Cytochrome P450 1B1 polymorphism drives cancer cell stemness and patient outcome in head-and-neck carcinoma.

BACKGROUND: Cytochrome P450 1B1 (CYP1B1) is mostly expressed in tumours and displays unusual properties. Its two polymorphic forms were differently associated with anticancer drug sensitivity. We decipher here the role of this polymorphism in anticancer drug efficacy in vitro, in vivo and in the clinical setting. METHODS: From head-and-neck squamous cell carcinoma cell lines not expressing CYP1B1, we generated isogenic derivatives expressing the two forms. Proliferation, invasiveness, stem cell characteristics, sensitivity to anticancer agents and transcriptome were analysed. Tumour growth and chemosensitivity were studied in vivo. A prospective clinical trial on 121 patients with advanced head-and-neck cancers was conducted, and a validation-retrospective study was conducted. RESULTS: Cell lines expressing the variant form displayed high rates of in vitro proliferation and invasiveness, stemness features and resistance to DNA-damaging agents. In vivo, tumours expressing the variant CYP1B1 had higher growth rates and were markedly drug-resistant. In the clinical study, overall survival was significantly associated with the genotypes, wild-type patients presenting a longer median survival (13.5 months) than the variant patients (6.3 months) (p = 0.0166). CONCLUSIONS: This frequent CYP1B1 polymorphism is crucial for cancer cell proliferation, migration, resistance to chemotherapy and stemness properties, and strongly influences head-and-neck cancer patients' survival.

Phenotypical characterization of peanut allergic children with differences in cross-allergy to tree nuts and other legumes.

BACKGROUND: Peanut allergy in children is often associated with allergies to tree nuts and/or legumes. The aim of this study was to analyze in cluster a cohort of children allergic to peanuts and assessed for cross-reactivity to nuts and legumes and to identify different phenotypes. METHODS: We included retrospectively 317 children with peanut allergy evaluated at the Allergy Unit of the Saint Vincent Hospital of Lille in the last 12 years. A complete workup for peanut allergy and nuts and legumes was carried out for each patient. A hierarchical agglomerative clustering method was used to search for clusters of individuals in the evaluated cohort. RESULTS: Cross-allergy to TN and/or other legumes was identified in 137 patients (43.2%), atopic dermatitis being a major risk factor (adjusted OR = 16 [95% CI: 7.4-37]; p < 0.001). Three phenotypes emerged from cluster analysis. Cluster 1 (72 patients) is characterized by high level of rAra h 2, low threshold reactive doses for peanut and high proportion of asthma; Cluster 2 (93 patients) is characterized by high threshold reactive doses for peanut and the lowest proportion of cross-allergy to TN and/or legumes; Cluster 3 (152 patients) has a high risk of cross-allergy to TN and/or legumes and most patients suffer from eczema. CONCLUSIONS: The three phenotypes highlighted by this study could be useful to identify children with high risk of cross-allergic reaction to TNs and legumes early after PA diagnosis.

Synthesis and biological activity of ferrocenyl indeno[1,2-c]isoquinolines as topoisomerase II inhibitors.

Three series of indeno[1,2-c]isoquinolines bearing a ferrocenyl entity were synthesized and evaluated for DNA interaction, topoisomerase I and II inhibition, and cytotoxicity against breast human cancer cell lines. In the first and second series, the ferrocenyl scaffold was inserted as a linker between the two nitrogen atoms. In the last series, it was introduced at the end of the carbon chain. The present study showed that the ferrocenyl entity enhanced the topoisomerase II inhibition. Most compounds showed a potent growth inhibitory effect on MDA-MB-231 cell line with the IC50 in µM range.

Activity of the polyamine-vectorized anti-cancer drug F14512 against pediatric glioma and neuroblastoma cell lines.

