FTY720Induces Apoptosis And Enhances Sensitivity To Chemotherapy In Colon Cancer Cell Lines

Objectives:FTY720, a synthetic sphingosine analogue of myriocine derived from culture filtrates of Isaria sinclairii, has been extensively applied in prevention of kidney graft rejection and treatment of relapsing multiple sclerosis. Recent studies indicated that FTY720could also inhibit proliferation and induce apoptosis in many cancer cells. However, little is known about the effects of FTY720on colon cancers. This study aims to explore the effects and mechanisms of FTY720on inducing growth inhibition and potentiating cytotoxicity of anticancer drugs in HCT-8human colon cancer cell line and its multidrug subline HCT-8/5-Fu.Methods:①Cell viability and sensitivity to chemotherapeutic drugs (e.g.5-Fluorouracil, cisplatin; doxorubicin, and etoposide) under different concentrations of FTY720was tested by MTT assay;②Annexin V/PI flow cytometry was adopted to evaluate cell apoptosis after treatment with FTY720alone or in combination with chemotherapeutic drugs as above for24hours;③Western blot was adopted to evaluate protein expression of nyclear factor-κB (NF-κB), and quantitative real-time polymerase chain reaction (QRT-PCR) was used to demonstrate expression levels of involved genes such as xIAP and cIAP2;④Changes in P-glycoprotein (P-gp) and multidrug resistance protein1(MRP1) were determined at the mRNA and functional level and the protection role of pertussis toxin in this process was also assessed.Results:①MTT assay indicated that FTY720alone could inhibit growth in HCT-8and HCT-8/5-Fu cells in a time-and dose-dependent manner, and IC50-24h values were12.99±0.54μM,13.67±0.45, respectively;②Flow cytometry also showed that FTY720could induce apoptosis in a time-and concentration-dependent manner using annexin V/PI staining;③10μM FTY720combined with chemotherapeutic drugs stated above could remarkably decrease the IC50-24h values (5-Fu:HCT-84.79±0.64μg/ml VS.198.67±12.32μg/ml, HCT-8/5-Fu228.36116.58μg/ml VS.1479.28±35.26μg/ml; CP: HCT-80.59±0.07μg/ml VS.5.64±0.93μg/ml,HCT-8/5-Fu1.23±0.23μg/ml VS.24.64±3.65μg/ml;DOX:HCT-80.24±0.07μg/ml VS.1.70±0.25μg/ml, HCT-8/5-Fu 2.15±0.54μg/ml VS.14.00±0.11μg/ml; VP16:HCT-80.6710.01μg/ml VS.5.13±0.43μg/ml, HCT-8/5-Fu4.67±0.05μg/ml VS.36.51±0.07μg/ml) and enhance apoptosis;④FTY720alone could significantly decrease phosphorylation of p65in nucleus and some downstream genes of NF-κB (xIAP, cIAP-2, cFLIPL, cFLIPS and Bcl-xL, etc.);⑤FTY720alone or in combination with anticancer drugs significantly inhibited P-gp and MRP1expression by blocking their gene transcription. And it also remarkably increased the intracellular accumulation and retention of the fluorescent substrates (doxorubicin, DiOC2(3) and Fluo3-AM). Moreover, pertussis toxin (PTx) could not reverse the above effects.Conclusions:In this study, the effects of FTY720on human colon cancer cell line HCT-8, and its multidrug resistant variant HCT-8/5-Fu were, for the first time, systematically evaluated. FTY720could inhibit cell proliferation, induce apoptosis and enhance sensitivity to5-Fu, cisplatin, doxorubicin and etoposide in a time-and dose-dependent manner. Furthermore, this study indicated for the first time that FTY720could inhibit the function and mRNA expression of P-gp and MRP1not via the sphigosine-1-phosphate (S1P) receptor1signaling pathway. These data provide preclinical support for chemotherapeutic approach with FTY720for the treatment of colon cancers especially chemo-resistant ones.