IM-9 Cells Research Articles

Monosodium Urate Crystals Bind with Idiotype Protein and Promote Dendritic Cells to Induce Cytotoxic T Cells in Vitro

Monosodium urate (MSU) crystals have been studied to act as a key substance in local immunoreactions. MSU released from damaged cells works as an endogenous danger signal to antigen-presenting cells. MSU crystals evoke specific cell immunity and work as an adjuvant in a mouse model. The crystals also have another unique characteristic to bind with positively charged proteins, which could help to deliver some antigens into human dendritic cells (DCs). We focused on the application of MSU crystals as a not only an adjuvant but also as a carrier of positively charged antigenic protein to induce human cytotoxic T cells (CTLs) efficiently in vitro. We confirmed that MSU crystals facilitated human DCs to express the maturation marker, CD83, deliver (Fab′)2 attaching to the crystals. In order to determine whether MSU crystals facilitate the T-cell proliferation activity of DCs, the proliferative effects of DCs on allogeneic CD4+ cells were investigated. DCs pulsed with MSU crystals significantly facilitated the proliferation of allogeneic CD4+ cells when compared to DCs alone. The stimulation index (SI) was 2.5 ± 0.1 and 1.7 ± 0.1, respectively. When using DCs pulsed with the Fab attached to MSU crystals, the proliferation of CD4+ cells was significantly greater than when using DCs pulsed with Fab alone. The SI was 2.6 ± 0.2 and 1.9 ± 0.1, respectively. No significant differences were seen in the proliferation of allogeneic CD4+ cells between DCs pulsed with the Fab attached to MSU and DCs pulsed with MSU alone. We selected the multiple myeloma IM-9 cell line and its product idiotype (Id) protein as an ideal pair of target cells and positively charged tumor-specific antigen, respectively. After sensitizing DCs derived from HLA-A matched volunteers pulsed with tumor-specific monoclonal IgG-Fab fragments (IM-9 Fab) attached to MSU crystals, the CD8+ T cells stimulated by the DCs killed significantly more target cells (38.5 ± 3.5%, n=4) than those stimulated by DCs pulsed with IM-9 Fab alone (3.5 ± 7.5%). These cytotoxic effects of CD8+ cells stimulated by the DCs pulsed with IM-9 Fab attached to MSU crystals were reduced (3.6 ± 1.7%) when MSU crystals were pre-coated with fetal bovine serum to block to bind with IM-9 Fab. For efficient induction of CTLs, it is necessary for Id proteins to attach to MSU crystals. MSU crystals have some advantages of a protein carrier binding with positively charged proteins and delivering antigenic protein into DCs, as well as an adjuvant promoting DC maturation and inducing CTLs.

Galiximab (anti-CD80 mAb)-Induced Cell Triggering Results in the Inhibition of Constitutive NF-Kb Activity in Raji: Contrasting Roles of Snail and RKIP in the Regulation of Drug/Immune Resistance

The CD80 antigen, also called B7.1, is the natural ligand for the T cell receptor CD28 and which maintains T cell and B cell adhesion. Galiximab (anti-CD80 mAb) is a primatized (human IgG1 constant regions and Cynomolgous macaque variable regions) mAb that binds CD80 on lymphoma cells and has been shown in vitro to inhibit tumor cell proliferation, upregulate apoptosis and induce ADCC. A phase I/II trial as single agent Galiximab with dose escalation demonstrated that it is well tolerated and produced modest clinical activity. Also, a phase I/II trial evaluated the combination of Rituximab and Galiximab in patients with relapse refractory follicular NHL. The combination produced an overall response rate of 66% with a median PFS of 12.4 months. We have reported that Galiximab sensitized Raji and IM-9 cells to drug-induced apoptosis. The present study extends these findings and examines the underlying molecular mechanism by which Galiximab sensitizes NHL cells to apoptosis by cytotoxic drugs. We hypothesized that Galiximab inhibits intracellularly cell survival anti-apoptotic pathways such as constitutively activated NF-kB, leading to sensitization to drug-induced apoptosis. We have used CD80-expressing Raji cells as a model for our studies. We demonstrate that following treatment of Raji with Galiximab (25–50 μg/ml) for 24 hours, cell lysates were assessed for various gene products of the NF-kB pathway by Western. There were significant downregulation of both p65 and phospho-p65, both IkB-α and phospho-IkB-α and downstream inhibition of Bcl-2 and BclXL and induction of Bak. In addition, there was a strong induction of the metastasis suppressor and immune surveillance cancer gene product Raf-1 kinase inhibitor protein (RKIP) and downregulation of the inactive and phosphorylated form of RKIP. The induction of RKIP by Galiximab was, in part, the result of NF-kB-induced inhibition downstream of the metastasis inducer and RKIP transcription repressor Snail. Galiximab also inhibited downstream both the Fas and DR5 transcription repressor Yin-Yang 1 (YY1) concomitantly with upregulation of Fas and DR5. These findings establish a molecular mechanism by which Galiximab sensitizes tumor cells to drug/immune-induced apoptosis via inhibition of NF-kB and Snail and induction of RKIP expression. We have previously reported that Rituximab modifies intracellular pathways including NF-κB and sensitizes B-NHL to apoptosis (Jazirehi and Bonavida, Oncogene , 24:2121, 2005). Thus, the combination treatment with Rituximab and Galiximab, through common and complementary mechanisms, may result in the reversal of CD20+/CD80+ B-NHL tumor cell resistance. The studies also suggest the potential combination treatment of Galiximab and non-toxic chemotherapeutic drug or immunotherapeutic drug (example: TRAIL) in the treatment of refractory CD80+ B cell malignancies.

