Discovery of a New Molecular Marker, Prohibitin, and Development of Candidates for New Chemotherapeutic Agents Targeting Prohibitin in Primary AML Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3342-3342
Author(s):  
Hye-Ran Kim ◽  
Myung-Geun Shin ◽  
Sun-Woo Lee ◽  
Hee-Nam Kim ◽  
Il-Kwon Lee ◽  
...  
Keyword(s):  
Molecular Marker ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Mitochondrial Protein ◽  
Genetic Mutation ◽  
Chemotherapeutic Agents ◽  
Leukemic Cells ◽  
Mitochondrial Proteins ◽  
Dependent Manner ◽  
Morphological Transformation

Abstract In light of the recent studies that showed significant causal relationship between mitochondrial genetic mutation and apoptosis in pathology of various tumors and degenerative diseases, mitochondrial proteins have been interesting targets for the study of apoptosis in leukemia and other malignancies. This observation prompted us to analyze mitochondrial proteins and develop new anti-proliferative agents targeting them. Mitochondria were isolated from AML cells by density-gradient ultracentrifugation using swelling buffer and sucrose buffer. We identified 48 spots corresponding to 38 proteins in primary AML cells using 2-DE and mass spectrometry (MALDI-TOF/TOF technology), the expression of which were altered significantly compared to that of normal hematopoietic cells. Out of these deregulated proteins, 12 and 20 proteins were observed in up- or down-regulated spots, respectively. Interestingly, prohibitin (PHB) (gi4505773) was highly expressed in primary AML cells, which was confirmed by Western blot, immunohistochemistry and immunofluorecenct study in the primary AML bone marrow cells and sections. To assess the functional significance of aberrant prohibitin expression, we applied siRNA delivery for silencing of prohibitin. Transduction with a siRNA 11867 construct resulted in 75% decrease of AML cells as compared to the nonsilencing control construct after two days. Potent chemical substances that can alkylate PHB in AML cells, cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU), were synthesized in our laboratory. Time and dose dependent manner of proliferation suppression when treated with CCEU and ICEU was observed in leukemic cell lines including THP-1, K-562 and Kasumi-1. Moreover, notable morphological transformation of leukemic cells was observed when treated with 50 – 200 umol of CCEU and ICEU for 24 hours. Cell cycle analysis of CCEU-and-ICEU-treated- THP-1 and Kasumi-1 cell lines showed a remarkable increase of the sub-G1 phase. Immunoblotting experiment revealed the change of cytoplasmic and nucleoplasmic PHB in K-562 cell line. Expression of cleaved caspase3 and poly ADP-ribose polymerases were also observed to have increased in primary AML cells and cell lines. By analyzing AML cell mitochondrial protein we discovered a new molecular marker, PHB, characteristically overexpressed in AML cells and developed new anti-cancer agents such as CCEU and ICEU that target against PHB in AML cells.

Prohibitin Serves as a Novel Target Mitochondrial Protein of Phenyl-Chloroethyl Ureas Involved In Their Antiproliferative Effect on Multiple Myeloma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3002-3002
Author(s):  
Hye-Ran Kim ◽  
Ha-Young Eom ◽  
Dong Min Kim ◽  
Il-Chul Kim ◽  
Manian Rajesh Kumar ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Mitochondrial Protein ◽  
Mitochondrial Proteins ◽  
Leukemia Cells ◽  
Dependent Manner ◽  
Morphological Transformation ◽  
Novel Target

