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Cancer Chemotherapy and Pharmacology Aug 2014The prognosis of patients with advanced-phase chronic myeloid leukemia (CML) remains dismal despite the availability of targeted therapies and allogeneic stem cell...
In vitro testing of drug combinations employing nilotinib and alkylating agents with regard to pretransplant conditioning treatment of advanced-phase chronic myeloid leukemia.
PURPOSE
The prognosis of patients with advanced-phase chronic myeloid leukemia (CML) remains dismal despite the availability of targeted therapies and allogeneic stem cell transplantation (allo-SCT). Increasing the antileukemic efficacy of the pretransplant conditioning regimen may be a strategy to increase remission rates and duration. We therefore investigated the antiproliferative effects of nilotinib in combination with drugs that are usually used for conditioning: the alkylating agents mafosfamide, treosulfan, and busulfan.
METHODS
Drug combinations were tested in vitro in different imatinib-sensitive and imatinib-resistant BCR-ABL-positive cell lines. A tetrazolium-based MTT assay was used for the assessment and quantification of growth inhibition after exposure to alkylating agents alone or to combinations with nilotinib. Drug interaction was analyzed using the median-effect method of Chou and Talalay, and combination index (CI) values were calculated according to the classic isobologram equation.
RESULTS
Treatment of imatinib-sensitive, BCR-ABL-positive K562 and LAMA84 cells with nilotinib in combination with mafosfamide, treosulfan, or busulfan resulted in synergistic (CI < 1), additive (CI ~ 1), and predominantly antagonistic (CI > 1) effects, respectively. In imatinib-resistant K562-R and LAMA84-R cells, all applied drug combinations were synergistic (CI < 1) at higher growth inhibition levels.
CONCLUSIONS
Our in vitro data warrant further investigation and may provide the basis for nilotinib-supplemented conditioning regimens for allo-SCT in advanced-phase CML.
Topics: Antineoplastic Combined Chemotherapy Protocols; Benzamides; Busulfan; Cell Proliferation; Cyclophosphamide; Drug Interactions; Drug Resistance, Neoplasm; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; In Vitro Techniques; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Piperazines; Pyrimidines; Transplantation Conditioning; Tumor Cells, Cultured
PubMed: 25038611
DOI: 10.1007/s00280-014-2533-6 -
RSC Medicinal Chemistry Jan 2024Aldehyde dehydrogenase 1A1 (ALDH1A1) is an isoenzyme that catalyzes the conversion of aldehydes to acids. However, the overexpression of ALDH1A1 in a variety of...
Aldehyde dehydrogenase 1A1 (ALDH1A1) is an isoenzyme that catalyzes the conversion of aldehydes to acids. However, the overexpression of ALDH1A1 in a variety of malignancies is the major cause of resistance to an anti-cancer drug, cyclophosphamide (CP). CP is a prodrug that is initially converted into 4-hydroxycyclophosphamide and its tautomer aldophosphamide, in the liver. These compounds permeate into the cell and are converted as active metabolites, , phosphoramide mustard (PM), through spontaneous beta-elimination. On the other hand, the conversion of CP to PM is diverted at the level of aldophosphamide by converting it into inactive carboxyphosphamide using ALDH1A1, which ultimately leads to high drug inactivation and CP resistance. Hence, in combination with our earlier work on the target of resistance, , ALDH1A1, we hereby report selective ALDH1A1 inhibitors. Herein, we selected a lead molecule from our previous virtual screening and implemented scaffold hopping analysis to identify a novel scaffold that can act as an ALDH1A1 inhibitor. This results in the identification of various novel scaffolds. Among these, on the basis of synthetic feasibility, the benzimidazole scaffold was selected for the design of novel ALDH1A1 inhibitors, followed by machine learning-assisted structure-based virtual screening. Finally, the five best compounds were selected and synthesized. All synthesized compounds were evaluated using enzymatic assay against ALDH1A1, ALDH2, and ALDH3A1. The results disclosed that three molecules A1, A2, and A3 showed significant selective ALDH1A1 inhibitory potential with an IC value of 0.32 μM, 0.55 μM, and 1.63 μM, respectively, and none of the compounds exhibits potency towards the other two ALDH isoforms ALDH2 and ALDH3A1. Besides, the potent compounds (A1, A2, and A3) have been tested for cell line assay in combination with mafosfamide (analogue of CP) on two cell lines A549 and MIA-PaCa-2. All three compounds show significant potency to reverse mafosfamide resistance by inhibiting ALDH1A1 against these cell lines.
