Shikonin Induces Apoptosis by Inhibiting Phosphorylation of IGF-1 Receptor in Myeloma Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4858-4858
Author(s):  
Takehiro Kimura ◽  
Tomonori Nakazato ◽  
Takatsune Shimizu ◽  
Yasuo Ikeda ◽  
Masahiro Kizaki
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cellular Growth ◽  
Great Promise ◽  
High Dose ◽  
Dependent Manner ◽  
Human Malignant Melanoma ◽  
Myeloma Cells ◽  
Induction Of Apoptosis ◽  
Induced Apoptosis

Abstract Multiple myeloma is an incurable hematological malignancy of plasma cells despite advances in conventional chemotherapy or high dose chemotherapy with stem cell transplantation. Recent advances in the biological treatment such as thalidomide and bortezomib will offer great promise to improve the outcome of refractory or relapsed patients with multiple myeloma; therefore, more novel biologically based therapies targeting both myeloma cells and its bone marrow microenvironment are urgently required. Shikonin, a Chinese herb derived from purple gromwell, Boraginaceae Lithospermum erythrorhizon, is previously known to have anti-inflammatory, anti-tumor, and anti-pyretic properties. Several studies have shown that Shikonin induces p53-mediated cell cycle arrest and apoptosis in human malignant melanoma cells, and induces apoptosis in human myeloid leukemic HL60 cells. However, none of the studies have concerned in the effects of Shikonin on myeloma cells. In this study, we investigated the possibilities to develop Shikonin as a novel promising agent for multiple myeloma. Shikonin suppressed the cellular growth of RPMI8226 and IM9 myeloma cells, via induction of apoptosis in a dose (0–1 μM)- and time (0–24 h)-dependent manner. Apoptotic cell death was induced in both RPMI8226 and IM9 cells within 3 h for treatment with only 0.5 μM of Shikonin, and finally 78% of RPMI8226 cells and 91% of IM9 cells were dead by apoptosis within18 h. Treatment with 0.5 μM Shikonin rapidly increased the population of cells in the G0/G1 phase with reduction of cells in the S phase, and then induction of apoptosis was confirmed by the appearance of cells in the sub-G1 fraction. Shikonin-induced apoptosis was in association with the loss of mitochondrial transmembrane potentials, and activation of caspase-3. Shikonin-induced apoptosis was completely blocked by the treatment with pan-caspase inhibitor (20 μM, Z-VAD), suggesting that caspase plays an important role in Shikonin-induced cell death in myeloma cells. Expression of p53 and Bax proteins was increased with down-regulation of Mcl-1 protein, but no changes were confirmed of the expression of Bcl-2 and Bcl-XL. Recent investigations have shown that cytokines such as IL-6, insulin growth factor (IGF), VEGF, and TNF-α mediate myeloma cell growth, survival and migration. IGF-1 increases survival of myeloma cells by activating various down-stream signal transduction molecules. Recently, inhibitors of IGF-1 receptor demonstrate promising anti-multiple myeloma activity in preclinical studies. Shikonin has reported to be an inhibitor of protein tyrosine kinase such as EGFR, v-Src, and KDR/Flk-1. To address the mechanism of Shikonin-induced apoptosis in myeloma cells, we thus examined the effects of Shikonin on the phosphorylation of IGF-1 receptor. IGF-1 (100 ng/ml) stimulated the proliferation of both RPMI8226 and IM9 cells. Interestingly, Shikonin (0.5 μM) overcame IGF-1-induced cell proliferation, and inhibited proliferation of myeloma cells via induction of apoptosis. Shikonin inhibited phosphorylation of IGF-1 receptor as early as 30 min with inhibition of PI3K/Akt signaling. These results suggest that Shikonin-induced apoptosis in myeloma cells was mediated by inhibiting phosphorylation of IGF-1 receptor and modulating its down-stream signaling pathway. In conclusion, Shikonin inhibited cellular growth by inhibiting IGF-1 receptor signaling in myeloma cells, and may have a potential as a novel biologically based therapeutic agent for the patients with multiple myeloma..

