Gene Profile during Arsenic Trioxide-Induced Apoptosis in Multiple Myeloma: Strong Anti-Oxidantive Response and Pro-Apoptotic Imbalance.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5175-5175
Author(s):  
Alejo A. Morales ◽  
Delia Gutman ◽  
Pedro J. Cejas ◽  
Kelvin P. Lee ◽  
Lawrence H. Boise
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Death ◽  
Arsenic Trioxide ◽  
Cell Lines ◽  
Heme Oxygenase ◽  
Metal Ion ◽  
Time Course ◽  
Heme Oxygenase 1 ◽  
Induced Apoptosis

Abstract Arsenic Trioxide (ATO) has been successfully used for the treatment of acute promyelocytic leukemia (APL) and has promising activity in multiple myeloma (MM). The purpose of the present study was to evaluate the changes in the gene expression profile in four human MM cell lines (U266, MM.1S, RPMI 8226 and KMS11) following exposure to ATO in a 48H time-course to gain further insight into the mechanism of action in MM. U266 and 8226 show some resistance to ATO, while MM.1S and KMS11 are more sensitive to ATO-induced apoptosis. Two μM ATO resulted in 45.3, 74.2, 48.3 and 76.4 percent apoptosis for U266, MM.1S, 8226 and KMS11, at 48H respectively. Affymetrix Hu133 2.0 Plus Chips containing 54,675 probe sets were used for the mRNA expression profiling at 0, 6, 24 and 48H after ATO treatment. A total of 654, 478 and 600 common up or down-regulated genes showed a clustered expression pattern in response to ATO at 6, 24 and 48H, respectively. The pattern was consistent with strong anti-oxidative and heavy metal responses and pro-apoptotic imbalance. Altered cell processes included: protein folding, response to metal ion, cation and amino acids transport, heme metabolism, cell proliferation, cysteine metabolism, fatty acid metabolism, cell proliferation and apoptosis. Common up-regulated genes including clusterin (apolipoprotein J), ferritin, biliverdin reductase B, glutamate-cysteine ligase, heme oxygenase (decycling) 1, metallothionein 1, NAD(P)H dehydrogenase quinone 1, and solute carrier family 7 imply a clear anti-oxidative response. Heme oxygenase-1 (HO-1) and metallothioneins (MTs) expression has been suggested to be associated with ATO-resistance. HO-1 baseline expression is 5 fold higher in U266 and 8226 cell lines compared to MM.1S and KMS11, also suggesting a possible role of HO-1 in ATO-resistance. However, the strong up-regulation of this protein (greater than 50 fold in MM.1S and KMS11 as early as 6H) could not protect cells from ATO-induced apoptosis. Similarly the strong up-regulation of MT-1 did not correlate with cell survival. Moreover, up-regulation of HO-1 and MT-1 prior to ATO treatment (using Hemin and ZnCl2, respectively) only marginally and transiently protected MM.1S from ATO-induced apoptosis. Common down-regulated genes such as bcl-x, survivin and insulin induced gene 1 suggest a pro-apoptotic imbalance in the cell fate. Bcl-x was down-regulated in all the four cell lines during ATO-induced apoptosis while one of its ligands, Bmf, a BH3-only pro-apoptotic Bcl-2 family member was up-regulated significantly (2.5 and 3.0 fold) in MM.1S and KMS11 while to a lesser extent in U266 and 8226 (1.5 and 1.6 fold). Additionally, Bmf baseline expression is higher in MM.1S and KMS11 compared to U266 and 8226, while Bim is highly expressed in the four cell lines and did not show any transcriptional regulation during ATO-induced apoptosis. Bim and Bmf are induced following JNK activation. JNK1 is phosphorylated in all cell lines in response to ATO. Taken together these data indicate that while the cells initially respond to ATO treatment in a manner consistent with oxidative stress, the compensatory mechanisms are not sufficient to block cell death. The mechanism(s) of cell death are not completely clear although changes in the expression of Bcl-2 family members suggest that activation of the intrinsic apoptotic pathway is likely to be important.

