4-Ethynyl-4-hydroxycyclohexa-2,5-dien-1-one 5 undergoes cycloaddition reactions with a range of s... more 4-Ethynyl-4-hydroxycyclohexa-2,5-dien-1-one 5 undergoes cycloaddition reactions with a range of substituted azides in the presence of copper salts to form 1,4-disubstituted triazoles 8-11 bearing the 4-hydroxycyclohexa-2,5-dien-1-one (quinol) pharmacophore; one example of an isomeric 1,5-disubstituted triazole 12 was formed from 5 and benzyl azide in the presence of a ruthenium catalyst. Compounds were screened for growth-inhibitory activity against five cancer cell lines of colon, breast and lung origin, but were overall less potent than the benzothiazolyl- and indolyl-substituted quinols 2 and 3.
ABSTRACT ON01910.Na, a styryl benzylsulfone, is a Phase III stage, non-ATP competitive anti-cance... more ABSTRACT ON01910.Na, a styryl benzylsulfone, is a Phase III stage, non-ATP competitive anti-cancer agent. It is multi-targeted, promoting selective mitotic arrest and apoptosis in cancer cells. Extensive Phase II/III trials conducted in patients with solid tumors and hematological cancers have proved its excellent efficacy and impressive safety profile. However, incomplete understanding of mechanisms of action and relatively low drug oral bioavailability remain obstacles to development. By modifying the structure of ON01910.Na, a novel class of (E)-Styrylsulfonyl methylpyridines was designed and synthesized. We report herein the evaluation of a selected compound, TL-77, (E)-N-(2-methoxy-5-((2,4,6-trimethoxystyrylsulfonyl)methyl)pyridin-3-yl)methanesulfonamide, as a potent small molecule inhibitor targeting mitotic pathways. MTT assays were performed to assess in vitro anti-proliferative effects. In a panel of 10 tumor and 2 non-transformed human cell lines following 96 h exposure, TL-77 gave consistent GI50 values in the sub-micromolar range in tumor cells, whereas, it was non-toxic to non-transformed human mammary endothelial cells (HMECs). Cell cycle was analyzed by flow cytometry. TL-77 caused significant G2/M arrest ≥ 6 h, whereas ON01910.Na caused the same effect after longer exposure periods (≥ 12 h). An OD-based tubulin polymerization assay allowed us to explain obstruction of cell division caused by TL-77. TL-77 retarded tubulin polymerization between 1μM and 6μM, intriguingly, microtubules were stabilizes at TL-77 concentrations > 8μM. Western blot analyses of cell lysates following treatment of cells with TL-77 revealed dose-dependent reduction of phosphorylated Cdc25c, a known substrate of Plk1 and/or Chk1/2, indicating Plk1 and/or Chk1/2 inhibition. Finally, dose- and time-dependent apoptosis triggered by TL-77, detected by Annexin-V assays, was closely associated with induction of caspase 3/7 activity. Importantly, pharmacokinetics studies in mice revealed that the oral bioavailability of TL-77 was optimized to 56% compared with 9% for ON01910.Na. In summary, TL-77 is a potent anti-cancer agent. It disturbs tubulin during cell division, resulting in G2/M cell cycle arrest, followed by caspase-dependent apoptosis. TL-77 appears to share an analogous mechanism of action with ON01910.Na, but exhibits greater selectivity towards cancer cells and possesses superior oral bioavailability. Therefore, further evaluation of TL-77 as a promising anti-tumor agent is justified. Citation Format: Tiangong Lu, Shudong Wang, Charles Laughton, Tracey Bradshaw. Evaluation of (E)-Styrylsulfonyl methylpyridine: A novel kinase inhibitor targeting mitotic pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3406. doi:10.1158/1538-7445.AM2013-3406
Glioblastoma multiforme is the most common aggressive adult primary tumour of the central nervous... more Glioblastoma multiforme is the most common aggressive adult primary tumour of the central nervous system. Treatment includes surgery, radiotherapy and adjuvant temozolomide (TMZ) chemotherapy. TMZ is an alkylating agent prodrug, delivering a methyl group to purine bases of DNA (O6-guanine; N7-guanine and N3-adenine). The primary cytotoxic lesion, O6-methylguanine (O6-MeG) can be removed by methylguanine methyltransferase (MGMT; direct repair) in tumours expressing this protein, or tolerated in mismatch repair-deficient (MMR-) tumours. Thus MGMT or MMR deficiency confers resistance to TMZ. Inherent- and acquired resistance to TMZ present major obstacles to successful treatment. Strategies devised to thwart resistance and enhance response to TMZ, including inhibition of DNA repair mechanisms which contribute to TMZ resistance, are under clinical evaluation. Depletion of MGMT prior to alkylating agent chemotherapy prevents O6-MeG repair; thus, MGMT pseudosubstrates O6-benzylguanine and...
