Study Of The Effect Of Z-10 Derivative Z-39 On Anti-APL And The Effect Of Myrotoxin A On Inducing Leukemia Cell Apoptosis

Acute promyelocytic leukemia(APL)is a unique subtype of acute leukemia.The major pathogenesis mechanism of APL is the formation of PML-RARa fusion gene from chromosomal translocation between chromosomes 15(PML)and 17(RARa).Retinoid X receptor alpha(RXRa)forms heterotetramer with PML-RARa via interacting with the RARa moiety.The heterotetramer is then binds to DNA to exhibit the oncogenic effect.From forming heterotetramer,RXR stabilizes PML-RAR and promotes DNA binding.Clinical drugs ATRA and Arsenic cure most APL patients through inducing PML-RARa transactivation and/or degradation.However,there are still som major issues such as the adverse side effects and drug resistance in APL treatment.Thus,it is extremely important to find new APL therapeutic approaches and drugs.In the previous study,we first identified that nitrostyrene derivative Z-10 is a nitro ligand of RXRa.Z-10 induces the degradation of PML-RARa in NB4 cells-an APL cell line,and triggers NB4 cell apoptosis.Z-10 also induces the degradation of PML-RARa and apoptosis in NB4-LR1 and NB4-LR2 cells that are two ATRA resistant APL cell lines.In addition,we unraveled that Z-10 inhibits the interaction of PML-RARa and RXRa to promote PML-RARa degradation.In order to find less toxic and more effective compounds,we designed and synthesized a number of nitrostyrene derivatives,of which Z-39 and Z-40 were quite different from Z-10 in structure.Z-39 and Z-40 possess a biphenyl moiety while Z-10 has a naphthalene moiety.In our mammalian one-hybrid assay,we found that Z-3 9 but not Z-40 dose-and time-dependently induced Gal4/DBD-RXR?/LBD transactivation.Our result also showed that Z-39 was more potent than Z-10 on inducing Gal4/DBD-RXRa/LBD transactivation.Z-39 failed to induce the transactivation of Gal4/DBD-RAR?/LBD and Gal4/DBD-ERa/LBD,indicating its RXRa selectivity.The Kd value of Z-39 binding to RXRaLBD determined by our fluorescent titration assay was?14.87 ?M,which was of the same order of magnitude as Z-10.Similar to Z-10,Z-39 induced PML-RARa degradation in both time-and dose-dependent manners in NB4 cells.The potency of Z-39 in inducing PML-RARa degradation was higher than Z-10.In Cos-7 cells,overexpressed PML-RARa and RXRa formed complex,which was largely disrupted by Z-39.This suggested that Z-39 induced PML-RARa degradation through inhibiting the interaction of PML-RARa and RXRa.Z-39 did not induce the degradation of RARa or inhibiting the interaction of RARa and RXRa.Consistently,Z-39 was more potent than Z-10 in apoptotic induction of NB4 cells.Interestingly,we found that Z-39 strongly induced RXRa phosphorylation,which was partially inhibited by AMPK inhibitor.However,the function of Z-39-induced RXRa phosphorylation and the underlying mechanism as well as its correlation with PML-RARa degradation need further investigation.In all,our study confirmed our previously identified efficacy and the mechanism of Z-10 inducing PML-RARa degradation and NB4 cell apoptosis.Moreover,we identified an optimized Z-10 derivative Z-39 with distinct structure.Thus,our study provided a novel direction for nitrostyrene optimization.Leukemia is a type of malignant tumor with abnormal hematopoiesis,and the incidence rate ranks sixth among all the tumors.According to the pathogenesis,leukemia can be divided into acute leukemia and chronic leukemia.Acute leukemia is characterized by a rapid increase of immature blood cells.Patient often dies from massive hemorrhage.Chronic leukemia is of a slow onset and a long progression course.After entering the critical phase,chronic leukemia develops rapidly and can easily lead to death.At present,leukemia is mainly managed by chemotherapy,stem cell transplant,immunotherapy and radiation therapy.Although some leukemia such as APL and CML can be well treated by targeted drugs,there are still some problems need to be solved,such as drug resistance and severe side effects.Moreover,most of leukemia do not have effective treatments.Thus,discovering new therapeutic strategies and new drugs for leukemia treatment is extremely important.A-S-4-i-4(Myrotoxin A)was isolated from an endophyte of the herb Ajuga decumbens by Dr.Lin' group.In our study,we found that A-S-4-i-4 can inhibit more than 800%proliferation of some leukemic cells at extremely low concentrations(?1nM),while it required relatively higher concentrations to inhibit only 50%proliferation of some adherent cells.Thus,A-S-4-i-4 was more sensitive to suspension than adherent cells.We also found that A-S-4-i-4 strongly induced apoptosis and activated caspase proteins in some leukemia cell lines including of Jurkat,MOLT-4,NB4,and U937.At the same condition,A-S-4-i-4 had no obvious effect on the adherent cells.This further verified that A-S-4-i-4 was more sensitive to suspension cells than adherent cells.We also found that A-S-4-i-4 activated caspase-mediated apoptotic pathway in Jurkat and MOLT-4 cells in both dose-and time-dependent manners.Z-VAD-FMK largely blocked A-S-4-i-4-induced activation of caspase-3,accompanying by the inhibition of apoptosis.This indicated that A-S-4-i-4 induced leukemia cell apoptosis through the caspase-mediated pathway.Although Trichothecenes are toxic,they may turn into effective drugs in curing leukemia through reasonable therapeutic strategies and further optimization.Together,our study unraveled that trichothecenes selectively induced leukemia cell apoptosis,which warranted the further investigation of its apoptotic induction mechanisms and exploration of its future clinical application in leukemia.