NF Kappa B as a Therapeutic Target in AML.

Abstract NF Kappa B is an inducible eukaryotic transcription factor, which is highly conserved throughout nature, and regulates a large number of genes which are essential for immune and inflammatory responses. Inappropriate expression has been implicated in a number of inflammatory and malignant conditions. We have investigated the expression of NF Kappa B in normal mobilised peripheral blood cells, AML cell lines (HL60, NB4, K562, U937) and 27 primary AML cells, with a view with it being a target for NF Kappa B inhibition. The cells were also treated with a range of doses of LC1 (Leuchemix Inc) which includes NF-KB inhibition in its activity profile. The expression of NF Kappa B was determined by Affymetrix Gene Chip Array (22,283 genes) and confirmed by RT-PCR. Overall the leukaemic cells had a greater than a two-fold expression compared with normal cells. Expression was significantly greater in the FAB M2 and M4 subgroups and tended to increase with the degree of morphological differentiation, and with poor risk cytogenetics. There was no relationship between expression age, gender or FLT3 mutation status. Since increased NF Kappa B expression confers resistance to apoptosis, the level of spontaneous apoptosis of cells after 24 hours in vitro was assessed by annexin V expression. Apoptosis was inversely correlated with expression. LC1 (Leuchemix Inc) is a novel agent with NF Kappa B inhibition among its properties. Cells were treated with LC1 in a dose range of 0.002 to 20μmol, and the LC50 determined in an MTS assay. For the cell lines HL60, NB4 and U937 the average LC50 was 6.1μmol. K562 cells have a lower expression of NF Kappa B (40%) compared with the other cell lines and this was reflected in a lower in vitro sensitivity (LC50 > 100μmol). The LC50s for the 27 primary AML stem cell samples ranged from 0.61μmol to > 100μmol with a mode of 7.1μmol. The majority of the cases (24/27) were between 0.61 and 13.2μmol with a mean of 5.9μmol while the 3 outliers have an LC50 which was 4–10 fold higher. In order to assess whether LC1 enhance the cytotoxicity of Ara-C co-incubation experiments were undertaken using the MTS assay. Significant synergy was observed in 8 or 27 cases, and antagonism in 12 of 27 cases. These data suggest that NF Kappa B inhibition with LC1 merits further evaluation as targeted treatment alone, or in combination, in AML.

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Arsenic Trioxide Plus ABT-737 Is Synergistic to Induce Apoptosis in AML Cells by Repression of Mcl-1 Protein and Inactivation of Bcl-2 Protein

Blood ◽

10.1182/blood.v112.11.2653.2653 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2653-2653

Author(s):

Lijuan Xia ◽

Rui Wang ◽

Hao Qian ◽

Janice Gabrilove ◽

Samuel Waxman ◽

...

Keyword(s):

Arsenic Trioxide ◽

Cell Lines ◽

Single Agent ◽

Annexin V ◽

K562 Cells ◽

Parp Cleavage ◽

Key Factor ◽

Induced Apoptosis ◽

Apoptotic Effects

Abstract Arsenic trioxide as a single agent induces remission in acute promyelocytic leukemia (APL) patients with minimal toxicity but not in other types of acute myeloid leukemia (AML). In vitro, APL as compared to AML cells are more sensitive to arsenic trioxide-induced apoptosis. Arsenic trioxide-induced apoptosis in APL cells results from activation of a mitochondria-mediated pathway. The Bcl-2 antiapoptotic protein family members Bcl-2, Bcl-XL, Bfl-1 and Mcl-1 block mitochondria-mediated apoptosis. In studies of several AML cell lines, we detected high levels of Bcl-2 and Mcl-1 protein, but lower or no expression of Bcl-XL and Bfl-1. Arsenic trioxide treatment decreased the levels of Mcl-1 without inducing apoptosis in AML cells suggesting that Bcl-2 would be a key factor causing arsenic trioxide resistance. We therefore hypothesize that inhibitors of Bcl-2 might restore arsenic trioxide-induced programmed cell death. In this study, the apoptotic effects of arsenic trioxide, ABT-737 (a potent Bcl-2 inhibitor) and their combination were investigated in NB4, HL-60, U937 and K562 cells. Arsenic trioxide at 1–2 μM induced apoptosis only in NB4 cells but decreased the levels of Mcl-1 in all of the four cell lines. ABT-737 at concentrations lower than 5 μM induced apoptosis in NB4, HL-60 and U937 cells but not in K562 cells which had undetectable Bcl-2 levels. Arsenic trioxide (2 μM) plus ABT-737 (0.05–0.5 μM) synergistically induced apoptosis in HL-60 and U937 but not in K562 cells as determined by PARP cleavage and Annexin V staining. Our data suggest that inhibition of Mcl-1 expression by arsenic trioxide and inactivation of Bcl-2 activity by ABT-737 leads to the synergistic apoptosis observed with this combination and is the basis for a novel treatment for AML.