HIF-1 Supports the Survival of Multiple Myeloma Cells through the Induction of Survivin Gene.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 110-110 ◽  
Author(s):  
Keita Kirito ◽  
Hu Yongzhen ◽  
Kozue Yoshida ◽  
Toru Mitsumori ◽  
Kei Nakajima ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cancer Cells ◽  
Cell Lines ◽  
Dependent Manner ◽  
Survivin Mrna ◽  
Myeloma Cells ◽  
Survivin Gene ◽  
Growth And Survival ◽  
Induced Apoptosis

Abstract In spite of the recent development of therapeutic strategies, multiple myeloma (MM) still remains incurable. Several cytokines and chemokines contribute to progression of the disease and acquisition of resistance to chemotherapy. These humoral factors support the growth and survival of myeloma cells through the regulation of transcription factors including NF-κB, Stat3 and FOXO3a. Hypoxia inducible factor-1 (HIF-1) is an important transcription factor that is activated under low oxygen tension and controls dozens of genes involved in angiogenesis, energy production and resistance to apoptosis. Interestingly, HIF-1 is frequently activated in cancer cells even under normoxic condition and it is well established that HIF-1 expression and activation correlates with tumor progression and resistance to cancer treatments. In this study, we investigated whether HIF-1 is involved in the biology of multiple myeloma. To this end, we used three MM cell lines U266, RPMI8226 and KMM-1. After informed consent, we also prepared primary MM cells from bone marrow samples of patients (n=5) using anti-CD138 magnetic beads. Initially, we treated MM cells with insulin-like growth factor-1 (IGF-1) and IL-6, both of which are major growth and survival factors for myeloma cells. Treatment with IGF-1 and, to be a lesser degree, IL-6 clearly enhanced expression of HIF-1α, a subunit of HIF-1, in all three cell lines. Similar results were obtained from isolated primary MM cells. Based on several lines of evidence that survivin, a member of inhibitor of apoptosis (IAP) family protein, is transcriptionally regulated by HIF-1 in breast cancer cells, and that this anti-apoptotic factor is important for growth of MM cells, we examined whether HIF-1 supports the survival of MM cells through the induction of survivin. Quantitative RT-PCR assay revealed that IGF-1 increased survivin mRNA both in MM cell lines and in primary MM cells. In addition, IGF-1 activated survivin gene promoter containing a HIF-1-binding site. To confirm that IGF-1-induced activation of survivin gene is mediated by HIF-1, we treated MM cell lines with echinomycin, an inhibitor of DNA-binding activity of HIF-1. As expected, echinomycin inhibited IGF-1-induced survivin gene expression in a dose-dependent manner. The inhibitor also induced apoptosis of MM cells, and IGF-1 could not rescue the MM cells from echinomycin-induced apoptosis. Furthermore, echinomycin enhanced melphalan-induced apoptosis of MM cells. To further examine the involvement of HIF-1 in IGF-1-induced survivin gene expression, we generated three independent HIF-1α knockdown KMM-1 clones using siRNA system. Survivin mRNA was not detected in the HIF-1α knockdown cells, and these clones easily underwent apoptosis even in the presence of IGF-1, compared to the parental cells. Taken together, HIF-1 plays a pivotal role in survival of MM cells through the induction of survivin gene. In conclusion, HIF-1 might be an attractive therapeutic target for MM.

Interfering with Arginine Metabolism As a New Treatment Strategy for Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3005-3005
Author(s):  
Bjoern Jacobi ◽  
Lea Stroeher ◽  
Nadine Leuchtner ◽  
Hakim Echchannaoui ◽  
Alexander Desuki ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Caspase 3 ◽  
Xenograft Model ◽  
Myeloma Cell ◽  
Intracellular Protein ◽  
Myeloma Cells ◽  
Induction Of Apoptosis

