Proteomics of cancer cell lines resistant to microtubule-stabilizing agents

Abstract

Despite the clinical success of microtubule-interacting agents (MIA), a significant challenge for oncologists is the inability to predict the response of individual patients with cancer to these drugs. In the present study, six cell lines were compared by 2D DIGE proteomics to investigate cellular resistance to the class of MIAs known as microtubule-stabilizing agents (MSA). The human lung cancer cell line A549 was compared with two drug-resistant daughter cell lines, a taxol-resistant cell line (AT12) and an epothilone B (EpoB)-resistant cell line (EpoB40). The ovarian cancer cell line Hey was compared with two drug-resistant daughter cell lines, an EpoB-resistant cell line (EpoB8) and an ixabepilone-resistant cell line (Ixab80). All 2D DIGE results were validated by Western blot analyses. A variety of cytoskeletal and cytoskeleton-associated proteins were differentially expressed in drug-resistant cells. Differential abundance of 14-3-3σ, galectin-1 and phosphorylation of stathmin are worthy of further studies as candidate predictive biomarkers for MSAs. This is especially true for galectin-1, a β-galactose-binding lectin that mediates tumor invasion and metastasis. Galectin-1 was greatly increased in EpoB- and ixabepilone-resistant cells and its suppression caused an increase in drug sensitivity in both drug-sensitive and -resistant Hey cells. Furthermore, the growth medium from resistant Hey cells contained higher levels of galectin-1, suggesting that galectin-1 could play a role in resistance to MSAs.

Figure 1

Experimental design for proteomics of cancer cell lines resistant to MSAs studied by 2D DIGE and mass spectrometry. The study includes a total of six cell lines that were analyzed in triplicate on a total of nine gels. Each gel contained two samples and a pooled standard.

Figure 2

Alterations in tubulin isotype expression in drug-resistant cells. A charge-altering mutation in βI-tubulin was detected by 2D DIGE (A to D). (A) Silver stained 2D gel of A549 total lysates. (B) Silver stained 2DE image of tubulin-depleted A549 lysates for improved resolution of α- and β-tubulin. (C) 2D DIGE analysis of tubulin-depleted fractions of A549 (red) and EpoB40 (green). The arrows (1 and 2) indicate two spots that are increased in EpoB40. (D) Resolution of tubulin isotypes by isoelectrofocusing, adapted from Yang et al., MCT 4:987, 2005. (C and D) 1, mono-glutamylated mutant βI-tubulin; 2, mutant βI-tubulin; 3, wild type βI-tubulin. (E) Expression levels of βI-, βIII-, βIV- and βV-tubulin in the total lysates of drug sensitive and resistant cells were determined by Western blot analyses using isotype-specific anti-tubulin antibodies (Table S1). Polyclonal human anti-βV-tubulin antibody was developed in this laboratory (37). Lower panel: Ponceau-stained blot showing equal protein loading.

Figure 3

Expression of actins, keratins and vimentin in drug-resistant cell lines. See Fig. 1 for proteomic changes in drug-resistant cell lines in the high mass range (30 to 80 kDa). (A) Silver stained keratins and actins in 2D DIGE of A549 and AT12 cells; two spots representing significantly increased actin and keratin in AT12 cells. (B) Silver stained 2D gel of A549 total lysates. (C) Silver stained 2DE image of proteins released from a MT pellet by salt wash. (D) Silver stained vimentin in 2D DIGE of Hey and Ixab80 cells. (E) Expression of vimentin in the total lysate of drug-sensitive and -resistant cells was determined by Western blot analysis.

Figure 4

Expression and phosphorylation of stathmin in drug-resistant cell lines. See Fig. 1 for proteomic changes in drug-resistant cell lines in the low mass range (10 to 35 kDa). (A) Selected 2D DIGE region showing two spots identified as stathmin 1 (STMN1). (B) The same region after phospho-specific stain. (C) Expression of stathmin and Ser16-Phospho-stathmin in the total lysates of drug-sensitive and -resistant cells was determined by Western blot analysis.

Figure 5

Expression of 14-3-3σ and galectin-1 was increased differentially in drug-resistant cells and suppression of galectin-1 expression increased drug sensitivity in both drug-sensitive and -resistant Hey cells. (A) Expression of 14-3-3σ and galectin-1 in the total cell lysates was determined by Western blotting. (B) Hey and EpoB8 cells were transfected with vehicle only (C, dotted lines), 30 nM galectin-1 siRNA (solid lines), or 30 nM non-targeting siRNA (NT, dashed lines), followed by SRB cytotoxicity assays after 72 h. IC₅₀ values for vehicle only and for galectin-1 siRNA transfection are labeled. The statistical significance of experimental data were evaluated using student's t test where * and ** indicate that results were statistically significant at P<0.05 and P<0.01, respectively. (C) Galectin-1 level in the growth medium of Hey, EpoB8 and Ixab80 cells. Lower panel: proteins in the growth medium showing equal loading.