Combination with bortezomib enhances the antitumor effects of nanoparticle-encapsulated thiostrepton

Abstract

Bortezomib is well-known for inducing cell death in cancer cells, specifically through the mechanism of proteasome inhibition. Thiostrepton, a thiazole antibiotic, has also been described for its proteasome inhibitory action, although differing slightly to bortezomib in the proteasomal site to which it is active. Previously we had shown the synergic effect of bortezomib and thiostrepton in breast cancer cells in vitro, where sub-apoptotic concentrations of both proteasome inhibitors resulted in synergic increase in cell death when combined as a treatment. Here, we administered such a combination to MDA-MB-231 xenograft tumors in vivo, and found that the effect of complementary proteasome inhibitors reduced tumor growth rates more efficiently than compared with when administered alone. Increased induction of apoptotic activity in tumors was found be associated with the growth inhibitory activity of combination treatment. Further examination additionally revealed that combination-treated tumors exhibited reduced proteasome activity, compared with non-treated and single drug-treated tumors. These data suggest that this drug combination may be useful as a therapy for solid tumors.

Figure 1

Effect of bortezomib and micelle-thiostrepton treatment on MDA-MB-231-luc tumor xenograft models. (A) Biodistribution of rhodamine-labeled micelle-thiostrepton complexes in tumor-bearing mice. High micelle-associated fluorescence is apparent in tumors and in the liver 4 h post-injection. Value for each organ represents an average of n = 2–4. (B) Anticancer effects of bortezomib (0.5 mg/kg) alone, micelle-thiostrepton treatment (30 mg/kg) alone, a combination of bortezomib and micelle-thiostrepton on MDA-MB-231 xenograft volumes, after five treatments over 15 d, n = 6–8. All averages were considered significant, and the t-test p value of the non-treated and the V+T-treated tumors was calculated to be 0.011. Error bars represent SEM. (C) Change in tumor-associated bioluminescence in animals bearing breast cancer xenografts after continuous treatment with a combination of complementary proteasome inhibitors over 15 d. Luciferase expression demonstrates that the sizes of tumors treated with bortezomib and thiostrepton were much smaller in size than the non-treated controls. (D) Change in animal weight during treatment schedule. Only the animals receiving the combination of proteasome inhibitors demonstrated slight weight loss, compared with non-treated and single drug-treated animals.

Figure 2

Combination treatment with bortezomib and micelle-thiostrepton induces cell death in MDA-MB-231-luc tumor xenografts, as determined by promotion of apoptotic markers. (A) Immunhistochemical analysis of four individual tumors (two from non-treated and two from combination treated groups) for cleaved caspase-3 expression (counterstained with hematoxylin). Cleaved-caspase-3 is evidently enhanced in combination-treated tumors, identified by the presence of larger numbers of dark, DAB-stained clusters. Control slides were incubated with rabbit IgG primary antibodies. Blue bars represent 100 µm (20 x). (B) Protein gel blotting of a panel of individual tumor homogenates (five of non-treated and five of combination-treated) against cleaved PARP and β-actin. Cleaved PARP expression is increased in tumors treated with a combination of thiostrepton and bortezomib.

Figure 3

Combination of bortezomib and thiostrepton suppresses proteasome activity. (A) Proteasome activity assay showed that lysates from MDA-MB-231 tumors treated with a combination of proteasome inhibitors had decreased ability for proteasome substrate cleavage, compared with that from non-treated and single drug-treated tumors. Values represent averages of 6–8 tumors and error bars depict SEM p values are stated within the figure. (B) Proteasome activity assay showed that bortezomib and thiostrepton inhibit proteasome activities of purified proteasomes to a greater extent when used in combination.