The Mechanism Of Differentiation Induction By Wangzaozin A And P(HPMA-APMA)-ATRA In Human Promyelocytic Leukemia Cell HL-60

Plants belonging to the genus Labiatae Isodon are widely distributed in China.There are variety of compounds, which is isolated from Labiatae Isodon, apply toanti-tumor therapy based on their high efficiency and low toxicity. Ent-kaurenediterpenoids, which are the main active constituents against tumor, are rich in the gnusIsodon. Most of them are used as antitumor, antibacterial, and anti-inflammatoryherbs. In recent years, it was reprted that Oridonin, a kind of Ent-kaurene diterpenoids,have good curative effect on treatment of leukemia. Professor Chen Guoqiang et alisolated another antipodal kaurane diterpenoid gland two. Breviscapine fromRabdosia adenantha, found the two terpenes can induce leukemia cells differentiationon morphology and function, its differentiation mainly through binding with peroxidereductase PrxI and PrxII specific covalent, induced acute early fine grain (APL)differentiation of leukemia cell line NB4,However, the compounds which have similarmolecular structure of Ent-kaurene diterpenoids against leukemia have rarely reported.Many of them explore the antitumor mechanism through the apoptotic pathway.Wangzaozin Awas diterpenoids from Isodon racemosa (Hemsl) Hara., Wangzaozin Ahas the strongest bioactivity in20diterpenoids from sodon racemosa (Hemsl) Hara.There were less anti-tumor activity reports about Wangzaozin A induction ofdifferentiation signal pathway.Wangzaozin A, an ent-kaurane diterpenoid, is used as subjects compound to testthe effects on HL-60cell growth with trypan blue exclusion method and MTT. Theeffect of Wangzaozin A on proliferation was studied by FCM, Hoechst33258fluorescent staining and Giemsa staining were used to evaluate the production ofnucleus and morphology in HL-60cells, At the same time, the fluorescent particlephagocytosis and NBT method, flow cytometry cell surface CD11b testing the drugon cell differentiation, intracellular reactive oxygen species and the activity ofNADPH induced the differentiation process in medicine,The results as as follows:1. Wangzaozin A has stronger cytotoxicity on HL-60cells, restrains cell growth under lowconcentration, and has lethal effects under high concentration and a time-and dose-dependent manners. MTT assay show that IC50=0.765±0.08?mol/L?0.561±0.06?mol/L?.2. PI staining assays by flow cytometry displayed that the distribution of cellcycle could be changed with the treatment of Wangzaozin A. It could induce strong G1phase arrest in time-dependent. The fragment of DNA could not overflow. At last, thecells arrest G1phase. inhibition of cell growth, and induce cell differentiation.3. The results of Hoechst33258fluorescent staining showed that Wangzaozin Acould produce a large number of multicore.4. Flow cytometry was used to make a further investigation of phagocytosis offluorescent tracer effects by Wangzaozin A on human acute promyelocytic leukemiaHL-60cells respectively?The results indicated that the fluorescence intensityEnhanced with the concentration increase.5. NBT assay and expression of CD11b were used to make a furtherinvestigation of differentiation of cells effects by Wangzaozin A on human acutepromyelocytic leukemia HL-60cells respectively. The results indicated TheseWangzaozin A induced the generation of nitroblue tetrazolium (NBT)-reducingactivity, more HL-60cells became NBT positive and expression of CD11b thanATRAwith the increase of the concentration of.6. Flow cytometry and fluorescence microscope observation showed that theintracellular ROS, different concentrations of Wangzaozin A with different degree offormic acid can be caused by the increase of intracellular ROS level, the elevatedlevel of ROS was positively correlated with the concentration of the drug, whenWangzaozin A and NAC72h also treated cells, induced differentiation abilitycompared with the single Wangzaozin A Wangzaozin A cell, the expression level ofCD11b cell surface antigen decreased significantly, suggesting that the elimination ofROS can inhibit Wangzaozin Aon the differentiation of HL-60cells induced by.In short, Wangzaozin A on human promyelocytic leukemia cells have strongtoxicity, on HL-60cell, inhibit the cell growth, induced significant G1phase arrest,and have the time dose effect. At the same time, the compound can also enable cellsto produce much nucleus and nucleus; at the same time, HL-60cells were treated withcompound treatment, cells of fluorescent particles of P (HPMA-FMA) phagocytosisability increases with the concentration increased, the NBT reduction assay and cell surface CD11b detection results were increased, ROS level of intracellular drugaction increased significantly, the elimination of ROS by NAC, differentiation abilityof drugs on cells decreased significantly, showing the drug induced HL-60celldifferentiation, the differentiation pathway may be through an increase in theintracellular ROS level signal conditioning.P(HPMA-APMA)-ATRA was synthesized by free-radical solutionpolymerization with N-(2-hydroxypropyl methacrylamide)(HPMA), N-(3-aminopropyl) methacrylamide (APMA) and all-trans retinoic acid (ATRA) as rawmaterials. The structure of polymer was confirmed by1H NMR. Compared with themonomeric ATRA, the water-solubility of the polymer increased significantly andcould enter the cells through endocytosis. The inhibition of polymerized andmonomeric ATRA on HL-60cell growth was assessed byMTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide). Then flowcytometry was used to detect the effects of the two compounds on cell cycledistribution and expression of cell surface antigen CD11b in HL-60cell. At last, afurther experiment with NBT(nitroblue tetrazolium) was done to analysis the celldifferentiation of HL-60induced by the two compounds.With the results, we foundthe P-ATRA showed a stronger suppressive ability on cell growth than monomericATRA, the IC50were1.03?mol/L and4.09?mol/L, respectively. And also, theP-ATRA had a higher G0/G1phase cell block effect. At the concentration of1.2?mol/L, G0/G1phase cell rate induced by P-ATRA was17.7%higher than that ofmonomeric ATRA. The NBT reduction ability of HL-60induced by0.4?mol/LP-ATRA was similar with that of2.4?mol/L ATRA, the expression of cell surfaceantigen CD11b in HL-60cell induced by0.8?mol/L P-ATRAwas similar with that of2.4?mol/L ATRA, and the results indicated that the differentiation of HL-60cellinduced by P-ATRAincreased3to4folds compared to that ofATRA.