Abstract Introduction Starvation of tumor cells from the amino acid arginine has recently gained particular interest because of the downregulation of the rate-limiting enzyme argininosuccinate synthethase 1 (ASS1) in various cancer entities. ASS1-deficient cells cannot resynthesize arginine from citrulline and are therefore considered arginine auxotrophic. The arginine depleting enzyme arginine deiminase (ADI-PEG20, Polaris Pharmaceuticals) is currently tested in phase I-III clinical trials for different arginine auxotrophic cancers. The natural arginine analogue canavanine can compete with arginine for arginyl-tRNA-binding sites and consequently be incorporated into nascent proteins instead of arginine. Canavanine could therefore potentially further disturb intracellular protein homeostasis, especially under arginine deprivation. The sensitivity of myeloma cells towards arginine depletion strategies has not been analyzed so far. Methods Human myeloma cell lines and CD138-sorted primary human myeloma cells from patient bone marrow were screened for ASS1 expression by western blotting (WB). The cells were cultured in arginine free medium and assessed for proliferation and metabolic activity (CFSE/MTT assays), apoptosis (caspase-3 cleavage) and cell death (annexinV/propidium iodide). Canavanine was supplied in both arginine-sufficient and -deficient conditions. The level of intracellular protein stress was determined by WB and/or flow cytometry analysis for ubiquitinated proteins, phosphorylated eukaryotic initiation factor 2α (peIF2α) and the spliced isoform of the X-Box binding protein 1 (Xbp1s). Repetitive ADI-PEG20 ± canavanine application i.p. were tested in vivo in an U266 myeloma xenograft model in NOD/SCID/IL2Rcg-/- (NSG) mice. Arginine and canavanine levels in plasma were determined by HPLC. Tumor growth was measured, mice were assessed for survival, weight and side effects. Tumor tissues were analyzed for caspase-3 cleavage and Ki67 expression by immunohistochemistry. Results 5 of 6 myeloma cell lines were negative for ASS1. Also, ASS1 was either not or only weakly expressed in the majority of primary CD138+ myeloma patient samples. Arginine starvation induced an arrest of cell proliferation and/or metabolic activity of primary myeloma cells and myeloma cell lines after 18-24 h. Addition of citrulline could only rescue ASS1 positive myeloma cells due to the intracellular resynthesis of arginine. Arginine starvation alone led to delayed induction of apoptosis (e.g. 35% cell death of NCI-H929 cells after 72 h of treatment). Addition of 100 mM canavanine strongly increased cell death specifically in the context of arginine deficiency (e.g. cell death in NCI-H929 cells: 87% after 24 h, 100 % after 48h) while it was non-toxic and had no effect on cell viability under physiological arginine conditions. Co-application of canavanine induced ubiquitination of cellular proteins and led to the prolongation of a fatal unfolded protein response (UPR) as measured by markedly elevated Xbp1s levels. Prolonged UPR ultimately led to the induction of apoptosis as reflected by annexin V binding and caspase-3 cleavage. In an U266 myeloma NSG xenograft model, systemic arginine depletion by ADI-PEG20 suppressed tumor growth in vivo and significantly prolonged median survival of mice when compared with the control group (22±3 vs. 15±3 days). Canavanine treatment alone had no influence on viability (13±0 days). However, the combination of ADI-PEG20 and canavanine demonstrated the longest median survival (27±7 days). Histological examination of explanted tumors showed the highest rates of caspase-3 cleavage in the ADI-PEG20/canavanine group. Conclusion Myeloma cells are mostly arginine auxotrophic and can be selectively targeted by arginine starvation. Combination of arginine depletion with the arginine analogue canavanine leads to a highly efficient and specific tumor cell eradication and should be further optimized in multiple myeloma preclinical models. Disclosures Bomalaski: Polaris Pharmaceuticals Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

5HPP-33, a Novel Tubulin-Polymerization Inhibitor Derived from Thalidomide, Directly Inhibits Proliferation and Induces Apoptosis of Human Multiple Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1586-1586
Author(s):  
Toyotaka Iguchi ◽  
Sawako Oda-Nakazato ◽  
Tomomi Noguchi ◽  
Yuichi Hashimoto ◽  
Yutaka Hattori ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cellular Growth ◽  
Phase Cell ◽  
Tubulin Polymerization ◽  
Inhibiting Activity ◽  
Myeloma Cells ◽  
Polymerization Inhibitor ◽  
Induction Of Apoptosis ◽  
Tubulin Polymerization Inhibitor

Abstract Multiple myeloma is an incurable hematological malignancy despite the use of high-dose chemotherapy with hematopoietic stem cell transplantation. In addition, repeated episode of relapse may lead to refractory or resistant multiple myeloma; therefore, novel therapeutic approaches are desired in clinical settings. Recently, thalidomide has been introduced in the treatment of myeloma, and many clinical trials have since confirmed its efficacy in patients with relapsed, refractory and newly diagnosed multiple myeloma. Multiple mechanisms have been proposed for the anti-myeloma activity of thalidomide; however, its precise mechanism of action is still unclear, because thalidomide rapidly undergoes spontaneous, nonenzymatic, hydrolytic cleavage to numerous metabolites in vivo. In addition, side effects and teratogenic potential of thalidomide have often prevented its use in the direct treatment of myeloma. To address the exact anti-myeloma effect of thalidomide and develop the new derivatives without causing teratogenic effects, we have performed structural development studies of thalidomide and obtained various analogues with specific molecular properties. Among these derivatives, we report here for the first time that 2-(2,6-Diisopropylphenyl)-5-hydroxy-1H-isoindole-1,3-dione (5HPP-33) has most potent anti-myeloma effect with tubulin-polymerization-inhibiting activity. 5HPP-33 inhibited cellular growth of myeloma cell lines (RPMI8226, IM9, U266) and freshly isolated myeloma cells from patients in dose (0–50 μM)- and time (0–24 h)-dependent manners with an IC50 between 1–10 μM. In contrast, thalidomide itself did not inhibit cellular growth of RPMI8226 cells except exposure with high concentration (100 μM). Cultivation with 10 μM 5HPP-33 induces G2/M phase cell cycle arrest, and a strong induction of apoptosis 3 h after treatment. Immunofluorescent staining of RPMI8226 cells with anti-tubulin antibody revealed that 10 μM 5HPP-33 inhibits tubulin-polymerization. Induction of apoptosis was also confirmed in terms of both morphological changes and DNA ladder formation. Treatment with 5HPP-33 induced caspase-3 activity and PARP cleavage. Tubulin polymerization assay using microtubule protein from porcine brain revealed that 5HPP-33 showed potent tubulin-polymerization-inhibiting activity with an IC50 of 8.1 μM, comparable to that of known tubulin-polymerization inhibitors, rhizoxin and colchicines. In addition, its activity was more potent than a known thalidomide metabolite, 5-hydroxythalidomide. Interestingly, the structural requirement for activity was critical, because other analogues and derivatives of 5HPP-33 showed only slight tubulin-polimerization-inhibiting activity. To evaluate the effects of 5HPP-33 in vivo, we are currently doing the experiments to clarify the potency of tumor reduction using RPMI8226-transplanted NOD/SCID mice model. In conclusion, a novel tubulin-polymerization inhibitor, 5HPP-33, directly inhibits proliferation and induces apoptosis of myeloma cells in vitro and in vivo. In addition, one possible mechanism of growth inhibition by thalidomide might be its tubulin-polymerization inhibition activity in vivo. Taken together, 5HPP-33 is one of the promising candidates for the new therapeutic agent of multiple myeloma.