The poor prognosis of children with high-grade glioma (HGG) and high-risk neuroblastoma, despite multidisciplinary therapeutic approaches, demands new treatments for these indications. F14512 is a topoisomerase II inhibitor containing a spermine moiety that facilitates selective uptake by tumor cells via the Polyamine Transport System (PTS) and increases topoisomerase II poisoning. Here, F14512 was evaluated in pediatric HGG and neuroblastoma cell lines. PTS activity and specificity were evaluated using a fluorescent spermine-coupled probe. The cytotoxicity of F14512, alone or in combination with ionizing radiation and chemotherapeutic agents, was investigated in vitro. The antitumor activity of F14512 was assessed in vivo using a liver-metastatic model of neuroblastoma. An active PTS was evidenced in all tested cell lines, providing a specific and rapid transfer of spermine-coupled compounds into cell nuclei. Competition experiments confirmed the essential role of PTS in the cell uptake and cytotoxicity of F14512. This cytotoxicity appeared greater in neuroblastoma cells compared with HGG cells but appeared independent of PTS activity levels. In vivo evaluation confirmed a marked and prolonged antitumoral effect in neuroblastoma cells. The combinations of F14512 with cisplatin and carboplatin were often found to be synergistic, and we demonstrated the significant radiosensitizing potential of F14512 in the MYCN-amplified Kelly cell line. Thus, F14512 appears more effective than etoposide in pediatric tumor cell lines, with greater efficacy in neuroblastoma cells compared with HGG cells. The synergistic effects observed with platinum compounds and the radiosensitizing effect could lead to a clinical development of the drug in pediatric oncology.

Cilengitide targets pediatric glioma and neuroblastoma cells through cell detachment and anoikis induction.

The prognosis of children with high-grade glioma or high-risk neuroblastoma remains poor. Cilengitide is a selective antagonist of αvß3 and αvß5 integrins, which are involved in tumor growth and development of metastasis. We have evaluated the effects of cilengitide on pediatric glioma and neuroblastoma cell lines for the first time. Expression levels of αvß3 and αvß5 were determined by flow cytometry in three neuroblastoma and five pediatric glioma cell lines compared with adult U87-MG before and after irradiation. Cell detachment, cytotoxicity, and cell growth under nonadhesive conditions were measured using the MTS assay. Cell death and apoptosis were assessed by annexin-V/propidium iodide staining. The varying αvß3 and αvß5 expression levels were unrelated to tumor grade. Irrespective of the αvß5 expression level, the pediatric cells expressing αvß3 were dose dependently sensitive to cilengitide. UW479 cells expressed only αvß5 integrin and were not sensitive to cilengitide, suggesting that cilengitide's action largely depends on αvß3 inhibition. Cell detachment resulted in a higher cytotoxicity in pediatric glioma compared with U87-MG cells, which seem able to grow despite the significant cilengitide-induced cell detachment. Growth kinetics on polyHEMA showed that only pediatric glioma cells were sensitive to anoikis and so died after cilengitide-induced detachment. Furthermore, irradiation of glioma cells increased αvß3 expression slightly but not cilengitide sensitivity. Cilengitide's action on glioma and neuroblastoma cells appears to be dependent on αvß3 expression and sensitivity to anoikis. Cilengitide is able to target pediatric glioma and neuroblastoma cells in vitro directly and efficiently. Tumor context could validate these promising observations.

New selective carbonic anhydrase IX inhibitors: synthesis and pharmacological evaluation of diarylpyrazole-benzenesulfonamides.

Carbonic anhydrase (CA) IX expression is increased upon hypoxia and has been proposed as a therapeutic target since it has been associated with poor prognosis, tumor progression and pH regulation. We report the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors, 4-(5-aryl-2-hydroxymethyl-pyrazol-1-yl)-benzenesulfonamides. A molecular modeling study was conducted in order to simulate the binding mode of this new family of enzyme inhibitors within the active site of hCA IX. Pharmacological studies revealed high hCA IX inhibitory potency in the parameters nanomolar range. This study showed that the position of sulfonamide group in meta of the 1-phenylpyrazole increase a selectivity hCA IX versus hCA II of our compounds. An in vitro antiproliferative screening has been performed on the breast cancer MDA-MB-231 cell using doxorubicin as cytotoxic agent and in presence of selected CA IX inhibitor. The results shown that the cytotoxic efficiency of doxorubicin in an hypoxic environment, expressed in IC50 value, is restored at 20% level with 1µM CA IX inhibitor.

