Characterization of Gene Expression Associated with Cyclophosphamide Resistance in Chronic Myeloid Leukemia (CML).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1532-1532
Author(s):  
Fei Bao ◽  
Mary L. Nordberg ◽  
Paula Polk ◽  
Amanda Sun ◽  
David Murray ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Resistant Cell ◽  
Parental Line ◽  
Resistant Line ◽  
Rt Pcr

Abstract Cyclophosphamide (CP) is one of the alkylating agents collectively referred to as oxazaphosphorines that are used to treat many types of cancers including myeloid leukemia. Tumor cell drug resistance is an important factor for clinical treatment failure. The mechanisms of drug resistance are multifactorial and incompletely understood. KBM-7 human CML cell line was established from blast cells from a patient in the terminal phase of CML. In the CP resistance model, the B5-180 sub-line was isolated following exposure to the in vitro active CP analog 4HC. B5-180 cells were cross-resistant to busulfan and γ-radiation. Total RNA was extracted and hybridized to Affymetrix Genechip HG-U95Av2 arrays. Each array contains 12,386 probes corresponding to approximately 9000 known human genes. Each cell line was arrayed in triplicate. Quantitative RT-PCR, Fluorescence In-Situ Hybridization (FISH) and cytogenetic analysis were performed in both cell lines. Both the KBM-7/B5 parental line and B5-180 resistant sub-line expressed high-levels of BCR-ABL transcripts by real-time RT-PCR. FISH and cytogenetic analysis revealed multiple copies of t(9;22) translocation and other additional chromosomal abnormalities such as trisomy 8, and abnormalities of chromosome 18 in both cell lines. Gene array identified 794 gene transcripts that were more than twofold (range from 2-fold to 2675-fold) over-expressed or under-expressed in the resistant line relative to the parental line. ALDH1A1 (aldehyde dehydrogenase 1 family) showed the most differential expression between sensitive and resistant cell lines, ALDH1A1 was upregulated more than 2000-fold in the resistant sub-line. ALDH-2 (aldehyde dehydrogenase 2 family mitochondrial) was also expressed substantially higher in the resistant line. This finding is consistent with the established fact that elevated ALDH activity is an important factor in the resistance of B5-180 cells to 4HC. The remaining differentially expressed genes encode proteins with a wide variety of biochemical functions, which include 44 apoptosis and 7 anti-apoptosis-related genes, 56 genes related to cell cycle and cell growth, 6 DNA repair genes, 13 genes involved in hemopoiesis and B-cell activation. We also tested the expression of the hematopietic transcription factor PU-1 and PUB, a novel PU-1 binding factor. Interestingly, the expression of PU-1 was decreased and PUB increased in the resistant clone. In conclusion, we have identified a large number of differentially expressed genes in a CP resistant cell line derived from CML blast crisis by microarray technology. Our results suggest that CP resistance is a complex phenotype that involves multiple genes and a variety of mechanisms. Real-time RT-PCR analysis and further characterization of selected genes associated with CP resistance as well as the response in vitro to tyrosine kinase inhibitors are currently under investigation.

scholarly journals A Novel, Myeloid Transcription Factor, C/EBPε, Is Upregulated During Granulocytic, But Not Monocytic, Differentiation

Blood ◽  
1997 ◽  
Vol 90 (7) ◽  
pp. 2591-2600 ◽  
Author(s):  
Roberta Morosetti ◽  
Dorothy J. Park ◽  
Alexey M. Chumakov ◽  
Isabelle Grillier ◽  
Masaaki Shiohara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Hematopoietic Stem Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Nb4 Cells

