Screening Therapeutic Drugs For Adult T-cell Leukemia And The Mechanism Study

BackgroundAdult T-cell leukemia(ATL)is a malignant tumor of peripheral T-lymphocytes caused by human T-cell leukemia virus type I(HTLV-1).Compared with the invasive non-Hodgkin’s lymphoma,the prognosis is poor.Currently,there are a few treatment methods including bone marrow transplantation,immunotherapy,and chemotherapy.However,these methods have many drawbacks,such as high costs of treatments and serious complications,which have limited the widespread use of these treatment methods.Therefore,it is necessary to develop some new therapeutic drugs with targeted,low toxicity and high efficiency.ObjectiveThe purpose of this study is to screen the active components that can inhibit the proliferation of ATL cells in vitro and explore the possible molecular mechanism.It will provide theoretical and experimental basis for the prevention and treatment of ATL in clinic,and will have a broad prospect for the development of new drug targets.MethodsOn the one hand,CCK-8 assay was used to screen active compounds that come from different traditional Chinese medicines and naphthalimid derivatives which can inhibit the proliferation of ATL cells.On the other hand,the possible molecular mechanism of these active compounds was studied by different methodologies.First of all,Hoechst 33258 fluorescent staining was used to test morphological changes of cells treated by these compounds.And then the flow cytometry was applied to detect the cell cycle distribution of these cells and to find out the initial mechanism.Transmission electron microscopy(TEM)was also used to observe the ultrastructure of these cells.Secondly,a large number of technologies of molecular biology were applied for further studying the mechanism of these compounds.Western blot assay was used to detect the changes of Bax,Bcl-2,caspase-3 and other related proteins in cells.The interactions between drugs and Pan-caspase inhibitors were implemented to measure the changes in apoptotic cells.RT-PCR,cell transfection experiments and siRNA screening were used to detect the effects of drugs on gene expression of HTLV-1.Results1.According to the results of CCK-8 assay,andrographolide,2f,and 4pq were chosen for further study of the mechanism about inhibition the proliferation of ATL cells.After staining with Hoechst 33258,the nucleus of the control group was light blue,and the cells of experimental groups gradually turned bright blue,which indicated that the drug could induce apoptosis in a certain concentration.2.The data of flow cytometry showed that andrographolide could induce the apoptosis of ATL-ED,TL-om1,ATL-2 cells,and it blocked the G2 and M phase of ATL-ED cells in varying degrees and showed the sub diploid peak.While 2f and 4pq can induce apoptosis of ATL-2 cells,but G2 and M arrest of ATL-ED and TL-om1.3.The photos of TEM showed that the cells of the control group were oval in shape,intact organelles,obvious nucleoli,and uniform chromatin.After the treatment of andrographolide,chromatin condensation,and many vacuoles appeared in cytoplasm and mitochondria swelling.After treated with 2f and 4pq,the nuclear chromatin was agglomerated and the mitochondria blackened.4.From the results of Western blot,we found that andrographolide caused the shearing of Caspase-3,Caspase-9.In addition,the expression of PARP,Bax,Bad,and Cyt-c had been modulated by up-regulating,while Bcl-2 and Cyclin B were downregulating.When ATL-ED cells were pretreated with Pan-caspase inhibitor Z-VAD-FMK and then co-incubated with andrographolide,the apoptotic rate decreased.However,2f and 4pq reduced the expression of topoisomerase Ⅱ.5.Results of RT-PCR showed that andrographolide and 4pq significantly inhibited the expression of the HBZ gene in ATL-ED cells.The virus-encoded protein Tax was transferred into the cells without the Tax gene and then treated with andrographolide resulting in the proliferation of the cells to a certain extent.After knocking down the expression of Tax,the activity of cells decreased after the treatment with drugs.ConclusionsIn conclusion,andrographolide(a Chinese medicine monomer),2f and 4pq(naphthalimide derivatives)were selected to inhibit the proliferation of ATL cells and their mechanisms were also studied.Andrographolide mainly induced apoptosis of ATL cells through the Caspase dependent pathway,which is a mainly mitochondrial pathway and it can also induce DNA damage.Besides it also inhibited the proliferation of ATL by downregulating the expression of the HBZ gene.While 2f and 4pq were topoisomerase inhibitors,which inhibited the expression of topoisomerase II,thus causing the double-strand breaking of the genomic DNA of the cells to cause cell cycle arresting,thus affecting the mitotic process of ATL cells.