OBJECTIVE To investigate the mechanism of polyinosinic polycytidylic acid Poly(I:C) enhancing luteolin (LTN) to inhibit the proliferation of acute lymphoblastic leukemia cell line Nalm6.
METHODS Using CCK-8 method and flow cytometry to detect the proliferation and apoptosis of Nalm6 cells treated with LTN alone or combined with Poly(I:C), and explored the effect of programmed apoptosis inhibitor(ZVAD) and necrotic cell death inhibitor(Nec-1) on the combined treatment. Flow cytometry was performed to analyze the effects of LTN alone or combined with Poly(I:C) on cell apoptosis of Nalm6 cells at different time points, cell cycle states at 24 h, and TLR3 expression. The protein expression of p-IRF3, p-mTOR, p-NFκB-65, p-p70S6K, p-ERK1/2, PARP, Caspase 3 and Caspase 8 were detected by Western blotting.
RESULTS Compared with LTN group, Poly(I:C) combined with LTN group significantly inhibited the proliferation of Nalm6 cells (P<0.05); ZVAD could completely block the proliferation-inhibiting effects of Poly(I:C) combined with LTN in Nalm6 cells (P<0.05). Under Poly(I:C) combined with LTN treatment, cell apoptosis occurred at 3 h and reached the late stage of apoptosis after 24 h. Cell cycle results showed that Poly(I:C) significantly promoted LTN-induced DNA breakage (P<0.05). Neutralization of TLR3 pathway did not affect the apoptosis-enhancing effects of Poly(I:C) combined with LTN in Nalm6 cells. Western blotting analysis showed that compared with LTN alone, Poly(I:C) combined with LTN activated PARP, Caspase 3 and Caspase 8 to initiate apoptosis of Nalm6 cells for 6 h, but did not affect the NF-κB and PI3K-AKT signaling pathways. LTN alone activated p-ERK1/2 phosphorylation, inhibiting cell proliferation by activating the MEK/ERK signaling pathway.
CONCLUSION Poly(I:C) induced apoptosis by activating PARP, Caspase 3 and Caspase 8 rather than necrosis, to enhance the proliferation-inhibiting effects of LTN on proliferation of Nalm6 cell.
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