Nanopore Long-Read Transcriptomics Profiling Reveals Gene Expression Signatures of Mouse Tumor Endothelial Cells 2H-11

Abstract Background:Tumor endothelial cells (TECs) play an indispensable role in tumor growth and metastasis. Compared with normal endothelial cells (NECs), TECs exhibit unique phenotypic and functional heterogeneity in terms of metabolism, genetics, and transcriptomics. It is not only the key to coordinate tumor angiogenesis, but also an important factor of immune regulation in the tumor microenvironment. In recent years, the role of TECs in tumor metabolism and invasion has been continuously reported. However, the research on the mechanism behind the complex functions of TECs is still at the basic stage. We use Oxford Nanopore Technology (ONT) three-generation full-length transcriptome sequencing to detect all genetic structural changes in the transcriptome of mouse TECs 2H-11 and mouse NECs SVEC4-10.Results: In Tumor endothelial cells 2H-11,1847genes are up-regulated and 1202 genes are down-regulated. According to the Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs), we found that different functional trends related to metabolic processes, developmental processes, localization, immune system processes, and locomotion are the main reasons for the differences. DEGs are mainly enriched in signal pathways related to cancer, immunity and metabolism, involving Pathways in cancer,Antigen processing and presentation , Proteoglycans in cancer, Focal adhesion, MAPK signaling pathway ,Protein digestion and absorption，ECM-receptor interaction，PI3K-Akt signaling pathway and Glutathione metabolism. We also obtained the structural variation of transcripts such as alternative splicing, gene fusion, and alternative polyadenylation and accurately quantified the expression of the transcript. Some of our results have been confirmed in other documents. But other data have not been reported yet, which is the focus of our future exploration.Conclusion: We try to use transcriptomics and bioinformatics methods to characterize tumor endothelial cell-related genes and signaling pathways.It could help better understand the molecular mechanisms of tumor endothelial cells involved in tumorigenesis and development. DEGs in key pathways may be potential diagnostic markers or therapeutic targets of TECs. Our data also provide useful genetic resources for improving the genome and transcriptome annotations of TECs and NECs.

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Anticancer Effects of Emodin on HepG2 Cell: Evidence from Bioinformatic Analysis

BioMed Research International ◽

10.1155/2019/3065818 ◽

2019 ◽

Vol 2019 ◽

pp. 1-14 ◽

Cited By ~ 3

Author(s):

Rui-sheng Zhou ◽

Xiong-Wen Wang ◽

Qin-feng Sun ◽

Zeng Jie Ye ◽

Jian-wei Liu ◽

...

Keyword(s):

Signaling Pathway ◽

Hepg2 Cells ◽

Molecular Mechanisms ◽

Mapk Signaling ◽

Ion Concentration ◽

Receptor Interaction ◽

Pathway Enrichment Analysis ◽

Mrna Levels ◽

Hub Genes ◽

Positive Regulation

Hepatocellular carcinoma (HCC) is a primary cause of cancer-related death in the world. Despite the fact that there are many methods to treat HCC, the 5-year survival rate of HCC is still at a low level. Emodin can inhibit the growth of HCC cells invitroand invivo. However, the gene regulation of emodin in HCC has not been well studied. In our research, RNA sequencing technology was used to identify the differentially expressed genes (DEGs) in HepG2 cells induced by emodin. A total of 859 DEGs were identified, including 712 downregulated genes and 147 upregulated genes in HepG2 cells treated with emodin. We used DAVID for function and pathway enrichment analysis. The protein-protein interaction (PPI) network was constructed using STRING, and Cytoscape was used for module analysis. The enriched functions and pathways of the DEGs include positive regulation of apoptotic process, structural molecule activity and lipopolysaccharide binding, protein digestion and absorption, ECM-receptor interaction, complement and coagulation cascades, and MAPK signaling pathway. 25 hub genes were identified and pathway analysis revealed that these genes were mainly enriched in neuropeptide signaling pathway, inflammatory response, and positive regulation of cytosolic calcium ion concentration. Survival analysis showed that LPAR6, C5, SSTR5, GPR68, and P2RY4 may be involved in the molecular mechanisms of emodin therapy for HCC. A quantitative real-time PCR (qRT-PCR) assay showed that the mRNA levels of LPAR6, C5, SSTR5, GPR68, and P2RY4 were significantly decreased in HepG2 cells treated with emodin. In conclusion, the identified DEGs and hub genes in the present study provide new clues for further researches on the molecular mechanisms of emodin.

