NVP-BEZ235Inhbits Proliferation And Increase Immune Sensitization Of CD34~+CD38~-Human Acute Myeloid Leukemia Stem Cells

BackguroundThe incidence of leukemia is4-5/10million in china.In our country leukemia is the first disease lead teenagers to death. Leukemia is a malignant diease in hematopoietic system because of expansion of colony-forming cell on some develping stage. Recently, the incidence of leukemia has increased becaused of accumulative damage caused by exacerbated environment. The cure of leukemia is more difficulter than before. Leukemia is a common and highly aggressive hematopoietic malignancy, which is a kind of disorder of hematopoietic system. Now commonly treatments include radiation, chemotherapy and hematopoietic stem cell transplantation. But these treatments are faced with relapse. Bonnet D was the first person who had separated blood cell subset with phenotype of CD34+CD38" from acute myeloid leukemia (AML) patients. He transplant the cells with phenotype of CD34+CD38" to mice with severe combined immunodeficiency disease (NOD/SCID mice). He found that the cells with phenotype of CD34+CD38" could lead to acute myeloid leukemia (AML) except acute promyelocytic leukemia (M3). The same report could be repeated on the second transplant recipient mice. Then leukemia stem cells were supposed to exist in vivo of AML and it was the root cause of leukemia’s occurrence, development and recurrence after treatment.Leukemia stem cells were of long-term self-renewal ability, multi-differentiating potential, and strong proliferative ability. Acute myeloid leukemia (AML) accounted for60%of leukemia.Leukemia stem cells from M0-M5except M3have the same phenotype of CD34+CD38-,which is similar to normal bone marrow HSC surface features Leukemia stem cells are in the initial stage of cloning. Leukemia stem cells are resist to regular radiation and regular dose of drugs, even to total body radiation and lethal does drugs because leukemia stem cells are often on the rest stage of cell cycle. Currently the guiding ideology on the clinical management is enhance radiation and chemotherapy to improve leukemia cells number. This kind of treatment often do not have target.It not only have great blindness, and it is difficult to achieve really cure. It is the waste of medical resources and bring the patients and these family unnecessary spirtitual and economic pain. So only targeted leukemia stem cells or improve leukemia stem cell sensitivity to radiation and chemotherapy,can completely cure leukemia,preventing relapes after treatment, improve long-term survival rates, to achieve the true sense of biology. So looking forward to a new drug or treatment,which not only target to Leukemia stem cells,at the same time,but also can transform the turner cells more senstive to the body’s immune system and drug-resistance.PI3K/AKT/mTOR signaling pathways involved in cell proliferation, differentiation, apoptosis and the regulation of glucose transport and so on a variety of cellular functions. PI3K as a treatment target for tumor can be in the spotlight, mainly because it is on the key signal position of many important signaling pathways regulating cell function. phosphatidyinositol3-kinase(PI3K) is a lipid kinase family,curently most involves I type of PI3K. They can catalytic PIP2phosphorylation as PIP3and activate downstream protein serine threonine kinase (AKT). AKT phosphorylation mTORC1to promote protein synthesis and cell growth, mTORC1can raised hypoxia induced factor1α and raised vascular endothelial growth factor to promote angiogenesis.In addition AKT can also be activated by mTORC2. mTOR (mammalian target of rapamycim) are mammals rapamycin target protein, is a serine/threonine kinase. It is in the downstream of PI3K signaling pathways. mTOR exist in the form of mTORl and mTOR2catalytic subunit. These two kinds of compounds (mTOR1and mTOR2) Participate in cell gene transcription and protein translation initiation, ribosome biosynthesis, cell apoptosis, etc. In recent years, many studies had shown that the abnormal cell malignant transformation were related to the pathway abnormal activity PI3K/AKT/mTOR signaling pathway inhibitors as a new anti-tumor leukemia drugs is attracting worldwide attention due to PI3K/AKT/mTOR signaling pathways play an important role in the development of tumor. NVP-BEZ235is a kind of ATP competitive inhibitor with imidazole and quinoline,which is the dual kinase Inhibitor both inhibit PI3K and mTOR. At present this drug had already entered the stage Ⅰ/Ⅱ clinical trials in solid tumor. NVP-BEZ235showed high inhibition of PI3K by ATP competitive inhibition of PI3K, it selectively blocking PI3K catalytic subunit p110α and p110γ. NVP-BEZ235can blocking mTOR1and mTOR2by increasing concentration and Preventing ordinary PI3K mutations.NVP-BEZ235significantly reduce the level of mTOR activate P70S6kinase protein phosphorylation. There’s a variety of studies suggesting