTase inhibition in cell homogenates.
- Examined changes in H-ras and N-ras membrane localization using Western blots with specific antibodies.
Main Results:
- FPTase inhibition by alpha-hydroxyfarnesylphosphonate was effective in neuroblastoma cells, with ID50 values of 6.5 microM (SK-N-AS) and 3.4 microM (SK-N-SH).
- H-ras protein was significantly more sensitive to FPTase inhibition than N-ras protein.
- Complete disappearance of H-ras from the membrane fraction was observed after 24 hours of treatment with 20 microM alpha-hydroxyfarnesylphosphonate, while N-ras was only 50% affected.
Conclusions:
- FPTase inhibitors represent a potential therapeutic strategy for neuroblastoma.
- Differential sensitivity of H-ras and N-ras to FPTase inhibition suggests distinct requirements for their membrane localization.
- Achieving complete inactivation of N-ras in neuroblastoma may necessitate more potent inhibition or combination therapies compared to H-ras.