Ascorbic acid enhances arsenic trioxide–induced cytotoxicity in multiple myeloma cells

Blood ◽  
2001 ◽  
Vol 98 (3) ◽  
pp. 805-813 ◽  
Author(s):  
Jennifer M. Grad ◽  
Nizar J. Bahlis ◽  
Isildinha Reis ◽  
Marc M. Oshiro ◽  
William S. Dalton ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Ascorbic Acid ◽  
Arsenic Trioxide ◽  
Cell Lines ◽  
Plasma Cells ◽  
Radical Scavenging ◽  
B Cell Malignancy ◽  
Drug Efflux Pumps ◽  
Induced Apoptosis

Abstract Multiple myeloma (MM) is a clonal B-cell malignancy characterized by slow-growing plasma cells in the bone marrow (BM). Patients with MM typically respond to initial chemotherapies; however, essentially all progress to a chemoresistant state. Factors that contribute to the chemorefractory phenotype include modulation of free radical scavenging, increased expression of drug efflux pumps, and changes in gene expression that allow escape from apoptotic signaling. Recent data indicate that arsenic trioxide (As2O3) induces remission of refractory acute promyelocytic leukemia and apoptosis of cell lines overexpressing Bcl-2 family members; therefore, it was hypothesized that chemorefractory MM cells would be sensitive to As2O3. As2O3 induced apoptosis in 4 human MM cell lines: 8226/S, 8226/Dox40, U266, and U266/Bcl-xL. The addition of interleukin-6 had no effect on cell death. Glutathione (GSH) has been implicated as an inhibitor of As2O3-induced cell death either through conjugating As2O3 or by sequestering reactive oxygen induced by As2O3. Consistent with this possibility, increasing GSH levels with N-acetylcysteine attenuated As2O3 cytotoxicity. Decreases in GSH have been associated with ascorbic acid (AA) metabolism. Clinically relevant doses of AA decreased GSH levels and potentiated As2O3-mediated cell death of all 4 MM cell lines. Similar results were obtained in freshly isolated human MM cells. In contrast, normal BM cells displayed little sensitivity to As2O3 alone or in combination with AA. Together, these data suggest that As2O3 and AA may be effective antineoplastic agents in refractory MM and that AA might be a useful adjuvant in GSH-sensitive therapies.

VEGF Upregulates Mcl-1 Expression and Protects Multiple Myeloma Cells Against Starvation Induced-Apoptosis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 631-631
Author(s):  
Steven Le Gouill ◽  
Klaus Podar ◽  
Martine Amiot ◽  
Teru Hideshima ◽  
Dharminder Chauhan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Myeloid Cell ◽  
Fibroblast Cell ◽  
Transfected Cells ◽  
Murine Embryonic Fibroblast ◽  
Induced Apoptosis

Abstract Vascular endothelial growth factor (VEGF) induces proliferation of MM cells and induces interleukin-6 (IL-6) secretion in a paracrine loop involving MM cells and bone marrow stromal cells. In turn, IL-6 triggers multiple myeloma (MM) cell proliferation and also protects against apoptosis by upregulating Myeloid-cell-leukemia 1 (Mcl-1), a critical survival protein in MM cells. The goal of our study was to investigate the role of Mcl-1 in VEGF induced-proliferation and protection against apoptosis. Using two murine embryonic fibroblast cell lines as a model (a Mcl-1 deleted cell line and its wild type: Mcl-1Δ/null and Mcl-1wt/wt MEFs, respectively), we here demonstrate that deletion of Mcl-1 reduces fetal bovine serum (FBS), VEGF, and IL-6 induced-proliferation. In addition, we demonstrate that the percentage of cells in S phase is lower in Mcl-1Δ/null compared to Mcl-1wt/wt MEFs (21% (+/−1) versus 30% (+/− 3), respectively). Taken together, these results demonstrate that Mcl-1 is required to mediate VEGF, Il-6 and FBS-induced-proliferation and cell cycle progression. To highlight the key anti-apoptotic role of Mcl-1 in MM cells, humans MM1s cells were transfected with Mcl-1 siRNA. Specific inhibition of Mcl-1 was associated with decreased proliferation (42% and 61% decreases at 24 and 48 h, respectively) and induction of apoptosis (subG1 peak: 22% and 41% in Mcl-1 siRNA transfected cells versus 15% and 15 % in non-transfected cells at 24 and 48 h, respectively), confirming that Mcl-1 is critical for both proliferation and protection against apoptosis in MM cells. In 3 human MM cell lines (MM1s, U266 and MM1R) and MM patient cells we next showed that Mcl-1 protein expression, but not other bcl-2 family members, is upregulated by VEGF in a time and dose manner; and conversely that the pan-VEGF inhibitor GW654652, blocks VEGF induced-upregulation of Mcl-1. Furthermore using flow cytometry with a double staining (CD38-FITC and Apo 2.7-PE), we demonstrate that VEGF protects MM patient cells from FBS-starvation-induced-apoptosis: the percentage of apoptotic MM patient cells (CD38++ and Apo 2.7+) in non starved medium (RPMI 1640 supplemented with 10% FBS) was 15% versus 93% in starved medium (RPMI 1640 supplemented with FBS 2%), and 48% in starved medium supplemented with 25ng/ml VEGF. In conclusion, our study demonstrates that VEGF protects MM cells against apoptosis, and that VEGF-induced MM cell proliferation and survival is mediated via Mcl-1. these studies provide the preclinical framework for novel therapeutics targeting both Mcl-1 and/or VEGF to improve patient outcome in MM.