Inhibition of the Akt Pathway in MM Cell Lines by the Anti-TfR-IgG3-Avidin Fusion Protein (Anti-TfR-IgG3-Av): Role in Chemosensitization to CDDP-Induced Apoptosis

Multiple Myeloma (MM) is an incurable disease of monoclonal malignant plasma cells. Treatment of MM with conventional chemotherapeutic drugs has resulted in improved response rates, however, with no sufficient improvement in overall survival. Bortezomib has been recently used and results in significant clinical responses in refractory MM. However, many patients relapse and become refractory to cytotoxic therapies and, hence, the need for new therapies. We have generated an Anti-TfR-IgG3-Avidin Fusion Protein (Anti-TfR-IgG3-Av) that can bind MM which express high levels of transferrin receptor and can deliver biotinylated molecules into cancer cells (Ng et al PNAS 2002; 79:10706). We have reported that treatment of MM with Anti-TfR-IgG3-Av results in inhibition of cell proliferation and direct cytotoxicity in few cell lines. Further, we have also found that Anti-TfR-IgG3-Av can sensitize resistant MM cells to drugs (eg. CDDP)-induced apoptosis. Sensitization by Anti-TfR-IgG3-Av resulted in the inhibition of several anti-apoptotic gene products like XIAP, Bid, Bcl-2 and BclXL. Since these gene products are regulated by the NF-κB pathway, we hypothesized that Anti-TfR-IgG3-Av may inhibit the AKT pathway in MM cell lines. The AKT signaling inactivates several pro-apoptotic factors, such as Bad, which is phosphorylated and inhibits its binding and inactivation of BclXL. AKT also activates IκB kinase (IKK) to phosphorylate IκB (inhibitor of NF-κB ) and leading to its proteasomal degradation and NF-κB nuclear localization. The AKT and NF-κB pathways result in the transcription of many anti-apoptotic gene products like XIAP, Bcl-2, survivin and BclXL. Treatment of MM cell lines with Anti-TfR-IgG3-Av resulted in inhibition of phospho-AKT and inhibition of NF-κB activity and downstream inhibition of above anti-apoptotic gene products. We then examined the roles of AKT and NF-κB in Anti-TfR-IgG3-Av-induced sensitization of MM to CDDP-apoptosis. Treatment of IM-9 cells with siRNA AKT, not control siRNA, resulted in inhibition of AKT concomitantly with inhibition of Bcl-2 and survive in. The cells treated with si-RNA AKT were sensitized to CDDP-induced apoptosis. These findings suggested that Anti-Anti-TfR-IgG3-Av-induced sensitization to CDDP may be due, in part, to inhibition of AKT. Likewise, the role of NF-κB inhibition by Anti-TfR-IgG3-Av in the sensitization to CDDP was demonstrated by the use of the specific NF-κB inhibitor, DHMEQ. Thus, both inhibition of AKT and NF-κB pathways by Anti-TfR-IgG3-Av play a major role in Anti-TfR-IgG3-Av-induced sensitization to CDDP. The apoptosis achieved by the combination of Anti-TfR-IgG3-Av and CDDP resulted from the complementation of several gene products modified by each agent alone and resulting in the activation of caspases 9, 8 and 3 and apoptosis. The above findings provide an underlying mechanism of Anti-TfR-IgG3-Av-induced cell signaling modification that renders drug-resistant MM cells sensitive to apoptosis by drugs.

Binding of monosodium urate crystals with idiotype protein efficiently promote dendritic cells to induce cytotoxic T cells

Monosodium urate (MSU) crystals have been reported to evoke specific cell immunity and to work as an adjuvant in a mouse model. The crystals also have another unique characteristic to bind with positively charged proteins, which could help to deliver some antigens into human dendritic cells (DC). We focused on the application of MSU crystals as not only an adjuvant but also as a carrier of positively charged antigenic protein to induce human cytotoxic T cells (CTL) efficiently in vitro. We selected human leukocyte antigen (HLA)-A2 expressing the multiple myeloma IM-9 cell line and its product idiotype (Id) protein as one of the best pairs of target cells and positively charged tumor-specific antigen, respectively. Following the sensitization of DC derived from HLA-A2-positive volunteers pulsed with tumor-specific monoclonal immunoglobulin G-Fab fragments (IM-9 Fab) attached to MSU crystals, the DC-stimulated CD8(+) T cells killed significantly more target cells (40.1 +/- 1.7%) than those stimulated by DC pulsed with MSU crystals alone (6.2 +/- 8.6%, P < 0.01) or IM-9 Fab alone (4.7 +/- 8.1%, P < 0.01). These cytotoxic effects of the DC-stimulated CD8(+) cells were reduced when MSU crystals were precoated with fetal bovine serum. In addition, we confirmed that MSU crystals facilitated human DC to express the maturation marker, CD83 and deliver (Fab')(2), attaching to the crystals by flow cytometer analysis. MSU crystals have distinct advantages of a protein carrier binding with positively charged proteins and delivering antigenic protein into DC, as well as an adjuvant promoting DC maturation and inducing CTL.