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In Vitro and In Vivo Study of Synergistic Effect of Imatinib and Simvastatin in Chronic Myelogenous Leukemia.

Blood ◽

10.1182/blood.v114.22.2202.2202 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2202-2202

Author(s):

Bora Oh ◽

Dong Soon Lee ◽

Tae Young Kim ◽

Hyun Jung Min ◽

Yun Song Lee ◽

...

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Annexin V ◽

K562 Cells ◽

Myelogenous Leukemia ◽

Killing Effect ◽

Rna Transfection ◽

Imatinib Resistant

Abstract Abstract 2202 Poster Board II-179 Background: Statins (HMG-CoA reductase inhibitors) are known to show anti-proliferative effects and are anticipated as a potential drug in the treatment of malignancies. To investigate the effect simvastatin on chronic myelogenous leukemia cells, we treated simvastatin on 3 kinds of CML cell lines and CD34+ primary CML cells from patients: erythrocytic lineage (K-562), granulocytic lineage (KCL-22), erythroid-megakaryocytic (LAMA-84) cell line. Also, antiproliferative effect on imatinib-resistant CML cell lines was assessed. Mehtods: Anti-proliferative effect was assessed by CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI). Apoptosis was assessed by Annexin V and Western blot study. Killing effect was calculated by CalcuSyn does effect analysis software (Biosoft, Ferguson, MO). CD34+cells for patients with CML were purified using immunomagnetic bead column. Cell cycle analysis was done by flow cytometrc method. Si-RNA transfection study for p27 gene was performed for verification of killing mechanism. Change of intracellular location of BCR/ABL protein was observed by confocal microscopy. Cellular changes of proteins and tyrosin phosphorylation after treatment of simvastain was evaluated by 2 dimensional electrophoresis and MALDI-TOF/TOF mass spectrophotometer. In vivo effect of simvastain was evaluated in BALB/c-nude. Results: Simvastatin inhibited the proliferation of imatinib-sensitive and imatinib-resistant K562 cell line in a dose dependent manner. The IC50 values of simvastatin and imatinib in imatinib-sensitive K562 cells were 14.5 and 0.4 μM. Treatment of simvastain induced apoptosis both in capase-dependent and caspase-independent pathways in all 3 cell lines; apoptosis by Annexin V analysis and increased apoptotic proteins (cytochrome C, AIF, Smac/Diablo, caspase-3, and caspase-9) by western blot. Cell cycle analysis revealed the G1/S arrest on treatment of simvastatin and si-RNA transfection specific for p27 reversed the G1/S arrest, suggesting cell cycle arrest as one of anti-proliferative mechanism. Co-treatment of imatinib and simvastain showed synergistic killing interactions between simvastatin and imatinib in imatinib-resistant K562 cells (mean combination index values were 0.56, P< 0.001). Enhanced killing effect was observed in all 3 imatinib-resistant CML cell lines (K-562: 0.6, KCL-22: 0.42 , LAMA-84; 0.99). Co-treatment with imatinib and simvastatin decreased the amount of Bcr-Abl protein and stimulated the import of Abl protein in the nuclei in K562 cells. In CML cells, simvastatin inhibited tyrosine phosphorylation included protease, cytocrome-c reductase, DNA/RNA processing proteins, oxidoructase protein, chaperones, glycolysis protein, cytoskeleton proteins, microtubule protein. Treatment of simvastain reduced subcutaneous tumor mass in nude mice. Conclusion: We showed that simvastatin killed CML cells in vitro and in vivo animal model and killing effect occurred via the induction of apoptosis, cell cyle arrest via p27 and inhibited BCR/ABL tyrosine kinase (TK) activity. Simvastatin may be a potential candidate for the treatment of imatinib-resistant CML patients and the effective dose of imatinib could be reduced in a combined treatment with simvastatin. Disclosures: No relevant conflicts of interest to declare.

