In Vitro Study Of Effects And Mechanisms Of Celecoxib In Human Acute Promyelocytic Leukemia Cell Lines

Acute Promyelocytic Leukemia (APL) is a special subtype of acute myelogenous leukemia (AML) which is characterized for a specific translocation between chromosome 15 and 17 t (15; 17) and the expression of PML/RARα. fusion gene. Without therapy, APL is life threatening because of disseminated intravascular coagulation (D1C) and intracerebral hemorrhage. After use of all-trans-retinoic acid (ATRA) and arsenic trioxide (As₂O₃) in 1980s by Chinese hematologists, the complete remission (CR) rate of APL began to rise to 85% and APL became a curable subtype of AML. ATRA and As₂O₃ can induce APL cells differentiation and apoptosis, and their usage is a milestone in leukemia therapy. However, the therapeutic effect of ATRA is limited as a signal agent is frequently transient, and relapse of APL is often accompanied with resistance to ATRA. The long-time clinical use of As₂O₃ is also limited by serious systemic toxicity. New, economic, and effective methods and treatments are also urgently needed.Celecoxib, productive named Celebrex, is one of the specific inhibitors of cyclooxygenase-2 (COX-2). As a non-steroid anti inflammation drug (NASID), it has been widely used in bone and joint diseases, such as rheumatoid arthritis and osteoarthritis. In recent years, besides its effect of anti-inflammation, celecoxib has been reported to induce anti-neoplastic activity on many solid human tumor cell lines, such as gastric cancer, intestinal cancer, ovarian cancer, pancreatic cancer and osteosarcoma. It can inhibit cell proliferation, and prevent from relapse and metastasis. While there was few reports of the anti-tumor potential and mechanism of celecoxib in acute leukemic cells especially in acute myelogeneous leukemia. To investigate the effects of celecoxib on APL cells and its mechanism, we conducted this study to address the following questions: a) Whether celecoxib has the same effects on APL cell lines and primary APL cells? b) What is the mechanism? c) Whether celecoxib has effect on telomerase activity and how about its mechanism?Part I The Effects of Celecoxib on Cell Proliferation and Cell Apoptosis of Acute Promyelocytic Leukemia Cell Line and Primary APL CellsIn Part I, the effects of celecoxib on the APL cell lines NB4 and MR2 cell and primary APL cells were investigated. Using MTT assay we first investigated the effects of celecoxib on NB4 and MR2 cell proliferation. The results showed that treated with celecoxib (20-160μmol/L) for 12-48 h, the proliferation of NB4 and MR2 cells were inhibited significantly, in comparison with the control group (P<0.01). 50% growth inhibition (1C50) at 12h in NB4 and MR2 occurred at 102.70μmol/L and 86.61μmol/L; 24h at 86.24μmol/L and 80.93μmol/L; 36h at 78.30μmol/L and 80.69μmol/L; 48h at 60.39μmol/L and 71.72μmol/L. Trypan blue staining also indicated that celecoxib inhibited cell proliferation significantly. In addition, celecoxib also inhibited the growth of freshly isolated bone marrow cells from 2 APL patients with an IC50 (at 24h) of 75-80μmol/L. Human umbilical vein endothelial cell line eahy926 and freshly isolated human peripheral blood mono-nucleated cells were used as normal cell control, and after treated with celecoxib for 24-48h, MTT assay showed that celecoxib could inhibit cell proliferation, but IC50 concentration was far beyond the clinical usage. To explore the possible mechanism of the inhibition of celecoxib on APL cells, further investigation was carried out from the two stratifications of inducing apoptosis and cell signaling pathway.In order to analyze the cell apoptosis quantitatively and qualitatively exactly, some classic and specific and sensitive methods were adapted. Cell morphology was used to identify apoptosis. Compared with control, the nuclei of apoptosis cells were characterized by nuclear margination and condensation, and apoptotic bodies were easily found, after incubated with celecoxib (80-100μmol/L) for 24h. NB4 and MR2 cells following treatment with 120-160μmol/L celecoxib for 24h, a typical DNA ladder pattern of internucleosomal fragmentation was observed with DNA fragmentation analysis. Flow cytometry was also used to evaluate the DNA content, percentage of hypodiploid and translocation of phosphatidylserine (PS) at the outer surface of the cell plasma membrane. And it indicated that level increased following the augmehtation of the drug concentration. After treated with celecoxib for 24h, a progressive increase in sub-G₁ phase cells; and apoptosis induced by celecoxib was also confirmed by following annexinV / PI staining, NB4 cell exposure to 40-160μmol/L celecoxib for 24 h caused 11.00%, 29.10%, 39.30% apoptotic