scholarly journals Monocytoid differentiation of freshly isolated human myeloid leukemia cells and HL-60 cells induced by the glutamine antagonist acivicin

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1728-1737
Author(s):  
KE Nichols ◽  
SR Chitneni ◽  
JO Moore ◽  
JB Weinberg
Keyword(s):  
Amino Acid ◽  
Cell Lines ◽  
Essential Amino Acid ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
H2o2 Production ◽  
Isolated Cells ◽  
Dihydroxyvitamin D3

Previously we showed that starvation of HL-60 promyelocytic leukemia cells for a single essential amino acid induced irreversible differentiation into more mature monocyte-like cells. Although not an essential amino acid, glutamine is important in the growth of normal and neoplastic cells. The glutamine analogue, alpha S,5S-alpha-amino-3- chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) inhibits several glutamine-utilizing enzymes and therefore depletes cells of certain metabolic end products. The current study was designed to examine in vitro the effects of acivicin on growth and differentiation of several established human myeloid leukemia cell lines, including the HL-60 cell line, and of freshly isolated cells from patients with acute nonlymphocytic leukemia (ANLL). Four-day culture of HL-60 cells with acivicin at concentrations of 0.1 to 10.0 micrograms/mL (0.56 to 56 nmol/L) decreased cell growth by 33% to 88% as compared with untreated control cells. Viability of cells was greater than 92% for untreated cells and 93% to 41% for acivicin-treated cells. Cells treated with acivicin differentiated along a monocytic pathway as shown by increased H2O2 production and alpha-naphthyl butyrate esterase (NSE) content. Differentiation was time and dose dependent, and was irreversible. Changes in H2O2 production and NSE content were partially abrogated by co-culture with 10 mmol/L exogenous cytidine and guanosine but not by co-culture with other nucleosides or glutamine. At these concentrations of acivicin, differentiation was associated with expression of the N- formyl-methyl-leucyl-phenylalanine-receptor (FMLP-R) on 8% to 29% of cells as compared with 8% for control cells. Acivicin potentiated the differentiating effects of interferon-gamma, tumor necrosis factor, dihydroxyvitamin D3, dimethylsulfoxide, and retinoic acid. Culture of cells from the U937 (monoblastic), K562 (erythroleukemia), and KG-1 (myeloblastic) cell lines resulted in decreased growth and viability, but not consistently in differentiation. Acivicin decreased survival of freshly isolated ANLL cells and increased their H2O2 production and NSE content. These results suggest that the glutamine analogue acivicin may be useful as a differentiating agent with antileukemia activity in patients with ANLL.

scholarly journals Monocytoid differentiation of freshly isolated human myeloid leukemia cells and HL-60 cells induced by the glutamine antagonist acivicin

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1728-1737 ◽  
Author(s):  
KE Nichols ◽  
SR Chitneni ◽  
JO Moore ◽  
JB Weinberg
Keyword(s):  
Amino Acid ◽  
Cell Lines ◽  
Essential Amino Acid ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
H2o2 Production ◽  
Isolated Cells ◽  
Dihydroxyvitamin D3

Abstract Previously we showed that starvation of HL-60 promyelocytic leukemia cells for a single essential amino acid induced irreversible differentiation into more mature monocyte-like cells. Although not an essential amino acid, glutamine is important in the growth of normal and neoplastic cells. The glutamine analogue, alpha S,5S-alpha-amino-3- chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) inhibits several glutamine-utilizing enzymes and therefore depletes cells of certain metabolic end products. The current study was designed to examine in vitro the effects of acivicin on growth and differentiation of several established human myeloid leukemia cell lines, including the HL-60 cell line, and of freshly isolated cells from patients with acute nonlymphocytic leukemia (ANLL). Four-day culture of HL-60 cells with acivicin at concentrations of 0.1 to 10.0 micrograms/mL (0.56 to 56 nmol/L) decreased cell growth by 33% to 88% as compared with untreated control cells. Viability of cells was greater than 92% for untreated cells and 93% to 41% for acivicin-treated cells. Cells treated with acivicin differentiated along a monocytic pathway as shown by increased H2O2 production and alpha-naphthyl butyrate esterase (NSE) content. Differentiation was time and dose dependent, and was irreversible. Changes in H2O2 production and NSE content were partially abrogated by co-culture with 10 mmol/L exogenous cytidine and guanosine but not by co-culture with other nucleosides or glutamine. At these concentrations of acivicin, differentiation was associated with expression of the N- formyl-methyl-leucyl-phenylalanine-receptor (FMLP-R) on 8% to 29% of cells as compared with 8% for control cells. Acivicin potentiated the differentiating effects of interferon-gamma, tumor necrosis factor, dihydroxyvitamin D3, dimethylsulfoxide, and retinoic acid. Culture of cells from the U937 (monoblastic), K562 (erythroleukemia), and KG-1 (myeloblastic) cell lines resulted in decreased growth and viability, but not consistently in differentiation. Acivicin decreased survival of freshly isolated ANLL cells and increased their H2O2 production and NSE content. These results suggest that the glutamine analogue acivicin may be useful as a differentiating agent with antileukemia activity in patients with ANLL.