The interactions of small molecules with nucleic acids are of considerable interest for the desig... more The interactions of small molecules with nucleic acids are of considerable interest for the design of novel anticancer compounds. The physical properties and sequence specificity observed in the interactions between a group of flavonoids with proven antitumour activity and various nucleic acid structures are summarised. UV and fluorescence spectroscopy, together with competition dialysis, were used to assess the affinity of the drugs for the nucleic acid structures in the presence or absence of different metal ions. The effect of these compounds on breast and leukaemia cancer cell lines was evaluated using MTS and COMET assays and flow cytometry. The flavonoids studied are weak duplex DNA-binding ligands and the binding of flavonoids to DNA is affected by metal ions. Baicalein and quercetin display stronger affinity for triplex and quadruplex than for double-stranded DNA and offer interesting scaffolds for the design of novel, high order DNA-binding agents.
ON01910.Na [sodium (E)-2-(2-methoxy-5-((2,4,6-trimethoxystyrylsulfonyl)methyl)phenylamino)acetate... more ON01910.Na [sodium (E)-2-(2-methoxy-5-((2,4,6-trimethoxystyrylsulfonyl)methyl)phenylamino)acetate; Rigosertib, Estybon], a styryl benzylsulfone, is a phase III stage anticancer agent. This non-ATP competitive kinase inhibitor has multitargeted activity, promoting mitotic arrest and apoptosis. Extensive phase I/II studies with ON01910.Na, conducted in patients with solid tumors and hematologic cancers, demonstrate excellent efficacy. However, issues remain affecting its development. These include incomplete understanding of antitumor mechanisms, low oral bioavailability, and unpredictable pharmacokinetics. We have identified a novel (E)-styrylsulfonyl methylpyridine [(E)-N-(2-methoxy-5-((2,4,6-trimethoxystyrylsulfonyl)methyl)pyridin-3-yl)methanesulfonamide (TL-77)] which has shown improved oral bioavailability compared with ON01910.Na. Here, we present detailed cellular mechanisms of TL-77 in comparison with ON01910.Na. TL-77 displays potent growth inhibitory activity in vitro (GI50 ...
Specific chromosomal abnormalities are increasingly recognised to be associated with particular t... more Specific chromosomal abnormalities are increasingly recognised to be associated with particular tumour subtypes. These cytogenetic abnormalities define the sites of specific genes, the alteration of which is implicated in the neoplastic process. We used comparative genomic hybridisation (CGH) to examine DNA from different breast and ovarian cancer cell lines for variations in DNA sequence copy number compared with the same normal control. We also compared different sources of the MCF7 breast line by both CGH and cDNA expression arrays. Some of the differences between the subcultures were extensive and involved large regions of the chromosome. Differences between the four subcultures were observed for gains of 2q, 5p, 5q, 6q, 7p, 7q, 9q, 10p, 11q, 13q, 14q, 16q, 18p and 20p, and losses of 4q, 5p, 5q, 6q, 7q, 8p, 11p, 11q, 12q, 13q, 15q, 19p, 19q, 20p, 21q, 22q and Xp. However, few variations were found between two subcultures examined, 5 months apart, from the same initial source. Th...