Discovery and stereoselective synthesis of the novel isochroman neurokinin-1 receptor antagonist ‘CJ-17,493’

A novel central nervous system (CNS) selective neurokinin-1 (NK 1) receptor antagonist, (2 S,3 S)-3-[(1 R)-6-methoxy-1-methyl-1-trifluoromethylisochroman-7-yl]-methylamino-2-phenylpiperidine ‘CJ-17,493’ (compound (+)- 1), was synthesized stereoselectively using a kinetic resolution by lipase-PS as a key step. Compound (+)- 1 displayed high and selective affinity ( K i = 0.2 nM) for the human NK 1 receptor in IM-9 cells, potent activity in the [Sar 9, Met(O 2) 11]SP-induced gerbil tapping model (ED 50 = 0.04 mg/kg, sc) and in the ferret cisplatin (10 mg/kg, ip)-induced anti-emetic activity model (vomiting: ED 90 = 0.07 mg/kg, sc), all levels of activity comparable with those of CP-122,721. In addition, compound (+)-1 exhibited linear pharmacokinetics rather than the super dose-proportionality of CP-122,721 and this result provides a potential solution for the clinical issue observed with CP-122,721.

Structural Basis for the Lower Affinity of the Insulin-like Growth Factors for the Insulin Receptor

Insulin and the insulin-like growth factors (IGFs) bind with high affinity to their cognate receptor and with lower affinity to the noncognate receptor. The major structural difference between insulin and the IGFs is that the IGFs are single chain polypeptides containing A-, B-, C-, and D-domains, whereas the insulin molecule contains separate A- and B-chains. The C-domain of IGF-I is critical for high affinity binding to the insulin-like growth factor I receptor, and lack of a C-domain largely explains the low affinity of insulin for the insulin-like growth factor I receptor. It is less clear why the IGFs have lower affinity for the insulin receptor. In this study, 24 insulin analogues and four IGF analogues were expressed and analyzed to explore the role of amino acid differences in the A- and B-domains between insulin and the IGFs in binding affinity for the insulin receptor. Using the information obtained from single substituted analogues, four multiple substituted analogues were produced. A "quadruple insulin" analogue ([Phe(A8), Ser(A10), Thr(B5), Gln(B16)]Ins) showed affinity as IGF-I for the insulin receptor, and a "sextuple insulin" analogue ([Phe(A8), Ser(A10), Thr(A18), Thr(B5), Thr(B14), Gln(B16)]Ins) showed an affinity close to that of IGF-II for the insulin receptor, whereas a "quadruple IGF-I" analogue ([His(4), Tyr(15), Thr(49), Ile(51)]IGF-I) and a "sextuple IGF-II" analogue ([His(7), Ala(16), Tyr(18), Thr(48), Ile(50), Asn(58)]IGF-II) showed affinities similar to that of insulin for the insulin receptor. The mitogenic potency of these analogues correlated well with the binding properties. Thus, a small number of A- and B-domain substitutions that map to the IGF surface equivalent to the classical binding surface of insulin weaken two hotspots that bind to the insulin receptor site 1.

Pharmacological Characterization of T-2328, 2-Fluoro-4’-methoxy- 3’-[[[(2S,3S )-2-phenyl-3-piperidinyl]amino]methyl]-[1,1’-biphenyl]-4- carbonitrile Dihydrochloride, as a Brain-Penetrating Antagonist of Tachykinin NK1Receptor

The pharmacological properties of T-2328 were evaluated as an antagonist of the tachykinin neurokinin 1 (NK1) receptor. T-2328 inhibited the specific binding of [3H][Sar9,Met(O2)11]substance P to tachykinin NK1receptors in human lymphoblastic IM9 cells with Kiof 0.08 nM. In the same assay, Kifor aprepitant, a brain-penetrating NK1antagonist, was 1.3 nM. The antagonism of T-2328 is highly selective for the human NK1receptors since the affinities for human NK2, NK3receptors, and 13 other kinds of receptors and ion channels were >1000-fold lower than for NK1receptors. Reduction in Bmax with no change in affinity suggests the non-competitive nature of T-2328 interaction with the NK1receptor. T-2328 (0.03 –0.1 mg/kg, i.v.) and aprepitant (1 –3 mg/kg, i.v.) significantly prevented the GR73632 (i.c.v.)-induced foot tapping response in gerbils. The potencies of T-2328 in both in vitro and in vivo studies were more than 10 times greater than those of aprepitant. I.v. administration of T-2328 (0.1 –0.3 mg/kg) potently blocked both acute and delayed emetic responses induced by cisplatin (5 mg/kg, i.p.) in ferrets. It is concluded that T-2328 is a potent, centrally active NK1antagonist. T-2328 may have potential as a novel therapeutic agent for the treatment of chemotherapy-induced emesis.

