scholarly journals MiR-221/222 Promote Dexamethasone Resistance of Multiple Myeloma through Inhibition of Autophagy By Targeting ATG12

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4469-4469
Author(s):  
Jian Xu ◽  
Yan Su ◽  
Aoshuang Xu ◽  
Fengjuan Fan ◽  
Haifan Huang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Conflicts Of Interest ◽  
Combined Treatment ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Expression Levels

Abstract Dexamethasone (Dex) is the most widely used chemotherapeutic drug in the treatment of multiple myeloma (MM). Inherent or acquired resistance to Dex is broadly associated with poor prognosis in MM. Many microRNAs are aberrantly expressed in MM, including miR-221/222, which have been reported to act as oncogenes in many cancer types. Recently, accumulating evidence has shown that miR-221/222 are involved in the development of chemoresistance in a variety of cancers. However, there is still a lack of valuable data regarding the role of miR-221/222 in the chemoresistance of MM. Here, we first evaluated the expression levels of miR-221/222 in plasma cells (PCs) from MM patients by qRT-PCR analysis. The results showed that miR-221/222 were markedly upregulated in PCs from newly diagnosed MM patients compared to healthy donors, and they were further upregulated in PCs from patients with relapsed MM. In addition, we found that the expression levels of miR-221/222 were inversely correlated with Dex-sensitivity of human MM cell lines (HMCLs). Importantly, enforced expression of miR-221/222 dramatically reduced the sensitivity of Dex-sensitive HMCLs to Dex, while inhibition of miR-221/222 re-sensitized Dex-resistant HMCLs to Dex. Previous studies have shown that Dex-induced cell death in lymphoid leukemia is mediated through initiation of autophagy. To study whether autophagy was involved in Dex-induced cell death in MM cells, HMCLs were exposed to Dex, and then autophagy in these cells was evaluated by the transmission electron microscopy and western blot analysis. The results showed that Dex induced the occurrence of autophagy in Dex-sensitive HMCLs, but not in Dex-resistant HMCLs. Moreover, pharmacological inhibitors of autophagy could significantly reduce Dex-induced cell death in Dex-sensitive HMCLs. These results reveal that autophagy is critical for the induction of cell death following Dex treatment in MM. MicroRNAs have been reported to play an important role in regulating autophagy. We therefore examined whether miR-221/222 can regulate autophagy in MM cells. Low miR-221/222 expressing MM.1S (Dex-sensitive) or high miR-221/222 expressing MM.1R (Dex-resistant) cells were transfected with agomir-221/222 or antagomir-221/222, respectively, and then the level of autophagy was evaluated. The results showed that overexpression of miR-221/222 reduced the level of autophagy in MM.1S cells, while inhibition of miR-221/222 elevated the level of autophagy in MM.1R cells. Using microRNA target prediction bioinformatics tools and dual-luciferase reporter assay, we confirmed that autophagy-related gene 12 (ATG12) was a novel target gene of miR-221/222. Indeed, miR-221/222 could negatively regulate the expression of ATG12 at both the mRNA and protein levels in MM cells. In addition, knockdown of ATG12 by siRNA markedly reduced the autophagy-inducing and Dex-sensitizing activity of miR-221/222 antagomirs in MM.1R cells. Of note, in MM.1S cells, Dex treatment could further decreased the expression of miR-221/222, accompanied by upregulated expression of ATG12, whereas silencing the expression of ATG12 could significantly inhibited Dex-induced autophagy and cell death. Thus, these results suggest that ATG12 is a key player in miR-221/222-mediated autophagy inhibition and Dex-resistance. Next, we evaluated whether miR-221/222 could regulate autophagy and Dex-sensitivity of MM cells invivo. NOD/SCID mice were subcutaneously injected with MM.1R cells to establish Dex-resistant MM xenografts. Combined treatment with antagomir-221/222 plus Dex showed a remarkable reduction of tumor size compared to antagomir-221/222 or Dex alone (397.6±55.08 mm3 VS 895.8±72.44 mm3 VS 987.3±68.49 mm3). Immunohistochemistry and western blot analysis of the retrieved xenografted tumors showed that combination treatment with antagomir-221/222 plus Dex induced upregulation of ATG12, as well as extended autophagy with increased p62 degradation and Beclin-1 expression. In conclusion, our data reveal that upregulation of miR-221/222 promotes Dex resistance of MM cells through inhibition of autophagy by targeting ATG12. Therefore, miR-221/222-ATG12 autophagy-regulatory axis may potentially be applied in glucocorticoid resistance prediction and treatment. Disclosures No relevant conflicts of interest to declare.

