beta-lapachone,a novel plant product,overcomes drug resistance in human multiple myeloma cells

OBJECTIVE: To evaluate the anti-tumor potential of beta-lapachone in multiple myeloma (MM) cell lines (U266, RPMI8226, and MM.1S); MM cell lines resistant to dexamethasone (MM.1R), melphalan (RPMI8226/LR5), doxorubicin (RPMI8226/DOX40), and mitoxantrone (RPMI8226/ MR20); and MM cells from patients (MM1-MM4). MATERIALS AND METHODS: Cytotoxicity of beta-lapachone was assessed by MTT and [3H]-thymidine uptake assays. Apoptosis was analyzed using propidium iodide staining, DNA fragmentation, TUNEL assay, caspase-9 colorimetric assay, and immunoblotting for caspase-3, poly (ADP-ribose) polymerase (PARP), and caspase-8 cleavage products. Paracrine growth of MM cells was assessed by [3H]-thymidine uptake in cultures of bone marrow stromal cells (BMSCs) and MM cells. Interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) secretion in the culture supernatants was measured by specific enzyme-linked immunosorbent assays (ELISAs). RESULTS: beta-lapachone showed significant cytotoxicity in MM cells (IC(50): 4-8 microM). In contrast, normal peripheral blood mononuclear cells (PBMCs) and BMSCs from MM patients were relatively resistant (IC(50): 8-16 microM). IL-6 did not protect against beta-lapachone-induced apoptosis in MM.1S cells, and dexamethasone showed additive cytotoxicity. beta-lapachone also decreased binding of MM.1S cells to BMSCs; abrogated IL-6 and VEGF secretion triggered by adhesion of BMSCs to MM.1S cells; reduced proliferation of MM.1S cells adherent to BMSCs; and decreased intracellular adhesion molecule-1 (ICAM-1) expression on MM.1S cells. Furthermore, beta-lapachone induced typical PARP cleavage, increased caspase-9 proteolytic activity, and activation of caspase-3, without activation of caspase-8 in U266 cells. CONCLUSION: These studies provide a framework for clinical evaluation of beta-lapachone to improve the outcome for patients with MM.     

脑源性神经营养因子及其受体在多发性骨髓瘤细胞中的表达及意义

目的 研究多发性骨髓瘤（MM）脑源性神经营养因子（BDNF）及其受体的表达水平在MM发生与发展中的作用。方法 RT-PCR法、Western blot法检测人类MM细胞系（HMCLs）RPMI 8226、U266、KM3细胞BDNF及其受体P75^NTR和TrkB的表达;ELISA法检测HMCLs BDNF的分泌水平;免疫组织化学染色法检测MM患者骨髓活检切片中BDNF和TrkB的表达。采用四唑盐比色法观察BDNF对MM细胞增殖的影响;用改良的Boyden小室法观察BDNF对MM细胞迁移影响。结果 HMCLs不仅表达和分泌BDNF蛋白,也表达其高亲和力酪氨酸激酶受体TrkB。骨髓活检15例MM患者有12例瘤细胞BDNF阳性表达（80％）,15例TrkB阳性表达（100％）;而9例对照组正常造血细胞仅有2例和4例弱阳性表达。研究表明BDNF以剂量和时间依赖性方式促进MM细胞的增殖,并可明显促进MM细胞的迁移。结论 MM中存在着BDNF及其受体TrkB的异常表达,BDNF可能通过促进MM细胞的增殖、迁移参与MM的病理生理进程。     

Targeted inhibition of interleukin-6 with CNTO 328 sensitizes pre-clinical models of multiple myeloma to dexamethasone-mediated cell death

Interleukin (IL)-6-mediated signalling attenuates the anti-myeloma activity of glucocorticoids (GCs). We therefore sought to evaluate whether CNTO 328, an anti-IL-6 monoclonal antibody in clinical development, could enhance the apoptotic activity of dexamethasone (dex) in pre-clinical models of myeloma. CNTO 328 potently increased the cytotoxicity of dex in IL-6-dependent and -independent human myeloma cell lines (HMCLs), including a bortezomib-resistant HMCL. Isobologram analysis revealed that the CNTO 328/dex combination was highly synergistic. Addition of bortezomib to CNTO 328/dex further enhanced the cytotoxicity of the combination. Experiments with pharmacologic inhibitors revealed a role for the p44/42 mitogen-activated protein kinase pathway in IL-6-mediated GC resistance. Although CNTO 328 alone induced minimal cell death, it potentiated dex-mediated apoptosis, as evidenced by increased activation of caspases-8, -9 and -3, Annexin-V staining and DNA fragmentation. The ability of CNTO 328 to sensitize HMCLs to dex-mediated apoptosis was preserved in the presence of human bone marrow stromal cells. Importantly, the increased activity of the combination was also seen in plasma cells from patients with GC-resistant myeloma. Taken together, our data provide a strong rationale for the clinical development of the CNTO 328/dex regimen for patients with myeloma.     