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Alkaloids exhibit a meaningful function as anticancer agents by restraining cellular signaling pathways

Mini-Reviews in Medicinal Chemistry ◽

10.2174/1389557521666211007114935 ◽

2021 ◽

Vol 21 ◽

Author(s):

Wen Xu ◽

Bei Wang ◽

Yisong Gao ◽

Yuxuan Cai ◽

Jiali Zhang ◽

...

Keyword(s):

Signaling Pathways ◽

Signaling Pathway ◽

Anticancer Agents ◽

Molecular Mechanisms ◽

Cellular Signaling ◽

Biological Effects ◽

Enzymatic Reaction ◽

Mapk Signaling ◽

Research Progress ◽

Signal Pathways

: Alkaloids are nitrogen-containing organic compounds widely found in natural products, which play an essential role in clinical treatment. Cellular signaling pathways in tumors are a series of enzymatic reaction pathways that convert extracellular signals into intracellular signals to produce biological effects. The ordered function of cell signaling pathways is essential for tumor cell proliferation, differentiation, and programmed death. This review describes the antitumor progression mediated by various alkaloids after inhibiting classical signaling pathways; related studies are systematically retrieved and collected through PubMed. We selected the four currently most popular pathways for discussion and introduced the molecular mechanisms mediated by alkaloids in different signaling pathways, including the NF-kB signaling pathway, PI3K/AKT signaling pathway, MAPK signaling pathway, and P53 signaling pathway. The research progress of alkaloids related to tumor signal transduction pathways and the realization of alkaloids as cancer prevention drugs by targeting signal pathways remains.

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Integrated mRNA-seq and miRNA-seq analysis of goat fibroblasts response to Brucella Melitensis strain M5-90

PeerJ ◽

10.7717/peerj.11679 ◽

2021 ◽

Vol 9 ◽

pp. e11679

Author(s):

Baobao Li ◽

Si Chen ◽

Chengqiang Wang ◽

Qiaoling Chen ◽

Churiga Man ◽

...

Keyword(s):

Signaling Pathway ◽

Molecular Mechanisms ◽

Defense Mechanism ◽

Brucella Melitensis ◽

Pearson Correlation ◽

Killer Cell ◽

Mapk Signaling ◽

Differentially Expressed ◽

Receptor Interaction ◽

Bacterial Disease

Brucellosis is a globally zoonotic bacterial disease of humans and various animals including goats, sheep, and cattle. Brucella melitensis M5-90, a live attenuated vaccine strain, has been widely used to prevent brucellosis in goats and sheep. However, the molecular mechanisms governing protective immunity response in non-professional phagocytes infected with B. melitensis M5-90 have not been fully investigated, especially in goats. In our research, goat fibroblasts were used as in vitro models to determine these mechanisms by transcriptome analysis. After incubating with B. melitensis M5-90 3 h, the infected goat fibroblasts were collected at 0 h, 4 h, 24 h, 48 h and 72 h for RNA-seq. The results indicated that there were totally 11,819 differentially expressed genes (DEGs) and 777 differentially expressed (DE) miRNAs found in experiment groups compared with the control groups (|log2(Foldchange)|≥1, FDR<0.05). GO and KEGG enrichment analyses revealed that down-regulated genes were involved in the riboflavin metabolism and positive regulation of IL-8 secretion pathway. The up-regulated genes were mainly involved in adaptive immunity, including TNF signaling pathway, MAPK signaling pathway and JAK/STAT pathway. Additionally, cytokine-cytokine receptor interaction, natural killer cell mediated cytotoxicity and toll-like receptor signaling pathway, which associated with innate immunity pathways, were also induced. Based on the Pearson correlation coefficients and prediction results of TargetScan and miRanda, the miRNA-mRNA networks of NFKB1, IFNAR2 and IL10RB were constructed and verified in goat fibroblasts by qPCR, which demonstrated that goat fibroblasts displayed immunomodulatory properties. Our findings provide a deeper insight into the host miRNA-driven B. melitensis defense mechanism and reveal the transcriptome changes involved in the innate and adaptive immune response of the goats to B. melitensis infection.

