We previously reported that chaetocin has potent and selective anti-myeloma activity attributable to reactive oxygen species (ROS) induction imposed by inhibition of the redox enzyme thioredoxin ...reductase; we now detail its effects in solid tumours.
Cellular assays, transcriptional profiling and the NCI60 screen were used to assess the effects of chaetocin in solid tumour and endothelial cells.
NCI-60 screening demonstrated chaetocin to even more potently inhibit proliferation in solid tumour than in haematological cell lines; transcriptional profiling revealed a signature consistent with induction of inflammatory response and cell death pathways. Chaetocin induced ROS, oxidative damage to cellular proteins and apoptosis, with 2-10 nM IC(50)s (24 h exposures) in all tested solid tumour cell lines. The pan-caspase inhibitor zVAD-fmk did not block chaetocin-induced cell death despite inhibiting mitochondrial membrane depolarisation and apoptosis. Further, Molt-4 rho(0) cells lacking metabolically functional mitochondria were readily killed by chaetocin; in addition chaetocin-induced cytotoxicity was unaffected by autophagy inhibitors or hypoxia and consequent HIF-1α upregulation. Moreover, chaetocin inhibited SKOV3 ovarian cancer xenografts producing less vascular tumours, and inhibited human umbilical vein endothelial cell proliferation.
Chaetocin has intriguing and wide-ranging in vitro and in vivo anticancer effects, and is an attractive candidate for further preclinical and clinical development.
The binding of small molecules to distinctive three-dimensional structures in mRNA provides a new dimension in RNA control, previously limited to the targeting of secondary structures with antisense ...and RNA interference; such targeting can modulate mRNA function and rates of protein biosynthesis. Small molecules that selectively bind the iron-responsive element (IRE), a specific three-dimensional structure in the noncoding region of the ferritin mRNA model that is recognized by the iron-regulatory protein repressor, were identified by using chemical footprinting. The assay used involved an oxoruthenium(IV) complex that oxidizes guanine bases in RNA sequences. Small molecules that blocked oxidation of guanines in the internal loop region were expected to selectively increase the rate of ferritin synthesis, because the internal loop region of the ferritin IRE is distinctive from those of other IREs. The natural product yohimbine was found (based on gel mobility shifts) to block cleavage of the internal loop RNA site by >50% and seemed to inhibit protein binding. In the presence of yohimbine, the rate of biosynthesis of ferritin in a cell-free expression system (rabbit reticulocyte lysate) increased by 40%. Assignment of the IRE-yohimbine interaction as the origin of this effect was supported by a similar increase in synthesis of luciferase protein in a chimera of the IRE and luciferase gene. The identification of a small, drug-like molecule that recognizes a naturally occurring three-dimensional mRNA structure and regulates protein biosynthesis rates raises the possibility that small molecules can regulate protein biosynthesis by selectively binding to mRNA.
Polypyridyl complexes of Co decorated with 350-Da polyether chains (Co350 2+) form molten phases of nucleic acids when paired with DNA counterions (Co350DNA) or 25-mer oligonucleotides. Analysis of ...voltammetry and chronoamperometry of mixtures of these phases with complexes having ClO4 - counterions (Co350(ClO4)2) and no other diluent provides charge transport rates from the oxidation and reduction currents for the complexes. As the mole fraction of the Co350(ClO4)2 complex in the mixture is varied from ca. 0.25 to 1, the physical diffusion constants derived from the CoIII/II wave increase from 1 × 10-11 cm2/s to 5 × 10-10 cm2/s, and apparent diffusion constants dominated by the CoII/I electron self-exchange increase from 1 × 10-10 cm2/s to 2 × 10-8 cm2/s. Pure Co350DNA melts, containing no Co350(ClO4)2 complex, do not exhibit recognizable voltammetric waves; DNA suppresses the CoII/I electron transfer reactions of Co complexes for which it is the counterion. There are therefore two microscopically distinct kinds of Co350 complexes, those with DNA and those with ClO4 - counterions, with respect to their CoII/I electron-transfer dynamics, leading to percolative behavior in their mixtures. The electron-transfer rates of the CoII/I couple are controlled by the diffusive relaxation of the ionic atmosphere around the reaction pair, and the inactivity of the bound Co complexes can be attributed to the very low mobility of the anionic phosphate groups in the DNA counterion. Substitution of sulfonated polystyrene for DNA produced similar results, suggesting that this phenomenon is general to other polymer counterions of low mobility. We conclude that the measured CoII/I charge transport and electron-transfer rate constants reflect more the diffusive mobility of the perchlorate counterion than the intrinsic CoII/I electron hopping rate.