Dexamethasone plus retinoids decrease IL-6/IL-6 receptor and induce apoptosis in myeloma cells

Interleukin 6 (IL-6) is the most important known growth factor for multiple myeloma, and IL-6 signalling pathways are potential targets for therapy. We hypothesized that interfering with the IL-6 signalling pathway at more than one level would be more effective than a single block in inhibiting proliferation of myeloma cells. Accumulating data support the concept that glucocorticoids down-regulate IL-6, whereas retinoic acid derivatives (RA) down-regulate IL-6R in myeloma. We found that all- trans RA (ATRA), 13- cis -RA and 9- cis -RA each similarly inhibited growth of RPMI 8226 myeloma cells and that addition of dexamethasone (DEX) added to RA growth inhibition. The major effects of retinoids were to reduce the proliferative fraction and induce apoptosis whereas DEX increased the apoptotic fraction. When combined, apoptosis was enhanced. Effects of RA + DEX were also least able to be overcome by exogenous IL-6. RA decreased IL-6R levels and addition of DEX to RA delayed recovery of IL-6R levels compared with RA alone. Since RPMI 8226 cells have undetectable IL-6, we investigated U266B1 cells and found that RA and DEX decreased both IL-6 secretion and IL-6 RNA levels. Mechanistically, IL-6R down-regulation by RA was enhanced by DEX, whereas IL-6 protein and RNA levels were reduced by DEX and by RA. In summary, combinations of RA + DEX were not only more effective in inhibiting myeloma cells growth by the dual mechanisms of decreasing proliferative fraction and increasing apoptotic fraction, but were also less able to be overcome by IL-6.     

Analysis of histone deacetylase inhibitor, depsipeptide (FR901228), effect on multiple myeloma

Multiple myeloma (MM) is a neoplastic proliferation of plasma cells and remains an incurable disease because of the development of drug resistance. Histone deacytylase (HDAC) inhibitors are a new class of chemotherapeutic reagents that cause growth arrest and apoptosis of neoplastic cells. Depsipeptide, a new member of the HDAC inhibitors, was found to be safe in humans and has been shown to induce apoptosis in various cancers. In order to evaluate the effects of depsipeptide, a MM cell line, U266 [interleukin (IL)-6 dependent], was analysed for viability and apoptosis. The combined effect of depsipeptide with melphalan and changes in BCL-2 family proteins (BCL-2, BCL-XL, BAX and MCL-1) were also investigated. In addition, the RPMI 8226 cell line (IL-6 independent), and primary patient myeloma cells were also analysed for apoptosis after depsipeptide treatment. Depsipeptide induced apoptosis in both U266 and RPMI 8226 cell lines in a time- and dose-dependent fashion, and in primary patient myeloma cells. We also demonstrated that depsipeptide had an additive effect with melphalan (10 micromol/l). BCL-2, BCL-XL and MCL-1 showed decreased expression in depsipeptide-treated samples. Based on recent clinical trials demonstrating minimal clinical toxicity, our study supports the future clinical utilization of depsipeptide in the management of MM.     