PubMed: 38283216
DOI: 10.1039/d3md00543g -
Magnetic Resonance in Medicine Sep 2016To facilitate decision making in the oncology clinic, technologies have recently been developed to independently inject and assess multiple anticancer agents directly in... (Comparative Study)
Comparative Study
PURPOSE
To facilitate decision making in the oncology clinic, technologies have recently been developed to independently inject and assess multiple anticancer agents directly in a patient's tumor. To increase the flexibility of this approach beyond histological readouts of response, contrast-enhanced MRI was evaluated for the detection of cell death in living tumors after injection.
METHODS
A six-needle arrayed microinjection device designed to provide head-to-head comparisons of chemotherapy responses in living tumors was used. Xenografted non-Hodgkin lymphoma tumors in athymic Nude-Foxn1(nu) mice were injected either with different doses of vincristine or with one needle each of vincristine, doxorubicin, bendamustine, prednisolone, mafosfamide, and a vehicle control. To assess drug responses, measurements of enhancement by T1-weighted contrast-enhanced MRI were made for individual sites at 24, 48, and 72 h after injection. For comparison, histological evaluations of cell death were obtained after tumor resection.
RESULTS
Measurements of MRI enhancement at injection sites showed a significant (P < 0.001) positive regression slope as a function of vincristine dose. Average MRI measurements were closely correlated with cell death by hematoxylin and eosin staining (R = 0.81; P = 0.001).
CONCLUSION
Contrast-enhanced MRI has the potential to replace or augment histological analyses of tumor responses to microinjected doses of chemotherapy agents with potential application in selecting optimal chemotherapy regimens. Magn Reson Med 76:946-952, 2016. © 2015 Wiley Periodicals, Inc.
Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Drug Monitoring; Lymphoma, Non-Hodgkin; Magnetic Resonance Imaging; Mice; Mice, Nude; Microinjections; Reproducibility of Results; Sensitivity and Specificity; Treatment Outcome
PubMed: 26362018
DOI: 10.1002/mrm.25978 -
Cancer Biology & Medicine Aug 2021Promotion of the proliferative expansion of CD4Foxp3 regulatory T cells (Tregs) is one of the side effects that limits the use of bleomycin (BLM) in the treatment of...
OBJECTIVE
Promotion of the proliferative expansion of CD4Foxp3 regulatory T cells (Tregs) is one of the side effects that limits the use of bleomycin (BLM) in the treatment of tumors. In this study, we examined the hypothesis that cyclophosphamide (CY), a chemotherapeutic agent with the capacity to eliminate tumor infiltrating Tregs, abrogated BLM-induced expansion of Tregs and consequently resulted in a better anti-tumor effect.
METHODS
The effects of BLM, with or without mafosfamide (MAF, the active metabolite of CY), on both TGF-β-induced differentiation of Tregs (iTregs), and TNF-induced expansion of naturally occurring Tregs (nTregs) were assessed. The effect of low doses of BLM and CY on tumor-infiltrating Tregs, as well as on the growth of mouse B16-F10 melanomas, was also studied.
RESULTS
treatment with BLM promoted the differentiation of iTregs, as well as TNF-induced expansion of nTregs. These effects of BLM were completely abrogated by MAF. Furthermore, in the mouse B16-F10 melanoma model, treatment with low doses of BLM increased the number of tumor-infiltrating Tregs, and this effect of BLM was also abrogated by CY. Importantly, combination therapy with low doses of BLM and CY showed synergistic anti-tumor effects.
CONCLUSIONS
CY abrogated the effect of BLM on the expansion of Tregs. The combination of these 2 chemotherapeutic agents may represent a safer and more effective therapy in the treatment of cancer patients, and thus merits future clinical evaluation.
PubMed: 34378880
DOI: 10.20892/j.issn.2095-3941.2021.0027 -
Genome Medicine May 2016The genetic origins of chemotherapy resistance are well established; however, the role of epigenetics in drug resistance is less well understood. To investigate...