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Abstract 549: Characterization of Mesenchymal Stromal Cell-derived Exosomes From Patients With Improved Function Outcome After Ischemic Cardiomyopathy: A Biorepository Evaluation of the Focus-CCTRN Trial

Circulation Research ◽

10.1161/res.127.suppl_1.549 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Lourdes I Chacon ◽

Fernanda Mesquita ◽

Hassan Virk ◽

Camila Hochman-mendez ◽

Doris A Taylor

Keyword(s):

Endoplasmic Reticulum ◽

Signaling Pathway ◽

Ischemic Cardiomyopathy ◽

Antigen Processing ◽

Molecular Mechanisms ◽

Autologous Transplantation ◽

Cardiac Repair ◽

Gene Set Enrichment Analysis ◽

Receptor Interaction ◽

Modest Improvement

Mesenchymal stromal cells derived from bone marrow (BM-MSCs) have been considered promisingcandidates for regenerative medicine therapies for more than 20 years, but to date cardiovascular clinicaltrials of MSC-based therapies have demonstrated only modest improvement. However, the mechanismsunderlying why some patients improve and others do not remain undefined. Although MSC survival aftertransplantation remains a challenge, several studies have reported that BM-MSC-mediated paracrineeffects and delivery of exosomes may be therapeutically relevant. We hypothesized that MSC-derivedexosomes from patients with cardiac functional improvement contain factors that promote cell survival,proliferation, and cardiac protection. Here, we compared exosomes (derived from MSCs that had beenexpanded from BM-MNCs used for autologous transplantation) from two cohorts of disease- and age-matched consented patients with ischemic cardiomyopathy enrolled in the FOCUS-CCTRN trial—thosewith improved (cohort1, n=3) or worsened (cohort2, n=3) functional outcome in three primary endpoints:left ventricular (LV) ejection fraction, LV end-systolic volume, and maximal oxygen consumption.MicroRNA-seq analysis identified 16 upregulated and 12 downregulated miRNAs in cohort 1 whencompared to cohort 2. Using DIANA-Mir Path v.3.0, we identified 3,522 experimentally validated genetargets. Gene set enrichment analysis identified enrichment for fatty acid biosynthesis and metabolism,Hippo signaling pathway, protein processing in endoplasmic reticulum, adherent junction, cell cycle,endocytosis, RNA transport and degradation, estrogen-signaling pathway, regulation of actincytoskeleton, Prolactin signaling pathway, Focal adhesion, Notch-signaling pathway, p53-signalingpathway, insulin-signaling pathway and HIF-1-signaling pathway in the targets of upregulated miRNAs.The downregulated miRNAs were related to extracellular matrix-receptor interaction, protein processingin endoplasmic reticulum, cytokine-cytokine receptor interaction and antigen processing andpresentation. Thus, the identified miRNAs differentially expressed in patients with an improved functionaloutcome that may play a role in the molecular mechanisms that underlie in cardiac repair in patients withischemic cardiac disease. Moreover, these finding indicate that not all autologous cell products may becompetent therapy candidates for cardiac repair These results warrant further investigation to confirmthe effects of the identified differences on the target genes, which could improve the prognosis and unveilnew therapeutic approaches.

