Effect of nucleolin on adriamycin resistance via the regulation of B-cell lymphoma 2 expression in Burkitt's lymphoma cells

Abstract

Nucleolin (NCL, C23) is an important nucleocytoplasmic multifunctional protein. Due to its multifaceted profile and high expression in cancer, NCL is considered to be a marker of drug resistance associated with chemotherapy. However, the biochemical mechanisms in which NCL suppresses drug sensitivity in several cancers have yet to be fully elucidated. This study aims to explore the effect of NCL on drug sensitivity and its potential mechanism in CA46 Burkitt's lymphoma (BL) cells. CA46 BL cells were transfected with lentiviruses carrying the NCL gene (CA46-NCL-overexpression, CA46-NCL-OE), or shRNA sequences that target the endogenous NCL gene (CA46-NCL-knockdown, CA46-NCL-KD). Adriamycin (ADM) IC50 levels for CA46-NCL-overexpressed (OE), CA46-NCL-OE control (OEC), CA46-NCL-knockdown (KD), and CA46-NCL-KD control (KDC) cells were 0.68 ± 0.06 μg/ml, 0.68 ± 0.06 μg/ml, 0.68 ± 0.06 μg/ml, and 0.30 ± 0.04 μg/ml, respectively. Apoptosis rates were significantly increased following NCL KD, whereas the opposite effect was noted in OE cells. A significant reduction of B-cell lymphoma 2 (Bcl-2) mRNA and protein levels in KD cells was observed, while OE cells displayed the opposite effect. The stability of Bcl-2 mRNA was influenced by NCL levels, the half-life of which was extended after NCL-OE, whereas it was reduced in KD cells. Finally, results of RNA-immunoprecipitation assays indicated that NCL could bind to Bcl-2 mRNA in CA46 cells. Taken together, these results suggested that NCL could mediate Bcl-2 expression and stability, and thus enhance ADM resistance in CA46 BL cells.

Keywords: Bcl-2; Burkitt's lymphoma; CA46; adriamycin resistance; nucleolin.

Figure 1

Effect of NCL KD and OE on drug sensitivity of adriamycin in human Burkitt's lymphoma cell line CA46. CA46, nontransduced CA46 cells; CA46‐NCL‐KDC, CA46 cells transduced with lentivirus‐mediated scramble shRNA as control; CA46‐NCL‐KD, CA46 cells transduced with lentivirus‐mediated NCL shRNA; CA46‐NCL‐OEC, CA46 cells transduced with empty lentiviral vector as control; CA46‐NCL‐OE, CA46 cells transduced with lentivirus‐mediated NCL overexpression. (a) qRT‐PCR was applied to detect NCL mRNA expression. Relative mRNA expression was normalized to CA46. (b) Western blot analysis was applied to detect NCL protein expression. β‐Actin was used as a loading control. (c) Drug sensitivity of adriamycin was determined using the MTS assay. The results were are presented as IC50 values for each group. Data were are shown as mean ± SD for at least three independent experiments. KD, knockdown; NCL, nucleolin; OEC, overexpression control; qRT‐PCR, quantitative reverse‐transcription polymerase chain reaction; shRNA, short hairpin RNA. *p < .05, **p < .01

Figure 2

Effect of NCL knockdown and overexpression on induction of apoptosis in CA46 cells. The induction of apoptosis was determined using flow cytometry. (a) Knockdown of NCL increased the induction of apoptosis in CA46 cells. (b) Overexpression of NCL decreased the induction of apoptosis in CA46 these cells. The data indicate mean ± SD for at least three independent experiments. (c) Apoptosis was measured by DAPI staining. (d) Caspase3 protein were examined by western blot analysis. DAPI, 4′,6‐diamidino‐2‐phenylindole; NCL, nucleolin. *p  < .05, **p < .01 [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Effect of NCL knockdown and overexpression on Bcl‐2 mRNA and protein expression in CA46 cells. (a) qRT‐PCR was applied to detect Bcl‐2 mRNA. Relative mRNA expression was normalized to CA46. (b) Western blot analysis was applied to detect Bcl‐2 protein expression levels. Data indicated mean ± SD for at least three independent experiments. Bcl‐2, B‐cell lymphoma 2; NCL, nucleolin; qRT‐PCR, quantitative reverse‐transcription polymerase chain reaction. *p < .05, **p < .01

Figure 4

NCL modulated the stability of Bcl‐2 mRNA via binding directly to the mRNA transcript. (a) Decay of Bcl‐2 mRNA in the different CA46 groups following treatment with actinomycin D (2 µg/ml) for the indicated time periods. qRT‐PCR was performed to detect Bcl‐2 mRNA. The results were are expressed as percentage of the Bcl‐2/β‐actin ratio at the indicated time points compared with the Bcl‐2/β‐actin mRNA ratio at time 0 hr (CA46‐NCL‐KD vs. CA46‐NCL‐KDC, CA46‐NCL‐OE vs. CA46‐NCL‐OEC, *p < .05, **p < .01). (b) Bcl‐2 mRNA was enriched by RIP assay with an anti‐NCL antibody. Bcl‐2 mRNA was measured by qRT‐PCR. M: molecular marker. NCL: Bcl‐2 mRNA was immunoprecipitated with an anti‐NCL antibody. IgG: negative control; Bcl‐2 mRNA was immunoprecipitated with a normal mouse IgG antibody. Data indicated mean ± SD for at least three independent experiments. Bcl‐2, B‐cell lymphoma 2; KDC, knockdown control; NCL, nucleolin; OEC, overexpression control; qRT‐PCR, quantitative reverse‐transcription polymerase chain reaction; RIP, RNA‐immunoprecipitation [Color figure can be viewed at wileyonlinelibrary.com]