Insights into the Genetic Signature Associated with Cell Death Induced by an Antibody-Avidin Fusion Protein Specific for Transferrin Receptor.

We have previously developed an anti-human transferrin receptor IgG3-avidin fusion protein (anti-hTfR IgG3-Av) that exhibits a potent anti-proliferative/pro-apoptotic activity against malignant B cells. This effect has been attributed, at least in part, to the internalization and rapid degradation of the hTfR by anti-hTfR IgG3-Av, resulting in lethal iron deprivation. However, in a panel of eight malignant B cells treated with anti-hTfR IgG3-Av we observed varying levels of sensitivity among the cell lines, where IM-9 (a human lymphoblastoid cell line) showed high sensitivity, while U266 (a human multiple myeloma cell line) was significantly resistant; although both cell lines express high levels of hTfR. To elucidate the mechanisms behind resistance and sensitivity of these human malignant B cells we conducted a global gene expression comparison treating the two cell lines for 0, 1, 3, 9, and 24 hours with anti-hTfR IgG3-Av or buffer control. Samples of total RNA were collected in duplicate at each time point and gene expression levels analyzed using HumanRef-8 v2 Expression BeadChips (Illumina Inc. San Diego, CA). Microarray data were preprocessed using quantile normalization and the difference in levels of gene expression was established using Bayesian statistics. Out of 8,095 array probes detected at time zero in both IM-9 and U266 cell lines, 167 had a greater than four-fold significant difference (regularized bayesian t-test p<0.05). With the genes selected by this global comparison, we performed a gene ontology analysis to identify genes associated with functional processes that may be involved in the differential sensitivity to anti-hTfR IgG3-Av. We found that genes facilitating apoptotic signaling through RAS, MAPK, and P53 (such as PIK3CD and CBLB) were expressed at significantly higher levels in IM-9 compared to U266, while genes involved in cell cycle including the cyclin-dependent growth regulator CDKN2C and a number of core histones were more highly expressed in U266 compared to IM-9. Of all the genes present in both cell lines, a time course study of 24 hour treatment with anti-hTfR IgG3-Av identified 74 genes in IM-9 cells and 10 genes in U266 cells with significant changes (p<0.05) at greater than 1.5-fold different expression levels compared to buffer control treated cells. IM-9 cells treated with anti-hTfR IgG3-Av showed increased expression of a number of apoptosis related genes including GADD45A, IKIP, DDB2, and TP53I3 at 24 hours. In contrast, U266 cells treated with anti-hTfR IgG3-Av showed decreased expression of genes involved in apoptosis induction and cell death such as PLEKHF1, OKL38, and CDC14B. Although further studies are needed to validate the expression changes, these results illustrate the complexity of the mechanism responsible of cell death induced by anti-hTfR IgG3-Av, and suggest that the molecular pathway involving p53 may be associated with cell death in the sensitive cell line. These studies are expected to pave the way to fully elucidate the mechanisms responsible for cell death induced by anti-hTfR IgG3-Av and the identification of a genetic signature responsible for resistance to this therapeutic.

Monosodium Urate Crystal as a Carrier for Idiotype Tumor Vaccine.

Monosodium urate (MSU) crystal works as a danger signal to evoke specific cell immunity. It stimulates dendritic cells (DCs) maturation and enhances the generation of cytotoxic T cells (CTL), so that it could help specific immune therapy for tumor. On the other hand, MSU crystals are found to attach IgG in gouty arthritis and has been reported that the site of attachment is the Fab moiety of IgG. We focused on the application of MSU as a carrier of IgG idiotype protein to induce cytotoxic T cells to cell line producing the idiotype protein. Multiple myeloma cell line, IM-9, which is characterized by having HLA-A2 and producing monoclonal IgG-k protein, was used as a target cell. Monoclonal Fab fragments were used as idiotype protein; those were made by digesting with papain of IgG collected from the medium harvesting IM-9 cells. MSU crystals were made by sitting at refrigerator of uric acid dissolved by sodium hydroxide solution with boiling. Allogenic dendritic cells were derived from monocytes in peripheral blood of HL-A matched volunteer by treatment with GM-CSF and IL-4. They were pulsed by MSU attached with Fab fragments (MSU-Fab), by Fab fragments (Fab), or by none. Following education by pulsed DCs to CD3+ T cells collected from peripheral blood of same volunteer, CD8+ T cells were collected and were used as effector cells. Cell cytotoxic ability of the effector cells was determined by lactate dehydrogenase release assay under mixed culture of the target cells and the effector cells (CD8+ cells: IM-9 cells = 5: 1). Compared to Fab, MSU-Fab were easily phagocytosed by DCs. DCs pulsed with MSU-Fab or MSU were induced to express CD83 molecule, a marker for mature human blood dendritic cells, whereas neither Fab-pulsed nor untreated-control DCs did not express CD83 molecule. CD8+ T cells educated by DCs pulsed with MSU-Fab significantly killed more IM-9 myeloma cells than CD8+ T cells educated by DCs pulsed with MSU, DCs pulsed with Fab or DCs alone. These cytotoxic effect of CD8+ T cells educated by DCs pulsed with MSU-Fab were reduced when MSU crystals were bathed 10% fetal bovine serum solution before mixing with Fab in order to block MSU to be attached with Fab. The present study revealed that MSU crystals work as carrier of idiotype tumor vaccine not only by facilitating the utilization of the vaccine protein in DCs but also by enhancing the ability to induce tumor specific CTL. The safety and efficacy of MSU-Fab idiotype vaccine will be determined by clinical setting in patients with multiple myeloma.

