The Experts below are selected from a list of 81 Experts worldwide ranked by ideXlab platform
Robert Bases - One of the best experts on this subject based on the ideXlab platform.
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enhanced content of igg in burkitt s lymphoma cells after treatment with the topoisomerase ii inhibitor Lucanthone
Journal of Bioanalysis & Biomedicine, 2017Co-Authors: Robert Bases, Rukmani Lekhraj, Xudong Tang, Jinghang Zhang, Zhi Duan, Jennifer T Aguilan, Edward NievesAbstract:Burkitt’s lymphoma cells (CRL-1647) which had survived treatment with Lucanthone contained 3.6 fold more IgG than untreated cells, although most of the cellular immunoglobulins were still IgM. DNA activation induced cytidine deaminase (AID) was increased 5 fold in these surviving cells, consistent with active Class Switch Recombination (CSR). Progeny of the small fraction of cells which had survived 20 h exposure to 8 μM Lucanthone before rescue were cloned. 1.5 × 108 cloned cells contained ~1 μg of cytidine DNA deaminase, as determined from affinity column isolation of the enzyme, assayed by digestion of a 30 nt 32P labeled specific DNA substrate. Before Lucanthone treatment, little AID could be detected. After the second treatment, a six fold increase in AID was found. In confirmation, Western blot determinations of AID from lysates of Lucanthone treated cells showed 5 fold increased AID content. These results suggest that Lucanthone led to increased IgG content of surviving cells, consistent with their increased AID activity. The surviving cells were also more resistant to the standard Lucanthone treatment, as determined in clonogenic assays. IgG could not be detected in the cell membranes of CRL cells before or after Lucanthone by immunostaining and flow cytometry, but both cell types secreted 80 kDa and 25 kDa immunorelated protein. Lucanthone, formerly used to safely to treat hundreds of thousands of schistosomiasis patients, might be considered as a means to promote IgG synthesis in macroglobulinemia patients.
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abasic sites in dna of hela cells induced by Lucanthone
Cancer Investigation, 2002Co-Authors: Frances Mendez, Joshua D Goldman, Robert BasesAbstract:Abasic sites in HeLa cell DNA were increased in frequency by exposing the cells to Lucanthone. Cell growth in the presence of Lucanthone caused progressive accumulation of abasic sites and loss of cellular DNA. After 2 hr in 8 μM Lucanthone, the abundance of abasic sites was 2.4 fold greater than the background of 9.9±2.0 SE abasic sites/106 nucleotides; 80 μM Lucanthone in the growth medium increased the level 12.6±2.5 SE fold and decreased the DNA content in HeLa cells to one-half of the value obtained in untreated cells. The frequency of abasic sites in cellular DNA was determined by the aldehyde reactive probe method, with reference to abasic sites created in plasmid pBR322.The ability of Lucanthone to inhibit the normal repair of abasic sites might reflect inhibition of apurinic/apyrimidinic endonuclease (HAP1) by the drug, thereby preventing an early step in the base excision repair pathway. Unrepaired abasic sites prevalent after ionizing radiation are cytotoxic lesions that promote DNA strand brea...
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ACCELERATED REGRESSION OF BRAIN METASTASES IN PATIENTS RECEIVING WHOLE BRAIN RADIATION AND THE TOPOISOMERASE II INHIBITOR, Lucanthone
International Journal of Radiation Oncology Biology Physics, 1999Co-Authors: John D. Del Rowe, Robin J. Mitnick, Christopher Filippi, Frances Mendez, Justin K Moran, Brij Sood, Katherine Freeman, Jacqueline A. Bello, Robert BasesAbstract:Abstract Purpose: To determine if Lucanthone crossed the blood-brain barrier in experimental animals; and to determine accelerated tumor regression of human brain metastases treated jointly with Lucanthone and whole brain radiation. Methods and Materials: The organ distribution of 3 H Lucanthone in mice and 125 I Lucanthone in rats was determined to learn if Lucanthone crossed the blood-brain barrier. Size determinations were made of patients' brain metastases from magnetic resonance images or by computed tomography before and after treatment with 30 Gy whole brain radiation alone or with Lucanthone. Results: The time course of Lucanthone's distribution in brain was identical to that in muscle and heart after intraperitoneal or intravenous administration in experimental animals. Lucanthone, therefore, readily crossed the blood-brain barrier in experimental animals. Conclusion: Compared with radiation alone, the tumor regression in patients with brain metastases treated with Lucanthone and radiation was accelerated, approaching significance using a permutation test at p = 0.0536.
