Effects of Lenalidomide Alone or Combination with Bortezomib on Proliferation and Apoptosis of Mantle Cell Lymphoma Cells in Vivo and Vitro
|Course||Department of Hematology|
|Keywords||bortezomib lenalidomide lymphoma, mantle cell proliferation apoptosis nude|
Mantle Cell Lymphoma (MCL) is a clinical treatment challenging B cells malignant non-hodgkin’s lymphoma(NHL), accounts for about 5-10 percent of adult NHL.The characteristics of high incidence in the elderly, occult onset and rapid progress led to only 3 to 4 years of survival time. Despite recent high-dose chemotherapy combined with stem cell transplantation for treatment of MCL, the total effective rate is considerable, but because of the reaction of short duration and the high intensity chemotherapy brings into the inevitable high treatment-related mortality, the treatment of MCL remains a great challenge. It is necessary to explore a new high-efficiency but low-toxicity combination treatment strategy.New molecular targeted drugs in clinical treatment has demonstrated a good efficacy,such as immunomodulatory drugs lenalidomide (LEN) and proteasome inhibitor bortezomib (BTZ) as a representative of this class of drugs, although the mechanisms are not yet completely clear, but the application of these drugs in the treatment of hematologic malignancies have been gradually attached importance and expanded. Following the approval lenalidomide for multiple myeloma (MM) treatment by the FDA of USA in 2006, people found that it has equally effect in the treatment of NHL. In NCCN guidelines bortezomib had been recommended as a second-line drugs in the treatment of MCL, its effectiveness had also been acknowledged by the clinical trials of MCL treatment, but the clinical trial about combination of LEN and BTZ which may have different mechanisms is just beginning, and its effectiveness and action mechanism have not been reported. This study investigated the anti-MCL effect of LEN alone or combination with BTZ and the mechanisms of action which can provides a valuable preclinical data for future clinical application of LEN-based combination therapy strategy. Objective:1. To observe the effects of LEN alone or combined with BTZ on proliferation and apoptosis of mantle cell lymphoma cell line Jeko-1 cells in vitro, and to explore the possible mechanism of those effects. 2. To establish nude mouse model of human mantle cell lymphoma, and observing the effects of the drugs on tumor proliferation.Methods:Human mantle cell lymphoma cell line Jeko-1 was cultured in vitro. Methyl thiazolyl tetrazolium (MTT) colorimetric assay was used to determine the growth inhibition rate of the cell line which was con-cultured with LEN (10,20,50,100μmol/L) alone for 1-7d, or BTZ (2.5,5.0,7.5 , 10.0nmol/L) alone for 24h, 48h, or their combination (LEN/BTZ) for 24h, 48h; The growth of cells were observed by inverted microscope; flow cytometry was used for detection of cell cycle changes and apoptosis; semi-quantitative RT-PCR assay was used for detection of Caspase-3, Bcl-2, Noxa, VEGF mRNA expression. The nude mice were inoculated subcutaneously in the axilla with human mantle cell lymphoma cell line Jeko-1. And the volume of tumor was measured every 3 days. The nude mouse was killed after 5 weeks, then comparing the inhibition rate of tumor which were affected by different ways .Results:(1) MTT assay indicated that the inhibitory effect of LEN on the proliferation of Jeko-1 cells was found in a time-and-does dependent manner after 3 days; (2) From the micro perspective, the cell density of LEN group was lower than control group, but obvious apoptosis or death phenomenon was not observed. However, the cell of BTZ group and the combination group grow sparsely and obvious apoptosis and necrosis could be observed; (3) Flow cytometry analysis showed that compared with the control group, with the concentration and action time of LEN increased, the percentage of cells in G0/G1 phase was increased gradually, and even the concentration of 100μmol/L for 7 days, LEN can not induce cell apoptosis. Compared with single drug BTZ group, combination of 100μmol/L LEN with 2.5nmol/L, 5.0nmol/L of BTZ exposure to Jeko-1 cells in 48h respectively, resulted in significant sub-G1 peak, and showed additive effect of apoptosis (P<0.05). (3) RT-PCR was used to detect the expression of messenger RNA. After increasing concentrations of BTZ were exposed to cells for 48 hours, we found Bcl-2, VEGF mRNA decreased, and caspase 3, Noxa mRNA expression increased significantly, but in the LEN group, except for VEGF mRNA increased follow by the concentration and time changes, Caspase-3, Bcl-2, Noxa mRNA do not change obviously. In the combination group after 48 hours, the expression of VEGF mRNA was significantly decreased (P<0.05), but compared with BTZ alone, the Caspase-3、Bcl-2 and Noxa mRNA levels have No significant differences; (4) In the study of human mantle cell lymphoma tumor-bearing mice, the inhibition rate of tumor weight in BTZ group, LEN group and LEN+BTZ group was 32.7%, 49.3% and 58.9% at 5 weeks, respectively. Compared with the control group, LEN+BTZ group can significantly slow down the tumor growth volume of the human MCL-bearing mice (P<0.05).Conclusion 1. Lenalidomide could inhibit the growth of Jeko-1 cells in a time and concentration-dependent manner, but it can not induce Jeko-1 cells apoptosis; 2. Combination of Lenalidomide and bortezomib can effectively suppress Jeko-1 cells proliferation and induced apoptosis; 3. Lenalidomide alone or combined with bortezomib have anti-tumor effect in human mantle cell lymphoma bearing nude mice.