Transiently Elevated AST/LDH Are Associated with Clinical Response to Recombinant Circularly Permuted TRAIL (CPT) Plus Thalidomide in Patients with Relapsed and/or Refractory Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3478-3478
Author(s):  
Wenming Chen ◽  
Peng Wei ◽  
Shifang Yang ◽  
Xiangjun Zheng ◽  
Lugui Qiu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Time Course ◽  
Single Agent ◽  
Release Rates ◽  
Refractory Multiple Myeloma ◽  
Before And After ◽  
Clinical Responses ◽  
Thalidomide Treatment

Abstract Introduction: Circularly permuted TRAIL (CPT), a recombinant mutant of human Apo2L/TRAIL, is a promising anti-tumor candidate. In previous phase1/2 clinical trials of single-agent CPT in patients with relapsed and/or refractory multiple myeloma (RRMM), transient elevations of serum AST and LDH were observed early after CPT treatment in most response patients, but not in the non-respondent. Positive correlations were found between increased AST/LDH on day 2 or 3 (24 or 48 hours) after CPT initial dosing and the clinical responses to CPT. Objective: To determine whether transiently elevated AST/LDH is predictive of responses to CPT plus thalidomide in thalidomide-relapsed or refractory MM patient and the time course of AST or LDH elevation. Methods: We retrospectively analyzed the data of a phase 2 study of CPT plus thalidomide. The changes of serum AST, LDH or ALT were analyzed before treatment and on days 2, 3 and 6 after initial dosing. Relationship was evaluated between ΔAST, ΔLDH, ΔALT (ratio of ASTD2, LDHD2 or ALTD2to baseline value) and the best clinical responses. Four MM cell lines (RPMI 8226, NCI-H929, MM.1S, MM.1R) sensitive to CPT were used to detect the concentrations of AST, ALT and LDH in the cytoplasm or the medium of CPT-treated cells, with the purpose of determining whether CPT-induced cell death could result in an elevation of AST or LDH. Results: Of 41 efficacy-evaluable patients, 9(22.0%) achieved a partial response (PR) or better and 14 (34.1%) achieved a minimal response (MR) or better. The serum ASTD2 and LDHD2 levels were dramatically increased from baseline in patients with ≥PR or ≥MR, but not in those with NC/PD. However, serum ALTD2 was comparable to baseline value either in response patients (≥PR or ≥MR) or in non-response ones (NC/PD). Consequently, the median ΔAST or ΔLDH of patients with ≥PR or ≥MR was significantly higher than that of patients with NC/PD (Table 1). The elevation of AST or LDH was transient with a peak on day 2 after treatment, then dramatically declined on day 3, and usually disappeared within one week (at most two weeks for LDH) (Figure 1), which was uniquely observed in the first treatment cycle. A univariate logistic-regression analysis showed that ΔASTwas predictive of achieving responses of ≥MR or not (P=0.04). Indeed, patients with higher level of ΔAST had higher probability of achieving responses of ≥MR (72.7% in patients with ΔAST>1.35 vs. 26.3% in patients with ΔAST≤1.35). In the cytoplasm of MM cell lines, abundant AST and LDH but only detectable level of ALT was observed. There was no significant change in the release rates of AST and LDH with CPT incubation for 1, 2, 3, 4 or 6 hours, even though the cell viabilities at 6h had already declined to about 10-20% of the control cells by ATP chemiluminescent assay. Remarkable increase in the release rates of AST and LDH occurred at 24h, with no evident change of ALT, which was consistent with what was observed in patients in the clinical study. It was suggested that the transient elevation of AST or LDH in RRMM patients was most likely resulted from CPT-induced myeloma cell death. Conclusion: The early transient elevations of serum AST and LDH after CPT plus thalidomide treatment were positively correlated with the clinical responses to CPT plus thalidomide, and were possibly resulted from CPT-induced cell death. ΔAST on day2 could be a surrogate response biomarker for CPT treatment in RRMM patients. Figure 1 Representative time course of AST, ALT or LDH changes before and after CPT plus thalidomide treatment in response patients Figure 1. Representative time course of AST, ALT or LDH changes before and after CPT plus thalidomide treatment in response patients Table 1 Differences in ΔAST, ΔALT and ΔLDH between response patients (≥PR or ≥MR) and non-response patients (NC/PD) Table 1. Differences in ΔAST, ΔALT and ΔLDH between response patients (≥PR or ≥MR) and non-response patients (NC/PD) Disclosures Wei: Beijing Sunbio Biotech Co., Ltd.: Employment. Yang:Beijing Sunbio Biotech Co., Ltd.: Employment. Zheng:Beijing Sunbio Biotech Co., Ltd.: Employment. Pang:Beijing Sunbio Biotech Co., Ltd.: Employment.