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Anti-K562 cell monoclonal antibody RTF8X recognizes tumor-associated antigen of erythroid lineage

Blood ◽

10.1182/blood.v72.5.1487.1487 ◽

1988 ◽

Vol 72 (5) ◽

pp. 1487-1491 ◽

Cited By ~ 1

Author(s):

T Sakurai ◽

H Hara ◽

K Nagai

Keyword(s):

Monoclonal Antibody ◽

Cell Lines ◽

Surface Antigen ◽

K562 Cell ◽

K562 Cells ◽

Erythroid Differentiation ◽

Enzyme Treatment ◽

Normal Peripheral Blood ◽

Marrow Cells

Abstract A new anti-K562 cell monoclonal antibody, RTF8X, a cytotoxic IgM, recognized a surface antigen on erythroblasts from patients with erythroleukemia and polycythemia vera. RTF8X, which is highly specific to K562 cells, did not react with the other 14 hematopoietic cell lines and the seven nonhematopoietic cell lines. RTF8X antigen was not detected in normal peripheral blood, but was found in less than 1% of normal marrow cells. RTF8X did not inhibit in vitro colony formation of CFU-E and BFU-E in a complement-dependent cytotoxicity assay. Cell- sorting analysis showed that, morphologically, the RTF8X-positive marrow cells from the patients and normal volunteers contained more than 60% erythroblasts and that CFU-E and BFU-E were not demonstrated in cells with RTF8X antigen. Enzyme treatment suggested that RTF8X antigen was a sialoglycolipid. These results indicate that RTF8X may recognize the surface antigen found increasingly in association with tumors of erythroid lineage. RTF8X should be useful for studies of erythroid differentiation and proliferation in patients.

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PEG Graft Polymer Carriers of Antioxidants: In Vitro Evaluation for Transdermal Delivery

Pharmaceutics ◽

10.3390/pharmaceutics12121178 ◽

2020 ◽

Vol 12 (12) ◽

pp. 1178

Author(s):

Justyna Odrobińska ◽

Magdalena Skonieczna ◽

Dorota Neugebauer

Keyword(s):

Cell Lines ◽

Graft Copolymers ◽

Biological Response ◽

Annexin V ◽

Dermal Fibroblasts ◽

In Vitro Tests ◽

Side Chains ◽

Skin Cell ◽

Active Substances

The in vitro biochemical evaluation of the applicability of polymers carrying active substances (micelles and conjugates) was carried out. Previously designed amphiphilic graft copolymers with retinol or 4-n-butylresorcinol functionalized polymethacrylate backbone and poly(ethylene glycol) (PEG) side chains that included Janus-type heterografted copolymers containing both PEG and poly(ε-caprolactone) (PCL) side chains were applied as micellar carriers. The polymer self-assemblies were convenient to encapsulate arbutin (ARB) as the selected active substances. Moreover, the conjugates of PEG graft copolymers with ferulic acid (FA) or lipoic acid (LA) were also investigated. The permeability of released active substances through a membrane mimicking skin was evaluated by conducting transdermal tests in Franz diffusion cells. The biological response to new carriers with active substances was tested across cell lines, including normal human dermal fibroblasts (NHDF), human epidermal keratinocyte (HaCaT), as well as cancer melanoma (Me45) and metastatic human melanoma (451-Lu), for comparison. These polymer systems were safe and non-cytotoxic at the tested concentrations for healthy skin cell lines according to the MTT test. Cytometric evaluation of cell cycles as well as cell death defined by Annexin-V apoptosis assays and senescence tests showed no significant changes under action of the delivery systems, as compared to the control cells. In vitro tests confirmed the biochemical potential of these antioxidant carriers as beneficial components in cosmetic products, especially applied in the form of masks and eye pads.

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Dasatinib (BMS-354825) Overcomes Multiple Mechanisms of Imatinib Resistance in Chronic Myeloid Leukemia (CML).

Blood ◽

10.1182/blood.v106.11.1994.1994 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1994-1994 ◽

Cited By ~ 10

Author(s):

Francis Y. Lee ◽

Mei-Li Wen ◽

Rajeev Bhide ◽

Amy Camuso ◽

Stephen Castenada ◽

...

