Multiple Myeloma Cell U266 Research Articles

Curcumin in combination with bortezomib synergistically induced apoptosis in human multiple myeloma U266 cells

Growth of multiple myeloma cells is controlled by various factors derived from host bone marrow microenvironments. Interaction between multiple myeloma cells and bone marrow stromal cells (BMSCs) plays an important role in the expression of adhesive molecules and secretion of growth factors involved in multiple myeloma (MM) cell growth, survival, and resistance to anticancer drugs. Recently, the possibility of developing novel anti-cancer therapeutic strategies targeting both MM cells and MM cell–BMSC interactions has been discussed. Here we present data showing that curcumin, a major constituent of turmeric compounds extracted from the rhizomes of the plant Curcuma longa, effectively reduced the growth of MM cells and BMSCs. Upon treatment with curcumin, IL-6/sIL-6R-induced STAT3 and Erk phosphorylation was dramatically reduced in the co-cultured cells. In addition, curcumin inhibited the production of pro-inflammatory cytokines and VEGF, factors that are associated with the progression of multiple myeloma, from both MM cells and BMSCs. In a combination treatment with curcumin and bortezomib, IL-6/sIL-6R-induced STAT3 and Erk phosphorylation was effectively inhibited. Moreover, this combination treatment synergistically inhibited the growth of MM cells co-cultured with BMSCs as compared to controls. Taken together, these results indicate that curcumin potentiates the therapeutic efficacy of bortezomib in MM suggesting this combination therapy to be of value in the clinical management of MM.

Liposomal delivery improves the growth-inhibitory and apoptotic activity of low doses of gemcitabine in multiple myeloma cancer cells

Gemcitabine-loaded pegylated unilamellar liposomes (200 nm) were proposed for the treatment of multiple myeloma cancer disease. Physicochemical and technological parameters of liposomes were evaluated by using laser light scattering and gel permeation chromatography. The growth-inhibitory activity of gemcitabine-loaded liposomes compared to the free drug was assayed in vitro on U266 (autocrine, interleukin-6-independent) and INA-6 (IL-6-dependent) multiple myeloma cell lines. Liposomes noticeably improved the growth-inhibitory activity of gemcitabine in terms of both dose-dependent and incubation-time effects. Liposomal delivery of gemcitabine consistently and significantly increased induction of apoptosis and caused a complete inhibition of proliferation. Liposomes were able to interact with multiple myeloma cells as demonstrated by confocal laser scanning microscopy and hence to improve the intracellular gemcitabine delivery. Gemcitabine-loaded liposomes were much more effective in vitro than the free drug. This formulation may offer even more in vivo advantages both in terms of drug pharmacokinetic and biodistribution.

Abrogation of U266 Multiple Myeloma Cell Proliferation Via Inhibition of NF-κB Activation by Curcumin

Background: Curcumin is a naturally occurring biologically active compound, and it has been shown to possess potent anti-inflammatory, anti-tumor and anti-oxidative properties. It is known for its anti-pro- liferative and proapoptotic effects in several cancer cells. Curcumin's effects on the mechanisms of cell survival and the expression of various cytokines were investigated in U266 cells and the in vivo effects of curcumin were examined using an animal model. Methods: Cell proliferation assay and flow cytometry were used to examine cell proliferation, along with cell cycle analysis. The protein expressions were analyzed by Western blotting and the expressed levels of cytokines were analyzed by the ELISA method. Results: Curcumin inhibited U266 cell growth in a dose-dependent and time-dependent manner. Cell cycle analysis showed an increased sub-G1 phase, a down regulated cyclinD1 expression and an induced p21 expression. Apoptosis induced a down regulated procaspase 3 expression and it induced cleaved PARP. Curcumin inhibited the IL (interleukin)-6 induced cell signal pathway via decreasing the STAT1 an 3, Erk cyclinD1 and c-myc expressions. Also, administration of 25mg/kg curcumin to a U266 animal model inhibited cancer cell engraftment in the bone marrow and it decreased the IL-6, sIL-6R and IL-8 ex- pression levels. Conclusion: Curcumin induced cell cycle arrest and apoptosis and it inhibited the IL-6 mediated signal transduction pathways in U266 cells. Similar to the in vitro results, curcumin inhibited cancer cell pro- liferation and the expression of cytokine in vivo. (Korean J Hematol 2008;43:19-27.)

The Role of AKT and ERK in Regulating D-Cyclin Translation Inhibition and G1 Arrest in Multiple Myeloma Cells Treated with mTOR Inhibitors: Rationale for Combination Thereapy.