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topoisomerase inhibition by Lucanthone an adjuvant in radiation therapy
International Journal of Radiation Oncology Biology Physics, 1997Co-Authors: Robert Bases, Frances MendezAbstract:Purpose: To determine whether Lucanthone can inhibit human topoisomerases in vitro. Methods and Materials: Lucanthone was incubated with human topoisomerases II and I together with their plasmid substrates, to determine if Lucanthone interfered with the catalytic activities of topoisomerases and if it enhanced the formation of DNA strand breaks, as determined by agarose gel electrophoresis of the resultant plasmid forms. Results: Incubation of the enzymes with Lucanthone inhibited the catalytic activity of topoisomerases II and I. With topoisomerase II, it increased the abundance of DNA double strand breaks (cleavable complexes). Conclusion: Lucanthone, like actinomycin D, inhibited topoisomerases II and I. It may act to enhance the yield of DNA double strand breaks in cells through a mechanism of topoisomerase II inhibition.
Pankaj Chaudhary - One of the best experts on this subject based on the ideXlab platform.
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Lucanthone and Its Derivative Hycanthone Inhibit Apurinic Endonuclease-1 (APE1) by Direct Protein Binding
2016Co-Authors: Mamta D. Naidu, Louis A. Peña, Rakhi Agarwal, Mihaly Mezei, Min Shen, Yuan Liu, Zina Sanchez, Luis Cunha, David M, Pankaj ChaudharyAbstract:Lucanthone and hycanthone are thioxanthenone DNA intercalators used in the 1980s as antitumor agents. Lucanthone is in Phase I clinical trial, whereas hycanthone was pulled out of Phase II trials. Their potential mechanism of action includes DNA intercalation, inhibition of nucleic acid biosyntheses, and inhibition of enzymes like topoisomerases and the dual function base excision repair enzyme apurinic endonuclease 1 (APE1). Lucanthone inhibits the endonuclease activity of APE1, without affecting its redox activity. Our goal was to decipher the precise mechanism of APE1 inhibition as a prerequisite towards development of improved therapeutics that can counteract higher APE1 activity often seen in tumors. The IC50 values for inhibition of APE1 incision of depurinated plasmid DNA by Lucanthone and hycanthone were 5 mM and 80 nM, respectively. The KD values (affinity constants) for APE1, as determined by BIACORE binding studies, were 89 nM for Lucanthone/10 nM for hycanthone. APE1 structures reveal a hydrophobic pocket where hydrophobic small molecules lik
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graphene nanoribbons as a drug delivery agent for Lucanthone mediated therapy of glioblastoma multiforme
Nanomedicine: Nanotechnology Biology and Medicine, 2015Co-Authors: Sayan Mullick Chowdhury, Louis A. Peña, Zina Sanchez, Pankaj Chaudhary, Michael J Waring, Cassandra Surhland, M Suresh A Kumar, Stephen Lee, Balaji SitharamanAbstract:Abstract We report use of PEG-DSPE coated oxidized graphene nanoribbons (O-GNR-PEG-DSPE) as agent for delivery of anti-tumor drug Lucanthone (Luc) into Glioblastoma Multiformae (GBM) cells targeting base excision repair enzyme APE-1 (Apurinic endonuclease-1). Lucanthone, an endonuclease inhibitor of APE-1, was loaded onto O-GNR-PEG-DSPEs using a simple non-covalent method. We found its uptake by GBM cell line U251 exceeding 67% and 60% in APE-1-overexpressing U251, post 24 h. However, their uptake was ~ 38% and 29% by MCF-7 and rat glial progenitor cells (CG-4), respectively. TEM analysis of U251 showed large aggregates of O-GNR-PEG-DSPE in vesicles. Luc-O-GNR-PEG-DSPE was significantly toxic to U251 but showed little/no toxicity when exposed to MCF-7/CG-4 cells. This differential uptake effect can be exploited to use O-GNR-PEG-DSPEs as a vehicle for Luc delivery to GBM, while reducing nonspecific cytotoxicity to the surrounding healthy tissue. Cell death in U251 was necrotic, probably due to oxidative degradation of APE-1. From the Clinical Editor This study reports on the utility of PEG-DSPE coated oxidized graphene nanoribbons as anti-tumor drug delivery agents of Lucanthone into Glioblastoma Multiformae cells targeting base excision repair enzyme APE-1, demonstrating promising anti-tumor effects with good preservation of healthy cells.
