| Background:Acute promyelocytic leukemia(Acute promyelocytic leukemia,APL)is a special subtype of acute myeloid leukemia(Acute myeloid leukemia,AML),accounting for about 15%-20%of AML.It is the only AML that can be really cured so far.At present,the standardized treatment of APL mainly includes strong supportive managment,inducing differentiation and promoting apoptosis,in which arsenic trioxide(Arsenic trioxide,ATO)and all-trans retinoic acid(All trans retinoicacid,ATRA)have become the backbone drugs for the first-line treatment of APL patients.ATO has excellent efficacy in the initial diagnosis and recurrent APL,and occupies a very important position in all stages of treatment,but ATO itself also has drug resistance,especially its own toxicity.Previous work in this study group showed that serum selenium levels were significantly reduced in patients with acute promyelocytic leukemia,and with the further decrease of arsenic acid after the application,it was also lower than normal after achieving complete remission,suggesting that trace element selenium deficiency is of concern in APL patients.Existing studies have shown that selenium and selenium compounds can play a dual role in anti-tumor and organ protection in anti-tumor treatment,the role in APL is related to concentration,high concentrations can play a role in promoting apoptosis,at low concentrations,simultaneous use with arsenic can play an antagonistic effect,and sodium selenite has dose-dependent toxicity to normal tissues,this study attempts to explore whether the efficacy of ATO against APL after pretreatment of sodium selenite is affected.In order to provide theoretical reference value for selenium supplementation in patients with selenium deficiency in the interval between anti-APL treatment.Objective:This study aims to explore the effect of sodium selenite pretreatment on the efficacy of ATO against APL,and explore its potential mechanism,so as to provide new therapeutic ideas and strategies for selenium supplementation in follow-up in vivo studies and clinical treatment of APL.Methods:1、CCK-8 detected the effect of sequential ATO on the proliferation of NB4 cells after sodium selenite pretreatment:NB4 cells were pretreated with 0.1 μM,0.5μM,1.0μM,2.0μM,4.0μM,and 10.0μM sodium selenite for 48h,and 7.5μM ATO was incubated sequentially for 24h,and the effect of sequential ATO on NB4 cells was detected by CCK-8 after pretreatment with different sodium selenite concentrations.NB4 cells were pretreated with 0.5μM sodium selenite for 24h,48h,and 72h,respectively,and 7.5μM ATO was incubated sequentially for 24h,and the effect of sequential ATO on the proliferation of NB4 cells was detected by CCK-8 with different sodium selenite pretreatment times.2、Flow cytometry detection of NB4 cell death induced by sequential ATO after sodium selenite pretreatment:NB4 cells were pretreated with 0.5μM sodium selenite for 72 h and then sequentially administered 7.5 μM ATO for 24 h,and NB4 blank control group(NB4)was set up,72h group(72h 0.5μM Se)was treated with 0.5μM sodium selenite alone,24h group(24h 7.5μM As)was treated with 7.5μM ATO alone.Cells were washed once with PBS and cell counted 2X105/100 μl,and NB4 cell death was induced by sequential ATO after sodium selenite pretreatment with Annexin V APC/7-AAD.3、Western-Blot detected the expression of exogenous apoptosis-related proteins in NB4 cells by sequential ATO after sodium selenite pretreatment:NB4 cells were pretreated with 0.5μM sodium selenite for 72h,and 7.5μM ATO was incubated sequentially for 24h,and NB4 blank control group(NB4)was set up,0.5μM sodium selenite alone was treated for 72h(72h 0.5μM Se),and 7.5μM ATO was treated for 24h(24h 7.5μM As)alone.NB4 cells were collected,the proteins of the cells were extracted,and the expression levels of Fas,Caspase 3,Cleaved-Caspase 3,Caspase 8,and Cleaved-Caspase 8 proteins were detected by WB.4、qPCR detected the expression of pyroptosis GSDME mRNA in NB4 cells by sequential ATO after sodium selenite pretreatment:NB4 cells were pretreated with 0.5μM sodium selenite for 72 h,and 7.5μM ATO was incubated sequentially for 24 h,and NB4 blank control group(NB4)was set up,0.5μM sodium selenite alone was treated for 72 h(72h 0.5μM Se),and 7.5μM ATO alone was treated for 24h(24h 7.5μM As).qPCR was used to detect the expression of pyroptosis-related mRNA GSDME in NB4 cells by sequential ATO after sodium selenite pretreatment.Results:1、The results of CCK-8 showed that 0.1μM,0.5μM,1.0μM,2.0μM,4.0μM,10.0μM sodium selenite pretreatment,and sequential ATO treatment did not affect or even aggravate the cytostatic effect of ATO on NB4 cells(P<0.01,P<0.001),compared with the control group(7.5μM ATO),which was statistically significant.0.5μM sodium selenite was pretreated for 24h,48h and 72h,respectively,and the results showed that the inhibition of NB4 cells by ATO was exacerbated by sodium selenite pretreatment time-dependently,and the(P<0.001)was statistically significant compared with the control group(7.5μM ATO).2、The flow cytometry results showed that sequential ATO enhancement of sodium selenite pretreatment induced apoptosis on NB4 cells,and sodium selenite-induced late apoptosis accounted for a large proportion of total apoptosis,suggesting that sodium selenite-induced NB4 cell death had other diaphragmal death methods,including pyroptosis,necrosis,middle and late apoptosis,etc.,24h 7.5μM As VS 72h 0.5μM Se+24h 7.5μMAs,*,P<0.05,***,P<0.001,which was statistically significant..3、The results of Werstern-Blot showed that the sequential ATO after sodium selenite pretreatment significantly upregulated the expression of exogenous apoptosis pathway-related proteins Fas,Cleaved-Caspase 3 and Cleaved-Caspase 8 proteins in NB4 cells,activating the Fas/Caspase 8 signaling pathway,24h 7.5μM As VS 72h 0.5μM Se+24h 7.5μM As,P<0.05,P<0.01,which was statistically significant.4、The qPCR results showed that although the expression of pyroptosis GSDME mRNA in NB4 cells after pretreatment was lower than that of pyroptosis GSDME mRNA in NB4 cells with sodium selenite alone,it was more able to promote the expression of pyroptosis GSDME mRNA in NB4 cells than ATO alone,,NB4 VS 72h 0.5μM Se,P<0.001,72h 0.5μM Se VS 72h 0.5μM Se+24h 7.5μM As,P<0.05,24h 7.5μM As VS 72h 0.5μM Se+24h 7.5μM As,P<0.001,statistically significant.Conclusion:1、There was a synergistic effect between the pretreatment of sodium selenite and arsenic on NB4 cells.2、This study shows that the mechanism of synergistic effect between sodium selenite and ATO on NB4 cells mainly stems from the exogenous pathway induced apoptosis induced by Fas/Caspase 8 and possibly Caspase 3/GSDME induced pyroptosis. |
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