Abstract Abstract 3002 Background: Prohibitin (PHB) is localized to the mitochondria where it might have a role in the maintenance of mitochondrial function. The diverse function of PHB, together with the emerging evidence that its function can be modulated specifically in certain diseases, implies that PHB would be a potential target for new therapeutics. Materials and Methods: We analyzed mitochondrial proteins and develop new anti-proliferative agents targeting multiple myeloma (MM) cells. Mitochondria were isolated from primary leukemia and MM cell lines (RPMI8226, ARH77, U266 and IM9) by density-gradient ultracentrifugation using swelling buffer and sucrose buffer. Dysregulated mitochondrial protein was identified using 2-DE and mass spectrometry (MALDI-TOF/TOF technology). Results: Out of 38 remarkable deregulated mitochondrial proteins in MM cell lines, prohibitin (PHB) (gi4505773) was highly expressed in primary MM and leukemia cells, which was confirmed by Western blot, immunohistochemistry and immunofluorecenct study in the primary bone marrow cells and sections. Potent chemical substances that can alkylate PHB, two molecules of phenyl-chloroethyl urea family such as cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU), were synthesized independently from the reaction with 2-ethylisocyanate and 4-cyclohexylaniline and 4-iodoaniline, respectively. They were characterized by 1H NMR and 13C NMR. Time and dose dependent manner of proliferation suppression when treated with CCEU and ICEU was observed in MM and leukemia cells. Moreover, notable morphological transformation of MM cells was observed when treated with 10 – 100 umol of CCEU and ICEU for 24 hours. The half of maximal inhibitory concentration (IC50) was 25umol of most MM cell lines. Cell cycle analysis of CCEU-and-ICEU-treated- MM cells showed a remarkable increase of the sub-G1 phase. Immunoblotting experiment revealed the change of cytoplasmic and nucleoplasmic PHB. Expression of cleaved caspase3 and poly ADP-ribose polymerases were also observed to have increased in MM cell lines. Conclusions: By analyzing mitochondrial protein in leukemia and MM cell, we discovered a new molecular marker, PHB, characteristically overexpressed in leukemia and MM cells and developed new anti-cancer agents such as CCEU and ICEU that target against PHB in MM cells. Disclosures: No relevant conflicts of interest to declare.

Development Of Novel Alkylating Chemotherapeutics, Phenyl-Chloroethyl Urea Family, Targeting Mitochondrial Prohibitin For Selective Killing Of Leukemic Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1325-1325
Author(s):  
Hye-Ran Kim ◽  
Hwan-Young Kim ◽  
Trang Nguyen Thi Dai ◽  
Il-Kwon Lee ◽  
Hyeoung-Joon Kim ◽  
...  
Keyword(s):  
Cell Lines ◽  
Conflicts Of Interest ◽  
Mitochondrial Protein ◽  
Leukemic Cells ◽  
Mitochondrial Proteins ◽  
Acute Myelocytic Leukemia ◽  
Dependent Manner ◽  
Mice Model ◽  
Morphological Transformation

Abstract Background Mitochondrial aberrations have been associated with chronic degenerative diseases, aging and cancer. Mitochondrial proteins are interesting targets for the development of selective anticancer drugs in leukemia and other malignancies. We analyzed mitochondrial proteins to develop novel anti-cancer agents targeting selective leukemic cell, especially acute myelocytic leukemia (AML) cells. Materials and Methods Mitochondria were isolated from primary AML cells and AML cell lines (THP-1 and KG-1) by density-gradient ultracentrifugation using swelling buffer and sucrose buffer. Dysregulated mitochondrial protein were identified using 2-DE and mass spectrometry (MALDI-TOF/TOF technology). For in vivo experiments, AML cell grafts were established in 6-week-old Balb/c mice by subcutaneous injection of 1x107 THP-1 AML cells in the right flank at day 0. Results Out of these deregulated proteins, totally 12 and 20 proteins were observed in up- or down-regulated spots, respectively. Interestingly, prohibitin (gi4505773) was highly expressed in all type of leukemic cells, especially primary AML cells (Fig. 1), which is confirmed by immunoblot and immunofluorecenct methods. Aberrant expressed prohibitin induced growth suppression and repressed E2F-mediated transcription. We synthesized potent chemical substances that can alkylate PHB, two molecules of phenyl-chloroethyl urea family such as cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU) from the reaction with 2-ethylisocyanate and 4-cyclohexylaniline and 4-iodoaniline, respectively. They were characterized by 1H NMR and 13C NMR. Time and dose dependent manner of proliferation suppression when treated with CCEU and ICEU was observed in primary AML and AML cell lines. Notably morphological transformation of AML cells was observed when treated with 10 - 100 umol of CCEU and ICEU for 24 hours. The half maximal inhibitory concentration (IC50) was 25umol of most AML cell lines. Cell cycle analysis of CCEU-and-ICEU-treated- AML cells showed a remarkable increase of the sub-G1 phase. Immunoblotting experiment revealed the change of cytoplasmic and nucleoplasmic PHB. The increment expression of cleaved caspase3 and poly ADP-ribose polymerases were also observed in AML cell lines. Moreover, CCEU and ICEU selectively killed AML cells in mouse model (Fig. 2). Conclusion This study showed the development of novel alkylating chemotherapeutics, phenyl-chloroethyl urea family (CCEU and ICEU), targeting mitochondrial prohibitin for selective killing of leukemic cells. Selective eradication effects of these novel chemotherapeutics were further confirmed using in vivo mice model. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Silver Nanoparticles Induce Mitochondrial Protein Oxidation in Lung Cells Impacting Cell Cycle and Proliferation