Keyword(s):

Cell Lines ◽

Kinase Inhibitor ◽

Leukemic Cell ◽

K562 Cells ◽

Src Family Kinases ◽

Imatinib Resistance ◽

Over Expression ◽

Imatinib Resistant

Abstract Resistance to imatinib is a growing concern in CML, particularly in advanced disease. The most common cause of resistance is mutations in BCR-ABL, but other mechanisms have also been identified, including over-expression of BCR-ABL, activation of SRC family kinases and the P-glycoprotein (PGP) efflux pump (via MDR1 over-expression). Dasatinib (BMS-354825) is a novel, oral, multi-targeted tyrosine kinase inhibitor that targets BCR-ABL and SRC kinases. Dasatinib has 325-fold greater potency versus imatinib in cell lines transduced with wild-type BCR-ABL and is active against 18 out of 19 BCR-ABL mutations tested that confer imatinib resistance (Shah et al, Science305:399, 2004; O’Hare et al, Cancer Res65:4500–5, 2005), and preliminary results from a Phase I study show that it is well tolerated and has significant activity in imatinib-resistant patients in all phases of CML (Sawyers et al, J Clin Oncol23:565s, 2005; Talpaz et al, J Clin Oncol23:564s, 2005). We assessed the ability of dasatinib to overcome a variety of mechanisms of imatinib resistance. First, the leukemic-cell killing activity of dasatinib was tested in vitro in three human imatinib-resistant CML cell lines (K562/IM, MEG-01/IM and SUP-B15/IM). Based on IC50 values, dasatinib had >1000-fold more potent leukemic-cell killing activity compared with imatinib versus all three cell lines. Furthermore, in mice bearing K562/IM xenografts, dasatinib was curative at doses >5 mg/kg, while imatinib had little or no impact at doses as high as 150 mg/kg, its maximum tolerated dose. We determined that the MEG-01/IM and SUP-B15/IM cell lines carried BCR-ABL mutations known to confer imatinib resistance to imatinib clinically (Q252H and F359V, respectively). In K562/IM cells, BCR-ABL mutations or BCR-ABL over-expression were not detected, but the SRC family member FYN was over-expressed. PP2, a known inhibitor of SRC family kinases but not BCR-ABL, could reverse the imatinib resistance in these cells. Together, these data suggest that activation of FYN may be a cause of imatinib resistance in K562/IM. Based on cell proliferation IC50, we found that the anti-leukemic activity of dasatinib in K562/IM cells was 29-fold more potent compared with AMN107 (a tyrosine kinase inhibitor that inhibits BCR-ABL but not SRC family kinases). Given that the human serum protein binding of dasatinib, imatinib and AMN107 were 93, 92 and >99% respectively, the difference in potency between dasatinib and AMN107 in vivo may be far greater than the simple fold-difference in the in vitro IC50 values. Finally, in K562 cells over-expressing PGP (K562/ADM), we found that dasatinib was only 6-fold less active than in parental K562 cells. Because of the extreme potency of dasatinib in K562 cells, this reduced potency still afforded an IC50 of 3 nM, which is readily achievable in vivo. Indeed, in mice bearing K562/ADM xenografts, dasatinib was curative at 30 mg/kg, with significant anti-leukemic activity at 15 mg/kg. In conclusion, the rational design of dasatinib as a multi-targeted kinase inhibitor allows this agent to overcome a variety of mechanisms of resistance to imatinib in CML, including mechanisms that are not overcome by agents with a narrower spectrum of inhibition, such as AMN107. Dasatinib is currently in Phase II evaluation in imatinib-resistant/-intolerant patients in the ‘START’ program, and in Phase I evaluation in solid tumors.

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A New Abl Kinase Inhibitor (AMN107) Has In Vitro Activity on Chronic Myeloid Leukaemia (CML) Ph+ Cells Resistant to Imatinib.

Blood ◽

10.1182/blood.v106.11.2004.2004 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2004-2004 ◽

Cited By ~ 2

Author(s):

Giovanni Martinelli ◽

Alberto M. Martelli ◽

Tiziana Grafone ◽

Irina Mantovani ◽

Alessandra Cappellini ◽

...