Interleukin-6 sensitizes multiple myeloma cell lines for apoptosis induced by interferon-alpha

OBJECTIVE: Interleukin-6 (IL-6) is a multifunctional cytokine affecting growth and survival of normal B cell lineage and multiple myeloma cells. To test the hypothesis that IL-6, as well as other hematopoietic growth factors, may enhance apoptosis of target cells, we investigated the effect of IL-6 on myeloma cells in the presence of IFN-alpha, which is prescribed for patients with multiple myeloma. MATERIALS AND METHODS: Four myeloma cell lines, PCM6, NOP-2, U266, RPMI8226 were tested. We determined the induction of apoptosis by flow cytometry, using an FITC-Annexin V. RESULTS: IFN-alpha induced apoptosis on myeloma cell lines, and this apoptosis was further enhanced in the presence of IL-6, via activation of caspase 3. During induction of this apoptosis, the expression of c-Myc and Fas increased. The addition of IL-6 further increased the expression of Fas, but not that of c-Myc. Bcl-2, Bcl-x, and p53 were not affected by the addition of IL-6 and/or IFN-alpha. Addition of a PI-3-K inhibitor interfered with the enhancing effect of IL-6 on the apoptosis induced by IFN-alpha. CONCLUSION: We propose that IL-6 has the death signal, as well as growth promoting effects, and that PI-3-K may play a key role in the induction of apoptosis by IL-6.     

Antitumor activity of fludarabine against human multiple myeloma in vitro and in vivo

Fludarabine, a nucleoside analogue, plays a major role in the treatment of B-cell lymphocytic leukemia, hairy cell leukemia, and indolent lymphomas. There is a controversy about antitumor activity of fludarabine in multiple myeloma (MM). The aim of this study was to evaluate the activity of fludarabine against human myeloma cells both in vivo and in vitro. We demonstrated that myeloma cell line RPMI8226 was efficiently inhibited by fludarabine, concomitantly with decreased phosphorylation of Akt, down-regulation of the inhibitor of apoptosis proteins (IAP) family, including XIAP and survivin, and induction of apoptosis related to activation of caspase cascade. Contrary to dexamethasone, the effect of fludarabine on RPMI8226 cells was independent of interleukin-6. Fludarabine also induced cytotoxicity in dexamethasone-sensitive (MM.1S) and -resistant (MM.1R) cells at 48 h with IC50 of 13.48 microg/mL and 33.79 microg/mL, respectively. In contrast, U266 cells were resistant to fludarabine. Moreover, RPMI8226 myeloma xenograft model was established using severe combined immunodeficient mice. The tumors treated with fludarabine at 40 mg/kg increased less than 5-fold in 25 d comparing with approximately 10-fold in the control tumors, demonstrating the antitumor activity of fludarabine in vivo. These results suggest that fludarabine may be an important therapeutic option for MM patients who are resistant to dexamethasone.     

Suitable drug combination with bortezomib for multiple myeloma under stroma-free conditions and in contact with fibronectin or bone marrow stromal cells

Several clinical trials have demonstrated the effectiveness of bortezomib in combination with various anti-myeloma agents; however, no definitive information is available regarding drugs best suited for use in combination with bortezomib. Using isobologram analysis, we investigated the combined effects of bortezomib with four key anti-myeloma drugs (melphalan, cyclophosphamide, doxorubicin and lenalidomide), which represent components of major bortezomib-based regimens with corticosteroids, in three myeloma cell lines (U266, RPMI8226 and KMS-12BM) under various conditions. Melphalan showed the best performance with bortezomib under all culture conditions tested (liquid culture, on fibronectin-coated plates, and co-culture with bone marrow stromal cells), whereas cyclophosphamide was antagonistic with bortezomib especially in the presence of stromal cells. Doxorubicin showed additive effects under stroma-free conditions and in contact with fibronectin, but was rather antagonistic in the presence of stromal cells. In contrast, lenalidomide exerted the most favorable effect with bortezomib in contact with stromal cells. Consistent with these results, caspase-3 was activated more strongly by melphalan than by other agents in combination with bortezomib. Moreover, bortezomib-induced up-regulation of CHOP was readily enhanced by lenalidomide in contact with stromal cells. The present findings may provide fundamental information for the selection of bortezomib-based regimens for myeloma patients.     

