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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 -
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 -
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 -
Postepy Higieny I Medycyny... Mar 2013Chronic lymphocytic leukemia (CLL) remains incurable; therefore searching for new therapeutic strategies in this disease is necessary. An important mechanism of tumor...
INTRODUCTION
Chronic lymphocytic leukemia (CLL) remains incurable; therefore searching for new therapeutic strategies in this disease is necessary. An important mechanism of tumor development is neoangiogenesis. A potent antiangiogenic factor, bevacizumab (Avastin, AVA), has been poorly explored in CLL so far. In the current study we assessed cytotoxic activity of AVA alone or in combinations with drugs routinely used in this disease.
MATERIALS AND METHODS
Cells isolated from 60 CLL patients were treated with AVA alone or in combination with anti-CD20 monoclonal antibody (MoAb), rituximab (RIT), anti-CD52 MoAb, alemtuzumab (ALT), 2-CdA (2-chlorodeoxyadenosine), FA (fludarabine), MAF (mafosfamide) or RAPA (rapamycin). Cytotoxicity was assessed by propidium iodide staining. Apoptosis was evaluated using annexin-V and TUNEL assays. Additionally, a drop of mitochondrial potential (DYm) as well as expression of apoptosis-regulating proteins Bax, Bak, Bid, Bad, Bcl-2, Mcl-2, XIAP, FLIP, Akt and Bcl-2-A1 were determined by flow cytometry.
RESULTS
At the dose of 40 μg/ml, after 48 hours of incubation, AVA induced significant cytotoxicity against CLL cells. The drug triggered apoptosis, with activation of caspase-3 and -9, but not caspase-8, along with a drop of DYm. Incubation with AVA induced significant overexpression of proapoptotic Bak and Bad as well as downregulation of antiapoptotic Mcl-2 and Akt proteins. Combination of AVA with RIT, ALT or RAPA significantly increased cytotoxicity when compared with the effects of single drugs.
DISCUSSION
In conclusion, this is the first report showing proapoptotic activity of AVA against CLL cells. Combination of AVA with RIT, ALT or RAPA may be a promising therapeutic strategy, which requires confirmation in further studies.
Topics: Alemtuzumab; Angiogenesis Inhibitors; Antibodies, Monoclonal, Humanized; Antibodies, Monoclonal, Murine-Derived; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bevacizumab; Caspase 3; Caspase 9; Cladribine; Cyclophosphamide; Down-Regulation; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Proteasome Inhibitors; Rituximab; Sirolimus; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vidarabine
PubMed: 23475487
DOI: 10.5604/17322693.1038349 -
Toxins Feb 2013Shiga toxin 1 (Stx1), produced by pathogenic Escherichia coli, targets a restricted subset of human cells, which possess the receptor globotriaosylceramide...
Shiga toxin 1 (Stx1), produced by pathogenic Escherichia coli, targets a restricted subset of human cells, which possess the receptor globotriaosylceramide (Gb3Cer/CD77), causing hemolytic uremic syndrome. In spite of the high toxicity, Stx1 has been proposed in the treatment of Gb3Cer/CD77-expressing lymphoma. Here, we demonstrate in a Burkitt lymphoma cell model expressing this receptor, namely Raji cells, that Stx1, at quasi-non-toxic concentrations (0.05-0.1 pM), inhibits the repair of mafosfamide-induced DNA alkylating lesions, synergistically potentiating the cytotoxic activity of the anticancer drug. Conversely, human promyelocytic leukemia cells HL-60, which do not express Gb3Cer/CD77, were spared by the toxin as previously demonstrated for CD34+ human progenitor cells, and hence, in this cancer model, no additive nor synergistic effects were observed with the combined Stx1/mafosfamide treatment. Our findings suggest that Stx1 could be used to improve the mafosfamide-mediated purging of Gb3Cer/CD77+ tumor cells before autologous bone marrow transplantation.
Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Cyclophosphamide; DNA Repair; Drug Synergism; HL-60 Cells; Human Umbilical Vein Endothelial Cells; Humans; Protein Synthesis Inhibitors; Shiga Toxin 1
PubMed: 23430607
DOI: 10.3390/toxins5020431 -
Journal of Neuro-oncology Sep 2012A pilot study to investigate the feasibility of the addition of intrathecal (IT) mafosfamide to a regimen of concomitant multi-agent systemic chemotherapy followed by... (Clinical Trial)
Clinical Trial
Pilot study of systemic and intrathecal mafosfamide followed by conformal radiation for infants with intracranial central nervous system tumors: a pediatric brain tumor consortium study (PBTC-001).
A pilot study to investigate the feasibility of the addition of intrathecal (IT) mafosfamide to a regimen of concomitant multi-agent systemic chemotherapy followed by conformal radiation therapy (RT) for children <3 years with newly diagnosed embryonal CNS tumors was performed. Ninety-three newly diagnosed infants and children (<3 years) with embryonal CNS tumors were enrolled. Twenty weeks of systemic multi-agent chemotherapy commenced within 35 days of surgery. Patients without CSF flow obstruction (n = 71) received IT mafosfamide (14 mg) with chemotherapy. Localized (M(0)) patients with SD or better subsequently received RT followed by 20 additional weeks of chemotherapy. Second look surgery was encouraged prior to RT if there was an incomplete surgical resection at diagnosis. 71 evaluable patients with normal CSF flow received IT Mafosfamide with systemic chemotherapy; patients with M + disease were removed from protocol therapy at 20 weeks and those with PD at the time of progression. One and 5-year progression free survival (PFS) and overall survival (OS) for the cohort of 71 evaluable patients were 52 ± 6.5 % and 33 ± 13 %, and 67 ± 6.2 % and 51 ± 11 %, respectively. The 1-year Progression Free Survival (PFS) for M0 patients with medulloblastoma (MB, n = 20), supratentorial primitive neuroectodermal tumor (PNET, n = 9), and atypical teratoid rhabdoid tumor (ATRT, n = 12) was 80 ± 7 %, 67 ± 15 % and 27 ± 13 % and 5-year PFS was 65 ± 19 %, 37 ± 29 %, and 0 ± 0 %, respectively. The addition of IT mafosfamide to systemic chemotherapy in infants with embryonal CNS tumors was feasible. The PFS for M0 patients appears comparable to or better than most prior historical comparisons and was excellent for those receiving conformal radiotherapy.
Topics: Antineoplastic Agents; Brain Neoplasms; Cyclophosphamide; Disease-Free Survival; Feasibility Studies; Female; Humans; Infant; Injections, Spinal; Male; Neoplasm Staging; Pilot Projects; Radiotherapy, Conformal
PubMed: 22790443
DOI: 10.1007/s11060-012-0929-x -
The Journal of Biological Chemistry Dec 2011Human aldehyde dehydrogenases (ALDHs) comprise a family of 17 homologous enzymes that metabolize different biogenic and exogenic aldehydes. To date, there are relatively...
Human aldehyde dehydrogenases (ALDHs) comprise a family of 17 homologous enzymes that metabolize different biogenic and exogenic aldehydes. To date, there are relatively few general ALDH inhibitors that can be used to probe the contribution of this class of enzymes to particular metabolic pathways. Here, we report the discovery of a general class of ALDH inhibitors with a common mechanism of action. The combined data from kinetic studies, mass spectrometric measurements, and crystallographic analyses demonstrate that these inhibitors undergo an enzyme-mediated β-elimination reaction generating a vinyl ketone intermediate that covalently modifies the active site cysteine residue present in these enzymes. The studies described here can provide the basis for rational approach to design ALDH isoenzyme-specific inhibitors as research tools and perhaps as drugs, to address diseases such as cancer where increased ALDH activity is associated with a cellular phenotype.
Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Aldehyde Dehydrogenase, Mitochondrial; Aldehydes; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclophosphamide; Enzyme Inhibitors; Humans; Kinetics; Mass Spectrometry; Molecular Structure; Protein Structure, Secondary; Retinal Dehydrogenase
PubMed: 22021038
DOI: 10.1074/jbc.M111.293597