Pierre Cuq

Enhancement of 5‐fluorouracil cytotoxicity by human thymidine‐phosphorylase expression in cancer cells: In vitro and In vivo study

Transferring a gene into cancer cells in order to sensitize them to drugs is an important approach in human cancer gene‐therapy research. Thymidine phosphorylase (TP) is the first enzyme in the metabolic activation pathway of 5‐fluorouracil (5‐FU) to fluorodeoxyribonucleotides, thus, it could be used to increase the sensitivity of cancer cells to this anti‐pyrimidine agent. In this study, an expression vector containing the human TP cDNA was transfected into C26 murine colon‐carcinoma cells. Stable transfectants were selected; all showed increased TP activity, ranging from 2‐ to 10‐fold when compared with wild‐type cells. The in vitro sensitivity of transfectants to 5‐FU and 5′‐deoxy‐5‐fluorouridine (5′‐DFUR) was enhanced, in agreement with the observed increase in TP activity. Then, tumors were generated by s.c. injection of TP‐transfected or wild‐type C26 cells in syngeneic BALB/c mice. 5‐FU (25 mg/kg, i.p.) induced a growth delay of TP‐transfected C26 tumors as compared with C26 wild‐type tumors. These data suggest that TP could be transfected in tumor cells to increase the sensitivity to 5‐FU for subsequent cancer gene therapy. Int. J. Cancer 80:465–470, 1999.

Synthesis and biological evaluation of Fotemustine analogues on human melanoma cell lines

Two new analogues of Fotemustine have been synthesized and tested on two melanoma cell lines. Compounds 4 and 8 proved to be more potent than the reference compound on A375 cell line which express the MGMT enzyme involved in the chemoresistance of tumoral cells.

New imidazoquinoxaline derivatives: Synthesis, biological evaluation on melanoma, effect on tubulin polymerization and structure-activity relationships.

Microtubules are considered as important targets of anticancer therapy. EAPB0503 and its structural imidazo[1,2-a]quinoxaline derivatives are major microtubule-interfering agents with potent anticancer activity. In this study, the synthesis of several new derivatives of EAPB0503 is described, and the anticancer efficacy of 13 novel derivatives on A375 human melanoma cell line is reported. All new compounds show significant antiproliferative activity with IC50 in the range of 0.077-122μM against human melanoma cell line (A375). Direct inhibition of tubulin polymerization assay in vitro is also assessed. Results show that compounds 6b, 6e, 6g, and EAPB0503 highly inhibit tubulin polymerization with percentages of inhibition of 99%, 98%, 90%, and 84% respectively. Structure-activity relationship studies within the series are also discussed in line with molecular docking studies into the colchicine-binding site of tubulin.

Differential Responsiveness to Contractile Agents of Isolated Smooth Muscle Cells from Human Colons as a Function of Age and Inflammation

To study the involvement of age and inflammationin motor colonic activity in man, contractile responsesto CCK, carbachol, and KCl of isolated colonic smoothmuscle cells (SMC) from normal and inflamed human colons were evaluated; the incidence ofsex and smoking on contraction was also analyzed.Contractile responses to the three agonists weresignificantly lower in tissues with a low degree ofinflammation than in tissues with high level of inflammationor normal tissues. This reduction in cell responsivenessappears to be nonspecific and nonreceptor mediated. Apositive correlation of the contractile responses to the three stimulants with the age ofpatients was observed. In contrast, no association wasfound between sex, smoking, and cell contraction. Inconclusion, contractions of SMC due to CCK, carbachol, and KCl were significantly modified duringlife; inflammation of the colon led to a loss of SMCresponsiveness.

Differential involvement of glutathione S-transferase mu 1 and multidrug resistance protein 1 in melanoma acquired resistance to vinca alkaloids

On account of its extreme intrinsic resistance to apoptosis and of its strong ability to become chemoresistant after a primary response to drugs, malignant melanoma (MM) is still a therapeutic challenge. We previously showed that glutathione S‐transferase mu 1 (GSTM1) acts in synergy with multidrug resistance protein 1 (MRP1) to protect GSTM1‐transfected human CAL1 melanoma cells from toxic effects of vincristine (VCR). Herein, we investigated the role of these proteins in the acquired resistance of CAL1 cells to vinca alkaloids (VAs). Resistant lines were established by continuous exposure (>1 year) of parental CAL1‐wt cells to VCR, vindesine (VDS), or vinorelbine (VRB): CAL1R‐VCR, CAL1R‐VDS, CAL1R‐VRB, respectively. All resistant lines displayed more than 10‐fold increase in resistance to their selection VA, and specifically expressed GSTM1. Suggesting a direct interaction between this protein and VAs, each VA specifically decreased the GSTM1‐mediated glutathione conjugation activity in cell lysates. Curcumin (GSTM1 inhibitor), BSO (glutathione synthesis inhibitor), and MK571 (MRP1 inhibitor) considerably reversed the acquired resistance to VCR and VDS, but not to VRB. Microarray data analysis revealed similar gene expression patterns of CAL1R‐VCR and CAL1R‐VDS, and a distinct one for CAL1R‐VRB. These data suggest a differential involvement of GSTM1 and MRP1 in acquired resistance to VAs. A coordinated expression and activity of GSTM1 and MRP1 is required to protect CAL1 cells from VCR and VDS, while the simple expression of GSTM1 is sufficient, possibly by a direct drug/protein interaction, to confer resistance against VRB.