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Expression profile analysis of Differentially Expressed Genes in Human Coronary Artery Endothelial Cells Induced by Serum from Kawasaki Disease: A preliminary study

10.21203/rs.3.rs-54163/v1 ◽

2020 ◽

Author(s):

Xue Fan ◽

Meng Li ◽

Min Xiao ◽

Cong Liu ◽

Mingguo Xu

Keyword(s):

Endothelial Cells ◽

Coronary Artery ◽

Kawasaki Disease ◽

Signaling Pathway ◽

Differentially Expressed Genes ◽

Differentially Expressed ◽

Receptor Interaction ◽

Healthy Children ◽

Rna Seq ◽

Human Coronary Artery

Abstract Background: Kawasaki disease (KD) leads to coronary artery damage and the etiology of KD is unknown. The present study was designed to explore the differentially expressed genes (DEGs) in KD serum-induced human coronary artery endothelial cells (HCAECs) by RNA-sequence (RNA-seq). Methods: HCAECs were stimulated with serum (15% (v/v)), which were collected from 20 healthy children and 20 KD patients, for 24 hours. DEGs were then detected and analyzed by RNA-seq and bioinformatics analysis. Results: The expression of SMAD1, SMAD6, CD34, CXCL1, PITX2, and APLN was validated by qPCR. 102 genes, 59 up-regulated and 43 down-regulated genes, were significantly differentially expressed in KD groups. GO enrichment analysis showed that DEGs were enriched in cellular response to cytokines, cytokine-mediated signaling pathway, and regulation of immune cells migration and chemotaxis. KEGG signaling pathway analysis showed that DEGs were mainly involved in cytokine−cytokine receptor interaction, chemokine signaling pathway, and TGF−β signaling pathway. Besides, the mRNA expression levels of SMAD1, SMAD6, CD34, CXCL1, and APLN in the KD group were signiﬁcantly up-regulated compared with the normal group, whilePITX2 was significantly down-regulated. Conclusion: 102 DEGs in KD serum-induced HCAECs were identified, and six new targets were proposed as potential indicators of KD.

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Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

International Journal of Molecular Sciences ◽

10.3390/ijms20030458 ◽

2019 ◽

Vol 20 (3) ◽

pp. 458 ◽

Cited By ~ 9

Author(s):

Fernanda Ursoli Ferreira ◽

Lucas Eduardo Botelho Souza ◽

Carolina Hassibe Thomé ◽

Mariana Tomazini Pinto ◽

Clarice Origassa ◽

...

Keyword(s):

Endothelial Cells ◽

Signaling Pathway ◽

Molecular Mechanisms ◽

Transforming Growth Factor ◽

Umbilical Vein ◽

Transforming Growth Factor Β ◽

Selective Inhibition ◽

Mesenchymal Transition ◽

Best Response ◽

Endothelial To Mesenchymal Transition

The endothelial-to-mesenchymal transition (EndMT) is a biological process where endothelial cells (ECs) acquire a fibroblastic phenotype after concomitant loss of the apical-basal polarity and intercellular junction proteins. This process is critical to embryonic development and is involved in diseases such as fibrosis and tumor progression. The signaling pathway of the transforming growth factor β (TGF-β) is an important molecular route responsible for EndMT activation. However, it is unclear whether the anatomic location of endothelial cells influences the activation of molecular pathways responsible for EndMT induction. Our study investigated the molecular mechanisms and signaling pathways involved in EndMT induced by TGF-β2 in macrovascular ECs obtained from different sources. For this purpose, we used four types of endothelial cells (coronary artery endothelial cells, CAECs; primary aortic endothelial cells PAECs; human umbilical vein endothelia cells, HUVECs; and human pulmonary artery endothelial cells, HPAECs) and stimulated with 10 ng/mL of TGF-β2. We observed that among the ECs analyzed in this study, PAECs showed the best response to the TGF-β2 treatment, displaying phenotypic changes such as loss of endothelial marker and acquisition of mesenchymal markers, which are consistent with the EndMT activation. Moreover, the PAECs phenotypic transition was probably triggered by the extracellular signal–regulated kinases 1/2 (ERK1/2) signaling pathway activation. Therefore, the anatomical origin of ECs influences their ability to undergo EndMT and the selective inhibition of the ERK pathway may suppress or reverse the progression of diseases caused or aggravated by the involvement EndMT activation.