The Diterpenoid Lactone Andrographolide (ANDRO) Stimulates Autophagy and Induces Reactive Oxygen Species (ROS)-Dependent Apoptosis in Lymphoma Cell Lines

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4991-4991
Author(s):  
Shuo Yang ◽  
Andrew M. Evens ◽  
Sheila Prachand ◽  
Leo I. Gordon
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Cell Lines ◽  
Redox State ◽  
Annexin V ◽  
Lymphoma Cell ◽  
Time Dependent ◽  
Cellular Redox ◽  
Forkhead Transcription Factors ◽  
Induced Apoptosis

Abstract ANDRO is a diterpenoid lactone isolated from Andrographis paniculata (King of Bitters), an important herbal medicine used in China. It has been reported to have anti-inflammatory, anti-hypertensive, anti-viral and immunostimulant properties. It has also been shown to inhibit cancer cell proliferation and induce apoptosis in HL-60 (leukemia), PC-3 (prostatic adenocarcinoma), MDA-MB-231 (breast cancer), HepG2 (liver cancer), HeLa (cervical cancer) and HCT116 (colorectal cancer) cell lines. The diterpenoids have been found to generate ROS and may increase apoptosis by altering the cellular redox state. We hypothesized that ANDRO would lead to cell death in lymphoma cell lines and that the effect may be related to altered cellular redox state. We studied the Burkitt p53 mutated Ramos cell line, the mantle cell lymphoma line Granta and L428, a resistant EBV-negative Hodgkin lymphoma cell line. We found that after incubation with increasing concentrations of ANDRO, there was dose and time-dependent cell death as measured by MTT. The IC50 (concentration that achieved 50% cell proliferation inhibition) at 48h was 20μM for Ramos, 40μM for Granta, and 50μM for L428. ROS was measured by oxidation of 2’7’dichlorofluorescein diacetate (DCFDA) to dichlorofluorescein (DCF) and analyzed by fluorescence-activated cell sorting (FACS) following incubation at 1hour (h), 2h, 3h, 5h, 38h, and 48h with ANDRO (20–80μM). ANDRO increased ROS production in all lymphoma cell lines, which was abrogated by the antioxidant N-acetyl-L-cysteine (NAC). Maximum ROS generation with ANDRO was seen at 48h for Ramos (1.7 fold), 5h for Granta (1.6 fold), and 38h for L428 (2.4 fold). To determine the mechanism of cell death, we measured apoptosis by Annexin-V/propidium iodide (PI), and detected by flow cytometry (FACS). Cells were treated with ANDRO in the presence or absence of the reduced glutathione (GSH) depleting agent buthionine sulfoximine (BSO) (100μM) for 28h, 48h, and 72h. We found that the AC50 (concentration that achieved 50% apoptosis) was 40μM for Ramos at 72h, 40μM for Granta at 48h and >80μM for L428 at 48h, while in the presence of BSO it was <10μM for Ramos at 72h, between 30–40μM for Granta at 28h and between 30–40μM for L428 at 48h. Apoptosis was completely blocked, by NAC, both in the presence and absence of BSO. Further, ANDRO induced PARP cleavage and activation of caspases 3, 8, and 9 in Granta and Ramos. Next, we explored the relationship of ANDRO and Forkhead transcription factors. ANDRO caused dephosphorylation of FOXO3a or FOXO1, in a dose- and time-dependent manner, and this was reversible by NAC. Downstream proteins of FOXO3a, Bim, p27kip1 and the isoforms of the autophagy-related protein LC3B were upregulated, and this was reversed by NAC. The LC3B isoform-II, which is cleaved from LC3B-I, is a marker of autophagy activation. To determine the role of autophagy in cell death related to ANDRO, we inhibited autophagy with 3-methyladenine (1–2mM) and found significant enhancement of ANDRO-induced apoptosis in Granta and Ramos. Finally, ANDRO induced apoptosis (>60% Annexin-V+/PI+) in malignant B-cells from a patient with chronic lymphocytic leukemia/small lymphocytic lymphoma (trisomy 12, peripheral blood absolute lymphocyte count 95.2 K/uL, bulky adenopathy) very low concentrations (5μM at 18h) in vitro, which was also reversible with NAC. We conclude that ANDRO induces ROS-dependent apoptosis in lymphoma cell lines and in a primary tumor sample, which is enhanced by depletion of GSH and inhibited by the antioxidant NAC. These effects appear to proceed through caspase activation and inhibition of autophagy, and are in part dependent on signaling through forkhead transcription factors and altered cellular redox pathways. Further studies of diterpenoids as single agents or in combination with other anti-lymphoma agents are warranted.

Triggering of Toll-Like Receptor-4 In Human Multiple Myeloma Cells Promotes Proliferation and Alters Cell Responses to Immune and Chemotherapy Drug Attack

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1905-1905
Author(s):  
Zhen Cai ◽  
Hanying Bao ◽  
Peilin Lu ◽  
Lijuan Wang ◽  
Donghua He ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
T Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Response To Chemotherapy ◽  
Tlr4 Ligand ◽  
Induced Apoptosis

