The potent bisphosphonate ibandronate does not induce myeloma cell apoptosis in a murine model of established multiple myeloma

Abstract Azacytidine (AZA), a DNA methyltransferase inhibitor, has been shown to inhibit cell growth and induce apoptosis in some cancer cells. We determined the impact of AZA on a panel of human myeloma cell lines (HMCL); KMS 12PE, KMS 18, LP-1, NCI-H929, OPM-2, RPMI-8226 and U266 and in an in vivo murine model of multiple myeloma (5T33 model). Dose responsiveness to AZA was determined via MTS assays with a range of AZA doses (1–10mM) for 72 hours. FACS and cell cycle analysis were used to evaluate the profile of the cells after exposure to AZA for 72 hours. MTS assays demonstrated a dose and time dependent AZA-induced inhibition of HMCL viability with effective concentrations of AZA ranging from 1–10 mM. This was associated with accumulation of cells in the Go/G1 phase with decreasing number of cells in the S and G2/M phases. Western Blot analysis using antibodies against caspases 3,8,10, PARP, phospho-ERK, ERK, Stat3 and phospho -Stat3 were performed to help characterize the mechanism(s) of cell killing. Cleavage of caspases 3,8,10 and PARP within 24 hours of AZA treatment confirmed early AZA-induced HMCL apoptosis. phospho-ERK which was absent in untreated U266 appeared after 48 hours exposure to 5mM AZA. Similarly inhibitors of caspases 3,8 and 9 were used to determine which apoptotic pathway was being preferentially activated by AZA. Inhibitors of both caspase 3 and 9 effectively abrogated AZA-induced apoptosis in U266 and NCI-H929. In contrast caspase 8 inhibitor was less effective which is consistent with AZA acting via the mitochondrial apoptotic pathway. Reactivation of p16 gene by AZA-induced hypomethylation was assessed with methylation specific PCR. MSP-PCR of the p16 gene indicated a loss of methylation and up-regulated transcription after 48 hours treatment with 5 mM AZA. The level of IL-6 in conditioned media from U266 cells treated with AZA was determined by ELISA assay and demonstrated a rapid fall in autocrine IL-6 production. RT-PCR demonstrated rapid AZA-induced cessation of IL-6 transcription temporarily associated with the disappearance of upstream phospho -Stat3. Addition of exogenous IL-6 did not rescue U266 from AZA-induced apoptosis. AZA was also administered to a 5T33 murine model of multiple myeloma at increasing concentrations (1, 3, 10 mg/kg). At 10 mg/kg the median survival of vehicle versus AZA treated mice was 28 days versus 30+ days (p=0.003). These findings justify further evaluation of AZA as a potential therapeutic agent for multiple myeloma.

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Pentameric IgM Monoclonal Antibodies Specific for Human β2-Microglobulin Are More Potent at Inducing Apoptosis in Multiple Myeloma Cells

Blood ◽

10.1182/blood.v112.11.5177.5177 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5177-5177

Author(s):

Jing Yang ◽

Yabing Cao ◽

Sungyongl Hong ◽

Haiyan Li ◽

Larry W. Kwak ◽

...

Keyword(s):

Multiple Myeloma ◽

Monoclonal Antibodies ◽

Mhc Class I ◽

Cell Apoptosis ◽

Myeloma Cell ◽

Class I ◽

Cross Link ◽

Tumoricidal Activity ◽

Myeloma Cells ◽

Mhc Class I Molecules

Abstract Multiple myeloma is still an incurable plasma malignancy because patients are prone to quickly relapse after conventional treatment or even high-dose chemotherapy. At the present time, monoclonal antibodies (mAbs) are being successfully used to treat cancers. We recently discovered that anti-β2-microglobulin (β2M) mAbs (IgG1 isotype) induced myeloma cell apoptosis (Yang et al., Cancer Cell2006; 10:295–307). The mAbs bind, cross-link, and recruit surface β2M/MHC class I molecules on myeloma cells to lipid rafts where downstream signaling pathways are activated. Therefore, it is possible that enhancing the capacity of the mAbs to cross-link surface MHC class I could further improve the efficacy of mAb-induced myeloma cell apoptosis. To examine this hypothesis, we generated anti-β2M mAbs of IgM isotype with a pentameric structure. By using Annexin V and TUNEL assays, we showed that, compared with monomeric IgG mAbs, IgM anti-β2M mAbs exhibit stronger tumoricidal activity on all six myeloma cell lines and primary myeloma cells isolated from five myeloma patients in dose- and time-dependent manner. About 80% to 90% of myeloma cells were apoptotic when treated with IgM mAb at a concentration as low as 20 mg/mL in a 12-hour culture. Furthermore, IgM anti-β2M mAbs, which at the same dose had stronger therapeutic efficacy than IgG mAbs in vivo, had greatly reduced tumor burdens and retarded tumor growth in SCID mice. To examine whether the pentameric structure plays an important role in IgM mAb-mediated tumoricidal activity, IgM mAbs were treated with β-mercaptoethanol (2ME), a specific agent that breaks the J-chain of IgM antibody leading to irreversible disruption of the IgM pentameric structure. By using native gel electrophoresis, we confirmed that without 2ME treatment, IgM mAbs were pentamers with one band of 950 kDa. After treatment with 2ME, IgM mAbs displayed a strong band in 175 kDa (monomer), indicating that 2ME completely broke pentamers to monomers. Furthermore, we found that the addition of 2ME-pretreated IgM mAbs to cell culture induced much weaker myeloma cell apoptosis than pentameric IgM mAbs. By using Western blot analysis, we further showed that 2ME-pretreated IgM mAbs induce weaker JNK activation and caspase-9, -8, -3, and PARP cleavage. By using confocal microscopy, we showed that 2ME-pretreated IgM mAbs are much less efficient at recruiting β2M/MHC class I molecules into lipid rafts on myeloma cells, although 2ME-pretreated IgM mAbs bound well to myeloma cell surface. Thus, our findings indicate that enhancing cross-linking of surface β2M/MHC class I molecules may be a novel approach to improve the antimyeloma efficacy of the mAbs. This study also suggests that our IgM anti-β2M mAbs may be a better therapeutic agent for future clinical application.

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