Triptolide inhibits cell growth and induces G0- G1 arrest by regulating P21wap1/cip1 and P27 kip1 in human multiple myeloma RPMI-8226 cells

2010 ◽  
Vol 22 (2) ◽  
pp. 141-147 ◽  
Author(s):  
Yuan Liu ◽  
Ling-lan Zeng ◽  
Yan Chen ◽  
Fei Zhao ◽  
Rui Li ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
G1 Arrest ◽  
Human Multiple Myeloma ◽  
Rpmi 8226 ◽  
P27 Kip1

Growth Inhibition of Human Multiple Myeloma Cells by a CD40 Ligand (CD40L, CD154) Transgene - Oncolytic Virus Construct.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3427-3427
Author(s):  
Margret S. Rodrigues ◽  
Erica M. Gomes ◽  
Reuben Hernandez ◽  
Jack Chang ◽  
Joe Kansopon ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Annexin V ◽  
Normal Fibroblast ◽  
Distribution Analysis ◽  
Untreated Culture ◽  
Human Multiple Myeloma ◽  
Rpmi 8226

Abstract Human multiple myeloma (MM) remains incurable despite recent advances in induction therapy. To explore biotherapeutic approaches that may potentially enhance the efficacy of conventional treatment, we examined the growth regulatory properties of CD40L (CD154), the natural ligand for the myeloma cell surface receptor, CD40. Based on our previous findings that the recombinant CD40L protein effectively inhibited human MM cell growth, a conditional-replicative adenoviral construct, AdEHCD40L was used for targeted delivery of the CD40L transgene. AdEHCD40L incorporates tumor/tissue specific promoters that limit viral and transgene expression to HIF (hypoxia inducing factor)-1α overexpressing cells, which are prevalent in the human bone marrow compartment. Conditional expression of the early adenoviral E1A gene and the CD154 transgene was validated in the IL-6 independent MM line RPMI 8226 (62% and 66%, respectively), and the IL-6 dependent cell line Kas-6/1 (32.68% and 30%, respectively). Further, treatment with AdEHCD40L at a multiplicity of infection (MOI) of 1 resulted in pronounced growth inhibition for both cell lines (95.5±2.1% and 80.5±9.8%, mean±SD, respectively). AdEHCD40L treatment was more effective than the parental construct without the CD154 transgene (AdEHNULL) in both cell lines (p=0.04). Both AdEHNULL and AdEHCD40L were minimally cytotoxic to normal peripheral blood mononuclear cells (0% at 48hrs) and normal fibroblast cells IMR-90 (2.8±0.3%). The in vivo antitumor activity of AdEHCD40L was examined with a subcutaneous RPMI 8226 heterotransplant model in SCID mice. Intratumoral injection of AdEHCD40L (5x107 pfu, x 5) reduced xenograft growth by 53% at day 29 (4.8±0.9 mm, vs. 10.5±1.2 mm in mock-treated animals; p=0.002), and was more effective than AdEHNULL (7.6±1.1 mm; p=0.03). Adenoviral hexon and CD40L expression was detectable at 29 days post-viral treatment, based on immunohistochemical analysis. Hence intratumoral treatment with AdEHCD40L likely involved oncolytic viral replication. To further characterize cellular events that accompany MM cell growth inhibition, apoptotic activity was measured by annexin V and propidium iodide incorporation. A marked elevation of the annexin V+ subset (21.3±6.5%, vs. 7.7±1.9% in untreated culture; p=0.007) was accompanied by decreased cell viability (52.8±1%, vs. 76.1±11% in untreated cultures; p = 0.04) following AdEHCD40L treatment. Cell cycle distribution analysis demonstrated a corresponding increase in the subG0/G1 compartment (AdEHCD40L, 23.9±3.6%; vs. untreated, 9.1±2.3%) that was consistent with elevated apoptosis. Further, AdEHCD40L increased S phase accumulation by 72 hrs (68.0±2.4%, vs. 54.0±5.5% in AdEHNULL and 48.9±4.6% in untreated culture; p<0.003). Currently, gene expression array analyses are underway to define molecular events that are pertubated by CD154 transgene activity and by viral oncolysis. These findings will further elucidate the mechanism of action of the CD154+oncolytic viral approach for experimental gene therapy of human MM.