Circulating thrombospondin 1 level as a surrogate marker in patients receiving cyclophosphamide-based metronomic chemotherapy.

Several previous reports suggest that thrombospondin (TSP-1) may be a mediator of the antiangiogenic effects of low-dose metronomic cyclophosphamide-based chemotherapy (MC). We conducted a randomized phase II trial evaluating megestrol acetate (n = 44) versus MC (n = 44) in patients having exhausted all standard treatments. We measured the TSP-1 levels at baseline and D15. We did not observe significant differences in TSP-1 at baseline in the two arms (p = 0.07). TSP-1 levels decreased in patients receiving metronomic cyclophosphamide (from 16.6 ± 7.2 µg/ml to 12.8 ± 7.4 µg/ml; p = 0.057). The TSP-1 level was stable in patients receiving megestrol acetate. Nevertheless, the TSP-1 level driven by MC did not correlate to clinical benefit.

Synthesis, cytotoxicity and topoisomerase inhibition properties of multifarious aminoalkylated indeno[1,2-c]isoquinolin-5,11-diones.

A number of mono- or diaminoalkylated indeno[1,2-c]isoquinolin-5,11-diones analogs of 1 were synthesized and evaluated for their DNA binding affinities, topoisomerase inhibition properties and antiproliferative activities against human cancer cell lines (HL60). Impact of the side chain connected to the aromatic D ring and to the N6 lactam position on the biological profile will be discussed.

Justification of the starting dose as the main determinant of accrual time in dose-seeking oncology phase 1 trials.

INTRODUCTION: New drug development is a time- and resource-consuming process. Phase 1 trials constitute a major key-step of this development. Shortening the accrual time is of major importance. METHODS: 292 published phase-1-trials were retrospectively reviewed to establish the determinants of accrual time using Log-rank test and then Cox Model. RESULTS: Out of 292 trials (1997-2008), only 107 reports (36%) described the accrual time (median: 20 months, 5-72). Phase-2-recommended dose was established in 87 studies (81%). Most studies investigated regimens including cytotoxic drugs (77%) or molecular targeted therapies (29%). Under univariate analysis, two parameters shortened the accrual time: studies conducted in USA vs. other places (19 vs. 21 months p = 0.03) and regimen with more than 2 dose-escalated drugs (13 vs. 21 months, p = 0.003). One parameter was significantly associated with longer accrual time: starting dose justified by animal toxicology data vs. previous clinical trials (22 vs. 19 months, p = 0.03). Most of parameters did not significantly affect the accrual time: nature of investigated drugs, duration of treatment cycle, phase 1 dedicated to specific tumoral subtypes, number of centers, method of drug escalation (classical 3+3 vs. accelerated titration design), type of increment (modified Fibonacci method vs. others) and presence of expansion of cohort at the phase-II-recommended dose. Cox model analysis retained one determinant: starting dose justified by animal toxicology data: HR = 2.00 [1.45-5.20], p = 0.047. CONCLUSION: Few parameters influence the accrual time of dose-escalation phase-1 trials. Real first-in-man phase 1 studies based on starting dose estimated from animal toxicological data require longer accrual time.

Montmorillonite K-10 catalyzed cyclization of N-ethoxycarbonyl-N'-arylguanidines: access to pyrimido[4,5-c]carbazole and pyrimido[5,4-b]indole derivatives.

Two new heterocycles, pyrimido[4,5-c]carbazole and pyrimido[5,4-b]indole, were prepared in three steps from 3-aminocarbazole and 3-aminoindole, respectively. The key Friedel-Crafts intramolecular cyclization was realized under microwave irradiation using montmorillonite K-10 clay as a catalyst. The pyrimido[4,5-c]carbazole derivative shows significant micromolar IC(50) against cancer cell lines. Unlike similar carbazole and indolocarbazole compounds, the molecule does not interfere with topoisomerase activity.

Synthesis and biological evaluation of new penta- and heptacyclic indolo- and quinolinocarbazole ring systems obtained via Pd(0) catalysed reductive N-heteroannulation.