Novel 2-(4-aminophenyl)benzothiazoles (e.g., compounds 1 and 2) possess highly selective, potent ... more Novel 2-(4-aminophenyl)benzothiazoles (e.g., compounds 1 and 2) possess highly selective, potent antitumor properties in vitro and in vivo. Elucidation of the mechanism of action of this structurally simple class of compounds has occurred in parallel with selection of a candidate clinical agent. Antitumor benzothiazoles induce and are biotransformed by cytochrome P 450 1A1 to putative active, as well as inactive metabolites. Metabolic inactivation of the molecule has been thwarted by isosteric replacement of hydrogen with fluorine atoms at positions around the benzothiazole nucleus. Amino acid conjugation to the exocyclic primary amine function of 2-(4-aminophenyl)benzothiazoles has been used to overcome limitations posed by drug lipophilicity. Water soluble, chemically stable prodrugs rapidly and quantitatively revert to their parent amine in mice, rats, and dogs in vivo. Plasma concentrations of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (2) regenerated from the lysylamide p...
Phortress is a novel, potent, and selective experimental antitumor agent. Its mechanism of action... more Phortress is a novel, potent, and selective experimental antitumor agent. Its mechanism of action involves induction of CYP1A1-catalyzed biotransformation of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) to generate electrophilic species, which covalently bind to DNA, exacting lethal damage to sensitive tumor cells, in vitro and in vivo. Herein, we investigate the effects of DNA adduct formation on cellular DNA integrity and progression through cell cycle and examine whether a relevant pharmacodynamic end point may be exploited to probe the clinical mechanism of action of Phortress and predict tumor response. Single cell gel electrophoresis (SCGE) was applied to quantify DNA damage and cell cycle analyses conducted upon 5F 203 treatment of benzothiazole-sensitive MCF-7 and inherently resistant MDA-MB-435 breast carcinoma cells. Following treatment of xenograft-bearing mice and mice possessing hollow fiber implants containing MCF-7 or MDA-MB-435 cells with Phortress (20 m...
Treatment for glioblastoma multiforme includes the alkylating agent temozolomide combined with io... more Treatment for glioblastoma multiforme includes the alkylating agent temozolomide combined with ionizing radiation. Persistent O6-guanine methylation by temozolomide in O6-methylguanine methyl transferase negative tumors causes cytotoxic lesions recognized by DNA mismatch repair, triggering apoptosis. Resistance (intrinsic or acquired) presents obstacles to successful temozolomide treatment, limiting drug efficacy and life expectancy. Two glioma cell lines, SNB19 and U373, sensitive to temozolomide (GI(50) values 36 and 68 microM, respectively) were exposed to increasing temozolomide concentrations (1-100 microM). Variant cell lines (SNB19VR, U373VR) were generated that displayed acquired temozolomide resistance (GI(50) values 280 and 289 microM, respectively). Cross-resistance to mitozolomide was observed in U373VR cells only. In clonogenic and MTT assays, methylguanine methyltransferase (MGMT) depletion using O6-benzylguanine sensitized U373VR cells to temozolomide, indicating the resistance mechanism involves MGMT re-expression. Indeed, Western blot analyses revealed MGMT protein in cell lysates. In SNB19VR cells, down-regulation of MSH6 message and protein expression may confer temozolomide tolerance. Inhibition of poly(ADP-ribose) polymerase-1 (a key base excision repair (BER) enzyme) partially restored sensitivity, and DNA repair gene arrays demonstrated up-regulation (>5-fold) of BER gene NTL1 in SNB19VR cells. In conclusion, we have developed two glioma cell lines whose distinct mechanisms of acquired resistance to temozolomide, involving expression of MGMT, or inactivation of DNA mismatch repair and recruitment of BER enzymes, are consistent with clinical observations. These cell lines provide valuable models for the development of strategies to combat temozolomide resistance.
... Samantha Wrigley, Carol J. McCall, Peter Lelieveld, § Iduna Fichtner, and Malcolm FG Stev... more ... Samantha Wrigley, Carol J. McCall, Peter Lelieveld, § Iduna Fichtner, and Malcolm FG Stevens* . Cancer Research Laboratories, Department of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD, UK, Pharmaceutical Sciences Institute, Aston ...
