Effect Of ATRA Combined With G-CSF On The Growth,Differentiation And RARα2Expression Of Myeloma Cells

Backgroud and Objective:Multiple myeloma (MM) is a malignant tumor originates in plasma cells and overally it is a disease remains incurable. Studies showed that1.0umol/L ATRA (all trans-retinoicacid) can induce apoptosis of myeloma cells and acute non lymphocytic leukemia (AML) cells, which express RARα2+.1000U/mL G-CSF (granulocyte colony stimulating factor) can increase the expression of RARα2+in AML cells and have synergistic effect of inducing apoptosis with ATRA. But it’s not enough in research with the combination of the two drugs when it comes to MM in at home or abroad. Our study observed influence of ATRA combined with G-CSF on the growth, apoptosis and differentiation of myeloma cells, detected the expression of RARa2in myeloma cell lines and primary myeloma cells. So as to explore the relation between them, and to provide experimental evidence for more efficient drugs treating MM.Methods:Myeloma cell lines OPM2and U266were treated with ATRA (1.0μM) in the presence or absence of G-CSF (1000U/ml or2000U/ml). The methods of MTT, inverted microscope observation, Annexin V-PI staining and RT-PCR were used to study various biological responses; morphology change was evaluated by Wright-Giemsa staining; CD49e expression were analyzed by flow cytometry. Mononuclear cells were separated from bone marrow of26MM patients using lymphocytes separation, than detect the mRNA expression of RARa2of all cases using semi-quantitative RT-PCR.Results:Cell viability of OPM2cells in2000U/mlG-CSF,1.0uM ATRA single and their combination group were100.75%,89.77%and86.41%of control group at48h, respectively, while the corresponding cell viability of U266cells were94.21%,96.73%and96.06%of control group, respectively. The proliferation of OPM2cells (P<0.05) could be inhibited by ATRA at48h. The growth inhibition rate of single G-CSF groups<0, and the growth inhibition rates of combined groups were12.52%and13.59%, respectively, which were more than their single groups (p<0.05).While in U266cells the above effect was not significant (p>0.1). Compared with the control group, obvious morphology changes could be observed under the inverted microscope. Annexin-V/PI staining confirmed that24h incubation with2000U/mL G-CSF and1.0umol/L ATRA induced apoptosis in (8.1±2.0)%OPM2cells and (4.5±1.4)%U266cells. Annexin-V/PI staining confirmed that48h incubation with2000U/mLG-CSF and lumol/L ATRA induced apoptosis in (11.6±2.4)%OPM2cells and (7.5±3.7)%U266cells. Expression of RARa2mRNA in OPM2cells:G-CSF+ATRA combination groups and single ATRA group were higher than the control group (P<0.05); G-CSF+ATRA combination groups were higher than the single ATRA group (P<0.05); the control group and single G-CSF groups had no difference (P>0.05). Expression of RARa2mRNA in U266cells:the control group and single G-CSF groups almost had no expression;2000U/mL G-CSF+l.Oumo/L ATRA group and single ATRA group had weak expression. The OPM2cell stained by Wright-Giemsa in ATRA groups showed that, the cell nucleus became smaller, chromatin condensed, number of nucleolus reduced, the volume of cytoplasm increased and the cytoplasm became dark blue. Expression rates of CD49e in2000U/mL G-CSF+1.0umol/L ATRA group and the control group of OPM2cells were (5.1+0.23)%and (2.4+0.47)%, respectively. In our26cases,55.6%(10) of18newly diagnosed cases and50.0%(4) of8retreated cases expressed RARa2.Conclusion:ATRA can induce proliferation inhibition in myeloma cells which express RARα2. ATRA can stimulate expression of RARα2in myeloma cells, this effect can be enhanced by G-CSF; and G-CSF alone has no effect as ATRA as above. ATRA also plays a certain role in promoting differentiation of RARα2+myeloma cells. Some patients in clinical can be detected the expression of RARa2.