Nocodazole Induces Multiple Myeloma Cell Death and Reduces Tumor Growth through Sequential Microtubular Network Damage and C-Jun N-Terminal Kinase-Mediated Bcl-2 Phosphorylation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 296-296
Author(s):  
Rentian Feng ◽  
Huihui Ma ◽  
Noriyoshi Kurihara ◽  
Shirong Li ◽  
Judy A Ziegler ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Death ◽  
Tumor Growth ◽  
Murine Model ◽  
Immunofluorescent Staining ◽  
Solid Cancer ◽  
Dependent Manner ◽  
Microtubular Network ◽  
Induced Apoptosis

Abstract Abstract 296 Background: Benzimidazoles, originally categorized as broad-spectrum anthelmintic drugs, have been recently reported to induce growth arrest and apoptosis in some solid cancer models (e.g. colorectal and lung). We performed a multiplex drug-screening assay that identified benzimidazoles as potential anti-multiple myeloma (MM) agents. Methods and Results: In this study, we demonstrate that one of the benzimidazole members, nocodazole, inhibited proliferation and induced apoptosis in MM cell lines and primary MM cells alone and in co-culture with bone marrow stromal cells. The resistant phenotype of those MM cells resistant to conventional therapies could be completely reversed by nocodazole. The IC50 values were 60 nM (RPMI8226-S), 25 nM (RPMI8226-Dox40), 80 nM (RPMI8226-MR20), 60 nM (RPMI8226-LR5), 65 nM (MM.1S) and 60 nM (MM.1R). Viability of primary cells decreased by 66% in CD138+ cells and 7% in CD138− mononucleated bone marrow cells after 48 hour treatment. Cell cycle analysis revealed a G2/M arrest and subsequent cell death induced by nocodazole. Nocodazole also caused morphologic elongation in MM cells in a dose-dependent manner during prometaphase. The morphologically changed cells exhibited a microtubular network disarray as evidenced by microtubular immunofluorescent staining. Signaling studies indicated that increased expression of Bim protein and reduced XIAP and Mcl-1 levels were involved in nocodazole-induced apoptosis. Further investigation showed Bcl-2 phosphorylation as a critical mediator of cell death, which was triggered by the activation of JNK, instead of p38 kinase or ERKs. Treatment with JNK inhibitor SP600125 completely inhibited Bcl-2 phosphorylation at Ser70 and Thr56 induced by nocodazole. Nocodazole-induced cell death subsequently decreased from 79% to 28% after pretreatment with SP600125. Combination of nocodazole with dexamethasone induced significantly stronger induction of cell death at either drug dose. Dexamethasone at 20 nM, nocodazole at 15 or 30 nM could only induce 19%, 10.3% and 16% cell death, respectively. However, their combinations resulted in 67% and 92% nuclear fragmentation, respectively. Based on our in vitro data, we analyzed nocodazole in a SCID xenograft murine model. Nocodazole alone (5 and 20 mg/kg) or combined with dexamethasone (2 mg/kg) at a lower dose of 12 mg/kg significantly inhibited H929 tumor growth and prolonged survival in a SCID xenograft murine model. Conclusions: Our studies demonstrate that nocodazole has a potent anti-MM activity and might be a promising new treatment approach for MM. * Supported by a grant from the Multiple Myeloma Research Foundation. Disclosures: Roodman: Acceleron: Consultancy; Novartis: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy; Celgene: Consultancy. Lentzsch:Celgene: Consultancy, Speakers Bureau; Pfizer: Consultancy.