In a previous study, we showed that heightened AKT activity sensitized multiple myeloma (MM) cells to the in vivo anti-tumor effects of CCI-779. To test the mechanism of AKT's regulatory role, we studied isogenic U266 MM cell lines transfected with an activated AKT allele or empty vector. The AKT-transfected cells were markedly more sensitive to cytostasis induced in vitro by rapamycin or in vivo by CCI-779. In contrast, cells with quiescent AKT were completely resistant. The ability of rapamycin and CCI-779 to inhibit D-cyclin expression was also significantly greater in AKT-transfected MM cells and this was, in part, due to a greater ability to curtail cap-independent translation and internal ribosome entry site (IRES) activity of D-cyclin transcripts. As ERK/p38 activity can facilitate IRES-mediated translation of some transcripts, we investigated ERK/p38 as regulators of rapamycin sensitivity. AKT-transfected cells demonstrated significantly decreased ERK and p38 activity, suggesting their involvement. However, only an ERK inhibitor prevented D-cyclin IRES activity in resistant “low AKT” myeloma cells while a p38 inhibitor had no effect. Furthermore, the combination of rapamycin and the ERK inhibitor successfully sensitized myeloma cells to rapamycin in terms of down regulated D-cyclin protein expression and G1 arrest. These data support a scenario where ERK facilitates D-cyclin IRES function and heightened AKT activity down regulates this ERK-dependent phenomenon. Thus ERK and AKT activity are potential predictors of responsiveness to mTOR inhibitors.

IL-6 Enhances C-myc Translation in Myeloma Cells Via Activation of the Myc Internal Ribosomal Entry Site (IRES) and an IRES-Binding Trans Factor, hnRNP A1.

Dysregulated c-myc expression in multiple myeloma (MM) can be due to a translocation with juxtaposition of the myc gene to the Ig enhancer resulting in constitutive transcription. However, myc protein levels can also be increased in MM cells when it is due to heightened translation. Activated translation can be mediated via an mTOR-mediated, cap-dependent mechanism, thus named because the translation initiation complex utilizes the cap structure on mRNA to recruit the ribosomal subunit. However, an alternative mechanism is cap-independent where the ribosomal subunit binds a transcript via an IRES in the RNA's 5′UTR. IRES function is regulated by IRES trans activating factors (ITAFs) which bind to the 5′UTR. We studied myc expression in ANBL-6 wild type and activated K-RAS-transfected MM cells which proliferatively respond to IL-6 but do not contain myc translocations. IL-6 increased myc protein expression in both cell lines in a concentration and time-dependent fashion but RNA expression was only enhanced in the wild type cells. The increased protein expression in K-RAS mutant cells was unaffected by rapamycin, indicating independence from mTOR and cap-dependent mechanisms. Using an IRES-dependent reporter vector, reporter expression was shown to be increased by IL-6, confirming IL-6 activates myc IRES activity. This was also demonstrated in the IL-6 autocrine U266 MM cell line where myc IRES-dependent reporter expression was ablated following exposure to anti-IL-6 antibodies. In addition, primary MM cells treated with IL-6 also demonstrated an increase in myc IRES activity. Since a yeast three-hybrid screen for proteins specifically binding to the myc IRES identified several clones encoding hnRNP A1, we evaluated hnRNP A1 as a mediator of myc IRES activity. IL-6 increased the cytoplasmic localization of hnRNP A1 in ANBL-6 MM cells. Furthermore, in MM cells where hnRNP A1 expression was knocked down, myc IRES activity was greatly diminished and there was no enhancement with addition of IL-6. These results indicate that IL-6 can enhance c-myc translation in MM cells via activation of the myc IRES and that hnRNP A1 is a critical myc ITAF involved in the IL-6 response. hnRNP A1 may serve as a new therapeutic target in myeloma.

The Role of Iron in the Cytotoxicity Induced in Hematopoietic Malignant Cell Lines by an Anti-Transferrin Receptor IgG3-Avidin Fusion Protein and Gambogic Acid.

The human transferrin receptor (hTfR) is a cell surface glycoprotein responsible for iron uptake, which is required for several cellular reactions including respiration and DNA synthesis. In cancer cells, the hTfR is over-expressed due to the high amount of iron required to maintain the uncontrolled cell proliferation. This makes the cancer cells highly sensitive to death induced by iron starvation. In addition to iron uptake, the TfR may also play a role in cell growth signaling. We have developed a mouse/human chimeric anti-human transferrin receptor IgG3-avidin fusion protein (anti-hTfR IgG3-Av) that induces cell death in human malignant B-cells such as IM9 and ARH-77 (B lymphoblastoid cell lines). The TfR is constitutively internalized and recycled back to cell surface. Anti-hTfR IgG3-Av binding to the hTfR disrupts this cycling and redirects the hTfR to lysosomal compartments where hTfR is presumably degraded. This promotes a state of iron starvation that induces cancer cell death by apoptosis, which can be blocked by iron supplementation. However, the cytotoxic effect induced by anti-hTfR IgG3-Av was minor or absent in U266 (multiple myeloma cell line). Gambogic acid (GA), a natural compound from traditional Chinese medicine, was previously shown to bind the TfR, blocking its internalization and inducing apoptosis in an iron independent manner in Jurkat (acute T cell leukemia) and HL-60 (acute promyelocytic leukemia) cell lines. The goal of the present work is to explore the cytotoxic effect of anti-hTfR IgG3-Av and GA alone or in combination in a panel of hematopoietic malignant cell lines and understand the relevance of iron in the cytotoxicity. We demonstrate that GA inhibits the proliferation of Ramos, HS-sultan, Raji (Burkitt's lymphomas), IM9, U266, Jurkat and HL-60 cell lines. Our results showed that 0.3 μM GA at 24 h decreases cell proliferation by 70–90%, and 95–99.9% at 48 h, in all cell lines tested. We also observed morphological changes in the cells treated with GA consistent with cell death. Importantly, using ferric ammonium citrate as an iron supplement, we confirmed that the cytotoxic effect of GA in the cell lines tested is iron independent. This work also demonstrates, for the first time, that anti-hTfR IgG3-Av has a potent cytotoxic effect in the panel of leukemia and lymphoma cell lines tested above. Additionally, we confirmed that anti-hTfR IgG3-Av induces cell death in an iron dependent manner. Since both anti-hTfR IgG3-Av and GA target the TfR, we tested the effect of the combination of these components. Despite a potential antagonistic effect due to the fact that anti-hTfR IgG3-Av decreases the cell surface levels of the hTfR and GA blocks its internalization, the combination of anti-hTfR IgG3-Av plus GA enhanced the cytotoxicity, including cases of synergy, of either one alone in the cell lines tested. Studies are currently underway to understand the role of iron in the enhancement of toxicity. Our results suggest that anti-hTfR IgG3-Av and GA, alone or in combination, could be an effective treatment option for various hematopoietic malignancies.

