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Journal of Medicinal Chemistry Jan 2014Aldehyde dehydrogenase enzymes irreversibly oxidize aldehydes generated from metabolism of amino acids, fatty acids, food, smoke, additives, and xenobiotic drugs....
Aldehyde dehydrogenase enzymes irreversibly oxidize aldehydes generated from metabolism of amino acids, fatty acids, food, smoke, additives, and xenobiotic drugs. Cyclophosphamide is one such xenobiotic used in cancer therapies. Upon activation, cyclophosphamide forms an intermediate, aldophosphamide, which can be detoxified to carboxyphosphamide by aldehyde dehydrogenases (ALDH), especially ALDH1A1 and ALDH3A1. Consequently, selective inhibition of ALDH3A1 could increase chemosensitivity toward cyclophosphamide in ALDH3A1 expressing tumors. Here, we report detailed kinetics and structural characterization of a highly selective submicromolar inhibitor of ALDH3A1, 1-[(4-fluorophenyl)sulfonyl]-2-methyl-1H-benzimidazole (CB7, IC50 of 0.2 μM). CB7 does not inhibit ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, or ALDH2 activity. Structural, kinetics, and mutagenesis studies show that CB7 binds to the aldehyde binding pocket of ALDH3A1. ALDH3A1-expressing lung adenocarcinoma and glioblastoma cell lines are sensitized toward mafosfamide (MF) treatment in the presence analogues of CB7, whereas primary lung fibroblasts lacking ALDH3A1 expression, are not.
Topics: Aldehyde Dehydrogenase; Antineoplastic Agents; Benzimidazoles; Cell Line, Tumor; Crystallography, X-Ray; Cyclophosphamide; Drug Screening Assays, Antitumor; Drug Synergism; Fibroblasts; Humans; Kinetics; Lung; Models, Molecular; Mutation; Structure-Activity Relationship
PubMed: 24387105
DOI: 10.1021/jm401508p -
Postepy Higieny I Medycyny... Dec 2013Ranpirnase (onconase; ONC) is an endoribonuclease obtained from the frog Rana pipiens. This enzyme exhibits anticancer properties mediated by degradation of cellular RNA...
Ranpirnase (onconase; ONC) is an endoribonuclease obtained from the frog Rana pipiens. This enzyme exhibits anticancer properties mediated by degradation of cellular RNA and induction of apoptosis. In this study we assessed cytotoxicity of ONC in combination with currently used anticancer drugs on a human diffuse large B-cell lymphoma (DLBCL)-derived cell line (Toledo). Cytotoxic activity was measured by the exclusion of propidium iodide assay while apoptosis was assessed using the annexin-V binding method. Additionally, flow cytometry was used to assess the decline of mitochondrial potential and to determine activation of caspases 3, 8 and 9. It was observed that in vitro treatment with ONC in combination with rituximab, mafosfamide, vincristine, doxorubicin, and dexamethasone (drugs corresponding with elements of R-CHOP regimen) resulted in increased cytotoxicity. As a result ONC showed marked cytotoxicity against Toledo cells. Importantly, in combination of ONC with drugs imitating the R-CHOP regimen, this effect was significantly intensified. The main mechanism responsible for this event was induction of apoptosis along a mitochondrial dependent pathway. In conclusion, these data indicate that further preclinical and eventually clinical studies assessing activity of ONC+R-CHOP treatment are warranted.
Topics: Antibodies, Antineutrophil Cytoplasmic; Antibodies, Monoclonal, Murine-Derived; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line; Cyclophosphamide; Doxorubicin; Humans; Lymphoma, Large B-Cell, Diffuse; Prednisone; Ribonucleases; Rituximab; Treatment Outcome; Vincristine
PubMed: 24379257
DOI: 10.5604/17322693.1078386 -
PloS One 2013Regulatory T cells (Treg) play a pivotal role in the immune system since they inhibit the T cell response. It is well known that cyclophosphamide applied at low dose is...
