The Mechanism Of Apoptosis In Monocytic Leukemia Cell THP-1 Induced By The Activation Of TLR4

Objective: The Toll-like acceptors (TLRs) are a kind of importantmolecules which participate in the non-specific immunity and they are thebridges connect the non-specificity immunity and the specificityimmunity. Recently people discovered that many microorganismcomponents exert anti-cancer immunity effect by activating TLR signalpathway. In this experiment TLR4 was activated by LPS to induceapoptosis in monocytic leukemia THP-1 cells, and with this cut-in pointwe studied the molecular mechanism of apoptosis in monocytic leukemiacells induced by activation of TLR4, in order to provide the theory basisfor the monocytic leukemia treatment by choosing TLR as the target.Methods: (1) RT-PCR is selected to detect the gene expression inTHP-1 cells. (2) FACS is used to detect the protein expression on thesurface of THP-1 cells. (3) By using of ELISA, the cytokine protein inthe supernatant is measured. (4) PI-Annexin V staining tests theapoptosis level of THP-1 inducing by LPS. (5) Neutralization experimentby antibody is used to verify the involvement of cytokines in theinduction of apoptosis of THP-1 cells induced by LPS. (6) By using ofwestern blotting, we assay the activation of signal transduction moleculessuch as P38, ERK, JNK and NF-κB in THP-1 cells.Results: RT-PCR results showed that the TLRs except TLR3 wereexpressed on the surface of THP-1 cells. Proteins of CD14 TLR2 andTLR4 were expressed on THP-1 cell by FACS, and the expression ofthese proteins was up-regulated when the THP-1 cells were stimulatedwith TLR4 ligand LPS. And LPS induced the phophorylation of NF-κBand MAPK family protein ERK, JNK, P38, which belong to the TLR4signal transduction pathway, suggesting THP-1 cells expressed functionalTLR4. Moreover, the activation of TLR4 by LPS induced apoptosis inTHP-1 cells with dose and time dependent manner by PI-Annexin Vstaining. The apoptosis related cytokines TNFαand TRAIL involved the induction of apoptosis, because TNFαand TRAIL neutralizing antibodydecreased the apoptosis induced by LPS. In addition, LPSdown-regulated the expression of Bcl-2, and up-regulated the expressionof BAK, suggesting not only receptor apoptosis pathway but alsomitochondria apoptosis pathway involved in the induction of apoptosisinduced by LPS. This findings was further proved by the detection ofactivation of both caspase 8 and caspase 9. Moreover, the activation ofTLR4 by LPS down-regulated the expression of caspase 8 inhibitorprotein FLIP_L and thus promoted the induction of apoptosis in THP-1cells.Conclusions: TLR4 Activated by LPS could induce the apoptosisof THP-1 cells. Both death receptor apoptosis pathway and mitochondriaapoptosis pathway involved in the induction of apoptosis in THP-1cells. The activation of TLR4 by LPS down-regulated the expression ofFLIP_L, which promoted the apoptosis of THP-1 cell.