Investigation Of Tamibarotene On Inducing Acute Promyelocytic Leukemia Cell Line NB4Differentiation

Objective:Tamibarotene (Am80) is a new synthetic retinoic acid derivative. This studyaimed to investigate the influence of NB4cell proliferation responsed to Am80andAm80-induced NB4cell differentiation, and further explored the effect mechanism ofAm80.Methods:Four groups were classified，Am80as the experimental group,all trans retinoicacid as the positive control group, adding the same concentration of ethanol as anegative control group, adding nothing drugs as the control group. Cell morphologywas observed by Wright stain. The cell growth was tested by CCK-8assay. At thesame time, the expression of cell surface differentiation antigen and apoptosis wasdetected using flow cytometry and the expression of retinoid acid induced gene-Gwas tested by real-time PCR.Results:1. The NB4cells trended to differentiate to mature granulocytes after treatedwith various concentration of Am80and these changes were in a dose-dependentmanner. When the concentration was below0.1μmol/L, there were no significantdifferences at cell morphology (0.001μmol/L and0.01μmol/L) during the96h. A fewdifferentiated cells were observed at0.1μmol/L, substantial myelo of metagranuloc-ytes were observed at1μmol/L group and many segmented cells were observedbesides metagranulocytes in10μmol/L Am80. However, the cells treated with drugswere ruptured at10μmol/L after96h. Cell differentiation became earlier and muchmore significant in1μmol/L Am80group than1μmol/L ATRA group, while therewere no no significant difference between the solvent and empty control group.Thecell proliferation inhibition ratios of various concentrations Am80were (11.87±1.33)%,(15.90±0.94)%,(20.50±1.02)%,(26.78±1.10)%,(32.91±0.32)%respectively (P<0.05) after72h, which was in a dose-dependent manner.2. The cell proliferation inhibition ratios of various concentrations Am80 were(11.87±1.33)%,(15.90±0.94)%,(20.50±1.02)%,(26.78±1.10)%,(32.91±0.32)%respectively (P <0.05) after72h, which was in a dose-dependent manner.3. The expression rates of CD11b(+) cells were (75.50±0.66)%,(75.03±1.51)%in Am80and ATRA group after48h(P=0.616),(79.35±0.77)%vs (76.34±0.73)%after96h (P<0.005) and (90.82±1.63)%vs (79.35±1.22)%after144h day(P=0.000) in the two groups.The CD16expression each group was<10%after48h, while(27.83±1.32)%vs(14.27±1.20)%after96h(P<0.005) and(92.47±0.81)%vs(27.83±1.32)%after144h(P<0.005) in Am80and ATRA group.After144h the CD13expression decreased to (6.45±0.69)%from the(40.30±1.22)%of48h in Am80group, but(25.68±1.07)%from the(28.85±1.08)%in ATRAgroup (P=0.000).Am80could downregulate CD13expression more significantly thanATRA.4. The apoptosis rate induced by the Am80was (36.51±1.33)%after48h、(36.05±1.43)%after96h, which was significantly higher than the the ATRA Group((23.76±1.79)%of48h、(33.32±1.08)%of96h)(P＝0.00, P＝0.03). However, theapoptosis rate of ATRA group after144h was higher than the Am80group((61.28±1.75)%vs (43.32±1.44)%,p=0.000), which might indicate that ATRAinduced earlier the apoptosis.5. The RT-PCR results showed that RIG-G expression of Am80and ATRAgroups was higher than the empty control group(p=0.028,0.008), while no differencebetween the Am80and ATRA groups (P＝0.353).Conclusion：1. Am80could promote the NB4cell to different from the inchoate cell tometamyelocyte, and this effect of Am80became earlier than ATRA.2. Am80could inhibit proliferation of NB4cell.3. Am80had a strong impact on inducing NB4cell differentiation. Theexpression of CD11b and CD16of NB4were upregulated after treated with Am80,the CD13was decreased. Compared with the ATRA, Am80was an efficient induced-differentiation agent.4. Am80could induce earlier apoptosis in contrast to the ATRA. 5. The potential mechanism of Am80in cell proliferation, differentiation andapoptosis was that it could induce the expression of RIG-G.