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Cell Cycle Progression ◽

Kinase Inhibitor ◽

K562 Cells ◽

Sh2 Domain ◽

Regulatory Subunit ◽

Imatinib Resistance ◽

First Line Therapy

Abstract Imatinib mesylate (Novartis Pharma), an inhibitor of the bcr/abl tyrosine kinase, has rapidly become the first-line therapy for CML. Imatinib has proved remarkably effective at reducing the number of leukaemia cells in individual CML patients and promises to prolong life substantially in comparison with earlier treatments. However, in patients in advanced phases of the disease, the development of resistance to this drug is a frequent setback. Therefore, new inhibitors of bcr/abl are needed. Very recently, a new bcr/abl inhibitor, AMN107 (Novartis Pharma), has been developed. We have tested AMN107 on human leukaemia cell lines and on blasts isolated from imatinib-resistant CML patients. After a 24 h incubation, AMN107 (10 nM) blocked K562 cells in the G1 phase of the cell cycle. To obtain the same effect with imatinib, a 200 nM concentration was required. AMN107 had no affect on cell cycle progression of bcr/abl-negative cell lines such as HL60 and NB4, even if the concentration was raised to 500 nM. After 48 h incubation, AMN107 (10 nM) was capable of inducing a massive apoptosis of K562 cells whereas, once again, 200 nM imatinib was required to obtain the same effect. Western blot analysis with phosphospecific antibodies revealed that in K562 cells AMN107 (50 nM) markedly down-regulated autophosphorylation of bcr/abl Tyr177 and Tyr412, whereas autophosphorylation of Thr735 was unaffected. In contrast, imatinib even if used at 200 nM, did not diminish phosphorylation of either bcr/abl Tyr177 or Tyr412. This finding seems particularly important because recent evidence has demonstrated that the signalling pathway emanating from Tyr177 plays a major role in the pathogenesis of CML. Indeed, phosphorylated Tyr177 forms a high-affinity binding site for the SH2 domain of the adapter Grb2. The main effectors of Grb2 are Sos and Ras, however Grb2 also recruits the scaffolding adapter protein Gab2 to bcr/abl via a Grb2-Gab2 complex, which results in activation of phosphoinositide 3-kinase (PI3K)/Akt and Erk signalling networks. Consistently, we found by immunoprecipitation decreased levels of bcr/abl-associated Gab2, Grab2, and p85 regulatory subunit of PI3K in AMN107-treated cells. AMN107 treatment of K562 cells also caused a reduction of STAT5, cCBL, CRKL, and Akt phosphorylation levels, as well as Bcl-XL expression. AMN107 (5 μM for 24h) significantly increased the apoptosis rate of CML blasts isolated from patients resistant to imatinib. Therefore, AMN107 might represent a new bcr/abl selective inhibitor useful for overcoming imatinib resistance.

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ABT-737, an Inhibitor of Bcl-2/Bcl-XL Proteins, Is Cytotoxic in Multiple Myeloma Cells.

Blood ◽

10.1182/blood.v106.11.1589.1589 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1589-1589

Author(s):

Michael Kline ◽

Terry Kimlinger ◽

Michael Timm ◽

Jessica Haug ◽

John A. Lust ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Tumor Microenvironment ◽

Cell Lines ◽

Plasma Cells ◽

Annexin V ◽

Significant Activity ◽

Rpmi 8226 ◽

Dose Dependent

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that is incurable with the currently available therapeutics. New therapies based on better understanding of the disease biology are urgently needed. MM is characterized by accumulation of malignant plasma cells predominantly in the bone marrow. These plasma cells exhibit a relatively low proliferative rate as well as a low rate of apoptosis. Elevated expression of the anti-apoptotic Bcl-2 family members has been reported in MM cell lines as well as in primary patient samples and may be correlated with disease stage as well as resistance to therapy. ABT-737 (Abbott Laboratories, Abbott Park, IL) is a small-molecule inhibitor designed to specifically inhibit anti-apoptotic proteins of the Bcl-2 family and binds with high affinity to Bcl-XL, Bcl-2, and Bcl-w. ABT-737 exhibits toxicity in human tumor cell lines, malignant primary cells, and mouse tumor models. We have examined the in vitro activity of this compound in the context of MM to develop a rationale for future clinical evaluation. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity of ABT-737 was measured using the MTT viability assay. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Flow cytometry was also used to measure BAX: Bcl-2 ratios after ABT-737 treatment and cell permeabilization with FIX & PERM (Caltag Laboratories, Burlingame, CA) Results: ABT-737 exhibited cytotoxicity in several MM cell lines including RPMI 8226, KAS-6/1, OPM-1, OPM-2, and U266 with an LC50 of 5-10μM. The drug also had significant activity against MM cell lines resistant to conventional agents such as melphalan (LR5) and dexamethasone (MM1.R) with similar LC50 (5-10 μM), as well as against doxorubicin resistant cells (Dox40), albeit at higher doses. Furthermore, ABT-737 retained activity in culture conditions reflective of the permissive tumor microenvironment, namely in the presence of VEGF, IL-6, or in co-culture with marrow-derived stromal cells. ABT-737 was also cytotoxic to freshly isolated primary patient MM cells. Time and dose dependent induction of apoptosis was confirmed using Annexin V/PI staining of the MM cell line RPMI 8226. Flow cytometry analysis of cells treated with ABT-737 demonstrated a time and dose dependent increase in pro-apoptotic BAX protein expression without significant change in the Bcl-XL or Bcl-2 expression. Ongoing studies are examining the parameters and mechanisms of ABT-737 cytotoxicity to MM cells in more detail. Conclusion: ABT-737 has significant activity against MM cell lines and patient derived primary MM cells in vitro. It is able to overcome resistance to conventional anti-myeloma agents suggesting a different mechanism of toxicity that may replace or supplement these therapies. Additionally, it appears to be able to overcome resistance offered by elements of the tumor microenvironment. The results of these studies will form the framework for future clinical evaluation of this agent in the clinical setting.