Synergistic action of the novel HSP90 inhibitor NVP‐AUY922 with histone deacetylase inhibitors,melphalan, or doxorubicin in multiple myeloma

Heat shock protein 90 (HSP90) is a promising target for tumor therapy. The novel HSP90 inhibitor NVP‐AUY922 has preclinical activity in multiple myeloma, however, little is known about effective combination partners to design clinical studies. Multiple myeloma cell lines, OPM‐2, RPMI‐8226, U‐266, LP‐1, MM1.S, and primary myeloma cells were exposed to NVP‐AUY922 and one of the combination partners histone deacetylase inhibitor NVP‐LBH589, suberoylanilide hydroxamic acid (SAHA), melphalan, or doxorubicin, either simultaneously or in sequential patterns. Effects on cell proliferation and apoptosis were determined. Synergistic effects were evaluated using the method of Chou and Talalay. Combined sequential incubation with NVP‐AUY922 and SAHA showed that best synergistic effects were achieved with 24 h preincubation with SAHA followed by another 48 h of combination treatment. Combination of NVP‐AUY922 with SAHA, NVP‐LBH589, melphalan, or doxorubicin resulted in synergistic inhibition of viability, with strong synergy (combination index < 0.3) in the case of melphalan. Importantly, resistance of the RPMI‐8226 cell line and relative resistance of some primary myeloma cells against NVP‐AUY922 could be overcome by combination treatment. These data show impressive synergistic action of the novel HSP90 inhibitor NVP‐AUY922 with melphalan, doxorubicin, NVP‐LBH589, and SAHA in multiple myeloma and build the frame work for clinical trials.     

The bortezomib/proteasome inhibitor PS-341 and triterpenoid CDDO-Im induce synergistic anti-multiple myeloma (MM) activity and overcome bortezomib resistance

The synthetic triterpenoid 2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid (CDDO) induces apoptosis in leukemic cells. Here we show that CDDO and its new derivative CDDO-imidazolide (CDDO-Im) trigger apoptosis in multiple myeloma (MM) cells resistant to conventional therapies including melphalan (LR-5), doxorubicin (Dox-40), and dexamethasone (MM.1R, U266, RPMI 8226) without affecting the viability of normal cells. CDDO-IM also triggers apoptosis in bone marrow stromal cells (BMSCs) and decreases interleukin-6 (IL-6) secretion induced by MM cell adhesion to BMSCs. Moreover, CDDO-Im-induced apoptosis in MM cells is not blocked by IL-6 or insulin growth factor-1 (IGF-1). Importantly, CDDO-Im and bortezomib/proteasome inhibitor PS-341 trigger synergistic apoptosis in MM cells associated with loss of mitochondrial membrane potential, superoxide generation, release of mitochondrial proteins cytochrome c/second mitochondria-derived activator of caspases (cytochrome c/Smac), and activation of caspase-8, -9, and -3. Conversely, the pancaspase inhibitor Z-VAD-fmk abrogates the CDDO-Im + bortezomib-induced apoptosis. Low doses of CDDO-Im and bortezomib overcome the cytoprotective effects of antiapoptotic proteins Bcl2 and heat shock protein-27 (Hsp27) as well as nuclear factor-kappa B (NF-kappaB)-mediated growth/survival and drug resistance. Finally, combining CDDO-Im and bortezomib induces apoptosis even in bortezomib-resistant MM patient cells. Together, these findings provide the framework for clinical evaluation of CDDO-Im, either alone or in combination with bortezomib, to overcome drug resistance and improve patient outcome in MM.     

The FA/BRCA pathway is involved in melphalan-induced DNA interstrand cross-link repair and accounts for melphalan resistance in multiple myeloma cells

Melphalan, a DNA cross-linker, is one of the most widely used and effective drugs in the treatment of multiple myeloma (MM). In this report, we demonstrate that enhanced interstrand cross-link (ICL) repair via the Fanconi anemia (FA)/BRCA pathway contributes to acquired drug resistance in melphalan-resistant myeloma cell lines, and disruption of this pathway reverses drug resistance. Using the alkaline comet assay (single-cell gel electrophoresis), we observed that melphalan-resistant cells have reduced ICL formation and enhanced ICL repair compared with melphalan-sensitive cells. Cell-cycle studies demonstrated that enhanced ICL repair released cells from melphalan-induced cell-cycle delay. Using siRNA to knock down FANCF in 8226/LR5 and U266/LR6 drug-resistant cells demonstrated a direct relationship between ICL repair capacity and drug sensitivity. Overexpression of FANCF in 8226/S and U266/S drug-sensitive cells partially reproduced the drug-resistant phenotype. These data show that enhanced DNA repair via the Fanconi anemia/BRCA pathway is involved in acquired melphalan resistance. Our findings provide for a new target to enhance response to DNA cross-linking agents in cancer treatment.     