Human C/EBPε is a newly cloned CCAAT/enhancer-binding transcription factor. Initial studies indicated it may be an important regulator of human myelopoiesis. To elucidate the range of expression of C/EBPε, we used reverse transcription-polymerase chain reaction (RT-PCR) analysis and examined its expression in 28 hematopoietic and 14 nonhematopoietic cell lines, 16 fresh myeloid leukemia samples, and normal human hematopoietic stem cells and their mature progeny. Prominent expression of C/EBPε mRNA occurred in the late myeloblastic and promyelocytic cell lines (NB4, HL60, GFD8), the myelomonoblastic cell lines (U937 and THP-1), the early myeloblast cell lines (ML1, KCL22, MDS92), and the T-cell lymphoblastic leukemia cell lines CEM and HSB-2. For the acute promyelocytic leukemia cell line NB4, C/EBPε was the only C/EBP family member that was easily detected by RT-PCR. No C/EBPε mRNA was found in erythroid, megakaryocyte, basophil, B lymphoid, or nonhematopoietic cell lines. Most acute myeloid leukemia samples (11 of 12) from patients expressed C/EBPε. Northern blot and RT-PCR analyses showed that C/EBPε mRNA decreased when the HL60 and KG-1 myeloblast cell lines were induced to differentiate toward macrophages. Similarly, Western blot analysis showed that expression of C/EBPε protein was either unchanged or decreased slightly as the promyelocytic cell line NB4 differentiated down the macrophage-like pathway after treatment with a potent vitamin D3 analog (KH1060). In contrast, C/EBPε protein levels increased dramatically as NB4 cells were induced to differentiate down the granulocytic pathway after exposure to 9-cis retinoic acid. Furthermore, very early, normal hematopoietic stem cells (CD34+/CD38−), purified from humans had very weak expression of C/EBPε mRNA, but levels increased as these cells differentiated towards granulocytes. Likewise, purified granulocytes appeared to express higher levels of C/EBPε mRNA than purified macrophages. Addition of phosphothiolated antisense, but not sense oligonucleotides to C/EBPε, decreased clonal growth of HL-60 and NB4 cells by about 50% compared with control cultures. Taken together, our results indicate that expression of C/EBPε is restricted to hematopoietic tissues, especially myeloid cells as they differentiate towards granulocytes and inhibition of its expression in HL-60 and NB4 myeloblasts and promyelocytes decreased their proliferative capacity. Therefore, this transcriptional factor may play an important role in the process of normal myeloid development.

scholarly journals Jianpi Yangwei decoction promotes apoptosis and suppresses proliferation of 5-fluorouracil resistant gastric cancer cells in vitro and in vivo

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Huijuan Tang ◽  
Wenjie Huang ◽  
Qiang Yang ◽  
Ying Lin ◽  
Yihui Chen ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Drug Resistance ◽  
Cell Line ◽  
Real Time ◽  
Mtt Assay ◽  
Xenograft Tumor ◽  
Rt Pcr ◽  
Induced Apoptosis

Abstract Background The exploration of new therapeutic agents targeting 5-Fu resistance may open a new opportunity to gastric cancer treatment. The objective is to establish a 5-Fu resistant gastric cancer cell line and observe the effect of Jianpi Yangwei decoction (JPYW) on its apoptosis and drug-resistance related proteins. Methods MTT assay was used to measure the effect of JPYW on the BGC823 cells proliferation, and the apoptosis was observed by flow cytometry and Hoechst fluorescence staining. The BGC823 xenograft tumor nude mice models were established, the apoptosis was detected by Tunel method. BGC-823/5-Fu was established by repeated low-dose 5-Fu shocks, the drug resistance index and proliferation were detected by the MTT assay; MDR1 mRNA was detected by real-time RT-PCR; Western blot was used to detect the ratio of p-AKT to AKT; The BGC823/5-Fu xenograft tumor nude mice models were established and apoptosis was measured. The expressions of MRP1, MDR1, ABCG2, AKT, p-AKT, caspase-3 and bcl-2 were detected by immunohistochemistry and the AKT mRNA expression was detected by real-time RT-PCR. Results JPYW induced apoptosis in BGC823 cells; Drug-resistant cell line BGC-823/5-Fu was sucessfully established; JPYW induced apoptosis of BGC823/5-Fu cells, down-regulated the expression of MRP1, MDR1 and ABCG2 in vitro and in vivo, and further decreased MDR1 expression when combined with pathway inhibitor LY294002 (P < 0.05); JPYW down-regulated the ratio of p-AKT to AKT in vitro in a dose-dependent manner, the same as after the combination with LY294002 (P < 0.05). Conclusion JPYW can induce apoptosis of BGC823 and BGC823/5-Fu cells, and down-regulate the expression of MDR1, MRP1, ABCG2 in vitro and in vivo. Its in vitro effect is related to the PI3K/AKT signaling pathway.

