Comparative mitochondrial proteomic analysis of Rji cells exposed to adriamycin

Abstract

The antitumor mechanisms of adriamycin (ADR) have been thought to contribute to induction of apoptosis and inefficiency of DNA repair, processes that are to a large extent mediated by mitochondria. This study aimed to investigate characteristics of ADR, including its antineoplastic activity, drug resistance, and unexpected toxicity in non-Hodgkin lymphoma (NHL) Raji cells at the mitochondrial proteomic level. The alterations of the mitochondrial proteome of Raji cells treated with ADR were analyzed by two-dimensional differential in-gel electrophoresis (2D-DIGE) coupled with linear ion trap quadrupole-electrospray ionization tandem mass spectrometry (LTQ-ESI-MS/MS).The altered patterns of three identified proteins were validated by Western blot and analyzed by pathway studio software. The results showed that 34 proteins were downregulated and 3 proteins upregulated in the study group compared with the control group. The differentially expressed proteins distributed their functions in reduction-oxidation reactions, DNA repair, cell cycle regulation, transporters and channels, and oxidative phosphorylation. Furthermore, heat shock protein 70 (HSP70), ATP-binding cassette transporter isoform B6 (ABCB6), and prohibitin (PHB) identified in this study may be closely related to chemoresistance and could serve as potential chemotherapeutic targets for NHL. Collectively, these results suggest that specific mitochondrial proteins are uniquely susceptible to alterations in abundance following exposure to ADR and carry implications for the investigation of therapeutic and prognostic markers. Further studies focusing on these identified proteins will be used to predict treatment response and reverse apoptosis resistance,and to explore drug-combination strategies associated with ADR for NHL therapy.

Figure 1

Inhibition of growth rate of Raji cells by ADR administered at various doses and times. NHL Raji cells (4 × 10⁵) were exposed to ADR (0.2, 0.5, 1.0, 1.5, and 2.0 μg/mL) for the various incubation times indicated (24 h, 48 h, and 72 h). Inhibition of growth rate was measured with MTT and reported as a percentage.

Figure 2

(A) Separated mitochondria were identified by Janus green B staining. The isolated mitochondria were stained as bluish green round particles. (B) Validation of Raji cell mitochondrial purity by Western blotting. An equal amount of proteins (20 μg) were loaded onto a 10% SDS-PAGE with indicated antibodies against marker proteins from mitochondria (mito) and nuclei and lysosomes (nuclei/lysosome). Antibodies against COXIV were used as a marker specific for mitochondria. Antibodies against PCNA and cathepsin D were used as markers for nuclear and lysosome proteins, respectively. The results showed that COXIV was specifically detected in the purified mitochondrial fraction, and this fraction lacked any detectable contamination by abundant nuclear and lysosome proteins such as PCNA and cathepsin D.

Figure 3

(A) Graphical presentation of altered mitochondrial proteins of Raji cells treated with ADR; 26 spots were analyzed by mass spectrometry and resulted in 37 unique proteins. Among these, 34 proteins decreased and 3 proteins increased. The magnitude ratio of changes ranged from 1.93 multiple upregulation (O75947) and to 4.71 multiple downregulation (Q9NX63). (B) Distribution of function, pI, molecular mass, and GRAVY of the identified mitochondrial proteins; (1) function distribution, (2) pI distribution. (3) molecular mass distribution, (4) GRAVY distribution.

Figure 4

Comparison of HSP 70, PHB, and ABCB6 protein expression levels measured by DIGE and Western blotting. The selected spots were displayed as three-dimensional images and as a partial view of the 2D-DIGE. Verification by Western blotting of HSP70, PHB, and ABCB6 were used to validate the MS results. (A and B) HSP70 (70 kDa) and PHB (32 kDa). Upregulated expression of HSP70 and PHB, respectively. (C) ABCB6 (93 kDa). Downregulated expression of ABCB6. (D) β-actin (43 kDa). β-Actin as an internal marker.

Figure 5

Pathways and networks associated with HSPA9, PHB, and ABCB6. Pathway Studio software was used to map the above three identified proteins onto characterized human pathways and networks that are associated with these proteins based on known protein-to-protein interactions, mRNA expression studies, and other biochemical interactions previously described. Proteins belonging to different structural and functional families had PHB as a common target and were involved in processes such as mitogenesis, defense response, germination, inflammation, proliferation, and apoptosis. Most of the diseases associated with these three proteins were cancer derived from various organs.