Inhibition of lymphocyte function associated antigen 1 by LFA878 induces apoptosis in multiple myeloma cells and is associated with downregulation of the focal adhesion kinase/phosphatidylinositol 3 kinase/Akt pathway

Multiple myeloma (MM) is still an incurable disease and adhesion of MM cells to bone marrow stromal cells is one of the hallmarks of the disease. Lymphocyte function associated antigen 1 (LFA-1) is an adhesion molecule that mediates lymphocyte adhesion, but its role in MM is only poorly understood. The aim of the presented study was to improve knowledge on LFA-1 and associated pathways in MM for the development of molecular targeted therapies. We demonstrate that LFA-1 is expressed in U266, RPMI-8226, OPM-2, and NCI-H929 MM cell lines and in primary cells of eight tested patients. The LFA-1 inhibitor LFA878 induces apoptosis in all four cell lines as revealed by annexin V staining and caspase 3 cleavage. Apoptosis is not hampered by adhesion to stromal cells. Additionally, the soluble ligand, intracellular adhesion molecule 1 (ICAM-1), which is increased in the serum of MM patients, does not protect from melphalan-induced apoptosis. Western blots demonstrate downregulation of FAK, PI3-K, and Akt upon LFA878 treatment. Additionally, sequential inhibition of the pathway by simultaneous application of Src family kinase or PI3-K inhibitors significantly increases LFA878 induced apoptosis. We conclude that LFA-1/FAK/PI3-K/Akt is a survival pathway in MM and that targeted inhibition may provide new therapeutic options.

A Novel, Mercaptoketone-Based HDAC Inhibitor, KD5170 Exerts Marked Inhibition of Osteoclast Formation and Anti-Myeloma Activity In Vitro.

Multiple Myeloma (MM) is characterized by the malignant proliferation of plasma cells and high osteoclast (OCL) activity resulting in increased bone destruction and the development of lytic lesions. Ideally, novel therapeutic agents directed at MM should not only target the myeloma cell, but also OCL activity. Multiple, chemically distinct, Histone Deacetylase (HDAC) inhibitors have shown promising anti-myeloma activity in pre-clinical studies. The majority of the HDAC inhibitors in clinical development employ either benzamide or hydroxamate chemistry. Through an uHTS biochemical screen of 600,000 compounds and subsequent medicinal chemistry efforts, a novel mercaptoketone series of HDAC inhibitors has been developed. The most potent compounds in this series exhibit nanomolar activity in both biochemical and cell-based assays. To determine the clinical potential of this series in treating MM we have profiled the lead compound, KD5170 in assays that monitor osteoclastogenesis and proliferation and apoptosis of MM cells.To evaluate the effects of KD5170 on the formation of OCL from mononuclear hematopoietic precursors, we utilized a human bone marrow culture system from MM patients to generate OCLs by in vitro RANKL/M-CSF stimulation. KD5170 treatment resulted in a significant dose dependent (0.5–8 10−6M) inhibition of osteoclast formation. In addition, RPMI-8226 cell proliferation was significantly suppressed by KD5170 at 3 10−6M as measured by [3H]-Thymidine-incorporation. Furthermore, we observed that KD5170 induced dose- (0.05–1 10−6M) and time-dependent (2–24h) apoptosis of U266 MM cells, as measured by Hoechst/PI double staining to identify distinct stages of apoptosis and to differentiate between apoptotic and necrotic cell death. Significantly, less than 5% of the cells were necrotic (PI-positive) indicating active engagement of the apoptotic pathway. KD5170 treatment (1 10−6M, 72 h) also markedly decreased the viability of primary CD138+ bone marrow cells from MM patients, as determined by trypan blue exclusion. In contrast, the viability of CD138− and mononuclear bone marrow cells from MM patients and normal healthy donors were unaffected, suggesting targeted selectivity.Our studies identify KD5170 as a promising new HDAC inhibitor that inhibits both osteoclast formation as well as the proliferation of MM cells. Furthermore, pronounced anti-proliferative activity of KD5170 on the MM cell line U266, suggests that a subset of MM may be the ideal target of this agent. U266 harbors a translocation involving 11q13 raising the possibility that this chromosomal alteration and Cyclin D1 dysregulation may have a role in mediating KD5170 sensitivity and as such will be a focus of future studies.