Abstract Abstract 1905 Multiple myeloma (MM) is a fatal plasma cell malignancy mainly localized in the bone marrow. The clonal expansion of tumor cells is associated with the disappearance of normal plasma cells and with a marked depression in the production of normal immunoglobulin (Ig). This makes MM patients highly vulnerable to bacterial, fungal and viral infections and recurrent infections remain to be a major cause of death in MM patients. It has been shown that most primary myeloma cells and cell lines express multiple Toll-like receptors (TLRs). Among them, TLR4 is most frequently expressed. To investigate TLR-initiated responses in MM cells including proliferation, anti-apoptosis and immune escape, we first screened four commonly used human myeloma cell line (HMCL) for the expression of major TLRs by RT-PCR. Surprisingly, all the HMCL expressed multiple TLRs. We also examined primary myeloma cells from 4 patients with MM and our results showed that TLR4 was expressed by all the tumor cells. We incubated myeloma cells with LPS, the natural ligand for TLR4, and found that cell proliferation increased significantly. Targeting TLRs on malignant B cells can induce resistance to chemotherapeutic agents but can also be exploited for combined therapeutic approaches. As mechanisms involved in the resistance to apoptosis play a major role in MM escape to therapies, we sought to determine the capacity of TLR4 ligand to promote the survival of HMCL cells. Myeloma cells were pretreated for four hours with LPS before being induced apoptosis by adriamycin. Results showed that LPS pretreatment partially protected the cells from adriamycin-induced apoptosis. The TLR signaling pathway activates several signaling elements, including NF-kB and ERK/JNK/p38 MAPKs, which regulate many immunologically relevant proteins. Time-dependent MAPK phosphorylation was measured to assess the activation of these kinases upon treatment with LPS in cell lines. ERK1/2, p38, and JNK phosphorylation and NF-kB were significantly up-regulated following LPS treatment. Moreover, our findings demonstrated that LPS-induced cell proliferation was dependent on JNK, ERK and p38 signaling. IL-18, a recently described member of the IL-1 cytokine superfamily, is now recognized as an important regulator of innate and acquired immune responses. In this study, we found that LPS induced IL-18 secretion and activated MAPK and NF-kB signaling simultaneously. Therefore, our results suggest that activation of the MAPK signaling and secretion of IL-18 are interconnected. Tumors evade immune surveillance by multiple mechanisms, including the production of factors such as TGF-β and VEGF, which inhibit and impair tumor-specific T cell immunity. Our study also showed that T cell proliferation induced by allostimulatory cells decreased when the HMCL were pre-treated with LPS. Moreover, immunoregulatory molecules on HMCL, such as B7-H1, B7-H2 and CD40, were upregulated after treatment with LPS, suggesting that TLR4 ligand LPS facilitates tumor cell evasion of the immune system. Our results show that TLRs are functional on myeloma tumor cells, and the ligands to these TLRs have a functional role in affecting myeloma cell proliferation, survival, and response to chemotherapy and immune attacks. Disclosures: No relevant conflicts of interest to declare.

Role of Nuclear Heme Oxygenase 1 (HO-1) in Bortezomib Induced Cell Death and Genomic Instability of Multiple Myeloma (MM) Cells

2015 ◽  
Vol 15 ◽  
pp. e241
Author(s):  
D. Tibullo ◽  
I. Barbagallo ◽  
C. Giallongo ◽  
P. La Cava ◽  
N. Parrinello ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Genomic Instability ◽  
Heme Oxygenase ◽  
Heme Oxygenase 1

Combination of Akt/PKB Inhibition (Perifosine) and Farnesyl Transferase Inhibition (Tipifarnib) Results in Increased Cell Death in Myeloma Cells Lines.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1568-1568 ◽  
Author(s):  
Rajni Sinha ◽  
Ebenezer David ◽  
Emily Zeilter ◽  
Claire Torre ◽  
Jonathan L. Kaufman ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Death ◽  
Combination Therapy ◽  
Cell Line ◽  
Cell Lines ◽  
Single Agent ◽  
Myeloma Cell ◽  
Myeloma Cell Line

