The Mechanism Of Flavonoid Effects On Acute Myelogenous Leukemia In Vivo And In Vitro

ObjectiveIn HL60, K562, HL60/ADR, K562/ADR cell lines, AML primary cells,Leukemia tumor-burdened mice and Tg (fli: EGFP) zebrafish models forthe in vivo and in vitro studies, systematic research on flavonoid effectson leukemia cells growth inhibition, induction of apoptosis, collaborativesensitizing mechanism combined with adrimycin and anti-angiogenicmechanism, in order to fully explore the feasibility of flavonoid treated ofleukemia.Methods and ResultsPart1: Research on flavonoid and combined with adriamycininduced apoptosis and antiproliferation in myeloid cell linesMethods: HL60, K562, HL60/ADR, K562/ADR cells were treatedwith flavonoid and adriamycin, counting kit was used to detect theIC50values and growth inhibition rate of flavoniod monotherapy andcombination therapy for AML leukemia cell lines and primary cells,apoptosis rate of cell lines was detected by flow cytometry, morphological changes were observed by light microscopy. Results:Flavonoid could significantly inhibit the proliferation of cell linesin12h,24h,48h with a dose-and time-dependent manner. The inhibitoryeffect on cell proliferation was enhanced while cells were treatedwith combined use of different concentrations of flavonoid andadriamycin (IC20), showing a synergistic and additive effect. Flavonoid at75μg/mL and100μg/mL could induce cell apoptosis in cell lines, whenin combination use with adriamycin(IC20) apoptosis was increased. Aftermononuclear cells of normal person was added flavonoid has no effect oncell growth. Five cases of newly diagnosed AML patients, primary cellsafter adding flavonoid, cell growth was inhibited in a time-anddose-dependent.Part2: The mechanism of flavonoid and combined with adriamycininduced apoptosis in myeloid leukemia cell linesMethods: Using microarry to analyze changes of signaling pathway inthe four cell lines after adding flavonoid. The protein changes ofapoptosis signaling pathways and leukemia resistance proteins weredetected by western blot and flow cytometry respectively in leukemia celllines and AML primary cells with flavonoid and combined withadriamycin in24h and48h. Results: The apoptosis signaling pathwaysand regulating angiogenesis factor pathway were taken place changes inleukemia cell lines after using flavonoid. The Rh123fluorescence intensity increased significantly in leukemia cell lines with flavonoid andcombined with adriamycin in24h and48h. Extrinsic and intrinsicapoptosis pathway involved the cleavage of caspase-8and caspase-3canoccur using flavonoid and combined with adriamycin. Flavonoid andcombined with adriamycin decreased the rxpression of anti-apoptoticBcl-2and Bcl-xL, increased significantly the expression of pro-apoptoticprotein Bim, Bad and Bax, phosphorylated P-P38MAPK and P-JNKprotein expression levels were increased, while P-ERK protein wasdecreased significantly. MDR and MRP1protein of K562/ADR andHL60/ADR were changed adding flavonoid and combined withadriamycin in24h and48h.Part3: Study the role of of flavonoid and combined withadriamycin in tumor bearing mice modelsMethods: BALB/c male nude mice, HL60tumor-bearing mice modelwas established. Divided into four groups: No dosing group, flavonoidsgroup, doxorubicin, adriamycin flavonoid group, intraperitoneal injectionof the drug two weeks. The tumor-bearing mice model was divided intofour groups: control group, flavonoid group, adriamycin group, flavonoidcombined with adriamycin group, intraperitoneal injection of the drug fortwo weeks. The weight and tumor size of tumor-bearing mice model weremeasured twice weekly, the blood IgM of tumor-bearing mice weredetected by ELISA method before and after using drugs. After tumor removal, neutral formalin-fixed, make immunohistochemistry whenumor-bearing mice were sacrificed. Results: HL60cells2x107weresubcutaneously injected into BALB/c nude mice, tumorigenicity rateswere100%, the tumorigenicity latence were14days. Tumor-bearingmice in each group after the administration found that the body weightand tumor volume were significantly reduced. Flavonoid group wassignificantly elevated IgM, a significant difference (P <0.01) with theprevious medication. Immunohistochemical of tumor block found theexperimental groups promote apoptosis, caspase activation significantly,Bcl-2, Bcl-XL expression were decreased, Bim, Bad and Bax wereincreased significantly, phosphorylated P-P38MAPK and P-JNKexpression were significantly increased, while P-ERK decreased.Part4: The anti-angiogenesis mechanism of flavonoid in vivo and invitroMethods: Angiogenesis factors gene of VEGF, VEGFR1, VEGFR2,Notch1, Delta like4were detected using qRT-PCR in HL60cells withdifferent concentrations of flavonoid, while extracting cellular proteinsand were tested by western blot. Tg (fli1: EGFP) zebrafish has beenconstructed by laboratory and zebrafish embryos were picked out at14hours post fertilization (hpf) treated with different concentrations offlavonoid from38hpf and62hpf. The blood vessel was observed underfluorescence inverted microscopy to measure the number of complete intersegmental vessels (ISVs) and angiogenic sprouts by manualcounting.At14hpf,38hpf and62hpf these embryos were harvested inTRIzol reagent, respectively, and their RNA was extracted andreversely transcribed to single-stranded cDNA, followed by qTR-PCRdetect the expression of angiogenic factors. In tumor-bearing mice model,tumor angiogenesis factor were tested by immunohistochemistry.Results: After treatment with different concentrations of flavonoid (0,50,75, and100μg/ml) for12hr,24hr and48hr, the mRNA expressions ofVEGF, VEGFR2, DLL4were significantly down-regulated, comparedwith those of control group, and the mRNA expressions of VEGFR1and Notch1were decreased at24hr and48hr, the protein levels ofVEGF, VEGFR2, DLL4, VEGFR1and Notch1revealed a similarreduction tendency. The immunohistochemistry of tumor-bearing micemodel showed that the treatment groups was significantly inhibited tumortissue VEGF, VEGFR1, VEGFR2, DLL4and Notch1expression. TheTg(fli1EGFP) zebrafish embryos added flavonoids solution found thatVEGF, VEGFR2, Notch1and DLL4mRNAs were down-regulation in aconcentration dependent manner. The mRNA expression of VEGFR1wasdecreased in62hpf. When Tg(fli1:EGFP) transgenic zebrafish embryoswere analyzed at38hpf and62hpf, time points where all intersegmentalvessels (ISVs) in the vehicle control group have fully extended to formthe dorsal longitudinal anastomotic vessels (DLAVs) and to result in significant reductions in the number of complete ISVs and angiogenicsprouts.ConclusionsFlavonoid can inhibit the growth of leukemia cell lines and AML primarycells, flavonoid combined with adriamycin inhibited more obvious andwas synergistic or additive effect and induced apoptosis of leukemia cells.Flavonoid can change the apoptosis signaling pathway and the regulationof angiogenesis factor pathway. Flavonoid and combined with adriamycincan decrease mitochondrial membrane potential loss, activate caspasepathway, regulate Bcl-2family proteins, down-regulation ERK pathwayand up-regulation P38MAPK and JNK pathway, and verified intumor-bearing mice in vivo. Flavonoid have increased in nude micehumoral immune function, enhanced serum IgM. While the flavonoid andcombined with adriamycin, and can reverse the expression K562/ADRHL60/ADR resistance protein. Flavonoid have anti-angiogenic factorsrole in HL60cell lines, it has been confirmed in Tg (fli1: EGFP)zebrafish and HL60tumor-bearing mice model.