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Lucanthone and its derivative hycanthone inhibit apurinic endonuclease 1 ape1 by direct protein binding
PLOS ONE, 2011Co-Authors: Mamta D. Naidu, Louis A. Peña, Rakhi Agarwal, Luis F Cunha, Mihaly Mezei, Min Shen, David M Wilson, Yuan Liu, Zina Sanchez, Pankaj ChaudharyAbstract:Lucanthone and hycanthone are thioxanthenone DNA intercalators used in the 1980s as antitumor agents. Lucanthone is in Phase I clinical trial, whereas hycanthone was pulled out of Phase II trials. Their potential mechanism of action includes DNA intercalation, inhibition of nucleic acid biosyntheses, and inhibition of enzymes like topoisomerases and the dual function base excision repair enzyme apurinic endonuclease 1 (APE1). Lucanthone inhibits the endonuclease activity of APE1, without affecting its redox activity. Our goal was to decipher the precise mechanism of APE1 inhibition as a prerequisite towards development of improved therapeutics that can counteract higher APE1 activity often seen in tumors. The IC50 values for inhibition of APE1 incision of depurinated plasmid DNA by Lucanthone and hycanthone were 5 µM and 80 nM, respectively. The KD values (affinity constants) for APE1, as determined by BIACORE binding studies, were 89 nM for Lucanthone/10 nM for hycanthone. APE1 structures reveal a hydrophobic pocket where hydrophobic small molecules like thioxanthenones can bind, and our modeling studies confirmed such docking. Circular dichroism spectra uncovered change in the helical structure of APE1 in the presence of Lucanthone/hycanthone, and notably, this effect was decreased (Phe266Ala or Phe266Cys or Trp280Leu) or abolished (Phe266Ala/Trp280Ala) when hydrophobic site mutants were employed. Reduced inhibition by Lucanthone of the diminished endonuclease activity of hydrophobic mutant proteins (as compared to wild type APE1) supports that binding of Lucanthone to the hydrophobic pocket dictates APE1 inhibition. The DNA binding capacity of APE1 was marginally inhibited by Lucanthone, and not at all by hycanthone, supporting our hypothesis that thioxanthenones inhibit APE1, predominantly, by direct interaction. Finally, Lucanthone-induced degradation was drastically reduced in the presence of short and long lived free radical scavengers, e.g., TRIS and DMSO, suggesting that the mechanism of APE1 breakdown may involve free radical-induced peptide bond cleavage.
Louis A. Peña - One of the best experts on this subject based on the ideXlab platform.