The CXCR4 Antagonist AMD3100 Has Dual Effects, Initially Enhancing and Later Inhibiting Survival and Proliferation, in Myeloid Leukemia Cells in Vitro.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1721-1721
Author(s):  
Ha-Yon Kim ◽  
Ji-Young Hwang ◽  
Seong-Woo Kim ◽  
Gak-Won Yun ◽  
Young-Joon Yang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Dependent Manner ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Leukemia Cell Lines ◽  
Number Of Cells

Abstract Abstract 1721 Poster Board I-747 AMD3100, a small bicyclam antagonist for chemokine receptor CXCR4, induces the peripheral mobilization of hematopoietic stem cells. It also induces the segregation of leukemia cells in the bone marrow microenvironment, which should enhance the chemosensitivity of the cells. Based on these observations, AMD3100 is being considered for clinical use. However, AMD3100 activates G-protein coupled with CXCR4 and acts as a partial CXCR4 agonist. In this study, we explored whether AMD3100 affects the proliferation and survival of myeloid leukemia cells. As demonstrated previously, both AMD3100 and T140, another CXCR4 antagonist, markedly inhibited stromal cell-derived factor-1 (SDF-1)-induced chemotaxis and induced the internalization of CXCR4 in myeloid leukemia cell lines (U937, HL-60, MO7e, KG1a, and K562 cells) and CD34+ primary human acute myeloid leukemia (AML) cells. SDF-1 alone did not stimulate the proliferation of these leukemia cells, nor did it rescue the cells from apoptosis induced by serum deprivation. By contrast, AMD3100, but not T140, stimulated the proliferation of all five leukemia cell lines and primary AML cells in a dose-dependent manner in serum-free conditions for up to 5 days (∼ 2-fold increases at a concentration of 10-5M), which was abrogated by pretreating the cells with pertussis toxin. AMD3100 binds to CXCR7, another SDF-1 receptor, and all of the cells examined in this study expressed CXCR4 on the cell surface to some extent. The proliferation-enhancing effects of AMD3100 were not changed by knocking-down CXCR7 using the siRNA technique, whereas knocking-down CXCR4 significantly delayed the enhanced proliferation induced by AMD3100. Neither AMD3100 nor T140 induced the phosphorylation of Akt, Stat3, MAPK p44/p42, or MAPK p38, which are involved in SDF-1 signaling. In extended cultures of these cells for up to 14 days, AMD3100, but not T140, induced a marked decrease in the number of cells, compared to the control, after incubation for 5-7 days. Adding SDF-1 at the beginning and middle of the incubation did not affect the early increase or later decrease in the number of cells. AMD3100 reduced the apoptosis of these cells to a modest degree over the first 5-7 days and then markedly increased it. Consistent with the proliferation assay, AMD3100 increased the number of leukemia cell colonies during the early period of the assay, while it markedly decreased the number and size of the colonies in the later period of the assay. In conclusion, AMD3100 exerts dual effects, initially enhancing and subsequently inhibiting the survival and proliferation, in myeloid leukemia cells in vitro. Disclosures No relevant conflicts of interest to declare.

