The Roles Of AF1Q Protein Degradation And The Anti-acute Myeloid Leukemia Effect Of Nitidine Chloride

Section IThe role of chaperone mediated autophagy in the degradation of AF1Q proteinBackground:Leukemia is the most common hematological malignancy. Mixed-lineage leukemia (MLL) gene rearrangements are common chromosomal abnormalities in human leukemia. The AF1Q gene is initially identified as a mixed lineage leukemia (MLL) fusion partner in two infant patients diagnosed with acute myelomonocytic leukemia, which carried a t(1;11)(q21; q23) translocation. In hematopoietic tissues, AF1Q expression is abundant in normal lineage committed hematopoietic progenitor cells and thymus, but is undetectable in peripheral hematopoietic tissues such as peripheral blood and spleen. AFlq cooperates with the Notch signaling pathway to foster the emergence of bone marrow prothymocytes and drive subsequent intrathymic specification toward the T-cell lineage. It is confirmed that elevated AF1Q expression is a poor prognostic biomarker for pediatric acute myeloid leukemia (AML), adult AML with normal cytogenetic and adult myelodysplastic syndrome. The biological function of AF1Q has not been completely characterized, but increasing evidence has shown a potentially proto-oncogenic function of this protein in solid tumors such as thyroid oncocytic tumors, breast cancer and testicular germ cell tumors. In addition to the proposed proto-oncogenic role of AF1Q, this protein may participate in the regulation of apoptosis in tumor cells.It has been shown that AF1Q expression can be directly regulated by microRNA-29b; the catabolism of AF1Q protein is ubiquitin-mediated degradation by the proteasome in the centrosomal area. However, mechanisms underlying the regulation of AFIQ and its degradation pathway are not understood. The ubiquitin-proteasome system and the autophagy-lysosome system comprise the two major intracellular proteolytic systems in mammalian cells. Autophagy is an intracellular degradation system for long-lived cytoplasmic constituents using lysosomes. Based on the known mechanisms that substrates of autophagy are delivered into lysosomes, three major forms of autophagy have been described in mammalian cells including macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). Compared to other forms of autophagy, CMA is a selective lysosomal pathway best characterized in the degradation of individual cytoplasmic proteins. For degradation via CMA, cytoplasmic proteins bear the KFERQ motif or a closely related sequence are recognized by the heat shock70kDa protein8(HSPA8) chaperone. Following binding of the chaperone-substrate complex, the proteins are delivered to a lysosomal membrane receptor-lysosome-associated membrane protein2A (LAMP-2A), and then translocated into the lysosomal lumen and degraded.Objective:The aim of this study was to investigate whether lysosome pathway took part in AFIQ degradation, and to clarify the molecular mechanisms by which CMA recognized and degraded AF1Q.Materials and methods:1. Cell treatment:When appropriate, K562leukemia cells and HEK-293cells were treated with the following agents:MG132, chloroquine (CQ),3-MA,6-AN. For starvation, cells were switched to Hanks balanced salt solution (HBSS). Western blot and real-time RT-PCR were carried out after treatment.2. Cell transfection:To overexpress or down knock down a protein, transient transfection of cells was performed with lipofectamine2000. 3. Western blot analysis:Cells treated under different experimental conditions were lysed in Cell lysis buffer for Western and IP. Protein concentrations in the supernatant were quantified with the bicinchoninic acid assay protein reagent kit. The whole cell lysates were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or Tricine-SDS-PAGE. The protein levels were determined by immunoblotting methods.4. Real-time RT-PCR:Cells treated under different experimental conditions were collected, and total RNA was extracted from cells using TRIzol reagent. The mRNA expression of AF1Q was evaluated by Real-time RT-PCR.5. Immunofluorescence microscopy:After transfected with specific plasmids for48h, cells were plated onto coverslips and cultured for24h. Cells were fixed and incubated with primary antibodies. Images were acquired by laser scanning confocal microscope.6. Co-immunoprecipitation:The whole cell lysates were incubated with anti-AF1Q and further incubated with protein A+G agarose. These precipitates were fractionated by SDS-PAGE. The protein was determined by immunoblotting methods.7. Statistical analysis:Statistical analysis was conducted using Student’s t-test or one-way analysis of variance. P<0.05was considered significant.Results:1. The effects of lysosome inhibitor or activator on the levels of AF1Q protein:(1) Intracellular AF1Q levels increased when K562leukemia cells and HEK-293cells were treated with total lysosome inhibitor CQ or proteasome inhibitor MG132.(2) Intracellular AF1Q was not increased when K562leukemia cells and HEK-293cells were treated with macroautophagy inhibitor3-methyladenine.(3) In both K562and HEK-293cells, CMA activator6-AN treatment decreased the levels of AF1Q.