Emerging of Valproic Acid as an Anti-Myeloma Agent.

Multiple myeloma (MM) expands in a manner dependent on the bone marrow microenvironment, and develops devastating bone destruction. MM is still an incurable disease, and preferentially arises in the elderly. Therefore, novel well-tolerated therapeutic alternatives are wanted especially for elderly patients with MM. Valproic acid (VPA), a well-tolerated and safe anti-epileptic agent with extensive clinical experience, has recently been shown to be a class I- and IIa-specific HDAC inhibitor, and induce cytotoxic effects on various types of tumor cells. In the present study, we evaluated the impact of VPA on MM cell growth and survival as well as MM-induced bone marrow microenvironment. VPA reduced viable cell numbers to less than 50 % from the baseline at day 2 in all MM cell lines (5/5) as well as primary CD138-positive MM cells (4/4) tested, and a portion of B cell (2/5) and T cell (1/3) lines, but not in AML cell lines (0/4) at 100 microg/ml, a therapeutic concentration for epilepcy, which raises a possibility for VPA as a therapeutic agent against MM. Interestingly, CD138-negative non-MM bone marrow cells remained intact and CFU-GM numbers were not affected by VPA, suggesting tumor-specific actions of VPA. VPA induced death receptor- but not mitochondrial pathway-mediated apoptosis with down-regulation of cellular FLICE-inhibitory protein (c-FLIP) and cleavage of caspase 8 in RPMI8226 MM cells. Furthermore, VPA down-regulated cyclin D1, and up-regulated the cyclin-dependent kinase inhibitor p21(Cip1) with accumulation of MM cells at G0/G1 phase, suggesting the involvement of cell cycle arrest in anti-proliferation actions of VPA. Notably, VPA at therapeutically relevant concentrations potentiated the induction of apoptosis by dexamethasone which triggers the release of Smac from mitochondria. However, VPA did not enhance the cytotoxic effects of cell cycle-specific agents including doxorubicine and melphalan. The VPA-induced tumor cell dormancy may reduce the susceptibility of MM cells to such cell cycle-specific agents. In parallel with MM progression, angiogenesis as well as osteoclastogenesis are enhanced in the bone marrow. We previously demonstrated that MM cell-osteoclast (OC) interactions enhance the growth and survival of MM cells as well as angiogenesis. Therefore, we next investigated the effects of VPA on MM cell-OC interactions and angiogenesis. Although VPA showed no significant effects on osteoclastogenesis induced by MM cells, VPA suppressed the growth and survival of RPMI8226 and U266 MM cells in the presence of OCs generated from monocytes to the levels similar to those without OCs. Furthermore, VPA potently inhibited in vitro vascular tubule formation enhanced by conditioned media from co-cultures of MM cells and OCs. Such anti-angiogenic effects of VPA was further potentiated in concert with thalidomide. Collectively, the present study suggests that VPA exerts multi-factorial anti-MM actions and may serve as a novel well-tolerated therapeutic alternative against MM.

AKT Kinase Addiction in Multiple Myeloma.

Cancer cells with activating kinase mutations are singularly hypersensitive to drugs that target these kinases, a phenomenon referred to as kinase or oncogene addiction. Although the AKT kinase is not known to be mutated in multiple myeloma (MM), it is frequently hyperactivated due to mutations in upstream signaling proteins. Previous work indicated that MM cells with heightened AKT activity were hypersensitive to drugs targeted to mTOR, a downstream substrate of AKT, suggesting an addiction to this pathway. “Kinase addiction” would theortically render the cell dependent on the kinase for growth. This dependency implies that other cascades of viability and/or proliferation may become downregulated subsequent to kinase activation. To test for AKT kinase addiction in MM we stably transfected the U266 MM cell line that is known to be protected against apoptosis by an autocrine Il-6/IL6R pathway (Caitlett-Falcone et al., Immunity 10, 105–115, 1999) with a constitutively active AKT construct (U266-AKT). AKT-transfected MM cells were protected against fas-induced apoptosis even when the autocrine IL-6 protective pathway was paralyzed by anti-IL6 and anti-IL6 receptor antibodies. However, as expected for an AKT addicted cell line, the U266-AKT cell line was more sensitive to agents that target the PI3/AKT/mTOR pathway, including two AKT inhibitors and the PI3K inhibitors LY294002 and wortmanin in terms of increased level of apoptosis and/or growth inhibition in comparison to the control U266 cell line. This “kinase-addicted” state was associated with down regulation of several components of the autocrine anti-apoptotic pathway of U266 cells. Expression of IL6, the IL6 receptor subunits gp80 and gp130 and STAT3 were all markedly silenced following AKT transfection. This down regulation of the survival pathway was relatively specific as expression of the fas receptor and VEGF receptors were relatively unaffected. The decrease in protein expression was also reflected in the decrease of mRNA for the receptor subunits, suggesting a transcriptional mechanism for down regulation. As expected from the decrease in autocrine IL-6 signaling, AKT-transfected cells also demonstrated a marked inhibition of Jak1 and MAPK 42/44 phosphorylation and enzymatic activity. In summary, over expression of activated AKT results in an AKT-addicted state whereby the MM cell becomes dependent on AKT for survival as other growth-promoting and anti-apoptotic cascades become paralyzed. These data support AKT as an important target for therapeutic intervention of MM and suggest such therapy would be most successful in patients whose MM cells contain the highest amount of activated AKT.