Antioxidants ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 552 ◽  
Author(s):  
Reetta J. Holmila ◽  
Stephen A. Vance ◽  
S. Bruce King ◽  
Allen W. Tsang ◽  
Ravi Singh ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Silver Nanoparticles ◽  
Ionizing Radiation ◽  
Cell Lines ◽  
Protein Oxidation ◽  
Mitochondrial Protein ◽  
Antibacterial Properties ◽  
Dependent Manner ◽  
Mitochondrial Redox State ◽  
And Function

Silver nanoparticles (AgNPs) are widely used nanomaterials in both commercial and clinical biomedical applications, due to their antibacterial properties. AgNPs are also being explored for the treatment of cancer in particular in combination with ionizing radiation. In this work, we studied the effects of AgNPs and ionizing radiation on mitochondrial redox state and function in a panel of lung cell lines (A549, BEAS-2B, Calu-1 and NCI-H358). The exposure to AgNPs caused cell cycle arrest and decreased cell proliferation in A549, BEAS-2B and Calu-1, but not in NCI-H358. The mitochondrial reactive oxygen species (ROS) and protein oxidation increased in a time- and dose-dependent manner in the more sensitive cell lines with the AgNP exposure, but not in NCI-H358. While ionizing radiation also induced changes in the mitochondrial redox profiles, in general, these were not synergistic with the effects of AgNPs with the exception of NCI-H358 and only at a higher dose of radiation.

scholarly journals Anti-MY9-blocked-ricin: an immunotoxin for selective targeting of acute myeloid leukemia cells

Blood ◽  
1991 ◽  
Vol 77 (11) ◽  
pp. 2404-2412 ◽  
Author(s):  
DC Roy ◽  
JD Griffin ◽  
M Belvin ◽  
WA Blattler ◽  
JM Lambert ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Leukemic Cells ◽  
Short Exposure ◽  
Continuous Exposure ◽  
Dependent Manner ◽  
Acute Myeloid

