ALBERT

Description: Abstract 3084 With modern intensive chemotherapy, 78% to 93% of adult patients with acute lymphoblastic leukemia (ALL) achieve complete remission (CR). However, the disease-free survival rate is only 30% to 40% due to the high rate of relapse. A part of relapsed patients can achieve second remission (CR2) with salvage therapy, and allogeneic hematopoietic stem cell transplantation (HSCT) in CR2 will be the only curative strategy. Prognosis after relapse in adult patients with ALL is considered to be extremely poor, but reports as to the outcome after relapse have been limited. To elucidate the outcome of relapsed patients and prognostic factors after relapse, we retrospectively collected and analyzed clinical data from 69 institutions in Japan on patients with Philadelphia-chromosome (Ph) negative ALL, aged 16–65 years, who relapsed after first CR (CR1) between 1998 and 2008. A total of 332 patients were included in this study. The median age of them was 35 years, and 165 patients were male. Median duration of CR1 was 290 days (range 15–7162 days), and median follow-up time after relapse was 319 days (range 3–3689 days). Fifty-eight and 4 of them relapsed after allogeneic and autologous HSCT in CR1, respectively. The overall survival (OS) rate was not significantly different between patients who relapsed after allogeneic HSCT in CR1 and those who relapsed after chemotherapy only (50.0% vs. 43.4% at 1 year and 10.6% vs. 16.3% at 5 year, respectively). Among 270 patients who relapsed after chemotherapy only, 234 patients received salvage chemotherapy after relapse, and 123 patients achieved CR2 (52.5%). Sixty-two patients out of those 123 patients underwent allogeneic HSCT in CR2. Median duration between the achievement of CR2 with salvage chemotherapy and allogeneic HSCT in CR2 was 76 days. OS rate was significantly better in patients who underwent allogeneic HSCT in CR2 following salvage chemotherapy than those who did not (74.1% vs. 55.1% at 1 year and 44.7% vs. 11.6% at 5 year, respectively) by a landmark analysis limiting patients who were surviving without disease at 76 days after the achievement of CR2. In multivariate analysis of factors that included allogeneic HSCT in CR2 following salvage chemotherapy as a time-dependent covariate, lower white blood cell count at relapse (less than 10000/μl) and allogeneic HSCT in CR2 were associated with better OS rate among patients who achieved CR2 following salvage chemotherapy. Forty-six patients underwent allogeneic HSCT in non-CR after receiving salvage chemotherapy. A part of them survived long, and 5 year OS rate was 20.9%. In conclusion, the prognosis of adult patients with relapsed Ph-negative ALL is poor. Allogeneic HSCT after first relapse could improve the prognosis. Disclosures: No relevant conflicts of interest to declare.

Description: Rituximab has greatly improved the prognosis of B cell malignancies (BCM). However, several resistance mechanisms have been reported, including the inhibition of apoptosis, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) as well as modulation of CD20 antigen and increased complement-related cytotoxicity inhibition factor (CD55). The calicheamicin-conjugated anti-CD22 monoclonal antibody (mAb), CMC544, is one of the new promising agents, and effective on several types of BCM in vitro, especially when used in combination with rituximab. However, details regarding the cytotoxic effects of CMC544 and mechanisms triggering cell death, particularly when it is combined with rituximab, have not been elucidated. We studied cell cycle features of CMC544 on BCM used alone or in combination with rituximab, and analyzed quantitative alteration of target molecules such as CD20, CD22 and CD55.(Materials and Methods) The cell lines used in this study were CD22-positive Daudi and Raji cells, and CD22-negative Jurkat and NB4 cells. Cells obtained from 8 patients with BCM were also used. CMC544, unconjugated anti-CD22 mAb (G5/44), and free NAC-calicheamicin DMH were provided by Wyeth Pharmaceutical Co., Ltd. The effects of CMC544 were analyzed by incubating the cells at the concentrations of 0–100μg/ml for 0–96h, then by measuring cell count, cell viability, 3H-thymidine incorporation and cell cycle distribution on flow cytometry (FCM) as well as by video microscopic observation. The amounts of CD20, CD22 and CD55 antigens were analyzed by FCM, laser scanning microscope. The process of CMC544-induced cell death was assessed by Apocyto®. Three possible mechanisms of the combined effect of CMC544 and rituximab were investigated separately by direct effect of the mAb, CDC and ADCC. (Results) Analysis of the cell cycle distribution indicated that CD22-positive cells were temporally arrested at the G2/M phase after 6–12h incubation with CMC544, and the hypodiploid proportion increased after 24–72h. CD22-positive cells enlarged after 12–24h incubation with CMC544. Apoptotic morphological changes such as bleb formation and cell shrinkage were observed in 35–57% of the cells. These changes were not observed after incubation with G5/44. Apocyto® stain showed a continuous pattern from early to late apoptosis. Similar effects were observed in clinical samples that expressed CD22. The amounts of CD22 and CD55 were significantly reduced 3h after incubation with CMC544, while the CD20 expression was maintained 24h after incubation with CMC544. Incubation of the cells for 12–24h with CMC544 and then for 12hr with rituximab induced 1.2–2.1 times more anti-proliferation, apoptosis, CDC and ADCC than simultaneous incubation.(Conclusion) The effect of CMC544 was clearly observed as G2/M arrest following increase of the hypodiploid proportion in the cell cycle distribution, and this was a useful method to investigate the effect of CMC544. Combination of CMC544 and rituximab enhanced the cytotoxic effect, and sequential administration was more effective. The reduction of CD55 and continued expression of CD20 after the incubation with CMC544 supported the above observation. Further investigations are required to find the most effective treatment regimen to overcome drug resistance of B cell malignancies.

