engleski

Dual inhibition of sphingosine kinase 1/2 enhances antitumor activity of 5-fluorouracil in human hepatocellular carcinoma cells in vitro

Hepatocellular carcinoma (HCC) is a major challenge for medical oncologists because of its inherent chemoresistance. Common chemotherapy drugs used for the treatment of advanced HCC such as 5-fluorouracil (5-FU) show low response rates. Novel targeted agent recently introduced into the HCC treatment, namely multikinase inhibitor sorafenib, also exhibited moderate efficacy and adverse events in clinical studies. Therefore, there is an urgent need for innovative strategies that would provide significant clinical benefit to patients with advanced HCC. Sphingosine kinase (SphK) is a key regulator of sphingolipid rheostat which balances between the two major bioactive interconvertible sphingolipids with opposing functions, namely pro-apoptotic ceramide and pro-proliferative sphingosine-1- phosphate. Importantly, SphK is found to be overexpressed in a variety of solid human tumours, which makes it a suitable target for pharmacological intervention. In the present work, we investigated the effect of dual sphingosine kinase isoenzyme inhibitor SK1-II (4-[[4-(4-chlorophenyl)-1, 3-thiazol-2- yl]amino]phenol on the response of human hepatocellular carcinoma cells HepG2 to 5-FU. Initially, IC50 values were determined for an each single agent and then for their combinations followed by the assessment of their combinatorial effects using CompuSyn software. We found that SphK inhibition reduces cell viability and enhances the sensitivity of HepG2 cells to 5-FU. Combinations of SphK inhibitor with 5-FU at their sub-lethal concentrations resulted mainly in additive or slightly synergistic cytotoxicity. Furthermore, the combination of SK1-II and 5-FU at concentrations exerting synergistic effect had more pronounced effect on apoptosis induction versus 5-FU alone, especially after 48-hour treatment. Additional Western blot analysis revealed that improved antitumor efficacy of 5- FU combined with SK1-II in comparison with single-agent therapy with 5-FU could be attributed to inhibition of IGF1R signalling. Consequently, this led to reduced activity of Akt kinase and down-regulation of anti- apoptotic protein NF-κB and ligand for the receptor activator of NF-κB (RANKL), which promoted apoptosis. In parallel, combination therapy resulted in the abrogation of Ras/MAPK pathway as evidenced from the reduced activity of c-Raf, ERK1/2 and p38 MAPK, which could account for reduced cell growth and proliferation. Finally, we observed a marked decline in the activity of focal adhesion kinase (FAK) in combination versus single-agent therapy with 5-FU, a major regulator of survival signalling. Collectively, our results indicate that combination therapy with 5-FU and SphK inhibitor has the potential to further advance therapeutic options for HCC.

sphingosine kinase ; 5-fluorouracil ; hepatocellular carcinoma

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