Evaluation of influence of Ap4A analogues on Fhit-positive HEK293T cells; cytotoxicity and ability to induce apoptosis.

Fragile histidine triad (Fhit) protein encoded by tumour suppressor FHIT gene is a proapoptotic protein with diadenosine polyphosphate (Ap(n)A, n=2-6) hydrolase activity. It has been hypothesised that formation of Fhit-substrate complex results in an apoptosis initiation signal while subsequent hydrolysis of Ap(n)A terminates this action. A series of Ap(n)A analogues have been identified in vitro as strong Fhit ligands [Varnum, J. M.; Baraniak, J.; Kaczmarek, R.; Stec, W. J.; Brenner, C. BMC Chem. Biol.2001, 1, 3]. We assumed that in Fhit-positive cells these compounds might preferentially bind to Fhit and inhibit its hydrolytic activity what would prolong the lifetime of apoptosis initiation signalling complex. Therefore, several Fhit inhibitors were tested for their cytotoxicity and ability to induce apoptosis in Fhit-positive HEK293T cells. These experiments have shown that Ap(4)A analogue, containing a glycerol residue instead of the central pyrophosphate and two terminal phosphorothioates [A(PS)-CH(2)CH(OH)CH(2)-(PS)A (1)], is the most cytotoxic among test compounds (IC(50)=17.5±4.2 µM) and triggers caspase-dependent cell apoptosis. The Fhit-negative HEK293T cells (in which Fhit was silenced by RNAi) were not sensitive to compound 1. These results indicate that the Ap(4)A analogue 1 induces Fhit-dependent apoptosis and therefore, it can be considered as a drug candidate for anticancer therapy in Fhit-positive cancer cells and in Fhit-negative cancer cells, in which re-expression of Fhit was accomplished by gene therapy.

[FHIT--tumor suppressor protein involved in induction of apoptosis and cell cycle regulation]. / FHIT--bialko supresorowe zaangazowane w indukcje apoptozy i regulacje cyklu komórkowego.

The fragile histidine triad (Fhit) protein is a diadenosine triphosphate hydrolase belonging to the histidine triad family of nucleotide-binding proteins. Fhit is a tumor suppressor protein which plays an important role in pro-apoptotic signalling, cell cycle control and sensitivity to DNA damaging agents. The Ap3A--hydrolase activity of Fhit is not required for exertion of its tumor suppressor function. The FHIT gene is located in FRA3B region--the most common fragile site in the human genome. Loss of FHIT is observed in a variety of tumors and in premalignant states. Re-expression of FHIT in human cancer cells leads to inhibition of tumorogenecity and restoration of caspase-dependent apoptosis. Viral-mediated FHIT gene therapy induces apoptosis and suppresses tumors in preclinical models. Therefore, treatment with the FHIT gene may constitute a novel clinical approach for gene therapy of human cancers.