scholarly journals Specific killing of multiple myeloma cells by (-)-epigallocatechin-3-gallate extracted from green tea: biologic activity and therapeutic implications

Blood ◽  
2006 ◽  
Vol 108 (8) ◽  
pp. 2804-2810 ◽  
Author(s):  
Masood A. Shammas ◽  
Paola Neri ◽  
Hemanta Koley ◽  
Ramesh B. Batchu ◽  
Robert C. Bertheau ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Green Tea ◽  
Fas Ligand ◽  
Mononuclear Cells ◽  
Apoptotic Cell ◽  
Growth Arrest ◽  
Laminin Receptor ◽  
Peripheral Blood Mononuclear ◽  
Myeloma Cells

AbstractEpigallocatechin-3-gallate (EGCG), a polyphenol extracted from green tea, is an antioxidant with chemopreventive and chemotherapeutic actions. Based on its ability to modulate growth factor-mediated cell proliferation, we evaluated its efficacy in multiple myeloma (MM). EGCG induced both dose- and time-dependent growth arrest and subsequent apoptotic cell death in MM cell lines including IL-6-dependent cells and primary patient cells, without significant effect on the growth of peripheral blood mononuclear cells (PBMCs) and normal fibroblasts. Treatment with EGCG also led to significant apoptosis in human myeloma cells grown as tumors in SCID mice. EGCG interacts with the 67-kDa laminin receptor 1 (LR1), which is significantly elevated in myeloma cell lines and patient samples relative to normal PBMCs. RNAi-mediated inhibition of LR1 resulted in abrogation of EGCG-induced apoptosis in myeloma cells, indicating that LR1 plays an important role in mediating EGCG activity in MM while sparing PBMCs. Evaluation of changes in gene expression profile indicates that EGCG treatment activates distinct pathways of growth arrest and apoptosis in MM cells by inducing the expression of death-associated protein kinase 2, the initiators and mediators of death receptor-dependent apoptosis (Fas ligand, Fas, and caspase 4), p53-like proteins (p73, p63), positive regulators of apoptosis and NF-κB activation (CARD10, CARD14), and cyclin-dependent kinase inhibitors (p16 and p18). Expression of related genes at the protein level were also confirmed by Western blot analysis. These data demonstrate potent and specific antimyeloma activity of EGCG and provide the rationale for its clinical evaluation.

Effects of Integrin-Linked Kinase (ILK) Inhibitor QLT0267 on Multiple Myeloma Cells and Evaluation of ILK as a Therapeutic Target in Multiple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3529-3529
Author(s):  
Thorsten Stühmer ◽  
Torsten Steinbrunn ◽  
Evelyn Grella ◽  
Ralf C. Bargou
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Cells ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Treatment Options ◽  
Therapeutic Window ◽  
Culture Dish ◽  
Peripheral Blood Mononuclear ◽  
Myeloma Cells

Abstract Multiple myeloma (MM) is a fatal plasma cell tumor that accounts for about 1% of cancers. A hallmark of the disease is its location in the bone marrow where the tumor cells receive prosurvival support from the microenvironment and cause extensive osteolytic damage. Novel drugs are currently being developed into a range of new treatment options. However, because the problems of cancer relapse and eventual selection of therapy-resistant offspring remain, additional therapeutic targets should still be investigated. ILK is a multifunctional protein that, as an adaptor and/or as a kinase, may relay adhesion- and growth factor receptor-mediated signals to downstream signaling cascades that promote growth and survival. We have analysed the expression of ILK in MM cells and have tested the effects of a novel small molecule ILK-inhibitor (QLT0267; QLT Inc., Vancouver, Canada) in MM cell lines, primary MM tumor cells and healthy cells, respectively. ILK expression at either cDNA or protein level was detectable in virtually every MM sample tested. Treatment with QLT0267 for up to 3 days resulted in extensive apoptotic death in MM cell lines (EC50 values below 10 microM in 8/9 MM cell lines tested) and in a majority of primary (anti-CD138-purified) MM samples (EC50 values below 10 microM in 8/14 primary MM samples tested). Drug treatment led to rapid decreases in the levels of phospho-STAT3, phospho-GSK3beta and total Akt protein, whereas levels of ILK and of phospho-ERK were unaffected or, in the latter case, showed a slight increase. Similar to other current pharmacologic approaches, targeting ILK may have several detrimental impacts on the signaling network that sustains MM cells. Such pleiotropic effects could prove valuable for combination treatments. The survival of peripheral blood mononuclear cells and of bone marrow stromal cells (BMSCs) at 20 microM QLT0267 was just slightly affected, indicating that the scope for establishment of a therapeutic window in MM might exist. High (20 microM) concentrations of QLT0267 gradually (and reversibly) promoted detachment of BMSCs from the culture dish, indicating that the drug might be useful to temporarily impair their effectiveness to support myeloma cells. Taken together, these experiments provide a rationale to further explore the utility of ILK-inhibition for the treatment of MM.