cells, which was more than that of the untreated group 2.60%(P<0.01). MR2 cell exposure to 40-160μmol/L celecoxib for 24 h caused 9.59%, 24.00%, 36.10% apoptotic cells, which was more than that of the untreated group 2.84%(P<0.01).Part II The Mechanisms of Cell Apoptosis of Acute Promyelocytic Leukemia CellLines Induced by CelecoxibIn order to explore the possible pathway of the signal conduction of the cell apoptosis induced by celecoxib, we using western blot to detect the change of caspase-8,-9,-3 and PARP in NB4 and MR2 cells. Following treatment with 40-160μmoI/L celecoxib for 24h, typical caspase-9,-3 and PARP cleavage were detected, while no significant cleavage was observed with caspase-8Cell cycle analyzed by flow cytometry with Pl staining showed 40-160μmol/L celecoxib led to cell cycle arrest in G]/S phase. Compared with control group, cells in G₀/G₁ phase were increased, while numbers of cells in S+G₂/M phase were decreased (P<0.01). And western-blot was used to detect the expression of cell-cycle-regulating proteins. After incubated with celecoxib for 24h, the expression of CyclinD1 and CyclinE decreased, accompanied with upregulation of P21^waf/cip1 P27^KIPP16^INK4aThe expression of mRNA of fusion gene PML/RARα and Survivin, bcl-2/bax, CIAP1 and CIAP2 was assessed by reverse transcription polymerase chain reaction (RT-PCR), and β-actin was used as inner control. The results indicated there were no significant changes in fusion gene PML/RARα of NB4 and MR2 cells (P>0.05) . Expression of survivin and CIAP2 was down-regulated with treatment with celecoxib, and bcl-2/bax was also down-regulated. And there was no significant change with the expression of CIAP1.RT-PCR indicated the expression level of COX-2 mRNA in NB4 and MR2 cells were low and there was no significant change after treatment of celecoxib.Since vascular endothelial growth factor (VEGF) is important growth factor of many leukemia cells, we detected the expression of VEGF mRNA level by RT-PCR in NB4 and MR2 cells. The result showed that celecoxib at 80-160μmol/L could significantly down-regulated the expression of VEGF mRNA. Then we examined the expression of VEGF protein by western blot, the result showed that celecoxib could down regulate the expression of VEGF in a dose-dependent manner.Part III The Effects and Mechanisms of Celecoxib on Telomerase Activity and SignalConduct PathwayIn order to investigate the other possible mechanisms and signal conduct pathway of the cells apoptosis induced by celecoxib. The telomerase activity of NB4 and MR2 cells was analyzed by PCR-ELISA. NB4 and MR2 cells were culture with celecoxib (80-160μmol/L) for 24h, and then analyzed the telomerase activity by ELISA. The results indicated that celecoxib could inhibit the telomerase activity of APL cell lines, and the inhibition was time and concentration dependent.The expression of hTERT mRNA and c-myc mRNA was assessed by RT-PCR, and it showed the expression of both mRNA were down-regulated by celecoxib in dose-dependent manner.Phosphorylation was important to the function of telomerase. We further studied the protein expression of Akt and phosphorylated Akt in APL cells with or without celecoxib treatment. After cells were cultured with celecoxib for 24 h, as the concentration increased, western blot showed that the expression of p-Akt was down regulated, but Akt was stable. It suggested that the celecoxib could inhibit activity of phosphorylation pathway in APL cells, and it might be one of the mechanisms of apoptosis induction.Summary: (1) Celecoxib inhibited the growth of APL cell lines NB4 and MR2 and primary APL cells in dose and time dependent manners. Celecoxib has no significant effect on normal human peripheral blood mono-nucleated cells; (2) The effect of celecoxib was accompanied with cell cycle arrest at G]/S phase and down-regulation of cell cycle regulating protein CyclinD1 and CyclinE, and up-regulation of P21^waf/cip1 , P27^KIP, P16^INF4a; (3) Celecoxib not only had directly toxicity in APL cells, but also inhibited the level of VEGF in APL cells; (4) Treatment with (80-160μmol/L) celecoxib induced APL cells apoptosis in dose- and time- dependent manners; (5) The celecoxib-induced apoptosis of APL cells was associated with the cleavage of caspase-9 and -3 and PARP, and down-regulation of Survivin and C1AP2, and up-regulation of bcl-2/bax; (6) Celecoxib induced APL cells apoptosis without significant effects on the expression of PML/RARα fusion gene and COX-2 gene; (7) Celecoxib inhibited activity of telomerase of APL cells and down regulated the expression of hTERT and c-myc; (8) Celecoxib also inhibited the phosphorylation of Akt, which may be one of the mechanisms of celecoxib induced the apoptosis in APL cells.