Cetuximab directly inhibits P-glycoprotein function in vitro independently of EGFR binding.

PURPOSE: Cancer chemotherapy typically combines anticancer drugs from different mechanisms of action. However, cancer cells could become resistant to chemotherapy via P-gp or other ATP binding cassette proteins. The objective of this study was to evaluate whether cetuximab, monoclonal antibody directed toward epidermal growth factor receptor, could increase intracellular concentration of conventional chemotherapy by interacting with P-gp. METHODS: Two human ovarian carcinoma (IGROV1) and two human embryonary kidney (HEK) cell lines, overexpressing or weakly expressing P-gp, were used. Their EGFR expressions were compared. Cetuximab effect on P-gp functionality was evaluated by measuring doxorubicin (P-gp fluorescent substrate) intracellular accumulation. Cetuximab ability to increase doxorubicin cytotoxicity was evaluated by MTT test. A quaternary structure model of the P-gp-Cetuximab complex was established. RESULTS: Exposure of cetuximab in therapeutic concentrations range with doxorubicin led to significant doxorubicin accumulation and reversion of doxorubicin resistance in P-gp expressing cells lines. Molecular modeling of P-gp-cetuximab interactions showed that cetuximab is able to bind P-gp extracellular part. CONCLUSIONS: Cetuximab increases a P-gp substrate intracellular accumulation in both P-gp expressing cell lines, independently of their EGFR expression. One hypothesis is that cetuximab binding on P-gp could hamper the conformational changes that occur during drugs efflux. Our results offer new possibilities of research on monoclonal antibodies influence in MDR phenomena.

Disposition of everolimus in mdr1a-/1b- mice and after a pre-treatment of lapatinib in Swiss mice.

The aim of this study was to document the in vivo transport of everolimus (inhibitor of mTOR) by P-glycoprotein (P-gp), and to investigate the influence of lapatinib (inhibitor of P-gp) on everolimus disposition. Pharmacokinetics of everolimus (0.25mg/kg) has been investigated after oral administration in mdr1a-/1b- mice compared to the wild type. Also, everolimus pharmacokinetics was characterized after oral administration on Swiss mice either alone or after 2 days of pre-treatment of lapatinib (200mg/kg). The influence of lapatinib pre-treatment on intestinal P-gp expression was investigated by Western blot analysis. The non-compartimental analysis was performed using Winonlin professional version 4.1 software (Pharsight, Mountain View, CA). The areas under the plasma concentration-time curve (AUC) were compared using Bailer's method. A significant 1.3-fold increase of everolimus AUC observed in mdr1a-/1b- mice suggested that everolimus is transported in vivo by intestinal P-gp in mice. In addition, a 2.6-fold significant increase of everolimus AUC with lapatinib pre-treatment as compared with the everolimus alone group was noticed. The elimination half-life was comparable (t(1/2)=5.3h vs. t(1/2)=4h). A 38.5% significant decrease of P-gp expression was observed in duodenum segment in lapatinib pre-treated group as compared with control group. In conclusion, lapatinib enhanced everolimus absorption by decreasing intestinal P-gp expression. An inhibition of CYP 450 could not be excluded. These results confirm the necessity of a therapeutic monitoring of everolimus combined with an inhibitor of the P-gp and CYP 450 like lapatinib in a future anti-tumor treatment.

Thyroid oxidase (THOX2) gene expression in the rat thyroid cell line FRTL-5.

A cDNA encoding an NADPH oxidase flavoprotein was isolated from the rat thyroid gland. The predicted 1517-residue polypeptide was 82.5% identical to the human THOX2/DUOX2 and 74% similar to THOX1/DUOX1. Rat THOX2 lacks a stretch of 30 residues, corresponding to one exon in the human gene sequence. THOX2 mRNA was found to be expressed in cultured FRTL-5 cells. The level of THOX2 mRNA was increased by cAMP in these cells and it was decreased in the thyroids of rats treated with the antithyroid drug methimazole, unlike the TPO and NIS mRNAs. Since it was found in the intestine, duodenum, and colon, in addition to thyroid, we suggest that it be called LNOX, the new family of long homologs of NOX flavoproteins rather than THOX and/or DUOX.

