Synergistic Anti-cancer Eeffect Of Proteasone Inhibitor With Auranofin

Background:Ubiquitin-proteasome system(UPS) is a major intracellular pathway for the degradation of proteins. Multiple proteins which govern important life processes of the cell are degraded by the UPS. The proteasome has become an important target for many diseases,including cancer, therefore, it is of great significance to find new effective proteasome inhibitors. Generally, proteasome inhibitors are classified as natural proteasome inhibitors and synthetic proteasome inhibitiors. The natural proteasome inhibitiors are mainly composed of Lactacystin, CsA, Epoxomicin and DCI, etc., and synthetic proteasome inhibitiors include dipeptidy aldehyde inhibitiors(MG132, MLN519) and dipeptidy boronic acid inhibitiors(Bortezomib, 2-aminobenzyls etc.). Bortezomib (PS341) has been approved by FDA for clinical treatment of myeloma. Clinical studies have shown that Bortezomib (PS341) has a good anti-tumor effect but its side effects could not be ignored. In order to get the best curative effects, it is common to combine two or more drugs to have a less toxic and a more powerful anti-tumor effect during clinical treatment. Baesed on the combination therapy theory, it will be of great clinical significance to find a drug whichcould increase PS341's anti-tumor effect.Objective:Based on our previous screening study, an anti-rheumatic drug Auranofin (Aur) was used as a sensitizer of proteasome inhibitiors to observe the combination effect on tumor growth both in vitro and in vivo...Methods:1. Cell viability assay: MTS assay was employed to detect the effects of proteasome inhibitior, Auranofin and the combination on cellular proliferation and cell viability. 2. Cell death assay: Fluorescence microscope was used to detect the morphological changes and dead cells double stained with propidium idodide (PI) and annexin V.3. Protein assay: Western blot was applied to assess the protein levels of Caspase-3, Caspase-8, Caspase-9 and PARP.4. In vivo anti-cancer assay: H22 xenograft mouse model was used to detect the effects of proteasome inhibitior PS341 , Auranofin and the combination on tumor growth in mice.Results:(I) The effect of proteasome inhibitors and Auranofin on cell viability of breast cancer MCF-7 cells in vitro 1. Effect of the combination treatment on MCF-7 cell proliferationVarious doses of MG132 (2μmol/L, 3μmol/L and 4μmol/L) were combined with Auranofin (0.2μmol/L, 0.3μmol/L and 0.4μmol/L). Compared with MG132- or Aur-treated group alone, cell viability significantly decreased in the following combination-treated groups: 2μmol/L MG132 + 0.3μmol/L Aur, 2μmol/L MG132 + 0.4μmol/L Aur, 3μmol/L MG132 + 0.2μmol/L Aur, 3μmol/L MG132 + 0.3μmol/L Aur, 3μmol/L MG132 + 0.4μmol/L Aur, 4μmol/L MG132 + 0.2μmol/L Aur. .2. Effects of the combination treatment on cell death in MCF-7 cell Cell death increased progressively treated with the combination of MG132 (3μmol/L, 4μmol/L) and Auranofin (0.3μmol/L, 0.4μmol/L). The results showed that the combination of 3μmol/L MG132 with 0.4μmol/L Aur induced the largest number of PI- and Annexin V-positive cells in cell morphology.3. Effects of the combination treatment on caspase activation and PARP cleavage in MCF-7 cells.Either proteasome inhibitor or Auranofin alone did not induce dramatic changes in pro-caspase-3 , pro-caspase-8 , pro-caspase-9 and PARP while the combination treatment induced typical caspase activation and PARP cleavage in MCF-7 cells treated with MG132 (3μmol/L) combined with 0.2μmol/L or 0.3μmol/L of Aur repectively for 24 hour.(II) The anti-cancer effect of the combination in a mouse H22 xenograft model The tumor weight in PS341 group, Aur group and the combination group were statistically less than in the control group; the tumor weight in the combination group was obviously less than in the PS341- or the Aur-treated group alone. The inhibition rate in the combination group was higher than in the non-combined group.Conclusions:1,Auranofin could increase the anti-tumor effect of proteasome inhibitior(MG132,PS341).2,The synergistic effects of proteasome inhibitior(MG132,PS341) and Auranofin was associated with the activation of the caspase system.