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Lucanthone and Its Derivative Hycanthone Inhibit Apurinic Endonuclease-1 (APE1) by Direct Protein Binding
2016Co-Authors: Mamta D. Naidu, Louis A. Peña, Rakhi Agarwal, Mihaly Mezei, Min Shen, Yuan Liu, Zina Sanchez, Luis Cunha, David M, Pankaj ChaudharyAbstract:Lucanthone and hycanthone are thioxanthenone DNA intercalators used in the 1980s as antitumor agents. Lucanthone is in Phase I clinical trial, whereas hycanthone was pulled out of Phase II trials. Their potential mechanism of action includes DNA intercalation, inhibition of nucleic acid biosyntheses, and inhibition of enzymes like topoisomerases and the dual function base excision repair enzyme apurinic endonuclease 1 (APE1). Lucanthone inhibits the endonuclease activity of APE1, without affecting its redox activity. Our goal was to decipher the precise mechanism of APE1 inhibition as a prerequisite towards development of improved therapeutics that can counteract higher APE1 activity often seen in tumors. The IC50 values for inhibition of APE1 incision of depurinated plasmid DNA by Lucanthone and hycanthone were 5 mM and 80 nM, respectively. The KD values (affinity constants) for APE1, as determined by BIACORE binding studies, were 89 nM for Lucanthone/10 nM for hycanthone. APE1 structures reveal a hydrophobic pocket where hydrophobic small molecules lik
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graphene nanoribbons as a drug delivery agent for Lucanthone mediated therapy of glioblastoma multiforme
Nanomedicine: Nanotechnology Biology and Medicine, 2015Co-Authors: Sayan Mullick Chowdhury, Louis A. Peña, Zina Sanchez, Pankaj Chaudhary, Michael J Waring, Cassandra Surhland, M Suresh A Kumar, Stephen Lee, Balaji SitharamanAbstract:Abstract We report use of PEG-DSPE coated oxidized graphene nanoribbons (O-GNR-PEG-DSPE) as agent for delivery of anti-tumor drug Lucanthone (Luc) into Glioblastoma Multiformae (GBM) cells targeting base excision repair enzyme APE-1 (Apurinic endonuclease-1). Lucanthone, an endonuclease inhibitor of APE-1, was loaded onto O-GNR-PEG-DSPEs using a simple non-covalent method. We found its uptake by GBM cell line U251 exceeding 67% and 60% in APE-1-overexpressing U251, post 24 h. However, their uptake was ~ 38% and 29% by MCF-7 and rat glial progenitor cells (CG-4), respectively. TEM analysis of U251 showed large aggregates of O-GNR-PEG-DSPE in vesicles. Luc-O-GNR-PEG-DSPE was significantly toxic to U251 but showed little/no toxicity when exposed to MCF-7/CG-4 cells. This differential uptake effect can be exploited to use O-GNR-PEG-DSPEs as a vehicle for Luc delivery to GBM, while reducing nonspecific cytotoxicity to the surrounding healthy tissue. Cell death in U251 was necrotic, probably due to oxidative degradation of APE-1. From the Clinical Editor This study reports on the utility of PEG-DSPE coated oxidized graphene nanoribbons as anti-tumor drug delivery agents of Lucanthone into Glioblastoma Multiformae cells targeting base excision repair enzyme APE-1, demonstrating promising anti-tumor effects with good preservation of healthy cells.
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Lucanthone and its derivative hycanthone inhibit apurinic endonuclease 1 ape1 by direct protein binding
PLOS ONE, 2011Co-Authors: Mamta D. Naidu, Louis A. Peña, Rakhi Agarwal, Luis F Cunha, Mihaly Mezei, Min Shen, David M Wilson, Yuan Liu, Zina Sanchez, Pankaj ChaudharyAbstract:Lucanthone and hycanthone are thioxanthenone DNA intercalators used in the 1980s as antitumor agents. Lucanthone is in Phase I clinical trial, whereas hycanthone was pulled out of Phase II trials. Their potential mechanism of action includes DNA intercalation, inhibition of nucleic acid biosyntheses, and inhibition of enzymes like topoisomerases and the dual function base excision repair enzyme apurinic endonuclease 1 (APE1). Lucanthone inhibits the endonuclease activity of APE1, without affecting its redox activity. Our goal was to decipher the precise mechanism of APE1 inhibition as a prerequisite towards development of improved therapeutics that can counteract higher APE1 activity often seen in tumors. The IC50 values for inhibition of APE1 incision of depurinated plasmid DNA by Lucanthone and hycanthone were 5 µM and 80 nM, respectively. The KD values (affinity constants) for APE1, as determined by BIACORE binding studies, were 89 nM for Lucanthone/10 nM for hycanthone. APE1 structures reveal a hydrophobic pocket where hydrophobic small molecules like thioxanthenones can bind, and our modeling studies confirmed such docking. Circular dichroism spectra uncovered change in the helical structure of APE1 in the presence of Lucanthone/hycanthone, and notably, this effect was decreased (Phe266Ala or Phe266Cys or Trp280Leu) or abolished (Phe266Ala/Trp280Ala) when hydrophobic site mutants were employed. Reduced inhibition by Lucanthone of the diminished endonuclease activity of hydrophobic mutant proteins (as compared to wild type APE1) supports that binding of Lucanthone to the hydrophobic pocket dictates APE1 inhibition. The DNA binding capacity of APE1 was marginally inhibited by Lucanthone, and not at all by hycanthone, supporting our hypothesis that thioxanthenones inhibit APE1, predominantly, by direct interaction. Finally, Lucanthone-induced degradation was drastically reduced in the presence of short and long lived free radical scavengers, e.g., TRIS and DMSO, suggesting that the mechanism of APE1 breakdown may involve free radical-induced peptide bond cleavage.