Abstract The use of immunotoxins (IT) to selectively destroy acute myeloid leukemia (AML) cells in vivo or in vitro is complicated by both the antigenic similarity of AML cells to normal progenitor cells and the difficulty of producing a sufficiently toxic conjugate. The monoclonal antibody (MoAb) anti-MY9 is potentially ideal for selective recognition of AML cells because it reacts with an antigen (CD33) found on clonogenic AML cells from greater than 80% of cases and does not react with normal pluripotent stem cells. In this study, we describe an immunotoxin that is selectively active against CD33+ AML cells: Anti- MY9-blocked-Ricin (Anti-MY9-bR), comprised of anti-MY9 conjugated to a modified whole ricin that has its nonspecific binding eliminated by chemical blockage of the galactose binding domains of the B-chain. A limiting dilution assay was used to measure elimination of HL-60 leukemic cells from a 20-fold excess of normal bone marrow cells. Depletion of CD33+ HL-60 cells was found to be dependent on the concentration of Anti-MY9-bR and on the duration of incubation with IT at 37 degrees C. More than 4 logs of these leukemic cells were specifically depleted following short exposure to high concentrations (10(-8) mol/L) of Anti-MY9-bR. Incubation with much lower concentrations of Anti-MY9-bR (10(-10) mol/L), as compatible with in vivo administration, resulted in 2 logs of depletion of HL-60 cells, but 48 to 72 hours of continuous exposure were required. Anti-MY9-bR was also shown to be toxic to primary AML cells, with depletion of greater than 2 logs of clonogenic cells following incubation with Anti- MY9-bR 10(-8) mol/L at 37 degrees C for 5 hours. Activity of Anti-MY9- bR could be blocked by unconjugated Anti-MY9 but not by galactose. As expected, Anti-MY9-bR was toxic to normal colony-forming unit granulocyte-monocyte (CFU-GM), which expresses CD33, in a concentration- and time-dependent manner, and also to burst-forming unit-erythroid and CFU-granulocyte, erythroid, monocyte, megakaryocyte, although to a lesser extent. When compared with anti-MY9 and complement (C′), Anti- MY9-bR could be used in conditions that provided more effective depletion of AML cells with substantially less depletion of normal CFU- GM. Therefore, Anti-MY9-bR may have clinical utility for in vitro purging of AML cells from autologous marrow when used at high IT concentrations for short incubation periods. Much lower concentrations of Anti-MY9-bR that can be maintained for longer periods may be useful for elimination of AML cells in vivo.

scholarly journals All-trans retinoic acid enhances, and a pan-RAR antagonist counteracts, the stem cell promoting activity of EVI1 in acute myeloid leukemia

2019 ◽  
Vol 10 (12) ◽  
Author(s):  
Chi Huu Nguyen ◽  
Katharina Bauer ◽  
Hubert Hackl ◽  
Angela Schlerka ◽  
Elisabeth Koller ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Retinoic Acid ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Acute Myeloid

AbstractEcotropic virus integration site 1 (EVI1), whose overexpression characterizes a particularly aggressive subtype of acute myeloid leukemia (AML), enhanced anti-leukemic activities of all-trans retinoic acid (atRA) in cell lines and patient samples. However, the drivers of leukemia formation, therapy resistance, and relapse are leukemic stem cells (LSCs), whose properties were hardly reflected in these experimental setups. The present study was designed to address the effects of, and interactions between, EVI1 and retinoids in AML LSCs. We report that Evi1 reduced the maturation of leukemic cells and promoted the abundance, quiescence, and activity of LSCs in an MLL-AF9-driven mouse model of AML. atRA further augmented these effects in an Evi1 dependent manner. EVI1 also strongly enhanced atRA regulated gene transcription in LSC enriched cells. One of their jointly regulated targets, Notch4, was an important mediator of their effects on leukemic stemness. In vitro exposure of leukemic cells to a pan-RAR antagonist caused effects opposite to those of atRA. In vivo antagonist treatment delayed leukemogenesis and reduced LSC abundance, quiescence, and activity in Evi1high AML. Key results were confirmed in human myeloid cell lines retaining some stem cell characteristics as well as in primary human AML samples. In summary, our study is the first to report the importance of EVI1 for key properties of AML LSCs. Furthermore, it shows that atRA enhances, and a pan-RAR antagonist counteracts, the effects of EVI1 on AML stemness, thus raising the possibility of using RAR antagonists in the therapy of EVI1high AML.

scholarly journals Environmentally Relevant Concentrations of Bisphenol A Interact with Doxorubicin Transcriptional Effects in Human Cell Lines

Toxics ◽  
2019 ◽  
Vol 7 (3) ◽  
pp. 43 ◽  
Author(s):  
Edna Ribeiro ◽  
Mariana Delgadinho ◽  
Miguel Brito
Keyword(s):  
Bisphenol A ◽  
Cell Lines ◽  
Cancer Biology ◽  
Cellular Response ◽  
Chemotherapeutic Agents ◽  
Transcriptional Analysis ◽  
Dependent Manner ◽  
Continuous Increase ◽  
Worldwide Production ◽  
Endocrine Disruptor Chemicals