that NVP-BEZ235induce apoptosis and arrest cell cycle in GO/G1phase in solid tumor.Schult C et al found that the dual kinase inhibitor NVP-BEZ235in combination with cytotoxic durg exerts anti-proliferative active towards Acute Lymphoblastic Leukemia Cells. Baumann P et al found that NVP-BEZ235in combination with cytotoxic durg exerts inhibits growth and proliferation in multiple myeloma and it have.synergy and accumulative effect in combination with bortezomib, melphalan and adriamycin, etc. However the NVP- BEZ235’s effect on CD34+CD38-the role of myeloid leukemia stem cells have not be reported. Therefore,we study the infulance of NVP-BEZ235on CD34+CD38-KG la cell lines of myeloid leukemia stem cells biological characteristics,provided the laboratory basis s for the treatment of acute myeloid leukemia.Leukemian cells can escape immune surveillance through various mechanism including gene mutation especially leukemia stem cells is one of the importent reasons caused leukemia.The surivival leukemia stem cells through immune obtain proliferation for colony and develop into leukemia. Natural killer cells (NK cells) are bone marrow sources of large granular lymphocyte. About5%to10%of the total number of human peripheral blood lymphocytes are natural killer cells. NK cells are the body’s first line of defense against tumor, played important roles in monitoring tumor. NK cells adoptive cellular immunotherapy has been an important treatment on cancer therapies. In virus infection, malignant transformation, inflammatory reaction and chemical stress-inducible heat shock factor (HSF) involved in transcription regulation to promote MICA/B, ULBP1, ULBP2molecules’s expression and be recognized by receptors. NK cell killing effect will be activated after receptor activation. NKG2D have MICA/B, ULBP1, ULBP2ligands which is NK cells’s killer activatory receptors. Studies have shown that a variety of blood tumor cell surface expression of certain level of NKG2D ligands, and the killing ability of NK cells links to the expression of NKG2D level. Improve NKG2D mediated NK cells immune clearance rate target to leukemia stem cells is important to treat leukemia and prevent recurrence. It next research direction to find the a drug role reversal leukemia stem cells’s resist of the immune system to maximize removal of leukemia stem cells.It will be the focus of our rsearch how to know NVP-BEZ235can or not improve the body’s immune cells’s sensitivie to KG1a cells. This study would be discussed that the biological characteristics of CD34+CD38-KG la cell lines influences by NVP-BEZ235and the NVP-BEZ235weather regulate CD34+CD38-KG1a cells more sensitive to the immune killers.So as to provide the date of NVP-BEZ235against acute myeloid leukemia in vitro research to find out whether NVP-BEZ235can enhance the sensitivety of KG1a cells to killer cells.Part Ⅰ The biological characteristics of CD34+CD38-human acute myeloid leukemia stem cells influenced by NVP-BEZ235[Objective] To explore the effect of NVP-BEZ235, a dual phosphatidylinositol3-kinase/mammalian target of rapamycin inhibitor, on proliferation, cell cycle percentage,apoptosis and colony forming capability of CD34+CD38-human acute myeloid leukemia KG1a cells.[Methods] Flow cytometry was used to dectect the expresstion of CD34and CD38on the surface of human acute myeloid leukemia KG1a cells used in the experiment. Trypan blue assay was used to analyse the effect of NVP-BEZ235with various concertrations (0μmol/L、0.125μmol/L.0.25μmol/L.0.5μmol/L、1μmol/L) on proliferation of KG1acells. Flow cytometry was performed to examine the cell cycle and apoptosis of KG1a cells after various concertrations (0μmol/L、0.125μmol/L、0.25μmol/L、0.5μmol/L、1μmol/L) NVP-BEZ235treatment. Soft agar colony-forming experiment was used to detect the colony forming ability of KGla cells treated with NVP-BEZ235at various concertrations (0μmol/L、0.125μmol/L、0.25μmol/L、0.5μmol/L、1μmol/L)SPSS13.0software was used for data analysis and The experiment data were representded as the mean±standard deviation. the inhibition of NVP-BEZ235to KG1a cells proliferation and clone formation test were used analysis of variance, LSD comparisons were used between groups multiple comparisons when variance together. When different concentrations of NVP-BEZ235lead to the cell cycle and apoptosis changes t test was used to compared between groups. [Result] Flow cytometry detected the KG1a cells used in experiment, the CD34expression rate (%) was98.6±0.17, CD38expression rate (%) was00.01±0.00. The development is on the stage of stem cell of leukemia. DMSO (0umol/L) for CD34+CD38-KGla had no effect on cell proliferation (P>0.05). NVP-BEZ235(0.125-1umol/L) inhibited the proliferation of KGlacells at a time-and does-dependent manner (P<0.05) and the50%inhibition concentrations (IC50) at24h and48h were0.597μmol/L and0.102μmol/L. At the same time point every concentration groups compered with0umol/L group the