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Radiation Resistance in Glioma Cells Determined by DNA Damage Repair Activity of Ape1/Ref-1
Journal of Radiation Research, 2010Co-Authors: Mamta D. Naidu, Hua Fung, James M. Mason, Raymond V. Pica, Louis A. PeñaAbstract:Since radiation therapy remains a primary treatment modality for gliomas, the radioresistance of glioma cells and targets to modify their radiation tolerance are of significant interest. Human apurinic endonuclease 1 (Ape1, Ref-1, APEX, HAP1, AP endo) is a multifunctional protein involved in base excision repair of DNA and a redox-dependent transcriptional co-activator. This study investigated whether there is a direct relationship between Ape1 and radioresistance in glioma cells, employing the human U87 and U251 cell lines. U87 is intrinsically more radioresistant than U251, which is partly attributable to more cycling U251 cells found in G2/M, the most radiosensitive cell stage, while more U87 cells are found in S and G1, the more radioresistant cell stages. But observed radioresistance is also related to Ape1 activity. U87 has higher levels of Ape1 than does U251, as assessed by Western blot and enzyme activity assays (~1.5–2 fold higher in cycling cells, and ~10 fold higher at G2/M). A direct relationship was seen in cells transfected with CMV-Ape1 constructs; there was a dose-dependent relationship between increasing Ape1 overexpression and increasing radioresistance. Conversely, knock down by siRNA or by pharmacological down regulation of Ape1 resulted in decreased radioresistance. The inhibitors Lucanthone and CRT004876 were employed, the former a thioxanthene previously under clinical evaluation as a radiosensitizer for brain tumors and the latter a more specific Ape1 inhibitor. These data suggest that Ape1 may be a useful target for modifying radiation tolerance.
Frances Mendez - One of the best experts on this subject based on the ideXlab platform.
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abasic sites in dna of hela cells induced by Lucanthone
Cancer Investigation, 2002Co-Authors: Frances Mendez, Joshua D Goldman, Robert BasesAbstract:Abasic sites in HeLa cell DNA were increased in frequency by exposing the cells to Lucanthone. Cell growth in the presence of Lucanthone caused progressive accumulation of abasic sites and loss of cellular DNA. After 2 hr in 8 μM Lucanthone, the abundance of abasic sites was 2.4 fold greater than the background of 9.9±2.0 SE abasic sites/106 nucleotides; 80 μM Lucanthone in the growth medium increased the level 12.6±2.5 SE fold and decreased the DNA content in HeLa cells to one-half of the value obtained in untreated cells. The frequency of abasic sites in cellular DNA was determined by the aldehyde reactive probe method, with reference to abasic sites created in plasmid pBR322.The ability of Lucanthone to inhibit the normal repair of abasic sites might reflect inhibition of apurinic/apyrimidinic endonuclease (HAP1) by the drug, thereby preventing an early step in the base excision repair pathway. Unrepaired abasic sites prevalent after ionizing radiation are cytotoxic lesions that promote DNA strand brea...
