The Antitumor Activities Of Marine Natural Product Stellettin B On Chronic Myeloid Leukemia Cells And The Molecular Mechanism

Objective:The in vitro anti-leukemia activity and molecular mechanism of Stellettin B(Stel B),a triterpenoid we isolated from marine sponge Jaspis stellifera,on human CML cells was recently investigated,aiming to provide evidence for development of such kind of drug candidate for therapy of chronic myeloid leukemia(CML).Methods: 1.The effects of Stel B on the growth of K562,KU812,as well as normal PBMCcells were determined using WST-8 assay.Since K562 cell exhibited much higherresponse than KU812,we further investigated the antitumor effect of Stel B onK562 cells by use of soft agar colony formation assay.2.In synergisitc assay,we first determined antiproliferative activities of Stel B andImatinib with MTT assay,and calculated the IC50 values.3.Flow cytometry was used to analyze the effect of Stel B on cell cycle distribution,cell apoptosis,intracellular ROS level and mitochondrial membrane potential(MMP,??m)4.Western blot was analyzed the expression of apoptosis regulators such ascaspase-9,caspase-3,PARP,Bax,Bcl-2 and Bad.We examined the effect of StelB on expression level of PI3K/Akt pathway in K562 cells.5.Small interfering RNA(siRNA)assay was used to knock down Stat5,Westernblot was determined the effect on Stat5 and PI3K/Akt pathway in K562 cells.6.We performed synergisitc assay by using Chou and Talalay's method to examinethe combination of Stel B and Imatinib.Fa-CI data were analyzed by CalcuSynsoftware to determine drug combination effect.Results: 1.Growth inhibitory effect of Stel B on CML cells.WST assay showed that Stel Breduced K562,KU812 cell viability in a dose-dependent manner.The IC50 valuewas calculated to be 0.035,0.95 ?M.Also,with a low cytotoxicity of Stel B onnormal PBMC cells,the IC50 value more than 39 ?M.Soft agar assay showed thattreatment with Stel B both number and size of cell colonies were decreased,suggesting Stel B inhibited tumorigenicity of CML K562 cells.2.Stel B does not affect cell cycle distribution of K562 cells.3.Stel B induces apoptosis in K562 cells.The apoptotic cell population wasdetermined by Annexin V-FITC/PI double staining,afeter treatment with Stel Bthe apoptotic cell population was 7.5%,22.44%,26.1%,32.2%,44.6%.Also,with increase of Bad and Bax,decrease of Bcl-2 and increased cleavage ofcaspase-9,caspase-3,PARP.After treatment with Stel B,the amount of ROSincreased and the relative MMP reduced dose dependently.4.Stel B inhibited expression of 4 PI3 K catalytic isoforms,the regulatory subunitPI3K-p85,as well as the phosphorylation of Stat5,PDK1,Akt,mTOR and p70S6K.After treatment with Stat5 inhibitor SH-4-54 and Stat5 siRNA,thephosphorylation of PDK1,Akt were reduced,accompanied with downregulationof 4 PI3 K catalytic isoforms and regulatory subunit PI3K-p85,while PI3K-p110?has significant downregulation;co-treatment with Stel B enhanced the aboveeffect,further supporting that the inhibition against Stat5 might contribute to theeffect on PI3K/Akt pathway.5.Synergisitc assay showed that combination of Stel B with Imatinib with ratio asIC50 Stel B ? 5 × IC50 Imatinib led to synergistic effect.Combination indexes(CI)werethen calculated,with CI < 1,suggesting synergistic effect of Stel B and Imatinibon K562 cells.Soft agar assay showed that combination significantly decreasedthe number of colonies,compared with either single treatment.Also,co-treatmentwith Stel B and Imatinib led to a highly enhanced apoptosis-inducing effect.Inaddition,increased cleavage of caspase-3 and PARP was also observed for thecombination.Our result indicated that combination of Stel B and Imatinib couldshow better therapeutic efficacy,showing a growth inhibitory effect and inducesapoptosis in K562 cells.Conclusions: 1.The in vitro anti-leukemia activity of Stel B on human CML cells wasinvestigated.The activities and molecular mechanism of Stel B on CML cellsmight be regulate Stat5 and PI3K/Akt pathway.The antileukemia effect of Stel Bon CML has fistly reported to date.2.Combination with Imatinib at ratio of IC50 Stel B : 5 × IC50 Imatinib led to synergismin the antiproliferative effect and apoptosis-inducing effect.These results willprovide guide and theory for CML therapy alone or together with Imatinib.