Regulatory T cells (Treg) play a pivotal role in the immune system since they inhibit the T cell response. It is well known that cyclophosphamide applied at low dose is able to stimulate the immune response while high dose cyclophosphamide exerts inhibitory activity. Data obtained in mice indicate that cyclophosphamide provokes a reduction in the number of Treg and impairs their suppressive activity, resulting in immune stimulation. Here, we addressed the question of the sensitivity of human Treg to cyclophosphamide, comparing Treg with cytotoxic T cells (CTL) and T helper cells (Th). We show that Treg are more sensitive than CTL and Th to mafosfamide, which is an active derivative of cyclophosphamide, which does not need metabolic activation. The high sensitivity of Treg was due to the induction of apoptosis. Treg compared to CTL and Th were not more sensitive to the alkylating drugs temozolomide and nimustine and also not to mitomycin C, indicating a specific Treg response to mafosfamide. The high sensitivity of Treg to mafosfamide resulted not only in enhanced cell death, but also in impaired Treg function as demonstrated by a decline in the suppressor activity of Treg in a co-culture model with Th and Helios positive Treg. Treatment of Treg with mafosfamide gave rise to a high level of DNA crosslinks, which were not repaired to the same extent as observed in Th and CTL. Also, Treg showed a low level of γH2AX foci up to 6 h and a high level 24 h after treatment, indicating alterations in the DNA damage response. Overall, this is the first demonstration that human Treg are, in comparison with Th and CTL, hypersensitive to cyclophosphamide, which is presumably due to a DNA repair defect.
Topics: Apoptosis; Cyclophosphamide; Dose-Response Relationship, Drug; Humans; Immunosuppressive Agents; Interleukin-2 Receptor alpha Subunit; Necrosis; T-Lymphocytes, Regulatory
PubMed: 24376696
DOI: 10.1371/journal.pone.0083384 -
Indian Journal of Experimental Biology Aug 2013Oxazaphosphorines belong to a group of alkylating agents. Mafosfamide cyclohexylamine salt (D-17272), 4-hydro-peroxy-cyclophosphamide (D-18864) and glufosfamide...
Oxazaphosphorines belong to a group of alkylating agents. Mafosfamide cyclohexylamine salt (D-17272), 4-hydro-peroxy-cyclophosphamide (D-18864) and glufosfamide (D-19575, beta-D-glucose-isophosphoramide mustard) are new generation oxazaphosphorines. The objective of the present study was to compare the cytotoxic action of these oxazaphosphorine compounds against human histiocytic lymphoma U937 cells. The chemical structures of the oxazaphosphorines were responsible for the different responses of U937 cells. The cytotoxic effects of D-17272, D-18864, and D-19575 on U937 cells depended on the agent tested, its dose, and the time intervals after the oxazaphosphorine application. Among the oxazaphosphorine agents, D-18864 appeared to be the most cytotoxic, and D-19575 was characterized by the lowest cytotoxicity. The in vitro cytotoxic activities of the oxazaphosphorines were strongly associated with their cell death inducing potential.
Topics: Antineoplastic Agents, Alkylating; Apoptosis; Cell Proliferation; Cyclophosphamide; Flow Cytometry; Glucose; Humans; Ifosfamide; Lymphoma, Large B-Cell, Diffuse; Membrane Potential, Mitochondrial; Necrosis; Phosphoramide Mustards; Tumor Cells, Cultured
PubMed: 24228385
DOI: No ID Found -
Science Translational Medicine Nov 2013High-dose, posttransplantation cyclophosphamide (PTCy) is an effective strategy for preventing graft-versus-host disease (GVHD) after allogeneic blood or marrow...