Resistance to the Novel Translation Inhibitor Silvestrol Is Mediated by Elevated Mcl-1 Expression.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1737-1737
Author(s):  
David M. Lucas ◽  
Ellen J. Sass ◽  
Ryan B. Edwards ◽  
Li Pan ◽  
Gerard Lozanski ◽  
...  
Keyword(s):  
Drug Resistance ◽  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Multi Drug Resistance ◽  
Parental Line

Abstract Abstract 1737 Poster Board I-763 We previously reported the efficacy and B-cell selectivity of the natural product silvestrol in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), using both primary cells and B-cell lines. We also showed that silvestrol inhibits translation, resulting in rapid depletion of the short half-life protein Mcl-1 followed by mitochondrial damage and apoptosis. Cencic et al. reported that silvestrol directly blocks translation initiation by aberrantly promoting interaction of eIF4A with capped mRNA (PLoS One 2009; 4(4):e5223). However, the loss of Mcl-1 in breast and prostate cancer cell lines is delayed relative to what we observe in B-leukemias (48 hr vs. 4-6 hr in CLL and ALL cells). Additionally, silvestrol does not reduce Mcl-1 expression in normal T-cells to the same extent that it does in B-cells, potentially explaining in part the relative resistance of T-cells to this agent. We therefore investigated cell-type differences, as well as the importance of Mcl-1, in silvestrol-mediated cytotoxicity. We incubated the ALL cell line 697 with gradually increasing concentrations of silvestrol to generate a cell line (697-R) with resistance to 30 nM silvestrol (IC50 of parental 697 < 5 nM). No differences between 697-R and the parental line were detected upon detailed immunophenotyping. However, cytogenetic analysis revealed a balanced 7q;9p translocation in 697-R not present in the parental 697 cell line that may be related to the emergence of a resistant clone. We also detected no difference in expression of multi-drug resistance proteins MDR-1 and MRP, which can contribute to resistance to complex amphipathic molecules such as silvestrol. In contrast, we found that baseline Mcl-1 protein expression is strikingly increased in 697-R cells relative to the parental line, although these cells still show similar percent-wise reduction in Mcl-1 upon re-exposure to 80 nM silvestrol. To investigate whether this resistance to silvestrol is reversible, 697-R cells were maintained without silvestrol for 6 weeks (∼18 passages). During this time, viability remained near 99%. Cells were then treated with 30 nM silvestrol. Viability was 94% at 48 hr post-treatment and returned to 99% within a week, while parental 697 cells with the same treatment were completely dead. Baseline Mcl-1 levels remained elevated in 697-R even with prolonged silvestrol-free incubation. These results indicate that the resistance phenotype is not rapidly reversible, as is seen with transient upregulation of multi-drug resistance or stress-response proteins. Additionally, silvestrol moderately induces the transcription of several pro-apoptotic Bcl-2 family members and results in elevated levels of these proteins despite its translation inhibitory activity. Interestingly, no such activity is detected in silvestrol-treated normal T-cells. Together, these results support the hypothesis that in B-cells, silvestrol induces cell death by altering the balance of pro- and anti-apoptotic factors, and that increased Mcl-1 protein can force the balance back toward survival. This work further underscores the importance of Mcl-1 in silvestrol-mediated cytotoxicity. We are now investigating the mechanism of Mcl-1 upregulation in 697-R cells to identify a factor or pathway that can be targeted therapeutically to circumvent resistance. Silvestrol is currently undergoing preclinical pharmacology and toxicology investigation by the U.S. National Cancer Institute Drug Development Group at the Stage IIA level to facilitate its progression to Phase I clinical testing. Disclosures No relevant conflicts of interest to declare.

Deregulated Apoptotic Pathways Point to Effectiveness of IAP Inhibitor Therapy in Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1275-1275
Author(s):  
Sonja C Lück ◽  
Annika C Russ ◽  
Konstanze Döhner ◽  
Ursula Botzenhardt ◽  
Domagoj Vucic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Gene Set Enrichment Analysis ◽  
Core Binding Factor ◽  
Binding Factor ◽  
Acute Myeloid