Pterostilbene: A Novel Histone Deacetylase 1 Inhibitor (HDACi1) Demonstrating Efficacy in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3844-3844
Author(s):  
Haiming Chen ◽  
Rui Hao ◽  
Eric Sanchez ◽  
Jing Shen ◽  
Mingjie Li ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Death ◽  
Western Blot ◽  
Histone Acetylation ◽  
Tumor Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Anti Cancer ◽  
Mts Assay

Abstract Abstract 3844 Poster Board III-780 Pterostilbene (3,5-Dimethoxy-4'-hydroxy-trans-stilbene) is a key compound found predominantly in blueberries and grapes. Pterostilbene has been shown to exhibit potential anti-cancer characteristics, anti-hypercholesterolemia and anti-hypertriglyceridemia properties. However, the mechanism of anti-cancer effects has not been elucidated and this compound has not been previously evaluated in multiple myeloma (MM). In this study, we first examined the HDAC binding ability of pterostilbene in the RPMI8226 multiple myeloma (MM) cell line and 293 HEK cells by using a novel HDAC screening assay. Briefly, RPMI8226 cells or 293 HEK cells were lysed with M-PER supplemented with protease and phosphatase inhibitors. The lysates were diluted and incubated with pterostilbene in concentrations ranging from 1 to 200 μM or without drug. Proteolysis was performed using thermolysin and aliquots were removed every 5 minutes and western blot analysis completed using antibodies against different HDACs or GAPDH. The results showed that pterostilbene prevents specifically HDAC1 digestion by thermolysin in both RPMI8226 and 293 cells. Next, we examined H4 acetylation of lysine residues in RPMI8226 cells following treatment with pterostilbene using western blot analysis. Increases in histone acetylation in RPMI8226 cells following exposure to pterostilbene treatment occurred in a concentration-dependent manner. Specifically, 1 to10μM of the pterostilbene markedly induced histone acetylation in RPMI8226 cells within 24 hrs. We also analyzed pterostilbene's effect on MM tumor cell proliferation using the MTS assay and apoptosis with flow cytometric analysis following Annexin V staining, in cells from the RPMI8226 and MM1S MM cell lines and freshly obtained tumor cells from MM patients. Pterostilbene alone showed a 50% growth inhibition (IC50) of cells from the RPMI8226 and MM1S lines as well as fresh bone marrow tumor cells from MM patients treated for 48 hours at approximately the same concentration (5 μM) as showing anti-MM effects in the MTS assay. Notably, the combination of pterostilbene and melphalan or the proteasome inhibitor bortezomib showed markedly increased inhibition of MM cell growth compared to treatment of the cells with the drugs alone. Since this effect may have resulted from decreased cell proliferation due to cell cycle arrest or increased cell death, we further determined apoptosis in cells from RPMI8226, U266, and MM1S cell lines as well as fresh tumor cells from MM patients following treatment with pterostilbene. Our data demonstrated that pterostilbene induced apoptotic cell death in a concentration dependent fashion. We also evaluated the combination of this compound with bortezomib (1-6 nM) and showed marked increases in apoptosis using this combination compared to either drug alone. These studies establish pterostilbene as a novel HDACi with potent anti-MM activity alone and also show its ability to enhance the anti-MM effects of chemotherapeutic agents and bortezomib. Currently, we are evaluating pterostilbene alone and in combination treatments using our SCID-hu murine models of human MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Expression and subcellular localization of BNIP3 in hypoxic hepatocytes and liver stress

2009 ◽  
Vol 296 (3) ◽  
pp. G499-G509 ◽  
Author(s):  
Mallikarjuna R. Metukuri ◽  
Donna Beer-Stolz ◽  
Rajaie A. Namas ◽  
Rajeev Dhupar ◽  
Andres Torres ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Ischemia Reperfusion ◽  
Redox Stress ◽  
No Donor ◽  
Interacting Protein

