Evodiamine Induces Apoptosis Of Leukemia Cell Line K562 Via Modulation Of TRIB2/AKT Pathway

Naturally derived compounds are extremely important for finding new clinical drugs,especially anti-cancer drugs.More than half of cancer chemotherapy drugs are natural compounds and their derivatives.Evodiamine(EVO),isolated from Evodia,is a famous alkaloid with a quinazoline skeleton.EVO is traditionally used to treat headaches,amenorrhea,postpartum hemorrhage and gastrointestinal diseases.The biological activity of EVO has been extensively studied,including anti-obesity,anti-inflammatory,anti-atherosclerosis,neuroprotection,and anti-gastrointestinal motility and anti-proliferative effect.Among them,the multi-molecular targeted anti-proliferative activity of EVO has attracted the most attention.In a variety of cancers,including lung cancer,liver cancer,colon cancer,breast cancer,ovarian cancer,cervical cancer and other cancers,EVO has an excellent anti-tumor effect active.In leukemia,EVO can play an anti-tumor effect by inducing cell apoptosis and blocking cell cycle progression.In fact,AKT plays an extremely important role in the process of EVO inhibiting a variety of cancers Akt participates in the regulation of many important biological processes.It plays a key role in inhibiting apoptosis and promoting proliferation in cells by influencing a variety of downstream effector molecules.It is closely related to the occurrence and development of many human tumors.Therefore,further elucidating the specific mechanism of EVO regulating AKT activity is of great significance to the clinical anti-tumor application of EVO.Various growth factors and cytokines can activate AKT through the activation of the homologous receptor tyrosine kinase,cytokine receptor,and lipid kinase PI3K.PI3 K converts PIP2 to PIP3.PIP3 can bind to the PH domain of AKT and then transfer to the cell membrane,where it is phosphorylated by PDK1 at Thr308,and phosphorylated by PDK2 on Ser473 to fully activate AKT.In addition,there are also reports that CDK2 can phosphorylate AKT at Ser477 and Thr479,and cause AKT to enter the nucleus to function.In short,for AKT,its enzyme activity is mainly determined by Thr308 and Ser473.The Tribbles pseudokinase protein family is composed of TRIB1,TRIB2 and TRIB2,and is an important component of eukaryotic pseudoenzymes.As signal transduction mediators and scaffold proteins,TRIBs can cause changes in protein stability and activity,thereby affecting a variety of tumor cell processes,such as proliferation,differentiation,cell cycle,and apoptosis.For example,TRIB1 can directly bind to the ubiquitin ligase COP1 of the tumor suppressor protein C/EBPα,promote the degradation of C/EBPα,and accelerate the occurrence and development of acute myeloid leukemia.TRIB2 is also an oncogenic protein whose transcription is regulated by Wnt/TCF,which can inhibit the activity of FOXO protein and affect the occurrence and development of melanoma and leukemia.More importantly,TRIB2 has been reported to form a protein complex with AKT to promote the hyperphosphorylation of Ser473 and ultimately induce drug resistance in tumor cells.Clinical studies have also shown that the expression level of TRIB2 is related to the prognosis of patients.Numerous studies have reported that EVO can induce apoptosis of tumor cells by inhibiting the PI3K/AKT pathway,but whether it is possible in leukemia cells to induce cell apoptosis by inhibiting the AKT pathway,and the specific role of TRIB2 in EVO’s inhibition of the AKT signaling pathway remains unclear.Therefore,this study will explore the effect of EVO on leukemia cell apoptosis and the specific role of AKT in vivo and in vitro,and will preliminarily analyze the role of TRIB2 in EVO’s regulation of AKT activity.Objective: In the first part of this study,the human leukemia(Leukemia)cell line K562 was used as the research object to detect the effect of EVO on the growth of K562 cells in the subcutaneous tumor model of nude mice,and to explore the effect of EVO on the apoptosis of K562 cells in vitro.The second part is to explore the potential mechanism of apoptosis of K562 cells induced by EVO.Methods:The first part: the tumor growth model of BALB/c nude mice was established by subcutaneous injection of K562 cells.Mice were treated with different doses of EVO.the body weight and tumor volume of mice were recorded.At the end of the experiment,the tumor weight