The Combination of the mTOR Inhibitor Rapamycin and Proteasome Inhibitor Bortezomib Is Synergistic In Vitro in Multiple Myeloma.

Background: The PI3K and NFk-B/proteasome pathways are major regulators of survival in Multiple Myeloma (MM). Previous studies have demonstrated clinical efficacy of bortezomib in MM; however, not all patients responded to this agent. mTOR inhibitors have demonstrated significant in vitro and in vivo activity in MM, specifically clinical trials with the mTOR inhibitor CCI-779 (Wyeth) in MM. Therefore, we examined whether inhibition of the PI3K pathway by mTOR and proteasome inhibitors may lead to synergistic activity in MM.Methods: MM cell lines (MM.1S, RPMI, U266, OPM2) were treated with rapamycin 1–5nM (Sigma Aldrich), bortezomib 2.5–10nM (Millenium, MA), or the combination. Cytotoxicity was measured by the MTT assay at 48 hrs; DNA synthesis was measured using thymidine uptake assay; apoptosis was studied using Apo2.7 by flow cytometry, and cell cycle regulation was determined using flow cytometry. To determine whether these agents can overcome the growth advantage conferred by bone marrow stromal cells (BMSCs), we co-cultured cell lines with stromal cells. Normal peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers. Determination of the additive or synergistic effect of the combination was calculated using the CalcuSyn software (Biosoft, MO) based on the Chou-Talalay method, with synergistic activity determined as a combination index (CI) of <1.0.Results: Rapamycin induced dose-dependent cytotoxicity from 0.1nM to 1nM, with an IC50 of 5nM in MM.1S and OPM2. Interestingly, higher doses did not induce further cytotoxicity, confirming that low doses of rapamycin are as effective as higher doses. RPMI and U266 MM cell lines were less sensitive to rapamycin, with 5nM inducing 40% and 20% decrease in survival, respectively. Bortezomib induced significant inhibition of survival in all MM cell lines with an IC50 of 2.5nM, as previously reported. The combination of agents induced significant inhibition of proliferation as compared to each agent alone, specifically with the combination of 5nM rapamycin with 5nM of bortezomib. In the DNA synthesis assay, the combination of bortezomib and rapamycin was significantly cytotoxic compared to each agent alone, specifically at the dose of 5nM rapamycin and bortezomib 2.5nM. The combination of rapamycin 1 to 5 nM and bortezomib 5 to 10 nM were synergistic with a CI index less than 1.0, as in RPMI (CI=0.4) and U266 (CI=0.2) cell lines. The combination of rapamycin and bortezomib at serial concentrations did not trigger cytotoxicity in PBMCs from normal volunteers, indicating significant cytotoxicity in malignant cells, with lack of toxicity in normal PBMCs and suggesting a therapeutic index. The combination of bortezomib and rapamycin demonstrated a significant inhibitory effect on the growth of MM cell lines even in coculture with stromal cells. Cell cycle analysis demonstrated G1 arrest at 24 and 48 hrs in MM.1S cells. Similar results were obtained using primary CD138+ myeloma cells from patients.Conclusion: The combination of rapamycin and bortezomib resulted in synergistic in vitro cytotoxicity in MM cells. These results provide the framework for clinical trials evaluating the combination of CCI-779 and bortezomib in MM.

A novel isocoumarin derivative induces mitotic phase arrest and apoptosis of human multiple myeloma cells

The isocoumarin NM-3 reverses resistance of human multiple myeloma (MM) cells to dexamethasone and is in clinical trials. In the present work, the NM-3 analog, 185322, has been studied for activity against MM cells. Human U266, RPMI8226 and primary MM cells were analyzed for the effects of 185322 on cell cycle distribution, tubulin polymerization and induction of apoptosis. We show that, in contrast to NM-3, treatment with 185322 is associated with a marked arrest of MM cells in M phase. The results also demonstrate that treatment with 185322 is associated with a rapid decrease in tubulin assembly and an increase in Bcl-2 phosphorylation, consistent with disruption of mitosis. Our results further demonstrate that mitotic failure induced by 185322 results in activation of an apoptotic response in MM cell lines and primary MM cells. By contrast, 185322 had little if any effect on growth and survival of human carcinoma cells. These findings identify a novel inhibitor of microtubule assembly that induces mitotic arrest and apoptosis of MM cells.