We have previously demonstrated that the Bcl-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bcl-2 family, is upregulated in vitro and in vivo in both experimental and clinical settings of redox stress and that nitric oxide (NO) downregulates its expression. In this study we sought to examine the expression and localization of BNIP3 in murine hepatocytes and in a murine model of hemorrhagic shock (HS) and ischemia-reperfusion (I/R). Freshly isolated mouse hepatocytes were exposed to 1% hypoxia for 6 h followed by reoxygenation for 18 h, and protein was isolated for Western blot analysis. Hepatocytes grown on coverslips were fixed for localization studies. Similarly, livers from surgically cannulated C57Bl/6 mice and from mice cannulated and subjected to 1–4 h of HS were processed for protein isolation and Western blot analysis. In hepatocytes, BNIP3 was expressed constitutively but was upregulated under hypoxic conditions, and this upregulation was countered by treatment with a NO donor. Surprisingly, BNIP3 was localized in the nucleus of normoxic hepatocytes, in the cytoplasm following hypoxia, and again in the nucleus following reoxygenation. Upregulation of BNIP3 partially required p38 MAPK activation. BNIP3 contributed to hypoxic injury in hepatocytes, since this injury was diminished by knockdown of BNIP3 mRNA. Hepatic BNIP3 was also upregulated in two different models of liver stress in vivo, suggesting that a multitude of inflammatory stresses can lead to the modulation of BNIP3. In turn, the upregulation of BNIP3 appears to be one mechanism of hepatocyte cell death and liver damage in these settings.

Pan-BCL-2 Family Small Molecule Inhibitor GX015-070 (GeminX, Inc.) Exhibits Single Agent Cytotoxicity, Is Synergistic with Select Anti-Leukemia Cytotoxic Agents, and Induces Apoptosis in Cell Lines with t(4;11)(q21;q23) Translocations.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3368-3368 ◽  
Author(s):  
Jessicca M. Rege ◽  
Blaine W. Robinson ◽  
Manish Gupta ◽  
Jeffrey S. Barrett ◽  
Peter C. Adamson ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Family Member ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Single Agent ◽  
Cytotoxic Agents ◽  
Response Surface Modeling

Abstract Background: Leukemias with MLL translocations, especially t(4;11), often are resistant to common chemotherapeutic agents, which may be due to abnormal apoptosis regulation. Pro- and anti-apoptotic BCL-2 family member interactions govern initiation of the intrinsic apoptosis pathway. GX015-070, which currently is in Phase I/IIA clinical trials, mimics the BH3 domain on pro-apoptotic BCL-2 family proteins and can bind the BH3 binding pocket of anti-apoptotic BCL-2 family members and modulate apoptosis. We performed comprehensive protein expression profiling of BCL-2 family member proteins and evaluated in vitro activity and mechanism of action of GX015-070 in cell lines with t(4;11). Methods: Baseline expression of BCL-2 family proteins was determined by Western blot analysis. Cytotoxicity was assessed by MTT after a 3 day exposure of RS4:11, SEM-K2 and MV4-11 cells in log phase growth to single agent GX015-070 at concentrations from 5 nM to 7.5 μM. Combined effects of fixed-concentration GX015-070 with cytotoxic agents over a range of concentrations were assayed by MTT, and the results were analyzed by pharmacostatistical response surface modeling. Disruption of specific pro- and anti-apoptotic BCL-2 family member interactions was investigated by co-immunoprecipitation/Western blot analysis. Flow cytometry and/or Western blot analysis of Caspase-3 activation, and a FACS TUNEL assay, were used to assess apoptosis in GX015-070 treated and untreated cells. Results: The three cell lines had similar baseline levels of expression of BCL-2 family proteins. BCL-2 and BAX were most abundant followed by PUMA, BAK, BCL-XL, BIM-EL, MCL-1, BIK and NOXA. Results of assays of GX015-070 activity and mechanism of action are in shown in the table. Conclusions: These data indicate that GX015-070 has potent cytotoxic activity in cell lines with t(4;11) as a single agent and that the cytotoxicity results from apoptosis. Response surface modeling in RS4:11 cells suggested ability to achieve effective doses with GX015-070 combined with cytosine arabinoside (Ara-C), dexamethasone (Dex) or doxorubicin (ADR) that are lower than projected from the single agents, but synergy was not suggested when GX015-070 was combined with etoposide, methotrexate or 6-thioguanine. The co-IP experiments give proof of principle that GX015-070 disrupts pro- and anti-apoptotic BCL-2 family protein interactions in cell lines with t(4;11). Additional pre-clinical experiments directed at overcoming drug resistance from abnormal cell death regulation in leukemias with t(4;11) using GX015-070 are in progress. These studies provide a framework to understand the cell death/survival machinery in primary leukemias with t(4;11) translocations more completely and manipulate that machinery to achieve better treatments. GX015-070 Activity and Mechanism Cell Line Single Agent Activity Synergy Inhibition Caspase-3 Activation TUNEL RS4:11 IC50=43.5 nM Ara-C, Dex, ADR Mcl1:Bak; Bcl2:Bak + + SEM-K2 IC50=156 nM In progress Mcl1:Bak; Bcl2:Bak + In Progress MV4-11 IC50=123 nM In progress Mcl1:Bak In progress +