High-dose, posttransplantation cyclophosphamide (PTCy) is an effective strategy for preventing graft-versus-host disease (GVHD) after allogeneic blood or marrow transplantation (alloBMT). However, the mechanisms by which PTCy modulates alloimmune responses are not well understood. We studied early T cell reconstitution in patients undergoing alloBMT with PTCy and the effects of mafosfamide, a cyclophosphamide (Cy) analog, on CD4(+) T cells in allogeneic mixed lymphocyte reactions (MLRs) in vitro. Patients exhibited reductions in naïve, potentially alloreactive conventional CD4(+) T cells with relative preservation of memory CD4(+)Foxp3(+) T cells. In particular, CD4(+)CD45RA(-)Foxp3(+hi) effector regulatory T cells (Tregs) recovered rapidly after alloBMT and, unexpectedly, were present at higher levels in patients with GVHD. CD4(+)Foxp3(+) T cells from patients and from allogeneic MLRs expressed relatively high levels of aldehyde dehydrogenase (ALDH), the major in vivo mechanism of Cy resistance. Treatment of MLR cultures with the ALDH inhibitor diethylaminobenzaldehyde reduced the activation and proliferation of CD4(+) T cells and sensitized Tregs to mafosfamide. Finally, removing Tregs from peripheral blood lymphocyte grafts obviated PTCy's GVHD-protective effect in a xenogeneic transplant model. Together, these findings suggest that Treg resistance to Cy through expression of ALDH may contribute to the clinical activity of PTCy in preventing GVHD.
Topics: Adult; Aldehyde Dehydrogenase; Antineoplastic Agents, Alkylating; Bone Marrow Transplantation; Cyclophosphamide; Female; Humans; Lymphocyte Culture Test, Mixed; Male; Middle Aged; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory
PubMed: 24225944
DOI: 10.1126/scitranslmed.3006960 -
Laboratory Investigation; a Journal of... Oct 2013Chondrosarcomas are cartilage-forming, poorly vascularized tumors. They represent the second malignant primary bone tumor of adults after osteosarcoma, but in contrast...
Chondrosarcomas are cartilage-forming, poorly vascularized tumors. They represent the second malignant primary bone tumor of adults after osteosarcoma, but in contrast to osteosarcoma they are resistant to chemotherapy and radiotherapy, surgical excision remaining the only therapeutic option. Few cell lines and animal models are available, and the mechanisms behind their chemoresistance remain largely unknown. Our goal was to establish new cell lines and animal cancer models from human chondrosarcoma biopsies to study their chemoresistance. Between 2007 and 2012, 10 chondrosarcoma biopsies were collected and used for cell culture and transplantation into nude mice. Only one transplanted biopsy and one injected cell line has engrafted successfully leading to conventional central high-grade chondrosarcoma similar to the original biopsies. In culture, two new stable cell lines were obtained, one from a dedifferentiated and one from a grade III conventional central chondrosarcoma biopsy. Their genetic characterization revealed triploid karyotypes, mutations in IDH1, IDH2, and TP53, deletion in CDKN2A and/or MDM2 amplification. These cell lines expressed mesenchymal membrane markers (CD44, 73, 90, 105) and were able to produce a hyaline cartilaginous matrix when cultured in chondrogenic three-dimensional (3D) pellets. Using a high-throughput quantitative RT-PCR approach, we observed that cell lines cultured in monolayer had lost expression of several genes implicated in cartilage development (COL2A1, COMP, ACAN) but restored their expression in 3D cultures. Chondrosarcoma cells in monolayer were sensitive to several conventional chemotherapeutic agents but became resistant to low doses of mafosfamide or doxorubicin when cultured in 3D pellets, in parallel with an altered nucleic accumulation of the drug. Our results indicate that the cartilaginous matrix produced by chondrosarcoma cells may impair diffusion of several drugs and thus contribute to chemoresistance. Therefore, 3D chondrogenic cell pellets constitute a more relevant model to study chondrosarcoma chemoresistance and may be a valuable alternative to animal experimentations.
Topics: Adult; Aged; Animals; Antineoplastic Agents; Biopsy; Bone Neoplasms; Cell Line, Tumor; Chondrogenesis; Chondrosarcoma; Disease Models, Animal; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Gene Expression Profiling; Humans; Male; Mice; Mice, Nude; Mutation; Neoplasm Grading; Tumor Cells, Cultured; Xenograft Model Antitumor Assays
PubMed: 23958880
DOI: 10.1038/labinvest.2013.101 -
Clinical Cancer Research : An Official... Oct 2013Despite advances in the treatment of chronic lymphocytic leukemia (CLL), the disease remains incurable with standard therapies and relapse is inevitable. A growing body...