scholarly journals Characteristics of a Novel Target Antigen against Myeloma Cells for Immunotherapy

2020 ◽  
Author(s):  
Maiko Matsushita ◽  
Saku Saito ◽  
Shinya Yokoe ◽  
Daiju Ichikawa ◽  
Yutaka Hattori
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Target Antigen ◽  
Rt Pcr ◽  
Peripheral Blood Mononuclear ◽  
Myeloma Cells ◽  
Demethylating Agents ◽  
Specific Ctls

Despite the availability of therapeutic treatments, multiple myeloma is an incurable haematological disorder. In this study, we aimed to clarify the role of CXorf48 as a therapeutic target in multiple myeloma. Based on a previously identified HLA-A*24:02-restiricted epitope from this novel cancer/testis antigen, we characterized the activities of cytotoxic T lymphocytes (CTLs) specific to this antigen against myeloma cells and evaluated the effects of demethylating agents in increasing antigen expression and enhancing the cytotoxic activity of CTLs. CXorf48 expression was examined by RT-PCR using nine myeloma cell lines. Cell lines with low CXorf48 expression were treated by demethylating agents (DMAs), 5-azacytidine (5-aza), and 5-aza-2'-deoxycytidine (DAC) to evaluate gene expression using quantitative RT-PCR. Furthermore, CXorf48-specific CTLs were induced from peripheral blood mononuclear cells of HLA-A*24:02-positive healthy donors to evaluate antigen recognition using ELISpot and 51Cr cytotoxicity assays. CXorf48 was widely expressed in myeloma cells and gene expression was significantly increased by DMAs. Furthermore, CXorf48-specific CTLs recognized DMA-treated myeloma cells. These findings suggest that CXorf48 is a useful target for immunotherapy, such as vaccination, in combination with demethylating agents for the treatment of patients with myeloma.

scholarly journals Characteristics of a Novel Target Antigen against Myeloma Cells for Immunotherapy

Vaccines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 579
Author(s):  
Maiko Matsushita ◽  
Saku Saito ◽  
Shinya Yokoe ◽  
Daiju Ichikawa ◽  
Yutaka Hattori
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Target Antigen ◽  
Rt Pcr ◽  
Peripheral Blood Mononuclear ◽  
Myeloma Cells ◽  
Demethylating Agents ◽  
Specific Ctls

Despite the availability of therapeutic treatments, multiple myeloma is an incurable haematological disorder. In this study, we aimed to clarify the role of CXorf48 as a therapeutic target in multiple myeloma. Based on a previously identified HLA-A*24:02-restiricted epitope from this novel cancer/testis antigen, we characterized the activities of cytotoxic T lymphocytes (CTLs) specific to this antigen against myeloma cells and evaluated the effects of demethylating agents in increasing antigen expression and enhancing the cytotoxic activity of CTLs. CXorf48 expression was examined by reverse transcription polymerase chain reaction (RT-PCR) using nine myeloma cell lines. Cell lines with low CXorf48 expression were treated by demethylating agents (DMAs), 5-azacytidine (5-aza), and 5-aza-2’-deoxycytidine (DAC) to evaluate gene expression using quantitative RT-PCR. Furthermore, CXorf48-specific CTLs were induced from peripheral blood mononuclear cells of HLA-A*24:02-positive healthy donors to evaluate antigen recognition using ELISpot and 51Cr cytotoxicity assays. CXorf48 was widely expressed in myeloma cells, and gene expression was significantly increased by DMAs. Furthermore, CXorf48-specific CTLs recognized DMA-treated myeloma cells. These findings suggest that CXorf48 is a useful target for immunotherapy, such as vaccination, in combination with demethylating agents for the treatment of patients with myeloma.