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Priming Effect with G-CSF Enhances In Vitro Apoptosis by Treatment with Ara-C and VP-16 in Leukemic Cell Line.

Blood ◽

10.1182/blood.v110.11.4333.4333 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4333-4333

Author(s):

Jun-ichi Kitagawa ◽

Takeshi Hara ◽

Hisashi Tsurumi ◽

Nobuhiro Kanemura ◽

Masahito Shimizu ◽

...

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Priming Effect ◽

Low Dose ◽

Combined Treatment ◽

Annexin V ◽

Leukemic Cell ◽

Leukemic Cells ◽

Significant Difference

Abstract Introduction: We have recently reported that the effectiveness of low dose Ara-C, VP-16 and G-CSF (AVG therapy) for elderly AML patients who were ineligible for intensive chemotherapy (Hematol Oncol, in press). G-CSF has been reported to potentiate in vitro anti-leukemic effect of Ara-C. The mechanism of the potentiation is assumed to recruit quiescent G0 leukemic cells into cell cycle. We hypothesized that the enhanced cytotoxicity was due to the apoptosis by the effect of the priming of G-CSF, and the effect was depended on the cell cycle. In order to afford proof of this hypothesis, we assayed proliferation, apoptosis, and cell cycle in leukemic cell lines. Materials: Ara-C, VP-16, G-CSF was provided by Nippon Shinyaku, Nihonkayaku, Chugai pharmacy, respectively, Tokyo, Japan. 32D and HL-60 were obtained from RIKEN Bioresource Center Cell Bank (Ibaragi, Japan), Ba/F3 was generous gifts from Dr. Kume, Jichi medical school, Tochigi, Japan. Methods: 5 x 105/ml HL60, 32D and Ba/F3 were cultured with various concentrations of Ara-C and/or VP-16 in the presence or absence of G-CSF 50ng/ml for 3 days. At the end of the culture, cell proliferation and viability were determined by using the trypan blue. The Annexin V-binding capacity of treated cells was examined by flow cytometry using ANNEXIN V-FITC APOPTOSIS DETECTION KIT I purchased from BD Pharmingen™. Cell cycle analysis was done with BrdU Flow KIT purchased from BD Pharmingen™. The incorporated BrdU was stained with specific anti-BrdU fluorescent antibodies, and the levels of cell-associated BrdU are then measured by flow cytometory. Result: Ara-C and VP-16 inhibited proliferation and decreased viability of leukemic cell lines dose-dependently. Half killing concentration (IC50) was redused in combination of Ara-C and VP-16 than Ara-C or VP-16 alone. In G-CSF dependent cell line (32D), IC50 was redeced in the presence of G-CSF than absence of G-CSF at G-CSF, and there was no significant difference between with and without G-CSF in G-CSF independent cell lines (HL-60, Ba/F3) (p<0.05). In combined treatment of low dose Ara-C (10−7M) and VP-16 (10−7M), the percentage of apoptotic cells were increased to 20.67% from 13.04% by addition of G-CSF in 32D, and there was no significant differencebetween with and without G-CSF in HL-60 and Ba/F3 (p<0.05). At combined treatment of low dose Ara-C and VP-16, the percentage of G0/G1 phase cells were decreased to 43.94% from 35.63% and S phase cells were increased to 29.50% from 24.05% in 32D by addition of G-CSF, and there was no significant difference between with and without G-CSF in HL-60 and Ba/F3 (p<0.05). Discussion: We first showed a combination effect of Ara-C and VP-16. Next we demonstrated that the potentiation of the cytotoxicity was mediated through the mechanism of apoptosis, and apoptosis played an important role for eradicating leukemic cells by low dose Ara-C and VP-16. And G-CSF recruited cells G0/G1 phase into S phase in G-CSF dependent cells by addition of G-CSF. These results suggest that priming effect of G-CSF significantly potentiate the cytotoxicity mediated by AVG chemotherapy. Conclusion: The priming effect of G-CSF might be admitted at least of a part in AML cells.