Abstract Purpose Based upon promising preclinical and phase 1 trial results, combined flavopiridol and cisplatin therapy was evaluated in patients with ovarian and primary peritoneal cancers. Methods ...A two cohort phase 2 trial of cisplatin (60 mg/m2 IV) immediately followed by flavopiridol (100 mg/m2 IV, 24 h infusion; 21 day cycles) was undertaken in patients with recurrent platin-sensitive or platin-resistant disease (progression > vs. ≤ 6 months following prior platin-based therapy). Measurable disease (RECIST) - or evaluable disease plus CA125 > 2X post-treatment nadir - and ECOG performance ≤ 2 were required. Results Forty-five patients were enrolled between December 23, 2004 and February 25, 2010: 40 platin-resistant (Group 1), and 5 platin-sensitive (Group 2). In Group 1 , the median number of treatment cycles was 3 (range 2–12). Only 10% of patients incurred grade 4 toxicities, but grade 3 toxicities were common (65%): neutropenia (17.5%); nausea (12.5%); vomiting, fatigue, thrombosis, anemia (10% each). Seven patients (17.5%) achieved a confirmed response (1 CR, 6 PR; median duration 118 days); ten additional patients (25%) attained maintained stable disease. Median time to progression was 4.3 months; overall survival was 16.1 months. Pilot translational studies assessed ascites flavopiridol level; surrogate marker studies were uninformative. In Group 2 , although 4 of 5 patients responded (2 confirmed PRs with median time to progression, 10.8 months and median overall survival 20.6 months) the cohort was closed due to poor accrual. Conclusions The assessed flavopiridol and cisplatin regimen displayed clinical activity in platin resistant and sensitive ovarian/primary peritoneal cancers, meriting further study.
The effect of DNA bending on nucleobase electron transfer was investigated by studying the oxidation of double-stranded sequences containing seven repeats of the known bent sequence ...d(GGCA1A2A3A4A5A6C) where 7-deazaadenine (zA) was substituted at the A3 position. Native gel electrophoresis was used to show that the sequence remained bent upon substitution of zA, which provides for oxidation of the sequence by Ru(bpy)3 3+ (bpy = 2,2‘-bipyridine). The Ru(III) oxidant was generated by photolysis of Ru(bpy)3 2+ in the presence of ferricyanide, and the oxidation was visualized by high-resolution gel electrophoresis of the radiolabeled DNA sequence following base treatment. Cleavage of the DNA strand at the guanine residues and at the zA residues was observed. Comparison of the oxidation of zA in bent DNA versus the normal B form showed that hybridization of the B form sequence to its Watson−Crick complement produced a reduction in cleavage by a factor of 5.19 ± 0.46 while hybridization of the bent sequence only reduced cleavage by a factor of 1.58 ± 0.23. This result implies that the zA in the double-stranded, bent sequence is much more solvent-exposed than in normal B-form DNA. When the zA occurred in a B-form 5‘-zA-G doublet, the reactivity was 6.63 ± 0.10 times higher for the zA compared to the G. This implies an even greater effect of a 3‘-guanine on the oxidation potential of zA than in the well-known 5‘-GG doublet.
Abstract
Abstract #2149
Background: Inhibitors of the insulin-like growth factor 1 receptor (IGF-1R) are currently undergoing clinical testing. Preclinical investigations have identified IGF-1 ...signaling as a key mechanism for breast cancer growth and resistance to clinically useful therapies, including tamoxifen and trastuzumab. Thus, agents targeting IGF-1R have promise in the treatment of breast cancer. Determining mechanisms that can confer resistance to these agents may aid their clinical development. Methods: To understand factors may be important in predicting sensitivity to targeting the IGF-1 signaling pathway, we developed a cell line (MCF-7R4) that is resistant to BMS-554417, a small molecule, dual-kinase inhibitor of IGF-1R and insulin receptor (InsR). Compared with the parental MCF-7 cells, MCF-7R4 cells are 40- to 50-fold resistant to BMS-554417 and cross-resistant to the similar compound BMS-536924. The expression profiles of MCF-7R4 and that of MCF-7 were compared using Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays. Intracellular concentrations of BMS-536924 were examined by reverse phase high performance liquid chromatography. BMS-536924 cellular accumulation in vitro was visualized by fluorescence microscopy using a DAPI filter set. MCF-7 cells stably transfected with either the empty mammalian expression vector pcDNA (MCF-EV) or full length BCRP (MCF-BCRP) were examined for sensitivity to BMS 536924 by MTS assays. Results: Compared to MCF-7 cells, BCRP expression was increased 9-fold in MCF-7R4, which was highly statistically significant by t-test (p= 7.13E-09). Little change was observed in other ABC transporter proteins, including ABCB1. No change was observed in IGF-1R or InsR expression. BCRP overexpression in MCF-7R4 cells was confirmed by western blotting. MCF-7R4 cells had significantly lower intracellular accumulation of BMS-536924 compared to MCF-7 cells. Confirming these results, MCF-BCRP cells were significantly less sensitive to the cytoxic effects of BMS-536924 cells than MCF-EV cells. Conclusions: BCRP expression was stimulated by prolonged exposure of MCF-7 cells to BMS-554417. Upregulation of BCRP is one of the most significant changes observed in MCF-7R4 cells in comparison to parental cells. BCRP expression decreased cellular exposure to BMS-536924 and was sufficient to confer resistance. These data suggest that BSM-536924 is a substrate for BCRP-mediated efflux. Expression of BCRP may be important in de novo and acquired resistance to benzimidazole –based inhibitors of IGF-1R/InsR. Supported in part by the Mayo Clinic Breast SPORE (CA116201-03), NIH K12 (CA090628-05) and the Mayo Clinic Cancer Center (CA15083).
Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2149.
Molten salts of Ni(bpy350)3 2+ and single stranded oligonucleotides were prepared (bpy350 = 4,4‘-(CH3(OCH2CH2)7.24OCO)2-2,2‘-bipyridine). Photoinduced electron transfer was observed between small ...amounts of Ru(bpz)3 2+* (E 2+*/1+ = 1.35 V vs SCE, bpz = 2,2‘-bipyrazine) added to the melt and guanine in the nucleic acid, by measuring the emission lifetime of the ruthenium complex in melts containing varying amounts of guanine-containing nucleic acid. Electrochemically determined diffusion coefficients in these media were <1 × 10-11 cm2/s, showing that the Ru(bpz)3 2+* diffuses less than 0.4 Å during the measured excited-state lifetime. A linear relationship was observed between the fraction of Ru(bpz)3 2+* that was quenched (calculated as (τ° − τ)/τ) and the mole fraction of guanine-containing oligonucleotides. This relationship supports a quenching mechanism that does not involve diffusion of the ruthenium complex. The average electron-transfer rate constant at full guanine loading was 7 × 106 s-1, which implies an average electron-transfer distance of 12.5 Å (center-to-center).
Abstract
Background: Based upon preclinical synergy and prior phase 1 study results, the clinical efficacy of flavopiridol combined with cisplatin was assessed in patients with recurrent ovarian and ...primary peritoneal cancers.
Methods: A two cohort phase 2 trial of cisplatin (60 mg/m2 IV) followed by flavopiridol (100 mg/m2 IV, 24 h continuous infusion; 21 day cycles) was undertaken in patients with recurrent platin-sensitive or platin-resistant ovarian/primary peritoneal cancers (defined by disease progression > vs. <6 months following platin-based therapy). Measurable disease (RECIST criteria) – or evaluable disease plus CA125 >2X the post-treatment nadir – was required, as was ECOG performance <2 and exposure to only one prior treatment regimen.
Results: Forty-five patients were enrolled between April 20, 2004 and March 4, 2010 – 40 platin-resistant patients (Group 1), and 5 platin-sensitive patients (Group 2). In Group 1, the median number of treatment cycles was 3 (range 2-12); 39 of the 40 eligible patients have now discontinued treatment. While only 10% of all patients incurred grade 4 toxicities, grade 3 toxicities were seen in the majority (65%). The most frequent grade 3 and 4 toxicities were neutropenia (all grade 3, 17.5%); nausea (12.5%); vomiting, fatigue, thrombosis, anemia (10% each). Sensory neuropathy, grade 1 or 2, was observed in 75% of all patients – with grade 3 and 4 neuropathy not observed primarily due to pre-specified aggressive dose reductions. Six patients (15%) in Group 1 achieved a confirmed response (1 CR, 5 PR), with a median response duration of 119 days (range 84-212). Ten additional Group 1 patients (32.5%) experienced maintained stable disease. Median Group 1 overall time to progression was 3.7 months; overall survival was 17.2 months. Pilot assessment of attained ascites flavopiridol level and sensitivity of patient ascitic tumor cells to flavopiridol confirmed that patient flavopiridol levels were consistent with observed clinical antitumor efficacy. In Group 2, although 2 of 5 patients also responded (40%; 2 PR), the cohort was closed due to poor accrual.
Conclusions: The combination of flavopiridol and cisplatin has promising clinical activity in both platin-sensitive and platin-resistant ovarian and primary peritoneal cancers.
Supported in part by NCI CA097129, CA15083 and CM62205; clinicaltrials.gov identifier NCT00083122
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4712. doi:10.1158/1538-7445.AM2011-4712
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