Abstract Introduction Multiple myeloma is a clonal plasma cell malignancy characterized by proliferation and accumulation of plasma cells in the bone marrow. Most patients are incurable with the current treatment modalities. Clearly novel agents are needed to improve the outcome for patients with myeloma. We have previously shown that the combination of bortezomib and tipifarnib results in synergistic myeloma cell death. This increase in apoptosis is associated with down regulation of phosphorylated AKT, a potent anti-apoptotic signaling molecule. Therefore, agents that target AKT represent ideal compounds for further study in myeloma. Perifosine is a novel, oral bioavailable alkylphospholipid. Perifosine has displayed apoptotic and antipropliferative activity in vitro and in vivo in several human cancer models including leukemia. Perifosine exerts its actions by interfering with key intracellular pathways including AKT, MAPK, JNK, p21waf1. Our hypothesis is that targeting AKT via multiple upstream pathways will result in increased myeloma cell apoptosis. Therefore, we assessed the effects of single agent perifosine with and without tipifarnib on multiple myeloma cell lines. Method The myeloma cell line RPMI8226 was used. Cell viability and proliferation were assessed using MTT assays. Cells were incubated with increasing concentrations of both agents alone and in combination. Cell proliferation was assayed at 24, 48 and 72 hours. Western blots were then carried out to evaluate the effects of the intracellular protein PDK1, one of the critical signaling molecules that phosphorylates and activates AKT. Results As we and others have previously shown, tipifarnib at concentrations that can be achieved clinically is associated with minimal cytotoxicity. At 5 μM, tipifarnib decrease proliferation by only 20%. In contrast, there is a potent dose response effect of single agent perifosine (Fig. 1). These results were apparent as early as 24 hours. When tipifarnib at 5 μM is used in combination with a subtherapeutic dose of perifosine (2 μM), there is a marked decrease in cell proliferation (Fig. 2). In addition, combination therapy resulted in a reduction in the phosphorylated form of PDK1, a critical finding that was not seen with either drug alone. Conclusion Combination therapy with tipifarnib and perifosine results in less cell proliferation compared to either agent used alone in the RPMI8226 myeloma cell line. The dosages employed in these in-vitro studies are lower than those used in previously published data and are clinically achievable. Studies targeting other cell lines including MM.1R, MM.1S, and U266 are in progress. Analysis of AKT, Caspase 3, 8 and 9 are being explored to help delineate the mechanism of this novel combination. The goal is to develop further effective treatment options for patients with myeloma. Figure 1 Figure 1. Figure 2 Figure 2.

Arsenic Trioxide Induces Up-Regulation of BMF and NOXA and Overcomes an Anti-Oxidative Response To Induce Apoptosis in Myeloma Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3419-3419
Author(s):  
Alejo A. Morales ◽  
Delia Gutman ◽  
Pedro J. Cejas ◽  
Kelvin P. Lee ◽  
Lawrence H. Boise
Keyword(s):  
Arsenic Trioxide ◽  
Cell Lines ◽  
Transcriptional Repression ◽  
Mrna Level ◽  
Detoxification Enzymes ◽  
Heme Oxygenase 1 ◽  
Down Regulation ◽  
Apoptotic Pathway ◽  
Oxidative Response ◽  
Induced Apoptosis