CD40 activation mediates p53-dependent cell cycle regulation in human multiple myeloma cell lines

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 1039-1046 ◽  
Author(s):  
G. Teoh ◽  
Y.-T. Tai ◽  
M. Urashima ◽  
S. Shirahama ◽  
M. Matsuzaki ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Growth Arrest ◽  
Myeloma Cell ◽  
Luciferase Reporter ◽  
Temperature Sensitive ◽  
Multiple Myeloma Cell ◽  
Cd40 Activation ◽  
Human Multiple Myeloma ◽  
Rpmi 8226

It has been reported that the activation of multiple myeloma (MM) cells by CD40 induces proliferation, growth arrest, and apoptosis. To determine whether the biologic sequelae of CD40 activation in MM cells depends on p53 function, we identified temperature-sensitive p53 mutations in the RPMI 8226 (tsp53E285K) and the HS Sultan (tsp53Y163H) MM cell lines. These cells were then used as a model system of inducible wtp53-like function because wild-type-like p53 is induced at permissive (30°C) but not at restrictive (37°C) temperatures. Using p21-luciferase reporter assays, we confirmed that CD40 induces p53 transactivation in RPMI 8226 and HS Sultan cells cultured under permissive, but not restrictive, conditions. Furthermore, CD40 activation of these MM cells under permissive, but not restrictive, temperatures increased the expression of p53 and p21 mRNA and protein. Importantly, CD40 activation induced the proliferation of RPMI 8226 and HS Sultan cells at restrictive temperatures and growth arrest and increased subG1 phase cells at permissive temperatures. These data confirmed that CD40 activation might have distinct biologic sequelae in MM cells, depending on their p53 status.

Inhibition of ERK1/2 Activity by the MEK1/2 Inhibitor AZD6244 (ARRY-142886) Induces Human Multiple Myeloma Cell Apoptosis in the Bone Marrow Microenvironment: A New Therapeutic Strategy for MM.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3460-3460 ◽  
Author(s):  
Yu-Tzu Tai ◽  
Xian-Feng Li ◽  
Iris Breitkreutz ◽  
Weihua Song ◽  
Peter Burger ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Cell Lines ◽  
Caspase 3 ◽  
Parp Cleavage ◽  
Dependent Manner ◽  
Growth And Survival ◽  
Cyclin E1 ◽  
Human Multiple Myeloma

Abstract Activation of the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) signaling pathway mediates tumor cell growth in many cancers, including human multiple myeloma (MM). Specifically, this pathway mediates MM cell growth and survival induced by cytokines/growth factors (i.e. IL-6, IGF-1, CD40, BAFF) and adhesion to bone marrow stromal cells (BMSCs), thereby conferring resistance to apoptosis in the bone marrow (BM) milieu. In this study, we therefore examined the effect of the MEK1/2 inhibitor AZD6244 (ARRY-142886), on human MM cell lines, freshly isolated patient MM cells and MM cells adhered to BMSCs. AZD6244, inhibits constitutive and cytokine (IL-6, IGF-1, CD40)-stimulated ERK1/2, but not AKT phosphorylation. Importantly, AZD6244 inhibits the proliferation and survival of human MM cell lines, regardless of sensitivity to conventional chemotherapy, as well as freshly isolated patient MM cells. AZD6244 induces apoptosis in patient MM cells even in the presence of BMSCs, as evidenced by caspase 3 activity and PARP cleavage at concentrations as low as 20 nM. AZD6244 overcomes resistance to apoptosis in MM cells conferred by IL-6 and BMSCs, and inhibits IL-6 secretion induced by MM adhesion to BMSCs. AZD6244 suppresses MM cell survival/growth signaling pathways (i.e., STAT3, Bcl-2, cyclin E1, CDK1, CDK3, CDK7, p21/Cdc42/Rac1-activated kinase 1, casein kinase 1e, IRS1, c-maf) and up-regulates proapoptotic cascades (i.e., BAX, BINP3, BIM, BAG1, caspase 3, 8, 6). AZD6244 also upregulates proteins triggering cell cycle arrest (i.e. p16INK4A, p18INK4C, p21/WAF1 [Cdkn1a], p27 [kip1], p57). In addition, AZD6244 inhibits adhesion molecule expression in MM cells (i.e. integrin a4 [VLA-4], integrin b7, ICAM-1, ICAM-2, ICAM-3, catenin a1, c-maf) associated with decreased MM adhesion to BMSCs. These pleiotropic proapoptotic, anti-survival, anti-adhesion and -cytokine secretion effects of AZD6244 abrogate BMSC-derived protection of MM cells, thereby sensitizing them to both conventional (dexamethasone) and novel (perifosine, lenalidomide, and bortezomib) therapies. In contrast, AZD6244 has minimal cytotoxicity in BMSCs and does not inhibit DNA synthesis in CD40 ligand-stimulated CD19 expressing B-cells derived from normal donors at concentrations toxic to MM cells (between 0.02–2 mM). Furthermore, AZD6244 inhibits the expression/secretion of osteoclast (OC)-activating factors (i.e., macrophage inflammatory protein (MIP)-1a, MIP-1b, IL-1b, VEGF) from MM cells. It also downregulates MM growth and survival factors (IL-6, BAFF, APRIL) in OC cultures derived from MM patient peripheral blood mononuclear cells (PBMCs). Significantly, AZD6244 inhibits OC differentiation from MM PBMCs (n=10) in a dose-dependent manner. Together these results provide the preclinical basis for clinical trials with AZD6244 (ARRY-142886) in MM.