Clathrin Assembly Protein CALM Is Necessary for Leukemia Cell Proliferation and Erythroid Development Via Regulating Transferrin Receptor Endocytosis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 244-244
Author(s):  
Yuichi Ishikawa ◽  
Manami Maeda ◽  
Min Li ◽  
Sung-Uk Lee ◽  
Julie Teruya Feldstein ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Leukemia Cell Lines ◽  
Erythroid Development

Abstract Abstract 244 Clathrin assembly lymphoid myeloid leukemia (CALM) protein is implicated in clathrin dependent endocytosis (CDE) and the CALM gene is the target of the t(10;11)(p13;q14-21) CALM/AF10 translocation, which is observed in multiple types of acute leukemia. Although the translocation generally dictates poor prognosis, the molecular mechanisms by which the fusion protein exerts its oncogenic activity remains elusive. To determine the role of CALM and CDE in normal hematopoiesis and leukemogenesis, we generated and characterized both conventional (Calm+/−) and conditional (CalmF/FMx1Cre+) Calm knockout (KO) mutants. Furthermore, we determined the impact of Calm loss on leukemia cell growth in vitro and in vivo employing a series of leukemia cell lines and leukemia mouse models. Hematopoietic-specific Calm knockout mice (CalmF/FMx1Cre+) exhibited a hypocromatic anemia with increased serum iron levels. We observed significant reduction in mature erythroblasts/erythrocytes (TER119+CD71-) with concomitant increase in immature erythroblasts (TER119+CD71+) in the spleen of CalmF/FMx1Cre+ mice. The frequencies of erythroblasts in S phase were lower and the proportions of apoptotic (cleaved PARP positive) erythroblasts were increased in CalmF/FMx1Cre+ mice. Surface transferrin receptor 1 (Tfr1, CD71) levels were significantly up-regulated in Calm-deficient hematopoietic progenitors, and uptake of Alexa647-conjugated transferrin was abrogated in Calm-deficient erythroblasts, revealed by immunofluorescence analysis. Freez-etch electron microscopy analysis showed a defective clathrin coated vesicle (CCV) formation in Calm-deficient erythroblasts, indicating that Calm is indispensable for iron-bound transferrin internalization by regulating CCV formation, thereby critical for erythroid differentiation and hemoglobinization. CALM was highly expressed in leukemia/lymphoma cell lines and primary acute myeloid leukemia samples, although its expression was limited to erythroblasts in normal hematopoietic lineage cells. Treatment of leukemia cell lines with Desferoxamine (DFO), an iron chelator, led to a significant increase in Calm mRNA levels, suggesting that Calm expression is regulated by intracellular iron levels. Since highly proliferative leukemia cells demand iron as a cofactor for ribonucleotide reductase (RNR), we hypothesized that Calm is required for leukemia cell proliferation by regulating iron-bound transferrin internalization. To determine the effect of Calm inactivation in leukemia cells, we transduced a series of leukemia cell lines with a lentivirus-based ShRNA vector (pLKO-GFP), which allowed shRNA-expressing cells to be traced by green fluorescent protein (GFP). Calm shRNA transduced cells, but not cells transduced with scrambled shRNA, showed a proliferative disadvantage compared to non-transduced cells. To determine the effect of Calm deletion in leukemia cells in vivo, the CALM/AF10 oncogene was retrovirally transduced into either wild type (WT) or CalmF/FMx1Cre+ bone marrow (BM) cells and the cells were subsequently transferred to lethally-irradiated recipient mice. The Calm gene was deleted in donor cells via pIpC injections one month after transplant (before leukemia development) and survival curves generated. The recipients transplanted with the BM cells from CalmF/FMx1Cre+ mice showed a significantly delayed onset of leukemia and longer survivals compared to control (p=0.001), indicating that Calm is necessary for the development of CALM/AF10-induced leukemia. We next assessed whether Calm is required for the “maintenance” of leukemia in vivo. Leukemia cells were harvested from the primary recipients transplanted with the CALM/AF10-transduced CalmF/FMx1Cre+ BM cells (in which the endogenous Calm genes were intact) and transferred to the secondary recipients. The leukemic secondary recipient mice were then injected with pIpC and survival curves generated. Calm inactivation significantly delayed leukemia progression by blocking leukemia cell proliferation. Taken together, our data indicate that Calm is essential for erythroid development and leukemia cell proliferation by regulating TFR1 internalization. Since Calm inactivation significantly blocked the leukemia cell proliferation in vitro and in vivo, our findings may provide new therapeutic strategies for acute myeloid leukemia. Disclosures: Naoe: Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.

scholarly journals Anti-leukemia properties of cadmium nanoparticles in the in vitro and in vivo condition: A chemobiological study