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NPI-2358, a Novel Vascular Disrupting Agent Blocks Growth and Angiogenesis in Multiple Myeloma Cells.

Blood ◽

10.1182/blood.v114.22.3842.3842 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3842-3842

Author(s):

Dharminder Chauhan ◽

Ajita V. Singh ◽

Madhavi Bandi ◽

Noopur Raje ◽

Robert L Schlossman ◽

...

Keyword(s):

Cell Lines ◽

Annexin V ◽

Vascular Endothelial ◽

Caspase 9 ◽

Migration Assays ◽

Induced Apoptosis ◽

Rpmi 8226 ◽

Anti Tumor Activity

Abstract Abstract 3842 Poster Board III-778 Background and Rationale Vascular disrupting agents (VDAs) act via selectively disrupting established tumor vasculature and have shown remarkable clinical success as anti-cancer therapies. NPI-2358 is a novel VDA with a distinct structure and mechanism of action from other available VDAs. NPI-2358 binds to the colchicine-binding site of beta-tubulin preventing polymerization and disrupting the cytoplasmic microtubule network, thereby causing loss of vascular endothelial cytoskeletal function, and inducing cytotoxicity in cancer cells. Here, we examined the anti-angiogenic and anti-tumor activity of NPI-2358 in multiple myeloma (MM) cells using both in vitro and in vivo model systems. Material and Methods We utilized MM.1S, MM.1R, RPMI-8226, U266, and INA-6 human MM cell lines, as well as purified tumor cells from MM patients relapsing after prior anti-MM therapies. Cell viability/apoptosis assays were performed using MTT, trypan blue exclusion, and Annexin V/PI staining. Angiogenesis was measured in vitro using Matrigel capillary-like tube structure formation assays: Since human vascular endothelial cells (HUVECs) plated onto Matrigel differentiate and form capillary-like tube structures similar to in vivo neovascularization, this assay measures anti-angiogenic effects of drugs/agents. Migration assays were performed using transwell insert assays. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, PARP, Bcl-2, Bax, pJNK and GAPDH. Statistical significance was determined using a Student t test. Results Treatment of MM.1S, RPMI-8226, MM.1R, INA-6, and KMS-12BM with NPI-2358 for 24h induces a dose-dependent significant (P < 0.005) decrease in viability of all cell lines (IC50 range: 5-8 nM; n=3). To determine whether NPI-2358-induced decrease in viability is due to apoptosis, MM cell lines were treated with NPI-2358 for 24h; harvested, and analyzed for apoptosis using Annexin V/PI staining. A significant increase in NPI-2358-induced apoptosis was observed in all MM cell lines (% Annexin V+/PI- apoptotic cells: MM.1S, 48 ± 2.3%; MM.1R, 46.6 ± 3.1%; RPMI-8226, 61.7 ± 4.5%; and INA-6, 59.9 ± 3.2%; P < 0.05; n=3). Importantly, NPI-2358 decreased viability of freshly isolated MM cells from patients (IC50 range: 3-7 nM; P < 0.005), without affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index for NPI-2358. Examination of in vitro angiogenesis using capillary-like tube structure formation assay showed that even low doses of NPI-2358 (7 nM treatment for 12h; IC50: 20 nM at 24h) significantly decreased tubule formation in HUVECs (70-80% decrease; P < 0.05). Transwell insert assays showed a marked reduction in serum-dependent migration of NPI-2358-treated MM cells (42 ± 2.1% inhibition in NPI-2358-treated vs. control; P < 0.05). NPI-2358 at the concentrations tested (5 nM for 12h) in the migration assays did not affect survival of MM cells (> 95% viable cells). A similar anti-migration activity of NPI-2358 was noted against HUVEC cells (48 ± 1.7% decrease in migration; P < 0.05). Mechanistic studies showed that NPI-2358-induced apoptosis was associated with activation of caspase-8, caspase-9, caspase-3 and PARP. Importantly, treatment of MM.1S cells with NPI-2358 (5 nM) triggered phosphorylation of c-Jun amino-terminal kinase (JNK), a classical stress response protein, without affecting Bcl-2 family members Bax and Bcl-2. Blockade of JNK using dominant negative strategy markedly abrogated NPI-2358-induced apoptosis. Conclusion Our preclinical data provide evidence for remarkable anti-angiogenic and anti-tumor activity of NPI-2358 against MM cells, without significant toxicity in normal cells. Ongoing studies are examining in vivo anti-MM activity of NPI-2358 in animal models. Importantly, a Phase-1 study of NPI-2358 as a single agent in patients with advanced malignancies (lung, prostrate and colon cancer) has already established a favorable pharmacokinetic, pharmacodynamic and safety profile; and, a Phase-2 study of the combination of NPI-2358 and docetaxel in non-small cell lung cancer showed encouraging safety, pharmacokinetic and activity data. These findings, coupled with our preclinical studies, provide the framework for the development of NPI-2358-based novel therapies to improve patient outcome in MM. Disclosures: Chauhan: Nereus Pharmaceuticals, Inc: Consultancy. Lloyd:Nereus Pharmaceuticals, In: Employment. Palladino:Nereus Pharmaceuticals, Inc: Employment. Anderson:Nereus Pharmaceuticals, Inc: Consultancy.