Nutlin-3A, an MDM2 Antagonist, Induces p53-Dependent Cell Cycle Arrest and Apoptosis in Hodgkin Lymphoma (HL).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2506-2506
Author(s):  
Elias Drakos ◽  
Athanasios Thomaides ◽  
Jiang Li ◽  
Marina Konopleva ◽  
L. Jeffrey Medeiros ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Death ◽  
Western Blot ◽  
Cell Cycle Arrest ◽  
Small Molecule ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
P53 Gene ◽  
Cycle Arrest

Abstract p53 is the most frequently mutated tumor suppressor gene in human cancer. However, in Hodgkin lymphoma (HL) p53 is mutated only in a small subset of cases suggesting that modulation of wild-type-p53 (wt-p53) levels in Hodgkin and Reed-Sternberg (HRS) cells may have therapeutic implications in these patients. MDM2 (HDM2 in humans) is a physiologic negative regulator of p53 levels through a well-established auto-regulatory feedback loop. Nutlin-3A is a recently developed small molecule, which antagonizes mdm2 through disruption of p53-MDM2 interaction resulting in p53 stabilization. We hypothesized that nutlin 3A may stabilize p53 in HRS cells carrying wt-p53 gene, thus leading to p53-dependent apoptosis and G1-S cell cycle arrest. We used two novel classical HL cell lines recently established in our Institution, MDA-V and MDA-E, which have been shown to carry wt-p53 gene. As a control, we used a HL cell line L-428 harboring a mutant p53 (mt-p53) gene product (deletion at exon 4). We investigated effects on apoptosis and cell cycle arrest after treatment of cultured HRS cells with nutlin-3A or a 150-fold less active enantiomere, nutlin-3B. Treatment with nutlin-3A resulted in substantial cell death (up to 65%) in a concentration-dependent manner associated with increased apoptosis as shown by apoptotic morphology (DAPI immunofluorescence), annexin V binding (flow cytometry) and caspase activation (Western blot analysis) in MDA-V and MDA-E cells, but not in L-428 cells. Nutlin-3A-induced apoptotic cell death was accompanied by stabilization of p53 protein as detected by western blot analysis and immunofluorescence and up-regulation of pro-apoptotic Bax, a known target of p53. Inhibition of nuclear export by leptomycin B stabilized p53 at a similar level as compared to nutlin-3A treatment in these cells, suggesting that nutlin-3A stabilized p53 through inhibition of MDM2-mediated degradation of the protein. By contrast, no changes in cell viability, growth or apoptosis were seen after treatment with the inactive nutlin-3B small molecule. Treatment with nutlin-3A also resulted in a significant decrease (up to 85%) of cells in S-phase and a dose-dependent increase of cells in G1 phase of cell cycle as detected by flow cytometry, in MDA-V and MDA-E cells, but not in L-428 cells. Cell cycle arrest was associated with up-regulation of the cyclin-dependent kinase inhibitor p21, a transcriptional target of p53. In contrast, treatment of HRS cells with nutlin-3B had no effects on the cell cycle irrespective of p53 mutation status. Furthermore, combined treatment with nutlin-3A and doxorubicin revealed synergistic effects and enhanced cytotoxicity in HRS cells with wt-p53 gene. Targeting MDM2 with the specific antagonist nutlin-3A that leads to non-genotoxic p53 activation, apoptosis induction and cell cycle inhibition may provide a new therapeutic approach for patients with HL.

HDAC and LSD1 Inhibitors Synergize to Induce Cell Death in Acute Leukemia Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1427-1427 ◽  
Author(s):  
Lorimar Ramirez ◽  
Melissa Singh ◽  
Joya Chandra
Keyword(s):  
Cell Death ◽  
Monoamine Oxidase ◽  
Acute Leukemia ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Hdac Inhibitors ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Leukemia Cell Lines

