Laurence Vian

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.

Benzophenone-3: rapid prediction and evaluation using non-invasive methods of in vivo human penetration

The study described in this paper constitutes a practical assay system to evaluate in vivo drug penetration using two complementary non-invasive methods. An electrical capacitance test was first applied to the skin on the forearm to evaluate the hydration of the skin, and check the integrity of the stratum corneum. In the first step, the percentage absorption was measured using an occlusive and difference method; following benzophenone-3 application any residual formulation was washed off and the amount removed analyzed. In the second step, the tape stripping method-a useful procedure for selectively removing the skins outermost layer, the stratum corneum, and measuring the stratum corneum adsorption-was performed. Under these conditions the human skin permeation of this UV-filter over four hours was near to 35% of the applied dose with the occlusive method. The amount of topically applied benzophenone-3 found in the stratum corneum after 30 min exposure using the stripping procedure was evaluated at 4% to the applied dose.

The influence of alcohol, propylene glycol and 1,2-pentanediol on the permeability of hydrophilic model drug through excised pig skin

Alcohol and glycol including 1,2-pentanediol, a new product in this field, were examined for their transdermal penetration enhancing in vitro properties using pig skin and caffeine as a model drug. In order to investigate a possible influence of these compounds, we followed diffusion from an aqueous solution with caffeine followed by a series of different vehicles, their compositions were: (1) in water as a control; (2) in propylene glycol/ethanol/water (25:25:48; v/v/v); (3) in 1,2-pentanediol/water (2.5:95.5, v/v); (4) in 1,2-pentanediol/water (5:93, v/v); in propylene glycol/water (5:93; v/v); and in ethanol/water (5:93; v/v). The stratum corneum/vehicle partition coefficients (K(m)), maximum flux (J), enhancement factor (EF), 24-h receptor concentration (Q(24h)) were determined and compared to control values (caffeine in water). Permeation was also expressed in percentage of the applied dose absorbed in the different compartments. In all test models, caffeine was released and penetrated into pig skin. The 1,2-pentanediol was presented as the most effective enhancer; with a low proportion of this compound (only 5%), caffeine penetrated the skin quicker and in a greater extent. While this compound showed promise as penetration enhancer, further study was required to determine its effectiveness with others drugs and its irritation potential.

On quantification of CD1a, HLA-DR, and HLA class I expression on viable human Langerhans cells and keratinocytes.

In order to determine precisely the cellular density of surface molecules that are critical for antigen presentation in human epidermis, we utilized a quantitative immunofluorescence indirect assay and performed flow cytometric analysis of human epidermal cell (EC) suspensions. We first demonstrated that Tricolor-labeled streptavidin coupled to Cy-5 (SA-TC) was a reliable marker for non viable EC and that SA-TC+ EC accounted for the frequent nonspecific background of fluorescence due to isotype controls binding, although Langerhans cells (LC) and Keratinocytes (Kc) express Fc receptors for IgG on their surfaces. These results indicate that quantification of cell surface antigens on human EC requires the concomitant use of a marker of viability. Multicolor flow cytometric analysis allowed us to quantify CD1 molecules and major histocompatibility complex (MHC) antigens on viable human LC and Kc. Our results demonstrated a weak expression of MHC class I molecules on viable LC (163 +/- 19 x 10(3) molecules/cell) compared to viable Kc (785 +/- 110 x 10(3) molecules/cell). Mean antigen density of HLA-DR and CD1a molecules on viable LC were 579 +/- 82 x 10(3) molecules/cell and 1600 +/- 133 x 10(3) molecules/cell, respectively. Quantitative flow cytometry of viable EC may be proposed to evaluate the number of membrane antigens whose level of expression is related to cellular maturation or activation that occurs in skin diseases.

Irradiation of skin and contrasting effects on absorption of hydrophilic and lipophilic compounds.