A Fully Human Antagonist Anti-CD40 Antibody Triggers Significant Antitumor Activity Against Human Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2414-2414
Author(s):  
Yu-Tzu Tai ◽  
Xian-Feng Li ◽  
Xia Tong2 ◽  
Laurence Catley ◽  
Daniel Santos ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Dependent Manner ◽  
Peripheral Blood Mononuclear ◽  
Specific Lysis ◽  
Growth And Survival ◽  
Human Multiple Myeloma ◽  
Dose Dependent

Abstract We previously demonstrated that CHIR-12.12, a fully human anti-CD40 mAb (IgG1) generated in XenoMouseÒ mice (Abgenix, Inc), blocks CD40/CD40 ligand (CD40L) interactions and has more potent anti-lymphoma activity than Rituximab both in vivo and in vitro (abstract #2386, ASH, San Diego, Dec. 2003). In this study, we assess the efficacy of CHIR-12.12 against human multiple myeloma (MM) using CD40-expressing MM cell lines and purified CD138+ patient cells. CHIR-12.12 binds to purified CD138+ MM cells in >80% (10/12) of patient samples, as measured by flow cytometry: the mean fluorescence intensity (MFI) range was 1 to 20 for CHIR-12.12 vs 0.2–0.9 for control human IgG1. We next examined the antagonist activity of CHIR-12.12 in MM cells. CHIR-12.12 blocked CD40L-mediated proliferation of CD40-expressing MM lines and purified CD138+ patient cells from 2 MM patients in a dose-response manner. In contrast, CHIR-12.12 alone did not alter constitutive MM cell proliferation. Immunoblotting analysis demonstrated that PI3-K/AKT, NF-kB, and ERK activation induced by hCD40L in the 12BM MM cell line was significantly inhibited by CHIR-12.12 (5 μg/ml). Adhesion of MM cells to bone marrow stromal cells (BMSCs) confers growth and survival benefit for tumor cells. Since CD40 activation, either by stimulatory mouse anti-CD40 mAb G28.5 or formaldehyde-fixed CHO cells expressing hCD40L, induces MM cell adhesion to fibronectin (FN) or BMSCs, we next asked whether antagonist CHI12.12 abrogates this process. CHIR-12.12 inhibited CD40L-induced adhesion of MM cell lines to FN in a dose dependent manner (0.001-10 μg/ml), whereas control human IgG did not. Moreover, CHIR-12.12 (1 μg/ml) blocked hCD40L-induced adhesion of freshly isolated patient MM cells to BMSCs. Adhesion of MM cells to BMSCs induces IL-6 secretion, an important growth and survival cytokine for MM cells, and treatment of MM cells with hCD40L further augmented adhesion-induced IL-6 secretion. Conversely, pretreatment of CD40-expressing MM cell lines with CHIR-12.12 significantly decreased IL-6 secretion triggered by coculture of MM cells with BMSCs. We next examined whether CHIR-12.12 stimulates antibody-dependent cellular cytotoxicity (ADCC) against CD40-expressing MM cells. Human peripheral blood mononuclear cells and purified NK cells (CD56+CD3−) were used as effector cells. CHIR-12.12 triggered MM cell lysis in a dose dependent manner, as measured in CD40-expressing MM cell lines. The maximum specific lysis of 20–70 % was achieved at 10 μg/ml concentration of CHIR-12.12. CHIR-12.12 mediated lysis was specific to CD40-expressing MM cells, as CHIR-12.12 did not induce ADCC against CD40-negative MM cells. Importantly, CHIR-12.12 induced ADCC against CD138+ cells isolated from 2 MM patients. These results provide preclinical rationale for clinical evaluation of CHIR-12.12 with the goal of improving patient outcome in MM.

