Epinephrine Stimulates Cell Proliferation and Induces Chemoresistance in Myeloma Cells through the β-Adrenoreceptor in vitro

Objectives: To explore the effect of the β-adrenoreceptor signaling pathway on myeloma cells. Methods: The myeloma U266 cell line was treated with epinephrine and propranolol. Cell proliferation was analyzed by MTS assay. Apoptosis was detected by flow cytometry. The β-receptor subtype and the key enzyme of epinephrine were identified by reverse transcription polymerase chain reaction (RT-PCR). Results: Epinephrine (5-50 μM) promoted U266 cell growth in a dose-dependent manner and neutralized the inhibition effect of bortezomib (25 and 50 ng/mL) in vitro. Cell proliferation was inhibited by a β-receptor antagonist, propranolol, at a concentration of 50-200 μM. The proportions of early and late apoptotic cells were enhanced after treatment with propranolol. The expression of caspase 3/7, 8, and 9 was elevated in propranolol-treated myeloma cells. Both β1- and β2-adrenoceptor mRNAs were expressed in the U266 cell line. Key enzymes dopamine hydroxylase and tyrosinehydroxylase were identified in myeloma cells. Conclusions: Our results reveal that epinephrine stimulates myeloma cell growth in vitro while the β-blocker propranolol has an antiproliferative effect, indicating that stress hormones may trigger the progression of myeloma.

Insulin-Like Growth Factor Binding Protein-3 (IGFBP3) Gene Expression In Myeloma Cell Lines and Patients

Abstract 2489 Introduction: Epigenetic aberrations play an important role in the development and progression of Multiple Myeloma (MM). 5-aza-2-deoxycytidine (5Aza-dC) and Trichostatin A (TSA) have been studied in reactivating the expression of epigenetically-silenced genes. IGFBP3 gene is a member of the insulin-like growth factor binding protein (IGFBP) family and can regulate cell growth and death by the ability to bind insulin-like growth factors (IGFs) as well as its IGF-independent effects involving binding to other molecules. Several in vitro, in vivo studies as well as clinical evidence point to IGFBP-3 as an anti-cancer molecule. In our study we analyzed global changes in gene expression profiles of MM cell lines, responding to 5Aza-dC and TSA and we evaluated the IGFBP3 expression in three myeloma cell lines and in samples from myeloma patients. Methods: Human MM cell lines U266 and H929 were treated either with 0.5 micromol/L 5Aza-dC for 7 days or with 100 ng/mL TSA for 24 h or with the combination of 0.5 micromol/L 5Aza-dC for 7 days and 100 ng/mL TSA for additional 24 h. Control cells received no drug treatment. Applied Biosystems microarray platform ABI 1300 was used for carrying out microarray profiling and analysis. To classify up-regulated genes into functional categories the PANTHER Classification System was used (http://www.pantherdb.org). Stained or unstained bone marrow slides were collected from archival samples of 179 myeloma patients. For validation of microarray results, real-time reverse transcription-PCR (RT-PCR) was performed using Taqman Gene Expression Assays (Applied Biosystems). Results: After treatment with 5Aza-dC there was up regulation of gene expression in 698 genes in H929 cell line and 258 genes in U266 cell line. After treatment with TSA 719 genes were up regulated in H929 cell line and 742 genes in U266 cell line. The exposure to the combination of 5Aza-dC/TSA resulted in up-regulation of 921 genes in H929 cell line and 615 genes in U266 cell line. By using Panther classification system we classified up-regulated genes into functional categories and we identified several 5Aza-dC or TSA up-regulated genes that are involved in important cancer-related pathways including cell cycle, apoptosis, cell adhesion, oncogenesis and cell metabolism, including DNA repair and nucleosome assembly. Between these genes we particularly found interesting the expression changes of IGFBP-3 which was up regulated after treatment with 5-azacitidine in U266 cell line (level of induced expression was 5,16). The microarray data were validated in 3 MM cell lines and 179 patients samples by analysis of relative changes in IGFBP3 expression through ΔΔCt method. We found that the expression levels of IGFBP3 were significantly lower in U266 and RPMI myeloma cell lines, but not in H929 cell line and interestingly levels were lower in 54 percent of patients (Fig 1). It has been demonstrated decreased IGFBP-3 expression is associated with cancer progression and in our study we have shown that the down-regulation of this gene may be involved also in myeloma pathogenesis and mediate progression events, hence levels of IGFBP3 may be considered as a biomarker for disease staging. From a therapeutic point of view, up-regulation of IGFBP3 might be considered as target therapeutic strategy for monoclonal gammopathies/ smouldering Myelomas at high risk of progression to active myeloma. Disclosures: No relevant conflicts of interest to declare.

