Autophagy Inhibition Enhances Doxotubicin-induced Apoptosis In Myeloma Cells

Background and Aim: As a major intracellular degradation system that is found ubiquitously in eukaryotes, autophagy plays a key role in maintaining protein metabolic equilibrium and cell homeostasis, and in regulating cellular constituent recycleing, cell development and differentiation. Autophagy is one of research hot spots in cytobiology at present, furthermore the relationship between autophagy and tumor is attracting great attention. Since Beth Levine ea al demonstrates that human cells that carry monoallelic deletions of the Beclin1/ATG6 gene are tumorigenic, autophagy has been considered as a tumor suppressor, and inducing autophagy is attempted to cancer therapy. But further investigations find that autophagy has dual role in cancer, in some circumstances, inducing autophagy may inhibit tumor cell growth, and result in autophagic cell death; but in most circumstances, autophagy may function as a cytoprotective mechanism by responsing to stress situations including hypoxia, low energy, oxidative stress and damaged mitochondria. Furthermore autophagy is tumor cell type- and stress dependent, that is, In different cell type or different stress mode, the role or function of autophagy changes. Myeloma cells are featured by exceedingly active protein metabolism such as synthesis and secretion of immunoglobulin, and as one of major protein metabolism pathways, what`s the function of autophagy in myeloma cells? There is no past experience at present. Based on the background above, in this study, by means of the model of myeloma cells (RPMI8226 and H929 and primary cells) , we try to explore the effects of autophagy on cell apoptosis induced by DOX and the correlateing mechanism.Methods and Results: 1. In our work, MTT assays showed that autophagy inhibitor hydroxychloroquine (HCQ) could inhibit proliferation of RPMI8226 and H929 cells by dose-dependent manner in vitro. While autophagy inducer rapamycin had no effects on myeloma cell proliferation.2. Flow cytometer (FCM) displayed that autophagy inhibition by HCQ, 3-methlyadenine(3-MA) or bafilomycin A1 could enhance doxorubicin(DOX), cisplatin(DDP), mitoxantone(MIT), or melphalan(MEL) induced myeloma cell apoptosis repectively ( P < 0.05), while could not enhance vincristine(VCR), etoposide(VP-16) induced cell apoptosis(P>0.05).3. Autophagic morphology of myeloma cells was monitored by Electron microscopy and fluorescence microscopy.â‘ By electron microscopy, it showed that the autophagosome amounts in untreated, 3-MA treated and 3-MA combined with DOX treated cells were scanty, moderate in HCQ ,BAF, or DOX singly treated cells, and abundent in HCQ or BAF combined with DOX treated cells, and also abundant in positive control: rapamycin treated cells.â‘¡LC3-FITC had been used to monitor autophagy by immunofluorescence. It showed that there are a high level of LC3 puncta in HCQ+DOX or BAF +DOX treated cells, and in rapamycin treated cells, moderate level in HCQ, BAF, or DOX singly treated cells, and low level in 3-MA+DOX, and there was diffuse fluorescence in untreated or 3-MA treated cells. 4. Western blot showed:â‘ the amount of autophagic proteins Beclin1,Atg5,LC3-II increased gradually after RPMI8226 and H929 cells were treated with DOX for 0, 4, 8, 12h respectively, which indicated that autophagy was induced by DOX;â‘¡By means of analysis of express levels of Beclin1,Atg5,LC3-II after autophagy inhibitor HCQ, BAF or 3-MA combined with DOX, It showed that chemical sensitization of HCQ and BAF was due to post-sequestration step inhibition(by blocking fusion autophagosome with lysosome) to autophagy of myeloma cells, while 3-MA was due to sequestration inhibition.â‘¢Express levels of apoptosis protein caspase-3 and PARP treated by autophagy inhibitor HCQ, BAF or 3-MA combined with DOX increased compared with treated by DOX singly, which indicated that autophagy inhibition enhanced apoptosis induced by DOX. 5. The apoptosis induced by DOX also was enhanced by specific Beclin1 siRNA or Atg5 siRNA in H929 and RPMI8226 cells. 6. It was demonstrated in myeloma mouse model that in vivo autophagy inhibition also enhanced inhibition of tumor growth induced by DOX.Conclusions: 1.Autophagy as a protective mechanism responses to the apoptosis induced by DOX . 2. Autophagy inhibition by chemical inhibitor or specific siRNA, could enhance the apoptosis induced by DOX in myeloma cell lines or primary cells; 3. In myeloma mouse models, autophagy inhibition also enhanced inhibition of tumor growth induced by DOX.