2-(4-Amino-3-methylphenyl)-5-fluorobenzothiazole (5F203, NSC 703786) lysylamide belongs to a nove... more 2-(4-Amino-3-methylphenyl)-5-fluorobenzothiazole (5F203, NSC 703786) lysylamide belongs to a novel mechanistic class of antitumor agents. It elicits activity against ovarian, breast, kidney and colorectal cancer models. In sensitive breast cancer cells, 5F203 activates aryl hydrocarbon receptor (AhR) signaling. Herein, we evaluate the role of AhR in 5F203 activity in two ovarian cancer cell lines: IGROV-1 (sensitive to 5F203), SKOV-3 (resistant to this agent). In addition, cancer cells have been isolated from ascites fluid of ovarian cancer patients; sensitivity to 5F203 and concurrent AhR signal transduction has been examined in ascites-isolated ovarian cancer patients' cells. 5F203 induced enhanced CYP1A1 expression, AhR translocation and ROS formation in IGROV-1 cells and ascites-isolated ovarian cancer cells that were sensitive to 5F203. In IGROV-1 cells 5F203-induced ROS formation was accompanied by JNK, ERK and P38MAPK phosphorylation, DNA damage and cell cycle arrest prior to apoptosis. In contrast, 5F203 failed to induce CYP1A1 expression, AhR translocation or oxidative stress in 5F203-resistant SKOV-3 cells, or in ovarian cancer ascites cells inherently resistant to this agent. We propose that AhR may represent a new molecular target in the treatment of ovarian tumors and 5F203 may exemplify a potential novel treatment. Furthermore, putative biomarkers of sensitivity to this agent have been identified.
ABSTRACT The copper-catalysed (10 mol-% CuBr center dot SMe2, CuCN center dot LiCl or CuI/PPh3) a... more ABSTRACT The copper-catalysed (10 mol-% CuBr center dot SMe2, CuCN center dot LiCl or CuI/PPh3) addition of RMgBr to the pentafulvene 1-(cyclopenta-2,4-dien-1-ylidenemethyl)-2-methoxybenzene allows the formation of cyclopentadienyl derivatives with -CHR(2-MeOPh) sidechains (R = Me, Et, nBu, iBu, allyl, Ph) without H- transfer. The deprotonation of these sec-alkyl-substituted cyclopentadienyls followed by the addition of TiCl4 allows the isolation of TiCl2{5-C5H4CHR(2-OMePh)} as rac/meso mixtures that show activity against human colon, breast and pancreatic cell lines (GI50 2.3-42.4 mu M).
The aryl hydrocarbon receptor (AhR) is a cellular signaling molecule infamous for mediating the t... more The aryl hydrocarbon receptor (AhR) is a cellular signaling molecule infamous for mediating the toxicity of dioxins and related compounds. The aim of this review is to provide a background of AhR and to examine critically its role in chemical toxicity, in physiological systems, and its interaction with drugs and other compounds. The AhR is essential for the toxicity of dioxins and related chemicals. The AhR mediates the exquisite sensitivity of animals to dioxins, where as little as 2 ng/kg/day can yield striking adverse effects. PHYSIOLOGICAL ROLE OF AHR: The wide variety of adverse effects of dioxin argues for an important role of the AhR in a variety of physiological systems. Recent investigations have highlighted the role of AhR in the development of the brain and vasculature. DRUGS AND OTHER CHEMICAL ACTIVATORS OF AHR: The development of AhR agonists during drug development programs is sometimes inadvertent, but sometimes the target of development, and is yet further confirmation of the likely importance of AhR signaling in constitutive physiology. The presence of AhR agonists in the diet such as indolo-(3,2-b)-carbazole and 3,3'-diindolylmethane (metabolized from indole 3-carbinol), flavonoids, and sulforaphane and of endogenous activators of this signaling system such as eicosanoids, indirubin, bilirubin, cAMP, and tryptophan are suggestive that AhR activation is a normal physiological process and that it is the persistent and high-level stimulation of AhR by dioxins that is responsible for toxicity. AhR-mediated toxicity and physiology are highly relevant to clinical toxicology and drug development.
... requests for reprints should be addressed, at Cancer Research Laboratories, School of Pharmac... more ... requests for reprints should be addressed, at Cancer Research Laboratories, School of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD ... Shi DF., Bradshaw TD, Wrigley S., McCall CJ, Lelieveld P., Fichtner I., Stevens MFG Antitumor ...
