Bortezomib inhibits Burkitt's lymphoma cell proliferation by downregulating sumoylated hnRNP K and c-Myc expression

Abstract

Bortezomib (Velcal) was the first proteasome inhibitor to be approved by the US Food and Drug Administration to treat patients with relapsed/refractory multiple myelomas. Previous studies have demonstrated that bortezomib inhibits tumor cell proliferation and induces apoptosis by blocking the nuclear factor (NF)-κB pathway. However, the exact mechanism by which bortezomib induces cancer cell apoptosis is still not well understood. In this study, we found that bortezomib significantly inhibited cell proliferation in both human Burkitt's lymphoma CA46 and Daudi cells. Through proteomic analysis, we found that bortezomib treatment changed the expression of various proteins in distinct functional categories including unfolding protein response (UPS), RNA processing, protein targeting and biosynthesis, apoptosis, and signal transduction. Among the proteins with altered expression, hnRNP K, hnRNP H, Hsp90α, Grp78, and Hsp7C were common to both Daudi and CA46 cells. Interestingly, bortezomib treatment downregulated the expression of high-molecular-weight (HMw) hnRNP K and c-Myc but upregulated the expression of low-molecular-weight (LMw) hnRNP K. Moreover, cell proliferation was significantly correlated with high expression of HMw hnRNP K and c-Myc. HMw and LMw hnRNP K were identified as sumoylated and desumoylated hnRNP K, respectively. Using transient transfection, we found that sumoylated hnRNP K increased c-Myc expression at the translational level and contributed to cell proliferation, and that Lys422 of hnRNP K is the candidate sumoylated residue. Our results suggest that besides inhibiting the ubiquitin-proteasome pathway, bortezomib may inhibit cell proliferation by downregulating sumoylated hnRNP K and c-Myc expression in Burkitt's lymphoma cells.

Keywords: Bortezomib; Burkitt’s lymphoma; c-Myc; hnRNP K; sumoylation.

Figure 1. Inhibitory effects of bortezomib on the proliferation of human Burkitt's lymphoma cells

A. Daudi and B. CA46 cells were treated with various concentrations of bortezomib for different periods, and the viable cells were determined by MTT assay. The data are presented as mean ± S.E. of three independent experiments.

Figure 2. Proteomic analysis of altered proteins in bortezomib-treated human Burkitt's lymphoma cells

Daudi cells were treated with 10 nM bortezomib (Bz) for 12 h, total cellular proteins were subjected to two-dimensional electrophoresis, and altered proteins were identified by MALDI-Q-TOF. A. Upregulated and B. downregulated proteins. Ctrl, control.

Figure 3. Changes in high- and low-molecular-weight hnRNP K protein expression in bortezomib-treated human Burkitt's lymphoma cells

(A, B) Daudi cells were treated with A. various concentrations of bortezomib (Bz) for 12 h, or B. 10 nM bortezomib for the indicated periods. Total cellular proteins were collected to detect protein expression by western blotting. HMw, high-molecular-weight; LMw, low-molecular-weight. The numbers below the lanes indicate the relative intensities of HMw to control (defined as 1.0) proteins.

Figure 4. Downregulation of sumoylated hnRNP K expression in bortezomib-treated human Burkitt's lymphoma cells

A. Daudi cells were treated with 10 nM bortezomib (Bz) for 12 h. Total cellular proteins were collected to detect protein expression by immunoprecipitation (IP) and immunoblotting (IB). Solid arrowhead, sumoylated hnRNP K (Sumo-hnRNP K); open arrowhead, heavy chain of the IgG antibody (IgG). B. Daudi cells were transfected with the control plasmid (pcDNA 3) or the SRa-HA-SUMO plasmid (HA-Sumo) by electroporation. After 48 h of transfection, the cells were treated with 10 nM bortezomib for 12 h, and total cellular proteins were collected to detect protein expression by western blotting. C. Daudi cells were transfected with 5 and 10 μg of the SRa-HA-SUMO plasmid (HA-Sumo) by electroporation. After 48 h of transfection, total proteins were collected to detect protein expression by western blotting. D. Daudi cells were transfected with wild-type (w) and mutant (m) plasmids of SENP2 by electroporation. After 48 h of transfection, total proteins were collected to detect protein expression by western blotting. HMw, high-molecular-weight; LMw, low-molecular-weight. The numbers below the lanes indicate the relative intensities of HMw to control (defined as 1.0) proteins.

Figure 5. Effects of nutrition and starvation on the protein expression of sumoylated hnRNP K and c-Myc in human Burkitt's lymphoma cells

A. Daudi cells were starved for 24 h and then treated with 10% fetal bovine serum (FBS) for the indicated periods. B. Daudi cells were starved for the indicated periods. Total cellular proteins were collected to detect protein expression by western blotting. HMw, high-molecular-weight; LMw, low-molecular-weight. The numbers below the lanes indicate the relative intensities of HMw to control (defined as 1.0) proteins.

Figure 6. Downregulation of c-Myc protein expression in bortezomib-treated human Burkitt's lymphoma cells

(A, B) Daudi cells were treated with various concentrations of bortezomib (Bz), or with 10 nM bortezomib for the indicated periods. A. Total RNA was extracted to detect mRNA expression by RT-PCR, and B. total cellular proteins were collected to detect protein expression by western blotting.

Figure 7. Upregulation of c-Myc protein expression by sumoylated hnRNP K in human Burkitt's lymphoma cells

A. Schematic representation of the configuration of the pGL3-Myc-5′ UTR-Luc reporter plasmid. B. Daudi cells were transfected with 10 μg of the pGL3-Myc-5′ UTR-Luc reporter plasmid and the purl-TK plasmid (as an internal control) by electroporation. After 36 h of transfection, the cells were treated with various concentrations of bortezomib (Bz) for 12 h. Total cell lysates were collected to detect luciferase activities as described in “Materials and Methods”. C. Daudi cells were transfected with either SRa-HA-SUMO-1, Flag-SENP1, or Flag-SENP2 and hnRNP K as well as pGL3-Myc-5′ UTR-Luc and phRL-TK (as an internal control) by electroporation. After 48 h of transfection, total cell lysates were collected to detect luciferase activities as described in “Materials and Methods” and to analyze protein expression by western blotting. D. Daudi cells were transfected with hnRNP K and SRa-HA-SUMO-1 by electroporation. After 36 h of transfection, the cells were treated with 5 nM bortezomib (Bz) for 12 h and the viable cells were determined by MTT assay. Values are presented as the mean ± S.E. of triplicate tests. ^#p < 0.05 vs. lane 1; *p < 0.05 vs. lane 2. HMw, high-molecular-weight.

Figure 8. Lysine 422 is the potential sumoylation site in human hnRNP K

A. The possible sumoylation sites of hnRNP K and the scores predicted by the SUMOplot Analysis Program. B. Daudi cells were transfected with the wild-type hnRNP K (w) or hnRNP K K422R mutant (m) plasmids by electroporation. After 48 h of transfection, total cell lysates were collected to detect protein expression by western blotting. HMw, high-molecular-weight. The numbers below the lanes indicate the relative intensities of HMw to control (defined as 1.0) proteins.