Expression of Cancer Testis (CT) Antigens NY-ESO-1 and LAGE-1 in Multiple Myeloma (MM): Could a Vaccine against the Former Antigen Be Effective against the Latter?

Abstract 4887 Introduction In recent years, the expression of CT antigens has been studied in various malignant neoplasias. Based on their tumor-associated expression and due to their ability to elicit an immune response in the autologous host, CT antigens are potential targets for vaccine-based immunotherapy of cancer. Since different CT antigens can be expressed simultaneously in the same tumor, CT antigens are possible targets for polyvalent vaccines. Immunotherapy trials employing MAGE-A3 and NY-ESO-1 in MM patients are in progress. We previously reported frequent expression of LAGE-1 on mRNA level in MM patients (∼50%). Due to the high similarity between NY-ESO-1 and LAGE-1 (94% in mRNA and 84% in protein sequences) and the more prevalent presence of the latter, it is interesting to speculate if anti-NY-ESO-1 vaccine might elicit LAGE-1 immunity and hence may be efficient in patients with LAGE-1-positive tumors. Objectives To evaluate mRNA and protein expression of NY-ESO-1 and LAGE-1 in MM patients and to explore the possibility of an anti-NY-ESO-1 vaccine eliciting immunity to LAGE-1. Materials and methods The expression of NY-ESO-1 and LAGE-1 was studied by RT-PCR in 18 normal tissues, 28 bone marrow MM samples and U266 cell line. NY-ESO-1 and LAGE-1 protein expression were analyzed by immunohistochemistry in the MM specimens using monoclonal antibody (mAb) E978 and mAb 219-510-23 respectively. The protein extracts from U266 (NY-ESO-1+/LAGE-1+), H1299 (only NY-ESO-1+), SKBR3 (only LAGE-1+) and HaCat (double negative) cell lines were analyzed by Western Blot with mAb E978 (1:5,000). Results RT-PCR was positive for both genes in testis, placenta and U266 cell line. All other normal tissues were negative. In MM, LAGE-1 was positive in 36% (10/28) and NY-ESO-1 in 18% (5/28) of total bone marrow samples. However, only 2 patients (7%) were positive for protein expression by immunohistochemistry (both cases were also positive by RT-PCR). Both NY-ESO-1 mRNA-positive cell lines U266 and H1299 were positive by Western Blot for mAb E978, but this anti-NY-ESO-1 mAb was unable to identify LAGE-1 protein in U266 and SKBR3 cell line extracts. Conclusions In this small group of MM patients, NY-ESO-1 and LAGE-1 were expressed on mRNA level in 18% and 36% respectively, while both antigens were present on protein level in only 7%. Discrepancies between RNA and protein expression has been described in other tumors for CT antigens previously. The incidence of up to 36% LAGE-1 positive cases suggest that multiple myeloma might be susceptible to LAGE-1 and/or NY-ESO-1 vaccine-based immunotherapy. Also it is may be possible that, due to the high similarity between the proteins, anti-NY-ESO-1 vaccine might elicit LAGE-1 immunity and hence may be efficient in patients with LAGE-1-positive tumors. Disclosures No relevant conflicts of interest to declare.