Abstract Arsenic Trioxide (ATO) has been successfully used for the treatment of acute promyelocytic leukemia (APL) and has shown promise as a therapeutic agent in multiple myeloma (MM). We have been characterizing the response of MM cell lines to ATO and have demonstrated that four human MM cell lines, including U266, MM.1s, 8226/S and KMS11 were sensitive to ATO. Affymetrix Hu133 2.0 Plus arrays were used for expression profiling at 0, 6, 24 and 48 h after ATO treatment. The pattern observed in all four cell lines was consistent with anti-oxidative responses and an imbalance of Bcl-2 family members favoring apoptosis. Most notably expression changes were associated with a response driven by the Nrf2-Keap1 pathway. Nrf2 is expressed at the mRNA level however the protein is not observed in untreated cells. Consistent with inactivation of Keap1, Nrf2 protein is stabilized within 6 h of ATO treatment. Importantly, this up-regulation is associated with Nrf2 accumulation in the nucleus where it functions. The activation of the Nrf2 is associated with the changes observed during ATO treatment, including up-regulation of the following genes: metallothionein-1 (MT-1), phase II detoxification enzymes (NQO-1), anti-oxidant enzymes [heme oxygenase-1 (HO-1), thioredoxin reductase, glutathione reductase and γ-glutamate cysteine ligase], NADPH-generating enzymes (malic enzyme), chaperone proteins (Hsp70, Hsp40, Hsp27KDa and Hsp105KDa) and the transcriptional repression of genes involved in the cholesterol/lipid biosynthesis pathway. HO-1 and MT-1 expression has been previously suggested to be associated with ATO-resistance in myeloma. However, the up-regulation of this gene (greater than 50 fold in MM.1s and KMS11 as early as 6 hrs) could not protect cells from ATO-induced apoptosis. Similarly up-regulation of MT-1 did not correlate with cell survival. Moreover, up-regulation of HO-1 or MT-1 prior to ATO treatment had no effect on ATO-induced apoptosis in U266 or MM.1s cells, consistent with each of these being components of a larger anti-oxidative response. Bcl-x, was down-regulated in all the four cell lines during ATO-induced apoptosis while one of its ligands, Bmf, a BH3-only pro-apoptotic Bcl-2 family member was up-regulated 1.5–3.0 fold at the mRNA level and correlated with response. An additional BH3-only protein Noxa was up-regulated in three out four cell lines at the mRNA and protein level. In U266, Noxa is expressed constitutively and not affected by ATO. Interestingly however, ATO induced a down-regulation of Mcl-1, the Bcl-2 anti-apoptotic partner of Noxa in U266 cells. Taken together the Bmf/Bcl-x and Noxa/Mcl-1 ratios favored an apoptotic response following addition of ATO. Additionally Noxa up-regulation was enhanced when glutathione was depleted with BSO and blocked by addition of N-acetylcysteine. Taken together these data indicate that while the cells initially respond to ATO treatment in a manner consistent with a strong anti-oxidative response, these protective mechanisms are not sufficient to block cell death induced by the up-regulation of two BH3-only proteins (Bmf and Noxa) and the down-regulation of Bcl-x and Mcl-1 resulting in the activation of the intrinsic apoptotic pathway as part of the ATO-induced apoptotic mechanism(s). New arsenical compounds or combined therapies, could avoid this protective anti-oxidative pathway activated by ATO increasing its activity.