Description: Several new agents have been introduced for the treatment for B cell malignancies (BCM) to overcome resistance to rituximab. Inotuzumab ozogamicin (CMC544), a humanized anti-CD22 mAb conjugated to N-acetyl-g-calicheamicin demethyl hydrazide (NAC-calicheamicin DMH), binds CD22, leading to internalization and delivery of calicheamicin inside the cells. We have been studying gemtuzumab ozogamicin (CMA676), another calicheamicin-conjugated mAb targeting CD33, and we have reported several new findings regarding multi-drug resistance (MDR) and modification of surface antigens. In this study, we attempted to clarify the effect of CMC544 on BCM cells in relation to MDR, and we investigated the restoration effect of the MDR modifier. We also analyzed the effect of CMC544 in relation to CD22 and P-glycoprotein (P-gp) in the samples from BCM. (Materials and Methods) The cell lines used in this study were CD22-positive parental Daudi and Raji, and their P-gp positive sublines, Daudi/MDR and Raji/MDR, respectively. CD22-negative Jurkat, K562 and NB4 cells, and cells obtained from 11 patients with BCM, were also used. CMC544, unconjugated anti-CD22 mAb (G5/44), CMA676, and unconjugated NAC-calicheamicin DMH were kindly provided by Wyeth Pharmaceutical Co., Ltd. The amount of cell surface antigens and P-gp were analyzed by flow cytometry (FCM). For P-gp analysis, cells underwent a reaction with biotinylated MRK16 mAb or with a subclass-matched control mAb and were then stained with streptavidin-Cy-7. P-gp function was determined by intracellular rhodamine-123 (Rh123) accumulation and its enhancement by MDR modifiers, PSC-833 (Novartis) and MS209 (Mitsui), as previously described. The effect of CMC544 was analyzed by cell count, cell viability, and cell cycle distribution on FCM. It was also determined with or without a MDR modifier. Relationships between the CMC544 effect and the amount of P-gp or CD22 were examined statistically. (Results) A dose-dependent, selective cytotoxic effect of CMC544 was observed in cell lines that expressed CD22. CMC544 is not effective on P-gp-expressing MDR sublines, compared with parental cell lines. MDR modifiers restored the cytotoxic effect of CMC544 in P-gp-expressing sublines. In clinical samples, the cytocidal effect of CMC544, estimated from the fraction of cells in the hypo-diploid portion of the cell cycle, was inversely related to the amount of P-gp estimated by MRK16 mAb (P=0.04), and to the P-gp function assessed by intracellular Rh123 accumulation in the presence of PSC833 or MS209 as MDR modifier (P=0.02 and P=0.01, respectively). Additionally, these MDR modifiers reversed CMC544 resistance in P-gp-expressing CD22-positive cells. On the other hand, the cytotoxic effect of CMC544 was positively correlated with the amount of CD22 (P=0.01). (Conclusion) This study demonstrates that the CMC544 effect depends on the amounts of P-gp and CD22. We might be able to predict the clinical effects of this drug based on these factors. The treatment of BCM has progressed extremely rapidly, and we can now select particular drugs more specifically among many promising agents. Our results contribute to the development of new strategies to treat BCM. In CD22-positive BCM with P-gp, combined use of CMC544 and MDR modifiers may be more beneficial.