2021 ◽  
Author(s):  
Yudi Miao ◽  
Behnam Mahdavi ◽  
Mohammad Zangeneh
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Aqueous Extract ◽  
Myeloid Leukemia ◽  
Antioxidant Properties ◽  
Leukemia Cell ◽  
Sem Images ◽  
Acute Myeloid

IntroductionThe present study investigated the anti-acute myeloid leukemia effects of Ziziphora clinopodides Lam leaf aqueous extract conjugated cadmium nanoparticles.Material and methodsTo synthesize CdNPs, Z. clinopodides aqueous extract was mixed with Cd(NO3)2 .4H2O. The characterization of the biosynthesized cadmium nanoparticles was carried out using many various techniques such as UV-Vis. and FT-IR spectroscopy, XRD, FE-SEM, and EDS.ResultsThe uniform spherical morphology of NPs was proved by FE-SEM images with NPs the average size of 26.78cnm. For investigating the antioxidant properties of Cd(NO3)2, Z. clinopodides, CdNPs, and Daunorubicin, the DPPH test was used. The cadmium nanoparticles inhibited half of the DPPH molecules in a concentration of 196 µg/mL. To survey the cytotoxicity and anti-acute myeloid leukemia effects of Cd(NO3)2, Z. clinopodides, CdNPs, and Daunorubicin, MTT assay was used on the human acute myeloid leukemia cell lines i.e., Murine C1498, 32D-FLT3-ITD, and Human HL-60/vcr. The IC50 of the cadmium nanoparticles was 168, 205, and 210 µg/mL against Murine C1498, 32D-FLT3-ITD, and Human HL-60/vcr cell lines, respectively. In the part of in vivo study, DMBA was used for inducing acute myeloid leukemia in mice. CdNPs similar to daunorubicin ameliorated significantly (p≤0.01) the biochemical, inflammatory, RBC, WBC, platelet, stereological, histopathological, and cellular-molecular parameters compared to the other groups.ConclusionsAs mentioned, the cadmium nanoparticles had significant anti-acute myeloid leukemia effects. After approving the above results in the clinical trial studies, these cadmium nanoparticles can be used as a chemotherapeutic drug to treat acute myeloid leukemia in humans.

scholarly journals The Vitamin E Derivative Gamma Tocotrienol Promotes Anti-Tumor Effects in Acute Myeloid Leukemia Cell Lines

Nutrients ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 2808 ◽  
Author(s):  
Ghanem ◽  
Zouein ◽  
Mohamad ◽  
Hodroj ◽  
Haykal ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Vitamin E ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Apoptotic Cell ◽  
Leukemia Cell ◽  
Therapeutic Effects ◽  
Apoptotic Pathway ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is a blood cancer characterized by the formation of faulty defective myelogenous cells with morphological heterogeneity and cytogenic aberrations leading to a loss of their function. In an attempt to find an effective and safe AML treatment, vitamin E derivatives, including tocopherols were considered as potential anti-tumor compounds. Recently, other isoforms of vitamin E, namely tocotrienols have been proposed as potential potent anti-cancerous agents, displaying promising therapeutic effects in different cancer types. In this study we evaluated the anti-cancerous effects of γ-tocotrienol, on AML cell lines in vitro. For this purpose, AML cell lines incubated with γ-tocotrienol were examined for their viability, cell cycle status, apoptotic cell death, DNA fragmentation, production of reactive oxygen species and expression of proapoptotic proteins. Our results showed that γ-tocotrienol exhibits time and dose-dependent anti-proliferative, pro-apoptotic and antioxidant effects on U937 and KG-1 cell lines, through the upregulation of proteins involved in the intrinsic apoptotic pathway.

In Vitro and In Vivo Monitoring of Valproic Acid Effects on Gene Expression Signatures in Adult Acute Myeloid Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2605-2605
Author(s):  
Lars Bullinger ◽  
Konstanze Dohner ◽  
Richard F. Schlenk ◽  
Frank G. Rucker ◽  
Jonathan R. Pollack ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Serum Levels ◽  
Leukemia Cell Lines ◽  
Acute Myeloid ◽  
Myeloid Leukemia Cell