4-Ethynyl-4-hydroxycyclohexa-2,5-dien-1-one 5 undergoes cycloaddition reactions with a range of s... more 4-Ethynyl-4-hydroxycyclohexa-2,5-dien-1-one 5 undergoes cycloaddition reactions with a range of substituted azides in the presence of copper salts to form 1,4-disubstituted triazoles 8-11 bearing the 4-hydroxycyclohexa-2,5-dien-1-one (quinol) pharmacophore; one example of an isomeric 1,5-disubstituted triazole 12 was formed from 5 and benzyl azide in the presence of a ruthenium catalyst. Compounds were screened for growth-inhibitory activity against five cancer cell lines of colon, breast and lung origin, but were overall less potent than the benzothiazolyl- and indolyl-substituted quinols 2 and 3.
ABSTRACT ON01910.Na, a styryl benzylsulfone, is a Phase III stage, non-ATP competitive anti-cance... more ABSTRACT ON01910.Na, a styryl benzylsulfone, is a Phase III stage, non-ATP competitive anti-cancer agent. It is multi-targeted, promoting selective mitotic arrest and apoptosis in cancer cells. Extensive Phase II/III trials conducted in patients with solid tumors and hematological cancers have proved its excellent efficacy and impressive safety profile. However, incomplete understanding of mechanisms of action and relatively low drug oral bioavailability remain obstacles to development. By modifying the structure of ON01910.Na, a novel class of (E)-Styrylsulfonyl methylpyridines was designed and synthesized. We report herein the evaluation of a selected compound, TL-77, (E)-N-(2-methoxy-5-((2,4,6-trimethoxystyrylsulfonyl)methyl)pyridin-3-yl)methanesulfonamide, as a potent small molecule inhibitor targeting mitotic pathways. MTT assays were performed to assess in vitro anti-proliferative effects. In a panel of 10 tumor and 2 non-transformed human cell lines following 96 h exposure, TL-77 gave consistent GI50 values in the sub-micromolar range in tumor cells, whereas, it was non-toxic to non-transformed human mammary endothelial cells (HMECs). Cell cycle was analyzed by flow cytometry. TL-77 caused significant G2/M arrest ≥ 6 h, whereas ON01910.Na caused the same effect after longer exposure periods (≥ 12 h). An OD-based tubulin polymerization assay allowed us to explain obstruction of cell division caused by TL-77. TL-77 retarded tubulin polymerization between 1μM and 6μM, intriguingly, microtubules were stabilizes at TL-77 concentrations > 8μM. Western blot analyses of cell lysates following treatment of cells with TL-77 revealed dose-dependent reduction of phosphorylated Cdc25c, a known substrate of Plk1 and/or Chk1/2, indicating Plk1 and/or Chk1/2 inhibition. Finally, dose- and time-dependent apoptosis triggered by TL-77, detected by Annexin-V assays, was closely associated with induction of caspase 3/7 activity. Importantly, pharmacokinetics studies in mice revealed that the oral bioavailability of TL-77 was optimized to 56% compared with 9% for ON01910.Na. In summary, TL-77 is a potent anti-cancer agent. It disturbs tubulin during cell division, resulting in G2/M cell cycle arrest, followed by caspase-dependent apoptosis. TL-77 appears to share an analogous mechanism of action with ON01910.Na, but exhibits greater selectivity towards cancer cells and possesses superior oral bioavailability. Therefore, further evaluation of TL-77 as a promising anti-tumor agent is justified. Citation Format: Tiangong Lu, Shudong Wang, Charles Laughton, Tracey Bradshaw. Evaluation of (E)-Styrylsulfonyl methylpyridine: A novel kinase inhibitor targeting mitotic pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3406. doi:10.1158/1538-7445.AM2013-3406
Glioblastoma multiforme is the most common aggressive adult primary tumour of the central nervous... more Glioblastoma multiforme is the most common aggressive adult primary tumour of the central nervous system. Treatment includes surgery, radiotherapy and adjuvant temozolomide (TMZ) chemotherapy. TMZ is an alkylating agent prodrug, delivering a methyl group to purine bases of DNA (O6-guanine; N7-guanine and N3-adenine). The primary cytotoxic lesion, O6-methylguanine (O6-MeG) can be removed by methylguanine methyltransferase (MGMT; direct repair) in tumours expressing this protein, or tolerated in mismatch repair-deficient (MMR-) tumours. Thus MGMT or MMR deficiency confers resistance to TMZ. Inherent- and acquired resistance to TMZ present major obstacles to successful treatment. Strategies devised to thwart resistance and enhance response to TMZ, including inhibition of DNA repair mechanisms which contribute to TMZ resistance, are under clinical evaluation. Depletion of MGMT prior to alkylating agent chemotherapy prevents O6-MeG repair; thus, MGMT pseudosubstrates O6-benzylguanine and...