Description: Abstract 3381 [Background and Aims] MAPK pathways control the cellular processes such as proliferation, differentiation and apoptosis. Abnormal MAPK signaling plays a key role in the development and progression of cancers. The dual-specificity MAPK phosphatases (DUSPs) act as negative regulators of MAPK activity in normal tissues. DUSP1 (dual-specificity MAPK phosphatase-1) is able to inactivate the ERK, p38, and JNK in resulting the cell cycle and apoptosis, and it has been reported to be lost in a variety advanced cancers. However, its function is little known in CML cells. In this study, we have investigated the function of DUSP1 in CML cell proliferation. [Methods] The cells used in this study were human CML cell lines, K562, Meg01 and SHG3 cells. Primary CML cells (ALDHhi cells) were obtained from the bone marrow of CML (CP) patients (n=12). Human normal ALDHhi cells were isolated from bone marrow of healthy volunteers after obtaining informed consents. For analysis of DUSP1 mRNA expression, quantitative RT-PCR was performed in all cell lines treated with Abl kinase inhibitors (STI571, AMN107, and BMS354825). For cell survival analysis and the levels of p-ERK1/2, p-p38, and p-ATF2 in CML cells, MTT assays, western blot and cell cycle analysis were performed in all cell lines transfected with DUSP1 shRNA or cDNA. For colony analysis, the colonies of CFU-GEMM, CFU-GM, and BFU-E were counted in CML stem/progenitor cells transfected with DUSP1 cDNA or shiRNA, or treated with Abl kinase inhibitors. [Results] In CML cell lines, the expressions of DUSP1 mRNA and protein were significantly increased by treatment with Abl kinase inhibitors or transfection with Bcr-Abl shRNA. In CML cells treated with Abl kinase inhibitors or transfection with Bcr-Abl shRNA, CML cell proliferation was inhibited, and the phosphorylation levels of p-ERK1/2, p-p38, and p-ATF2 were markedly decreased compared to the untreated cells. In addition, the overexpression of DUSP1 induced G1 cell cycle arrest indicating down regulation of cyclin D1 expression in CML cells. Moreover, in CML cells transfected with DUSP1 shRNA, the inhibition of CML cell proliferation by the Abl kinase inhibitors were weakened. In CML stem/progenitor cells (ALDHhi cells) obtained from patients with CML, the expression of DUSP1 mRNA was suppressed, and the transfection with Bcr-Abl shRNA or treatment with Abl kinase inhibitors increased the expression of DUSP1 mRNA and protein, and decreased the counts of CFU-GEMM, CFU-GM and BFU-E. [Conclusion] Our results demonstrated that the expression of DUSP1 was suppressed by Bcr-Abl in CML cells, and the depletion of DUSP1 promotes cell cycle progression through the induction of cyclin D1 expression, and stimulates CML cell proliferation through the continuous phosphorylation of ERK1/2, p38, and ATF2. Moreover, the induction of DUSP1 expression inhibited the proliferation of CML stem/progenitor cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4463 [Background and Aims] In various signaling pathways activated by Bcr-Abl, c-Myc promotes the Bcr-Abl transformed cell proliferation through activated Akt, Jak2 and NF-κB. We have previously shown that Bcr-Abl represses the expression of THAP11 and continuously expressed c-Myc, resulting in the proliferation of CML cells. However, the mechanism of c-Myc regulation is unclear in CML cell proliferation. In the present study, we investigated the mechanism of c-Myc transcription regulation in the Bcr-Abl+ progenitor cells derived from CML patients. [Methods] The cells used in this study were human CML cell lines, K562, Meg01 and SHG3 cells. Primary CML cells (ALDHhi cells) were obtained from the bone marrow of CML (CP) patients (n=12). Human normal ALDHhi cells were isolated from bone marrow of healthy volunteers after obtaining informed consents. For analysis of THAP11 mRNA expression, quantitative RT-PCR was performed in all cell lines treated with Abl kinase inhibitors (STI571, AMN107, and BMS354825). For cell survival analysis and the levels of c-Myc, Fbw7 and some CDKIs in CML cells, MTT assays, western blot and cell cycle analysis were performed in all cell lines transfected with THAP11 siRNA or cDNA. For colony analysis, the colonies of CFU-GEMM, CFU-GM, and BFU-E were counted in CML stem/progenitor cells transfected with THAP11 siRNA or cDNA, or treated with Abl kinase inhibitors. [Results] In CML cell lines, the expressions of THAP11 mRNA and protein were