Abstract Inhibitors of histone deacetylases (HDACIs) like valproic acid (VPA) display activity in murine leukemia models, and induce tumor-selective cytoxicity against blasts from patients with acute myeloid leukemia (AML). However, despite of the existing knowledge of the potential function of HDACIs, there remain many unsolved questions especially regarding the factors that determine whether a cancer cell undergoes cell cycle arrest, differentiation, or death in response to HDACIs. Furthermore, there is still limited data on HDACIs effects in vivo, as well as HDACIs function in combination with standard induction chemotherapy, as most studies evaluated HDACIs as single agent in vitro. Thus, our first goal was to determine a VPA response signature in different myeloid leukemia cell lines in vitro, followed by an in vivo analysis of VPA effects in blasts from adult de novo AML patients entered within two randomized multicenter treatment trials of the German-Austrian AML Study Group. To define an VPA in vitro “response signature” we profiled gene expression in myeloid leukemia cell lines (HL-60, NB-4, HEL-1, CMK and K-562) following 48 hours of VPA treatment by using DNA Microarray technology. In accordance with previous studies in vitro VPA treatment of myeloid cell lines induced the expression of the cyclin-dependent kinase inhibitors CDKN1A and CDKN2D coding for p21 and p19, respectively. Supervised analyses revealed many genes known to be associated with a G1 arrest. In all cell lines except for CMK we examined an up-regulation of TNFSF10 coding for TRAIL, as well as differential regulation of other genes involved in apoptosis. Furthermore, gene set enrichment analyses showed a significant down-regulation of genes involved in DNA metabolism and DNA repair. Next, we evaluated the VPA effects on gene expression in AML samples collected within the AMLSG 07-04 trial for younger (age<60yrs) and within the AMLSG 06-04 trial for older adults (age>60yrs), in which patients are randomized to receive standard induction chemotherapy (idarubicine, cytarabine, and etoposide = ICE) with or without concomitant VPA. We profiled gene expression in diagnostic AML blasts and following 48 hours of treatment with ICE or ICE/VPA. First results from our ongoing analysis of in vivo VPA treated samples are in accordance with our cell line experiments as e.g. we also see an induction of CDKN1A expression. However, the picture observed is less homogenous as concomitant administration of ICE, as well as other factors, like e.g. VPA serum levels, might substantially influence the in vivo VPA response. Nevertheless, our data are likely to provide new insights into the VPA effect in vivo, and this study may proof to be useful to predict AML patients likely to benefit from VPA treatment. To achieve this goal, we are currently analyzing additional samples, and we are planning to correlate gene expression findings with histone acetylation status, VPA serum levels, cytogenetic, and molecular genetic data.

scholarly journals JQ1 Inhibits Proliferation and Induces Apoptosis of Leukemia Cells Through BCL-2 Regulated Pathway

2021 ◽  
Author(s):  
Yifan Zeng ◽  
Xing-Hua Liang ◽  
Yong Xia ◽  
Wen-Yin He
Keyword(s):  
Cell Cycle ◽  
Tumor Cells ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
High Concentration ◽  
Apoptosis Pathway ◽  
Leukemia Cell Lines ◽  
Myeloid Leukemia Cell

Abstract Objective To explore the mechanism of JQ1 on leukemia cells. Methods This study takes two myeloid leukemia cell lines as a research model. Cells treated with high concentration of JQ1 were collected for quantitative real-time PCR, immunoblot and flow cytometry to verify the effects of JQ1 on myeloid leukemia tumor cells. Combined with mRNA sequencing of cell lines to identify the differences in mRNA expression of different cell lines. Results Two cell lines changed cell morphology under JQ1 treatment. The cell membrane appeared in varying degrees of wrinkled internal subsidence. K562 cell lines can maintain stable proliferation after being induced by a specific concentration of JQ1. However, JQ1 cannot induce the death of the K562 cells. Although the MYC and BCL2 gene expression decreased, JQ1 did not affect the c-Myc targeted genes to affect the cell cycle, nor did it trigger the BCL2-mediated apoptosis pathway. On the contrary, after JQ1 induced the MV-4-11 cells, the MYC-mediated cell cycle significantly slowed down and arrested at the G0/G1 phase. The death of MV-4-11 tumor cells through the apoptosis pathway regulated by BCL-2 family. Conclusion JQ1 has different pharmacological effects on two myeloid leukemia cell lines. For MV-4-11, JQ1 mainly inhibited cell cycle by regulating MYC pathway and induced BCL-2-mediated apoptosis to kill myeloid leukemia tumor cells and thus perform anti-tumor effects. K-562 cells showed drug resistance to JQ1 which confirmed that the K-562 cell line has a feedback mechanism that prevents JQ1-induced apoptosis.