The worldwide production of synthetic chemicals, including endocrine disruptor chemicals (EDCs), such as Bisphenol A (BPA) has increased significantly in the last two decades. Human exposure to BPA, particularly through ingestion, is continuous and ubiquitous. Although, considered a weak environmental estrogen, BPA can induce divergent biological responses through several signaling pathways, including carcinogenesis in hormone-responsive organs. However, and despite the continuous increase of tumor cell-resistance to therapeutic drugs, such as doxorubicin (DOX), information regarding BPA drug interactions is still scarce, although its potential role in chemo-resistance has been suggested. This study aims to assess the potential interactions between environmentally relevant levels of BPA and DOX at a therapeutic dosage on Hep-2 and MRC-5 cell lines transciptome. Transcriptional effects in key-player genes for cancer biology, namely c-fos, p21, and bcl-xl, were evaluated through qRT-PCR. The cellular response was analyzed after exposure to BPA, DOX, or co-exposure to both chemicals. Transcriptional analysis showed that BPA exposure induces upregulation of bcl-xl and endorses an antagonistic non-monotonic response on DOX transcriptional effects. Moreover, the BPA interaction with DOX on c-fos and p21 expression emphasize its cellular specificity and divergent effects. Overall, Hep-2 was more susceptible to BPA effects in a dose-dependent manner while MRC-5 transcriptional levels endorsed a non-monotonic response. Our data indicate that BPA environmental exposure may influence chemotherapy outcomes, which emphasize the urgency for a better understanding of BPA interactions with chemotherapeutic agents, in the context of risk assessment.

Cellular Aminopeptidase Inhibition as a Target for the Therapy of AML by the Novel Agent CHR 2797.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1608-1608
Author(s):  
Christopher Jenkins ◽  
Ken Mills ◽  
Chris Pepper ◽  
Burnett Alan
Keyword(s):  
Intact Cell ◽  
Myeloid Cells ◽  
Culture Conditions ◽  
Chemotherapeutic Agents ◽  
Molecular Therapy ◽  
Leukemic Cells ◽  
Therapeutic Window ◽  
Aminopeptidase Activity ◽  
Dependent Manner ◽  
Normal Cells

Abstract CHR 2797 is one of a new class of enzyme inhibitors with pleiotropic effects against cancer cells which is currently in Phase II clinical trials. It inhibits a number of the M1 family of metalloenzymes that include the Zn++-dependent aminopeptidases. Aminopeptidases catalyze the hydrolysis of the terminal amino acids from short chain polypeptides, and are involved in the continuous cycle of protein formation and degradation in cells. As malignant cells may be more dependent on protein cycling than normal cells, interrupting this pathway is, therefore, a potential therapeutic target for novel agents. The effect of CHR 2797 on AML cells in-vitro was investigated to determine its effectiveness and mode of action. Treatment of leukemic cells with CHR 2797 was performed under standard culture conditions at a concentration range of 0.0002 - 20μM. The WST-1 reagent was used to determine cell viability at the end of the culture and the IC50 values were calculated. Primary diagnostic AML samples (n=52) were analyzed and an IC50 range of between 0.01 and >40μM was established, with a median of 1.2μM. There were no significant differences between the different classes of AML, although there was a trend towards a better response in the monocytic leukemias and the good prognostic cytogenetic groups. The comparative effects of CHR 2797 on normal bone marrow samples (n=10) were also analyzed. Under identical culture conditions, an IC50 range between 6.2 and >40μM was found, with a median of 15μM. This 13-fold higher result demonstrates a significant potential therapeutic window for leukemic versus normal cells. The level of synergy or antagonism with conventional therapeutic agents was calculated using a combination index. Synergy was demonstrated in 69% of cell samples in combination with cytarabine, and 78% with bortezomib. Synergy was also shown in 62% of cells samples with ATRA, even in non-promyelocytic leukemia types. Annexin V staining and cell cycle analysis demonstrated apoptosis after treatment with CHR 2797 in many cases. A degree of differentiation of acute promyelocytic cells to mature myeloid cells was also stimulated with the treatment. To determine the mode of drug action, the effect of CHR 2797 on intact cell aminopeptidase activity was also measured. The activity of these enzymes can be measured by their conversion of the membrane-permeant substrate ala-MCA to MCA, which can be fluorimetrically detected. CHR 2797 was demonstrated to reduce intact cell aminopeptidase activity in a time- and dose-dependent manner. A reduction in aminopeptidase activity was shown with a concentration of CHR 2797 of less than 0.5μM; with 10μM drug the activity in many AML samples was reduced by >90%. CD13 is a cell surface protein which is expressed selectively on myeloid cells and is also classified as an aminopeptidase N. By comparison with the results obtained with the CD13 blocking antibody WM15, CHR 2797 was demonstrated to fully inhibit CD13 activity. Further investigations proved that other extracellular, cytoplasmic and nuclear aminopeptidase enzymes were also inhibited. In conclusion new treatments are needed for acute myeloid leukemia to improve survival and circumvent the toxicity of conventional therapy. This study demonstrates that CHR 2797 might be an effective molecular therapy for AML, either alone or in combination with other chemotherapeutic agents.