significantly increased by treatment with Abl kinase inhibitors or transfection with Bcr-Abl siRNA. In CML cells transfected with the THAP11 cDNA, it is shown that CML cell proliferation was inhibited, and the expression of c-Myc protein was decreased compared to the untransfected cells. In addition, the inhibition of Bcr-Abl by the Abl kinase inhibitors or depletion of Bcr-Abl induced the expression of Fbw7 in CML cells. In CML stem/progenitor cells (ALDHhi cells) obtained from patients with CML, the expression of THAP11 mRNA was suppressed, and the transfection with Bcr-Abl siRNA or treatment with Abl kinase inhibitors increased the expression of THAP11 and Fbw7. In CFU-GEMM, CFU-GM, and BFU-E, treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced the THAP11 and Fbw7 expression and reduced the c-Myc expression, and inhibited colony formation of Bcr-Abl+ progenitor cells. [Conclusion] In CML cells, we found that the depletion of THAP11 and Fbw7 expression. We showed that Bcr-Abl induced the expression of c-Myc by depletion of THAP11 and also inhibited the degradation of c-Myc by depletion of Fbw7. This study showed that Bcr-Abl promoted the CML cell proliferation through the THAP11 and Fbw7 mediated-c-Myc aberrant expression. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 5105 [Background and Aims] RKIP (Raf kinase inhibitor protein) regulates cell proliferation, apoptosis, and cell cycle through the inhibition of Raf-1, and it has been reported that the suppression of the RKIP expression promotes the proliferation of tumor cells. However, its function is little known in CML. We found that the expression of RKIP gene and protein was suppressed in CML cells. In this study, we have investigated the expression and the function of the RKIP in CML cell proliferation. [Methods] The cells used in this study were human CML cell lines, K562, Meg01, and SHG3 cells. Primary CML cells (ALDHhicells) were obtained from the bone marrow of CML (CP) patients (n=12). Human normal ALDHhi cells were isolated from bone marrow of healthy volunteers after obtaining informed consents. For analysis of RKIP mRNA expression, quantitative RT-PCR was performed in all cell lines treated with Abl kinase inhibitors (STI571, AMN107, and BMS354825). For proliferation analysis and the levels of Erk1/2 phosphorylation in CML cells, MTT assays, western blot and cell cycle analysis were performed in all cell lines transfected with Bcr-Abl, RKIP siRNA, or RKIP cDNA respectively. For colony analysis, the colonies of CFU-GEMM, CFU-GM, and BFU-E were counted in CML stem/progenitor cells transfected with RKIP siRNA or treated with Abl kinase inhibitors. [Results] In CML cell lines treated with Abl kinase inhibitors or transfection with Bcr-Abl siRNA, the expressions of RKIP mRNA and protein were significantly increased more than untreated cells. Moreover, in CML cells transfected with the RKIP siRNA, the cell proliferation inhibition by treatment of the Abl kinase inhibitors was weakened compared to RKIP siRNA-untransfected cells, and the phosphorylation levels of Erk1/2 and Raf-1 were markedly increased compared to theRKIP siRNA-untransfected cells. On the other hands, the overexpression of RKIP induced G1 cell cycle arrest through p27 and p21 accumulation, decreased the phosphorylation levels of ERK1/2 and Raf-1, and inhibited the proliferation in CML cells. In CML stem/progenitor cells obtained from patients with CML, the expression of RKIP mRNA was suppressed in 10/10 (100 %) of CML. The transfection with Bcr-Abl siRNA or treatment with Abl kinase inhibitors increased the expression of RKIP mRNA and protein, and the overexpression of RKIP decreased the counts of CFU-GEMM, CFU-GM and BFU-E. [Conclusion] Our results demonstrated that the Bcr-Abl suppressed the expression of RKIP, the depletion of RKIP induced Raf-1 activation, and induced the proliferation of CML cells through ERK1/2 phosphorylation. Moreover, in CML, the induction of RKIP expression inhibited the proliferation of CML stem/progenitor cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3255 Poster Board III-1 [Background and Purpose] THAP11 (thanatos-associated protein 11) is one of the zinc-dependent, sequence-specific DNA-binding factors, which regulate cell proliferation, apoptosis and cell cycle. It has been reported that THAP11 is ubiquitously expressed in normal tissues and frequently downregulated in several tumors. In CML cells, we found that THAP11 expression was