| Objective Our previous studies have shown that matrine(MAT) play the role of alleviating all-trans retinoic acid (ATRA)resistance in acute promyelocytic leukemia(APL). The differentiation capacity of NB4 and NB4-R1 was enhanced with increase of MAT, and peaked at 0.1mmol/l.Therefore, we further investigate the role of matrine(MAT) inducing the differentiation of all-trans retinoic acid (ATRA)resistance in acute promyelocytic leukemia(APL)and its mechanism.Methods ATRA sensitive strain of APL (NB4) and resistant strain (NB4-R1) were used in this study. ①Cell morphologic changes were observed under optical micr- oscope.②The influence of MAT combines with ATRA on the differentiation of the two cell strains was observed by nitro blue tetrazolium chioride (NBT) test.③Flow Cytometry Analysis of Cell Surface Antigens. ④The acute promyelocytic leukemia fusion protein PML-RARa expression was detected by Western blot. ⑤The protein expression of topoisomerase Ⅱβ isoform was detected by immunohistochemical assay. ⑥Using the ELISA method to detect the contents of intracellular cAMP and intra-cellular protein kinase A (PKA) activity.Results ①Under the light microscope,we can see that 0.1 mmol/l matrine combined 1μmol/1 ATRA, can make both NB4 and NB4-R1 differentiate into mature cells. ② NBT positive rate shows that compared with the ATRA group,0.1 mmol/1 matrine collaborative 1μmol/1 retinoic acid can significantly increase the NBT-positive rate for the NB4-R1 cells (12% VS 41.33%). But to NB4 cells, there was no obvious change between the ATRA group and the ATRA combined with matrine group (34.67±2.05% VS 39.67± 2.49%). ③ CD11b positive percentage shows that compared with blank control group (23.455%±5.933%), separate matrine (27.62%±3.720%) or separate ATRA (32.776%± 6.610%) can partly promote the expression of cell surface CD11b, while matrine combined with ATRA can significantly increase the expression of CDllb for NB4-R1 cells (23.455± 5.933% VS 46.888±7.847%, P< 0.05). For NB4 cell lines, matrine alone can only partly increase the expression of cell surface CD11b (19.097±7.576%). Compared with the blank control group, both single drug ATRA group and matrine combined with ATRA group could obviously increase the expression of cell surface CDllb (80.383%±5.917% VS 79.058%± 80.383%, P< 0.01, P< 0.01), but there was no obvious change between the ATRA group and the ATRA combined with matrine group.④The acute promyelocytic leukemia(APL) fusion protein PML-RARa expression was detected by Western blot before and after using matrine.The results showed that the PML-RARa protein expression levels of the blank group of both NB4 cells and NB4-R1 cells are basically the same. ATRA single-drug group can make the PML-RARa protein expression decreased obviously on NB4 cells, but have no obvious effect on NB4-R1. Matrine combined with ATRA can make PML-RARa protein expression decreased obviously on both NB4 cells and NB4-R1 cells.⑤There was no significantly effect for the Topo Ⅱ β protein expression while using 0.1mmol/l matrine on NB4 and NB4-R1. Retinoic acid combined with matrine can reduce intracellular Topo Ⅱ β protein expression of the NB4-R1 cell line (P< 0.05).⑥Using O.lmmol/1 Matrine on NB4 and NB4-R1,there was no obvious effect for intracellular content of cAMP and PKA activity; Retinoic acid combined with matrine can increase NB4-R1 intracellular content of cMAP (P< 0.05), and PKA activity (P<0.01).Conclusion ① Matrine combined with all-trans retinoic acid can make NB4-R1 differentiate into mature cell.② Mechanism of Matrine in vitro inducing NB4-R1 cell differentiation may be as follows:promoting the PML-RARa protein degradation, reducing the Topo II beta expression, improving the cAMP content and PKA activity inside the cell, adjust the cAMP-PKA signaling pathways... |
PDF Full Text Request