The Antitumor Effects On Acute Promyelocytic Leukemia NB4 Cells Of Selenium Chitosan And Its Possible Mechanisms

Over 95% of cases of Acute Promyelocytic Leukemia(APL) isassociated with a reciprocal translocation between chromsomes 15 and17 that produces a PML-RARαfusion gene which encodes PML-RARαfusionprotein. This fusion protein exhibits high activity of tyrosine kinase,which is considered essential for malignant transformation. So.Decreasing the fusion protein contents, dowing regulation its tyrosinekinase activity and blocking its possible signal pathways appear to bean attractive therapeutic strategy.selenium chitosan, a potent antioxidant and chemopreventive agent,which is synthesized with selenous acid and chitosan, is a kind oforganic selenium, here we demonstrated that selenium chitosan and sodiumselenite all could inhibite proliferation in a dose and time dependentmanner in a PML-RARαfusion protein positive APL cell line NB4. theirIC₅₀ is 103.7mg/L and 16.7umol/L respectively. The effect of seleniumchitosan is three times high than that of sodim selenite with the sameamount of selenium. The effects of selenium chitosan on the cell cyclephases and on inducing apotosis were studied by using Laser scan confoulmicroscope, flow cytometry. AO/EB fluorescent staining, transmissionelectron microscope and DNA fragmentation assay with gelelectrophoresis analysis. Its promoting differentiation effect wasmeasured by NBT reduced method and CD11b expression by using flowcytometry.An exposure of NB4 cells to 50—100mg/L of selenium chitosan for 24h produced a dose and time dependent increase in G0-G1 phase cells,several hallmarks of apoptosis including DNA laddering, chromatincondensation and fragmentation were also observed after the cells weretreated with selenium chitosan. Besides this, we also observed that50mg/h selenium chitosan could promote NB4 cells differentiation andselenium chitosan could increase GSH-PX activity,decrease SODactivity and MDA content.The synergistic antitumor effects of selenium chitosan with ADM onNB4 cells in vitro were studied with MTT method and Jin's formula wasused to analyse the effect of drug combination. It was found thatadminstration of selenium chitosan and ADM at the same time brought asynergistic effect but sequential administration of the drugs onlyproduced a undirectional synergistic effect.The PML-RARαfusion gene—encoded PML-NARαfusion protein whichpossesses high activity of tyrosine kinase, it is known to be a majorfactor for the cause and treatment of APL. In order to reveal themechanism by which selenium chitosan induces apoptosis and inhibitesproliferation. The effects of selenium chitosan on the expression ofPML-RARαfusion gene was studied by using RT-PCR method. The synthesisof PML-RARαfusion protein,cyclin D1,c-jun,MEK-1 and NF-KBwere studied by using western blot analysis using monoclonal antibodyagainst RARαprotein,cyclin D1 protein,c-junprotein, MEK-1 proteinand P65 protein respectively. The change of intracellular calcium,CD11b,cAMP,cGMP content were studied by using flow cytometry andradioimmunity method. It has been fond that selenium chitosanremarkably inhibited the expression of PML-RARαfusion protein in adose and time dependent manner in the same conditions above. But didnot affect the expression of PML-RARαfusion gene. selenium chitosan could inhibite the expression of cyclin D1,c-jun,MEK-1 and NF-KBprotein and down regualtion the Ras and NF-KB signal pathway. Moreover.selenium chitosan was able to stimulate the concentration ofintracellular calcium and CD11b, increase the cAMP content and decreasethe cGMP content in NB4 cells. These were possible mechanisms whichselenium chitosan could inhibite proliferation, induce apotosis andpromot differentiation.The results suggest that the inhibition of PML-RARαfusion proteinand intracellular cGMP. The stimulation of intracellular calcium andcAMP. the Ras and NF-KB signal pathways. Which are all involved inselenium chitosan mediated apoptotic cell death. The therapeuticpotential of selenium chitosan in human APL is worthy of furtherinvestigation.