Inhibition of Heat Shock Factor 1 (HSF1) Is More Effective At Sensitizing Myeloma Cells to Bortezomib Than Inhibition of Individual HSF1 Targets.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2953-2953
Author(s):  
Shardule P Shah ◽  
Sagar Lonial ◽  
Lawrence H. Boise
Keyword(s):  
Multiple Myeloma ◽  
Heat Shock ◽  
Heat Shock Response ◽  
Conflicts Of Interest ◽  
The United States ◽  
Myeloma Cell ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Shock Response ◽  
Induced Apoptosis

Abstract Abstract 2953 Multiple myeloma is a plasma cell disorder with an average incidence of 21,000 new cases per year in the United States. Recent advances in therapeutic approaches such as the use of proteasome inhibitors have resulted in a significant increase in the overall survival of myeloma patients. Myeloma cells maintain many of the characteristics of normal plasma cells, including constitutive immunoglobulin production and secretion, therefore management of ER stress plays a role in myeloma cell sensitivity to proteasome inhibition. However, myeloma cells also upregulate protective genes in response to the proteotoxic stress that can limit the therapeutic response. Previous groups have published on the importance of the heat shock response and the heat shock protein (HSP) family, supporting preclinical and clinical exploration of HSP inhibition in myeloma. Our group had interest in regulation of the HSP response and has evaluated the master regulator HSF1 as a potential therapeutic target. We found that siRNA-mediated silencing of HSF1 enhances bortezomib-induced apoptosis in a myeloma cell line. To define the effectors of the heat shock response important in regulating bortezomib response, we determined which heat shock response genes are induced by bortezomib in an HSF1-dependent manner. From a realtime PCR array of 84 HSP family genes, we found 21 genes that were induced greater than 2-fold by bortezomib. Of these 21 genes, 10 genes showed >50% reduction in HSF1-silenced cells. 7/10 genes were confirmed by independent qRT-PCR and western blot analysis. These genes include: CRYAB (alpha-crystallin B chain), DNAJB1 (HSP40 subfamily B), HSPA1A (HSP70-1A), HSPA1B (HSP70-1B), HSPB1 (HSP27), HSPH1 (HSP105/110), and HSP90AB1 (HSP90b1). To begin to determine which of these genes was important for the HSF1-dependent protective response we silenced the 7 genes individually and subsequently treated the cells with bortezomib. Surprisingly only 1 of the 7 genes silenced individually, DNAJB1, had an observable effect on bortezomib-induced death. However DNAJB1 silencing does not account for all the HSF1 activity as the increase in cell death due to bortezomib is only 48% of that observed with HSF1 silencing. Thus targeting HSF1 is more effective at sensitizing multiple myeloma cells to bortezomib-induced apoptosis than targeting individual HSPs. Moreover these data suggest that HSP90 inhibitors are functioning by inhibiting at least two members of this family to be effective as single agents. Therefore, while clinical trials for individual HSP and HSP in combination with bortezomib are being conducted, a more effective strategy for apoptosis induction is achieved through inhibition of HSP regulators such as HSF1 in combination with bortezomib. These results provide support for investigating HSP regulation in response to PI to increase the efficacy of myeloma therapy. Disclosures: No relevant conflicts of interest to declare.

TM-233 Exerts Anti-Myeloma Activity and Overcomes Bortezomib-Resistance In Human Multiple Myeloma Cells By Targeting NF-κB and JAK/STAT Dual-Signaling Pathways

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4427-4427
Author(s):  
Morihiko Sagawa ◽  
Tatsuki Tomikawa ◽  
Tomoe Anan ◽  
Takayuki Tabayashi ◽  
Reiko Watanabe ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Signaling Pathways ◽  
Conflicts Of Interest ◽  
Subcutaneous Administration ◽  
Cellular Growth ◽  
Therapeutic Effects ◽  
Dependent Manner ◽  
Grade 3

