The Effects And The Mechanisms Of Hdm2I On Leukemic Cells:In Vitro And In Vivo

Leukemia is a series of malignant cloning diseases derived from hematopoietic stem or progenitor cells and is one of the most common cancers with the incidence accounting the sixth place of all various tumors. Even with the conventional therapy, including combined chemotherapy、 retinoic acid and arsenic trioxide-induced differentiation therapy and hematopoietic stem cell transplantation has resulted in a complete remission rate of70-90%, there are still considerable drug side-effect induced by chemotherapy non-selectivity as well as commonplace drug resistance of refractory or relapsed leukemia.The incidence of Chronic Myeloid Leukemia accounts for about15%-20%of leukemia in adults. Approximately95%patients with CML occurs significantly genetic mutations. The cytogenetic hallmark of CML is the Philadephia chromosome, created by t (9;22)(q34; q11),encoding a hybrid fusion protein Bcr/Abl with exceptional raised trosine kinase activties. Approved to be listed in United States2001, the first tyrosine kinase inhibitors imatinib soon became the first-line treatment of patients with chronic phase CML. A latest phase III clinical retrospective trail of CML-CP for eight years showed that event-free survival (EFS) and progression-free survival (PFS)were 83%and93%respectively, and75%patients with cytogenetic aberrations achieved complete cytogenetic response (CCyR). In the last15years, targeted treatment of CML with imatinib has achieved great success, but almost35%of patients developed to drug resistance. The second generation of the TKI, including dasatinib and nilotinib can overcome the vast majority of point mutations causing drug-resistant, but is invalid for Abl-T315I mutation produces drug resistance. On treatment of Abl-T315I mutation, clinical research including dasatinib combined with other drugs and the third generation TKI(AP245234、 danusertib) is still in progress, tolerance and an optimal treatment regimen have yet to be ascertained. Therefore, targeted Bcr/Abl therapy and imatinib-resistance reversal requires further exploration.Autophagy is becoming one of the most heated topics in the field of cell biology research, especially the relationship between autophagy and cancer was widespread attention. Autophagy is a number of biological processes of degradation and recycling of long-lived protein and impaired organelles in eukaryotic cells. Autophagy is becoming one of the most heated topics in the field of cell biology research, especially the relationship between autophagy and cancer was widespread attention. Autophagy is a number of biological processes of degradation and recycling of long-lived protein and impaired organelles in eukaryotic cells. There are various types of autophagy, including micro-and macroautophagy, as well as chaperone-mediated autophagy, and they differ in their mechanisms and functions. Under stress conditions, such as lack of cytokines, nutrients and oxygen, autophagy can stable internal environment and maintenance cell survival, but excessive autophagy can lead to autophagic death, which is also known as type Ⅱ programmed cell death. Autophagy is regulated by several signal pathways, and beclin-1is found as a tumor suppressor of Autophagy-related gene in the past ten years, which is mapped to a tumor susceptibility locus on chromosome17q21that is monoallelically deleted in40%to75%of human cancers. Many studies have shown that up-expression of beclin-1plays an important role in Inhibition of tumor. Therefore, beclin-1gene may become a new target for tumor treatment. In this study, we investigate the effects and the mechanisms of HDM2i on Leukemic cells through in vivo and in vitro experiments.In this study, we investigate the effects and the mechanisms of HDM2i on Leukemic cells through in vivo and in vitro experiments.This study is divided into two parts:Part Ⅰ:the effects and the mechanisms of HDM2i on CML and AML cells;Part Ⅱ: in vitro and in vivo study of combination of SG511-BECN and HDM2i on CML cells and its mechanism of Autophagic death.Part1:The effects and the mechanisms of HDM2i on CML and AML cellsObjective: To study and compare the effects of growth-inhibition and apoptosis induced by HDM2i on cell lines of CML cells and AML cells, and