inhibited. We investigated the mechanisms of the suppression of THAP11 and its function in CML cell proliferation. [Methods] The cells used in this study were human CML cell lines, K562 and Meg01 cells. Primary CML cells were obtained from the bone marrow of CML (CP) patients. Human normal mononuclear cells (MNCs) were isolated from bone marrow of healthy volunteers after obtaining informed consents. For analysis of THAP11 mRNA expression, quantitative RT-PCR was performed in all cell lines treated with Abl kinase inhibitors (STI571, AMN107, and BMS354825). For proliferation analysis and the expression of c-Myc in CML cells, MTT assays, western blot and cell cycle analysis were performed in all cell lines transfected with Bcr-Abl siRNA, THAP11 siRNA, or THAP11 cDNA respectively. Moreover, THAP11 expression, c-Myc expression, and the colony counts of CFU-GEMM, CFU-GM, and BFU-E were analyzed in CML stem/progenitor cells transfected with Bcr-Abl siRNA or treated with Abl kinase inhibitors. [Results] In CML cell lines treated with Abl kinase inhibitors or transfected with Bcr-Abl siRNA, the expressions of THAP11 and c-Myc mRNA and protein were significantly increased compared to untreated cells. On the other hand, in CML cells transfected with the THAP11 cDNA, the c-Myc expression was suppressed. The overexpression of THAP11 induced G1 cell cycle arrest through p27 and p21 accumulation, and inhibited the CML cell proliferation. Moreover, in CML stem/progenitor cells obtained from patients with CML, the transfection with Bcr-Abl siRNA or treatment with Abl kinase inhibitors increased the expression of THAP11 mRNA and protein, and decreased the c-Myc expression and the counts of CFU-GEMM, CFU-GM and BFU-E. [Conclusion] Our results demonstrated that the Bcr-Abl suppressed the expression of THAP11, the depletion of THAP11 induced c-Myc expression, and induced the proliferation of CML cells through cell cycle progression. Moreover, induction of THAP11 expression inhibited the proliferation of CML stem/progenitor cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1199 [Background and Aims] CABLES1 (cyclin-dependent kinase (CDK)-5 and ABL enzyme 1) is a regulator of cell proliferation, apoptosis, and cell cycle, and it has been reported to be lost in a variety cancers. It has been also reported that knockout of the Cable1 gene has minimal to no effect on hematopoietic stem cells. However, we found that the expression of Cables1 gene and CABLES1 protein was suppressed in CML cells, and its function is little known in CML. In this study, we have investigated the function of CABLES1 in CML cell proliferation. [Methods] The cells used in this study were human CML cell lines, K562, Meg01 and SHG3 cells. Primary CML cells (ALDHhi cells) were obtained from the bone marrow of CML (CP) patients (n=12). Human normal ALDHhi cells were isolated from bone marrow of healthy volunteers after obtaining informed consents. For analysis of Cables1 mRNA expression, quantitative RT-PCR was performed in all cell lines treated with Abl kinase inhibitors (STI571, AMN107, and BMS354825). For cell survival analysis and the levels of p53 and some CDKIs in CML cells, MTT assays, western blot and cell cycle analysis were performed in all cell lines transfected with Cables1 shRNA or cDNA. For colony analysis, the colonies of CFU-GEMM, CFU-GM, and BFU-E were counted in CML stem/progenitor cells transfected with Cables1 cDNA or shiRNA, or treated with Abl kinase inhibitors. [Results] In CML cell lines, the expressions of Cables1 mRNA and CABLES1 protein were significantly increased by treatment with Abl kinase inhibitors or transfection with Bcr-Abl shRNA. In CML cells transfected with the Cables1 cDNA, it is shown that CML cell proliferation was inhibited, and the phosphorylation levels of p53, and the expression of BAX and p21 protein were markedly increased compared to the untransfected cells. In addition, the overexpression of CABLES1 induced G1 cell cycle arrest and reduced the DiOC6 fluorescence, indicating breakdown of the mitochondrial membrane potential in CML cells. On the other hand, the changes of p73 and p27 protein expression were not detected. Moreover, in CML cells transfected with Cables1 shRNA, the inhibition of CML cell proliferation by the Abl kinase inhibitors were weakened. In CML stem/progenitor cells (ALDHhi cells) obtained from patients with CML, the expression of Cables1 mRNA was suppressed, and the transfection with