Although the introduction of bortezomib and immunomodulatory drugs (IMiDs) has led to improved outcomes in patients with multiple myeloma (MM), the disease remains incurable. Bortezomib, a proteasome inhibitor, is widely used in the treatment of MM and has resulted in marked therapeutic effects; however, this therapy is often complicated by peripheral neuropathy (PN), of which grade ≥3 PN is dose-limiting toxicity and can necessitate cessation of therapy. Subcutaneous administration of bortezomib can reduce the incidence of PN; however, among cases of PN that still occur, 24% are grade 2 PN and 6% are grade 3 PN. These data suggest that the incidence of PN higher than grade 2 is not attenuated by the subcutaneous delivery of bortezomib. In addition, patients often become refractory to bortezomib after long-term use. In an effort to identify potent and well-tolerated agents, clinical trials of novel agents (e.g., carfilzomib, pomalidomide, and monoclonal antibody against CS-1) are being conducted both in patients with newly diagnosed MM and in those with relapsed/refractory disease. We previously reported that 1’-acetoxychavicol acetate (ACA) obtained from the rhizomes of the plant Languas galanga induces cell death of MM cells in vitro and in vivo through inhibition of NF-κB-related functions (Cancer Res, 2005; 65: 4417). Subsequently, we developed several ACA analogs based on quantitative structure-activity relationship (QSAR) analysis to develop more potent NF-κB inhibitors, and successfully synthesized a novel benzhydrol-type analog of ACA, named TM-233, that exerted potent growth inhibition against various MM cells (U266, RPMI8226, and MM-1S cells) in a dose- and time-dependent manner when compared with ACA (Chem Pharm Bull., 2008; 56: 1490). Further, TM-233 inhibited constitutive phosphorylation of JAK2 and STAT3 and down-regulated the expression of anti-apoptotic Mcl-1 protein. TM-233 directly bound and activated the transcription of the Mcl-1 gene promoter. Mcl-1 is the downstream molecule of STAT3; therefore, these results suggest that TM-233 induces cell death in MM cells with down-regulated Mcl-1 via modulation of the JAK/STAT pathway. In addition, we examined the DNA-binding activity of NF-κB in TM-233-treated MM cells and found that NF-κB was inhibited by TM-233. Further, Western blotting showed that TM-233 rapidly decreased the nuclear expression of NF-κB but increased the accumulation of NF-κB in the cytosol, suggesting that TM-233 inhibits the translocation of NF-κB from the cytosol to the nucleus. Immunohistochemical analysis confirmed that the p50/RelA dimer of NF-κB was located in the cytosol and not in the nucleus in TM-233-treated MM cells. We then examined the effects of TM-233 on bortezomib-resistant MM cells. Bortezomib-resistant MM cell lines (i.e., KMS-11/BTZ and OPM-2/BTZ) were established by limiting dilution. We found that these cells have a unique point mutation, G322A, in the gene encoding the proteasome β5 subunit (Leukemia 2010; 24: 1506). TM-233, but not bortezomib, inhibited cellular growth and induced cell death in KMS-11/BTZ and OPM-2/BTZ cells in a time- (0-48 hours) and dose- (0-5 μM) dependent manner. Furthermore, the combination of low-dose TM-233 (less than 2 μM) and bortezomib (10 nM) significantly induced cell death in bortezomib-resistant MM cells via inhibition of NF-κB activity. These results indicate that TM-233 could overcome bortezomib resistance in MM cells by acting via different mechanisms from those of bortezomib. In conclusion, TM-233 induced cell death in MM cells, and this effect was mediated through the JAK/STAT and NF-κB dual-signaling pathways. These data indicate that TM-233 might be a more potent and more specific NF-κB inhibitor than that of original compound (ACA), and might be able to overcome bortezomib-resistance in MM cells. Therefore, further studies investigating clinical approaches, including combination therapy, are warranted. Disclosures: No relevant conflicts of interest to declare.

Autocrine interleukin-6 production and highly malignant multiple myeloma: relation with resistance to drug-induced apoptosis

Blood ◽  
2001 ◽  
Vol 97 (2) ◽  
pp. 483-489 ◽  
Author(s):  
Maria Antonia Frassanito ◽  
Antonio Cusmai ◽  
Giuseppe Iodice ◽  
Franco Dammacco
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Death ◽  
Interleukin 6 ◽  
Mononuclear Cells ◽  
Myeloma Cell ◽  
Disease Stage ◽  
Drug Induced ◽  
Myeloma Cells ◽  
Induced Apoptosis