Abstract Abstract 1427 Histone deacetylase inhibitors (HDACi) are a class of emerging epigenetic therapies which are being used to treat cancer. Two HDACi (vorinostat and romidepsin) are FDA approved for cutaneous T-cell lymphoma. HDACi have been employed in clinical trials for acute leukemia, but single agent activity has been limited. Improved efficacy is observed when combined with other anticancer agents. In the current study we addressed acute leukemia models using vorinostat, a pan-HDACi that inhibits HDAC class I, II, and IV and entinostat, a newer HDACi that inhibits HDAC class I more specifically. These HDACi were combined with inhibition of another histone modifying enzyme: lysine specific demethylase 1 (LSD1). The LSD1 gene encodes a favin-dependent monoamine oxidase, which demethylates mono- and di-methylated lysines, specifically lysines 4 and 9 on histone 3 (H3K4 and H3K9), thus it is also involved in gene regulation through post-translational histone modification. LSD1 overexpression has been linked to human carcinogenesis in bladder carcinomas, lung cancer, and poorly differentiated neuroblastoma. However, it has not been studied in hematologic malignancies. Because LSD1 is structurally similar to monoamine oxidase (MAO), it has been shown that nonselective MAO inhibitors also inhibit LSD1. Here we employed tranylcypromine, a monoamine oxidase inhibitor (MAOi), as an irreversible LSD1 inhibitor. Recently published work from our laboratory has shown synergistic effects of combined HDAC and LSD1 inhibition in brain tumors (glioblastoma multiforme). Similar results have been published in breast cancer cells, but no work has been done in hematological malignancies. The objective of this study was to investigate the possible synergy of HDAC and LSD1 inhibitors in acute leukemia cells. LSD1 protein expression in several leukemia cells lines was analyzed by Western blot analysis. LSD1 was expressed in all leukemia cell lines tested, which included T-cell ALL (Jurkat, Sub-T1, MOLT4), B-cell ALL (JM-1,697), and Philadelphia chromosome positive ALL (Z33, Z119, Z181). To determine whether synergy exists between HDACi and LSD1 inhibitors, Jurkat cells were exposed to different concentrations of tranylcypromine and vorinostat or entinostat. After 24 hr, DNA fragmentation was assessed by propidium iodide (PI) staining followed by flow cytometric analysis. A combination index (CI) less than 1.0 is representative of synergism as measured by Calcusyn software. Results showed a synergistic effect on DNA fragmentation when combining the 2.5 μM dose of vorinostat with a range of tranylcypromine doses (1 mM CI= 0.78, 1.5 mM CI= 0.49, and 2 mM CI= 0.39). The same effect was observed with the combination of 2.5 μM entinostat with 2 mM tranylcypromine (CI=0.52). Viability studies performed with the same drug concentrations in conbination also showed statistically significant cell death. Additional acute leukemia cell lines, 697 and MOLT-4, also demonstrated significantly increased cell death with the combination relative to treatment with either agent alone. Since these agents inhibit histone deacetylation and lysine demethylation, we tested whether these histone modifications were promoted by combination treatment. Jurkat cell lysates were generated by acid extraction of histones and Western blot analysis was conducted. We demonstrated that in fact histone acetylation was increased with combination treatment, indicating that these modifications coordinately regulate each other in acute leukemia cells. A molecular target for LSD1 is p53, a tumor suppressor protein whose activity is regulated by lysine methylation and demethylation. Western blot analysis showed that p53 is downregulated in leukemia cells after exposure to the combination of HDAC and LSD1 inhibitors. Future studies will address if p53 downregulation is a trigger for the synergistic cell death. Taken together, our data shows the efficacy of combining LSD1 inhibitors with HDAC inhibitors in multiple acute leukemia models. Since tranylcypromine is also a FDA-approved agent, these results urge the design of a feasible and effective clinical trial combining LSD1 and HDAC inhibitors for acute leukemia. Disclosures: No relevant conflicts of interest to declare.

Detection and Quantification of Cereblon Protein and mRNA in Multiple Myeloma Cell Lines and Primary CD138+multiple Myeloma Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4043-4043
Author(s):  
Anita K Gandhi ◽  
Herve Avet-Loiseau ◽  
Michelle Waldman ◽  
Anjan Thakurta ◽  
Sharon L Aukerman ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Protein Expression ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Full Length ◽  
Employment Equity ◽  
Equity Ownership ◽  
Protein Variants