Combination of a Spicamycin Analogue KRN5500 and Chemotherapeutic Agents Synergistically Enhances Their Cytotoxicity in Human Myeloma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4783-4783
Author(s):  
Hirokazu Miki ◽  
Shuji Ozaki ◽  
Osamu Tanaka ◽  
Shiro Fujii ◽  
Shingen Nakamura ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Growth ◽  
Cell Lines ◽  
Therapeutic Potential ◽  
Flow Cytometric Analysis ◽  
Human Tumor ◽  
Chemotherapeutic Agents ◽  
Dependent Manner ◽  
Induced Apoptosis ◽  
Rpmi 8226

Abstract Multiple myeloma (MM) is a plasma cell malignancy characterized by the accumulation of neoplastic plasma cells in the bone marrow. Although new classes of agents such as thalidomide, lenalidomide, and bortezomib have shown marked anti-MM activity in clinical settings, MM remains an incurable disease due to increased resistance to these agents. Therefore, alternative approaches are necessary to overcome drug resistance in MM. KRN5500 is a new derivative of spicamycin produced by Streptomyces alanosinicus (Kirin Pharma, Tokyo, Japan). This drug potently decreases protein synthesis and inhibits cell growth in human tumor cell lines both in vitro and in vivo. Several phase I studies of KRN5500 were conducted in patients with solid tumors, which showed Cmax values of 1000–3000 nM at the maximum tolerated doses. However, no objective anti-tumor response to KRN5500 alone was observed in these patients. In this study, we examined the anti-tumor activity of KRN5500 against MM cells and evaluated its therapeutic potential in combination with other anti-MM agents. MM cell lines and freshly-isolated MM cells were incubated with various concentrations of KRN5500 for 24 hours. Cell proliferation assay showed marked inhibition of cell growth in MM cells such as RPMI 8226, KMS12-BM, and UTMC-2 (IC50 = 10–40 nM), and U266, MM.1S, and primary MM cells (IC50 = 500–1000 nM). Importantly, a chemotherapy-resistant subclone of RPMI 8226 had a similar sensitivity to KRN5500. Annexin V/propidium iodide staining confirmed that KRN5500 induced apoptosis of MM cells in a dose- and time-dependent manner. Moreover, cleavage of poly (ADP-ribose) polymerase (PARP) was detected after 24 hours with only modest activation of caspase-8, -9, and -3 by immunoblotting. Flow cytometric analysis of anti-apoptotic proteins revealed that apoptosis induced by KRN5500 was associated with down-regulation of Mcl-1 and Bcl-2 expression. To determine the effect of KRN5500 on the unfolded protein response (UPR), splicing of XBP-1 mRNA was analyzed by reverse transcription-polymerase chain reaction. In response to stimulation with KRN5500, splicing of XBP-1 mRNA occurred after 24 hours in RPMI 8226 cells, suggesting that KRN5500-induced apoptosis is mediated in part by the inhibition of UPR. Furthermore, synergistic effects on MM cells were observed when KRN5500 was combined with anti-MM agents including melphalan, dexamethasone, and bortezomib. These results suggest that KRN5500 induces apoptosis in MM cells mainly by the caspase-independent pathway and that its unique mechanism of action provides a valuable therapeutic option to overcome drug resistance in patients with MM.

