| 1 ObjectiveIn order to study the differentiation molecular mechanism of acute promyelocytic leukemia (APL) cells induced by TanⅡA in vitro, and to provide application rationales of TanⅡA to APL patient treatment, the differentiation expression in aspects of cell biology and molecular biology induced by TanⅡA were compared with that induced by all-trans retinoid acid (ATRA) and arsenic trioxide (As2O3).2 Materials and methods2.1 Cell cultureLogarithmically growing NB4 cell were cultured at a density of 4×105/ml in RPMI 1640 medium, including Tan ]I A at different concentration of 1.7μmol/L(0.5μg/ml),2.25μmol/L(0.75μg/ml) and 3.4μmol/L(1.0μg/ml), As2O3 at concentration of 0.1μmol/L and 1.0μmol/L, ATRA at concentration of 1.0μmol/L or 0.01%DMSO, respectively. These cells were harvested at 12, 24, 48, 72, 96, 120, 144, and 158 hours respectively for following examinations.2.2 Making NB4 cell growing curveNB4 cells treated with different agents as above mentinoned were harvested at different culture time, calculated and differentiated under microscopy, then the NB4 cell growing curves were made, and the NB4 cell differentiation rates were calculated.2.3 NB4 cell differentiation expression in morphology and in membrane differentiation antigensThe harvested NB4 cells were smeared on glass slides, stained, and observed morphology under microscopy. These cells washed with PBS twice, and respectively incubated at concentration of 106/ml with phycoerythrin-conjugated anti-CD33 monoclonal antibodies and with FITC-conjugated anti-CD11b monoclonal antibodies at room temperature, then analysed by flow cytometery on a BD FACSAfia Profile.2.4 PML/RAR a mRNA measurementThe total RNA of NB4 cells were isolated by Trizol's method according to the manufacturer's protocol.The PML/RARαmRNA in NB4 cells were assayed by real-time Q-PCR.2.5 PML-RARαfusion protein assayThe PML-RARαfusion protein in NB4 cells were assayed by Western blotting according conventional experiment steps.2.6 Nuclear bodies observation in NB4 cellsThe cell smear slide were made by cytospin, fixed by formalin, incubated with primary antibodies(goat anti-PML antibodies, and washed and then incubated with secondary antibody marked with FITC. Finally these cells were stained PI for 5 minutes to show the nuclei and then to observe and count scores of the nuclear bodies and microspeckles in the cells under fluorescence microscopy. 2.7 C/EBPβprotein in NB4 cells measurmentThe protein were collected from NB4 cell lysates, quantified and boiled in Laemmli sample buffer, and the loaded onto 10% SDS-polyacrylamide gels for electrophoresis. The fractionated proteins were transferred onto nitrocellulose paper and then probed with mouse monoclonal antibodies against C/EBPβprotein. The blot was incubated with peroxidase-cojugated sheep anti-mouse immunonglobulin and detected by enhanced chemiluminescence method. The bands were quantified with densitometer. GAPDH protein as a internal control was blotted concurrently. The expression values of these proteins calculated by ratio of grey matter value of target protein to the grey matter value of intemal standard.3 Results3.1 The proliferative inhibition of NB4 cells induced by Tan IIAThe degree of proliferative inhibition of NB4 cells induced by Tan IIA was increasing with the concentration and reaction time of Tan IIA. The inhibition effects on proliferation of NB4 cell induced by Tan IIA at three concentrations (1.70μmol/L, 2.55μmol/L and 3.40μmol/L) during first 48 hours were not different (p＞0.05) , but showed significant difference among the different concentration groups after 48 hours treated, the effect of the lower concentration group was weaker than that of other two higher concentration groups (p＜0.05). In higher concentration groups there were no difference,at all the treatment time points. The proliferative inhibition effects reached a plateau after 120 hours. These results suggested that 2.55μmol/L of Tan IIA is the optimal concentration to inhibit the NB4 cell proliferation and the optimal time to treat the cells with Tan II A was 120 hours. During the first 48 hour treating, the proliferative inhibition effects were no difference between two drug groups (2.55μmol/L of Tan II-A and 0.10μmol/L As2O3 in cell growth rate (p＞0.05), after 48 hour treatment ,the proliferative inhibition effect on NB4 cells induced by Tan IIA at concentration of 2.55μmol/L was higher than that of As2O3 at concentrations of 0.10μmol/L; There were no difference between the effects induced by Tan IIA at concentration of 2.55μmol/L and As2O3 at concentration of 1.0μmol/L (p＞0.05). Before 144 hours the inhibition effect induced by Tan IIA at concentration of 2.55μmol/L was lower than that of ATRA at concentration of 1.0μmol/L, after then these is no difference between the two drug groups.3.2 Morphologic changes of NB4 cells induced by Tan II AAfter 24 hour treatment with Tan II A, the differentiation expression of NB4 cells in morphology became gradually significant with time. These is no difference among three groups of Tan. II A before 72 hour treatment, after then the differentiation rate of NB4 cells induced by Tan II A at concentration of 1.70μmol/L was lower than that of other two groups of Tan II A (p＜0.05), but there was no difference in differentiation rate at all time points between two groups of Tan II A at concentrations of 2.55μmol/L and 3.40μmol/L (p＞0.05). The differentiation rate of NB4 cells was increasing with the dosage and treatment