PU.1 Is Downregulated in a Subset of Multiple Myeloma by the Methylation of Its -17 kb Upstream Regulatory Region and the Promoter.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3417-3417
Author(s):  
Yutaka Okuno ◽  
Hiro Tatetsu ◽  
Shikiko Ueno ◽  
Hiroyuki Hata ◽  
Yasuhiro Yamada ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Cell Lines ◽  
Promoter Region ◽  
Growth Arrest ◽  
Myeloma Cell ◽  
Upstream Region ◽  
Cell Growth Arrest ◽  
Myeloma Cells ◽  
Expression Levels

Abstract It has been reported that disruption of transcription factors critical for hematopoiesis, such as C/EBPa and AML1, is involved in leukemogenesis. PU.1 is a transcription factor important for both myeloid and lymphoid development. We reported that mice in which the levels of PU.1 were 20% of that of wild-type developed acute myeloid leukemia, T cell lymphoma, and a CLL-like disease. These findings strongly suggest that PU.1 has tumor suppressive activity in multiple hematopoietic lineages. Last year, we reported that PU.1 is downregulated in a majority of multiple myeloma cell lines and and freshly isolated CD138 positive myeloma cells from certain number of myeloma patients, and that tet-off inducible exogenous expression of PU.1 in PU.1 negative myeloma cell lines induced cell growth arrest and apoptosis. Based on their PU.1 expression levels, we divided the myeloma patients into two groups, namely PU.1 high and PU.1 low-to-negative, (cutoff index of 25th percentile of the PU.1 expression level distribution among all patients). The PU.1 low-to-negative patients had a significantly poorer prognosis than the PU.1 high patients. To elucidate the mechanisms of downregulation of PU.1, we performed sequence and epigenetic analysis of the promoter region and the -17 kb upstream region that is conserved among mammalians and important for proper expression of PU.1. There are no mutations in these regions of all five myeloma cell lines. In contrast, the -17 kb upstream region was highly methylated in 3 of 4 PU.1 negative myeloma cell lines, while the promoter region was also methylated to various levels in all five myeloma cell lines including one PU.1 positive cell line. These data suggested that the downregulation of PU.1 in myeloma cell lines might be dependent on the methylation of both regulatory regions of PU.1 gene, especially the -17 kb upstream region. We also evaluated the mechanisms of cell growth arrest and apoptosis of myeloma cell lines induced by PU.1. Among apoptosis-related genes, we identified that TRAIL was upregulated after PU.1 induction. To evaluate the effect of upregulation of TRAIL, we stably introduced siRNA for TRAIL into myeloma cell lines expressing PU.1, and we found that apoptosis of these cells was partially suppressed by siRNA for TRAIL, suggesting that apoptosis of myeloma cells induced by PU.1 might be at least partially due to TRAIL upregulation. We are currently performing DNA microarray analysis to compare the expression levels of genes between before and after PU.1 induction, in order to further elucidate the mechanisms of cell growth arrest and apoptosis.

scholarly journals NEK2 Inhibition Enhances the Efficacy of PD-1/PD-L1 Blockade in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2671-2671
Author(s):  
Yan Cheng ◽  
Fumou Sun ◽  
Huojun Cao ◽  
Dongzheng Gai ◽  
Bailu Peng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
B Cells ◽  
Cell Lines ◽  
Immune Checkpoint ◽  
Mononuclear Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Gene Set Enrichment Analysis ◽  
Myeloma Cells