A short route, involving a tetramolecular condensation reaction and a Pd/C catalyst-H(2)-mediated reductive N-heteroannulation as the key-steps, has been found for the synthesis of some new penta- and heptacyclic indolo- (12), quinolino- (13) and indoloquinolinocarbazole (11) derivatives. HF-DFT (B3LYP) energy profiles and NMR calculations were carried out to help in the understanding of the experimental results. N-Alkylated indoloquinolinocarbazoles (16b, 17a, 17b and 18) were prepared and screened essentially toward some cancer-(G-quadruplex, DNA, topoisomerase I) and CNS-related (kinases) targets. Biological results evidenced 13 as a potent CDK-5 and GSK-3ß kinases inhibitor, while di- or triaminopropyl-substituted indoloquinolinocarbazoles 17b or 18 targeted rather DNA-duplex or telomeric G-quadruplex structures, respectively.

Indeno[1,2-c]isoquinolin-5,11-diones conjugated to amino acids: Synthesis, cytotoxicity, DNA interaction, and topoisomerase II inhibition properties.

Three series of indeno[1,2-c]isoquinolin-5,11-dione-amino acid conjugates were designed and synthesized. Amino acids were connected to the tetracycle through linkers with lengths of n=2 and 3 atoms using ester (series I), amide (series II), and secondary amine (series III) functions. DNA binding was evaluated by thermal denaturation and fluorescence measurements. Lysine and arginine substituted derivatives with n=3 provided the highest DNA binding. Arginine derivative 32 (n=2, series II) and glycine derivative 34 (n=2, series III) displayed high topoisomerase II inhibition. Incrementing the length of the N-6 side chain from two to three methylene units provided a significant increase in DNA affinity but a substantial loss in topoisomerase II inhibition. The most cytotoxic compounds toward HL60 leukemia cells were 19, 33, and 34 displaying micromolar IC(50) values. When tested with the topoisomerase II-mutated HL60/MX2 cell line, little variation of IC(50) values was found, suggesting that topoisomerase II might not be the main target of these compounds and that additional targets could be involved.

A universal formula based on cystatin C to perform individual dosing of carboplatin in normal weight, underweight, and obese patients.

PURPOSE: It has recently been shown that it is possible to improve the prediction of carboplatin clearance by adding plasma cystatin C level (cysC), an endogenous marker of glomerular filtration rate, to the other patient characteristics routinely used for carboplatin individual dosing, namely serum creatinine (Scr), actual body weight (ABW), age, and sex. This multicenter pharmacokinetic study was done to evaluate prospectively the benefit of using cysC for carboplatin individual dosing. EXPERIMENTAL DESIGN: The 357 patients included in the study were receiving carboplatin as part of established protocols. A population pharmacokinetic analysis was done using NONMEM program. Seven covariates studied were as follows: Scr, cysC, age, sex, ABW, ideal body weight, and lean body mass. RESULTS: The best covariate equation was as follows: carboplatin clearance (mL/min) = 117.8. (Scr/75)(-0.450). (cysC/1,00)(-0.385). (ABW/65)(+0.504). (age/56)(-0.366). 0.847(sex), with Scr in micromol/L, cysC in mg/L, ABW in kilograms, age in years, and sex = 0 for male. Using an alternative weight descriptor (ideal body weight or lean body mass) did not improve the prediction. This final covariate model was validated by bootstrap analysis. The bias (mean percentage error) and imprecision (mean absolute percentage error) were +1% and 15%, respectively, on the total population, and were of a similar magnitude in each of the three subgroups of patients defined according to their body mass index. CONCLUSION: For the first time, a unique formula is proposed for carboplatin individual dosing to patients, which is shown to be equally valid for underweight, normal weight, and obese patients.

Optimized synthesis and enhanced efficacy of novel triplex-forming camptothecin derivatives based on gimatecan.

Sequence-specific camptothecins are useful tools to inhibit specifically gene expression. The camptothecins are attached to the 3' end of triplex-forming oligonucleotides (TFO), sequence-specific DNA ligands that position the camptothecin moiety exclusively in proximity to their binding site. We studied here different gimatecan derivatives or analogues, a potent lipophilic camptothecin compound in clinical trials. We optimized the synthesis procedure in order to increase the yields and the purity and obtain the conjugates on a large scale. The greatly improved synthesis is now based on the conjugation of a bromoalkyl analogue of gimatecan to the 3' phosphorothioate of the TFO. We showed that the most efficient conjugate, both in vitro and in HeLa cells, bears the TFO on position 7 of the gimatecan analogue, and it is more efficient than the previous camptothecin conjugates. In addition, the gimatecan-like moiety at the 3' end of the TFO protects from nuclease degradation.