IL-7 along with Optimized CD3/CD28 Artificial APC Beads Promotes Ex Vivo Expansion of Cord Blood T Cells towards Adoptive Immunotherapy.

Background: Viral infections are the leading cause of non-relapse mortality after unrelated umbilical cord blood transplants (UCBT). Post-transplant lymphopenia, lack of established antiviral memory, and lack of cytotoxic function among infused UCB lymphocytes are jointly responsible for ineffective immunity. We have previously demonstrated ex vivo T cell expansion and maturation starting from a very small fraction (<5%) of the graft that could be employed post transplant as donor leukocyte infusion (DLI). Naïve UCB T cells proliferate when stimulated with IL-2 + CD3/CD28 co-stimulatory beads that function as artificial antigen presenting cells (APCs). However, high rate of apoptosis (>10%) has hindered T cell expansion. We hypothesized1.that the presence of cytokines essential for T cell homeostasis, in particular IL-7 may aid expansion and survival,2.beads with different densities of CD3/CD28 antibodies may impact UCB T cell expansion.Research Design: From frozen/thawed specimens we tested 2 different sources of CD3/CD28 artificial beads (historical control beads vs ClinExVivo®, (Invitrogen Corporation), ± IL-7.Methods: Thawed UCB samples were centrifuged over Ficoll gradient. T cells were positively selected with EasySep®, (StemCell Technologies) and were incubated in gas permeable bags with CD3/CD28 beads in 5% serum replete media + 100u/ml IL-2 ± 10ng/ml of IL7. Media & cytokines were replenished to maintain a concentration of ∼0.75 ×106 cells/ ml. Automated cell count, trypan blue viability assessed overall cell growth and viability at each feeding . ‘Lyse no wash' FACS staining with Trucount® beads quantified viable CD3+ T cells. 4-color FACS was employed to characterize the evolution of surface and intracellular immunophenotype. Cytotoxicity profile of the day 0 and D12-14 progeny was tested by Europium release assay (Delfia® assay) against a Burkitt's lymphoma cell line (IM9). Two-tailed paired Student t-tests analyzed the impact of the experimental variables.Results: At the end of 12–14 day expansion periods, analyzing all cytokine conditions, we could demonstrate an average of 43-fold viable CD3+ T cell expansion using control APC beads (n=8), while T cells on ClinExVivo® beads expanded on average 94-fold (p=0.11, n=11). Addition of IL7 to the culture media afforded significantly better expansion with the control bead condition (mean 43 vs 26 fold, p=0.02). IL7 also enhanced T cell expansion with ClinExVivo® beads (mean 147 fold vs 49 fold, though statistically NS). There were significantly more viable T cells generated with ClinExVivo® beads when all timepoints were analyzed, irrespective of the absence (p=0.017) or presence (p=0.05) of IL7. We also analyzed the mechanism how IL7 may potentiate overall T cell expansion. T cells entering cell cycle was enhanced (though not significantly) as demonstrated with intracellular Ki-67 staining (65% T cells in cycle with IL7 versus 52% without, p=0.2). IL7's overall salutary effect on total expansion may be best explained by simultaneously reduced T cell apoptosis as quantified by activated ic Caspase-3 detection (9.3% versus 7.6% without vs with IL7, respectively, p=0.08) regardless of the source of beads. Granzyme A, B, perforin expression was enhanced comparably, while cytotoxicity was absent against IM9 cells regardless of ± IL-7.Conclusions: These results demonstrate significant expansion of UCB T cell that is further improved by utilizing ClinExVivo® clinical grade APC beads and IL7. These results pave the way to donor-derived UCB T cell expansion suitable for DLI to boost immunity.

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.

Galiximab Sensitizes Malignant Human B Cell Lines to Apoptosis by Chemotherapeutic Drugs.