KRN5500, a Spicamycin Derivative, Exerts Anti-Myeloma Effects through Impairing Both Myeloma Cells and Osteoclasts.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1669-1669
Author(s):  
Hirokazu Miki ◽  
Shuji Ozaki ◽  
Osamu Tanaka ◽  
Shingen Nakamura ◽  
Ayako Nakano ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Death ◽  
Cell Growth ◽  
Cell Lines ◽  
G1 Arrest ◽  
Dependent Manner ◽  
Osteolytic Bone Disease ◽  
Rpmi 8226

Abstract Multiple myeloma (MM) is a plasma cell malignancy characterized by devastating bone destruction due to enhanced bone resorption and suppressed bone formation. Although high-dose chemotherapy and new agents such as thalidomide, lenalidomide, and bortezomib have shown marked anti-MM activity in clinical settings, MM remains incurable due to drug resistance mediated by interactions with osteoclasts or stroma cells. Moreover, osteolytic bone disease continues to be a major problem for many patients. Therefore, alternative approaches are necessary to overcome drug resistance and inhibit osteoclasts activity in MM. KRN5500 is a new derivative of spicamycin produced by Streptomyces alanosinicus (Kirin Pharma, Tokyo, Japan), which potently inhibits protein synthesis and induces cell death in human tumor cell lines. Phase I studies of KRN5500 in patients with solid tumors such as colon cancer and gastric cancer showed acceptable toxicity with Cmax values of 1000––3000 nM. In this study, we investigated the effects of KRN5500 against MM cells and osteoclasts in vitro and in vivo. MM cell lines such as RPMI 8226, MM.1S, INA-6, KMS12-BM, UTMC-2, TSPC-1, and OPC were incubated with various concentrations of KRN5500 for 3 days. Cell proliferation assay showed marked inhibition of cell growth with G1 arrest in these MM cells (IC50: 4–100 nM). KRN5500 (100 nM) also induced 30–90% of cell death in primary MM cells (n=7). Annexin V/propidium iodide staining showed that KRN5500 induced apoptosis of MM cells in a dose- and time-dependent manner. Western blot analysis confirmed activation of caspase-8, -9, and −3, cleavage of poly (ADP-ribose) polymerase (PARP), and down-regulation of Mcl-1. We next examined the effect of KRN5500 against MM cell lines and primary MM cells in the presence of bone marrow stroma cells and osteoclasts. Co-culture of these cells enhanced viability of MM cells; however, KRN5500 still induced strong cytotoxicity to MM cells. Of interest, KRN5500 specifically mediated apoptosis in osteoclasts but not stroma cells as assessed by TUNEL staining. More than 90% of osteoclasts were killed even at a low concentration of KRN5500 (20 nM). Finally, we evaluated the effect of KRN5500 against MM cells and osteoclasts in vivo. Two xenograft models were established in SCID mice by either subcutaneous injection of RPMI 8226 cells or intra-bone injection of INA-6 cells into subcutaneously implanted rabbit bones (SCID-rab model). These mice were treated with intraperitoneal injection of KRN5500 (5 mg/kg/dose) or saline thrice a week for 3 weeks after tumor development. In a subcutaneous tumor model, KRN5500 inhibited the tumor growth compared with control mice (increased tumor size, 232 ± 54% vs 950 ± 422%, p&lt;0.001, n=6 per group). In a SCID-rab model, KRN5500 also inhibited MM cell growth in the bone marrow (increase of serum human sIL6-R derived from INA-6, 134 ± 19% vs 1112 ± 101%, p&lt;0.001, n=5 per group). Notably, the destruction of the rabbit bones was also prevented in the KRN5500-treated mice as evaluated by radiography. Therefore, these results suggest that KRN5500 exerts anti-MM effects through impairing both MM cells and osteoclasts and that this unique mechanism of action provides a valuable therapeutic option to improve the prognosis in patients with MM.