Exploring the cellular activity of camptothecin-triple-helix-forming oligonucleotide conjugates.

Topoisomerase I is a ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins (CPTs). These drugs stimulate DNA cleavage by topoisomerase I but exhibit little sequence preference, inducing toxicity and side effects. A convenient strategy to confer sequence specificity consists of the linkage of topoisomerase poisons to DNA sequence recognition elements. In this context, triple-helix-forming oligonucleotides (TFOs) covalently linked to CPTs were investigated for the capacity to direct topoisomerase I-mediated DNA cleavage in cells. In the first part of our study, we showed that these optimized conjugates were able to regulate gene expression in cells upon the use of a Photinus pyralis luciferase reporter gene system. Furthermore, the formation of covalent topoisomerase I/DNA complexes by the TFO-CPT conjugates was detected in cell nuclei. In the second part, we elucidated the molecular specificity of topoisomerase I cleavage by the conjugates by using modified DNA targets and in vitro cleavage assays. Mutations either in the triplex site or in the DNA duplex receptor are not tolerated; such DNA modifications completely abolished conjugate-induced cleavage all along the DNA. These results indicate that these conjugates may be further developed to improve chemotherapeutic cancer treatments by targeting topoisomerase I-induced DNA cleavage to appropriately chosen genes.

Synthesis, cytotoxic activity, and DNA binding properties of antitumor cis-1,2-dihydroxy-1,2-dihydrobenzo[b]acronycine cinnamoyl esters.

Monocinnamoyl esters at position 2 of (+/-)-cis-1,2-dihydroxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-7-one and their acetyl derivatives at position 1 were prepared as stabilized analogues of the anticancer alkylating agent S23906-1. Monocinnamoyl esters at position 2 were slower DNA alkylators than the reference 2-monoacetate. Mixed esters bearing an acetyl ester group at position 1 and a cinnamoyl ester group at position 2 alkylated DNA slower than S23906-1. A strong correlation was observed between cytotoxicity and DNA alkylation kinetics, with slower alkylators displaying more potent antiproliferative activities. The most cytotoxic compounds proved to be significantly active in vivo against murine C-38 adenocarcinoma implanted in mice, but less potent than S23906-1.

Synthesis, cytotoxicity, DNA interaction, and topoisomerase II inhibition properties of novel indeno[2,1-c]quinolin-7-one and indeno[1,2-c]isoquinolin-5,11-dione derivatives.

Indeno[2,1- c]quinolin-7-ones and 6 H-indeno[1,2- c]isoquinolin-5,11-diones, bearing two cationic aminoalkyl side chains, were synthesized and evaluated for DNA interaction, topoisomerases inhibition, and cytotoxicity against human cancer cell lines. They displayed strong interaction with DNA and one indeno[1,2- c]isoquinolin-5,11-dione bearing side chains at N-6 and C-8 positions ( 6a) was a potent human topoisomerase II inhibitor with high cytotoxicity toward HL60 cells. An increased topoisomerase II inhibition is found with (a) a cationic aminoalkyl side chain at the C-8 rather than at the C-9 position, (b) a dimethylaminoethoxy side chain at the C-8 position introduced on the N-6 monosubstituted derivative, going with suppression of topoisomerase I poisoning, and (c) a dimethylaminoethyl rather than a dimethylaminopropyl side chain at the N-6 position. The cytotoxicity was only partially reduced when using the topoisomerase II-mutated mitoxantrone-resistant HL60/MX2 cell line, suggesting that additional targets are involved in their mechanism of action. These indeno[1,2- c]isoquinolin-5,11-dione derivatives represent new DNA-topoisomerase II interfering anticancer molecules.

Synthesis of 3,5-bis(2-indolyl)pyridine and 3-[(2-indolyl)-5-phenyl]pyridine derivatives as CDK inhibitors and cytotoxic agents.

