Next-generation proteasome inhibitor MLN9708 sensitizes breast cancer cells to doxorubicin-induced apoptosis

Abstract

Doxorubicin (Dox), one of the most effective chemotherapy drug for cancer treatment, is limited by its severe side effects and chemoresistance. Dox induces DNA damage and leads to significant proteomic changes in the cancer cells, which makes the ubiquitin-proteasome system a potential target to enhance the efficacy of Dox therapy. The unsuccessful clinical trials of proteasome inhibitor PS-341 (bortezomib) in solid tumors led to the invention of MLN9708 (ixazomib), an orally bioavailable next-generation proteasome inhibitor with improved pharmacokinetic and pharmacodynamic features. In this preclinical study, we used eight human breast cancer cell lines, which represent the major molecular subtypes of breast cancer, to validate the cytotoxic effects of MLN9708, alone and in combination with Dox. We found that MLN9708 had cytotoxic effects, induced autophagy and MKP-1 expression, and enhanced Dox-induced apoptosis in these cell lines. MLN9708 also enhanced Dox-induced JNK and p38 phosphorylation and inhibited Dox-induced IκBα degradation. Our in vitro results suggest that MLN9708 has antitumor effects in breast cancer and can sensitize breast cancer cells to Dox treatment. This promising combination may be an effective and feasible therapeutic option for treating breast cancer and warrants clinical validation.

Figure 1. MLN9708 shows cytotoxic effect in breast cancer cells.

(a) Cytotoxic effect of MLN9708 on breast cancer cells. Eight human breast cancer cell lines (T-47D, MCF7, MDA-MB-361, SK-BR-3, HCC1954, MDA-MB-468, MDA-MB-231, and BT-549) were incubated with medium alone or were treated with MLN9708 (0.001 μM, 0.003 μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, or 10 μM) for 72 h and then were subjected to a Cell Counting Kit-8 (CCK-8) assay. The absorbance of each well was measured at 450 nm, and the cell viability curve was plotted. Data were represented as means ± standard deviations (SD). Median inhibitory concentration (IC₅₀) values of MLN9708 in breast cancer cell lines were listed. (b) Photographs of treated cells (magnification, ×200). (c) Colony formation of breast cancer cells treated with MLN9708. Cells were seeded in 6-well plates (5 × 10³ per well), were incubated with medium alone or with MLN9708 (0.1 μM or 0.3 μM) for 72 h, and then were cultured in drug-free medium for 2 weeks. The cell colonies were fixed, were stained with crystal violet, and were photographed.

Figure 2. MLN9708 suppresses anchorage-independent growth in breast cancer cells.

(a) Cell anchorage-independent growth ability was assessed by soft agar assay. Six breast cancer cell lines (T-47D, MCF7, MDA-MB-361, MDA-MB-468, MDA-MB-231, and BT-549) were incubated with MLN9708 (0 μM, 0.1 μM, or 0.3 μM) in soft agar plates for 3 weeks. Cells were then stained with crystal violet and were photographed. (b) The colonies were counted, and the data were plotted. Data were represented as means ± standard deviations (SD). *P < 0.05, **P < 0.01, and ***P < 0.001 (analysis of variance and Dunnett multiple comparison post-test).

Figure 3. MLN9708 induces apoptosis in breast cancer cells.

(a–h) Breast cancer cells (T-47D, MCF7, MDA-MB-361, SK-BR-3, HCC1954, MDA-MB-468, MDA-MB-231, and BT-549) were treated with MLN9708 (0.03 μM, 0.1 μM, 0.3 μM, or 1 μM) for 24 h. Untreated cells were used as controls. Whole-cell lysates were subjected to SDS-PAGE and were immunoblotted with antibodies against PARP and Caspase 3 (or Caspase 7) to detect apoptosis. β-actin was used as a loading control.

Figure 4. MLN9708 induces autophagy in breast cancer cells.

(a–h) Breast cancer cells (T-47D, MCF7, MDA-MB-361, SK-BR-3, HCC1954, MDA-MB-468, MDA-MB-231, and BT-549) were treated with MLN9708 (0.1 μM) for 24 h and then were cultured with drug-free medium for 24 h, 48 h, or 72 h. Then whole-cell lysates were subjected to SDS-PAGE and were immunoblotted with antibodies against LC3A/B-I/II to detect autophagy. β-actin was used as a loading control.

Figure 5. MLN9708 induces MKP-1 expression in breast cancer cells.

(a–h) Breast cancer cells (T-47D, MCF7, MDA-MB-361, SK-BR-3, HCC1954, MDA-MB-468, MDA-MB-231, and BT-549) were treated with MLN9708 (1 μM) for 4 h, 8 h, 16 h, or 24 h. Untreated cells were used as controls. Then whole-cell lysates were subjected to SDS-PAGE and were immunoblotted with antibodies against MKP-1. β-actin was used as a loading control.

Figure 6. MLN9708 enhances the cytotoxic effects of doxorubicin (Dox) in breast cancer cells.

(a–h) Breast cancer cells (T-47D, MCF7, MDA-MB-361, SK-BR-3, HCC1954, MDA-MB-468, MDA-MB-231, and BT-549) were treated with Dox at the indicated concentrations with or without MLN9708 (0.1 μM [0.3 μM for BT-549 cells]) for 48 h. Cell viability was then measured by Cell Counting Kit-8 (CCK-8) assay. Data were represented as means ± standard deviations (SD). *P < 0.05, **P < 0.01, and ***P < 0.001 (t-test).

Figure 7. MLN9708 enhances doxorubicin (Dox)-induced apoptosis in breast cancer cells.

(a–h) Breast cancer cells (T-47D, MCF7, MDA-MB-361, SK-BR-3, HCC1954, MDA-MB-468, MDA-MB-231, and BT-549) were treated with Dox (1 μM) alone or in combination with MLN9708 (0.1 μM) for 16 h or 24 h. Untreated cells were used as controls. Then whole-cell lysates were subjected to SDS-PAGE and were immunoblotted with antibodies against PARP and Caspase 3 (or Caspase 7) to detect apoptosis. β-actin was used as a loading control.

Figure 8. MLN9708 inhibits doxorubicin (Dox)-induced IκBα degradation in breast cancer cells.

(a–h) Breast cancer cells (T-47D, MCF7, MDA-MB-361, SK-BR-3, HCC1954, MDA-MB-468, MDA-MB-231, and BT-549) were treated with Dox (20 μM) alone or in combination with MLN9708 (1 μM) for 2 h, 3 h, or 4 h. Untreated cells were used as controls. Then whole-cell lysates were subjected to SDS-PAGE and were immunoblotted with antibodies against p-JNK, JNK, p-p38, p38, and IκBα. β-actin was used as a loading control.

Figure 9. Working model shows cytotoxic effects of MLN9708 in breast cancer.

(a) The balance between intracellular proteins and the functional proteasome in living cells. (b) Loss of balance in cells treated with proteasome inhibitor MLN9708. (c) Doxorubicin (Dox)-induced changes of intracellular proteins challenge the working capacity of the proteasome. (d) Combination of MLN9708 with Dox further stresses the cells and causes apoptosis.