difference has statistically significant (P<0.05). At the same concentration different time points compared with each other the difference has statistically significant (P<0.05).54.07±7.18%.43.47±9.60%、80.17±3.57%、83.2±3.80%.83.2±3.80%、80.03±1.23%KGla Cells were arrested at G0/G1phase after treated with Control,0μmol/L,0.125μmol/L,0.25μmol/L,0.5μmol/L,1μmol/L NVP-BEZ235for24h. There was no statistically significant difference when Control group compared with0umol/L group (P>0.05). Groups compared with0μmol/L group the difference has statistically significant (P<0.05). NVP-BEZ235can not induce KGla apoptosis. There are no statistically significant difference between groups (P>0.05). KGla cells treated with NVP-BEZ235(0～1μmol/L) for14days and21days, the number of colony decreased respectively from (375.67±21.46) per2500KGla cells and (706.33±87.31) per2500KGla cells to0, with statistical significance (P<0.05).The colony size of0.25μmol/L were much smaller than the control group. There was no colony formation only observed found debris under inverted microscope when treated with0.5μmol/L and1μmol/L NVP-BEZ235.[Conclusion]1、The development of KG1a is on the stage of leukemia stem cell.2、NVP-BEZ235can inhibit proliferation of CD34+CD38-human acute myeloid leukemia KG1a cells with does and time dependant manners.3、CD34+CD38-human acute myeloid leukemia KG1a Cells were arrested at G0/G1phase after treated with NVP-BEZ235. NVP-BEZ235inhibits KGla cells proliferation may associate with the arrestion of cycle at G0/G1phase.4、NVP-BEZ235can not induce KG1a apoptosis5、NVP-BEZ235significantly inhibit the cloning formation ability of KGla cells, especially for the cells with self-renewal and high potential proliferation. Part II The influence of NVP-BEZ235to the CD34+CD38"human acute myeloid leukemia KGla cells killed by the cytotoxicity of activated PBMC[Objective]To expore the cytolytic sensitivties and preliminary mechanism of untreated PBMC and the PBMC stimulated by IL-2,IL-5on CD34+CD38-human acute myeloid leukemia KGla cells and after treated by NVP-BEZ235.[Methods]Trypan blue assay was used to analyse the proliferation of NVP-BEZ235peripheral blood mononuclear cells. To find the difference btween untreated PBMC and stimulated by IL-2,IL15, LDH releasing assay was used to detect the kill rate of KG1a cells at different effector-to-target ratios(10:120:1). The expression of NKG2D ligands was assayed by Flow cytometry before and after treated by NVP-BEZ235for24h.SPSS13.0software was used for data analysis and The experiment data were representded as the mean±standard deviation. NVP-BEZ235impact on PBMC proliferation with paired t test.The factorial design analysis of variance was used to analysis the kill rate of PBMC on KG1a cells and NKG2D ligands using independent sample t test, P<0.05think difference was statistically significant.[Results] The24h、48h、72h inhibition rate of0.5and0μ mol/L NVP-BEZ235on PBMC were respectively0.67±0.29(%) vsl.17±0.76(%)、1.67±0.29(%) vs1.0±0.5(%)、1.83±0.70(%) vs1.83±0.29(%). Comparative differences between groups have no statistical significance (P>0.05).when effect target ratio come to10:1and20:1the cytotoxic rate of without induced PBMC to KG1a cells were4.94±0.54(%)、5.59±0.55(%) there was no statistically significant difference between them (P>0.05). On equal conditions the cytotoxic rate of induced PBMC to KG la cells were25.73±2.49(%)、35.05±3.39(%). The difference was statistically significant (P<0.05). when effect target ratio come to10:1and20:1the cytotoxic rate of without induced PBMC to KGla cells were4.94±0.54(%)、5.59±0.55(%).When KG1a cells treated by0.5μ mol/L NVP-BEZ235,the the cytotoxic rate of without induced PBMC to KGla cells were4.95±1.16(%).7.00±2.05(%) there was no statistically significant difference (P>0.05). when effect target ratio come to10:1and20:1the cytotoxic rate of induced PBMC to KGla cells were25.73±2.49(%).35.05±3.39(%).When KGla cells treated by0.5μ mol/L NVP-BEZ235,the the cytotoxic rate of induced PBMC to KGla cells were25.61±4.01(%)、47.49±1.65(%). NVP, BEZ235continuing role in KGla cells after24hours, the cell surface of NKG2D ligands expressed not seen significant change.[Conclusion]1、0.5μmol/L NVP-BEZ235had no significant effect on PBMC proliferation2、The cytotoxic activity of induced PBMC was obviously higher than not induced PBMC3、 NVP-BEZ235can enhance the cytotoxic activity sensitivity of PBMC on KG1A cell [Conclusion]1.NVP-BEZ235significantly inhibit proliferation and the cloning formation ability of KGla cells.NVP-BEZ235inhibits KGla cells proliferation may associate with the arrestion of cycle at G0/G1phase.2.0.5u mol/L NVP-BEZ235had no significant effect on PBMC proliferation3.NVP-BEZ235can enhance the cytotoxic activity sensitivity of PBMC on KGla cells4. NVP-BEZ235can be used to treat leukemia, monotherapy or combination drug therapy or combined with immune cells in treatment were still needed to be studied in the future.