BZ was purchased from Selleck Chemicals (Houston, TX, USA). Azithromycin dihydrate (AZM) was purchased from Tokyo Chemical Industry (Tokyo, Japan). PTX, VNR, and colchicine were obtained from Wako Pure Chemical Industries (Osaka, Japan). BZ was dissolved in dimethyl sulfoxide (DMSO) to make stock solution at a concentration of 1 mM. AZM and PTX were dissolved in 95% ethanol at a concentration of 5 mM as stock solutions. VNR was dissolved in distilled water to prepare a stock solution of 1 mM. Colchicine was dissolved in 95% ethanol at a concentration of 10 mM as stock solution.

MDA-MB-231 cells were a kind gift from Dr Keiichi Iwaya (Department of Basic Pathology, National Defense Medical College, Saitama, Japan), and MDA-MB-468 cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). A CHOP^−/− MEF cell line (CHOP-KO-DR) established from a 13.5-day-old CHOP^−/− mouse embryo by SV-40 immortalization and a CHOP^+/+ MEF cell line (DR-wild-type) established by SV-40 immortalization as a control cell line for CHOP-KO-DR were obtained from ATCC. MDA-MB-231, MDA-MB-468, CHOP-KO-DR, and DR-wild-type cells were cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Biowest, Kiltimagh, Ireland), penicillin (100 U/ml), and streptomycin (100 μg/ml). All cell lines were cultured in a humidified incubator containing 5% CO₂ and 95% air at 37°C.

The number of viable cells was assessed using CellTiter-Blue^®, a cell viability assay kit (Promega Co., Madison, WI, USA), with fluorescence measurements at 560 nm for excitation and 590 nm for emission.

For assessment of apoptosis, cells were stained with Annexin V and propidium iodide (PI) using APOPCYTO^TM Annexin V-Azami-Green Apoptosis Detection kit (MBL, code 4690, Nagoya, Japan) according to the manufacturer's instructions and subjected to flow cytometry using Attune^® Acoustic Focusing Cytometer (Life Technologies, CA, USA).

Immunoblotting was performed as previously described (15). Cells were lysed with RIPA lysis buffer (Nacalai Tesque, Kyoto, Japan) supplemented with a protease and phosphatase inhibitor cocktail (Nacalai Tesque). Cellular proteins were quantified using a DC Protein assay kit of Bio-Rad (Richmond, CA, USA). Equal amounts of proteins were loaded onto the gels, separated by SDS-PAGE, and transferred onto Immobilon-P membranes (Millipore Corp., Bedford, MA, USA). These membranes were probed with first antibodies (Abs) such as anti-ubiquitin (P4D1) mAb and anti-GAPDH (6C5) mAb purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Immunoreactive proteins were detected using horseradish peroxidase-conjugated second Abs and an enhanced chemiluminescence reagent (ECL) (Millipore). Densitometry was performed using a Molecular Imager ChemiDoc XRS System (Bio-Rad).

Real-time polymerase chain reaction (PCR) for expression analysis of ER stress-related genes was performed as previously described in detail (15).

Cells were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for 1 h. The samples were further fixed in 1% osmium tetroxide for 1 h, dehydrated in graded ethanol (30–100%), and embedded in Quetol 812 epoxy resin (Nisshin EM Co., Ltd., Tokyo, Japan). Ultrathin sections were cut using an Ultracut J microtome (Reichert Jung, Vienna, Austria). These sections were stained with lead nitrate and uranium acetate and subjected to electron microscopic analysis using a scanning electron microscope JEM-1200EX II (Jeol, Tokyo, Japan).

MDA-MB-231 cells were seeded on 13-mm glass coverslips in a 24-well culture plate in the presence or absence of BZ, PTX, and VNR and cultured for 24 h. Coverslips were washed twice with PBS and fixed for 15 min on ice in methanol. After washing twice with PBS, cells were permeabilized with 0.1% Triton X-100 in TBST for 5 min at room temperature. The coverslips were then washed with TBST and further incubated with TBST containing 10% normal goat serum (NGS; Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at room temperature for blocking nonspecific binding. Cells were incubated with a primary antibody such as mouse anti-vimentin (V9) mAb, mouse anti-ubiquitin (P4D1) mAb, and anti-p62/SQSTM1 (D-3) mAb (all from Santa Cruz), and diluted in TBST, 1.5% NGS, 0.1% bovine serum albumin (BSA) at 4°C overnight. The coverslips were washed three times for 5 min with TBST at room temperature and subsequently incubated with Alexa Fluor^® 488 conjugate-goat anti-mouse IgG (H+L) secondary antibody (Thermo Fisher Scientific), diluted in TBST containing 1.5% NGS and 0.1% BSA, for 45 min at 37°C. The coverslips were washed with TBST and mounted in ProLong^® Diamond Antifade Montant (Thermo Fisher Scientific). Nuclei were stained with DAPI (Sigma-Aldrich, D-9542), and the cells were imaged using a confocal laser scanning fluorescence microscope, LSM 700 (Carl Zeiss, Germany).