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miR-3188 Regulates Cell Proliferation, Apoptosis, and Migration in Breast Cancer by Targeting TUSC5 and Regulating the p38 MAPK Signaling Pathway

Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics ◽

10.3727/096504017x14953948675421 ◽

2018 ◽

Vol 26 (3) ◽

pp. 363-372 ◽

Cited By ~ 11

Author(s):

Xiaowen Chen ◽

Jianli Chen

Keyword(s):

Breast Cancer ◽

Cell Proliferation ◽

Signaling Pathway ◽

P38 Mapk ◽

Molecular Mechanisms ◽

Mapk Signaling ◽

Mapk Signaling Pathway ◽

Luciferase Reporter ◽

And Migration ◽

Mcf 7

This study intended to investigate the effects of miR-3188 on breast cancer and to reveal the possible molecular mechanisms. miR-3188 was upregulated and TUSC5 was downregulated in breast cancer tissues and MCF-7 cells compared to normal tissue and MCF-10 cells. After MCF-7 cells were transfected with miR-3188 inhibitor, cell proliferation and migration were inhibited, whereas apoptosis was promoted. Luciferase reporter assay suggested that TUSC5 was a target gene of miR-3188. In addition, miR-3188 overexpression increased the p-p38 expression, while miR-3188 suppression decreased the p-p38 expression significantly. miR-3188 regulated breast cancer progression via the p38 MAPK signaling pathway. In conclusion, miR-3188 affects breast cancer cell proliferation, apoptosis, and migration by targeting TUSC5 and activating the p38 MAPK signaling pathway. miR-3188 may serve as a potential therapeutic agent for the treatment of breast cancer.

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DEPTOR Deficiency-Mediated mTORc1 Hyperactivation in Vascular Endothelial Cells Promotes Angiogenesis

Cellular Physiology and Biochemistry ◽

10.1159/000488619 ◽

2018 ◽

Vol 46 (2) ◽

pp. 520-531 ◽

Cited By ~ 7

Author(s):

Yan Ding ◽

Lanlan Shan ◽

Wenqing Nai ◽

Xiaojun Lin ◽

Ling Zhou ◽

...

Keyword(s):

Endothelial Cells ◽

Signaling Pathway ◽

Molecular Mechanisms ◽

Gene Knockout ◽

Umbilical Vein ◽

Vascular Endothelial Cell ◽

Tube Formation ◽

Vascular Endothelial

Background/Aims: The mechanistic target of rapamycin (mTOR) signaling pathway is essential for angiogenesis and embryonic development. DEP domain-containing mTOR-interacting protein (DEPTOR) is an mTOR binding protein that functions to inhibit the mTOR pathway In vitro experiments suggest that DEPTOR is crucial for vascular endothelial cell (EC) activation and angiogenic responses. However, knowledge of the effects of DEPTOR on angiogenesis in vivo is limited. This study aimed to determine the role of DEPTOR in tissue angiogenesis and to elucidate the molecular mechanisms. Methods: Cre/loxP conditional gene knockout strategy was used to delete the Deptor gene in mouse vascular ECs. The expression or distribution of cluster of differentiation 31 (CD31), vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 alpha (HIF-1α) were detected by immunohistochemical staining or western blot. Tube formation assay was used to measure angiogenesis in vitro. Results: Deptor knockdown led to increased expression of CD31, VEGF and HIF-1α in heart, liver, kidney and aorta. After treatment with rapamycin, their expression was significantly down regulated. In vitro, human umbilical vein endothelial cells (HUVECs) were transfected with DEPTOR-specific small interfering RNA (siRNA), which resulted in a significant increase in endothelial tube formation and migration rates. In contrast, DEPTOR overexpression markedly reduced the expression of CD31, VEGF and HIF-1α. Conclusions: Our findings demonstrated that deletion of the Deptor gene in vascular ECs resulted in upregulated expression of CD31 and HIF-1α, and further stimulated the expression of VEGF which promoted angiogenesis, indicating that disruption of normal angiogenic pathways may occur through hyperactivation of the mTORC1/HIF-1α/VEGF signaling pathway.

