Next-generation proteasome inhibitor oprozomib enhances sensitivity to doxorubicin in triple-negative breast cancer cells

Abstract

Doxorubicin (DOX) is the most common chemotherapeutic drug for treatment of breast cancer but intrinsic and acquired resistance frequently occurs and severe side effects occur at high doses. DOX might induce activation of NF-κB causing this resistance, in which case proteasome inhibitors could inhibit activation of NF-κB by blocking inhibitory factor κB-alpha degradation. Triple-negative breast cancer (TNBC) is highly progressive and there are no established therapeutic targets against TNBC. Although some proteasome inhibitors have been shown to have antitumor effects in breast cancer, the effect of orally bioavailable proteasome inhibitor oprozomib on TNBC proliferation remains unclear. In the present study, we investigated the role of oprozomib in two TNBC lines, MDA-MB-231 and BT-549. Oprozomib had cytotoxic effects on TNBC cells and increased DOX-induced cytotoxic effects and apoptosis by enhancing DOX-induced JNK/p38 MAPK phosphorylation and inhibiting DOX-induced inhibitory factor êB alpha degradation. These results suggest that oprozomib has potent antitumor effects on TNBC in vitro and can sensitize TNBC cells to DOX treatment. The combination of DOX and oprozomib may be an effective and feasible therapeutic option for TNBC.

Keywords: Proteasome inhibitor; breast cancer; doxorubicin; drug resistance; oprozomib.

Figure 1

Oprozomib (OPZ) shows cytotoxic effects on TNBC cells. A. Two TNBC cell lines, MDA-MB-231 and BT-549, were treated with OPZ at concentrations of 0 μM, 0.001 μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, or 10 μM for 72 hours, then subjected to MTT assays. Absorbance of each well was measured at 540 nm and plotted for the cell viability curve. Data are represented as mean ± standard deviation. Half-maximal inhibitory concentration (IC50) values of OPZ in each cell line are listed on the right. B. Colony formation of breast cancer cells treated with OPZ. Two TNBC cell lines were seeded in 12-well plates at 2 × 10³ per well, then incubated with oprozomib at 0 μM, 0.03 μM, or 0.1 μM for 72 hours and cultured in drug-free medium for about 2 weeks. Cell colonies were fixed, stained with crystal violet, and photographed.

Figure 2

Oprozomib (OPZ) suppresses anchorage-independent growth ability of TNBC cells. Cell anchorage-independent growth ability was assessed using soft agar assays. (A) Two breast cancer cell lines, MDA-MB-231 and BT-549, were incubated with OPZ at concentrations of 0 μM, 0.03 μM, or 0.3 μM in soft agar plates for 3 weeks, stained with crystal violet, and photographed. (B) The colonies shown in (A) were counted and plotted. Data are represented as mean ± standard deviation. ***P < 0.001, by analysis of variance and Dunnett’s multiple comparison post-test.

Figure 3

Oprozomib (OPZ) induces apoptosis in TNBC cells. TNBC cell lines MDA-MB-231 (A) and BT-549 (B) were treated with OPZ at concentrations of 0 μM, 0.03 μM, 0.1 μM, 0.3 μM, or 1 μM for 24 hours. Then, whole cell lysates were subjected to SDS-PAGE and immunoblotted with antibodies against PARP and Caspase 3 to detect apoptosis. α-Tubulin was used as loading control.

Figure 4

Oprozomib (OPZ) enhances cytotoxic effect of doxorubicin (DOX) in TNBC cells. TNBC cell lines MDA-MB-231 (A) and BT-549 (B) were treated with DOX at the indicated concentrations with or without OPZ (0.05 μM) for 72 hours. Cell viability was then measured by MTT assay. Data are represented as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, by t-test.

Figure 5

Oprozomib (OPZ) strengthens doxorubicin (DOX)-induced apoptosis in TNBC cells. TNBC cell lines MDA-MB-231 (A) and BT-549 (B) were treated with DOX (1-μM) alone or combined with OPZ (0.2 μM or 0.4 μM) for 0, 24, or 16 hours. Whole cell lysates were then subjected to SDS-PAGE and immunoblotted with antibodies against PARP and caspase 3 to detect apoptosis. α-tubulin was used as loading control.

Figure 6

Oprozomib (OPZ) inhibits doxorubicin (DOX)-induced inhibitory factor κB alpha (IκBα) degradation in TNBC cells. TNBC cell lines MDA-MB-231 (A) and BT-549 (B) were treated with DOX (20 μM) alone or in combination with oprozomib (5 μM) for 0, 2, 3, or 4 hours. Whole cell lysates were then subjected to SDS-PAGE and immunoblotted with antibodies against phospho-(p-)SARP/JNK, SARP/JNK, p-p38 MAPK, p38 MAPK, and IκBα. α-Tubulin was used as loading control.