Abstract Introduction The development of new treatments for high-risk multiple myeloma (HRMM) are needed. The PD-1/PD-L1 axis is one of the chief inhibitory immune checkpoints in antitumor immunity. Despite the success of PD-1 (PDCD1) / PD-L1 (CD274) blockade in some neoplasms, use of it as a monotherapy has failed to improve outcome in RRMM. We have previously demonstrated that the cell-cycle-regulated serine-threonine kinase, NEK2 is elevated in HRMM and that inhibition of NEK2 can overcome drug-resistance and prolong survival of xenografted MM cells. Here, we aimed to investigate the possible role of NEK2 in regulating the immune checkpoint response in MM and development of possible anti-PD1/PDL1 combination therapies. Methods Gene expression profiles and pathway enrichment analyses were conducted on oligonucleotide microarray gene expression profiles from over 1000 primary MM samples to evaluate the correlation of NEK2 and immune checkpoint expression levels. To elucidate the underlying mechanism, we used Nek2 -/- mice crossed with EμMyc mice to generate B cell tumor mouse model with NEK2 deficiency. RNA-sequencing analyses of premalignant B cells was compared between EμMyc/Nek2 WT and EμMyc/Nek2 -/- mice. The hub molecular regulators in the NEK2 correlated pathways were further determined by western blot using NEK2 overexpressing and knockdown cell lines and then verified by co-immunoprecipitation with a NEK2 antibody. Lastly, to establish its clinic significance, the efficacy of INH1 (small compound NEK2 inhibitor), (D)-PPA 1 (peptide-based PD-1/PD-L1 interaction inhibitor) or a PD-L1 (monoclonal antibody) was tested in bone marrow BM mononuclear cells from primary MM patients in-vitro as well as in MM xenografts. Tumor burden and T cell immune responses were monitored by M-spike and mass cytometry. Results Gene expression profiles demonstrated that CD274 expression was significantly higher in the non-proliferative hyperdiploid (HY) subtype of MM, representing between 25-35% of all MM. NEK2 was negatively correlated with CD274 gene expression across all 7 MM subtypes. Gene set enrichment analysis showed that the IFN-γ signaling pathway, which can induce CD274 expression, was significantly enriched in the HY subtype as well as premalignant B cells from EμMyc/Nek2 -/- mice. Elevated expression of EZH2, a histone methyltransferase gene, is also highly correlated wirth NEK2 levels in primary MM. We found that NEK2 inhibition increases CD274 expression as well as reduced EZH2 expression and H3K27me3 levels in MM cell lines. In contrarst, myeloma cells overexpressing NEK2 showed increased expression and activity of EZH2 and H3K27me3 levels. Thus, NEK2 appears to regulate CD274/PD-L1 expression through EZH2-mediated histone methylation. Next we demonstrated that NEK2 and EZH2 directly interact and that overexpression of NEK2 leads to increased methylation of the CD274/PD-L1 gene. We treated BM mononuclear cells from primary MM with PD-1/PD-L1 inhibitor with and without a NEK2 inhibitor. The combination was most effective at eliminating CD138 + myeloma cells while having no effects on T, B and myeloid cell populations. Conclusion Our study showed that expression of CD274/PD-L1 is suppressed in primary HRMM and that CD274/PD-L1 expression is negatively regulated by NEK2 via EZH2-mediated methylation. Inhibition of NEK2 sensitizes myeloma cells to PD-1/PD-L1 blockade, showing either a synergistic or an additive effect in MM cell cytotoxicity. Disclosures No relevant conflicts of interest to declare.

Sphingolipids as a novel target for the treatment of multiple myeloma.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e19534-e19534
Author(s):  
Yubin Kang ◽  
Jagadish Kummetha Venketa
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Line ◽  
Cell Lines ◽  
Apoptotic Cell ◽  
Myeloma Cell ◽  
Sphingolipid Metabolism ◽  
Myeloma Cell Line ◽  
Myeloma Cells ◽  
Novel Target

e19534 Background: Multiple myeloma (MM) is the second most common hematological malignancy in the United States and accounts for ~10,600 deaths annually. MM remains an incurable disease and almost all patients will eventually relapse and become refractory to currently available therapeutic agents. There is an unmet need for better understanding the disease’s molecular pathways and for identifying novel therapeutic targets. Sphingolipid metabolism is being increasingly recognized as a key pathway in tumor cell proliferation and in tumor sensitivity to anticancer drugs. We hypothesize that altered sphingolipid metabolism plays an important role in the pathogenesis of MM, thus providing a novel target in the treatment of MM. Methods: We first assayed sphingolipid metabolism including sphingolipid metabolites and sphingolipid metabolizing genes in myeloma cell lines, in freshly isolated human primary CD138+myeloma cells, and in publically available dataset. We then tested the efficacy of the selective SK2 inhibitor (ABC294640) and the SK2 shRNA in killing myeloma cells in vitro. Results: 1) Compared to immortalized B cells, the levels of pro-apoptotic ceramides were decreased whereas the proliferative sphingosine 1-phosphate (S1P) was increased in myeloma cell lines. 2) The expression of several key sphingolipid-metabolizing genes including sphingosine kinase (SK) 1 and 2 was altered in freshly isolated human primary bone marrow myeloma cells and in publically available microarray dataset. 3) The selective SK2 inhibitor (ABC294640) induces apoptotic cell death and inhibits myeloma cell growth with an IC50of ~20 μM in 9 myeloma cell lines. 4) Interestingly, OPM-1 myeloma cell line was extremely sensitive to ABC294640 with an IC50of <5 µM whereas U266 myeloma cell line was resistant to ABC294640. SK2 shRNA induced apoptotic cell death in OPM-1, but not in U266 cells. We are currently investigating the molecular mechanisms underlying the resistance of U266 myeloma cells to ABC294640. Conclusions: Our data demonstrated that sphingolipid metabolism provides an attractive target in the treatment of refractory/relapased multiple myeloma.

