Up-regulation of p21 and TNF-alpha is mediated in lycorine-induced death of HL-60 cells

Abstract

Background: Leukemia is one of the most life-threatening cancers today, and acute promyelogenous leukemia (APL) is a common type of leukemia. Many natural compounds have already been found to exhibit significant anti-tumor effects. Lycorine, a natural alkaloid extracted from Amaryllidaceae, exhibited anti-leukemia effects in vitro and in vivo. The survival rate of HL-60 cells exposed to lycorine was decreased, cell growth was slowed down, and cell regeneration potential was inhibited. HL-60 cells exhibited typical apoptotic characteristic. Lycorine can suppress leukemia growth and reduce cell survival and inducing apoptosis of tumor cells. The purpose of this work is to elucidate the mechanism by which lycorine induces APL cells.

Results: When HL-60 cells were treated with different concentration of lycorine, the expression of p21 and TNF-alpha was up-regulated in a concentration-dependent manner as shown by real-time quantitative reverse transcriptase-polymerase chain reaction and Western blotting. Lycorine also down-regulated p21-related gene expression, including Cdc2, Cyclin B, Cdk2 and Cyclin E, promoted Bid truncation, decreased IkappaB phosphorylation and blocked NF-kappaB nuclear import. Cytochrome c was released from mitochondria as observed with confocal laser microscopy.

Conclusions: The TNF-alpha signal transduction pathway and p21-mediated cell-cycle inhibition were involved in the apoptosis of HL-60 cells induced by lycorine. These results contribute to the development of new lycorine-based anti-leukemia drugs.

Figure 1

Effects of lycorine on expression of p21 and downstream genes regulated by p21 in HL-60 cells. (A) Expression levels of p21 in HL-60 cells were analysed by real-time quantitative RT-PCR. Expression levels were standardised using expression of the housekeeping gene β-actin. (B) Effects of lycorine on expression of p21 protein, (C) Cdc2 and Cyclin B proteins, (D) Cdk2 and Cyclin E proteins. α-tubulin was used for normalisation and verification of the protein loading in B, C, and D in Western blotting. Lanes 1-4, HL-60 cells were treated with 0, 1.25, 2.5, and 5.0 μM lycorine, respectively. Results were presented as mean ± S.D. (n = 3, three independent experiments). Asterisk symbol (*) indicates significant difference (p < 0.05) compared with the control group.

Figure 2

Effects of lycorine on expression of TNF-α and truncation of Bid in HL-60 cells. (A) Expression levels of TNF-α in HL-60 cells were analysed by real-time quantitative RT-PCR. Expression levels were standardised using expression of the housekeeping gene β-actin. (B) Effect of lycorine on expression of TNF-α protein, (C) Bid and truncation of Bid. α-tubulin was used for normalisation and verification of the protein loading in B and C in Western blotting. Lanes 1-4, HL-60 cells were treated with 0, 1.25, 2.5, and 5.0 μM lycorine, respectively. Results were presented as mean ± S.D. (n = 3, three independent experiments). Asterisk symbol (*) indicates significant difference (p < 0.05) compared with the control group.

Figure 3

Effect of lycorine on cytochrome c release. Cells were fixed and labelled for cytochrome c (red) and DNA (blue). (A) HL-60 cells in control group. (B) Cells were treated with 5.0 μM lycorine for 24 h. Cell nuclei were observed by DNA staining with DAPI (A2, B2). The staining pattern of cytochrome c became diffusive in most cells (B1), consistent with a translocation of cytochrome c into the cytosol and nucleus (B3), whereas cytochrome c displayed a dotted pattern in untreated cells, consistent with its location within mitochondria (A1, A3). Images were obtained with a confocal microscope (×400).

Figure 4

Effects of lycorine on phosphorylation of IκB and nuclear import of NF-κB. (A) Effect of lycorine on expression of IκB and phosphorylation of IκB protein. (B) Effect of lycorine on expression of NF-κB protein and (C) distribution of NF-κB protein in nucleus. α-tubulin was used for normalisation and verification of the protein loading in A, B, and C in Western blotting. Lanes 1-4: HL-60 cells were treated with 0, 1.25, 2.5, and 5.0 μM lycorine, respectively. (D) Subcellular distribution of NF-κB detected by immunofluorescence staining. HL-60 cells were treated with 0 μM (D1, D2, D3) and 5.0 μM lycorine (D4, D5, D6), respectively. Cells were labeled for NF-κB (red) and DNA (blue). Cell nuclei were stained by DAPI (D2, D5). NF-κB was mainly distributed in the cytoplasm (D4, D6) in the HL-60 cells treated with lycorine, whereas NF-κB was mainly distributed in the nucleus in untreated cells (D1, D3). Results were presented as mean ± S.D. (n = 3, three independent experiments). Asterisk symbol (*) indicates significant difference (p < 0.05) compared with the control group.