Abstract Abstract 4043 Background: Cereblon (CRBN), a component of the DDB1-CUL4A-Roc1 ubiquitin ligase complex, has been identified as a target of the immunomodulatory agents thalidomide, lenalidomide, and pomalidomide (Lopez-Girona et al. Leukemia. 2012; Zhu et al. Blood. 2011; Ito et al. Science. 2010.). CRBN binding by these agents mediates their anti-proliferative effects in multiple myeloma (MM) cells (Lopez-Girona et al. Leukemia. 2012; Zhu et al. Blood. 2011). However, the role of CRBN quantification as a marker for disease responsiveness or resistance to these drugs remains to be fully defined. Furthermore, it is unclear whether measuring mRNA or protein expression is the best approach for development of a quantitative CRBN expression assay. In order to define the optimal assay approach, we have studied CRBN mRNA and protein expression in MM cell lines (n=20) and MM patient samples. Methods: CRBN isoform mapping was undertaken using a nested PCR approach and Sanger sequencing. Commercially available and newly generated rabbit anti-CRBN antibodies were characterized with recombinant human CRBN protein and MM cell line extracts via western blot analysis. Results: Our data show that in addition to the transcript for full length protein (GenBank Accession NM_016302.3), in MM cells there are at least 6 alternatively spliced isoforms of CRBN as depicted in Figure 1. Five of the 6 CRBN isoforms (CRBN-003, -004, -005, -006, and -007) contain novel splice junctions not previously described. In addition, 3 of the identified transcripts (CRBN-002, -003, and -005) contain in-frame ORFs, suggesting they encode variants of CRBN protein. Of note, exon 10, which contains a portion of the IMiD-binding domain, is not present in CRBN-002. The functional consequence of CRBN-002 remains to be elucidated, but may be a marker of drug resistance. In order to measure CRBN protein levels, we developed and characterized three rabbit monoclonal antibodies to CRBN including antibody CRBN65, which has the potential to discriminate between the different CRBN protein products, including CRBN-002 by western blot analysis. Additionally, we compared 8 commercially available CRBN antibodies. Western blot analysis of cell lines with commercial and newly developed antibodies identified full length protein at 51 kD. Most commercial antibodies also identified multiple bands of other sizes which may represent CRBN protein variants; however, many are likely non-specific bands as they are larger than full-length CRBN. Conclusion: We have identified novel splice variants of CRBN from MM cell lines and primary tumor samples. The structure of the isoforms and their potential ability to be translated into several protein variants of CRBN reflect the complex regulation of the CRBN gene. These data suggest that accurate quantification of CRBN mRNA level in clinical studies may require measurement of both full-length CRBN mRNA as well as other mRNA isoforms. Currently available primers and gene expression arrays are not capable of identifying and/or resolving the complex set of CRBN isoforms present in cells. These data also demonstrate that CRBN65 is a highly specific and sensitive antibody that could be used for detection of CRBN and its key variants. Taken together, our data emphasize the importance for developing standardized reagents and assays for both mRNA and protein level measurement of CRBN before using them as markers for clinical response or resistance. Disclosures: Gandhi: Celgene Corp: Employment, Equity Ownership. Waldman:Celgene Corp: Employment, Equity Ownership. Thakurta:Celgene Corp: Employment, Equity Ownership. Aukerman:Celgene Corp: Employment, Equity Ownership. Chen:Celgene Corp: Employment, Equity Ownership. Mendy:Celgene Corp.: Employment, Equity Ownership. Rychak:Celgene Corp: Employment, Equity Ownership. Miller:Celgene Corp: Employment, Equity Ownership. Gaidarova:Celgene Corp: Employment, Equity Ownership. Gonzales:Celgene Corp: Employment, Equity Ownership. Cathers:Celgene Corp: Employment, Equity Ownership. Schafer:Celgene: Employment, Equity Ownership. Daniel:Celgene Corporation: Employment. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership.

Rictor Overexpression and mTORC2 Signaling in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3884-3884 ◽  
Author(s):  
Gwen Jordaan ◽  
Wei Liao ◽  
Joe Gera ◽  
Sanjai Sharma
Keyword(s):  
Western Blot ◽  
Mantle Cell Lymphoma ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Parp Cleavage ◽  
Mantle Cell ◽  
Bcr Signaling ◽  
Mtorc2 Complex

