| Objective:The aim of this study was to investigate the change of NB4-R1 cells to the sensitivity of Tamibarotene(Am80) and all-trans retinoic acid(ATRA) after inhibiting the signaling pathway of acute promyelocytic leukemia ATRA resistant NB4-R1 cells PI3 K / Akt. Then, to explore the relationship between PI3 K / Akt signaling pathway and acute promyelocytic leukemia cell line resistant NB4-R1 cells, Meanwhile, finding new ideas to overcome drug and provides new insight into the mechanism of APL. Methods:CAL-101 was added to the NB4- R1 cell at different drug concentration(3?mol/L?5?mol/L?7?mol/L?9?mol/L?12?mol/L) for 72 h and different action timeand( 24 h ? 48 h ? 72 h ? 96 h ? 120h) with 9?mol/L, real-time quantitative(Quantative Real-time PCR), Western blot detection the change of PI3 KCA, Akt1 gene and protein expression. NB4-R1 cells were divided into blank group, group solvent(ethanol, DMSO), ATAR group, ATRA + CAL-101 group, Am80 group, Am80 + CAL-101 group(Am80 and ATRA drug concentration of 30?mol / L, CAL-101 9?mol / L) and co-cultured 48 h, 96 h, 144 h with the cells. Observing wright's staining cell morphology, flow cytometry cell surface differentiation antigen CD11 b, CD13, CD16 expression, real-time quantitative(Quantative Real-time PCR) m RNA detection of PML-RAR? fusion gene expression levels. Result:1 ? CAL-101 effectively inhibited NB4-R1 cells PI3 KCA, gene and protein expression of Akt1, the degree of inhibition with increased concentration of CAL-101 was positively correlated(P <0.05).Group of 72 hours 9?mol / L CAL-101 inhibition PICKCA relative expression was(0.437 ± 0.025); then, 3?mol / L CAL-101 inhibition relative Akt1 gene expression rate was(0.525 ± 0.043). 9?mol / CAL-101 L for 72 h Akt1 gene relative expression was(0.114 ± 0.025), compared with the control group,Group of 9?mol / L group Akt1 protein expression was significantly reduced(0.195 ± 0.037 VS 1.672 ± 0.103, p <0.05).2?9?mol / L CAL-101 acts on NB4-R1 cell line at different times, PI3 KCA, decreased Akt1 gene and protein expression levels were positively correlated with the duration of action., Difference was not significant between PI3 KCA gene expression 24 h group and control group(P> 0.05), Compare with the control group, 48 h group was reduced significance(P <0.05); Akt1 gene relative expression of each drug group were significantly decreased compare with the control group(P <0.05). Compare with the control group,PI3 K p110? protein 24 h group and 48 h group had no difference(P> 0.05); Akt1 protein amount of each drug group compared with the control group decreased significantly(p <0.05). 24 h, 48 h, 72 h group Akt protein expression was(0.816 ± 0.059),(0.531 ± 0.022) and(0.311 ± 0.107).3?It is concluded that the ATAR group, ATAR+ CAL-101 group, Am80 group, Am80+ CAL-101 group, blank group and solvent 48 h group were no significant changes in the morphology of NB4-R1 cells by morphological observation. But,late promyelocyte cells could be seen in each drug group after 96 hours and after 144 hours later, the cells have been divided into mature in each group;with period of time,the cell differentiation and maturation is more obvious in CAL-101+ATRA and CAL-101+Am80 group.4 ? Cell surface differentiation antigen was gradually decreased with the prolongation of the time of drug action, and the expression of CD11 b and CD16 increased gradually,but the expression of CD13 decreased gradually.Forty-eight hours later, there was no obvious expression of CD11 b in each drug group and there was no significant difference compared with blank group( P > 0.05);The positive expression rate of CD13 in each drug group was lower than in the control group(P < 0.05),but there was no significant difference between each drug group(P>0.05);in the same,there was no obvious positive expression of CD16 in each drug group(P>0.05).Compared with the blank group, the positive expression rate of CD11 b in each drug group was significantly higher after 96 hours later,(P < 0.05), and the expression rate of the two drug group was higher than that of single drug group(P < 0.05);The positive expression rate of CD13 in each drug group was close to zero, and the expression rate of CAL-101+Am80 group was higher than that of Am80 group and also as CAL-101+ATRA group compared with ATRA group(P < 0.05);Also, compared with the blank group,the positive expression rate of CD16 in each drug group was significantly higher after 96 hours later,and the expression rate of the two drug group was higher than that of single drug group(P < 0.05).The positive expression rate of CD11 b in each drug group was close to the maximum after 144 hours later, and the Am80 group was higher than that of the ATRA group and the positive expression rate of CD11 b in the Am80+CAL-101 group was higher than the Am80,the ATRA+CAL-101 and the ATRA group(P < 0.05).And there was no significant difference between the other groups( P > 0.05).The positive expression rate of CD13 was zero, and there was no significant difference between the each other groups(P > 0.05);The positive expression rate of CD16 were higher in the CAL-101+Am80 group and Am80 group than that in CAL-101+ATRA and ATRA groups(P < 0.05).5?The expression level of PML-RAR alpha gene decreased with the time of drug action, the most significant was seen in double drug groups.The relative expression of PML-RAR alpha gene in each drug group was decreased compared with the blank group after 48 hours later( P < 0.05),Compared with ATRA group,Am80 group and the ATRA group respectively, the Am80 group,CAL-101+AM80 and group CAL-101+ATRA were lower in Gene expression(P < 0.05).This phenomenon is also found in 96 hours later and 144 hours later.Conclusion:1. CAL- 101 can inhibit the PI3K/Akt pathway, decrease the expression of PI3 K and Akt gene, and the effect intensity was time-dependent and positively related to its concentration.2. Am80 could induce NB4-R1 differentiation and was better than ATRA.3. After inhibiting the PI3 K signaling pathway, the susceptibility of NB4-R1 cell line to Am80 and ATRA enhanced and the expression of cell surface differentiation antigen increased.4. After inhibiting the PI3 K signaling pathway, Am80 and ATRA induced the expression of PML-RAR? gene further reduced?5.The activation of PI3K/Akt signaling pathway inhibition of NB4- R1 cell differentiation. |
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