explore the apoptosis-independent molecular mechanism of HDM2i on CML cells preliminarily.Methods:Cell growth was measured by MTT assay, trypan blue staining method for the detection of cellular activity, colony culture method was used to observe cells cloning ability, hoechst staining and AV/PI flow cytometry technique were used for detecting apoptosis, real-time PCR assay for detection of Bcr/Abl mRNA expression level and western blot method to analysis the expression of caspase9/8/3, PARP, Bcr/Abl and Bcr/Abl downstream proteins. Statistical significance was defined as p<0.05. SPSS11.5statistic analysis software was used for analysis and CalcuSyn statistical software was used for CI (combined index) analysis.Results:1. HDM2i can inhibit the proliferation of AML cells through induction of apoptosis;2. the mechanism of HDM2i anti-CML is apoptosis-independent, but targets leukemia fusion protein Bcr/Abl and inhibits its downstream signal pathway (p-CRKL， JAK-Stat5. p-AKT and so on);3. HDM2i anti-CML effect is more obviously than traditional upregulation p53protein of small molecules inhibitors Nutlin3, and is not dependent on intracellular p53protein activity, so HDM2i has a stronger tumor-suppressor effect. More importantly, HDM2i strengthens traditional Bcr/Abl inhibitors PD180970downregulation of Bcr/Abl and induces obvious apoptosis, showing synergistic effect on CML cells.Part Ⅱ:in vitro and in vivo study of combination of SG511-BECN and HDM2i on CML cells and its mechanism of Autophagic death.Objective:to investigate the mechanism of autophagy induced by small molecule inhibitors HDM2i and enhanced the autophagy by oncolytic adenovirus SG511-BECN displays synergistic effect on CML cells, furthermore, we explore the autophagic death mechanism through in vivo and in vitro experiments, with a view to provide new methods and strategies for clinical practice.Methods:We adopted methods including AVO staining, transmission electron microscopy and LC3-GFP-K562cell fluorescence change to detect autophagy induced by SG511-BECN and/orHDM2i, and cell growth was measured by MTT assay, crystal violet staining method for the detection of cell activity, colony culture method was used to observe cells cloning ability, hoechst staining and AV/PI flow cytometry technique were used for detecting apoptosis, TCID50method to detect the virus amplification level, western blot to analysis the expression of caspase9/3, PARP and autophagy associated proteins, in vivo study of SG511-BECN and/or HDM2i on the tumor growth of K562xenograft-bearing SCID mice. Statistical significance was defined as p<0.05. SPSS11.5statistic analysis software was used for analysis and CalcuSyn statistical software was used for CI (combined index) analysis.Results:1. HDM2i can induce autophagy by upregulating beclin-1gene, and which plays a no-protective role on CML cells;2. the novel oncolytic adenovirus SG511-BECN can induce CML cells undergoing autophagic death;3. SG511-BECN enhances the autophagy induced by HDM2i through upregulating beclin-1protein expression on CML cells and the two have a synergistic effect on CML in vitro and vivo;4. the paper finally through a series of toxicological experiments, proves that SG511-BECN and/or HDM2i have non-toxic side effects on normal liver cells L02and normal stem(progenitor) cells from bone marrow, reminding us of the treatment for leukemia with SG511-BECN and/or HDM2i is a novel targeted cancer therapy.Conclusion:Through a series of studies(in vitro and in vivo experiments) in, we prove that the small molecule inhibitors HDM2i has p53-independent anti-leukemia effects, and have obvious growth inhibition effect on p53mutation cells:induces apoptosis on AML cells and down-regulates Bcr/Abl on CML cells, more importantly, HDM2i strengthens traditional Bcr/Abl inhibitors PD180970downregulation of Bcr/Abl and induces obvious apoptosis, showing synergistic effect on CML cells. SG511-BECN enhances the autophagy induced by HDM2i through upregulating beclin-1protein expression, subsequently induces autophagic death on CML cells, showing a synergistic effect on CML in vitro and vivo. We finally demonstrate that the treatment for leukemia with SG511-BECN and/or HDM2i is a novel targeted cancer therapy, with a view to provide new methods and strategies for clinical practice.