Galiximab is a primatized anti-CD80 antibody that is being investigated as a treatment for non-Hodgkin's lymphoma. Results to date from clinical trials indicate that galiximab is well-tolerated and suggest clinical activity both as monotherapy and in combination with rituximab (anti-CD20). CD80 (B7.1), expressed not only by malignant B cells but also by normal B cells, monocytes/macrophages, dendritic cells as well as T cells, plays an important role in the complex and dynamic regulation of adaptive immunity by interacting with either co-stimulatory (e.g. CD28) or co-inhibitory (e.g. CTLA-4, PD-L1) receptors expressed by T lymphocytes. Inhibition of anti-lymphoma immunity that exists within the tumor microenvironment may reflect selection of inhibitory immune regulation mediated through CD80. Galiximab may thus function by directly targeting malignant and possibly non-malignant CD80-expressing cells for antibody-dependent cytotoxicity, or by blocking CD80 interactions involved in suppressing anti-tumor immunity. In preclinical xenograft models, which do not assess the effect of galiximab on non-malignant cells given a lack of cross-reactivity with murine CD80, treatment with galiximab exhibited enhanced tumor growth inhibition when used in combination with either rituximab or with the chemotherapeutic drugs fludarabine and doxorubicin (Hariharan et al., abstract 3040, ASCO, 2007). However, the mechanisms by which galiximab enhances the cytotoxic effects of chemotherapy remain unclear. Rituximab has been shown by us to chemosensitize tumor cells by inhibiting intracellular survival pathways. Thus, we hypothesized that treatment of B lymphoma cells with galiximab facilitates the cytotoxic activity of chemotherapeutic drugs by modifying intracellular anti-apoptotic signaling pathways, resulting in the alteration of pro and anti-apoptotic gene products. The above hypothesis was examined using Raji (Burkitt lymphoma) and IM-9 (multiple myeloma) cell lines as models. Treatment with galiximab resulted in significant inhibition of cell growth and proliferation and modest apoptosis with high concentration. Both Raji and IM-9 are resistant to chemotherapeutic drugs; however, pretreatment with galiximab significantly sensitized the tumor cells to apoptosis induced by CDDP. The synergy achieved was found with subtoxic concentrations of CDDP (5–10 ug/mL) and galiximab. Analysis of the survival pathways and gene products regulating apoptosis following treatment with galiximab, CDDP, and combination will be presented. The present findings demonstrate that galiximab can synergistically enhance cytotoxicity mediated by chemotherapeutic drugs. These findings suggest that in vivo galiximab may have a chemosensitizing effect on resistant B cell malignancies, in addition to its direct cytotoxic effect via ADCC and CDC. The clinincal relevance of these findings will be discussed.

A Novel Putative Mechanism of Anti-Myeloma Activity Targeted Against Heat Shock Protein 27 by Derivative of Artemisinin, Artesunate.

Recent introduction of molecular targeted drugs such as bortezomib and IMIDs in the clinical settings has achieved the improved treatment outcome of multiple myeloma (MM). However, MM is still an incurable disease and these drugs also possess serious adverse reactions, therefore, safe and more effective therapy should be established. Artesunate (ART) is a semi-synthetic derivative of artemisinin and is widely used for the treatment of malaria as a salvage therapy. Recent in vitro studies showed that ART also has an anti-tumor activity against several cancer cell lines. We thus investigated whether ART could possess anti-myeloma activity and demonstrated that apoptosis of myeloma cells is strongly induced by ART. Interestingly, we also found that this activity is mediated through heat shock protein (Hsp) 27-dependent pathway. Several MM cell lines (IM9, OPM2, RPMI8226, U266) were treated with various concentrations of ART for 48 hours, and MTT assays were performed to assess the anti-myeloma activity. IM9, OPM2, RPMI8226 cells showed the striking reduction of viability (up to 40% at 1μM and up to 90% at 10μM) in a dose- and time-dependent manner, whereas ART has less inhibitory effect on U266 cell line. ART induced G1 arrest of IM9 cells, and apoptosis was confirmed by decreased mitochondrial membrane potential, and flow cytometric analysis using AnnexinV/PI staining. Colony assay showed that ART has no growth inhibitory effect on normal CD34-positive bone marrow cells even at a concentration of 10μM. Immunoblot analyses demonstrated the activation of caspases-3 and -9, and the decreased expression of pro-apoptotic protein Bid. Interestingly, heat shock protein (Hsp) 27 was downregulated in myeloma cell lines (IM9, RPMI8226) which are sensitive to the ART treatment. In contrast, downregulation of Hsp 27 was not observed in U266 cells which are resistant to ART. Other anti-apoptotic Hsps (Hsp70, 90), as well as Bcl-2 family proteins (Bcl-2, Bax, Bad, Bcl-xL), Akt, MDM2, p53 and MAPKs (SAPK/JNK, p38, ERK1/2) were not affected by ART. Quantitative RT-PCR analysis showed that ART did not influence the mRNA expression of Hsp27, suggesting that ART could downregulate the Hsp27 protein at a posttranscriptional or posttranslational levels. Overexpression of Hsp27 cDNA by transfection method in ART-sensitive myeloma cell lines demonstrated that these cell lines acquired resistance to ART and apoptosis was not induced at a concentration which ART is effective to the parental cells. Our preliminary data using knockdown procedure of Hsp27 mRNA by RNAi-expressing lentivirus showed the growth suppression of myeloma cells, which apparently suggested that Hsp27 could confer a critical function in the proliferation of myeloma cells and that downregulation of Hsp27 could result in induction of apoptosis in MM. Hsp27 has an important role in anti-apoptotic effect against various stresses to the cells. Our data implies a novel mechanism that downregulation of Hsp27 by ART could induce misfolding of several client proteins indispensable for the cell growth, which results in the induction of apoptosis in MM cells. In conclusion, ART could become a candidate of safe and effective therapeutic drug for MM, and Hsp 27 might be a potential molecular target for the treatment of MM.

