Proteasome inhibitors increase tubulin polymerization and stabilization in tissue culture cells: a possible mechanism contributing to peripheral neuropathy and cellular toxicity following proteasome inhibition

Abstract

Bortezomib (Velcade((R))), a proteasome inhibitor, is approved by the FDA for the treatment of multiple myeloma (MM). While effective, its use has been hampered by peripheral neurotoxicity of unexplained etiology. Since proteasome inhibitors alter protein degradation, we speculated that proteins regulating microtubule (MT) stability may be affected after treatment and examined MT polymerization in cells by comparing the distribution of tubulin between polymerized (P) and soluble (S) fractions. We observed increased MT polymerization following treatment of SY5Y and KCNR [neuroblastoma], HCN2, and 8226 [MM] cells, using five proteasome inhibitors; the baseline proportion of total alpha-tubulin in 'P' fractions ranged from approximately 41-68%, and increased to approximately 55-99% after treatment. Increased acetylated alpha-tubulin, a post-translational marker of stabilized MTs, was observed in the neural cell lines HCN1A and HCN2 and this was sustained up to 144 hours after the proteasome inhibitor was removed. Cell cycle analysis of three cell lines after treatment, showed approximately 50-75% increases in the G(2)M phase. Immunofluorescent localization studies of proteasome inhibitor treated cells did not reveal microtubule bundles in contrast to paclitaxel treated, suggesting MT stabilization via a mechanism other than direct drug binding. We examined the levels of microtubule associated proteins and observed a 1.4-3.7 fold increase in the microtubule associated protein MAP2, in HCN2 cells following treatment with proteasome inhibitors. These data provide a plausible explanation for the neurotoxicity observed clinically and raise the possibility that microtubule stabilization contributes to cytotoxicity.

Figure 1.

The fraction of polymerized tubulin is increased in neuroblastoma and cultured neural cells after treatment with proteasome inhibitors. Lysates from neuroblastoma cells, SY5Y and KCNR, (Fig. 1A) and neural derived HCN2 cells (Fig. 1B), were obtained from cells either treated or not for 18–22 hours, with proteasome inhibitors at the indicated concentrations. Lysates were separated into polymerized (P) or soluble (S) fractions by centrifugation at ~15,000 g at 22°C for 10 minutes. Aliquots of equal volume were separated by SDS-PAGE, the blots probed with anti α-tubulin and the percent of polymerized tubulin calculated for each ‘P’ and ‘S’ pair. Vertical lines indicate that tracks from different blots from the same experiment, exposed equivalently, are shown alongside each other, and the results represent that of a typical experiment.

Figure 2.

Acetylated α-tubulin and MAP2 are increased in HCN1A and HCN2 neural cells after treatment with proteasome inhibitors. Cells were treated or not for 24 hours with proteasome inhibitors and total cell lysates were obtained. For each sample, equal amounts of total protein were separated by SDS-PAGE. For HCN1A cells, the western blot was probed with anti acetylated tubulin and GAPDH (Fig. 2A), and for the HCN2 cells, the blots were also probed with anti MAP2 (Fig. 2B). The intensity of each band was quantitated by densitometry, normalized to the value for GAPDH, and the fold increase compared to the untreated sample.

Figure 3.

The increases in the fraction of polymerized tubulin and acetylated α-tubulin in HCN2 cells treated with Bortezomib are stable during drug washout. HCN2 cells were treated or not for 24 hours with either 100 or 25 nM of bortezomib prior to a rinse in PBS and incubation in drug free medium for 0, 5 , 24, 48, 120 or 144 hours. In the case of the cells incubated in 100 nM bortezomib, lysates were either separated into polymerized (P) or soluble (S) fractions by centrifugation at ~15,000 g at 22°C for 10 minutes. Aliquots of equal volume for each pair were separated by SDS-PAGE, the blot probed with anti α-tubulin and the percent of polymerized tubulin calculated for each ‘P’ and ‘S’ pair is indicated. Data from several washout time points is shown as is that for untreated cells harvested at 24 or 48 hour time points (Fig 3A). Total lysates from HCN2 cells that were treated or not far 24 hours with either 100 or 25 nM of bortezomib prior to incubation in drug free medium far 0, 5, 24, 48, 120 or 144 hours were separated by SDS-PAGE. Western blots were sequentially probed with mouse monoclonal antibodies to either acetylated α-tubulin or actin (Fig. 3B). The intensity of each band was quantitated by densitometry, normalized to the value for actin, and the fold increase compared to the untreated sample.

Figure 4.

The percentage of cells in the G₂M phase of the cell cycle is increased after treatment with proteaseome inhibitors. When compared to cells treated with no drug or with vehicle, an increase in the number of cells in the G₂M phase of the cell cycle was observed for SY5Y [diagonal stripe bars], KCNR [solid black bars] and 8226 cells [stippled bars] which were treated for 24 hours with proteasome inhibitors. The percentage of cells in the G₂M phase is displayed on the y-axis.

Figure 5.

Confocal immunofluroescenl localization of HDAC6, tubulin and vimentin in SY5Y and HCN2 cells after treatment with proteasome inhibitors. For SY5Y cells [upper] or HCN2 cells [lower], which were either untreated (No Drug), treated with poclitaxel (PTX), or proteasome inhibitors (MG-132, bortezomib) at the concentrations indicated in the left margin, separate panels display the immunlocalization of HDAC6, a marker for aggresomes, indicated by the red color (rhodamine secondary antibody) and α-tubulin [center] indicated by the green color (fluorescein secondary antibody). DAPI stain (blue) localizes to cell nuclei. The tricolor localization in each cell line of ‘Tublin/HDAC6/DAPI’ is shown by the superimposition of three confocal images and shown in the third column panels. The superimposition from other samples of three images for the tricolor ‘Vimentin/HDAC6/DAPI’ localization, are shown in the fourth column of panels.