Myeloma cells express Fas antigen/APO-1 (CD95) but only some are sensitive to anti-Fas antibody resulting in apoptosis

To find out which cytokines are involved in the pathogenesis of multiple myeloma, we investigated cytokine receptor expression on myeloma cells using a panel of monoclonal antibodies (MoAbs). Flow cytometric analysis of five myeloma cell lines (RPMI8226, ARH77, KMM-1, U266, and Hs) and myeloma cells freshly isolated from eight patients showed that interleukin-1 receptor (IL-1R) type I and type II, IL-2R alpha and beta chains, IL-4R, IL-6R, IL-7R, IL-8R, granulocyte macrophage colony-stimulating factor receptor (GM-CSFR), c-kit (stem cell factor receptor [SCFR]), membrane bound stem cell factor (MBSCF), and tumor necrosis factor (TNF) receptors type I and type II were not always detected on the myeloma cells. However, interferon-gamma receptor, gp130, and Fas antigen were constitutively expressed, except one sample. To determine the role of Fas antigen on myeloma cells, these cells were cultured with anti-Fas MoAb. Apoptotic changes characterized by loss of cell volume, membrane blebbing, fragmentation of nuclei, and condensed chromatin were observed in three of five myeloma cell lines. When bcl-2 expression was examined, it was seen in all the cell lines regardless of the sensitivity to anti-Fas MoAb. Furthermore, anti-Fas MoAb not only induced apoptosis of freshly isolated myeloma cells but also inhibited the DNA synthesis, although such effects varied from patient to patient. The data indicate that only some myeloma cells undergo apoptosis in response to the signal mediated by the Fas antigen.     

A phase 2, randomized,double‐blind,placebo‐controlled study of siltuximab (anti‐IL‐6 mAb) and bortezomib versus bortezomib alone in patients with relapsed or refractory multiple myeloma

We compared the safety and efficacy of siltuximab (S), an anti‐interleukin‐6 chimeric monoclonal antibody, plus bortezomib (B) with placebo (plc) + B in patients with relapsed/refractory multiple myeloma in a randomized phase 2 study. Siltuximab was given by 6 mg/kg IV every 2 weeks. On progression, B was discontinued and high‐dose dexamethasone could be added to S/plc. Response and progression‐free survival (PFS) were analyzed pre‐dexamethasone by European Group for Blood and Marrow Transplantation (EBMT) criteria. For the 281 randomized patients, median PFS for S + B and plc + B was 8.0 and 7.6 months (HR 0.869, P = 0.345), overall response rate was 55 versus 47% (P = 0.213), complete response rate was 11 versus 7%, and median overall survival (OS) was 30.8 versus 36.8 months (HR 1.353, P = 0.103). Sustained suppression of C‐reactive protein, a marker reflective of inhibition of interleukin‐6 activity, was seen with S + B. Siltuximab did not affect B pharmacokinetics. Siltuximab/placebo discontinuation (75 versus 66%), grade ≥3 neutropenia (49 versus 29%), thrombocytopenia (48 versus 34%), and all‐grade infections (62 versus 49%) occurred more frequently with S + B. The addition of siltuximab to bortezomib did not appear to improve PFS or OS despite a numerical increase in response rate in patients with relapsed or refractory multiple myeloma. © 2014 Wiley Periodicals, Inc. Am. J. Hematol. 90:42–49, 2015. © 2014 Wiley Periodicals, Inc.     

Overexpression of Annexin II affects the proliferation, apoptosis, invasion and production of proangiogenic factors in multiple myeloma

The abnormal expression of Annexin II (AnxA2, A2) has been associated with the development of tumors; however, its expression and function in multiple myeloma (MM) is less known. We compared the expression of AnxA2 in primary myeloma cells from MM patients with that in normal plasma cells from normal subjects and found that myeloma cells from patients had higher expression of AnxA2. Expression of AnxA2 was also significantly higher in MM cell lines U266 and RPMI8226, compared with other hematologic tumor cell lines. Transfecting U266 and RPMI8226 cells with the small interfering RNA (siRNA) that targets human AnxA2 led to significant downregulation of AnxA2 expression, which resulted in the decreased proliferation, invasive potential and increased apoptosis of U266 and RPMI8226 cell lines. Silencing AnxA2 gene by siRNA also inhibited the expression of pro-angiogenic molecules including VEGF-C, VEGF-R2, MMP-2, MMP-9, MT1-MMP and TIMP-2 in the two cell lines. Our data suggested that the AnxA2 is overexpressed in MM patients and myeloma cell lines U266 and RPMI8226, and that AnxA2 overexpression appeared to affect the proliferation, apoptosis, invasive potential and production of pro-angiogenic factors in MM cell lines U266 and RPMI8226.     