Mitochondrial-Mediated Apoptosis by the Sesquiterpene Oil α-Bisabolol and Its Synergism with Imatinib and Nilotinib In BCR-ABL+ Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4456-4456
Author(s):  
Massimiliano Bonifacio ◽  
Antonella Rigo ◽  
Elisabetta Cavalieri ◽  
Emanuele Guardalben ◽  
Christian Bergamini ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Mitochondrial Membrane ◽  
Single Agent ◽  
Leukemic Cells ◽  
Parp Cleavage ◽  
Potential Candidate ◽  
Dependent Manner ◽  
Ros Production ◽  
Dna Laddering

Abstract Abstract 4456 Background. The plant-derived agent α-bisabolol is a small oily sesquiterpene alcohol that has been demonstrated to be cytotoxic against human malignant non-hematological and leukemic cells (Bonifacio M et al, Blood, 2009 ASH annual meeting abstracts;114:4800). Here we tested its activity against BCR-ABL+ cell lines and primary cells from patients, alone or in combination with the Tyrosine-Kinase Inhibitors (TKIs) Imatinib and Nilotinib. Also, the mechanism of α-bisabolol cytotoxicity in BCR-ABL+ cells was assessed. Methods. We used the BCR-ABL+ K562, LAMA-84 and CML-T1 cell lines and primary leukemic cells from 14 patients with BCR-ABL+ Acute Lymphoblastic Leukemia at diagnosis. First, the citotoxicity of single-agent α-bisabolol was determined by MTT. Then, mitochondrial membrane potential of treated cells was evaluated by the JC-1 dye in flow cytometry and fluorescence microscopy. Permeabilized leukemic cells were assayed for oxygen consumption by measuring mitochondrial state 3 and uncoupled respiration. Reactive oxygen species (ROS) production in α-bisabolol treated cells were quantified in flow cytometry by oxidation of CM-H2DCFDA, measuring the fluorescence intensity of the DCF products. Apoptosis was studied by the poly(ADP-ribose) polymerase (PARP) cleavage and internucleosomal DNA laddering analysis. Finally, the combination effects between α-bisabolol and Imatinib or Nilotinib (kindly provided by Novartis) were analyzed according to the median-effect method of Chou and Talalay using the CalcuSyn software. Results. α-bisabolol reduced the viability of BCR-ABL+ cells in a dose-dependent manner. The mean IC50 values of α-bisabolol were 46±11 μ M for primary leukemic cells and ranged from 62 to 115 μ M in the cell lines. JC-1 staining of BCR-ABL+ primary leukemic cells treated with 40 μ M α-bisabolol for 3 to 5 hours demonstrated a dissipation of the mitochondrial transmembrane potential (ΔΨm), thus indicating the start of the apoptotic process. Moreover, NADH-supported state 3 respiration in α-bisabolol treated leukemic cells was significantly decreased in comparison with untreated leukemic controls (140.0±70.5 vs 280.7±11.9 pmol O2/min/106 cells; p<.05). Finally, PARP cleavage and DNA laddering followed α-bisabolol exposure of leukemic BCR-ABL+ blasts. The apoptosis induction was accompanied by ROS production. When tested in combination at constant ratio with Imatinib or Nilotinib, α-bisabolol showed overall slight to strong synergistic effects, without evidence for antagonism across a range of doses (Table 1). In 3 patients with mutation of BCR-ABL (T315I, E255V and Y253H, respectively) we observed full activity of α-bisabolol as single agent and confirmed the synergism between α-bisabolol and Imatinib. Conclusion. This study indicates that α-bisabolol is an effective pro-apoptotic agent for human acute BCR-ABL+ leukemia cells via induction of mitochondrial membrane damage. The combination of α-bisabolol with Imatinib or Nilotinib allows a dose reduction up to 90% of each drug to obtain the same cytotoxic effect, so indicating a clear synergism. α-bisabolol may be a potential candidate for the treatment of BCR-ABL+ leukemias and the effective dose of TKIs could be reduced in a combined treatment with α-bisabolol. Disclosures: No relevant conflicts of interest to declare.