time of Tan II A. The results as above also demonstrated that 2.55μmol/L of Tan IIA was the optimal concentration to induce the NB4 cell differentiation and that the optimal time to reach to the effect plateau was 120 hours. The differentiation effect of Tan II A at Concentration of 2.55μmol/L on NB4 cells was higher than that of As2O3 at two concentrations of 0.1μmol/L and 1.0μmol/L (p＜0.05), lower than that of ATRA group (p＜0.05) before 96 hours treatment, but after then there was no difference between two groups of Tan II A and ATRA.3.3 The. expression of CD11b and CD33 on NB4 cells induced by Tan II ASince 24h treated the expression of CD11b on membrane of NB4 cells induced by Tan II A was increasing with dosage and treating time of Tan II A. In contrast to CD11b expression the CD33 expression was decreasing. The up-regulating CD11b expression and down-regulating CD33 expression induced by Tan II A at concentration of 1.70μmol/L was weaker than that of higher two concentration groups (p＜0.05), but these was no difference between later two groups. The effects on regulation expression of CD11b and CD33 of NB cells in three concentration groups of Tan IIA reached a plateau after 120 hour treating. The effect of 2.55μmol/L Tan II-A on the expression rates of CD33 and CD11b was stronger than that of 0.1μmol/L As2O3 (p＜0.05) and no less than that of 1.0μmol/L As2O3 (p＞0.05), weaker than that of 1.0μmol/L ATRA before 96 hours (p＜0.05) , and after then was similar to that of ATRA at concentration of 1.0μmol/L.3.4 The effect of Tan II-A on the distribution of PML in NB4 cellBefore treatment, there were lot uncountable microspeckles in NB4 cells. Upon Tanshinone II A treatment, the microspeckles in cells were decreasing with dosage and treatment time, in contrast to the large dots, referred to as nuclear bodies (NB), were increasing, these changes began at 12 hour treating. After 72 hour treatment 15-30 large dots p.er nucleus as normal human neutrophil were found, which suggested that PML protein relocated to normal position. The effect of Tan IIA on the distribution of PML in NB4 cell was dose- and time-dependent.The change of microspeckles to big dots induced by both of ATRA and As203 group was quicker than that of Tan IIA, it began from 12 hour treatment.3.5 The effect of Tan IIA on PML/RARa mRNA expression in NB4 cellsReal time Q-PCR was used to determine the PML/RARa mRNA expression in NB4 cells. Ct values were ranged from 25.67±0.3 to 29.01±0.6 in three groups at different time points, there was no difference among them. The Ct values of expressions of PML/RARa mRNA in NB4 cells treated with two concentrations of As2O3 were ranged from 25.45±0.9 to 28.24±0.6. The Ct values of ATRA were from 26.57±0.8 to 28.72±0.4. The Ct values of All groups including DMSO at different treatment time points were no difference (p＞0.05).3.6 The effect of Tan IIA on PML-RARa fusion protein in NB4 cellsThe level of PML-RARa protein presenting with grey value was decreasing and the level of PML protein was increasing with a dose- and time- dependent manner after 12 hour treatment with Tan II A at concentrations of 1.7, 2.55 and 3.4μmol/L. The level of PML protein presenting with grey value in higher concentration groups was higher than that of lower concentration group of Tan II A. The change of PML-RARa protein and PML protein induced by ATRA expressed with the same manner as that of Tan II A. The level of PML-RARa protein was decreased after 24 hours treatment in two As2O3 groups, the effect of Tan II A on PML-RARa protein and PML was slightly weaker than.that of ATRA.3.7 The effect of Tan II-A on C/EBP 13 protein in NB4 cellsThe C/EBPβis a very important transcription factor, expressing in isoforms (LAP and LIP), related with cell differentiation. The level of C/EBPβLAP protein was increased after 12 hour treatment in three Tan II A groups. The level of C/EBPβLAP protein was increased after 24h in As2O3 groups, The level of C/EBPβLAP protein was quickly increased after 12 hour treatment with ATRA. The level of C/EBPβLAP protein in ATRA group was higher than other groups.4 conclusion4.1 The differentiation effect on NB4 cells induced by Tan IIA is increasing with a dose- and time- dependent manner. 2.55μmol/L of Tan II-A is the optimal concentration to induce the NB4 cell differentiation and that the optimal time to reach to the effect plateau is 120 hours. The differentiation effect is weaker than that of ATRA before 120 hour treatment, but stronger than that of As2O3.4.2 Tan IIA can relocated PML protein into its normal position in NB4 cell nuclei, and restore the normal structure of nuclear bodies of NB4 cells with a dose and time-dependent manner. The process is slower than that of ATRA group and two As2O3 groups before 72 hour treatment (p＜0.05) , but after then it is the same as that of ATRA group and two As2O3 groups.4.3. Tan IIA can degradate PML-RARa protein in NB4 cells and induce release Of PML from PML-RARa protein.4.4 PML-RARa mRNA level is not affected by Tan IIA, ATRA and Arsenic Trioxide in short term (7 days).4.5 During the early period of NB4 cell differentiation induced by Tan IIA, the C/EBPβprotein is expressed with two isoforms, LAP and LIP. Tan IIA enhances C/EBPβLAP protein expression and reduce C/EBPβLIP protein expression in NB4 cells. ATRA and As2O3 also induced the expression of C/EBPβLAP protein expression ,but can not induce C/EBPβLIP protein expression.
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