Effects Of PI3K/Akt Signaling Pathway On The Chemoresistance Of Acute Myeloid Leukemia Cells Co-cultured With Stromal Cells And Its Clinical Significance

Objective: To investigate the effects of PI3K/Akt signaling pathway on acute myeloid leukemia cells co-cultured with stromal cells and its clinical significance.Methods: A co-culture system composed of AML cell lines(HL60 cells and U937cells) cultured with human bone marrow stromal cell line(HS-5 cells) was developed.Sensitivity of leukemia cells to DNR and Ara-C was detected by CCK8. Protein contents of p-Akt Ser473, p-PDK1 and p-PTEN were detected by Western Bolt.Transcriptional levels of PTEN, CCND1, m TOR, RICTOR and FOXO1 gene were detected by real time PCR.Results: Killing effects of DNR and Ara-C to HL60 cells and U937 cells were significant and does-dependent. Adhesion to HS-5 cells could induce resistance to DNR(0.2μM) in the two cell lines, inhibitory rate reducing from(65.77±2.74) % to(40.13±2.19) % in HL60 cells, and from(53.07±3.54) % to(36.43±2.96) % in U937 cells. The resistance can be partially restored using treatment with LY294002,inhibitory rates raising from(40.13±2.19) % to(54.67±3.72) % in HL60 cells, and from(36.43±2.96) % to(45.97±2.71) % in U937 cells. Adhesion to HS-5 cells could also induce resistance to Ara-C(2.5μM) in the two cell lines, inhibitory rate reducing from(68.17±3.16) % to(48.23±2.82) % in HL60 cells, and from(56.77±4.78) % to(40.37±3.36) % in U937 cells. And the resistance could also be partially restored using treatment with LY294002, inhibitory rates raising from(48.23±2.82) % to(61.43±2.07) % in HL60 cells, and from(40.37±3.36) % to(51.37±3.87) % in U937 cells. The adhesion activated PI3K/Akt signaling both in HL60 cells and in U937 cells. And treatment with LY294002 could partly inhibit the activation. p-Akt Ser473 and p-PDK1 were up regulated, and p-PTEN was reduced in AML cells adhered to HS-5 cells. And treatment with LY294002 induced p-Akt Ser473 and p-PDK1 significantly reduced, and p-PTEN up regulated.Adhesion to HS-5 cells increased transcription levels of CCND1 and FOXO1,reduced m TOR and PTEN in HL60 cells. But the level of RICTOR had no significant change. And adhesion to HS-5 cells up regulated protein contents of CCND1, m TOR and RICTOR, and reduced PTEN in HL60 cells. Neither HL60 cells cultured alone nor adhered to HS-5 cells revealed expression of FOXO1. Referring to the expression of the five genes, there was only a difference existed between the two cell lines. That was after adhesion to HS-5 cells, U937 cells revealed no expression of CCND1 protein.In 23 cases of primary AML, m RNA levels of FOXO1, m TOR, PTEN and RICTOR showed low. And there was no significant difference between normal people and AML patients in the levels of CCND1. And the protein levels were not go along with the transcription levels. The positive rates of RITOR, PTEN, CCND1, FOXO1 and m TOR were 78.3%(18/23), 73.9%(17/23), 73.9%(17/23), 65.2%(15/23) and52.2%(12/23), respectively.Conclusion: Adhesion to HS-5 cells could enable both HL60 cells and U937 cells resistance to DNR and Ara-C. And the activation of PI3K/Akt signal pathway plays a significant part in the process. The m RNA expression and protein expression levels of CCND1, FOXO1, PTEN, m TOR and RICTOR are not consistent in primary AML cells.