Characterization of Double BCR-ABL–positive Cell Lines Established from a Patient with Blasic Crisis of Chronic Myeloid Leukemia Who Showed Clinical Resistant to Imatinib

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4244-4244
Author(s):  
Tsuyoshi Nakamaki ◽  
Norimichi Hattori ◽  
Hidetoshi Nakashima ◽  
Takashi Maeda ◽  
Hirotsugu Ariizumi ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Kinase Domain ◽  
Abl Kinase ◽  
Leukemia Cell Lines ◽  
Clinical Resistance

Abstract Pervious in vitro studies have shown that molecular alterations of BCR-ABL-positive leukemia cells such as amplification of BCR-ABL gene and/or mutation(s) of abl kinase domain cause resistant to imatinib. However recent study showed that alterations of imatinib bioavailability might be a important factor to cause clinical resistant in BCR-ABL-positive leukemia patients, showing a differences between in vivo and in vitro sensitivity to imatinib of BCR-ABL-positive cells. To analyze mechanism(s) of clinical resistance to imatinib and to overcome the resistance, we have sequentially established and characterized two leukemia cell lines from a patient with myeloid blastic crisis of chronic myeloid leukemia (CML) who showed progressively resistant to imatinib. Case report and establishment of cell lines: a 59-years-old women developed blastic crisis preceded by four years of chronic phase of CML. Increased blasts in crisis was positive for CD13, 33 and showed double Ph-chromosome in addition to complexed chromosomal alterations such as, add(3)(p13), add(3)(q11), add(5)(q11), der(19)(3;19) (p21;q13). After repeated courses of combination chemotherapy including, 600mg of imatinib was administered orally in combination with chemotherapeutic drugs. For a brief period Imatinib showed clinical effects and slowed the increase of BCR-ABL-positive cells, however myeloblast progressively increased in peripheral blood in spite of daily administration of imatinib and she died four months treatment with imatinib. Two myeloid leukemia cell lines, NS-1 and NS-2 were established, after obtaining informed consent, from peripheral blood at day 65 and day 95 after initiation of imatinib administration, respectively. Cell surface phenotype and karyotype of these cell lines were identical to original blasts. NS-1 and NS-2 cell lines were characterized compared with BCR/ABL-positive K562 erythroleukemia cell line as a control Quantitative analysis by real-time polymerase chain reaction showed that copy number of BCR-ABL transcript were 2.2 × 105 and 1.6 × 10 5/μg RNA in NS-1 and NS-2 respectively, showing slightly lower than those (5.8 × 105) in K562 cell line. Although nucleotide sequence analysis showed that a point mutation in abl kinase domain resulted in amino acid substitution pro310ser in NS-1 cell line, no additional mutation was found in NS-2 cell line. Western blot analysis showed levels of both 210 KD BCR-ABL protein and BCR-ABL phosphorylation were similar in NS-1, NS-2 and K562 cells. Although two hours incubation with 10 mM imatinibin vitro did not show any detectable difference in levels of phosphorylation of BCR-ABL protein between NS-1 and NS-2 cell lines, sensitivity to imatinib measured by MTT assay showed that IC50 was 0.1 mM, 0.5 mM and 1.0mMin NS-1, NS-2 and K562 cell lines respectively. The measured IC50 of both NH-1 and NH-2 cell lines were much lower than reported plasma concentrations achieved by oral administration of 600 mg of imatinib (above 10 μM). The present results suggest difference between in vivo and in vitro sensitivity to imatinib indicate that alteration of bioavailability of imatinib possibly involved in clinical resistance to this drug, accumulations of BCR-ABL gene amplification and/or mutation are not necessarily a major reason of progressive clinical resistance to imatinib in BCR-ABL positive leukemia.