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Gene Expression Profiles of Hydroxyurea Tolerant Cell Lines Identify Genetic Mechanisms That Influence Hydroxyurea Maximum Tolerated Dose

Blood ◽

10.1182/blood.v120.21.1005.1005 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1005-1005

Author(s):

Rosa Diaz ◽

Jonathan M Flanagan ◽

Thad A Howard ◽

Russell E. Ware

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Cell Lines ◽

Expression Profiles ◽

K562 Cells ◽

Maximum Tolerated Dose ◽

False Discovery ◽

Microarray Expression ◽

Tolerant Cell

Abstract Abstract 1005 Hydroxyurea has emerged over the past decade as an effective therapeutic agent for patients with sickle cell anemia (SCA). However, drug dosing and hematological responses can be highly variable; both %HbF response and maximum tolerated dose (MTD) vary widely among patients with SCA who receive hydroxyurea treatment. To obtain further insight into the cellular and molecular pathways, as well as genetic factors that might influence the hydroxyurea MTD, K562 erythroleukemia cells were exposed to hydroxyurea in vitro, to create cell lines that were highly drug tolerant to doses ranging from 250μM to 1500μM. Cell lines had dose-response curves that exhibited clear drug tolerance; naïve K562 showed 50% proliferation in the presence of 250μM hydroxyurea, while tolerant cell lines showed >90% proliferation at the same dose as measured by the BrdU Cell Proliferation Assay. In addition, the tolerant lines showed normal and equivalent progression through cell cycle by flow cytometry cell cycle analysis. After 15 weeks of continuous exposure, cells were harvested and mRNA microarray expression profiles were analyzed for naïve K562 (no hydroxyurea exposure) and cell lines tolerant to 500, 1000, or 1500μM hydroxyurea. Gene expression was measured on Affymetrix U133 Plus 2.0 chips. Differential expression between sample groups was determined using ANOVA, and p-values were corrected for multiple testing using the Benjamin-Hochberg false discovery rate (FDR) method to identify genetic profiles and genes consistently increased or decreased compared to naïve K562 cells. Using a threshold of 2-fold change compared to untreated cells and a false discovery rate <5%, a total of 864 genes were significantly altered in hydroxyurea tolerant cells, including 337 genes whose expression consistently correlated with increasing hydroxyurea dose (Pearson correlation p<.001). The PANTHER classification system was used to group genes into categories based on molecular functions. Of the genes that correlated significantly with increasing hydroxyurea dosing (n=337), there were 181 up-regulated genes and 156 down-regulated genes that had molecular functions including catalytic activity, binding, transcription regulator activity and transporter activity. Genes with transporter activity included SLC6A19, ATP6VOD1, ABCG2, ATP6V1B2 and KCNN4. Other genes of interest based on function included RRM2, PLS3, KCNAB2, UBE2A and SRI. Real-time quantitative reverse transcription (RT)-PCR then quantified the expression of 20 candidate genes to verify the accuracy of the microarray expression data. The next steps will include correlation of these findings with clinical data, specifically early reticulocyte mRNA expression and hydroxyurea MTD values obtained from children with SCA enrolled in the prospective Hydroxyurea Study of Long-term Effects (HUSTLE, NCT00305175). These data document that continuous in vitro exposure of K562 cells to hydroxyurea leads to tolerant cell lines that feature substantial changes in gene expression. Altered expression of certain genes present in erythroid cells including RRM2 and membrane transporters represent compensatory changes in response to hydroxyurea exposure, and may help explain the variability in hydroxyurea MTD observed among patients with SCA. Disclosures: Off Label Use: Hydroxyurea is not FDA approved for pediatric sickle cell patients. Howard:Baylor College of Medicine: Employment.

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