Plumbagin (5-Hydroxy-2-methyl-1,4-naphthoquinone) Suppresses NF-κB Activation and NF-κB-regulated Gene Products Through Modulation of p65 and IκBα Kinase Activation, Leading to Potentiation of Apoptosis Induced by Cytokine and Chemotherapeutic Agents

Plumbagin, derived from the medicinal plant Plumbago zeylanica, modulates cellular proliferation, carcinogenesis, and radioresistance, all known to be regulated by the activation of the transcription factor NF-kappaB, suggesting plumbagin might affect the NF-kappaB activation pathway. We found that plumbagin inhibited NF-kappaB activation induced by TNF, and other carcinogens and inflammatory stimuli (e.g. phorbol 12-myristate 13-acetate, H2O2, cigarette smoke condensate, interleukin-1beta, lipopolysaccharide, and okadaic acid). Plumbagin also suppressed the constitutive NF-kappaB activation in certain tumor cells. The suppression of NF-kappaB activation correlated with sequential inhibition of the tumor necrosis factor (TNF)-induced activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, p65 nuclear translocation, and the NF-kappaB-dependent reporter gene expression activated by TNF, TNFR1, TRAF2, NIK, IKK-beta, and the p65 subunit of NF-kappaB. Plumbagin also suppressed the direct binding of nuclear p65 and recombinant p65 to the DNA, and this binding was reversed by dithiothreitol both in vitro and in vivo. However, plumbagin did not inhibit p65 binding to DNA when cells were transfected with the p65 plasmid containing cysteine 38 mutated to serine. Plumbagin down-regulated the expression of NF-kappaB-regulated anti-apoptotic (IAP1, IAP2, Bcl-2, Bcl-xL, cFLIP, Bfl-1/A1, and survivin), proliferative (cyclin D1 and COX-2), and angiogenic (matrix metalloproteinase-9 and vascular endothelial growth factor) gene products. This led to potentiation of apoptosis induced by TNF and paclitaxel and inhibited cell invasion. Overall, our results indicate that plumbagin is a potent inhibitor of the NF-kappaB activation pathway that leads to suppression of NF-kappaB-regulated gene products. This may explain its cell growth modulatory, anticarcinogenic, and radiosensitizing effects previously described.

Cyclin D1 Overexpression Increases the Susceptibility of Human U266 Myeloma Cells to CDK Inhibitors through a Process Involving p130-, p107- and E2F-Dependent S-Phase Entry

Dysregulation of cyclin D1 expression is one of the most common genetic aberrations found in hematopoietic malignancies, including multiple myeloma. To address the effects of cyclin D1 overexpression might have on the response of malignant hematopoietic cells to CDK inhibitors, the impact of ectopic cyclin D1 overexpression on the response of human multiple myeloma U266 cells to various cyclin-dependent kinase (CDK) inhibitors was examined. Cyclin D1 overexpression markedly increased the apoptotic response of cells to the CDK inhibitors flavopiridol, roscovitine, and R-roscovitine. Ectopic expression of cyclin D1 resulted in p21CIP1 accumulation, an effect that was diminished by CDK inhibitor exposure. In pRb-null U266 cells, enforced overexpression of cyclin D1 diminished CDK inhibitor-mediated dephosphorylation of the pocket proteins p130 and p107, reduced binding of E2F1 and E2F4 to p130 and p107, and attenuated inhibition of E2F activity. Notably, CDK inhibitors failed to reduce the S-phase fraction in cyclin D1/U266 cells in contrast to effects in their wild-type counterparts. Finally, cyclin D1/U266 cells exhibited diminished basal NF-?B activity compared to controls, which was essentially completely abrogated by CDK inhibitor exposure. Together, these findings suggest that dysregulation of cyclin D1 sensitizes human myeloma cells to the actions of CDK inhibitors through mechanisms involving interference with p21CIP1 expression, dephosphorylation of pocket proteins and inactivation of E2Fs culminating in S-phase entry, as well as inactivation of NF-?B, leading to apoptosis rather than growth arrest.

Analysis of expression of heat shock protein-90 (HSP90) and the effects of HSP90 inhibitor (17-AAG) in multiple myeloma

Heat shock protein 90 (HSP90) is required for structural folding and maintenance of conformational integrity of various proteins, including several associated with cellular signaling. Recent studies utilizing 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90, demonstrated an antitumor effect in solid tumors. To test whether HSP90 could be targeted in multiple myeloma (MM) patients, we first investigated expression of HSP90 by immunofluorescence and flow cytometric analysis in a myeloma cell line (U266) and primary myeloma cells. Following demonstration of HSP90 expression in myeloma cells, archival samples of 32 MM patients were analysed by immunoperoxidase staining. Myeloma cells in all patients showed strong cytoplasmic expression of HSP90 in all samples and 55% also demonstrated concurrent nuclear immunopositivity. Treatment of U266 and primary MM cells with 17AAG resulted in significantly increased apoptosis compared to untreated control cells. Analysis of anti-apoptotic BCL2 family proteins and akt in MM cells incubated with 17-AAG revealed down-regulation of BCL-2, BCL-XL, MCL-1 and akt. Furthermore, although a low concentration of bortezomib resulted in no cell death, a combination of 17AAG and bortezomib treatment revealed a synergistic apoptotic effect on the U266 cell line. These data suggest that targeted inhibition of HSP90 may prove to be a valid and innovative strategy for the development of future therapeutic options for MM patients.

