The
ubiquitin-
proteasome and autophagy-lysosome pathways are two major self-digestive systems for cellular
proteins. Ubiquitinated misfolded
proteins are degraded mostly by
proteasome. However, when
ubiquitinated proteins accumulate beyond the capacity of
proteasome clearance, they are transported to the microtubule-organizing center (MTOC) along the microtubules to form aggresomes, and subsequently some of them are degraded by the autophagy-lysosome system. We previously reported that
macrolide antibiotics such as
azithromycin and
clarithromycin block autophagy flux, and that concomitant treatment with the
proteasome inhibitor bortezomib (BZ) and
macrolide enhances endoplasmic reticulum (ER) stress-mediated apoptosis in
breast cancer cells. As
ubiquitinated proteins are concentrated at the aggresome upon
proteasome failure, we focused on the microtubule as the scaffold of this transport pathway for aggresome formation. Treatment of metastatic
breast cancer cell lines (e.g., MDA-MB‑231 cells) with BZ resulted in induction of aggresomes, which immunocytochemistry detected as a distinctive eyeball-shaped
vimentin-positive inclusion body that formed in a perinuclear lesion, and that electron microscopy detected as a sphere of fibrous structure with some dense amorphous deposit.
Vinorelbine (VNR), which inhibits microtubule polymerization, more effectively suppressed BZ-induced aggresome formation than
paclitaxel (PTX), which stabilizes microtubules. Combined treatment using BZ and VNR, but not PTX, enhanced the cytotoxic effect and apoptosis induction along with pronounced ER stress loading such as upregulation of
GRP78 and CHOP/GADD153. The addition of
azithromycin to block autophagy flux in the BZ plus VNR-containing cell culture further enhanced the cytotoxicity. These data suggest that suppression of BZ-induced aggresome formation using an inhibitory drug such as VNR for microtubule polymerization is a novel strategy for metastatic
breast cancer therapy.