We here report the synthesis and biological evaluation of new 3,5-bis(2-indolyl)pyridine and 3-[(2-indolyl)-5-phenyl]pyridine designed as potential CDK inhibitors. Indole, 5-hydroxyindole, and phenol derivatives were used to generate three substitutions of the pyridine. The resulting skeletons were successively exploited to introduce various dimethylaminoalkyl side chains by Williamson type reactions. The synthesis includes Stille or Suzuki type reactions, which were realized on the 3,5-dibromopyridine. The preparation and the use of stannylindoles in mono or bis cross-coupling reactions were also described and each step was optimized and detailed. Kinase assays were realized and shown that nude compounds 7, 18, and 25 inhibited CDK1 in the 0.3-0.7 micromolar range with a good selectivity over GSK-3. Cytotoxicity against CEM human leukemia cells was evaluated with IC(50) values in the 5-15 micromolar range. Precise structure-activity relationships were delineated. Molecular modeling and docking solutions were proposed to complete the studies and to explain the observed SAR in the CDK assays.

Generation of replication-dependent double-strand breaks by the novel N2-G-alkylator S23906-1.

S23906-1, a new DNA alkylating agent that reacts with the exocyclic 2-NH2 group of guanine residues yielding monofunctional adducts, is currently under clinical evaluation in phase I trials. To investigate the mechanism of action of S23906-1, we compared parental KB-3-1 cells and KB/S23-500 cells that are 15-fold resistant to S23906-1. Cell death induced by 1 micromol/L S23906-1 in KB-3-1 cells was associated with their irreversible arrest in the G2-M phases of the cell cycle followed by apoptosis, whereas a proportion of the resistant KB/S23-500 cells were able to exit from the G2 arrest and divide, leading to a significantly lower rate of apoptosis. The attenuated apoptotic response was associated with decreased Chk2 protein phosphorylation, indicating that the DNA damage signaling pathways are more potently activated in the sensitive cells. However, similar rates of adduct formation and repair were measured in both cell lines. Exposure to S23906-1 induced a higher formation of DNA breaks, measured by the comet assay, in sensitive cells. In agreement, a histone H2AX phosphorylation assay revealed that S23906-1 induced double-strand breaks (DSB) in a dose- and time-dependent manner and that these were more persistent in the parental cells. These DSBs were found mainly in S-phase cells and inhibited by aphidicolin, suggesting that they are DNA replication-mediated DSBs. These results suggest that secondary DNA lesions play an important role in the cytotoxicity of this compound and make histone H2AX phosphorylation an attractive marker for monitoring the efficacy of S23906-1.

CoDE-seq, an augmented whole-exome sequencing, enables the accurate detection of CNVs and mutations in Mendelian obesity and intellectual disability.

OBJECTIVE: The molecular diagnosis of extreme forms of obesity, in which accurate detection of both copy number variations (CNVs) and point mutations, is crucial for an optimal care of the patients and genetic counseling for their families. Whole-exome sequencing (WES) has benefited considerably this molecular diagnosis, but its poor ability to detect CNVs remains a major limitation. We aimed to develop a method (CoDE-seq) enabling the accurate detection of both CNVs and point mutations in one step. METHODS: CoDE-seq is based on an augmented WES method, using probes distributed uniformly throughout the genome. CoDE-seq was validated in 40 patients for whom chromosomal DNA microarray was available. CNVs and mutations were assessed in 82 children/young adults with suspected Mendelian obesity and/or intellectual disability and in their parents when available (ntotal = 145). RESULTS: CoDE-seq not only detected all of the 97 CNVs identified by chromosomal DNA microarrays but also found 84 additional CNVs, due to a better resolution. When compared to CoDE-seq and chromosomal DNA microarrays, WES failed to detect 37% and 14% of CNVs, respectively. In the 82 patients, a likely molecular diagnosis was achieved in >30% of the patients. Half of the genetic diagnoses were explained by CNVs while the other half by mutations. CONCLUSIONS: CoDE-seq has proven cost-efficient and highly effective as it avoids the sequential genetic screening approaches currently used in clinical practice for the accurate detection of CNVs and point mutations.