BACKGROUND
The genetic origins of chemotherapy resistance are well established; however, the role of epigenetics in drug resistance is less well understood. To investigate mechanisms of drug resistance, we performed systematic genetic, epigenetic, and transcriptomic analyses of an alkylating agent-sensitive murine lymphoma cell line and a series of resistant lines derived by drug dose escalation.
METHODS
Dose escalation of the alkylating agent mafosfamide was used to create a series of increasingly drug-resistant mouse Burkitt's lymphoma cell lines. Whole genome sequencing, DNA microarrays, reduced representation bisulfite sequencing, and chromatin immunoprecipitation sequencing were used to identify alterations in DNA sequence, mRNA expression, CpG methylation, and H3K27me3 occupancy, respectively, that were associated with increased resistance.
RESULTS
Our data suggest that acquired resistance cannot be explained by genetic alterations. Based on integration of transcriptional profiles with transcription factor binding data, we hypothesize that resistance is driven by epigenetic plasticity. We observed that the resistant cells had H3K27me3 and DNA methylation profiles distinct from those of the parental lines. Moreover, we observed DNA methylation changes in the promoters of genes regulated by E2a and members of the polycomb repressor complex 2 (PRC2) and differentially expressed genes were enriched for targets of E2a. The integrative analysis considering H3K27me3 further supported a role for PRC2 in mediating resistance. By integrating our results with data from the Immunological Genome Project (Immgen.org), we showed that these transcriptional changes track the B-cell maturation axis.
CONCLUSIONS
Our data suggest a novel mechanism of drug resistance in which E2a and PRC2 drive changes in the B-cell epigenome; these alterations attenuate alkylating agent treatment-induced apoptosis.
Topics: Animals; Antineoplastic Agents; Basic Helix-Loop-Helix Transcription Factors; Burkitt Lymphoma; Cell Line, Tumor; Cyclophosphamide; DNA Methylation; Drug Resistance, Neoplasm; Epigenesis, Genetic; Histones; Humans; Mice; Polycomb Repressive Complex 2; Principal Component Analysis; Promoter Regions, Genetic
PubMed: 27146673
DOI: 10.1186/s13073-016-0305-0 -
Oncotarget Apr 2017The study of long noncoding RNAs (lncRNAs) is an emerging area of cancer research, in part due to their ability to serve as disease biomarkers. However, few studies have...
The study of long noncoding RNAs (lncRNAs) is an emerging area of cancer research, in part due to their ability to serve as disease biomarkers. However, few studies have investigated lncRNAs in chronic lymphocytic leukemia (CLL). We have identified one particular lncRNA, treRNA, which is overexpressed in CLL B-cells. We measured transcript expression in 144 CLL patient samples and separated samples into high or low expression of treRNA relative to the overall median. We found that high expression of treRNA is significantly associated with shorter time to treatment. High treRNA also correlates with poor prognostic indicators such as unmutated IGHV and high ZAP70 protein expression. We validated these initial findings in samples collected in a clinical trial comparing the nucleoside analog fludarabine alone or in combination with the alkylating agent cyclophosphamide in untreated CLL samples collected prior to starting therapy (E2997). High expression of treRNA was independently prognostic for shorter progression free survival in patients receiving fludarabine plus cyclophosphamide. Given these results, in order to study the role of treRNA in DNA damage response we generated a model cell line system where treRNA was over-expressed in the human B-CLL cell line OSU-CLL. Relative to the vector control line, there was less cell death in OSU-CLL over-expressing treRNA after exposure to fludarabine and mafosfamide, due in part to a reduction in DNA damage. Therefore, we suggest that treRNA is a novel biomarker in CLL associated with aggressive disease and poor response to chemotherapy through enhanced protection against cytotoxic mediated DNA damage.
Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Biomarkers; DNA Damage; Female; Gene Expression; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Prognosis; RNA, Long Noncoding; Treatment Outcome; ZAP-70 Protein-Tyrosine Kinase
PubMed: 28412730
DOI: 10.18632/oncotarget.15401 -
Molecular Medicine Reports May 2019Chronic lymphocytic leukemia (CLL) treatment is improving; however, some patients do not respond to therapy. Due to the high heterogeneity in disease development, there...