Downregulation of PU.1 Is Required for Subtype of Multiple Myeloma.

There are several recent reports that certain mutations in critical transcription factors could be associated with leukemogenesis; however, it remains unclear whether such mutations could be linked to the pathogenesis of multiple myeloma (MM). In the present study, we attempted to determine whether mutations in transcription factors could be found in the patients with MM, utilizing MM cell lines and freshly isolated MM cells, purified with CD138 antibody and magnetic beads. When we looked at the expression of Blimp-1, XBP-1, NF-kB (p65 and p50), and PU.1 using semi-quantitative RT-PCR, all except PU.1 were found expressed in all MM cell lines and freshly isolated MM cells examined. All the PCR products were sequenced, but no significant mutations were identified. As examined with RT-PCR, PU.1 was found completely downregulated in 6 of 8 MM cell lines, while only faint to no expression of PU.1 was seen in 12 of 41 freshly isolated MM cells. As PU.1 message was sequenced, no mutations were found in 29 PU.1-positive MM cells or 2 MM cell lines that had relatively high PU.1 expression. Considering that PU.1 is essential for myeloid and lymphoid differentiation and that mice with null mutation in the PU.1 gene have no granulocytes, monocytes/macrophages or B lymphocytes, together with a recent report by others that AML developed in mice with PU.1 downregulation by 20%, we asked whether PU.1 downregulation might be associated with the oncogenesis or maintenance of MM. To examine the effects of PU.1 expression on MM cells, we generated tet-off conditional PU.1 expression model of PU.1-negative U266 MM cell line. Surprisingly the induction of PU.1 expression resulted in cell cycle arrest and apoptosis of those cells. Cell cycle analysis using flow cytometry revealed that PU.1-expressing U266 cells were in the G1 arrest. May-Giemsa staining revealed that such cells were swollen, enlarged, and vacuolous. These changes were completely reverted when the expression of PU.1 was silenced by tetracycline addition. There was no change in expression levels of cell cycle-related genes (p27, cyclin D1, cyclin E) and apoptosis-related genes (Bcl-2, Bcl-XL, and A1), while caspase3 cleavage was observed by 72 hr following PU.1 induction. We are currently examining the mechanism(s) of PU.1 downregulation in MM cells and a downstream cascade of PU.1 expression during cell death.

2-(8-Hydroxy-6-methoxy-1-oxo-1Η-2-benzopyran-3-yl)propionic Acid, a Small Molecule Isocoumarin, Potentiates Dexamethasone-Induced Apoptosis of Human Multiple Myeloma Cells

2-(8-Hydroxy-6-methoxy-1-oxo-1Eta-2-benzopyran-3-yl)propionic acid (NM-3) is a small molecule isocoumarin derivative that has recently entered clinical trials as an orally bioavailable anticancer agent. NM-3 induces lethality of human carcinoma cells by both apoptotic and nonapoptotic mechanisms and potentiates the effects of cytotoxic chemotherapeutic agents. The present studies have evaluated the effects of NM-3 on human multiple myeloma (MM) cells. The results demonstrate that NM-3 potentiates dexamethasone-induced killing of both dexamethasone-sensitive MM1.S and dexamethasone-resistant RPMI8226 and U266 MM cells. We show that NM-3 enhances dexamethasone-induced release of the mitochondrial apoptogenic factors cytochrome c and Smac/DIABLO. The results also demonstrate that NM-3 enhances dexamethasone-induced activation of the intrinsic caspase-9->caspase-3 apoptotic pathway. In concert with these results, NM-3 potentiates dexamethasone-induced apoptosis of MM1.S cells. Moreover, NM-3 acts synergistically with dexamethasone in inducing apoptosis of the dexamethasone-resistant RPMI8226 and U266 MM cells. These findings indicate that NM-3 may be effective in combination with dexamethasone in the treatment of MM.

Depletion of the Intracellular Akt Kinase, and Its Downstream Signaling, Mediates Synergy between the Proteasome Inhibitor MG-132 and the Heat Shock Protein 90 Inhibitor 17-AAG in the Multiple Myeloma Cell Line U266.