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Network Pharmacology of Yougui Pill Combined with Buzhong Yiqi Decoction for the Treatment of Sexual Dysfunction

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2019/1243743 ◽

2019 ◽

Vol 2019 ◽

pp. 1-10 ◽

Cited By ~ 2

Author(s):

Yangyun Wang ◽

Wandong Yu ◽

Chaoliang Shi ◽

Wei Jiao ◽

Junhong Li ◽

...

Keyword(s):

Sexual Dysfunction ◽

Signaling Pathway ◽

Herbal Medicines ◽

Mapk Signaling ◽

Glycyrrhetinic Acid ◽

Network Pharmacology ◽

Receptor Interaction ◽

Chemical Components ◽

Target Proteins ◽

Yougui Pill

Purpose. We aimed to find the possible key targets of Yougui pill and Buzhong Yiqi decoction for the treatment of sexual dysfunction. Materials and Methods. The composition of Yougui pill combined with Buzhong Yiqi decoction was obtained, and its effective components of medicine were screened using ADME; the component target proteins were predicted and screened based on the TCMSP and BATMAN databases. Target proteins were cross-validated using the CTD database. We performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses for target proteins using the Cytoscape plugin ClueGO + CluePedia and the R package clusterProfiler, respectively. Subsequently, protein-protein interaction (PPI) analyses were conducted using the STRING database. Finally, a pharmacological network was constructed. Results. The pharmacological network contained 89 nodes and 176 relation pairs. Among these nodes, there were 12 for herbal medicines (orange peel, licorice, Eucommia, Aconite, Astragalus, Chinese wolfberry, yam, dodder seed, ginseng, Cornus officinalis, Rehmannia, and Angelica), 9 for chemical components (18-beta-glycyrrhetinic acid, carvacrol, glycyrrhetinic acid, higenamine, nobilin, quercetin, stigmasterol, synephrine, and thymol), 62 for target proteins (e.g., NR3C1, ESR1, PTGS2, CAT, TNF, INS, and TP53), and 6 for pathways (MAPK signaling pathway, proteoglycans in cancer, dopaminergic synapse, thyroid hormone signaling pathway, cAMP signaling pathway, and neuroactive ligand-receptor interaction). Conclusion. NR3C1, ESR1, PTGS2, CAT, TNF, INS, and TP53 may be important targets for the key active elements in the decoction combining Yougui pill and Buzhong Yiqi. Furthermore, these target proteins are relevant to the treatment of sexual dysfunction, probably via pathways associated with cancer and signal transduction.

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Small Molecule MIRA-1 Induces p53-Independent Apoptosis in Multiple Myeloma Cells Through Activation of the p38 MAPK Signaling Pathway.

Blood ◽

10.1182/blood.v120.21.2937.2937 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2937-2937

Author(s):

Manujendra N Saha ◽

Hua Jiang ◽

Yijun Yang ◽

Donna Reece ◽

Hong Chang

Keyword(s):