Cells were lysed with Triton X-100 lysis buffer (10 mM Tris-HCl, 150 mM NaCl, 2% Triton X-100, pH 7.8) supplemented with a protease inhibitor cocktail (Nacalai Tesqucollectede). The lysates were centrifuged at 12,000 g for 30 min at 4°C. The supernatant was then collected as a detergent-soluble fraction. The pellets (which contain insoluble proteins) were then resuspended in sodium dodecyl sulfate (SDS) lysis buffer (10 mM Tris-HCl, 150 mM NaCl, 2% SDS, pH 7.8) and sonicated for 30 sec using a tip sonicator VP-5S (Taitec, Saitama, Japan) to prepare a detergent-insoluble fraction. All resuspended pellets and supernatants were boiled for 5 min in the presence of an equal volume of SDS-PAGE sample buffer (125 mM Tris-HCl, 4% SDS, 20% glycerol, 0.002% BPB, pH 6.8), and the containing proteins were separated by 11.25% SDS-PAGE and immunoblotted with anti-ubiquitin mAb.

After trypsinization, cell suspensions were sedimented and fixed on the slide glass using a Cytospin 4 (Thermo). Preparations were stained with May-Grünwald-Giemsa and examined using a digital microscope BZ-8100 (Keyence Co., Osaka, Japan).

All the quantitative data were expressed as mean ± standard deviation (SD). Statistical analysis was performed with two-tailed nonpaired Student's t-test. The criterion for statistical significance was taken as p<0.05.

It was reported that PTX inhibited autophagy flux in breast cancer cell lines (3). We previously reported that simultaneous treatment with the proteasome inhibitor BZ and AZM, which has an inhibitory effect on autophagy flux, resulted in enhanced cytotoxicity along with ER stress loading in multiple myeloma cells (17). Therefore, we hypothesized that combined treatment using BZ and PTX instead of AZM might also enhance the cytotoxic effect in metastatic breast cancer cell lines. MDA-MB-231 and MDA-MB-468 cells were first treated with BZ alone at various concentrations for 24–72 h. Both cell lines exhibited cell growth inhibition in a dose- and time-dependent manner. Fifty percent cell growth inhibitory concentrations (IC₅₀s) at 48-h exposure to BZ were 36.3 nM in MDA-MB-231 cells and 10.8 nM in MDA-MB-468 cells (Fig. 1A). The cells were then treated with PTX as well as VNR in the presence or absence of 25 nM BZ in MDA-MB-231 cells and 7.5 nM in MDA-MB-468 cells. As illustrated in Fig. 1B, assessment of viable cells indicated significant enhancement of cell growth inhibition by combining 10 nM PTX and 25 nM BZ. Unexpectedly, pronounced cytotoxicity did not increase further and reached a plateau at higher concentrations of PTX up to 100 nM. In contrast with the PTX combination, VNR plus BZ resulted in more prominent enhancement of cell growth inhibition in the dose-dependent manner of VNR. This phenomenon was more pronounced with the addition of AZM in the cell culture medium. When the cells were treated with BZ and AZM in the presence of PTX or VNR, only the VNR combination exhibited considerable enhancement of cell growth inhibition in both cell lines (Fig. 1C). Flow cytometric analysis with Annexin V/PI double staining indicated a significant increase of Annexin V-positive/PI-negative cells (cells undergoing early-stage apoptosis) and Annexin V/PI double-positive cells (undergoing late-stage apoptosis) only after combined treatment using BZ plus VNR, but not using BZ plus PTX (Fig. 1D). These data indicate that VNR is superior to PTX in combination with BZ for apoptosis induction in breast cancer cell lines.

