Roles And Mechanisms Of Autophagy In The Treatment Of Hematological Malignancies

This dissertion contains the following two parts:Part Ⅰ. The cytotoxicity of rhArg and the role and mechanism of autophagy in rhArg-induced lymphoma cell death.Recently, targeting cellular metabolism especially arginine metabolism as an approach for cancer therapy is undergoing a renaissance. Arginase, an arginine-degrading enzyme, has gained increased attention recently as a new experimental therapeutics for a variety of malignant solid cancers. This study was proposed to investigate whether recombinant human arginase (rhArg) could kill Non-Hodgkin’s Lymphoma (NHL) cells in vitro, induce autophagy in these cells and the role and mechanism of autophagy in rhArg-induced NHL cell death.Raji and Daudi NHL cells were treated with rhArg or cultured in L-arginine-deficient medium, and cell viability was measured by MTT assay. Apoptosis and mitochondrial membrane potential were detected by flow cytometry after NHL cells were treated with rhArg. Autophagosomes were observed using transmission electron microscopy, LC3 Ⅱ flurescent spots were detected by confocal microscopy and LC3 Ⅰ/Ⅱ was detected by western blot in rhArg-treated NHL cells. Pan-caspase inhibitor z-VAD-fmk and siRNA (targeting caspase 3) were used to inhibit caspase 3. Autophagy inhibitors (3-MA or chloroquine) and siRNA (targeting ATG5 and Beclinl) were used to inhibit rhArg-induced autophagy.Results showed that 0.016-4 IU/mL of rhArg could significantly inhibit cell viability of Raji and Daudi cells through arginine deprivation.1 IU/mL of rhArg could result in caspase cleavage and induce apoptosis in Raji and Daudi NHL cells. rhArg-treatment resulted in the appearance of autophagosomes and up-regulation of LC3 Ⅱ, indicating that rhArg induced autophagy in lymphoma cells. Further study indicated that mTOR/S6K signaling pathway was involved in rhArg-induced autophagy in NHL cells. Moreover, blocking autophagy using pharmacological inhibitors (3-MA and CQ) or genetic approaches (siRNA targeting ATG5 and Beclinl) enhanced the cell groth inhibition and apoptosis induced by rhArg.These results demonstrated that rhArg has a potent cytotoxicity which could be improved by in combination with autophagic inhibitors, suggesting that rhArg, either alone or in combination with autophagic inhibitors, could be a potential novel therapeutics for the treatment of NHL.Part II. Simultaneously inhibiting hedgehog (Hh) pathway and autophagy ovcomes drug-resistance of Bcr-Abl+CML cells.Drug resistance is one of the biggest challenges in the treatment of chronic myeloid leukemia (CML). Hh signaling pathway and autophagy are related to the tumorigenesis, cancer therapy and drug resistance. This study was proposed to explore whether Hh pathway could regulate autophagy in CML cells and whether simultaneously regulating Hh pathway and autophagy could kill drug-sensitive or-resistant Bcr-Abl+CML cells.The imatinib-sensitive cell lines K562 and BaF3-Bcr-Abl (wild type, p210), imatinib-resistant cell lines BaF3-Bcr-AblT3151 and BaF3-Bcr-AblY253F were exposed to Hh inhibitor and autophagy inhibitor either alone or in combination. Autophagy was detected by transmission electronic microscopy, confocal microscopy and western blot. Cell viability was measured by CCK-8. Apoptosis was determined by Annexin V/PI staining or western blot.Results showed that inhibiting Hh pathway could induce autophagy in Bcr-Abl+ CML cells. Simultaneously inhibiting Hh pathway and autophagy could remarkably inhibit cell viability and induce apoptosis of imatinib-sensitive or -resistant Bcr-Abl+ cells. Moreover, the combination of vismodegib and CQ had little cytotoxocity on human peripheral blood mononuclear cells (PBMCs). Furthermore, cell death induced by simultaneouely inhibiting Hh pathway and autophagy was related to PARP cleavage, caspase 3 and caspase 9 cleavage, the collapse of mitochondrial membrane potential, down-regulation of mTOR pathway and inhibition of Bcr-Abl. In conclusion, simultaneously inhibiting Hh pathway and autophagy could potently kill imatinib-sensitive or imatinib-resistant Bcr-Abl+cells. This study suggested that simultaneously inhibiting Hh pathway and autophagy might be a potent new strategy to overcome CML drug resistance.