PURPOSE
Despite advances in the treatment of chronic lymphocytic leukemia (CLL), the disease remains incurable with standard therapies and relapse is inevitable. A growing body of evidence indicates that alterations in the adhesion properties of neoplastic cells play a pivotal role in the development and progression of CLL.
EXPERIMENTAL DESIGN
The expression of 71 cell surface molecules was examined on CLL peripheral blood mononuclear cells (PBMCs) over 3 weeks in culture. The most highly upregulated marker, CD62L, was examined further for expression on CD5(+)/CD19(+) CLL cells in vitro and in lymph node and bone marrow biopsies. The prosurvival role of CD62L was examined using a functional blocking antibody and therapeutic potential evaluated by comparison with current chemotherapy agents.
RESULTS
Blocking CD62L resulted in apoptosis of CLL cells but not PBMCs from healthy donors suggesting a novel role for CD62L in CLL cell survival. The beneficial effect of coculturing CLL cells with bone marrow stromal cells or endothelial cells does not protect CLL cells from anti-CD62L-related toxicity. Moreover, combining fludarabine or mafosfamide with the anti-CD62L in vitro produced an additive effect both with and without stromal cells.
CONCLUSION
This is the first reported data showing that blocking the activation and homing marker, CD62L, regulates CLL cell survival in vitro. These data also suggest that therapeutic antibodies against CD62L may provide additional clinical benefit to patients with CLL receiving current standard chemotherapy protocols.
Topics: Antibodies, Monoclonal; Antineoplastic Agents; Apoptosis; Bone Marrow; Cells, Cultured; Coculture Techniques; Gene Expression Regulation, Neoplastic; Humans; L-Selectin; Leukemia, Lymphocytic, Chronic, B-Cell; Lymph Nodes; Stromal Cells
PubMed: 23948971
DOI: 10.1158/1078-0432.CCR-13-1037 -
Folia Biologica 2013Mafosfamide cyclohexylamine salt (D-17272), 4-hydro-peroxy-cyclophosphamide (D-18864) and glufosfamide (D-19575, beta-D-glucose-isophosphoramide mustard) are new...
Mafosfamide cyclohexylamine salt (D-17272), 4-hydro-peroxy-cyclophosphamide (D-18864) and glufosfamide (D-19575, beta-D-glucose-isophosphoramide mustard) are new generation oxazaphosphorine agents. The present investigation was undertaken to determine the activity of these three oxazaphosphorines in human promyelocytic leukemia HL-60 cells. The research was conducted using the spectrophotometric MTT assay and the electronic Beckman Coulter and microscopy methods. Functional and morphological changes were observed after exposure of HL-60 cells to the oxazaphosphorine agents. The various patterns of temporary alterations in cell viability, size and count, and also in the frequency of leukemic cells undergoing mitotic catastrophe, apoptosis and necrosis, were shown. Different leukemic cell responses to the action of the three oxazaphosphorines were evaluated. These are the first data comparing the in vitro activity of D-17272, D-18864 and D-19575 against human promyelocytic leukemia cells.
Topics: Antineoplastic Agents; Dose-Response Relationship, Drug; HL-60 Cells; Humans; Molecular Structure; Phosphoramide Mustards
PubMed: 23767290
DOI: 10.3409/fb61_1-2.31 -
Pharmacological Reports : PR 2013The heterogeneity of chronic lymphocytic leukemia (CLL) is thought to be due to differences in the expression of factors that regulate apoptosis and cell cycle, giving... (Comparative Study)
Comparative Study Randomized Controlled Trial
BACKGROUND
The heterogeneity of chronic lymphocytic leukemia (CLL) is thought to be due to differences in the expression of factors that regulate apoptosis and cell cycle, giving rise to diverse apoptotic disturbances and tumor properties. Therefore, the primary goal in CLL treatment is to overcome resistance to apoptosis and efficiently trigger this process in leukemic cells.