Stereoselective synthesis of a novel 2-aza-7-oxabicyclo[3.3.0]octane as neurokinin-1 receptor antagonist

A novel neurokinin-1 receptor antagonist, (±)-(1 R ∗,3 S ∗,4 S ∗,5 S ∗)-4-[( N-(2-methoxy-5-trifluoromethoxybenzyl)amino]-3-phenyl-2-aza-7-oxabicyclo[3.3.0]octane ( 1), was synthesized stereoselectively using Padwa’s intramolecular 1,3-dipolar cycloaddition methodology as the key step. Compound (±)- 1 showed high affinity for the NK-1 receptors in human IM-9 cells with an IC 50 value of 0.22 nM. This new structural scaffold demonstrated significant in vivo antagonistic activity in the guinea pig ureter capsaicin-induced plasma extravasation model with an ED 50 value of 1–10 mg/kg, po.

Expression of a Buckwheat Trypsin Inhibitor Gene in &amp;lt;italic&amp;gt;Escherichia coli&amp;lt;/italic&amp;gt; and its Effect on Multiple Myeloma IM-9 Cell Proliferation

The gene of buckwheat trypsin inhibitor (BTI) has been cloned and expressed in Escherichia coli. The yield of this recombinant inhibitor was over 12 mg/L by using one-step purification on a Ni2+-NTA Sepharose column. Its molecular weight was 9322.1 Da, determined by mass spectrum analysis. The MTT and cytometry analyses showed that recombinant BTI could specifically inhibit the proliferation of IM-9 human B lymphoblastoid cells (from patient with multiple myeloma) in a dose-dependent manner. The test of recombinant BTI-induced apoptosis in IM-9 cells implied that the inhibitor might have potential application in the treatment of cancer.

OSU-03012, a Novel Celecoxib Derivative, Is Cytotoxic to Myeloma Cells and Acts through Multiple Mechanisms

OSU-03012 is a novel celecoxib derivative, without cyclooxygenase-2 inhibitory activity, capable of inducing apoptosis in various cancer cells types, and is being developed as an anticancer drug. We investigated the in vitro activity of OSU-03012 in multiple myeloma (MM) cells. U266, ARH-77, IM-9, and RPMI-8226, and primary myeloma cells were exposed to OSU-03012 for 6, 24, or 72 h. Cytotoxicity, caspase activation, apoptosis, and effects on intracellular signaling pathways were assessed. OSU-03012 was cytotoxic to MM cells with mean LC50 3.69 +/- 0.23 and 6.25 +/- 0.86 micromol/L and at 24 h for primary MM cells and cell lines, respectively. As a known PDK-1 inhibitor, OSU-03012 inhibited the PI3K/Akt pathway with downstream effects on BAD, GSK-3beta, FoxO1a, p70S6K, and MDM-2. However, transfection of MM cells with constitutively active Akt failed to protect against cell death, indicating activity against other pathways is important. Phospho (p)-signal transducers and activators of transcription 3 and p-MAP/ERK kinase 1/2 were down-regulated, suggesting that OSU-03012 also inhibited the Janus-activated kinase 2/signal transducer and activator of transcription 3 and mitogen-activated protein kinase pathways. Although expression of Bcl-2 proteins was unchanged, OSU-03012 also down-regulated survivin and X-linked inhibitor of apoptosis (XIAP), and also induced G2 cell cycle arrest with associated reductions in cyclins A and B. Finally, although OSU-03012 induced cleavage of caspases 3, 8 and 9, caspase inhibition did not prevent cell death. We conclude that OSU-03012 has potent activity against MM cells and acts via different mechanisms in addition to phosphoinositide-3-kinase/Akt pathway inhibition. These studies provide rationale for the clinical investigation of OSU-03012 in MM.

FLOW CYTOMETRIC EVALUATION OF SYNERGISTIC PRO‐APOPTOTIC EFFECTS OF STATINS AND CLOFIBRATES IN IM‐9 HUMAN LYMPHOBLASTS

1. In the present study, we evaluated fibrate-mediated potentiation of statin-induced apoptosis in IM-9 human lymphoblasts. 2. The pro-apoptotic effects of statin and fibrate were measured by flow cytometry with biotin-annexin V, followed by addition of avidin-fluorescein isothiocyanate and propidium iodide. Apoptosis was confirmed using karyopyknotic staining, as well as detection of DNA fragmentation and caspase 3 activation. 3. Incubation of IM-9 cells with both 0.1 micromol/L cerivastatin and 200 micromol/L clofibrate had a synergistic effect compared with 0.1 micromol/L cerivastatin alone or 200 micromol/L clofibrate alone. The magnitude of apoptosis induced by various combinations of statins and clofibrate were as follows: cerivastatin (0.1 micromol/L) + clofibrate (200 micromol/L) > atorvastatin (0.1 micromol/L) + clofibrate (200 micromol/L) > pravastatin (100 micromol/L) + clofibrate (200 micromol/L). Other fibrates (bezafibrate and clinofibrate) did not show any synergistic effect. Furthermore, karyopyknotic staining, caspase 3 activation and DNA fragmentation demonstrated synergistic pro-apoptotic effects of statin and fibrate. 4. The results of the present study suggest that simultaneous treatment with statins and clofibrate could provide improved therapeutic efficacy in leukaemia patients.