Abstract In this study, flow cytometry was used to evaluate interleukin-6 (IL-6) production by bone marrow mononuclear cells from 47 patients with multiple myeloma (MM) in different clinical stages and 15 patients with monoclonal gammopathy of undetermined significance. In patients with MM, autocrine IL-6 production paralleled the clinical disease stage. The largest proportion of syndecan-1+/IL-6+ cells was detected in patients with resistant relapse or primary refractory disease, suggesting that tumor progression involves expansion of myeloma cells producing IL-6. The authors assessed autocrine IL-6 production and in vitro proliferation and apoptosis of myeloma cells in 6 myeloma cell clones (MCCs) and in 2 myeloma cell lines, namely IM-9 and U-266-1970, which showed different sensitivities to the addition of exogenous IL-6. Autocrine IL-6 production was observed in IL-6–independent MCC-2, MCC-3, and MCC-5 cloned from patients with aggressive disease and in the IM-9 cell line. In contrast, IL-6–dependent MCC-1, MCC-4, and MCC-6 were syndecan-1+ and IL-6−. Blocking experiments with anti–IL-6 monoclonal antibody from clone AH65, which binds IL-6–IL-6Rα complexes, prevented cell proliferation of IL-6+ MCCs. Flow cytometry evaluations after propidium iodide staining revealed different susceptibilities of MCCs to cell death. IL-6–producing MCCs showed minimal spontaneous and dexamethasone-induced apoptosis, whereas a regular amplitude of apoptosis occurred in the IL-6− MCCs. These data provide evidence that autocrine IL-6 reflects a highly malignant phenotype of myeloma cells. In fact, autocrine IL-6 production and deregulated apoptosis may induce expansion of selective IL-6+ myeloma cells resistant to spontaneous and drug-induced cell death.

scholarly journals Inhibition of the MUC1-C oncoprotein induces multiple myeloma cell death by down-regulating TIGAR expression and depleting NADPH

Blood ◽  
2012 ◽  
Vol 119 (3) ◽  
pp. 810-816 ◽  
Author(s):  
Li Yin ◽  
Michio Kosugi ◽  
Donald Kufe
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Transmembrane Potential ◽  
Redox Balance ◽  
Myeloma Cell ◽  
Pentose Phosphate ◽  
Myeloma Cells ◽  
Multiple Myeloma Cell ◽  
Late Apoptosis ◽  
Induced Apoptosis

Abstract The MUC1-C oncoprotein is aberrantly expressed in most multiple myeloma cells. However, the functional significance of MUC1-C expression in multiple myeloma is not known. The present studies demonstrate that treatment of multiple myeloma cells with a MUC1-C inhibitor is associated with increases in reactive oxygen species (ROS), oxidation of mitochondrial cardiolipin, and loss of the mitochondrial transmembrane potential. The MUC1-C inhibitor-induced increases in ROS were also associated with down-regulation of the p53-inducible regulator of glycolysis and apoptosis (TIGAR). In concert with the decrease in TIGAR expression, which regulates the pentose phosphate pathway, treatment with the MUC1-C inhibitor reduced production of NADPH, and in turn glutathione (GSH) levels. TIGAR protects against oxidative stress-induced apoptosis. The suppression of TIGAR and NADPH levels thus contributed to ROS-mediated late apoptosis/necrosis of multiple myeloma cells. These findings indicate that multiple myeloma cells are dependent on MUC1-C and TIGAR for maintenance of redox balance and that targeting MUC1-C activates a cascade involving TIGAR suppression that contributes to multiple myeloma cell death.

Heparanase Regulates Response to Proteasomal Inhibition in Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4902-4902
Author(s):  
Vishnu Prakash C. Ramani ◽  
Ralph D. Sanderson
Keyword(s):  
Cell Death ◽  
Tumor Cells ◽  
Annexin V ◽  
Conditioned Media ◽  
High Dose ◽  
Apoptotic Cells ◽  
Myeloma Cells ◽  
Response To Chemotherapy ◽  
Proteasomal Inhibition ◽  
Induced Apoptosis