Bcr-Abl shRNA or treatment with Abl kinase inhibitors increased the expression of Cables1 mRNA and CABLES1 protein, and decreased the counts of CFU-GEMM, CFU-GM and BFU-E. [Conclusion] Our results demonstrated that the Bcr-Abl suppressed the expression of CABLES1, and the depletion of CABLES1 promotes cell cycle progression and p53-dependent apoptosis. Moreover, the induction of CABLES1 expression has the potentiality to eradicate CML stem/progenitor cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4814 Background and Aims We synthesized two phospha sugar derivatives, 2,3,4-tribromo-3-methyl-1-phenylphospholane 1-oxide (TMPP) by reacting 3-methyl-1-phenyl-2-phospholene 1-oxide with bromine, and we has reported their potential as an antileukemic agent in cell lines. In this study, we have investigated that TMPP have antileukemic effects as shown in various leukemia cell lines as well as primary AML patient specimens. Moreover, we have identified the target molecules of TMPP by using docking simulation. Methods In human leukemia cell lines (HL60, NB4, U937, NOMO-1, CEM, MOLT4, SUP-B15, and YRK2 cells), the anti-leukemic effects were elevated by cell proliferation assay, Cell cycle analysis, Western blotting. In AML cells derived from AML patients and normal hematopoietic progenitor cells derived from healthy volunteers, the changes of viability by treatment with TMPP were evaluated. By using docking simulation, the target molecules were identified in leukemia cells treated with TMPP. Results TMPP showed inhibitory effects on leukemia cell proliferation, with mean IC50 values of 10.2 nmol/L for TMPP, indicating that inhibition appeared to be dependent on the number of bromine atoms in the structure. Further, TMPP at 8.4 nmol/L induced G2/M cell cycle block in leukemia cells, and TMPP at 17.3 nmol/L induced apoptosis in these cells. TMPP treatment reduced cell cycle progression signals (FoxM1, KIS, Cdc25B, Cyclin D1, Cyclin A, and Aurora-B) and tumor cell survival (Bcl-2, p27Kip1 and p21Cip1). Further, treatment with TMPP significantly reduced the viability of AML specimens derived from AML patients, but only slightly reduced the viability of normal ALDHhi progenitor cells. We also found that TMPP showed the strong activity against Aurora kinase B and Bcl-2 by using docking simulation. Conclusion We demonstrate that TMPP inhibits Aurora kinase B and Bcl-2 and shows a dominant Aurora B kinase inhibition-related cell cycle arrest and the induction of mitochondrial potential catastrophe-related apoptosis in acute leukemia cells. Moreover, we identified Aurora kinase B and Bcl-2 as the target molecules of TMPP. We conclude that TMPP has great therapeutic potential in anticancer therapy in a wide range of cancers. Disclosures: No relevant conflicts of interest to declare.

Description: Cyclooxygenase-2 (COX-2) is reported to regulate apoptosis and to be an important cellular target for therapy. In this study, we demonstrated that etodolac, a COX-2 inhibitor, inhibited proliferation and induced apoptosis in myeloma cell lines (RPMI 8226 and MC/CAR cells), expressing the COX-2 enzyme. In both cell lines, etodolac more strongly induced apoptosis compared with thalidomide or meloxicam. Etodolac induced down-regulation of bcl-2 protein and mRNA, activation of caspase-9, -7 and -3, down-regulation of caspase inhibitors, cIAP-1 and survivin, and loss of mitochondrial membrane potential in a dose-dependent manner. In addition, our data demonstrated that when myeloma cells were coincubated with 50 mM etodolac on bone marrow stromal cells (BMSC), myeloma cell adhesion to BMSC was significantly inhibited compared with thalidomide or meloxicam coincubation, and the adhesion molecules VLA-4, LFA-1 (CD11a), CXCX4, and CD44 were suppressed on myeloma cells treated with etodolac. Moreover, we found that 100 mM R-etodolac, S-etodolac, and the combination of R- and S-etodolac, which are the stereoisomers of etodolac, slightly inhibited the proliferation of myeloma cells, while 50 to 100 mM etodolac significantly inhibited the proliferation of myeloma cells. In conclusion, our findings indicate that etodolac induced apoptosis via a bcl-2 dependent pathway, suppressed the expression of adhesion molecules, and inhibited myeloma cell adhesion to BMSC compared with thalidomide or meloxicam. Thus, the activities of etodolac potentially extend to the treatment of patients with myeloma resistant to standard chemotherapy, including thalidomide.