The Research of Phenylhexyl Isothiocyanate’s Influence on p16 Gene Methylation of Leukaemia Cell

Objective Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. The aim of this study was to study whether phenylhexyl isothiocyanate can reduce the methylation level of P16 gene. This study used a microarray-based method for quantificationally detecting changes of P16 gene methylation in leukemia patient and U266 cell line. And to simply discuss the effect of phenylhexyl isothiocyanate on tumor methylation. Methods This method used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. One set of oligonucleotide probes were designed to fabricate a DNA microarray to detect the methylation changes of P16 gene CpG islands. Each set contained a pair of methylated and unmethylated oligonucleotides for interrogating 3 CpG sites in close proximity. By TA cloning, PCR, sequencing, positive and negative DNA targets were required. Next drawing a standard curve by fluorescence intensity. Leukemia samples DNA were abstracted and bisulfite-modified. Sample DNA targets were required by PCR amplification and were hybridized with the microarry. Finally the microarry was scanned with ScanArray Lite microarray analysis systems. Results The linear relationship (R2=0.9660) was established and it could be used to eliminate background noise. The methylation level of U266 is hypermethylation, after cultured with PHI, the level reduce. Eleven patients have P16 gene hypermethylation in thrity. After culture with PHI, seven patients showed level reduce, one patient showed raise, three patients showed remaining. Conclusion PHI can reduce the methylation level of P16 gene in U266 cell line, and also can reduce the methylation level of P16 gene of leukemia patient samples in vitro culture.

The Pan-Bcl-2 Inhibitor GX15-O70 Induces Apoptosis in Human Myeloma Cells by Noxa Induction and Strongly Enhances Melphalan, Bortezomib or TRAIL-R1 Antibody Apoptotic Effect.

GX15-070 belongs to small molecule pan-Bcl-2 inhibitors that mimic BH3-only proteins, therefore it may bind the groove of anti-apoptotic Bcl-2 family members and induce cell death. Mcl-1, an anti-apoptotic protein from the Bcl-2 family, is an essential survival protein for Multiple Myeloma (MM) cells. Mcl-1 displays specific affinity for some BH3-only proteins. In viable MM cells, Mcl-1 strongly interacts with BH3-only Bim protein, keeping in check its pro-apoptotic properties. Consequently, we tested a panel of human MM cell lines (HMCL) (n=9) for GX15-070 apoptosis induction. Apoptosis of HMCL was induced at doses ranging from 0,15 μM to 5 μM. HMCL were differentially sensitive to GX15-07. For most HMCL the maximal apoptotic effect was observed at 2,5 μM of GX15-070. Thus we found 2 very sensitive HMCL (> 60% of death cells), 3 intermediate sensitive HMCL (>30% <60%) and 4 resistant HMCL (<30%). Sensitivity to GX15-070 was not correlated with IL-6 dependence. For further studies 4 HMCL were selected according to GX15-070 apoptosis induction, namely RPMI-8826 (71% ± 4), MDN (64% ± 4), LP1 (43% ± 3) and U266 (22%± 2). After 24 hours treatment, no substantial modifications of the anti-apoptotic proteins (Bcl-2, Bcl-xL and Mcl-1) were induced. Among the BH3-only pro-apopototic proteins examined, only Noxa was consistently induced by GX15-070 in all sensitive HMCL. In contrast, Noxa was not induced in the GX15-070 resistant U266 cell line. Pro-apoptotic multidomain molecules Bax and Bak were not modified by GX15-070. We also evaluated GX15-070 in combination with other drugs currently used in MM treatment. We wondered if GX15-070 could sensitize HMCL LP1 and/or L363 cells to either Melphalan or PS-341 since both HMCL are weakly sensitive to both agents. HMCL were pre-incubated with GX15-070 for 7h at doses ranging from 0,15 to 5 μM followed by either melphalan or Bortezomib during 24h. GX15-070 displayed an additive apoptotic effect either in combination with melphalan or with Bortezomib for both HMCL. Interestingly, GX15-070 had a synergistic apoptotic effect in combination with a fully human antibody directed against TRAIL-R1, Mapatumumab, Human Genome Sciences, Rockville, MD. Of note, GX15-070/Mapatumumab combination exhibited a synergistic apoptotic effect on the GX15-070 resistant U266 cell line. Ongoing studies are conducted to unravel the contribution of GX15-070 either on the intrinsic or the extrinsic apoptotic pathways and to explore the feasibility of GX15-070 as a MM therapeutic agent.