HDAC3 Maintains DNMT1 through Dual Mechanisms in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4472-4472 ◽  
Author(s):  
Takeshi Harada ◽  
Hiroto Ohguchi ◽  
Yohann Grondin ◽  
Shohei Kikuchi ◽  
Morihiko Sagawa ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Board Of Directors ◽  
Combination Treatment ◽  
Hdac Inhibitors ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Rpmi 8226 ◽  
Dnmt1 Expression

Abstract Histone deacetylases (HDACs) represent novel therapeutic targets for the treatment of multiple myeloma (MM). Although non-selective HDAC inhibitors demonstrate remarkable anti-MM activity, they also are associated with side effects. To avoid these adverse events without reducing anti-MM efficacy, we have been developing isoform- or class-selective HDAC inhibitors. Specifically, we showed that HDAC3 plays an important role in MM cell proliferation (Minami J, et al, Leukemia. 2014), and here delineate the mechanism whereby HDAC3 inhibition abrogates MM cell growth. We first carried out gene expression profiling before and after knocking down of HDAC3 in MM.1S cells. Among significantly downregulated genes (adjusted P values < 0.001, log fold change > 1.0), we selected DNA methyltranseferase 1 (DNMT1) for further studies. Downregulation of DNMT1 by HDAC3 knockdown was first confirmed by quantitative real time PCR (Q-PCR) and immunoblotting in both MM.1S and RPMI 8226 cells. HDAC3 selective inhibitor BG45 also downregulated DNMT1 expression. Importantly, knockdown of DNMT1triggers apoptosis in MM cells, suggesting that DNMT1 downregulation plays, at least in part, a role in HDAC3 inhibitor-induced MM cell growth inhibition. Previous studies show that HDAC inhibitors downregulate c-Myc expression (Hideshima T, et al. Blood Cancer J. 2015), and we confirmed that c-Myc was downregulated by genetic downregulation and pharmacological inhibition of HDAC3 by HDAC3 shRNA and BG45, respectively. Moreover, treatment of MM.1S cells with BG45 markedly increased c-Myc acetylation. Importantly, c-Myc was significantly degraded after treatment of MM.1S with HDAC3 inhibitor BG45 in the presence of cycloheximide (CHX), indicating that downregulation of c-Myc by HDAC3 inhibition is due to loss of protein stability. To determine whether DNMT1 expression is regulated by c-Myc, we next analyzed ChIP-Seq data in MM.1S cells (GSE36354) and found that c-Myc binds to DNMT1 promoter region. We confirmed downregulation of DNMT1 after knockdown of MYC in MM.1S and RPMI 8226 cells by Q-PCR and immunoblotting. These results suggest that HDAC3 inhibition downregulates DNMT1 through downregulation of c-Myc. A recent study reported that acetylation of DNMT1 leads to its ubiquitination, resulting in degradation of DNMT1 (Cheng J, et al. Nat Commun. 2015). We showed that treatment of MM.1S cells with BG45 in the presence of CHX triggered hyperacetylation of DNMT1, followed by its degradation. We further confirmed this association of acetylation and ubiquitination of DNMT1 protein using a dequbiquitination assay in 293T cells. As expected, HDAC3 blocked DNMT1 ubiquitination. Taken together, these results suggest that HDAC3 inhibition modulates DNMT1 via both c-Myc and by acetylation and thereby altering protein stability. Finally, Azacytidine (AZA) is used as a DNMT1 inhibitor in the treatment of acute myeloid leukemia and myelodysplastic syndrome. We therefore examined combination treatment of MM cells with BG45 combined with AZA. Importantly, this combination triggered synergistic downregulation of DNMT1 and growth inhibition through apoptosis in both MM cell lines and patient MM cells. Efficacy of combination treatment was confirmed in a murine xenograft MM model, evidenced by both tumor growth inhibition and prolonged overall host survival. Our results therefore provide the rationale for combination treatment with HDAC3 inhibitor and DNMT1 inhibitor to improve patient outcome in MM. Disclosures Mazitschek: Acetylon: Equity Ownership. Hideshima:Acetylon: Consultancy; C4 Therapeutics: Equity Ownership. Anderson:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Oncoprep: Equity Ownership; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Elimination of clonogenic stem cells from human multiple myeloma cell lines by a plasma cell-reactive monoclonal antibody and complement

