The Effects And Underlying Mechanisms Of HSP90 Inhibitor BIIB021 On Chronic Myeloid Leukemia

Section 1:The effects and mechanisms of BIIB021 related proliferation inhibition and apoptosis in CML cells.Objective:To detect the effects of proliferation inhibition and apoptosis induced by BIIB021 on CML cell lines and explore the underlying mechanisms.Methods:MTT assays were applied to investigate the effects of BIIB021 on the cellular proliferation of CML cells. Annexin V apoptosis assays were performed to detect BIIB021-related apoptosis. The expression levels of Caspase-3, Caspase-9, PARP and apoptosis proteins were evaluated by western blot. Besides, Real-Time PCR was used to examine the mRNA expression levels of BCR-ABL in response to BIIB021. Western blot was also applied to analyze the protein expressions of BCR-ABL, its downstream targets:Jak/Stat signaling, Akt/mTOR signaling, Erk signaling andβ-Catenin/c-Myc signaling. Furthermore, confocal microscopy analysis was performed to observe the level ofβ-Catenin.Results:(1) BIIB021 effectively inhibited the proliferation of CML cell lines in a dose-and time-dependent manner including K562, K562/G, 32Dp210 and 32Dp210-T315I. The 50%inhibition (IC50) of BIIB021 in K562, K562/Q 32Dp210 and 32Dp210-T315I cell lines at 48 h was 513.99,603.53,110.08 and 148.07 nM respectively. (2) Treatment with the drug for 24 h resulted in a marked increase in the number of apoptosis cells. The cleaved Caspase-3, Caspase-9 and PARP increased, pro-apoptotic protein Bak and Bad increased, Bid, Mcl-1 and Survivin decreased while Bcl-2, Bcl-XL and Bax remained the same. (3) BIIB021 could downregulate both BCR-ABL protein and phosphorylation of BCR-ABL protein on Tyr177. The drug did not decrease the level of mRNA in BCR-ABL. The proteasome inhibitor MG-132 could reverse the BIIB021-mediated decline in BCR-ABL while the lysosome inhibitor chloroquine couldn’t. (4) BIIB021 inhibited the phosphorylation of Stat3, Stat5, Erkl/2 and the total protein of Jak2, Akt. (5) Treatment with BIIB021 facilated the expression of β-Catenin and its effector molecules, c-Myc. Confocal microscopy and western blot analysis revealed that P-Catenin was significantly depleted both in cytoplasm and nucleus. There was no obvious difference between the distribution of β-Catenin in cytoplasm and nucleus.Section 2:The effects and mechanisms of BIIB021 triggered autophagy in CML cells.Objective:To investigate the mechanisms of BIIB021-induced autophagy in CML cell lines. To evaluate the effect of BIIB021 co-treatment with autophagy inhibitor and discuss the role autophagy played in the process of BIIB021-triggered apoptosis to provide a new therapy for CML treatment.Methods:The appearance of acidic vesiclar organelles were observed by monodansylcadaverine and acridine orange staining. After transduced with AdmRFP-GFP-LC3, cells were observed by confocal microscope to detect autophagosomes. The levels of LC3I/II, p62, Beclin-1 and mTOR-Ulkl signaling were examined by western-blot. MTT assays were used to investigate the effects on the cellular proliferation. Knock down the expression of Caspase-3 by lentiviral vectors containing shRNA against Caspase-3. What’s more, SPSS 20 statistic analysis software was used for analysis and statistical significance was defined as p<0.05.Results:(1) BIIB021 could induce occurrence of autophagosomes in CML cells. (2) After treatment with BIIB021, the conversion of LC3I to LC3II increased while the level of p62 decreased. (3) BIIB021 induced autophagy by regulating Akt-mTOR-Ulk1 pathway other than in a Beclin-1 dependent manner. (4) The percentage of apoptosis cells by co-treatment BIIB021 with autophagy inhibitor (3-MA or bafilomycin A) was significantly augmented than that in the condition of BIIB021 treatment alone. These findings illustrated that BIIB021 related autophagy protected CML cells from the coinciding apoptosis.Conclusions:A series results demonstrated that BIIB021 has the ability against leukemia cells. The drug can induce apoptosis in imatinib resistant cells containing those harboring BCR-ABL T315I mutation. BIIB021 could decrease the level of BCR-ABL in a proteasome dependent manner. Importantly, protective autophagy occurred in CML cells exposed to BIIB021 by regulating Akt-mTOR-Ulk1 pathway. Co-treatment with autophagy inhibitor and BIIB021 could augment apoptosis in CML cells. Consequently, all these findings make BIIB021 a promising candidate to cure CML, especially cells insensitive to IM.