Effect of wild type PTEN gene on proliferation and invasion of multiple myeloma

We explored the effect of the wild type PTEN gene on the proliferation, apoptosis and invasive ability of multiple myeloma (MM) cells from MM patients and RPMI 8226 cells (a human myeloma cell line), and the effect of the PTEN/focal adhesion kinase (FAK)/MMP signaling pathway on the invasion activity of RPMI 8226 cells. The proliferation of RPMI 8226 cells and purified myeloma cells from MM patients were markedly inhibited after these cells were transfected with recombinant adenovirus-PTEN vectors containing green fluorescent protein (Ad-PTEN-GFP). Maximum growth inhibition of RPMI 8226 cells and purified myeloma cells from MM patients by AD-PTEN-GFP was 42.01 and 24.75%, respectively. After transfection with PTEN-siRNA, the proliferation of RPMI 8226 cells was increased significantly compared with NS-siRNA transfected controls. The maximal survival rate was 141.55 ± 8.34% in PTEN-siRNA transfected RPMI 8226 cells. Apoptosis of RPMI 8226 cells or purified myeloma cells from MM patients in the Ad-PTEN-GFP group was increased significantly when compared with that in the Ad-GFP (adenovirus vectors only expressing green fluorescent protein) group (p < 0.01). The cell cycle of RPMI 8226 cells was arrested at the G2/M phase. Furthermore, the number of cells that migrated through the matrigel and filter from the upper chamber to the lower chamber in the transwell assay in the Ad-GFP group was significantly larger than that in the Ad-PTEN-GFP group (52.65 ± 7.39 vs. 23.50 ± 6.12, p < 0.01). In the PTEN-siRNA group, the cell number (79.50 ± 11.89) was significantly larger than that in the NS-siRNA group (47.17 ± 7.76, p < 0.01). When RPMI 8226 cells were transfected with Ad-PTEN-GFP or NS-siRNA, the expression level of PTEN mRNA was up-regulated, and the expression levels of FAK, MMP-2 and MMP-9 mRNA were down-regulated significantly compared with that of the Ad-GFP group and the PTEN-siRNA group (p < 0.01, p < 0.05). The protein levels of FAK and p-FAK, MMP-2 and MMP-9 in RPMI 8226 cells which were transfected with Ad-PTEN-GFP decreased significantly, but increased significantly in PTEN-siRNA transfected RPMI 8226 cells (p < 0.01, p<0.05). These results indicated that wild type PTEN, which inhibited FAK, MMP-2, and MMP-9, could suppress the proliferation and invasion ability of multiple myeloma cells. Modulating the expression of PTEN may be a potential strategy for the treatment of multiple myeloma.     

Paracrine rather than autocrine regulation of myeloma-cell growth and differentiation by interleukin-6

To explore the mechanisms involved in the pathogenesis of human multiple myeloma (MM), we investigated the potential role of interleukin-6 (IL-6), a B-cell differentiation factor in humans, and a growth factor for rat/mouse heterohybridomas and murine plasmacytomas. Using a heterohybridoma assay, we found that two well-documented human myeloma cell lines, RPMI 8226 and U266, did not secrete IL-6 and did not express RNA messengers for IL-6. Neutralizing antibodies to IL-6 did not inhibit their proliferation, and recombinant IL-6 did not stimulate it. Taken together, these data show that IL-6 is not the autocrine growth factor of these human myeloma cell lines. A high production of IL-6 was found in the bone marrows of patients with fulminating MM, compared with patients with inactive or slightly active MM, or to healthy donors. This IL-6 production was assigned to adherent cells of the bone-marrow environment but not to myeloma cells. A spontaneous proliferation of myeloma cells freshly isolated from patients was observed in short-term cultures. Recombinant IL-6 was able to amplify it two- to threefold. The spontaneous proliferation of the myeloma cells was inhibited by anti-IL-6 antibodies and reinduced by recombinant IL-6. After 2 to 3 weeks of culture, the myeloma-cell proliferation progressively declined and no IL-6-dependent myeloma cell lines could be obtained despite repeated additions of fresh IL-6 and costimulation with other cytokines such as tumor necrosis factor (TNF)beta, or IL-1 beta. These data demonstrated a paracrine but not autocrine regulation of the growth and differentiation of myeloma cells by IL-6.     