Increasing legal requirements for risk assessment and efficacy testing in the dermo‐cosmetic field have led to the development of alternative test methods. In this study, the porcine skin model was chosen to test the effect of irradiation on the penetration habits of UV filters and caffeine. For decades, the pig has been recognized as an experimental animal in biomedical research thanks to its morphological and physiological similarities to humans. In this study, we wanted to investigate the effect of UV irradiation on the absorption of octocrylene (OC) and benzophenone‐3 (B3) sunscreens used under those circumstances and a model hydrophilic molecule, caffeine (Caf). These particular compounds were chosen due to their different lipophilic profiles. The percutaneous penetration of the two UV filters and Caf was studied after two simulated solar radiation doses of 61.4 kJ m−2. After irradiation simulation, the total absorbed dose was increased for OC while for B3 and Caf it was lower. Thus, modifications in percutaneous absorption have been observed, and it appears that UV could play a crucial role in this process. Moreover, it has been observed that the lipophilic profile of the studied compounds affects percutaneous penetration when irradiated.

The Liverbeads® as a tool for the comet assay

Liverbeads, cryopreserved hepatocytes entrapped within an alginate matrix, were examined for their relevance in the comet assay. It was estimated by their capacity to activate the indirectly acting mutagens, cyclophosphamide (CP), benzo[a]pyrene (BP), dimethylbenzanthracene (DMBA) and 2-acetylaminofluorene (2-AAF), into DNA reactive metabolites. The comet assay performed in alkaline condition is a sensitive method for detecting strand breaks at the level of individual cells and allows use of quiescent cells. Experimental conditions as treatment time, cell density, beads dissociation and viability were investigated. Significant statistical positive results assessed by the tail extent moment (TEM) were observed with both human and rat Liverbeads after 12h duration incubation compared to metabolic non-competent cells, HeLa S3. Due to the maintenance of specific functions assessed by the observed capacity to metabolize xenobiotics, Liverbeads represent a suitable tool system, easy to handle, for the detection of promutagens using the comet assay.

Skin Constituents as Cosmetic Ingredients. Part II: A Study of Bio‐Mimetic Monoglycerides Behavior at the Squalene‐Water Interface by the “Pendant Drop” Method in a Dynamic Mode

This work aims at presenting the viscoelastic behavior of bio‐mimetic monoglycerides used as emulsifier in a mixture made of two non‐miscible liquids, squalene and water. The measurement of the interfacial tension, carried out by the “pendant drop” method in “dynamic” mode, made it possible to characterize these amphiphilic molecules according to the value of their elastic modulus, ϵ, as well as their relaxation time, τR. The analysis of these parameters, as well as those developed in the previous publication [L. Blasco et al. (2006) Skin constituents as cosmetic ingredients. Part I: A Study of bio‐mimetic monoglyceride behavior at the squalene‐water interface by the “pendant drop” method in a static mode. J. Dispers. Sci. Technol., 27(6).] shows that the hydrocarbon chain structure, such as its length, the presence of one or more unsaturations, hydroxyl function, affects the behavior of surfactant molecules at the squalene/water interface.

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.

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.

Skin Constituents as Cosmetic Ingredients. Part III: A Molecular Modeling Study of Bio‐Mimetic Monoglycerides Behavior at the Squalene‐Water Interface

The molecular modeling study described here revisits the experimental work that focused on the interfacial properties of different monoglycerides at the squalene/water interface.1 2 The use of molecular modeling software programs allowed the determination of new parameters and provided a better understanding of the behavior of those amphiphilic molecules at the interface of the two non‐miscible liquids. Each monoglyceride studied has been characterized by an HLVB value; the curvature radius, Rc; the area of the droplets formed, AG; the number of monoglyceride molecules required to form a droplet, N; and the distance separating surfactant molecules at the interface, d. The analysis of those different parameters shows that short hydrocarbon chain monoglycerides, such as those having one or more unsaturations and those having a grafted hydroxyl function in the middle of their chain, are excellent surfactants. These configurations promote the efficient reduction of surface tension existing at the squalene/water interface.