Involvement of Gαq/11 in the contractile signal transduction pathway of muscarinic M3 receptors in caecal smooth muscle

The nature of the pertussis toxin-insensitive G-protein involved in muscarinic-mediated phosphoinositides breakdown and contraction of isolated smooth muscle cells from the circular layer of the rabbit caecum was investigated. Immunoblotting of membrane proteins using affinity purified antibodies directed against different G-protein alpha-subunits revealed the expression of G alpha q/11, G alpha 11 and G alpha 12 in these cells. The carbachol-mediated [3H]inositol phosphates accumulation in saponin-permeabilized cells was abolished by anti-G alpha q/11-antibodies whereas anti-G alpha i1,2-antibodies were ineffective. Moreover, the carbachol-induced contraction of permeabilized cells, as determined by videomicrocopic measurements, was reversed by anti-G alpha q/11-antibodies but not affected by anti-G alpha i1,2-antibodies. From these data, we conclude that carbachol stimulates phosphoinositides hydrolysis and cell contraction through activation of specific muscarinic M3 receptors coupled to the pertussis toxin-insensitive G alpha q/11-protein. This is the first demonstration of G alpha q/11 implication in the contractile signal transduction pathway of muscarinic M3 receptors in smooth muscle cells.

Imidazo[1,2-a]pyrazine, Imidazo[1,5-a]quinoxaline and Pyrazolo[1,5-a]quinoxaline derivatives as IKK1 and IKK2 inhibitors

The transcription nuclear factor NF-κB plays a pivotal role in chronic and acute inflammatory diseases. Among the several and diverse strategies for inhibiting NF-κB, one of the most effective approach considered by the pharmaceutical industry seems to be offered by the development of IKK inhibitors. In a former study, two potential IKK2 inhibitors have been highlighted among a series of imidazo[1,2-a]quinoxaline derivatives. In order to enhance this activity, we present herein the synthesis of twenty-one new compounds based on the imidazo[1,2-a]pyrazine, imidazo[1,5-a]quinoxaline or pyrazolo[1,5-a]quinoxaline structures. Their potential to inhibit IKK1 and IKK2 activities is also tested.

Lysosomes and unfolded protein response, determinants of differential resistance of melanoma cells to vinca alkaloids

On account of its strong ability to become chemoresistant after a primary response to drugs, malignant melanoma (MM) remains a therapeutic challenge. This study focuses on acquired resistance to vinca alkaloids (VAs) using VA‐resistant MM cell lines (CAL1R‐VCR, CAL1R‐VDS, and CAL1R‐VRB), established by long‐term continuous exposure of parental CAL1‐wt cells to vincristine (VCR), vindesine (VDS), or vinorelbine (VRB), respectively. Transcriptomic profiling using rma and rdam methods led to distinguish two cell groups: CAL1R‐VCR and CAL1R‐VDS, CAL1R‐VRB, and CAL1‐wt. mgsa of the specifically altered genes in the first group evidenced the GO terms ‘lysosomal lumen’ and ‘vacuolar lumen’ linked to underexpressed genes, and ‘endoplasmic reticulum (ER) stress response’ associated with overexpressed genes. A specific reduction of lysosomal enzymes, independent of acidic vacuole organelle (AVO) turnover, was observed (LTG probe) in CAL1R‐VCR and CAL1R‐VDS cells. It was associated with the specific lowering of cathepsin B and L, known to be involved in the lysosomal pathway of apoptosis. Confirming gene profiling, the same groups (CAL1R‐VCR and CAL1R‐VDS, CAL1‐wt and CAL1R‐VRB) could be distinguished regarding the VA‐mediated changes on mean size areas and on acidic compartment volumes. These two parameters were reduced in CAL1R‐VCR and CAL1R‐VDS cells, suggesting a smaller AVO accumulation and thus a reduced sensitivity to lysosomal membrane permeabilization‐mediated apoptosis. In addition, ‘ER stress response’ inhibition by tauroursodeoxycholic acid induced a higher VA sensitization of the first cell group. In conclusion, lysosomes and unfolded protein response could be key determinants of the differential resistance of MM to VAs.

Fluorescence Study of Imidazoquinoxalines

The fluorescence properties of eleven novel derivatives based on the imidazo[1,2-a]quinoxaline structures have been studied. The absorption and emission spectra of these compounds have been recorded in dimethylsulfoxide solution. The phenyl substituting group on position 1 gives them particular properties thanks to the diverse hydroxy or methoxy decorating moieties, especially when they are multiplied or mixed. The investigated fluorescence auto-quenching revealed that the decreasing fluorescence intensity correlated only with the chemical structures of the aromatic compounds.

New IKK inhibitors: Synthesis of new imidazo[1,2-a]quinoxaline derivatives using microwave assistance and biological evaluation as IKK inhibitors

The inhibition of the NF-κB-dependent pathways by IKK inhibitors plays an important role in immunity, inflammation, and cancer. New imidazoquinoxalines tricyclic derivatives are prepared using microwave assistance and their biological activities as IKK inhibitors are described. Compounds 6a present a potent inhibition activity and selectivity for IKK2. Docking studies in the IKK2 binding site allowed identification of residues most likely to interact with theses inhibitors and explain their potent IKK2 inhibition activity and selectivity.