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1482-1489 ◽  
Author(s):  
AW Tong ◽  
JC Lee ◽  
JW Fay ◽  
MJ Stone
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Cell Lines ◽  
Plasma Cell ◽  
Colony Formation ◽  
Human Multiple Myeloma ◽  
Rpmi 8226 ◽  
Marrow Cells

Abstract The monoclonal antibody (MoAb) MM4 reacts with human multiple myeloma (MM) cell lines and bone marrow from patients with plasma cell dyscrasias but not with normal peripheral blood or bone marrow cells. Treatment with MM4 and rabbit complement (C') was cytotoxic to the plasma cell-derived cell lines GM 1312, RPMI 8226, and ARH-77, as demonstrated by chromium release microcytotoxicity and trypan blue exclusion assays. The same treatment eliminated greater than 99% of clonogenic myeloma stem cell colony formation of these cell lines, with less than 20% inhibition of normal human bone marrow pleuripotent progenitor colony formation in vitro. As an experimental model to explore the efficacy of MM4 + C' in purging MM-involved bone marrow, normal marrow cells were mixed with RPMI 8226 or GM 1312 cells in the ratio of 90:10 or 50:50 (marrow:myeloma cells). Colony growth assays indicated that MM4 + C' eliminated at least 2 logs of clonogenic myeloma stem cells in both 90:10 and 50:50 preparations, while sparing the majority of normal marrow progenitors (inhibition of CFU-C:10% to 13%; BFU-E:0%). The selectivity of MM4-mediated cytotoxicity may be useful for eliminating myeloma clonogenic stem cells from bone marrow of patients with multiple myeloma.

scholarly journals Investigation of the Proliferation, Apoptosis/Necrosis, and Cell Cycle Phases in Several Human Multiple Myeloma Cell Lines. Comparison ofViscum albumQuFrF Extract with Vincristine in anIn VitroModel

2010 ◽  
Vol 10 ◽  
pp. 311-320 ◽  
Author(s):  
Eva Kovacs
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Cytostatic Effect ◽  
Multiple Myeloma Cell ◽  
Cytocidal Effect ◽  
Human Multiple Myeloma ◽  
Cell Cycle Phases ◽  
Rpmi 8226

Multiple myeloma is a haematological disorder of malignant plasma cells. Interleukin-6 (IL-6) is a potent growth factor for the proliferation of these cells. Vincristine as a chemotherapeutic agent is used mainly in combination with other chemotherapeutic substances in the treatment of different haematological disorders.Viscum albumQuFrF (VAQuFrF) extract is an experimental drug that is not used in the treatment in tumour patients. It contains 2000 ng lectin and 10 µg viscotoxin in 10 mg extract. In this study, the effects of VAQuFrF extract were compared with those of vincristine in six human multiple myeloma cell lines (Molp-8, LP-1, RPMI-8226, OPM-2, Colo-677, and KMS-12-BM) using anin vitromodel. As parameters, the IL-6 production, proliferation, apoptosis/necrosis, and cell cycle phases of the cells were taken. To measure the IL-6 production, apoptosis/necrosis, and cell cycle phases, the substances were tested in dose ranges of 10, 50, and 100 µg/106cells. To measure the proliferation of the cells, the substances were tested in dose ranges of 1, 5, and 10 µg/105cells. The profile of the antitumour effects of the two substances is identical. (1) Neither VAQuFrF extract nor vincristine produced IL-6 in any cell line. (2) Both substances inhibited the proliferation of the cells (cytostatic effect), arrested the cell cycle phases, and increased the number of apoptotic/necrotic cells (cytocidal effect). At a dose of 10 µg/105cells, VAQuFrF more effectively inhibited the proliferation than vincristine (p< 0.01) in the cell lines Molp-8, LP-1, and RPMI-8226. (3) VAQuFrF affected the tumour cells mainly via cytostatic effect. Vincristine had a clear cytocidal effect. These findings indicate that VAQuFrF extract could be a novel drug in the treatment of multiple myeloma.

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