(4) Switching to Hanks balanced salt solution (HBSS) for12h slightly reduced AF1Q protein levels in K562cells, and AF1Q protein levels were markedly reduced for24h in both K562and HEK-293cells. (5) Real-time RT-PCR analysis demonstrated that the mRNA expressions of AP1Q were not change in K562and HEK-293cells treated with MG132、CQ、3-MA or6-AN for24h. Switching to HBSS for6h had no effect on AF1Q mRNA expressions, but AF1Q mRNA expressions were markedly increased for12and24h in both K562and HEK-293cells.2. The effects of downregulated or overexpression macroautophagy related gene on the levels of AF1Q protein:(1) There was a reduction in levels of AF1Q when endogenous ATG5or ATG7was knocked down.(2) Overexpression of ATG5or ATG7increased the protein levels of AF1Q.3. The effects of downregulated or overexpression CMA related gene on the levels of AF1Q protein:(1) There was an increase in levels of AF1Q when endogenous HSPA8or LAMP-2A was knocked down.(2) Overexpression of HSPA8or LAMP-2A decreased the protein levels of AF1Q.4. AF1Q Interacts with HSPA8and LAMP-2A:(1) AF1Q was co-localized with HSPA8and LAMP-2A in cells expressing exogenous AF1Q.(2) Exogenous AF1Q was co-IP with exogenous HSPA8and LAMP-2A in HEK-293cells.5. Identification of CMA-targeted motif in AF1Q protein:(1) Sequence alignment of the AF1Q amino acid (aa) sequence with a KFERQ motif using the Clustal Omega tool revealed that84-88aa of AF1Q contain the putative KFERQ-like motif.(2) Knockdown of LAMP-2A was ineffective in increasing protein levels of80-90aa deletion AF1Q in comparison with the full-length AF1Q.Conclusion:1. The degradation of AF1Q in lysosome was through CMA but not macroautophagy.2. HSPA8and LAMP-2A, two core components of the CMA machinery, had physiological interaction with AF1Q.3. AF1Q has a KFERQ-like motif which is recognized by HSPA8for CMA-dependent proteolysis. Section IIThe role of anti-acute myeloid leukemia effect of nitidine chloride Background:Acute myeloid leukemia (AML) is a life-threatening hematopoietic stem cell neoplasm characterized by an increase in the number of myeloid cells in the bone marrow and an arrest in their maturation. Despite the improvement in treatment protocols, a large number of AML cases have poor prognosis. Therefore, alternative treatments strategies remain to be developed to prevent AML progression and improve patient survival rates.Natural products, used in complementary and alternative medicine, are an extraordinary source of prevention and treatment for tumors including AML because of their extensive biological activities and comparatively low toxicity. Nitidine Chloride (NC) is a natural bioactive phytochemical alkaloid extracted from the root of Zanthoxylum nitidum. Recently, intensive studies have shown that NC possesses potent anti-tumor activities by inhibiting proliferation and inducing apoptosis in in many human solid cancer cells. However, the effects and mechanisms of NC on AML cells have not been reported.Objective:To investigate the effect and the underline mechanism of NC on the growth, cell cycle and apoptosis in AML cells; to clarify the effect of NC on signaling pathway in AML cells; to probe the possibility of NC used as an anti-AML drug.Materials and methods:1. Cell viability assay:Cell viability of treared cells was performed using CCK8assay.2. Cell cycle assay by flow cytometry:Cells treated with NC for12h, and cell cycle distribution was analysed using propidium iodide (PI) staining.3. Apoptosis assay by flow cytometry:Cells treated with NC for24h, and apoptosis induction was analysed using the Annexin V/PI Apoptosis Detection kit.4. Western blot analysis:Cells treated under different experimental conditions were lysed in Cell lysis buffer for Western and IP. Protein concentrations in the supernatant were quantified with the bicinchoninic acid assay protein reagent kit. The whole cell lysates were fractionated by SDS-PAGE. The protein levels were determined by immunoblotting methods.5. Statistical analysis:Statistical analysis was conducted using Student’s t-test or one-way analysis of variance. P<0.05was considered significant.Results:1. NC inhibited cell growth of AML cells: The growth inhibition effect of NC is significant in both dose-and time-dependent manners in all tested AML cell lines (HEL, NB4and THP-1).2. NC induced cell cycle arrest in AML cells:(1) NC induced an accumulation of AML cells in the G2/M and S-phase fractions.(2) NC significantly inhibited cyclin B1and CDK1expression, whereas increased p27expression in AML cells.3. NC induced apoptosis in AML cells:(1) Treatment with NC for24h resulted in increased apoptotic rates in AML cells.(2) PARP inactivation and Caspase-3activation were increased in NC-treated AML cells.(3) The expressions of pro-apoptotic factor Bax were increased and anti-apoptotic factor Bcl-2were decreased significantly in NC-treated AML cells4. The effects of NC on signaling pathway of AML cells:(1) Treatment with NC resulted in the inactivation of the AKT pathway in AML cells with decreases in the phosphorylation of AKT.(2) The phosphorylation of ERK in AML cells was also decreased after treated withNC. However, significant change in p-P38was not detected.(3) NC slightly decreased in p-JAK2, but no change in p-STAT3was found.5.The effect of AKT inhibitor on cell growth inhibition of NC: AKT inhibitor LY294002can significantly increase the sensibility of AML on NC.6. The effect of ERK inhibitor on cell growth inhibition of NC: ERK inhibitor PD98059can significantly increase the sensibility of AML on NC.Conclusion:1. NC inhibited the growth of AML cells via cell cycle arrest and apoptosis.2. Anti-proliferation effect of NC in AML cells is partially via inhibiting the phosphorylation of AKT and ERK.3. NC could be an effective therapeutic agent again AML.