Abstract Abstract 3884 Signaling via the B-cell receptor (BCR) stimulates growth and survival of CLL leukemic cells and inhibits apoptosis by phosphorylating immunoreceptor tyrosine based activation motifs. This signaling subsequently activates PI3 Kinase/AKT, mTOR, ERK and other pathways. Activation of Akt in turn requires phosphorylation by mTOR kinase, which assembles in two complexes mTORC1 and mTORC2 and it is the mTORC2 complex that phosphorylates and activates Akt. This phosphorylation of Akt in CLL specimen's upregulates anti-apoptotic proteins such as Mcl-1, Bcl-xl and XIAP. We have identified that Rictor, a component of mTORC2 complex is over-expressed in CLL specimens as compared to normal peripheral mononuclear B cells. This over-expression was noted by real time PCR that showed 1.5 to 4 fold upregulation (n=12). Western blot analysis also showed Rictor overexpression in all the twelve CLL specimens tested. Rictor overexpression was also seen in Mantle cell lymphoma cell lines and to study its role in BCR signaling, stable Mantle cell lymphoma lines with SiRNA mediated Rictor knockdown were established. Rictor knockdown resulted in a significant decrease in Akt activation as phosphorylation (phospho S473) of Akt both in unstimulated cells and when the cells were stimulated with BCR crosslinking was decreased. To determine the effect of Rictor and mTORC2 inhibition on CLL specimens, we tested the activity of three compounds isolated via yeast two hybrid drug screen designed to identify molecules that inhibit Rictor/mTOR interaction. When tested on CLL specimens in the presence of BCR crosslinking, these mTORC2 inhibitor compounds inhibited the downstream phosphorylation of Akt S473. Functionally the inhibitors also induced apoptosis in CLL cells with 40–60% of CLL cells undergoing apoptosis (1.0mM, cells treated for 48 hours). In comparison, rapamycin (mTORC1 inhibitor) and ppp242 (mTORC1, 2 inhibitor) were comparatively less active in CLL specimens as they were less effective in the induction of apoptosis. Western blot analysis of mTORC2 inhibitor treated cells also showed PARP cleavage and an increase in the pro-apoptotic protein BAD. Our data indicates that Rictor overexpression in CLL specimens is required for Akt phosphorylation activation and downstream BCR signaling. Inhibition of this pathway by mTORC2 inhibitors in CLL will be an effective therapeutic strategy. Disclosures: No relevant conflicts of interest to declare.

C-jun N-terminal kinase dependent autophagic cell death in cancer cells induced by Zanthoxylum fruit extract from Japanese pepper.

2017 ◽  
Vol 35 (4_suppl) ◽  
pp. 653-653
Author(s):  
Toru Kono ◽  
Reo Nozaki ◽  
Hiroki Bochimoto ◽  
Tsuyoshi Watanabe ◽  
Kaori Oketani ◽  
...  
Keyword(s):  
Cell Death ◽  
Drug Development ◽  
Western Blot ◽  
Anticancer Activity ◽  
Cancer Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Human Cancer ◽  
Fruit Extract ◽  
Cytoplasmic Vacuolization

653 Background: Natural products constitute a promising resource for drug development including an anticancer drug. Zanthoxylum fruit, obtained from the Japanese pepper plant (Zanthoxylum piperitum De Candolle), and its extract (Zanthoxylum fruit extract, ZFE) is an important component of Daikenchuto, which is a form of Japanese traditional medicine. Recently, we have reported that Daikenchuto has an anticancer activity in vivo, however precise mechanism is still unclear. Therefore, we investigated the potential anticancer activity of ZFE as an inducer of autophagic cell death (ACD). Methods: ZFE powder was provided by Tsumura (Japan). We investigated the effect of ZFE on the morphology of six types of human cancer cells and normal cells by using phase contrast microscopy and electron microscopy. Knockdown of autophagy-related gene 5 (ATG5), which is an essential gene for autophagy, by transfecting small interfering RNA was performed and confirmed by quantitative RT-qPCR and Western blot analysis. Effect of bafilomycin A1 (Baf A1), an inhibitor of vacuolar type H+-ATPases, on the anticancer activity of ZFE was investigated. Western blot analysis revealed LC3-II levels, a marker of autophagy. Results: ZFE caused remarkable autophagy-like cytoplasmic vacuolization with the inhibition of cell proliferation and subsequent induction of cell death in human cancer cell lines, DLD-1, HepG2 and Caco-2 cells but not in A549, MCF-7 or WiDr cells. ZFE increased LC3-II protein levels. Suppression of an ATG5 using siRNA inhibited ZFE-induced cytoplasmic vacuolization and cell death. Moreover, ZFE increased the phosphorylation of c-jun N-terminal kinase (JNK) in cancer cells which can be induced cell death by ZFE and JNK inhibitor SP600125 attenuated both vacuolization and cell death induced by ZFE. Instead, ZFE-induced cell death was neither apoptosis nor necrosis according to the morphological perspective and the marker of apoptosis or necrosis. And normal intestinal cell was not affected by ZFE. Conclusions: ZFE induces JNK-dependent ACD, which appears to be the main mechanism underlying its anticancer activity, suggesting a promising starting point for anticancer drug development.

Overcoming resistance to antiandrogens with a TGF-β RI inhibitor in preclinical mouse model of PCa.