Honokiol Overcomes Conventional Drug Resistance in Human Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1488-1488
Author(s):  
Kenji Ishitsuka ◽  
Teru Hideshima ◽  
Makoto Hamasaki ◽  
Raje Noopur ◽  
Kumar Shaji ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Flow Cytometric Analysis ◽  
Caspase 8 ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood ◽  
Human Multiple Myeloma ◽  
Induced Apoptosis

Abstract Honokiol is an active component isolated and purified from Magnolia, a plant used in traditional Chinese medicine. It is an anti-oxidant, and inhibits both xanthine oxidase and angiogenesis. In this study, we first examined the direct toxicity of honokiol against human multiple myeloma (MM) cell lines in vitro. Honokiol significantly inhibited growth of MM cell lines (RPMI8226, U266 and MM.1S) via induction of G1 growth arrest, followed by apoptosis, with IC50 values at 48h of 5 to 10 μg/ml. Moreover, honokiol similarly inhibited growth of doxorubicin (Dox)-resistant (RPMI-Dox40), melphalan resistant (RPMI-LR5), and dexamethasone (Dex)-resistant (MM.1R) cell lines. Furthermore, flow cytometric analysis demonstrated that honokiol (6–10 μg/ml, 48h) induced death of CD38+CD138+ tumor cells isolated from 5 patients with relapsed refractory MM. In contrast, no toxicity was observed in normal peripheral blood mononuclear cells or long term-cultured bone marrow stromal cells (BMSCs) treated with honokiol (≤20 mg/ml). Neither culture of MM cells with BMSCs nor interleukin-6 (IL-6) and insulin like growth factor-1 (IGF-1) protected against honokiol-induced cytotoxicity in MM.1S cells. We next delineated the mechanism of honokiol-triggered cytotoxicity. Honokiol triggered increased expression of Bax and Bad; down regulated Mcl-1 protein expression, followed by caspase-8/9/3 cleavage. Importantly, the pan-caspase inhibitor z-VAD-fmk only partially inhibited honokiol-induced apoptosis in MM.1S cells. Furthermore, honokiol induced apoptosis even in SU-DHL4 cells, which express low level of caspase-8 and -3 and are resistant to both conventional (doxorubicin, melphalan, dexamethason) and novel (bortezomib, revimid) drugs. These results suggest that honokiol may induce apoptosis via both caspase-dependent and -independent pathways. Finally, honokiol inhibited IL-6-induced phosphorylation of ERK1/2, STAT3, and Akt, known to mediate growth, survival, and drug resistance, respectively. Taken together, our results suggest that providing the rationale for clinical evaluation of honokiol to improve patient outcome in MM.

Daratumumab, a Novel Potent Human Anti-CD38 Monoclonal Antibody, Induces Significant Killing of Human Multiple Myeloma Cells: Therapeutic Implication.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 608-608 ◽  
Author(s):  
Yu-Tzu Tai ◽  
Michel de Weers ◽  
Xian-Feng Li ◽  
Weihua Song ◽  
Sabikun Nahar ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Clinical Development ◽  
Target Cells ◽  
Igg Antibody ◽  
Peripheral Blood Mononuclear ◽  
Binding Characteristics ◽  
Dose Dependent