Dose and drug changes in chronic lymphocytic leukemia cell response in vitro: A comparison of standard therapy regimens with two novel cyclin‑dependent kinase inhibitors.
Chronic lymphocytic leukemia (CLL) treatment is improving; however, some patients do not respond to therapy. Due to the high heterogeneity in disease development, there is an urgent need for personalization of therapy. In the present study, the response of leukemic mononuclear cells to anticancer drugs used for CLL treatment (cladribine + mafosfamide; CM or CM combined with rituximab; RCM) was compared with the response to new cyclin‑dependent kinase (CDK) inhibitors: BP14 and BP30. Viable apoptotic and necrotic cells were quantified by flow cytometry using propidium iodide and Yo‑Pro stains. CDK inhibitors were studied in several doses to determine the reduction of necrosis and simultaneous increase of apoptosis in leukemic cell incubations with anticancer agents. The distinct cell response to applied doses/anticancer agents was observed. Results obtained in the current manuscript confirmed that modulation of doses is important. This was particularly indicated in results obtained at 24 h of cells incubation with anticancer agent. While an important time for analysis of anticancer response efficacy (monitoring of apoptosis induction potential) seems to be 48 h of cells exposition to anticancer agents. High variability in response to the drugs revealed that both the nature and the dose of the anticancer agents could be important in the final effect of the therapy. The present findings support the thesis that personalized medicine, before drug administration in the clinic, could be important to avoid the application of ineffective therapy.
Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinases; Dose-Response Relationship, Drug; Female; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Protein Kinase Inhibitors; Tumor Cells, Cultured
PubMed: 30864706
DOI: 10.3892/mmr.2019.10007 -
PloS One 2016Approximately fifty percent of patients with acute myeloid leukemia can be cured with current therapeutic strategies which include, standard dose chemotherapy for...
BACKGROUND
Approximately fifty percent of patients with acute myeloid leukemia can be cured with current therapeutic strategies which include, standard dose chemotherapy for patients at standard risk of relapse as assessed by cytogenetic and molecular analysis, or high-dose chemotherapy with allogeneic hematopoietic stem cell transplant for high-risk patients. Despite allogeneic hematopoietic stem cell transplant about 25% of patients still succumb to disease relapse, therefore, novel strategies are needed to improve the outcome of patients with acute myeloid leukemia.
METHODS AND FINDINGS
We developed an immunotherapeutic strategy targeting the CD33 myeloid antigen, expressed in ~ 85-90% of patients with acute myeloid leukemia, using chimeric antigen receptor redirected T-cells. Considering that administration of CAR T-cells has been associated with cytokine release syndrome and other potential off-tumor effects in patients, safety measures were here investigated and reported. We genetically modified human activated T-cells from healthy donors or patients with acute myeloid leukemia with retroviral supernatant encoding the inducible Caspase9 suicide gene, a ΔCD19 selectable marker, and a humanized third generation chimeric antigen receptor recognizing human CD33. ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells had a 75±3.8% (average ± standard error of the mean) chimeric antigen receptor expression, were able to specifically lyse CD33+ targets in vitro, including freshly isolated leukemic blasts from patients, produce significant amount of tumor-necrosis-factor-alpha and interferon-gamma, express the CD107a degranulation marker, and proliferate upon antigen specific stimulation. Challenging ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells with programmed-death-ligand-1 enriched leukemia blasts resulted in significant killing like observed for the programmed-death-ligand-1 negative leukemic blasts fraction. Since the administration of 10 nanomolar of a non-therapeutic dimerizer to activate the suicide gene resulted in the elimination of only 76.4±2.0% gene modified cells in vitro, we found that co-administration of the dimerizer with either the BCL-2 inhibitor ABT-199, the pan-BCL inhibitor ABT-737, or mafosfamide, resulted in an additive effect up to complete cell elimination.
CONCLUSIONS
This strategy could be investigated for the safety of CAR T-cell applications, and targeting CD33 could be used as a 'bridge" therapy for patients coming to allogeneic hematopoietic stem cell transplant, as anti-leukemia activity from infusing CAR.CD33 T-cells has been demonstrated in an ongoing clinical trial. Albeit never performed in the clinical setting, our future plan is to investigate the utility of iC9-CAR.CD33 T-cells as part of the conditioning therapy for an allogeneic hematopoietic stem cell transplant for acute myeloid leukemia, together with other myelosuppressive agents, whilst the activation of the inducible Caspase9 suicide gene would grant elimination of the infused gene modified T-cells prior to stem cell infusion to reduce the risk of engraftment failure as the CD33 is also expressed on a proportion of the donor stem cell graft.