Molecular mechanism of bis(maltolato)oxovanadium(IV)-induced insulin signaling in 3T3-L1 and IM9 cells: impact of dexamethasone

The organovanadium compound bis(maltolato)oxovanadium(IV) (BMOV) enhanced the tyr-phosphorylation of major upstream insulin signaling proteins including the vital site-specific phosphorylation of insulin receptor beta (IRbeta) in IM9 and 3T3-L1 cells in dose- and time-dependent manners more efficiently than insulin. Nevertheless, insulin in general had a synergistic impact on those phosphorylations in both cell lines, while its presence was obligatory to induce Tyr(972)-phosphorylation of IRbeta in IM9 cells at 18-h treatment with BMOV. However, prolonged exposure of cells to BMOV caused depletion in IR level and using IM9 cells we found that this event was counteracted by insulin, where monensin, a monocarboxylic acid ionophore made an additive impact, suggesting that a novel mechanism is being involved in the recycling of internalized IR in BMOV-treated cells. On the other hand, dexamethasone elevated the IR level in both cell lines. However, no correlation was found between the cellular content and the degree of phosphorylation of IRbeta in cells receiving combined treatment of BMOV, and dexamethasone with short insulin post-exposure. BMOV also induced the phosphorylation of Thr(308) and Ser(473) of Akt in both cell lines receiving insulin post-treatment, while dexamethasone decreased those phosphorylations. However, this activation/deactivation of Akt did not correlate with the phosphorylation status of Ser(9) and Ser(259) of glycogen synthase kinase (GSK)-3beta and Raf respectively. Taken together, it is conceivable that BMOV and/or dexamethasone modulate insulin signaling by acting differentially on the components of the insulin signaling network. We also consider that the observed dexamethasone-mediated modulation of insulin receptor kinase in BMOV-treated 3T3-L1 cells probably occurs through the activation/deactivation of some mechanism which needs further studies for proper characterization.

Membrane expression of DR4, DR5 and caspase-8 levels, but not Mcl-1, determine sensitivity of human myeloma cells to Apo2L/TRAIL

The improved recombinant form of the death ligand Apo2L/TRAIL (Apo2L/TRAIL.0) is not cytotoxic for normal human cells and is a good candidate for the therapy of multiple myeloma (MM), a B-cell neoplasia that remains incurable. We have analyzed the molecular determinants of myeloma sensitivity to Apo2L/TRAIL.0 in a number of MM cell lines, the mechanisms of resistance and a possible way of overcoming it. Expression of one death receptor for Apo2L/TRAIL (DR4 or DR5) is sufficient to transduce death signals, though DR5 was more efficient when both receptors were present. Membrane expression of decoy receptors (DcR1, DcR2) and intracellular levels of c-FLIP L, XIAP and Mcl-1 were not predictive of resistance to Apo2L/TRAIL. Inhibition of Mcl-1 degradation did not prevent Apo2L/TRAIL-induced apoptosis. In IM-9 cells, resistance was associated to a reduced caspase-8 expression. U266 cells, though expressing significant levels of DR4 and caspase-8, were nevertheless resistant to Apo2L/TRAIL. This resistance could be overcome by co-treatment with valproic acid (VPA), a histone deacetylase inhibitor. VPA caused the redistribution of DR4 to plasma membrane lipid rafts and restored DR4 signaling. Overexpression of Mcl-1 in U266 cells did not prevent Apo2L/TRAIL cytotoxicity in VPA-sensitized cells. These results, taken together, support the possible use of Apo2L/TRAIL.0 in the treatment of MM.

Cyclooxygenase isozymes are expressed in human myeloma cells but not involved in anti‐proliferative effect of cyclooxygenase inhibitors

Considering possible tumorigenic activity of cyclooxygenase (COX) isozymes in myeloma, we examined expression levels of COX-1 and -2 in seven human myeloma cell lines (ARH-77, IM-9, RPMI-8226, HPC, HS-Sultan, TSPC-1, and U-266). As analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR), all the cell lines constitutively expressed COX-1, while COX-2 levels markedly varied among different cell lines. Induction of COX-2 by phorbol ester was observed in RPMI-8226 and HPC cells. In contrast, COX-2 was constitutively expressed in ARH-77 and IM-9 cells. Moreover, the high expression level of COX-2 protein in ARH-77 cells was verified by Western blotting. Intact cells of ARH-77 converted 14C-labeled arachidonic acid to prostaglandin E2, F2alpha, and D2, and this activity was dose-dependently inhibited by selective COX-2 inhibitors (SC-58125 and NS-398), a non-selective COX inhibitor (indomethacin), and relatively high concentrations of a selective COX-1 inhibitor (SC-560). These COX inhibitors also suppressed the proliferation of ARH-77 cells, but significant suppression was seen only at 100 microM, a much higher concentration than those sufficient for the COX inhibition. Moreover, proliferation of the myeloma cells lacking COX-2 was also suppressed by 100 microM of SC-58125. These results suggested that the anti-proliferative effect of the COX inhibitors is independent of the inhibition of COX-2.