Identification of the Epigenetic Reader BRD4 As a Novel Potential Target in Ph+ CML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1571-1571
Author(s):  
Barbara Peter ◽  
Gregor Eisenwort ◽  
Gabriele Stefanzl ◽  
Daniela Berger ◽  
Wolfgang R Sperr ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Drug Target ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Thymidine Uptake ◽  
Dependent Manner ◽  
Potential Drug ◽  
Ku812 Cells

Abstract Chronic myelogenous leukemia (CML) is a bone marrow-derived hematopoietic neoplasm in which BCR/ABL1 acts as a major driver of proliferation, differentiation and survival of leukemic cells. In a majority of all patients with CML, leukemic cells can be kept under control by BCR/ABL1 tyrosine kinase inhibitors (TKI), including imatinib, nilotinib, dasatinib, bosutinib, and ponatinib. Nevertheless, resistance or intolerance against one or more of these TKI may occur. Therefore, current research is focusing on novel potential drug targets in CML. A promising class of targets may be epigenetic regulators of cell growth, such as members of the bromodomain and extra-terminal domain (BET) family. The epigenetic reader and BET family member BRD4 has recently been identified as a novel potential drug target in acute myeloid leukemia (AML). However, so far, little is known about the expression and function of BRD4 in CML cells. The aims of the present study were to determine the expression of BRD4 and its downstream target MYC in CML cells and to explore whether BRD4 can serve as a novel drug target in this disease. As determined by qPCR, primary CML cells (chronic phase patients, n=7) as well as the CML cell lines KU812 and K562 expressed BRD4 mRNA. In addition, both CML cell lines stained positive for BRD4 in our immunocytochemistry staining experiments. In one patient with accelerated phase CML, putative leukemic (CD34+/CD38-) stem cells were sorted to near homogeneity and found to express BRD4 mRNA by qPCR. In order to examine the functional role of BRD4 in CML cells, a BRD4-specific shRNA was applied. In these experiments, the shRNA-induced knockdown of BRD4 in KU812 cells and K562 resulted in reduced growth compared to a control shRNA. Furthermore, the BRD4-targeting drug JQ1 was found to inhibit 3H-thymidine uptake and thus proliferation in KU812 cells in a dose-dependent manner (IC50: 0.25-0.75 µM). In addition, we were able to show that JQ1 inhibits growth of primary CML cells with variable IC50 values (0.1-5 µM). However, no substantial growth-inhibitory effects of JQ1 were seen in K562 cells (IC50: >5 µM). As determined by Annexin V/PI staining, JQ1 induced apoptosis in KU812 cells whereas no apoptosis-inducing effect of JQ1 was observed in K562 cells. Nevertheless, we were able to show that both CML cell lines as well as primary CML cells express MYC mRNA, and treatment of KU812 cells or K562 cells with JQ1 resulted in a decreased expression of MYC mRNA and MYC protein. Next, we analyzed whether MYC expression in CML cells can be blocked by BCR/ABL1 TKI. We found that imatinib, nilotinib, dasatinib, and ponatinib decrease MYC mRNA- and MYC protein expression in KU812 and K562 cells. Finally, we found that JQ1 cooperates with imatinib, nilotinib, ponatinib and dasatinib in inhibiting the proliferation of KU812 and K562 cells. Together, our data show that BRD4 serves as a potential new target in CML cells, and that the BRD4 blocker JQ1 cooperates with BCR/ABL1 TKI in inducing growth-inhibition. Whether BRD4 inhibition is a pharmacologically meaningful approach in patients with TKI-resistant CML remains to be determined in clinical trials. Disclosures Sperr: Ariad: Consultancy; Celgene: Consultancy. Zuber:Mirimus Inc.: Consultancy, Other: Stock holder; Boehringer Ingelheim: Research Funding. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria.

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