Topics: B7-H1 Antigen; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Caspase 9; Cell Engineering; Cell Line, Tumor; Cell Proliferation; Cellular Reprogramming; Clinical Trials as Topic; Cyclophosphamide; Cytotoxicity, Immunologic; Genetic Vectors; Humans; Interferon-gamma; Leukemia, Myeloid, Acute; Lysosomal-Associated Membrane Protein 1; Myeloid Cells; Nitrophenols; Piperazines; Primary Cell Culture; Receptors, Antigen, T-Cell; Recombinant Fusion Proteins; Sialic Acid Binding Ig-like Lectin 3; Sulfonamides; T-Lymphocytes; Tumor Necrosis Factor-alpha
PubMed: 27907031
DOI: 10.1371/journal.pone.0166891 -
Cell Death & Disease Oct 2018Activation of T cells, a major fraction of peripheral blood lymphocytes (PBLCS), is essential for the immune response. Genotoxic stress resulting from ionizing radiation...
Sensitivity of CD3/CD28-stimulated versus non-stimulated lymphocytes to ionizing radiation and genotoxic anticancer drugs: key role of ATM in the differential radiation response.
Activation of T cells, a major fraction of peripheral blood lymphocytes (PBLCS), is essential for the immune response. Genotoxic stress resulting from ionizing radiation (IR) and chemical agents, including anticancer drugs, has serious impact on T cells and, therefore, on the immune status. Here we compared the sensitivity of non-stimulated (non-proliferating) vs. CD3/CD28-stimulated (proliferating) PBLC to IR. PBLCs were highly sensitive to IR and, surprisingly, stimulation to proliferation resulted in resistance to IR. Radioprotection following CD3/CD28 activation was observed in different T-cell subsets, whereas stimulated CD34+ progenitor cells did not become resistant to IR. Following stimulation, PBLCs showed no significant differences in the repair of IR-induced DNA damage compared with unstimulated cells. Interestingly, ATM is expressed at high level in resting PBLCs and CD3/CD28 stimulation leads to transcriptional downregulation and reduced ATM phosphorylation following IR, indicating ATM to be key regulator of the high radiosensitivity of resting PBLCs. In line with this, pharmacological inhibition of ATM caused radioresistance of unstimulated, but not stimulated, PBLCs. Radioprotection was also achieved by inhibition of MRE11 and CHK1/CHK2, supporting the notion that downregulation of the MRN-ATM-CHK pathway following CD3/CD28 activation results in radioprotection of proliferating PBLCs. Interestingly, the crosslinking anticancer drug mafosfamide induced, like IR, more death in unstimulated than in stimulated PBLCs. In contrast, the bacterial toxin CDT, damaging DNA through inherent DNase activity, and the DNA methylating anticancer drug temozolomide induced more death in CD3/CD28-stimulated than in unstimulated PBLCs. Thus, the sensitivity of stimulated vs. non-stimulated lymphocytes to genotoxins strongly depends on the kind of DNA damage induced. This is the first study in which the killing response of non-proliferating vs. proliferating T cells was comparatively determined. The data provide insights on how immunotherapeutic strategies resting on T-cell activation can be impacted by differential cytotoxic effects resulting from radiation and chemotherapy.
Topics: Amino Acid Chloromethyl Ketones; Antibodies; Ataxia Telangiectasia Mutated Proteins; CD28 Antigens; CD3 Complex; Caspases; Cell Proliferation; Chromones; DNA-Activated Protein Kinase; Drug Resistance; Gamma Rays; Gene Expression Regulation; Humans; Isoxazoles; Lymphocyte Activation; MRE11 Homologue Protein; Morpholines; Primary Cell Culture; Pyrazines; Pyrones; Radiation Tolerance; Signal Transduction; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; Thiophenes; Thioxanthenes
PubMed: 30323167
DOI: 10.1038/s41419-018-1095-7