SDX-101, the R-enantiomer of etodolac, induces cytotoxicity, overcomes drug resistance, and enhances the activity of dexamethasone in multiple myeloma

In this study we report that R-etodolac (SDX-101), at clinically relevant concentrations, induces potent cytotoxicity in drug-sensitive multiple myeloma (MM) cell lines, as well as in dexamethasone (MM.1R)-, doxorubicin (Dox40/RPMI8226)-, and bortezomib (DHL4)-resistant cell lines. Immunoblot analysis demonstrates that R-etodolac induces apoptosis characterized by caspase-8, -9, and -3 and PARP (poly-ADP [adenosine diphosphate]-ribose polymerase) cleavage and down-regulation of cyclin D1 expression. Subcytotoxic doses of R-etodolac up-regulate myeloid cell leukemia-1 proapoptotic variant (Mcl-1S), while enhancing dexamethasone (Dex)-induced caspase activation and apoptosis. The combination of R-etodolac with Dex results in a highly synergistic cytotoxic effect. R-etodolac also induces apoptosis against primary cells isolated from patients with MM refractory to chemotherapy. Although interleukin 6 (IL-6) and insulin-like growth factor-1 (IGF-1) abrogate Dex-induced MM cell cytotoxicity, neither IL-6 nor IGF-1 protects against R-etodolac-induced cytotoxicity in MM cells. R-etodolac also inhibits viability of MM cells adherent to bone marrow stromal cells (BMSCs), thereby overcoming a mechanism of drug resistance commonly observed with other conventional chemotherapeutic agents. Our data, therefore, indicate that R-etodolac circumvents drug resistance in MM cells at clinically relevant concentrations, targets Mcl-1, and can be synergistically combined with Dex.     

All‐trans retinoic acid can intensify the growth inhibition and differentiation induction effect of rosiglitazone on multiple myeloma cells

Objective: Activation of PPARγ by its ligands has shown potential anti‐neoplastic effects in solid tumors. In this study, we investigate the effects of rosiglitazone (RGZ) alone as well as in combination with all trans‐retinoic acid (ATRA) on human myeloma cell lines and try to address its potential mechanism. Methods: U266, RPMI‐8226 and primary myeloma cells from patients were treated with different concentrations of RGZ in the presence or absence of ATRA and various biological responses were studied by the methods of [³H] thymidine incorporation, MTT, cell cycle analysis, Annexin V‐PI staining, Wright‐Giemsa staining, CD49e expression assay, light chain protein detection, RT‐PCR and caspase‐3 activity assay. Results: We report that exposure to RGZ induced proliferation inhibition and viability reduction in a dose‐dependent manner in both U266 and RPMI‐8226 cells. A similar exposure to RGZ also induced cell cycle arrest and cell apoptosis of myeloma cells. A combination of RGZ with ATRA enhanced the effects of RGZ and induced cell cycle arrest and apoptosis more profoundly in both cell lines. RGZ treated cells displayed morphological characteristics of cell differentiation, and more evident signs of differentiation were observed when combined with ATRA. These changes were confirmed by the detection of CD49e expression and light chain protein secretion. Similar cell apoptosis and differentiation were observed when primary CD138⁺ myeloma cells were treated with RGZ and ATRA. The mRNA expressions of FLIP, XIAP and survivin were detected in both cell lines and the levels decreased significantly after culture with RGZ. The addition of ATRA in culture medium made these changes more apparently. Caspase‐3 activity was increased upon exposure to RGZ in both U266 and RPMI‐8226 cells while combination of RGZ and ATRA brought out more effective activation of caspase‐3. Similar apoptosis and cell differentiation induced by RGZ and ATRA can also be observed in primary CD138⁺ cells from myeloma patients. Conclusion: Concomitant RXRα activation by ATRA enhanced the inhibitory effects of RGZ on myeloma cell proliferation, cell cycle, apoptosis and differentiation. Combination of RGZ and ATRA may be a useful therapy for human multiple myeloma.