Preclinical Development of Triptolide Derivative MRx102 as a Therapeutic Agent for Acute Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3271-3271
Author(s):  
John M. Fidler ◽  
Jinhua An ◽  
John H. Musser ◽  
Duncan H. Mak ◽  
Bing Carter ◽  
...  
Keyword(s):  
Drug Development ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Employment Equity ◽  
Toxicology Study ◽  
Leukemia Cell Lines ◽  
Equity Ownership ◽  
Anti Tumor Activity

Abstract Abstract 3271 Acute Myeloid Leukemia (AML) is the most common form of adult acute leukemia and the second most common childhood leukemia. AML has the lowest survival rate among leukemias, and the frequency is increasing as the population ages. Current therapies are inadequate, and a need exists for better therapeutic agents to treat AML, both as initial treatment for newly diagnosed patients and for those who have failed current therapy and relapsed. Natural products, such as taxol, have shown activities in a variety of disease states, including cancer. Triptolide is a natural product diterpenoid derived from Tripterygium wilfordii Hook f, and has shown anti-cancer activity in a broad range of solid tumors in preclinical models. It induces apoptosis in various leukemic cell lines and primary AML blasts (Carter, B et al, Blood 2006). Derivatives of triptolide with improved pharmacokinetics and bioavailability offer the opportunity to optimize the activity of triptolide for clinical application in AML. MRx102 is a triptolide derivative that is more hydrophobic than triptolide. It has potent in vitro cytotoxic activity with human tumor and leukemia cell lines, an unusual result for triptolide derivatives because they are usually much less active in vitro than the parent compound. Designed as a prodrug, MRx102 exerts cytotoxic activity with human AML cell lines and other human leukemia cell lines without pre-incubation with plasma esterases (IC50 of 51.0 and 37.1 nM with MV4-11 AML cells at 48 and 72 hours, respectively, ∼55% and ∼36% of the activity of triptolide, respectively). MRx102 decreases the viable CD34+ blasts of AML patient samples (a mean of 79.8 ± 8.8% specific apoptosis at 100 nM, n=3), and overcomes the apoptosis protection by co-cultivated stromal cells (with a similar mean of 74.1 ± 8.5%). MRx102 shows dose-dependent anti-tumor activity with the MV4-11 cell line in nude mouse human AML tumor xenografts. After 42 days of MRx102 dosing at 1.35 mg/kg/day i.p., tumor volume was inhibited by 99.7%. Tumors removed from several mice appeared to be Matrigel pellets rather than vascularized tumors, suggesting that many of the tumors were completely eliminated. In studies with the OCI-AML3 human AML cell line xenograft model, the group receiving MRx102 at 1.35 mg/kg/day i.p. showed similar high activity, with mean tumor volume reduced by as much as 99.2% on day 23 compared to the vehicle control group. Tumors of 7 of 10 mice were smaller than the day 0 volumes at the day 28 end of the study. As part of drug development, toxicology testing with MRx102 was initiated with an acute single dose rat toxicology study with no deaths and no adverse signs up to the top dose of 3.0 mg/kg MRx102 in DMSO/PBS administered i.v. The maximum tolerated dose (MTD) is greater than 3 mg/kg of MRx102, and the no observable adverse effect level (NOAEL) is at least 3 mg/kg. A 7-day subacute rat toxicology study of MRx102 showed no deaths and no adverse signs up to the top dose of 1.5 mg/kg/day MRx102 in DMSO/PBS administered daily i.v. for 7 days. The histopatholgy report shows no findings related to administration of the test article. The MRx102 MTD is greater than 1.5 mg/kg/day, and the NOAEL is at least 1.5 mg/kg/day. Previously observed NOAELs for related compounds have been less than 0.1 mg/kg/day. The current studies show potent anti-tumor activity as well as an unusually positive safety profile for MRx102 when compared to triptolide and other triptolide derivatives. Further MRx102 drug development is underway, with the intention of submitting an Investigational New Drug application to the Food and Drug Administration leading to clinical evaluation of MRx102 in AML patients. Updated results on current drug development activities will be presented at the meeting. This work is supported in part by NCI SBIR Contract HHSN261200900061C to MyeloRx LLC. Disclosures: Fidler: MyeloRx LLC: Employment, Equity Ownership, PI for an NCI Contract to MyeloRx LLC, Patents & Royalties. An:MyeloRx LLC: Employment, Equity Ownership, participant in research under an NCI SBIR Contract to MyeloRx LLC. Musser:MyeloRx LLC: Employment, Equity Ownership, Patents & Royalties, participant in research under an NCI SBIR Contract to MyeloRx LLC. Mak:MyeloRx LLC: participant in research under an NCI SBIR Contract to MyeloRx LLC. Carter:MyeloRx LLC: participant in research under an NCI SBIR Contract to MyeloRx LLC. Andreeff:MyeloRx LLC: Consultancy, participant in research under an NCI SBIR Contract to MyeloRx LLC.

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