The interactions of small molecules with nucleic acids are of considerable interest for the desig... more The interactions of small molecules with nucleic acids are of considerable interest for the design of novel anticancer compounds. The physical properties and sequence specificity observed in the interactions between a group of flavonoids with proven antitumour activity and various nucleic acid structures are summarised. UV and fluorescence spectroscopy, together with competition dialysis, were used to assess the affinity of the drugs for the nucleic acid structures in the presence or absence of different metal ions. The effect of these compounds on breast and leukaemia cancer cell lines was evaluated using MTS and COMET assays and flow cytometry. The flavonoids studied are weak duplex DNA-binding ligands and the binding of flavonoids to DNA is affected by metal ions. Baicalein and quercetin display stronger affinity for triplex and quadruplex than for double-stranded DNA and offer interesting scaffolds for the design of novel, high order DNA-binding agents.
ON01910.Na [sodium (E)-2-(2-methoxy-5-((2,4,6-trimethoxystyrylsulfonyl)methyl)phenylamino)acetate... more ON01910.Na [sodium (E)-2-(2-methoxy-5-((2,4,6-trimethoxystyrylsulfonyl)methyl)phenylamino)acetate; Rigosertib, Estybon], a styryl benzylsulfone, is a phase III stage anticancer agent. This non-ATP competitive kinase inhibitor has multitargeted activity, promoting mitotic arrest and apoptosis. Extensive phase I/II studies with ON01910.Na, conducted in patients with solid tumors and hematologic cancers, demonstrate excellent efficacy. However, issues remain affecting its development. These include incomplete understanding of antitumor mechanisms, low oral bioavailability, and unpredictable pharmacokinetics. We have identified a novel (E)-styrylsulfonyl methylpyridine [(E)-N-(2-methoxy-5-((2,4,6-trimethoxystyrylsulfonyl)methyl)pyridin-3-yl)methanesulfonamide (TL-77)] which has shown improved oral bioavailability compared with ON01910.Na. Here, we present detailed cellular mechanisms of TL-77 in comparison with ON01910.Na. TL-77 displays potent growth inhibitory activity in vitro (GI50 ...
Specific chromosomal abnormalities are increasingly recognised to be associated with particular t... more Specific chromosomal abnormalities are increasingly recognised to be associated with particular tumour subtypes. These cytogenetic abnormalities define the sites of specific genes, the alteration of which is implicated in the neoplastic process. We used comparative genomic hybridisation (CGH) to examine DNA from different breast and ovarian cancer cell lines for variations in DNA sequence copy number compared with the same normal control. We also compared different sources of the MCF7 breast line by both CGH and cDNA expression arrays. Some of the differences between the subcultures were extensive and involved large regions of the chromosome. Differences between the four subcultures were observed for gains of 2q, 5p, 5q, 6q, 7p, 7q, 9q, 10p, 11q, 13q, 14q, 16q, 18p and 20p, and losses of 4q, 5p, 5q, 6q, 7q, 8p, 11p, 11q, 12q, 13q, 15q, 19p, 19q, 20p, 21q, 22q and Xp. However, few variations were found between two subcultures examined, 5 months apart, from the same initial source. Th...