Abstract Abstract 4902 Multiple myeloma is an incurable hematologic malignancy. In patients, even after several rounds of high-dose chemotherapy, myeloma tumor cells survive, proliferate aggressively and re-establish the active disease. Understanding the molecular mechanisms that aid myeloma cells in evading chemotherapy is therefore critical for curing this deadly disease. Studies from our lab have established heparanase, an endoglycosidase expressed highly by myeloma tumor cells, as a master regulator of aggressive myeloma disease where it drives tumor growth, angiogenesis, invasion, and osteolysis. However, the effect of heparanase on response to chemotherapy is unknown. Addressing this question, we have made two striking discoveries: i) treatment with proteasomal inhibitors dramatically elevates heparanase expression and secretion in myeloma, and ii) high heparanase expression significantly reduces therapy-induced apoptosis and cell death in myeloma. Proteasomal inhibition by either bortezomib or MG132 dramatically elevated the expression of heparanase and its secretion into the conditioned media. When this conditioned media was added to tumor cells that had not been exposed to proteosome inhibitors, the heparanase was readily taken up by the cells. This is consistent with previous studies demonstrating that exogenous heparanase can be taken up by myeloma tumor cells resulting in activation of aggressive tumor behavior. Next, to test whether heparanase influences chemoresistance in myeloma, we evaluated the effect of bortezomib on inducing apoptosis of myeloma cells expressing either high (HPSE-high) or low (HPSE-low) levels of heparanase. Apoptotic cells were identified by staining for Annexin V and cell death was assessed by staining with propidium iodide. Results revealed that following treatment with bortezomib, HPSE-high cells had a significantly higher percentage of viable cells (Annexin V negative, PI negative) and a significantly smaller fraction of apoptotic cells (Annexin V positive) compared to HPSE-low cells. Similarly, even when myeloma cells were grown on fibronectin, cells expressing high levels of heparanase exhibited significantly decreased bortezomib-induced cell death. Consistent with the above findings, we found that several well established chemoresistant myeloma cell lines such as LR5, Dox 6 and Dox 40 all expressed high levels of heparanase. Together the above findings demonstrate that tumors respond to proteosome inhibitors by upregulating expression and secretion of heparanase and that tumor cells having high levels of heparanase resist proteosome inhibitor-induced apoptosis. These results indicate that heparanase may play an important role in chemoresistance of myeloma cells and that heparanase inhibitors used in tandem with other drugs may improve initial patient outcome and reduce incidence of relapse. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Escherichia coli Shiga-Like Toxins Induce Apoptosis and Cleavage of Poly(ADP-Ribose) Polymerase via In Vitro Activation of Caspases

2002 ◽  
Vol 70 (8) ◽  
pp. 4669-4677 ◽  
Author(s):  
Joyce C. Y. Ching ◽  
Nicola L. Jones ◽  
Peter J. M. Ceponis ◽  
Mohamed A. Karmali ◽  
Philip M. Sherman
Keyword(s):  
Escherichia Coli ◽  
Cell Death ◽  
Intracellular Signaling ◽  
Chromatin Condensation ◽  
Specific Inhibitor ◽  
Caspase 8 ◽  
High Dose ◽  
Dependent Manner ◽  
Induced Apoptosis ◽  
Dose Dependent

ABSTRACT Shiga-like toxin-producing Escherichia coli causes hemorrhagic colitis and hemolytic-uremic syndrome in association with the production of Shiga-like toxins, which induce cell death via either necrosis or apoptosis. However, the abilities of different Shiga-like toxins to trigger apoptosis and the sequence of intracellular signaling events mediating the death of epithelial cells have not been completely defined. Fluorescent dye staining with acridine orange and ethidium bromide showed that Shiga-like toxin 1 (Stx1) induced apoptosis of HEp-2 cells in a dose- and time-dependent manner. Stx2 also induced apoptosis in a dose-dependent manner. Apoptosis induced by Stx1 (200 ng/ml) and apoptosis induced by Stx2 (200 ng/ml) were maximal following incubation with cells for 24 h (94.3% ± 1.8% and 81.7% ± 5.2% of the cells, respectively). Toxin-treated cells showed characteristic features of apoptosis, including membrane blebbing, DNA fragmentation, chromatin condensation, cell shrinkage, and the formation of apoptotic bodies, as assessed by transmission electron microscopy. Stx2c induced apoptosis weakly even at a high dose (1,000 ng/ml for 24 h; 26.7% ± 1.3% of the cells), whereas Stx2e did not induce apoptosis of HEp-2 cells. Thin-layer chromatography confirmed that HEp-2 cells express the Stx1-Stx2-Stx2c receptor, globotriaosylceramide (Gb3), but not the Stx2e receptor, globotetraosylceramide (Gb4). Western blot analysis of poly(ADP-ribose) polymerase (PARP), a DNA repair enzyme, demonstrated that incubation with Stx1 and Stx2 induced cleavage, whereas incubation with Stx2e did not result in cleavage of PARP. A pan-caspase inhibitor (Z-VAD-FMK) and a caspase-8-specific inhibitor (Z-IETD-FMK) eliminated, in a dose-dependent fashion, the cleavage of PARP induced by Shiga-like toxins. Caspase-8 activation was confirmed by detection of cleavage of this enzyme by immunoblotting. Cleavage of caspase-9 and the proapoptotic member of the Bcl-2 family BID was also induced by Stx1, as determined by immunoblot analyses. We conclude that different Shiga-like toxins induce different degrees of apoptosis that correlates with toxin binding to the glycolipid receptor Gb3 and that caspases play an integral role in the signal transduction cascade leading to toxin-mediated programmed cell death.

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