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ACCELERATED REGRESSION OF BRAIN METASTASES IN PATIENTS RECEIVING WHOLE BRAIN RADIATION AND THE TOPOISOMERASE II INHIBITOR, Lucanthone
International Journal of Radiation Oncology Biology Physics, 1999Co-Authors: John D. Del Rowe, Robin J. Mitnick, Christopher Filippi, Frances Mendez, Justin K Moran, Brij Sood, Katherine Freeman, Jacqueline A. Bello, Robert BasesAbstract:Abstract Purpose: To determine if Lucanthone crossed the blood-brain barrier in experimental animals; and to determine accelerated tumor regression of human brain metastases treated jointly with Lucanthone and whole brain radiation. Methods and Materials: The organ distribution of 3 H Lucanthone in mice and 125 I Lucanthone in rats was determined to learn if Lucanthone crossed the blood-brain barrier. Size determinations were made of patients' brain metastases from magnetic resonance images or by computed tomography before and after treatment with 30 Gy whole brain radiation alone or with Lucanthone. Results: The time course of Lucanthone's distribution in brain was identical to that in muscle and heart after intraperitoneal or intravenous administration in experimental animals. Lucanthone, therefore, readily crossed the blood-brain barrier in experimental animals. Conclusion: Compared with radiation alone, the tumor regression in patients with brain metastases treated with Lucanthone and radiation was accelerated, approaching significance using a permutation test at p = 0.0536.
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topoisomerase inhibition by Lucanthone an adjuvant in radiation therapy
International Journal of Radiation Oncology Biology Physics, 1997Co-Authors: Robert Bases, Frances MendezAbstract:Purpose: To determine whether Lucanthone can inhibit human topoisomerases in vitro. Methods and Materials: Lucanthone was incubated with human topoisomerases II and I together with their plasmid substrates, to determine if Lucanthone interfered with the catalytic activities of topoisomerases and if it enhanced the formation of DNA strand breaks, as determined by agarose gel electrophoresis of the resultant plasmid forms. Results: Incubation of the enzymes with Lucanthone inhibited the catalytic activity of topoisomerases II and I. With topoisomerase II, it increased the abundance of DNA double strand breaks (cleavable complexes). Conclusion: Lucanthone, like actinomycin D, inhibited topoisomerases II and I. It may act to enhance the yield of DNA double strand breaks in cells through a mechanism of topoisomerase II inhibition.
Mamta D. Naidu - One of the best experts on this subject based on the ideXlab platform.
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Lucanthone and Its Derivative Hycanthone Inhibit Apurinic Endonuclease-1 (APE1) by Direct Protein Binding
2016Co-Authors: Mamta D. Naidu, Louis A. Peña, Rakhi Agarwal, Mihaly Mezei, Min Shen, Yuan Liu, Zina Sanchez, Luis Cunha, David M, Pankaj ChaudharyAbstract:Lucanthone and hycanthone are thioxanthenone DNA intercalators used in the 1980s as antitumor agents. Lucanthone is in Phase I clinical trial, whereas hycanthone was pulled out of Phase II trials. Their potential mechanism of action includes DNA intercalation, inhibition of nucleic acid biosyntheses, and inhibition of enzymes like topoisomerases and the dual function base excision repair enzyme apurinic endonuclease 1 (APE1). Lucanthone inhibits the endonuclease activity of APE1, without affecting its redox activity. Our goal was to decipher the precise mechanism of APE1 inhibition as a prerequisite towards development of improved therapeutics that can counteract higher APE1 activity often seen in tumors. The IC50 values for inhibition of APE1 incision of depurinated plasmid DNA by Lucanthone and hycanthone were 5 mM and 80 nM, respectively. The KD values (affinity constants) for APE1, as determined by BIACORE binding studies, were 89 nM for Lucanthone/10 nM for hycanthone. APE1 structures reveal a hydrophobic pocket where hydrophobic small molecules lik
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Lucanthone and its derivative hycanthone inhibit apurinic endonuclease 1 ape1 by direct protein binding
