| Research background and purpose.Acute myeloid leukemia(AML)is a type of hematological malignant that originates from myeloid hematopoietic stem cells and is highly heterogeneous in pathophysiological,clinical,cytogenetic and molecular biology,which is characterized by uncontrolled cloning proliferation of myeloid derived blasts leading to impaired normal hematopoietic function,causing severe infection,anemia,bleeding,and even bone marrow failure.AML is more common in middle-aged and elderly people,with an average age of 68 years.Although our research on AML has made new progress in recent decades and more and more new drugs are used in clinical practice,the overall prognosis value of patients is still poor,with the five years OS is about 20-25%.Therefore,to further study the pathogenesis and find a therapeutic drug become the focus of the current treatment of AML.Solasonine is a steroidal alkaloid derived from the traditional Chinese medicine Solanum nigrum L.,which has a good anti-tumor effect,especially for solid tumors,but there are few studies on hematological malignancies.Therefore,this research intends to explore the anti-AML effect of solasonine and potential therapeutic targets in vivo and in vitro,so as to provide theoretical basis and reference value for the treatment of AML.Research MethodsIn vitro experiment:Firstly,different concentrations of solasonine intervened in seven acute leukemia cell lines(THP-1,MV4-11,NB-4,HL-60,HEL,Raji and Jurkat)for 24 hours and the CCK-8 assay detected its inhibitory effect and calculated the IC50 value,which confirm that the most sensitive cell lines are THP-1 and MV4-11.Subsequently,the THP-1 and MV4-11 cell lines were intervened with solasonine for 24 h and 48 h.The inhibitory effect was tested by CCK-8 assay and the inhibition rate was calculated.Next-Generation RNA sequencing was used to detect the effects of solasonine on THP-1 cell gene expression,KEGG pathway enrichment and GO function enrichment.Then,Experiments of flow cytometry,Western Blotting and TUNEL were used to detect the effects of different concentrations of solasonine on the apoptosis of THP-1 and MV4-11 cell lines;Flow cytometry and Western Blotting experiments were used to detect the effects of difference concentration of solasonine on the cell cycle of THP-1 and MV4-11 cell lines.Finally,the effects of solasonine on AMPK/FOXO3A pathway related mRNA,protein expression and subcellular localization were detected by qRT-PCR,Western Blotting and immunofluorescence experiments.The AMPK inhibitor Compound C was used to interfere with THP-1 and MV4-11 cells pretreated with solasonine.Flow cytometry and Western Blotting were used to detect whether solasonine induces cycle arrest and apoptosis through AMPK/FOXO3A pathway.In vivo experiment:1.This study established a subcutaneous xenograft model of THP-1 cells in nude mice and received intraperitoneal injections of different concentrations solasonine to observe the growth inhibition to tumors.Western Blotting and immunohistochemical experiments were performed to observe whether the inhibitory effect of solasonine on subcutaneous tumors were through cycle arrest and apoptosis,and to preliminarily explore the correlation with AMPK/FOXO3 A axis.2.This study established zebrafish xenograft model of THP-1 cells and selected the method of immersion administration to explore the maximum safe dose of solasonine and to observe the inhibitory effect of solasonine on tumor cell growth in zebrafish xenograft models,and its effect on survival time.Research ResultsIn vitro experiment results:1.Solasonine inhibited the proliferation of acute leukemia cell lines,and particularly sensitive to AML cell lines(THP-1 and MV4-1).The inhibitory effect on AML cell lines was in a concentration and time dependent(P<0.05 or P<0.01).2.Solasonine induced AML cell lines apoptosis.Through flow cytometry experiment,it was detected that with the increase of solasonine concentration,the apoptosis rate was increased(P<0.05 or P<0.01).;through TUNEL experiment,it was observed that solasonine could stain the broken DNA double strands to red in the nucleus;Through the Western Blotting experiment,it was observed that solasonine could make the expression of pro-apoptotic protein Bax,cleaved-PARP,cleaved-caspased 9 and cleaved-caspased 3 increased,anti-apoptotic protein Bcl-2 decreased(P<0.05 or P<0.01).3.Solasonine arrested the AML cell cycle in G2/M phase.Flow cytometry assay found solasonine could increase the number of cells in the G2/M phase(P<0.05 or P<0.01);Western Blotting observed that with the increased of solasonine concentration,the expression of P-CDK1 increased and Cyclin B1 decreased(P<0.05 or P<0.01).4.Solasonine activated AMPK/FOXO3A pathway.Through Next-Generation RNA Sequencing,it was observed that when solasonine was treated with THP-1 cells,the KEGG pathway was mainly enriched in AMPK/FOXO3A;qRT-PCR and Western Blotting experiments were used to verify that solasonine can increase the AMPK and FOXO3A mRNA expression,AMPK protein expression and promote the transfer of FOXO3A from intracellular to nuclear(P<0.05 or P<0.01);immunofluorescence staining experiments once again confirmed that solasonine can make FOXO3A nuclear translocation.5.Solasonine induced AML cell lines apoptosis and cell cycle arrest through AMPK/FOXO3A pathway.The AMPK inhibitor Compound C(2 ?M)pretreated the THP-1 and MV4-11 cell lines for 2 h,and then solasonine(8 ?M)interfered with the cells for 24 h.It was observed by Western Blotting that Compound C reduced the protein expression of P-AMPK,inhibited solasonine-induced FOXO3A nuclear translocation,and at the same time reducesd the solasonine induced protein expression of Bax,cleaved-PARP,cleaved-caspased 9,and increases the protein expression of Bcl-2(P<0.05 or P<0.01).It was observed by flow cytometry that Compound C decreased the rate of apoptosis induced by solasonine and decreased the AML cells in G2/M stage induced by solasonine(P<0.05 or P<0.01).In vivo experiment results:1.Results of THP-1 cell Mouse Xenograft Model:With the concentration of solasonine increased,the size and weight of tumors decreased(P<0.05 or P<0.01).During the experiment,it was found that there was no obvious decline in the weight of the mouse(P>0.05).At the same time,Western Blotting and immunohistochemical staining experiments showed that solasonine increased the expression of Bax,P-CDK1 and P-AMPK and downregulation the expression of Bcl-2,Cyclin B1 and occurred the nuclear translocation of FOXO3A(P<0.05 or P<0.01).2.Results of THP-1 cell Zebrafish Xenograft model:Through the exploration of the maximum safe dose(MTD),we found that the maximum safe dose of solasonine to zebrafish was 10 ?M,and the cytarabine was 4 ?M.Both solasonine and the positive control drug cytarabine can prolong the survival time of Zebrafish Xenograft model,the survival rates were 60%and 75%,respectively(P<0.001).At the same time,solasonine reduced the fluorescence intensity of zebrafish xenograft models and the phenomenon of metastasis to the tail and whole body of zebrafish(P<0.05).Research Conclusions1.In vitro cell experiments verified the proliferation inhibitory effect of solasonine on AML cell lines of THP-1 and MV4-11,as well as the induction of apoptosis and cell cycle arrest;2.Through Next-Generation RNA Sequencing,it is preliminarily speculated that solasonine activated the AMPK/FOXO3A signaling pathway.Through inhibition of AMPK/FOXO3A,it is found that solasonine induces apoptosis and cycle arrest through the AMPK/FOXO3A signaling pathway;3.In vivo animal experiments verify the growth inhibitory effect of solasonine on subcutaneous transplanted tumors in nude mice and tumor-bearing zebrafish transplanted tumor cells. |
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