Abstract Abstract 608 CD38 is a type II transmembrane glycoprotein highly expressed in many hematological malignancies including multiple myeloma (MM), representing a promising target for monoclonal antibody (mAb)-based targeted immunotherapy. We here investigated the mechanisms of anti-MM activity of daratumumab, a novel anti-CD38 mAb currently in clinical development, to treat human MM. Daratumumab was generated in human Ig transgenic HuMab-mice® and was selected for its excellent binding characteristics. It strongly reacts against a panel of CD38-expressing MM cell lines (>94%, 17/18) that are sensitive or resistant to current treatments for MM: median fluorescence intensity (MFI) observed was 378-20764 and 15-140 for daratuzumab and isotype control Ab, respectively. U266 cells, which lack CD38 mRNA, did not bind daratuzumab. Significantly, daratuzumab induced NK-mediated antibody-dependent cellular cytotoxicity (ADCC) against CD38-expressing MM cells but not CD38-negative U266 cells, confirming its specific targeting. Daratuzumab-mediated MM cell lysis was dose-dependent, with maximum lysis of the CD38-expressing MM cell lines (n=17) at concentrations of 0.01-0.1μg/ml. We also determined potential effects of daratumumab on NK and peripheral blood mononuclear cells (PBMC) effector cells, since these cells express CD38, albeit at lower levels than MM cells. Overnight pretreatment of PBMCs or purified NK cells from 2 normal donors with daratumumab (2 μg/ml) did not significantly alter daratumumab-induced ADCC against MM1S target cells. The bone marrow (BM) microenvironment has been shown to protect MM cells against apoptosis and stimulate their growth; we therefore next asked whether daratumumab effectively induced ADCC against MM cells in the presence of BM stromal cells (BMSCs). Daratumumab induced a similar, dose-dependent, ADCC of dexamethasone-sensitive MM1S as well as dexamethasone-resistant MM1R cells both in the presence and absence of BMSCs, suggesting that daratumumab could indeed effectively induce ADCC in the BM microenvironment. Significantly, our findings translated to patient cells, since daratumumab induced ADCC of both allogeneic (n=2) and autologous (n=9) patient MM cells. We next turned our attention to complement-mediated cytotoxicity (CDC). In calcein AM-labeling assays in the presence of normal human serum, daratumumab induced dose-dependent CDC of LP-1 and XG-1 MM cells expressing low levels of CD59 and CD55 both in the absence or presence of BMSCs. Effective daratumumab-induced CDC of CD138-purified MM cells was seen for 6 of 9 patients. Daratumumab-induced CDC was not affected by the expression of complement inhibitor proteins in this cohort of patients. Finally, daratumumab induced apoptosis in Ramos and Daudi cells when crosslinked by anti-IgG antibody, and it induced cell death in CD138-purified patient MM cells, as assessed by annexin V/PI staining. Taken together, our results indicate that daratumumab is able to effectively kill patient MM tumor cells via several cytotoxic mechanisms, supporting clinical development of daratumumab to improve patient outcome in MM. Disclosures: de Weers: Genmab: Employment. Bakker:Genmab: Employment. Vink:Genmab: Employment. Jacobs:Genmab: Employment. Oomen:Genmab: Employment. Bleeker:Genmab: Employment. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. van de Winkel:Genmab: Employment. Parren:Genmab: Employment. Richardson:Keryx Biopharmaceuticals: Honoraria. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Green tea polyphenol EGCG induces lipid-raft clustering and apoptotic cell death by activating protein kinase Cδ and acid sphingomyelinase through a 67 kDa laminin receptor in multiple myeloma cells

2012 ◽  
Vol 443 (2) ◽  
pp. 525-534 ◽  
Author(s):  
Shuntaro Tsukamoto ◽  
Keisuke Hirotsu ◽  
Motofumi Kumazoe ◽  
Yoko Goto ◽  
Kaori Sugihara ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Protein Kinase ◽  
Lipid Raft ◽  
Apoptotic Cell ◽  
Acid Sphingomyelinase ◽  
Laminin Receptor ◽  
Myeloma Cells ◽  
Raft Clustering ◽  
Protein Kinase Cδ

EGCG [(−)-epigallocatechin-3-O-gallate], the major polyphenol of green tea, has cancer chemopreventive and chemotherapeutic activities. EGCG selectively inhibits cell growth and induces apoptosis in cancer cells without adversely affecting normal cells; however, the underlying molecular mechanism in vivo is unclear. In the present study, we show that EGCG-induced apoptotic activity is attributed to a lipid-raft clustering mediated through 67LR (67 kDa laminin receptor) that is significantly elevated in MM (multiple myeloma) cells relative to normal peripheral blood mononuclear cells, and that aSMase (acid sphingomyelinase) is critical for the lipid-raft clustering and the apoptotic cell death induced by EGCG. We also found that EGCG induces aSMase translocation to the plasma membrane and PKCδ (protein kinase Cδ) phosphorylation at Ser664, which was necessary for aSMase/ceramide signalling via 67LR. Additionally, orally administered EGCG activated PKCδ and aSMase in a murine MM xenograft model. These results elucidate a novel cell-death pathway triggered by EGCG for the specific killing of MM cells.

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