The tumorigenicity-promoting activity of C-FABP in prostate cancer cells depends on its fatty acid-binding ability



Malki, Mohammed Imad
The tumorigenicity-promoting activity of C-FABP in prostate cancer cells depends on its fatty acid-binding ability. Doctor of Philosophy thesis, University of Liverpool.

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Abstract

Cutaneous fatty acid-binding protein (C-FABP) or FABP5, is a FABP family member that can bind to long chain fatty acids with high affinity. C-FABP was identified by our research group as a gene involved in malignant progression of prostate cancer and able to promote the growth of primary tumours and induce metastasis when transfected into rat benign Rama 37 model cells. It was demonstrated that C-FABP was a prognostic marker for patient outcome and a target of tumour-suppression for prostate cancer. As an initial step to understanding the complex molecular mechanisms involved in the cancer promoting activity of C-FABP, this study investigated the possible relationship between tumorigenicity-promoting activity of C-FABP and its fatty acid-binding capability. After single and double mutations were generated in the fatty acid binding motif of the C-FABP cDNA, wild type and mutant C-FABP cDNAs were excised from the mammalian vector pIRES2-EGFP and inserted into pBluescript cloning vector to generate a complimentary restriction sites at both ends of the cDNAs. The C-FABP cDNAs were excised from the pBluescript vector with KpnI and PstI and inserted into pQEs expression vector to form three constructs that express the wild type and two mutant C-FABPs (C-FABP-WT, C-FABP-R109A and C-FABP-R109/129A), respectively. SDS-PAGE and sequence analysis confirmed the correct insertion of C-FABPs into expression vector pQE. The pure recombinant proteins were subsequently produced and purified. The importance of fatty acid-binding activity to the cancer promoting function was assessed by comparing the cancer promoting abilities exerted by C-FABPs with different fatty acid-binding capabilities. To test whether the recombinant proteins produced were biological active, the fatty acid binding ability of wild type and mutant C-FABPs were tested. When fatty acid binding ability of the wild type C-FABP is set at 1, the average fatty acid binding ability the C-FABP-R109A and C-FABP-R109/129A were significantly reduced to 0.32% and 0.09%, respectively (Student t-test, P0.5). The most significant change occurred in C-FABP-WT stimulated cells that produced more than a 6.7-fold (85%) increase in the number of colonies formed in soft agar when compared to controls (Student t-test, P

Item Type: Thesis (Doctor of Philosophy)
Additional Information: Date: 2011-12 (completed)
Subjects: ?? RC0254 ??
Divisions: Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology
Depositing User: Symplectic Admin
Date Deposited: 07 Aug 2012 08:12
Last Modified: 16 Dec 2022 04:36
DOI: 10.17638/00005213
Supervisors:
  • Ke, Youqiang
  • Foster, Christopher S
URI: https://livrepository.liverpool.ac.uk/id/eprint/5213