Anti-Estrogens Induce Apoptosis of Multiple Myeloma Cells

Blood ◽  
1998 ◽  
Vol 92 (5) ◽  
pp. 1749-1757 ◽  
Author(s):  
Steven P. Treon ◽  
Gerrard Teoh ◽  
Mitsuyoshi Urashima ◽  
Atsushi Ogata ◽  
Dharminder Chauhan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Survival ◽  
Cell Lines ◽  
High Dose ◽  
Er Positive ◽  
Dose Oral ◽  
American Society ◽  
Induced Apoptosis

Abstract Previous studies have suggested that multiple myeloma (MM) cells express estrogen receptors (ER). In the present study, we characterized the effects of estrogen agonists and antagonists (anti-estrogens [AE]) on growth of MM cell lines and MM patient cells. In addition to antagonizing estrogen binding to ER, AE can trigger apoptosis. Hence, we also determined whether estrogens or AE altered MM cell survival. Immunoblotting showed that ER- is expressed in 4 of 5 MM cell lines (ARH-77, RPMI 8226, S6B45, and U266, but not OCI-My-5 cells), as well as in freshly isolated MM cells from 3 of 3 patients. 17β-estradiol (E2) did not significantly alter proliferation of MM cell lines or MM patient cells. In contrast, two structurally distinct AE, tamoxifen (TAM) and ICI 182,780 (ICI), significantly inhibited the proliferation of all 5 MM cell lines and MM cells from 2 of 2 patients (IC50, 2 to 4 μmol/L). Proliferation of these cell lines was also inhibited by the hydroxylated TAM derivative, 4-hydroxytamoxifen (4HTAM), although this derivative was less potent than TAM (IC50, 3 to 25 μmol/L). In contrast, the dehalogenated TAM derivative toremifene (TOR) did not inhibit MM cell proliferation. We next examined the effects of these agents on MM cell survival. TAM, ICI, and, to a lesser extent, 4HTAM and TOR triggered apoptosis in both ER-–positive as well as ER-–negative MM cell lines and patient MM cells, evidenced both by fluorescence-activated cell sorting (FACS) analysis using propidium iodide staining and the TUNEL assay. TAM-induced growth inhibition and apoptosis of ER-–positive S6B45 MM cells was not blocked by coculture with excess E2. TAM-induced apoptosis of S6B45 MM cells was also unaffected by addition of exogenous interleukin-6. Importantly, both the inhibition of MM cell proliferation and the induction of MM cell apoptosis were achieved at concentrations of TAM (0.5 and 5.0 μmol/L) that did not significantly alter in vitro growth of normal hematopoietic progenitor cells. Similar plasma levels of TAM have been achieved using high-dose oral TAM therapy, with an acceptable toxicity profile. These studies therefore provide the rationale for trials to define the utility of AE therapy in MM. © 1998 by The American Society of Hematology.

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