Abstract Abstract 1275 Poster Board I-297 Core binding factor (CBF) leukemias, characterized by translocations t(8;21) or inv(16)/t(16;16) targeting the core binding factor, constitute acute myeloid leukemia (AML) subgroups with favorable prognosis. However, 40-50% of patients relapse, and the current classification system does not fully reflect the heterogeneity existing within the cytogenetic subgroups. Therefore, illuminating the biological mechanisms underlying these differences is important for an optimization of therapy. Previously, gene expression profiling (GEP) revealed two distinct CBF leukemia subgroups displaying significant outcome differences (Bullinger et al., Blood 2007). In order to further characterize these GEP defined CBF subgroups, we again used gene expression profiles to identify cell line models similar to the respective CBF cohorts. Treatment of these cell lines with cytarabine (araC) revealed a differential response to the drug as expected based on the expression patterns reflecting the CBF subgroups. In accordance, the cell lines resembling the inferior outcome CBF cohort (ME-1, MONO-MAC-1, OCI-AML2) were less sensitive to araC than those modeling the good prognostic subgroup (Kasumi-1, HEL, MV4-11). A previous gene set enrichment analysis had identified the pathways Caspase cascade in apoptosis and Role of mitochondria in apoptotic signaling among the most significant differentially regulated BioCarta pathways distinguishing the two CBF leukemia subgroups. Thus, we concluded that those pathways might be interesting targets for specific intervention, as deregulated apoptosis underlying the distinct subgroups should also result in a subgroup specific sensitivity to apoptotic stimuli. Therefore, we treated our model cell lines with the Smac mimetic BV6, which antagonizes inhibitor of apoptosis (IAP) proteins that are differentially expressed among our CBF cohorts. In general, sensitivity to BV6 treatment was higher in the cell lines corresponding to the subgroup with good outcome. Time-course experiments with the CBF leukemia cell line Kasumi-1 suggested a role for caspases in this response. Interestingly, combination treatment of araC and BV6 in Kasumi-1 showed a synergistic effect of these drugs, with the underlying mechanisms being currently further investigated. Based on the promising sensitivity to BV6 treatment in some cell lines, we next treated mononuclear cells (mostly leukemic blasts) derived from newly diagnosed AML patients with BV6 in vitro to evaluate BV6 potency in primary leukemia samples. Interestingly, in vitro BV6 treatment also discriminated AML cases into two distinct populations. Most patient samples were sensitive to BV6 monotherapy, but about one-third of cases were resistant even at higher BV6 dosage. GEP of BV6 sensitive patients (at 24h following either BV6 or DMSO treatment) provided insights into BV6-induced pathway alterations in the primary AML patient samples, which included apoptosis-related pathways. In contrast to the BV6 sensitive patients, GEP analyses of BV6 resistant cases revealed no differential regulation of apoptosis-related pathways in this cohort. These results provide evidence that targeting deregulated apoptosis pathways by Smac mimetics might represent a promising new therapeutic approach in AML and that GEP might be used to predict response to therapy, thereby enabling novel individual risk-adapted therapeutic approaches. Disclosures Vucic: Genentech, Inc.: Employment. Deshayes:Genentech, Inc.: Employment.

Partially or Fully BCR-ABL Independent Mechanisms of in Vitro Resistance to Ponatinib

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2481-2481 ◽  
Author(s):  
Qian Yu ◽  
Anna M Eiring ◽  
Matthew S. Zabriskie ◽  
Jamshid Khorashad ◽  
David J Anderson ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Synthetic Lethality ◽  
Immunoblot Analysis ◽  
Kinase Domain ◽  
Resistant Cell ◽  
Abl Kinase ◽  
Kinase Domain Mutation

