Drug-resistant Multiple Myeloma Cell Lines Research Articles

Chemosensitization of CDDP-Resistant Multiple Myeloma Cell Lines Following Treatment with Anti-TfR IgG3-Av.

Multiple myeloma (MM) is an incurable proliferative disorder of monoclonal malignant plasma cells. Multiple agents conventional dose chemotherapy regimens have resulted in improved response rates, however, there have been no significant improvements in overall survival. Recently, Bortezomib has been introduced for the treatment of MM and has resulted in significant response rates. Nevertheless, additional strategies for direct treatment of MM patients are urgently needed. In general, cancer cells have been shown to express significantly higher levels of the transferrin receptor (TfR) and thus, this receptor is an attractive target for cancer therapy. We have generated an anti-TfR IgG3-avidin fusion protein (anti-TfR-IgG3-Av) that can deliver biotinylated molecules into cancer cells and exhibits intrinsic pro-apoptotic activity against hematopoietic malignant cells including myeloma (Ng P. et al., 2002, PNAS 99:10706–11). We have also reported that certain monoclonal antibodies (e.g., rituximab) can sensitize tumor cells to various chemotherapeutic drug-induced apoptosis (Jazirehi and Bonavida, 2005, Oncogene 65:264–76). Thus, this study investigated whether anti-TfR IgG3-Av sensitizes drug-resistant MM cell lines to drug-induced apoptosis. We initially examined the CDDP-resistant lymphoblastoid IM-9 and ARH-77 cell lines for chemosensitization using a suboptimal dose of anti-TfR IgG3-Av. Several methods were used to assess the cytotoxic activity, namely viable cell recovery and apoptosis by both PI and caspase-3 activation. The findings demonstrate that treatment of tumor cells with anti-TfR IgG3-Av for 18 h following treatment with CDDP for an additional 18 h resulted in significant cytotoxicity and apoptosis and synergy was achieved. The combined treatment also resulted in significant mitochondrial membrane depolarization, suggesting that apoptosis was achieved through a type-II pathway. In comparison with tumor cell treatment with anti-TfR-IgG3-Av, the anti-TfR IgG3 was a more potent chemosensitizer, based on both the concentration used and the extent of apoptosis achieved. These findings demonstrate that anti-TfRIgG3-Av is a potential therapeutic antibody that will reverse MM drug resistance when used in combination with chemotherapeutic drugs.

Trisenox (Arsenic Trioxide) in the Treatment for Multiple Myeloma after Bone Marrow Transplantation.

Multiple myeloma (MM) remains an incurable disease, with median survival of 4–6 years despite various new therapeutic approaches including autologous and allogeneic bone marrow transplantation (BMT). New agents are studied and Arsenic Trioxide (ATO) seems to be a promising additional agent against progressive and refractory MM. Various mechanisms are described for the therapeutic effect of ATO: growth inhibition of myeloma cells; induction of apoptosis of drug resistant MM cell lines; inhibition of angiogenesis by blocking VEGF; interaction with BM microenvironment; immunologic mechanisms by a marked increase in lymphokine-activated Killer (LAK)-mediated killing and up-modulation of CD38 and CD54; inhibition of myeloma cells to stick to BM stromal cells by inhibiting IL-6. ATO is a form of arsenic, a naturally occurring element (As2O3), which was used for various therapeutic purposes for more than 2000 years. ATO was first approved by the FDA in 2000 for acute promyelocytic leukemia (APL), and is investigated for myelodysplastic syndrome (MDS) and other indications for ATO are currently under investigation.We report our preliminary data from our clinical experience with ATO for 4 patients with MM after BMT, who had evidence of active and progressive disease. Other therapeutic approaches failed or were not relevant. ATO was given at low doses of 0.15 mg/kg/dose twice weekly and after respond at 0.3 mg/kg/dose once weekly. ATO was given intravenously in slow infusion over 2 hours together with intravenous lipids 500 ml (20%) to reduce ATO toxicity. ECG, markers for myeloma and possible adverse effects were monitored carefully. Oral ascorbic acid 500 mg twice daily was added to improve the effect of ATO. Our patients showed evidence of response to ATO both at the level of residual tumor mass and M-protein secretion. Furthermore, in one patient with severe GvHD we observed significant improvement of the GvHD manifestations concomittantly with Graft versus Myeloma effect.Based on our data and the review of the literature regarding the use of ATO for MM, ATO might play in the future an established role in the treatment for MM. ATO might induce and augment immunomodulatory effects of Graft versus Myeloma. We discuss future research for the use of ATO in the treatment of MM.

The Proteasome Inhibitor PR-171 Inhibits Cell Growth, Induces Apoptosis, and Overcomes De Novo and Acquired Drug Resistance in Human Multiple Myeloma Cells.