Novel 2-(4-aminophenyl)benzothiazoles (e.g., compounds 1 and 2) possess highly selective, potent ... more Novel 2-(4-aminophenyl)benzothiazoles (e.g., compounds 1 and 2) possess highly selective, potent antitumor properties in vitro and in vivo. Elucidation of the mechanism of action of this structurally simple class of compounds has occurred in parallel with selection of a candidate clinical agent. Antitumor benzothiazoles induce and are biotransformed by cytochrome P 450 1A1 to putative active, as well as inactive metabolites. Metabolic inactivation of the molecule has been thwarted by isosteric replacement of hydrogen with fluorine atoms at positions around the benzothiazole nucleus. Amino acid conjugation to the exocyclic primary amine function of 2-(4-aminophenyl)benzothiazoles has been used to overcome limitations posed by drug lipophilicity. Water soluble, chemically stable prodrugs rapidly and quantitatively revert to their parent amine in mice, rats, and dogs in vivo. Plasma concentrations of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (2) regenerated from the lysylamide p...
Phortress is a novel, potent, and selective experimental antitumor agent. Its mechanism of action... more Phortress is a novel, potent, and selective experimental antitumor agent. Its mechanism of action involves induction of CYP1A1-catalyzed biotransformation of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) to generate electrophilic species, which covalently bind to DNA, exacting lethal damage to sensitive tumor cells, in vitro and in vivo. Herein, we investigate the effects of DNA adduct formation on cellular DNA integrity and progression through cell cycle and examine whether a relevant pharmacodynamic end point may be exploited to probe the clinical mechanism of action of Phortress and predict tumor response. Single cell gel electrophoresis (SCGE) was applied to quantify DNA damage and cell cycle analyses conducted upon 5F 203 treatment of benzothiazole-sensitive MCF-7 and inherently resistant MDA-MB-435 breast carcinoma cells. Following treatment of xenograft-bearing mice and mice possessing hollow fiber implants containing MCF-7 or MDA-MB-435 cells with Phortress (20 m...
Treatment for glioblastoma multiforme includes the alkylating agent temozolomide combined with io... more Treatment for glioblastoma multiforme includes the alkylating agent temozolomide combined with ionizing radiation. Persistent O6-guanine methylation by temozolomide in O6-methylguanine methyl transferase negative tumors causes cytotoxic lesions recognized by DNA mismatch repair, triggering apoptosis. Resistance (intrinsic or acquired) presents obstacles to successful temozolomide treatment, limiting drug efficacy and life expectancy. Two glioma cell lines, SNB19 and U373, sensitive to temozolomide (GI(50) values 36 and 68 microM, respectively) were exposed to increasing temozolomide concentrations (1-100 microM). Variant cell lines (SNB19VR, U373VR) were generated that displayed acquired temozolomide resistance (GI(50) values 280 and 289 microM, respectively). Cross-resistance to mitozolomide was observed in U373VR cells only. In clonogenic and MTT assays, methylguanine methyltransferase (MGMT) depletion using O6-benzylguanine sensitized U373VR cells to temozolomide, indicating the resistance mechanism involves MGMT re-expression. Indeed, Western blot analyses revealed MGMT protein in cell lysates. In SNB19VR cells, down-regulation of MSH6 message and protein expression may confer temozolomide tolerance. Inhibition of poly(ADP-ribose) polymerase-1 (a key base excision repair (BER) enzyme) partially restored sensitivity, and DNA repair gene arrays demonstrated up-regulation (>5-fold) of BER gene NTL1 in SNB19VR cells. In conclusion, we have developed two glioma cell lines whose distinct mechanisms of acquired resistance to temozolomide, involving expression of MGMT, or inactivation of DNA mismatch repair and recruitment of BER enzymes, are consistent with clinical observations. These cell lines provide valuable models for the development of strategies to combat temozolomide resistance.
... Samantha Wrigley, Carol J. McCall, Peter Lelieveld, § Iduna Fichtner, and Malcolm FG Stev... more ... Samantha Wrigley, Carol J. McCall, Peter Lelieveld, § Iduna Fichtner, and Malcolm FG Stevens* . Cancer Research Laboratories, Department of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD, UK, Pharmaceutical Sciences Institute, Aston ...
