The Mechanisms Whereby Aspirin Exerts Antimyeloma Action And Enhances Cytotoxicity Of Bortezomib In Multiple Myeloma

Background and Objective:During the past five years, there is increasing morbidity of multiple myeloma(MM) due to the aggravation of aging population worldwide, which makes MMbecoming the second hematological malignancies only following non-Hodkinlymphoma (NHL). In spite of the improved outcome by the immunomodulator andproteasome inhibitor, nearly all patients with MM experience relapse and refractorydisease. Up to now, MM remains uncurable. As the backbone agent, bortezomib(BTZ) has been incorporated into the induction, consolidation, salvage, andmaintenance therapy for MM. However, continual administration of BTZ triggerssecondary drug resistance in MM. Recently, aspirin (ASA) has been confirmed toexert anticancer activity in a wide range of cancers such as colorectal cancer,hepatocellular carcinoma，pancreatic cancer and ovarian cancer.The study aims at exploring the antimyeloma action of ASA in vivo and in vitroand the interaction between ASA and BTZ in MM cells, as well as the underlyingmechanisms, which will provide the rationale for clinical administration of ASA asthe antimyeloma and chemosensitive agent in MM treatment.Methods:1. MM1.S (dexamethasone-sensitive) and RPMI-8226(dexamethasone-resistant)cells were exposed to various concentrations of ASA (0,2.5,5,10mmol/L) within24~72h. The cell proliferation and cell cycle were examined by CCk-8andpropidium iodide (PI) staining to explore the antimyeloma of ASA.2. The effect of ASA on the apoptotic death, the activation of Caspase-3,-8,-9andthe level of PARP protein were detected via Annexin V-FITC/PI staining,colorimetric method, and Western blot analysis respectively, when MM1.S andRPMI-8226cells treated with various dose of ASA. 3. Real-time quantitative polymerase chanin reaction (PCR) and Western blot wasrespectively employed to examine the mRNA and protein of Bcl-2, Bax, andVEGF in MM1.S and RPMI-8226cells treated with ASA at various dose.4. The effect of ASA on the survivial time and tumor growth was observed inNOD/SCID mice bearing xenograft tumor of MM1.S and RPMI-8226cells,based on the establishment of animal model.5. The interaction of ASA and BTZ was analyzed through the effect of ASA alone,BTZ alone and ASA plus BTZ on myeloma cell proliferation and apoptosis viaCCK-8and Annexin V-FITC/PI staining.6. The underlying mechanisms whereby ASA augments the cytotoxicity of BTZ inmyeloma cells were investigated through the examination of the level of Aktphosphorylation and Survivin protein by Western blot ananlysis.Results:1. ASA displayed anti-proliferative action against MM1.S and RPMI-8226cells indose-and time-dependent fashion within the range of2.5~10mmol/L and24~72h.2. ASA triggered the apoptotic death of MM1.S and RPMI-8226cellsdose-dependently within the concentration of2.5~10mmol/L. The higherapoptosis rate was observed in MM1.S treated with ASA of2.5~5mmol/Lconcentration when compared with RPMI-8226. But10mmol/L ASA exposureresulted in the similar apoptotic death in the two kinds of cell lines.3. With exposure to various dose of ASA, MM1.S and RPMI-8226cellsexperienced cell cycle arrest of G1phase in dose-dependent manner, inconcurrent with the corresponding decrease of cells in S phase.4. Caspase-3activation of MM1.S and RPMI-8226cells was increasedconcentration-dependently when treated with various dose of ASA,accompanying with enhanced activation of Caspase-8and Caspase-9.5. In the range of2.5~10mmol/L, ASA initiated the cleavage of PARP protein inMM1.S and RPMI-8226cells dose-dependently.6. ASA exposure to MM1.S and RPMI-8226cells caused downregulation of Bcl-2and VEGF expression and upregulation of Bax level in dose-dependent fashion. 7. The animal model bearing xenograft tumor of myeloma cells was establishedsuccessfully via subcutaneous injection of MM1.S or RPMI-8226cells (1×107cells per mouse). Wrigh’s and H-E staining revealed the typical myeloma cells.The total rate of success xenograft was71.88%in NOD/SCID mices.8. In comparision with PBS-treated group, ASA treatment markedly delayed thetumor growth in NOD/SCID mice bearing myeloma xenograft of MM1.S andRPMI-8226cells.9. When compared with PBS-treated group, ASA administration significantlyprolonged the median survival time of NOD/SCID mice bearing myelomaxenograft of MM1.S and RPMI-8226cells respectively (37.5d vs21d，p=0.035；35.5d vs21d，p=0.035).10. ASA in combination with BTZ substantially inhibited the proliferation ofMM1.S and RPMI-8226cells in time-dependent fashion, in comparision withASA or BTZ alone. The interaction analysis revealed the additive action of ASAand BTZ in both myeloma cell lines.11. The apoptosis death rate induced by ASA plus BTZ was markedly higher thanthat of ASA or BTZ alone in MM1.S and RPMI-8226cells.12. In MM1.S and RPMI-8226cells, Akt phosphorylation (Thr308) was inhibited byASA alone, but was induced by BTZ alone. However, ASA combined with BTZalmost completely suppressed Akt phosphorylation of Thr308.13. ASA and BTZ displayed the inhibitory effect on the Akt phosphorylation (Ser473)in MM1.S and RPMI-8226cells. When compared with ASA or BTZ alone, ASAplus BTZ caused significantly inhibition of Akt phosphorylation (Ser473).14. In MM1.S and RPMI-8226cells, the level of Survivin protein was inhibited byASA alone, but was upregualted by BTZ alone. Cotreatment of ASA and BTZresulted in substantially downregulation of Survivin protein.Conclusions:1. ASA exerted anti-proliferative activity against myeloma cells in vitro and invivo.2. ASA induced apoptotic death of myeloma cells via mitochondrial pathway anddeath receptor pathway. 3. The antimyeloma action of ASA was independent of dexamethasonesensitiveness.4. ASA induced cell cycle arrest of G1phase in myeloma cells.5. ASA exhibited pro-apoptotic property in myeloma cells via suppression of Bcl-2and VEGF and upregulation of Bax.6. ASA augumented the cytotoxicity of BTZ against myeloma cells.7. The chemosensitive action of ASA on BTZ against myelma cells was partlyascribed to the inhibition of Akt phosphorylation induced by botezomib.8. The chemosensitivity of ASA towards BTZ in myeloma cells was attributed, inpart, to suppression of Survivin.