Abstract Abstract 2481 Ponatinib (AP24534) is a pan-BCR-ABL inhibitor developed for treatment-refractory chronic myeloid leukemia (CML) and has significant activity in patients who fail second-line dasatinib and/or nilotinib tyrosine kinase inhibitor (TKI) therapy. A pivotal phase II trial (clinicaltrials.gov NCT01207440) is underway. BCR-ABL kinase domain mutation-mediated ponatinib resistance has been investigated in vitro (Cancer Cell 16, 2009, 401). Here, we developed ponatinib-resistant, BCR-ABL+ cell lines lacking a kinase domain mutation and investigated mechanisms of resistance to ponatinib and other TKIs. Methods: Four BCR-ABL+ CML cell lines (K562, AR230, BV173, and 32D(BCR-ABL)) were maintained in liquid culture containing ponatinib (0.1 nM) for 10 days. The ponatinib concentration was increased in small increments for a minimum of 90 days, yielding corresponding ponatinib-resistant cell lines. BCR-ABL kinase domain sequencing of sensitive and resistant cells confirmed BCR-ABL to be unmutated. Real-time qPCR was used to compare the expression of BCR-ABL in ponatinib-sensitive and -resistant cell lines. Immunoblot analysis (total and tyrosine-phosphorylated BCR-ABL) was used to the compare levels of BCR-ABL protein and to determine whether resistance to ponatinib corresponded with reduced (partially BCR-ABL-independent) or complete inhibition of BCR-ABL tyrosine phosphorylation (fully BCR-ABL-independent). Cell proliferation assays were performed on resistant and sensitive cell lines in the presence of ponatinib, nilotinib, and dasatinib. A small-molecule inhibitor screen composed of >90 cell-permeable inhibitors that collectively target the majority of the tyrosine kinome as well as other kinases (Blood 116, 2010, abstract 2754) is currently being applied to the 32D(BCR-ABL)R cell line in the presence of 24 nM ponatinib to assess synthetic lethality, with results analyzed using a companion drug sensitivity algorithm. As a second strategy to generate resistant lines, N-ethyl-N-nitrosourea (ENU) mutagenesis was done to investigate BCR-ABL kinase domain-mediated resistance in myeloid K562, AR230, BV173, and 32D(BCR-ABL) cells. After ENU exposure, cells were washed and cultured in 96-well plates with escalating ponatinib. Results: The four BCR-ABL+ cell lines initially grew in the presence of 0.1 nM but not 0.5 nM ponatinib. Upon gradual exposure to escalating ponatinib, each of the cell lines exhibited a degree of adaptation to growth in the presence of the inhibitor (range: 10 to 240-fold). Real-time qPCR showed a modest two-fold increase in BCR-ABL expression level in K562R, AR230R and BV173R cell lines relative to the respective parental lines. Based on immunoblot analysis, cell lines segregated into two categories of ponatinib resistance: partially (K562R and AR230R) or fully BCR-ABL-independent (BV173R and 32D(BCR-ABL)R). Cell proliferation assays showed that ponatinib resistant cell lines also exhibited resistance to nilotinib and dasatinib. The 32D(BCR-ABL)R cell line exhibited a level of ponatinib resistance comparable to that of the Ba/F3 BCR-ABLE255V cell line, which carries the most ponatinib-resistant BCR-ABL mutation. BCR-ABL tyrosine phosphorylation was efficiently blocked by low concentrations of ponatinib (<5 nM) in the 32D(BCR-ABL)R cell line, yet these cells remained viable in the presence of up to 24 nM ponatinib. The effects of providing a second kinase inhibitor along with ponatinib (24 nM) in order to probe for synthetic lethality are under study. Possible involvement of a second, moderately ponatinib-sensitive target is suggested by the sharp ponatinib maximum at 24 nM; even 26 nM ponatinib is toxic to 32D(BCR-ABL)R cells. Thus far, ENU mutagenesis screens in human CML cell lines failed to yield resistant clones and only a few were recovered from the murine 32D(BCR-ABL)R cell line (3/1440 wells; the only BCR-ABL mutant recovered was BCR-ABLL387F). Conclusions: The ponatinib resistant, BCR-ABL+ cell lines described here exhibit either a partially or fully BCR-ABL independent mechanism of resistance. The molecular details of both processes will be reported, with an emphasis on the striking level of resistance (240-fold over starting conditions) exhibited by the 32D(BCR-ABL)R cell line. Our in vitro results indicate that BCR-ABL independent mechanisms may contribute to ponatinib resistance in myeloid CML cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hi-C detects novel structural variants in HL-60 and HL-60/S4 cell lines

2018 ◽  
Author(s):  
Elsie C. Jacobson ◽  
Ralph S. Grand ◽  
Jo K. Perry ◽  
Mark H. Vickers ◽  
Ada L. Olins ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Large Scale ◽  
Rna Seq ◽  
Structural Variants ◽  
The Stability ◽  
Different Sources ◽  
Identification And Characterization