METHODS
Mononuclear cells were obtained from the blood of CLL patients by Histopaque-1077 sedimentation. CLL cell samples from the blood of drug treated patients, (cladribine or fludarabine with cyclophosphamide; CC or FC), as well as the cell samples of untreated patients exposed to the used drug combinations (CM, FM) or mafosfamide alone for 48 h were fractionated into nuclear and cytoplasmic fractions or were lysed. DNA fragmentation was evaluated by agarose electrophoresis and also cytometrically as sub-G1 population. The expression of apoptosis related proteins and H1.2 histone translocation were evaluated in lysates and nuclear and cytoplasmic fractions, respectively with appropriate antibodies.
RESULTS
Cladribine (C) and fludarabine (F) combined with cyclophosphamide/mafosfamide in vivo, as well as ex vivo trigger apoptosis in CLL cells. These drug combinations (CC; FC/CM; FM) induce leukemic cell apoptosis confirmed by DNA fragmentation, sub-G1 cell number, down-regulation of anti-apoptotic proteins (Mcl-1, Bcl-2), and H1.2 histone translocation in comparison with appropriate control cells, however, to a different degree.
CONCLUSIONS
The kinetics and rate of drug-induced apoptosis in leukemic cells under ex vivo experiments differ between patients, mirroring the differences noticed during in vivo treatment. Individual model cell samples indicate comparable susceptibility to the used drug combinations under in vivo and ex vivo conditions.
Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cladribine; Cyclophosphamide; DNA Fragmentation; Electrophoresis, Agar Gel; Female; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Leukocytes, Mononuclear; Male; Middle Aged; Vidarabine
PubMed: 23744431
DOI: 10.1016/s1734-1140(13)71022-3 -
Oncogene Apr 2014Malignant melanoma is a cancer characterized by high chemoresistance although p53 is rarely mutated. Here, we show that p53 wild-type melanoma cells acquire resistance...
Malignant melanoma is a cancer characterized by high chemoresistance although p53 is rarely mutated. Here, we show that p53 wild-type melanoma cells acquire resistance to cell death induced by fotemustine (FM), which is a representative of alkylating DNA interstrand cross-linking agents used in melanoma therapy. We show that drug-induced resistance is a result of p53-dependent upregulation of the nucleotide excision repair (NER) genes xeroderma pigmentosum complementation group C (XPC) and damaged DNA-binding protein 2 (DDB2), which stimulate the repair of DNA interstrand cross-links (ICLs) arising from O(6)-chloroethylguanine. Consequently, TP53 mutated cells are unable to repair ICLs, leading to prolonged ATM, ATR and checkpoint kinase 1 (CHK1) activation, and finally apoptosis. The roles of p53 and NER in ICL-triggered cell death were confirmed by knockdown of p53 and XPC. Upregulation of XPC and DDB2 in p53wt cells following a single drug treatment is a robust and sustained response that lasts for up to 1 week. Pretreatment with an inducing dose followed by a high and toxic dose of FM provoked an adaptive response as the killing outcome of the challenge dose was reduced. Upregulation of XPC and DDB2 was also observed in a melanoma mouse xenograft model following systemic administration of FM. Additionally, XPC and DDB2 induction occurred upon treatment with other cross-linking anticancer drugs, such as cisplatin and mafosfamide, indicating it is a general response of cancer cells to this group of chemotherapeutics. Collectively, the data indicate that p53-dependent upregulation of XPC and DDB2 is a key mechanism upon genotoxic stress, whereby melanoma cells acquire resistance towards DNA cross-linking agents. To our knowledge, this is the first demonstration of upregulation of NER following a single dose of a DNA interstrand cross-linker, which is a robust and long-lasting effect that impacts the killing response of cancer cells to subsequent treatments.
Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; DNA Damage; DNA Repair; DNA-Binding Proteins; Drug Resistance, Neoplasm; Flow Cytometry; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Immunoblotting; Melanoma; Mice; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Tumor Suppressor Protein p53; Up-Regulation
PubMed: 23604128
DOI: 10.1038/onc.2013.141