2018 ◽  
Vol 36 (6_suppl) ◽  
pp. 288-288
Author(s):  
Channing Judith Paller ◽  
Hong Pu ◽  
Diane Begemann ◽  
Mary Nakazawa ◽  
Natasha Kyprianou
Keyword(s):  
Cell Proliferation ◽  
Mouse Model ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Epithelial Mesenchymal Transition ◽  
Combined Treatment ◽  
Prostate Tumor ◽  
Metastatic Progression ◽  
Mesenchymal Transition

288 Background: Epithelial-mesenchymal transition (EMT) is a significant contributor to PCa metastatic progression and therapeutic resistance in patients treated with the androgen receptor (AR) directed therapies. We previously demonstrated that aberrant TGF-β signaling accelerates prostate tumor progression in the TRAMP mouse model of tumorigenesis via selective effects on EMT. Methods: We hypothesize that the combination of the TGF-β receptor inhibitor, galunisertib (G), and enzalutamide (E) will perturb the interactive signaling between TGF-β and AR signaling affecting the phenotypic landscape of EMT. This perturbation may be exploited in our mouse model, towards enhanced anti-tumor efficacy in advanced castration-resistant PCa (CRPC). We treated 2-week old mice for two weeks with the G (75mg/kg) and/or E (30mg/kg) in combination and as single agents. Results: Treatment with G alone or in combination with E resulted in a significant reduction in prostate tumor weight without affecting total body weight. Immunohistochemical (IHC) and Western blot analysis showed that, while treatment with the G alone led to increased apoptosis and decreased cell proliferation, combination of G and E had significantly higher efficacy in inducing apoptosis and inhibiting cell proliferation than either E or G alone. As expected treatment with the G decreased the levels of nuclear Smad4 protein; the combination of G and E further decreased nuclear Smad4 expression. Furthermore the combination of G and E reversed phenotypic EMT to MET (mesenchymal-epithelial-transition), as assessed by the increase in E-cadherin among the prostate tumor cell populations. IHC and Western blot analysis also revealed that the combined treatment of G and E led to a significant decrease in nuclear AR levels compared to E-only-treated or vehicle-control tumors. Conclusions: These results provide significant insights as to the therapeutic impact of G to effectively impair the TGF-β signaling and overcome resistance of PCa patients to E by reversing EMT to potentially sensitize tumors to the antiandrogen effect. This study has major translational relevance; the combination of G and E may lead to synergistic anti-tumor impact in patients with CRPC.

Secretoneurin is a potential paracrine factor from lactotrophs stimulating gonadotropin release in the goldfish pituitary

2010 ◽  
Vol 299 (5) ◽  
pp. R1290-R1297 ◽  
Author(s):  
E. Zhao ◽  
Caleb L. Grey ◽  
Dapeng Zhang ◽  
Jan A. Mennigen ◽  
Ajoy Basak ◽  
...  
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Time Course ◽  
Pcr Analysis ◽  
Pituitary Cells ◽  
Mrna Levels ◽  
Paracrine Factor ◽  
Lh Release

Secretoneurin (SN) is a functional neuropeptide derived from the evolutionarily conserved part of precursor protein secretogranin II (SgII). In the time course study, SN (10 nM) stimulates luteinizing hormone (LH) production and secretion after 6 h of static incubation of goldfish pituitary cells. Due to the existence of SN-immunoreactivity (SN-IR) in goldfish lactotrophs, endogenous SN might exert a paracrine effect on LH in the pituitary. In an in vitro immunoneutralization experiment, coincubation with anti-SN antiserum reduces the stimulatory effect of salmon gonadotropin-releasing hormone (sGnRH) on LH release by 64%. Using Western blot analysis, we demonstrate that sGnRH significantly increases the expression of the major SgII-derived peptide (∼57 kDa, with SN-IR) and prolactin (PRL) after 12 h in the static culture of goldfish pituitary cells. Furthermore, there exists a significant correlation between the levels of these two proteins ( R = 0.76, P = 0.004). Another ∼30 kDa SgII-derived peptide containing SN is only observed in sGnRH-treated pituitary cells. Consistent with the Western blot analysis results, real-time RT-PCR analysis shows that a 12-h treatment with sGnRH induced 1.6- and 1.7-fold increments in SgII and PRL mRNA levels, respectively. SgII gene expression was also associated with PRL gene expression ( R = 0.66; P = 0.02). PRL cells loaded with the calcium-sensitive dye, fura 2/AM, respond to sGnRH treatment with increases in intracellular Ca2+ concentration level, suggesting a potential mechanism of GnRH on PRL cells and thus SgII processing and SN secretion. Taken together, endogenous lactotroph-generated SN, under the control of hypothalamic GnRH, exerts a paracrine action on neighboring gonadotrophs to stimulate LH release.

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