AbstractCancer cell lines often have large structural variants (SVs) that evolve over time. There are many reported differences in large scale SVs between HL-60 and HL-60/S4, two cell lines derived from the same acute myeloid leukemia sample. However, the stability and variability of inter- and intra-chromosomal structural variants between different sources of the same cell line is unknown. Here, we used Hi-C and RNA-seq to identify and compare large SVs in HL-60 and HL-60/S4 cell lines. Comparisons with previously published karyotypes identified novel SVs in both cell lines. Hi-C was used to characterize the known expansion centered on the MYC locus. The MYC expansion was integrated into known locations in HL-60/S4, and a novel location (chr4) in HL-60. The HL-60 cell line has more within-line structural variation than the HL-60/S4 derivative cell line. Collectively we demonstrate the usefulness of Hi-C and with RNA-seq data for the identification and characterization of SVs.

scholarly journals KP772 overcomes multiple drug resistance in malignant lymphoma and leukemia cells in vitro by inducing Bcl-2-independent apoptosis and upregulation of Harakiri

2021 ◽  
Author(s):  
Lisa Kater ◽  
Benjamin Kater ◽  
Michael A. Jakupec ◽  
Bernhard K. Keppler ◽  
Aram Prokop
Keyword(s):  
Drug Resistance ◽  
Cell Lines ◽  
Caspase 3 ◽  
Multiple Drug Resistance ◽  
Antineoplastic Agent ◽  
Resistant Cell ◽  
Leukemia Cells ◽  
Dependent Manner ◽  
Induced Apoptosis

AbstractDespite high cure rates in pediatric patients with acute leukemia, development of resistance limits the efficacy of antileukemic therapy. Tris(1,10-phenanthroline)tris(thiocyanato-κN)lanthanum(III) (KP772) is an experimental antineoplastic agent to which multidrug-resistant cell models have shown hypersensitivity. Antiproliferative and apoptotic activities of KP772 were tested in leukemia, lymphoma and solid tumor cell lines as well as primary leukemia cells (isolated from the bone marrow of a child with acute myeloid leukemia (AML). The ability to overcome drug resistances was investigated in doxorubicin- and vincristine-resistant cell lines. Real-time PCR was used to gain insight into the mechanism of apoptosis induction. KP772 inhibited proliferation and induced apoptosis in various leukemia and lymphoma cell lines in a concentration-dependent manner (LC50 = 1–2.5 µM). Primary AML cells were also sensitive to KP772, whereas daunorubicin showed no significant effect. KP772 induces apoptosis independently of Bcl-2, Smac, and the CD95 receptor and is also effective in caspase 3-deficient MCF7 cells, indicating that apoptosis is partly triggered independently of caspase 3. mRNA expression profiling revealed an upregulation of the BH3-only Bcl-2 protein Harakiri in the course of KP772-induced apoptosis. Remarkably, KP772 overcame drug resistance to doxorubicin and vincristine in vitro, and the apoptotic effect in resistant cells was even superior to that in non-resistant parental cells. In combination with vincristine, doxorubicin and cytarabine, synergistic effects were observed in BJAB cells. The cytotoxic potency in vitro/ex vivo and the remarkable ability to overcome multidrug resistance propose KP772 as a promising candidate drug for antileukemic therapy, especially of drug-refractory malignancies.Graphic abstract

scholarly journals A phospho-proteomic study of cetuximab resistance in KRAS/NRAS/BRAFV600 wild-type colorectal cancer

2021 ◽  
Author(s):  
Alexandros Georgiou ◽  
Adam Stewart ◽  
Georgios Vlachogiannis ◽  
Lisa Pickard ◽  
Nicola Valeri ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Signal Transduction ◽  
Drug Resistance ◽  
Cell Lines ◽  
Ex Vivo ◽  
Pi3k Pathway ◽  
Resistant Cell ◽  
Wild Type ◽  
Proteomic Study

Abstract Purpose We hypothesised that plasticity in signal transduction may be a mechanism of drug resistance and tested this hypothesis in the setting of cetuximab resistance in patients with KRAS/NRAS/BRAFV600 wild-type colorectal cancer (CRC). Methods A multiplex antibody-based platform was used to study simultaneous changes in signal transduction of 55 phospho-proteins in 12 KRAS/NRAS/BRAFV600 wild-type CRC cell lines (6 cetuximab sensitive versus 6 cetuximab resistant) following 1 and 4 h in vitro cetuximab exposure. We validated our results in CRC patient samples (n = 4) using ex vivo exposure to cetuximab in KRAS/NRAS/BRAFV600 cells that were immunomagnetically separated from the serous effusions of patients with known cetuximab resistance. Results Differences in levels of phospho-proteins in cetuximab sensitive and resistant cell lines included reductions in phospho-RPS6 and phospho-PRAS40 in cetuximab sensitive, but not cetuximab resistant cell lines at 1 and 4 h, respectively. In addition, phospho-AKT levels were found to be elevated in 3/4 patient samples following ex vivo incubation with cetuximab for 1 h. We further explored these findings by studying the effects of combinations of cetuximab and two PI3K pathway inhibitors in 3 cetuximab resistant cell lines. The addition of PI3K pathway inhibitors to cetuximab led to a significantly higher reduction in colony formation capacity compared to cetuximab alone. Conclusion Our findings suggest activation of the PI3K pathway as a mechanism of cetuximab resistance in KRAS/NRAS/BRAFV600 wild-type CRC.