PLOS ONE, 2011Co-Authors: Mamta D. Naidu, Louis A. Peña, Rakhi Agarwal, Luis F Cunha, Mihaly Mezei, Min Shen, David M Wilson, Yuan Liu, Zina Sanchez, Pankaj ChaudharyAbstract:Lucanthone and hycanthone are thioxanthenone DNA intercalators used in the 1980s as antitumor agents. Lucanthone is in Phase I clinical trial, whereas hycanthone was pulled out of Phase II trials. Their potential mechanism of action includes DNA intercalation, inhibition of nucleic acid biosyntheses, and inhibition of enzymes like topoisomerases and the dual function base excision repair enzyme apurinic endonuclease 1 (APE1). Lucanthone inhibits the endonuclease activity of APE1, without affecting its redox activity. Our goal was to decipher the precise mechanism of APE1 inhibition as a prerequisite towards development of improved therapeutics that can counteract higher APE1 activity often seen in tumors. The IC50 values for inhibition of APE1 incision of depurinated plasmid DNA by Lucanthone and hycanthone were 5 µM and 80 nM, respectively. The KD values (affinity constants) for APE1, as determined by BIACORE binding studies, were 89 nM for Lucanthone/10 nM for hycanthone. APE1 structures reveal a hydrophobic pocket where hydrophobic small molecules like thioxanthenones can bind, and our modeling studies confirmed such docking. Circular dichroism spectra uncovered change in the helical structure of APE1 in the presence of Lucanthone/hycanthone, and notably, this effect was decreased (Phe266Ala or Phe266Cys or Trp280Leu) or abolished (Phe266Ala/Trp280Ala) when hydrophobic site mutants were employed. Reduced inhibition by Lucanthone of the diminished endonuclease activity of hydrophobic mutant proteins (as compared to wild type APE1) supports that binding of Lucanthone to the hydrophobic pocket dictates APE1 inhibition. The DNA binding capacity of APE1 was marginally inhibited by Lucanthone, and not at all by hycanthone, supporting our hypothesis that thioxanthenones inhibit APE1, predominantly, by direct interaction. Finally, Lucanthone-induced degradation was drastically reduced in the presence of short and long lived free radical scavengers, e.g., TRIS and DMSO, suggesting that the mechanism of APE1 breakdown may involve free radical-induced peptide bond cleavage.
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Radiation Resistance in Glioma Cells Determined by DNA Damage Repair Activity of Ape1/Ref-1
Journal of Radiation Research, 2010Co-Authors: Mamta D. Naidu, Hua Fung, James M. Mason, Raymond V. Pica, Louis A. PeñaAbstract:Since radiation therapy remains a primary treatment modality for gliomas, the radioresistance of glioma cells and targets to modify their radiation tolerance are of significant interest. Human apurinic endonuclease 1 (Ape1, Ref-1, APEX, HAP1, AP endo) is a multifunctional protein involved in base excision repair of DNA and a redox-dependent transcriptional co-activator. This study investigated whether there is a direct relationship between Ape1 and radioresistance in glioma cells, employing the human U87 and U251 cell lines. U87 is intrinsically more radioresistant than U251, which is partly attributable to more cycling U251 cells found in G2/M, the most radiosensitive cell stage, while more U87 cells are found in S and G1, the more radioresistant cell stages. But observed radioresistance is also related to Ape1 activity. U87 has higher levels of Ape1 than does U251, as assessed by Western blot and enzyme activity assays (~1.5–2 fold higher in cycling cells, and ~10 fold higher at G2/M). A direct relationship was seen in cells transfected with CMV-Ape1 constructs; there was a dose-dependent relationship between increasing Ape1 overexpression and increasing radioresistance. Conversely, knock down by siRNA or by pharmacological down regulation of Ape1 resulted in decreased radioresistance. The inhibitors Lucanthone and CRT004876 were employed, the former a thioxanthene previously under clinical evaluation as a radiosensitizer for brain tumors and the latter a more specific Ape1 inhibitor. These data suggest that Ape1 may be a useful target for modifying radiation tolerance.