2-(4-Amino-3-methylphenyl)-5-fluorobenzothiazole (5F203, NSC 703786) lysylamide belongs to a nove... more 2-(4-Amino-3-methylphenyl)-5-fluorobenzothiazole (5F203, NSC 703786) lysylamide belongs to a novel mechanistic class of antitumor agents. It elicits activity against ovarian, breast, kidney and colorectal cancer models. In sensitive breast cancer cells, 5F203 activates aryl hydrocarbon receptor (AhR) signaling. Herein, we evaluate the role of AhR in 5F203 activity in two ovarian cancer cell lines: IGROV-1 (sensitive to 5F203), SKOV-3 (resistant to this agent). In addition, cancer cells have been isolated from ascites fluid of ovarian cancer patients; sensitivity to 5F203 and concurrent AhR signal transduction has been examined in ascites-isolated ovarian cancer patients' cells. 5F203 induced enhanced CYP1A1 expression, AhR translocation and ROS formation in IGROV-1 cells and ascites-isolated ovarian cancer cells that were sensitive to 5F203. In IGROV-1 cells 5F203-induced ROS formation was accompanied by JNK, ERK and P38MAPK phosphorylation, DNA damage and cell cycle arrest prior to apoptosis. In contrast, 5F203 failed to induce CYP1A1 expression, AhR translocation or oxidative stress in 5F203-resistant SKOV-3 cells, or in ovarian cancer ascites cells inherently resistant to this agent. We propose that AhR may represent a new molecular target in the treatment of ovarian tumors and 5F203 may exemplify a potential novel treatment. Furthermore, putative biomarkers of sensitivity to this agent have been identified.
ABSTRACT The copper-catalysed (10 mol-% CuBr center dot SMe2, CuCN center dot LiCl or CuI/PPh3) a... more ABSTRACT The copper-catalysed (10 mol-% CuBr center dot SMe2, CuCN center dot LiCl or CuI/PPh3) addition of RMgBr to the pentafulvene 1-(cyclopenta-2,4-dien-1-ylidenemethyl)-2-methoxybenzene allows the formation of cyclopentadienyl derivatives with -CHR(2-MeOPh) sidechains (R = Me, Et, nBu, iBu, allyl, Ph) without H- transfer. The deprotonation of these sec-alkyl-substituted cyclopentadienyls followed by the addition of TiCl4 allows the isolation of TiCl2{5-C5H4CHR(2-OMePh)} as rac/meso mixtures that show activity against human colon, breast and pancreatic cell lines (GI50 2.3-42.4 mu M).
The aryl hydrocarbon receptor (AhR) is a cellular signaling molecule infamous for mediating the t... more The aryl hydrocarbon receptor (AhR) is a cellular signaling molecule infamous for mediating the toxicity of dioxins and related compounds. The aim of this review is to provide a background of AhR and to examine critically its role in chemical toxicity, in physiological systems, and its interaction with drugs and other compounds. The AhR is essential for the toxicity of dioxins and related chemicals. The AhR mediates the exquisite sensitivity of animals to dioxins, where as little as 2 ng/kg/day can yield striking adverse effects. PHYSIOLOGICAL ROLE OF AHR: The wide variety of adverse effects of dioxin argues for an important role of the AhR in a variety of physiological systems. Recent investigations have highlighted the role of AhR in the development of the brain and vasculature. DRUGS AND OTHER CHEMICAL ACTIVATORS OF AHR: The development of AhR agonists during drug development programs is sometimes inadvertent, but sometimes the target of development, and is yet further confirmation of the likely importance of AhR signaling in constitutive physiology. The presence of AhR agonists in the diet such as indolo-(3,2-b)-carbazole and 3,3'-diindolylmethane (metabolized from indole 3-carbinol), flavonoids, and sulforaphane and of endogenous activators of this signaling system such as eicosanoids, indirubin, bilirubin, cAMP, and tryptophan are suggestive that AhR activation is a normal physiological process and that it is the persistent and high-level stimulation of AhR by dioxins that is responsible for toxicity. AhR-mediated toxicity and physiology are highly relevant to clinical toxicology and drug development.
... requests for reprints should be addressed, at Cancer Research Laboratories, School of Pharmac... more ... requests for reprints should be addressed, at Cancer Research Laboratories, School of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD ... Shi DF., Bradshaw TD, Wrigley S., McCall CJ, Lelieveld P., Fichtner I., Stevens MFG Antitumor ...
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