scholarly journals Inecalcitol and Decitabine Synergize to Inhibit Cell Proliferation and to Induce Differentiation of Human Acute Myeloid Leukemia Cell Lines

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5234-5234
Author(s):  
Enguerran Mouly ◽  
Emilie Rousseau ◽  
Cecile Planquette ◽  
Remi Delansorne
Keyword(s):  
Cell Proliferation ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Employment Equity ◽  
Elderly Aml ◽  
Equity Ownership ◽  
Inhibit Cell Proliferation ◽  
Stimulation Of

Abstract Decitabine (DAC) is a hypomethylating agent indicated as front-line therapy for de novo or secondary acute myeloid leukemia (AML) in newly diagnosed patients aged 65 years or older unfit for standard induction chemotherapy (Kantarjian et al., 2012, Malik & Cashen, 2014, Nieto et al., 2016, He et al., 2017). Its mechanism of action at the DNA level mostly results in inhibition of cell proliferation. Cellular differentiation can also be involved in some extent in a fraction of the leukemic cell population, as reported in initial pharmacological studies (Creusot et al., 1982; Pinto et al., 1984). Overall survival advantage is nevertheless limited to several more months and the next challenge is to combine DAC with other drugs to improve it further (Kubasch & Platzbecker, 2018). Inecalcitol (INE: 14epi-,19nor-,23yne-,1,25dihydroxy-cholecalciferol) is a vitamin D receptor agonist characterized by potent anti-proliferative and pro-differentiating general properties on cancer cells and by a low calcemic potential (Okamoto et al., 2012; Ma et al., 2013; Medioni et al. 2014), and especially on AML cell lines (AACR 2017, 2018). INE is currently being tested in combination with DAC in this category of elderly AML patients unfit for standard chemotherapy. The aim of the present report was to look for synergies in vitro between DAC and INE on four non-APL human AML cell lines (MOLM-13, U-937, THP-1, OCI-AML2) both on inhibition of proliferation and induction of differentiation. After 72 hours of incubation, cells were counted and labeled for CD11b and CD14 at the cell surface as biomarkers of monocytic/macrophagic differentiation. The range of DAC concentrations had to adapted to each cell line to avoid maximal cytotoxicity: 1.2 nM to 100 nM on MOLM-13, 3 nM to 250 nM on U-937 and THP-1, and 31 nM to 500 nM on OCI-AML2. The same range of 0.12 to 10 nM INE concentrations was tested on each cell line. Each concentration of INE was tested in combination with each concentration of DAC. Synergy was calculated as the excess over the highest single agent (HSA) using the open source Combenefit software (Di Veroli et al., 2016). The highest concentration of DAC alone (MOLM-13: 100 nM, U-937 and THP-1: 250 nM; OCI-AML2: 500 nM) induced a decrease in cell count of 30% of THP-1 cells, 50% of OCI-AML2 cells, 65% of U-937 cells and 80% of MOLM-13 cells. The highest concentration of INE alone (10 nM) induced a decrease in cell count of 20% of U-937 and THP-1 cells, 60% of OCI-AML2 cells and 70% of MOLM-13 cells. The antiproliferative effects of DAC and INE were at least additive in all combinations tested. Significant HSA synergy indexes were found for the decrease in cell number in all four cell lines, ranging from 12% to 23% depending on cell lines and combinations of concentrations. The highest concentration of DAC alone had no (U-937, THP-1) or limited activity (<+12% of labeled MOLM-13 or OCI-AML2 cells) to induce either CD11b or CD14 on the cell surface. By contrast, the highest concentration of INE alone (10 nM) stimulated the expression of CD11b and CD14 in up to 70% to 95% of the cells depending on the cell line (except the CD14 labeling of U-937 cells which remained < 8%). The respective EC50 of INE for CD11b and CD14 induction was 1 and 3 nM on THP-1 cells, 4 and 3 nM on MOLM-13 cells, 3 and 3 nM on OCI-AML2 cells and 1 nM on U-937 cells (50% not reached for CD14). There was no antagonistic effect of DAC towards the pro-differentiating properties of INE. A significant HSA synergy index in the 16% to 26% range was observed for both CD11b and CD14 in MOLM-13 cells and for CD14 in OCI-AML2 cells. A very high HSA synergy index of 75% was observed for the stimulation of CD14 in U-937 cells. In summary, DAC exerted more antiproliferative activity than INE which was more potent to induce monocytic/macrophagic differentiation of four non-APL human AML cell lines. The combination of DAC and INE systematically resulted in a synergy to inhibit cell proliferation, and the strong stimulation of cell differentiation induced by INE alone was in some cases boosted by DAC. These in vitro results provide the mechanistic basis for the potential interest of treating elderly AML patients with INE in addition to DAC in the ongoing double-blind placebo-controlled Phase II clinical trial (NCT02802267). Disclosures Mouly: Hybrigenics: Employment. Rousseau:Hybrigenics: Employment. Planquette:Hybrigenics: Employment, Equity Ownership, Patents & Royalties: inventor, but no royalties. Delansorne:Hybrigenics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: inventor, but no royalties.

