Immune killing effects of 1-methyltroptophan combined with peripheral blood mononuclear cells on human glioma cells

Abstract

Objective To investigate the immune killing effects of 1- methyltroptophan(1- MT) combined with peripheral blood mononuclear cells(PBMC) on human A172 glioma cells and the relative mechanism. Methods PBMC were isolated from healthy donors by Ficoll density- gradient centrifugation. A172 cells were co- cultured with or without PBMCin the presence or absence of 1- MT. Cell counting kit-8(CCK- 8)assay was used to test the inhibitory effect of 1- MT(0,25,125,250,500μmol/L;1,2,4, 8,10 mmol/L)combined with PBMC on growth of A172 cells.Annexin V/propidium iodide(PI)staining was used to detect the effect of 1- MT(500μmol/L)combined with PBMC on apoptosis of A172 cells. The expression of indoleamine 2,3- dioxygenase(IDO)protein in A172 cells was detected by Western blotting in different groups.The expression changes of forkhead helix transcription factor p3(Foxp3) mRNA in PBMC were investigated by real- time quantitative polymerase chain reaction(RT- qPCR). Results 1- MT reduced the growth rate of A172 glioma cells in a dose- dependent manner under the condition of a constant PBMCamount.The apoptosis rate of A172 cells induced by combined 1- MT and PBMC(45.0%) was significantly higher than that induced by either 1- MT(33.7%)or PBMCs(20.0%).Western blotting analysis showed that the expression of IDO was considerably lower in 1- MT+ PBMC group(0.182±0.015)than in PBMC group(0.313±0.017).1- MT group and A172 blank group showed no significant IDO expression.The mRNA level of Foxp3 was significantly higher in PBMC group(2.764±0.180)than in 1- MT+ PBMC group (2.189±0.179)and PBMC blank group(1.000±0.028)by RT- qPCR. Conclusion 1- MT can significantly increase the immune killing effects of PBMC by inhibiting the expression of IDO and Foxp3 and the differentiation of Treg cells in A172 cells. Key words: Indoleamine 2,3-dioxygenase; Glioma cells; Peripheral blood mononuclear cells; Apoptosis