Aggresome formation occurs when production of aggregation-prone misfolded protein exceeds the capacity of proteasome digestion. In aggresomes, protein aggregates are actively degraded by the autophagy-lysosome pathway(29,31,35). Hence, we next examined whether aggresome is induced in MDA-MB-231 cells in response to proteasome inhibition by BZ. Immunocytochemistry using anti-vimentin mAb exhibited a single eyeball-shaped dense body in the perinuclear lesion after 24-h treatment with BZ (Fig. 2A), which appears to indicate the marker of aggresome formation at MTOC, as previously reported (27,32). Although the intracellular structure of vimentin filaments changed greatly after exposure to BZ compared with untreated cells, we could detect only 20% of the spherical body-positive MDA-MB-231 cells during 24-h exposure to BZ. Extended BZ exposure time or increased BZ concentration resulted in increasing dead cells and losing optimal conditions for detecting a single spherical aggresome. Immunoblottings with anti-ubiquitin and anti-p62 mAbs exhibited the assembly of ubiquitinated proteins as well as p62 in the perinuclear lesion in response to BZ, but they were not as dense as the vimentin profile (Fig. 2A). Electron microscopy indicated that, in agreement with the spherical body stained with anti-vimentin Ab, a different density area was observed in the perinuclear lesion surrounded by mitochondria (Fig. 2B). At higher magnification, substantial condensation of fibrous structure with some dense amorphous deposits became evident in the spherical body area. These data indicate aggresome formation in response to BZ treatment in MDA-MB-231 cells.

PTX inhibits microtubule depolymerization, whereas VNR inhibits microtubule polymerization (2,3). As shown in Fig. 3, morphologic features at metaphase were different after PTX- and VNR-treatment. Since protein aggregates are transported along the microtubules to MTOC for aggresome formation, we next assessed the inhibitory effect of PTX and VNR on the blocking efficacy of aggresome formation. As indicated in Fig. 4, both PTX and VNR inhibited aggresome formation, along with the scattering of ubiquitinated protein and p62 in the cytoplasm. Although the fibrous structure appeared to be somewhat different between PTX and VNR treatments, quantitative assessment of the efficacy of aggresome inhibition was difficult using immunocytochemistry. Therefore, we performed immunoblotting using anti-ubiquitin (P4D1) mAb to detect the polyubiquitinated proteins in detergent-soluble and detergent-insoluble fractions (Fig. 5). After treatment of MDA-MB-231 cells with BZ, polyubiquitinated proteins in the detergent-insoluble fraction, which indicate aggresome, increased (lane 12) with some increase in the detergent-soluble fraction, compared with untreated control cells. Treatment of the cells using BZ plus AZM further enhanced aggresome in the detergent-insoluble fraction (lane 16), as well as the detergent-soluble ubiquitinated-proteins (lane 6). This condition appears to indicate overflowing of the ubiquitinated proteins by simultaneous inhibition of two major protein degradation systems. It is noteworthy that ubiquitinated proteins in the soluble fraction increased more with BZ plus VNR treatment than with BZ plus PTX (lanes 7 vs. 8). The fact that treatment with VNR alone did not increase ubiquitinated proteins in the soluble fraction (lane 5) confirms that VNR inhibits BZ-induced aggresome formation more efficiently than PTX. This phenomenon becomes more apparent in the presence of AZM for blocking autophagy flux (lanes 9 vs. 10).

Our previous report indicated that blocking BZ-induced aggresome by HDAC6 inhibition using SAHA resulted in ER stress loading, which led to CHOP-mediated apoptosis induction in myeloma and breast cancer cells (34,35). Therefore, we assessed ER stress-related gene expression after combined treatment using BZ and PTX or VNR in MDA-MB-231 cells. During 48-h exposure to these reagents, real-time PCR exhibited pronounced expression of GRP78 and CHOP with combined treatment using BZ plus VNR, but not using BZ plus PTX, compared with those treated using each reagent alone (Fig. 6). Thus, the gene expression profiles fit well with the result of the cell growth inhibition presented in Fig. 1B.

Since the proapoptotic transcription factor CHOP plays a central role in ER stress-related apoptosis induction (23,25), we next examined whether CHOP is involved in pronounced cytotoxicity by combined treatment with VNR and BZ+AZM using immortalized MEF cells derived from CHOP knockout mouse. VNR enhanced BZ+AZM-induced cytotoxicity in immortalized wild-type MEF in a dose-dependent manner, whereas CHOP^−/− MEF exhibited less sensitivity to BZ+AZM treatment and attenuated enhanced cytotoxicity by VNR (Fig. 7). Notably, as well as breast cancer cell lines, PTX exhibited no enhanced cytotoxicity in the presence of BZ+AZM in MEF cell line (Figs. 1C and 7). These data suggest that pronounced apoptosis induction by BZ and VNR is at least partially mediated through CHOP induction.