Proteasome inhibitors are a new class of drugs that have shown to be cytotoxic and induce apoptosis in tumor cells, particularly, multiple myeloma (MM). This led to the clinical evaluation and subsequent FDA approval of the proteasome inhibitor, bortezomib, for the treatment of refractory and relapsed multiple myeloma. However, continued occurrence of refractoriness, development of resistance, and the toxicity of bortezomib in treated patients has led to the recent development of novel agents with increased binding efficiency and specificity to the proteasome. PR-171 is a member of the epoxomicin-class of compounds that act as potent and selective irreversible inhibitors of the chymotrypsin-like protease activity of the 20S proteasome resulting in accumulation of ubiquinated proteins and apoptosis of tumor cells (Ming et al., PNAS 96:10403). In this study, we evaluated the effects of PR-171 on human MM cells' survival, proteasome activity and drug resistance. PR-171 induced growth inhibition and apoptosis in the drug sensitive human MM cell lines 8226/S, MM1, H929, and U266, with IC50 values ranging from 9 to 21nM. PR-171 retains activity on cells resistant to melphalan, mitoxantrone, and zarnestra, but has reduced activity on cells resistant to doxorubicin. Pretreatment with verapamil restored sensitivity of the doxorubicin resistant cells (8226/Dox40) to PR-171 back to levels seen in the parental, drug sensitive cell line (8226/S). Proteasome activity was inhibited by PR-171 in the drug sensitive and drug resistant MM cell lines, although much less inhibition was observed in the doxorubicin resistant cells, consistent with the reduced effects of PR-171 on cell viability. In freshly isolated MM cells from patients, PR-171 at doses of 10–250nM induced apoptosis in 9/9 samples tested from both newly diagnosed and relapsed refractory patients. To test the effects of PR-171 on Cell Adhesion Mediated Drug Resistance (CAM-DR), MM cell lines were treated with drug after adhesion to fibronectin. PR-171 at 1–100nM overcomes CAM-DR in 8226/S and H929 cell lines, but not in MM1. The ability of PR-171 to overcome CAM-DR was also seen in primary MM cells from patients in 6 out of 9 samples tested. In summary, we have shown that PR-171 directly inhibits proteasome activity, proliferation, and induces apoptosis in human MM sensitive and resistant cell lines, induces apoptosis in isolated MM cells from newly diagnosed and relapsed refractory patients, and overcomes de novo drug resistance. These studies provide the foundation for clinical investigation of PR-171 to improve the outcome of patients with MM and other hematological malignancies.

FTY720 Induces Apoptosis in Multiple Myeloma Cells and Overcomes Drug Resistance

The novel immunomodulator FTY720 down-modulates sphingosine-1-phosphate receptor 1 on lymphocytes at low nanomolar concentrations, thereby inhibiting sphingosine-1-phosphate receptor 1-dependent egress of lymphocytes from lymph nodes into efferent lymphatics and blood. At high micromolar concentration, FTY720 has been shown to induce growth inhibition and/or apoptosis in human cancer cells in vitro. In this study, we investigated the biological effects of FTY720 on multiple myeloma cells. We found that FTY720 induces potent cytotoxicity against drug-sensitive and drug-resistant multiple myeloma cell lines as well as freshly isolated tumor cells from multiple myeloma patients who do not respond to conventional agents. FTY720 triggers activation of caspase-8, -9, and -3, followed by poly(ADP-ribose) polymerase cleavage. Interestingly, FTY720 induces alterations in mitochondrial membrane potential (DeltaPsim) and Bax cleavage, followed by translocation of cytochrome c and Smac/Diablo from mitochondria to the cytosol. In combination treatment studies, both dexamethasone and anti-Fas antibodies augment anti-multiple myeloma activity induced by FTY720. Neither interleukin-6 nor insulin-like growth factor-I, which both induce multiple myeloma cell growth and abrogate dexamethasone-induced apoptosis, protect against FTY720-induced growth inhibition. Importantly, growth of multiple myeloma cells adherent to bone marrow stromal cells is also significantly inhibited by FTY720. Finally, it down-regulates interleukin-6-induced phosphorylation of Akt, signal transducers and activators of transcription 3, and p42/44 mitogen-activated protein kinase; insulin-like growth factor-I-triggered Akt phosphorylation; and tumor necrosis factor alpha-induced IkappaBalpha and nuclear factor-kappaB p65 phosphorylation. These results suggest that FTY720 overcomes drug resistance in multiple myeloma cells and provide the rationale for its clinical evaluation to improve patient outcome in multiple myeloma.

Azaspirane (N-N-diethyl-8,8-dipropyl-2-azaspiro [4.5] decane-2-propanamine) inhibits human multiple myeloma cell growth in the bone marrow milieu in vitro and in vivo

AbstractAzaspirane (N-N-diethyl-8,8-dipropyl-2-azaspiro [4.5] decane-2-propanamine; trade name, Atiprimod) is an orally bioavailable cationic amphiphilic compound that significantly inhibits production of interleukin 6 (IL-6) and inflammation in rat arthritis and autoimmune animal models. We here characterize the effect of atiprimod on human multiple myeloma (MM) cells. Azaspirane significantly inhibited growth and induced caspase-mediated apoptosis in drug-sensitive and drug-resistant MM cell lines, as well as patient MM cells. IL-6, insulin-like growth factor 1 (IGF-1), or adherence of MM cells to bone marrow stromal cells (BMSCs) did not protect against atiprimod-induced apoptosis. Both conventional (dexamethasone, doxorubicin, melphalan) and novel (arsenic trioxide) agents augment apoptosis induced by atiprimod. Azaspirane inhibits signal transducer activator of transcription 3 (STAT3) and a PI3-K (phosphatidylinositol 3-kinase) target (Akt), but not extracellular signal-regulated kinase 1 and 2 (ERK1/2), inhibits phosphorylation triggered by IL-6, and also inhibits inhibitorκBα (IκBα) and nuclear factor κB (NFκB) p65 phosphorylation triggered by tumor necrosis factor α (TNF-α). Of importance, azaspirane inhibits both IL-6 and vascular endothelial growth factor (VEGF) secretion in BMSCs triggered by MM cell binding and also inhibits angiogenesis on human umbilical vein cells (HUVECs). Finally, azaspirane demonstrates in vivo antitumor activity against human MM cell growth in severe combined immunodeficient (SCID) mice. These results, therefore, show that azaspirane both induces MM cell apoptosis and inhibits cytokine secretion in the BM milieu, providing the framework for clinical trials to improve patient outcome in MM.

Novel therapies for multiple myeloma.

Novel therapies for multiple myeloma.