Screening of 129 FDA approved anti-cancer drugs in colorectal cancer cell lines resistant to oxaliplatin or irinotecan (SN38).

2017 ◽  
Vol 35 (4_suppl) ◽  
pp. 642-642 ◽  
Author(s):  
Jan Stenvang ◽  
Christine Hjorth Andreassen ◽  
Nils Brünner
Keyword(s):  
Colorectal Cancer ◽  
Drug Resistance ◽  
Cell Viability ◽  
Cell Line ◽  
Cell Lines ◽  
Resistant Cell ◽  
Cancer Drug ◽  
Cancer Drugs ◽  
Drug Candidates ◽  
Anti Cancer

642 Background: In metastatic colorectal cancer (mCRC) only 3 cytotoxic drugs (oxaliplatin, irinotecan and fluorouracil (5-FU)) are approved and the first and second line response rates are about 50% and 10-15%, respectively. Thus, new treatment options are needed. Novel anti-cancer drug candidates are primarily tested in an environment of drug resistance and the majority of novel drug candidates fail during clinical development. Therefore, “repurposing” of drugs has emerged as a promising strategy to apply established drugs in novel indications. The aim of this project was to screen established anti-cancer drugs to identify candidates for testing in mCRC patients relapsing on standard therapy. Methods: We applied 3 parental (drug sensitive) CRC cell lines (HCT116, HT29 and LoVo) and for each cell line also an oxaliplatin and irinotecan (SN38) resistant cell line. We obtained 129 FDA approved anti-cancer drugs from the Developmental Therapeutics Program (DTP) at the National Cancer Institute (NCI) ( https://dtp.cancer.gov/ ). The parental HT29 cell line and the drug resistant sublines HT29-SN38 and HT29-OXPT were exposed to 3 concentrations of each of the anti-cancer drugs. The effect on cell viability was analyzed by MTT assays. Nine of the drugs were analyzed for effect in the LoVo and HCT116 and the SN38- and oxaliplatin-resistant derived cell lines. Results: None of the drugs caused evident differential response between the resistant and sensitive cells or between the SN38 and oxaliplatin resistant cells. The screening confirmed the resistance as the cells displayed resistance to drugs in the same class as the one they were made resistant to. Of the drugs, 45 decreased cell viability in the HT29 parental and oxaliplatin- or SN-38 resistant cell lines. Nine drugs were tested in all nine CRC cell lines and eight decrease cell viability in the nine cell lines. These included drugs in different classes such as epigenetic drugs, antibiotics, mitotic inhibitors and targeted therapies. Conclusions: This study revealed several possible new “repurposing” drugs for CRC therapy, by showing that 45 FDA-approved anti-cancer drugs decrease cell viability in CRC cell lines with acquired drug resistance.

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