was measured and the apoptosis level of tumor cells was detected by Tunel staining.In the in vitro experiment,CCK-8 assay was used to detect the effect of different concentrations of EVO on the proliferation of K562 cells,and Flow cytometry was used to detect the effect of EVO on K562 cell apoptosis.The second part: the expression of TRIB2、AKT、p-AKT、IκBα、p-IκBα、NF-κB p65、p-NF-κB p65、Caspase 3、Bcl-2 in K562 cells was detected by Western Blot,and the m RNA level of TRIB2 was detected by RT-PCR.K562 cells were pretreated with AKT activator SC79,apoptosis was detected by Flow cytometry,the expression of TRIB2,AKT,p-AKT,IκBα,NF-κB p65,NF-κB p65,Caspase 3,Bcl-2 was detected Western Blot.The expression of TRIB2 transcription factor TCF4 was inhibited by siRNA,apoptosis was detected by Flow cytometry,the expression of TRIB2,AKT,p-AKT,IκBα,NF-κB p65,NF-κB p65 was detected by Western Blot.K562 cells pretreated with Wnt activator SKL2001,Western Blot was used to detect the expression of TRIB2,AKT and p-AKT.Results: The first part1.The body weight records of mice showed that there was no significant difference in body weight among the control group,low dose EVO group(10 mg/Kg)and high dose EVO group(20 mg/Kg).2.The tumor volume record revealed that the tumor volume of the EVO group was significantly smaller than that of the control group,and the tumor volume of the high dose EVO group was smaller low dose EVO group.3.The tumor weight record showed that the tumor weight in the low dose group and the high dose group was significantly lower than that in the control group.4.The results of Tunel staining showed that both low and high doses of EVO could induce apoptosis of K562 cells in nude mice.5.The results of CCK-8 showed that EVO could significantly inhibit the growth of K562 cells in a concentration-dependent manner.6.Flow cytometry showed that EVO could induce apoptosis of K562 cells in a concentration-dependent manner.The second part1.Western Blot results showed that EVO could significantly inhibit the expression of Trib2、p-AKT、p-IκBα、p-NF-κB p65、Bcl-2 in K562 cells,but did not affect the expression of AKT、IκBα、NF-κB p65.The levels of Casepase3 were significantly up-regulated.2.The results of RT-PCR showed that EVO could significantly inhibit the m RNA level of TRIB2 in a concentration-dependent manner.3.The results of flow cytometry after pretreatment with AKT activator SC79 showed that SC79 could significantly inhibit the apoptosis of K562 cells induced by EVO.4.After cells were pretreated with SC79,an AKT activator,Western Blot showed that EVO alone could significantly inhibit TRIB2/AKT signal pathway,while in EVO combined with SC79 group,the expression of TRIB2 did not change,but the inhibitory effect of EVO on AKT signal pathway was significantly decreased.5.After the expression of TRIB2 transcription factor TCF4 was inhibited by siRNA,the Flow cytometry showed that the apoptosis level of K562 cells in EVO group,siTCF4 group and combined group was significantly higher than that in control group.6.After the expression of TRIB2 transcription factor TCF4 was inhibited by siRNA,Western Blot results showed that although EVO could not inhibit the expression of TCF4 protein,EVO group,siTCF4 group and combination group could effectively inhibit TRIB2/AKT signal pathway.7.After cells were pretreated with SKL2001,a Wnt activator,Western Blot showed that SKL2001 could significantly up-regulate the expression of TRIB2 and activate AKT signal pathway,while EVO could inhibit the up-regulation of TRIB2 expression by SKL2001 and the activation of AKT signal pathway.Conclusion1.In the tumor formation experiment of nude mice,different doses of EVO could inhibit the growth of tumor volume and tumor weight,and up-regulate the level of apoptosis without affecting the body weight of mice.In vitro,EVO could significantly inhibit the growth and induce apoptosis of K562 cells.2.EVO can induce apoptosis by inhibiting TRIB2/AKT signal pathway and down-regulating the transcriptional activity of NF-κB p65.SC79,an activator of AKT,can weaken the inhibitory effect of EVO on AKT signal pathway and reverse its ability to induce apoptosis of K562 cells.While inhibiting the expression of TCF4,a transcription factor of TRIB2,can also inhibit AKT signal pathway and induce apoptosis of K562 cells.EVO could also inhibit the up-regulation of TRIB2 expression and AKT activation induced by Wnt activator SKL2001.