ABSTRACT
Despite the development of promising cancer therapeutic drugs, multiple myeloma (MM) remains an incurable disease. Bufalin is a bufanolide steroid compound of the traditional Chinese medicine Chan Su that was previously shown to exert growth suppression effects on myeloma cell lines. Previous studies conducted by our group demonstrated that bufalin activated the AKT/mTOR pathway in myeloma cells, which is considered an essential pathway to disease progression and is related to drug resistance in MM. In view of the significant role of AKT in MM, the allosteric AKT inhibitor MK2206 was selected in order to enhance the antitumor effects of bufalin in different MM cell lines (NCI-H929, U266, LP-1 and RPMI8226). The data indicated that MK2206 enhanced the cytotoxicity of bufalin in MM cells, via the suppression of cellular proliferation and the induction of apoptosis, as demonstrated by cleavage of apoptosis-related proteins. This effect was further noted in the presence of exogenous interleukin-6 and/or following the co-culture of MM cells with bone marrow stromal cells (BMSC). This process was associated with the inhibition of the AKT/mTOR pathway. The combination of bufalin with MK2206 reduced the secretion of IL-6 in U266 cells. The combined treatment exhibited similar anti-MM effects in bortezomib-resistant cell lines (NCI-H929R, U266R). In addition to the in vitro cell line models, the synergistic effect was noted in primary MM cells and in MM xenografts of BALB-c and NOD-SCID mice. In conclusion, the data suggested that MK2206 significantly enhanced the cytocidal effects of bufalin in MM cells, regardless of the sensitivity to bortezomib, via the inhibition of the AKT/mTOR pathway. The study provided the basis of a promising treatment approach for MM.
Subject(s)
Bufanolides/pharmacology , Heterocyclic Compounds, 3-Ring/pharmacology , Multiple Myeloma/drug therapy , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/metabolism , Animals , Bufanolides/agonists , Cell Line, Tumor , Drug Synergism , Female , Heterocyclic Compounds, 3-Ring/agonists , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Mice, SCID , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Proto-Oncogene Proteins c-akt/genetics , Signal Transduction/genetics , TOR Serine-Threonine Kinases/genetics , Xenograft Model Antitumor AssaysABSTRACT
DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest.