While proteasome inhibitors are effective therapy for multiple myeloma (MM), their efficacy could be improved by synergistic targeting of apoptosis. The intracellular serine/threonine kinase Akt has been demonstrated to have a central role in MM cell growth, survival, and drug resistance. Akt is activated by extracellular cytokines such as IGF-1 and IL-6, and contributes to MM resistance by ameliorating the apoptotic effects of proteasome inhibition. Akt requires chaperone proteins for proper stability and function, including the 90 kD molecule Heat Shock Protein 90 (HSP-90). HSP-90 function is abrogated by geldanamycin and its derivative, 17-allylamino-17-demethoxygeldanamycin (17-AAG).In the U266 MM cell line, the IC50 of the proteasome inhibitor MG-132 and 17-AAG was 100 nM and 800 nM, respectively. Following exposure of U266 MM cells to either drug alone, or the combination at a fixed-ratio of their IC50s (1:8), apoptosis was determined by Annexin V staining and FACScan analysis. Synergy analysis was performed using Calcusyn (Biosoft, Cambridge, UK). We found that the combination index (CI) was synergistic (CI<1) throughout the dose range, with a CI = 0.449 ± 0.298 at the combination IC50 (highly significant). For example, the apoptotic effect of 50 nM MG-132 and 400 nM 17-AAG was 6 ±2 % and 23 ±3 %, respectively, whereas the 50:400 nM combination produced apoptosis in 68 ± 2 % of the cells.To analyze effects on Akt and its substrates, we incubated U266 MM cells with MG-132 (50 nM), 17-AAG (400 nM), or the combination. We harvested lysates after zero, two, six, and 24 hours incubation, and Western blot analysis was performed. Co-incubation with MG-132 and 17-AAG, but not either alone, depleted Akt by 24 hours post-therapy. Co-treatment also produced significantly greater upregulation of HSP-90 and HSP-70 than 17-AAG alone, thus demonstrating greater functional inhibition of Akt. The combination also demonstrated the greatest abrogation of Akt-mediated effects on mitochondrial apoptosis: Co-treatment produced the greatest expression of BAD, decreased BCL-XL expression, reduced phosphorylation of GSK-3, and produced the greatest activation of caspase 3.Monotherapy with 17-AAG upregulated HSP-90 and HSP-70, reduced BCL-XL expression, and activated caspase 9. MG-132 monotherapy produced none of these effects. These findings demonstrate that synergy between proteasome inhibitors and 17-AAG is mediated by Akt depletion and abrogation of Akt signaling, predominantly by MG-132 augmentation of 17-AAG-mediated decay of Akt. Down-regulation of Akt-mediated resistance allows dual-apoptotic signaling and synergistic effect of combination therapy. These findings demonstrate a mechanistic rationale for utilizing heat shock protein inhibitors in combination with proteasome inhibitors as therapy for MM.

Fatty Acid Synthase Inhibitor Induces Apoptosis and Inhibits Proliferation in Multiple Myeloma Cells.

Objective: To investigate the effect of fatty acid synthase (FAS) inhibitor (cerulenin) in multiple myeloma cells and its mechanism.Methods: Proliferatory inhibition rate of cerulenin in multiple myeloma cells U266 and RPMI8226 was assayed by MTT, apoptosis analysis and cell DNA cycle analysis by flow cytometry (FCM).Results: Treated with 5ug/ml~640ug/ml of cerulenin for 24h, the proliferation of U266 and RPMI8226 cells were obviously inhibited with dosage related effect at the same concentration, the inhibition rate of human skin fibroblast are all lower than 28%. When U266 and RPMI8226 are treated for 12h with 20ug/ml of cerulenin the apoptosis rate revealed by FCM are 51.3% and 48.8% respectively. Cell DNA cycles analysis show the cell DNA is blocked in G1 phase and S phase cells decreased in both of U266 and RPMI8226.Conclution: FAS cold induce MM cell apoptosis and inhibit MM cell proliferation. FAS might be a new potential target for MM therapy.

Expression of Heat Shock Protein 90 and the Effects of HSP90 Inhibitor (17-AAG) in Multiple Myeloma.

Heat Shock Protein 90 (HSP90) is required for structural folding and maintenance of conformational integrity of various proteins, including several associated with cellular signaling. Recent studies utilizing 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90, demonstrated antitumor effect in solid tumors. In order to test whether HSP90 could be targeted in multiple myeloma (MM) patients, we first investigated expression of HSP90 by immunofluorescence and flow cytometric analysis in a myeloma cell line (U266) and primary myeloma cells. Following demonstration of HSP90 expression in myeloma cells, archival samples of 32 MM patients were analyzed by immunoperoxidase staining. Myeloma cells in all patients showed strong cytoplasmic expression of HSP90 in all samples and 55 % also demonstrated concurrent nuclear immunopositivity. Treatment of U266 and primary MM cells with 17AAG resulted in significantly increased apoptosis compared to untreated control cells. Analysis of anti-apoptotic BCL2 family proteins in MM cells incubated with 17-AAG revealed down-regulation of BCL-2, BCL-XL and MCL-1. Furthermore, while low concentration of bortezomib had no cell death, combination of 17AAG and bortezomib treatment revealed a synergistic apoptotic effect on the U266 cell line. These data suggest that targeted inhibition of HSP90 may prove to be a valid and innovative strategy for the development of future therapeutic options for MM patients.