Signaling Pathway ◽

Research Funding ◽

Molecular Mechanisms ◽

Mapk Signaling ◽

Mapk Signaling Pathway ◽

Mutant P53 ◽

Wild Type ◽

Apoptotic Protein ◽

Induced Apoptosis ◽

Dose Dependent

Abstract Abstract 2937 Mutation of p53, a tumor suppressor protein, is relatively rare (∼10% in newly diagnosed patients) in multiple myeloma (MM). However, p53 mutations/deletions are important risk factors for predicting the resistant to chemotherapy and no treatment is currently available for this subgroup of patients. MIRA-1, a novel class of small molecules with the ability to restore wild type conformation and function to mutant p53, induces apoptosis in different types of solid tumors harboring mutant p53. However, its effect on MM cells is not known. In this study we examined the ability of MIRA-1 to induce cytotoxic and apoptotic response in MM cells and inhibit tumor growth in MM mouse xenograft model. In addition, we explored the molecular mechanisms of MIRA-1-induced apoptosis in MM cells. Treatment of MM cells with MIRA-1 resulted in a time- and dose-dependent decrease in survival and increase in apoptosis of MM cells harboring either wild type (MM.1S, H929) or mutant (U266, 8226, and LP1) p53 suggesting that MIRA-induced apoptosis in MM cells is independent of p53 status. The IC50 of MIRA-1 observed in these cells was ranged between 10 and 15 μM. In addition, MIRA-1 elicited a dose-dependent inhibition of myeloma cell growth in seven primary MM samples with an average IC50of 10 μM. Two of the seven patient samples harbors p53 mutations/deletions. In contrast, MIRA-1 did not have a significant inhibitory effect on the survival of bone marrow or peripheral blood mononuclear cells obtained from three healthy donors at the concentrations (10–20 μM) that induced apoptosis of MM cells, indicating a preferential killing of myeloma cells by this drug. Apoptosis induced by MIRA-1 in MM cells harbouring either wild type or mutant p53 was associated with time- and dose-dependent activation of caspas-8, caspase-3 and PARP with subsequent up-regulation of a pro-apoptotic protein, Noxa and down-regulation of an anti-apoptotic protein, Mcl-1. Interestingly, MIRA-1 did not significantly modulate the level of p53 expression, although immunoprecipitation studies confirmed the restoration of wild type conformation of mutant p53 in LP1 and 8226 cells. Importantly, genetic knockdown of p53 using siRNA against wild type or mutant p53 had only a little effect on apoptosis induction by MIRA-1 in MM.1S or LP1 cells, respectively, confirming that apoptosis induction by MIRA-1 in MM cells is independent of p53. Furthermore, the combination of MIRA-1 with current anti-myeloma agents, dexamethasone or doxorubicin displayed synergistic cytotoxic response in MM.1S or LP1 cells (CI<1; p<0.05). To delineate the molecular mechanisms of apoptosis in MM cells induced by MIRA-1, we performed RT2 profiler PCR array analysis for the differential expression of 84 genes related to mitogen activated protein kinase (MAPK) signaling pathway. A significant number of genes of the MAPK family including MAP3K: MAP3K2 (MEKK2), MAP3K4 (MEKK4), PAK1; MAP2K: MAP2K5 (MEK5); and MAPK: MAPK11 (p38bMAPK) as well as transcription factors such as c-Jun, c-FOS, EGR1, and MKNK1, whose expression is induced by MAPK signaling, were up-regulated by more than 2-fold in MIRA-1-treated 8226 cells. On the other hand, expression of the scaffolding/anchoring genes, MAPK8IP2 (JIP-1) was down-regulated by ∼2-fold. Up-regulations of c-Jun, c-Fos, and EGR1 at their protein levels were further confirmed by Western blot analysis of MM.1S and 8226 cells treated with MIRA-1. Importantly, Western blot analysis revealed that treatment of MIRA-1 resulted in a time- and dose-dependent increase of phosphorylated p38 MAPK level in both MM.1S and 8226 cells. Taken together, our data indicates that activation of the MAPK signaling pathway is, at least in part, associated with MIRA-1-induced apoptosis of MM cells. Finally, we evaluated anti-tumorigenic potential of MIRA-1 in MM xenograft SCID mouse models. 8266 cells were inoculated into SCID mice and the mice received i.p. injections of either 100 μL PBS (control) or 10 mg/kg MIRA-1 once daily for 18 days after tumor formation was evident. Administration of MIRA-1 resulted in significant inhibition of tumor growth (p<0.05) and increase in survival (p=0.007) of the mice with no apparent toxicity. Our study for the first time demonstrates potent in vitro and in vivo anti-myeloma activity of MIRA-1 and thus providing a framework for clinical evaluation of MIRA-1 either alone or in combination with current anti-myeloma agents. Disclosures: Reece: Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Merck: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Millennium Pharmaceuticals: Research Funding.

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