Purification of a novel flavoprotein involved in the thyroid NADPH oxidase. Cloning of the porcine and human cdnas.

Hydrogen peroxide is the final electron acceptor for the biosynthesis of thyroid hormone catalyzed by thyroperoxidase at the apical surface of thyrocytes. Pig and human thyroid plasma membrane contain a Ca(2+)-dependent NAD(P)H oxidase that generates H(2)O(2) by transferring electrons from NAD(P)H to molecular oxygen. We purified from pig thyroid plasma membrane a flavoprotein which constitutes the main, if not the sole, component of the thyroid NAD(P)H oxidase. Microsequences permitted the cloning of porcine and human full-length cDNAs encoding, respectively, 1207- and 1210-amino acid proteins with a predicted molecular mass of 138 kDa (p138(Tox)). Human and porcine p138(Tox) have 86.7% identity. The strongest similarity was to a predicted polypeptide encoded by a Caenorhabditis cDNA and with rbohA, a protein involved in the Arabidopsis NADPH oxidase. p138(Tox) shows also similarity to the p65(Mox) and to the gp91(Phox) in their C-terminal region and have consensus sequences for FAD- and NADPH-binding sites. Compared with gp91(Phox), p138(Tox) shows an extended N-terminal containing two EF-hand motifs that may account for its calcium-dependent activity, whereas three of four sequences implicated in the interaction of gp91(Phox) with the p47(Phox) cytosolic factor are absent in p138(Tox). The expression of porcine p138(Tox) mRNA analyzed by Northern blot is specific of thyroid tissue and induced by cyclic AMP showing that p138(Tox) is a differentiation marker of thyrocytes. The gene of human p138(Tox) has been localized on chromosome 15q15.

Protective effect of curcuminoids on epidermal skin cells under free oxygen radical stress.

Curcuminoids from Curcuma longa L. (Zingiberaceae) protected normal human keratinocytes from hypoxanthine/ xanthine oxidase injury. Since curcuminoids synergistically inhibited nitroblue tetrazolium reduction, a decrease in superoxide radical formation leading to lower levels of cytotoxic hydrogen peroxide was proposed as an explanation for this protective effect.

Barrier function of reconstructed epidermis at the air-liquid interface: influence of dermal cells and extracellular components.

To evaluate the epidermal barrier function of in vitro reconstructed epidermis, we measured the penetration of estradiol and water across human keratinocytes cultured in defined medium (DM), in the presence of proliferative fibroblasts (pF) or conditioned medium derived from pF, at the air-liquid interface on synthetic porous membrane, noncoated or coated with laminin, fibronectin, type I collagen or type IV collagen. Ultrastructural analysis showed a well-developed stratum corneum whatever the culture conditions. The permeability of reconstructed epidermis in DM on a noncoated porous membrane was 5- to 10-fold higher than human native epidermis, with both tracers. No significant change in barrier function was observed whatever the culture conditions.

Comparison of six different methods to assess UVA cytotoxicity on reconstructed epidermis. Relevance of a fluorimetric assay (the calcein-AM) to evaluate the photoprotective effects of alpha-tocopherol.

A three-dimensional culture of human keratinocytes exposed at the air-liquid interface has been developed and used in conjunction with fluorimetric, colorimetric and radioligand incorporation assays to assess the in vitro toxicity of UVA. The aims of the study were: (1) to compare the relevance of the neutral red uptake (NR), MTT metabolism, (35)S-methionine incorporation, IL1-alpha release and calcein-AM esterification assays for the evaluation of UVA injury; (2) to test the preventive protective effect of an emulsion containing 3% of tocopherol applied on the reconstructed epidermis, in comparison with an application of tocopherol 3% diluted in culture medium either on the apical compartment or in the underneath compartment of the skin culture insert. Viability measurement methods are based on different endpoints. None of the five endpoints measured produced LD(50) values (40 J/cm(2)) that differed significantly from the others. However, calcein-AM assay was relatively more reproducible and easier to handle than the others, and seemed to be a better choice for the evaluation of the protective effects of the tocopherol emulsion. Tocopherol diluted in culture medium under the epidermis 24 hr before irradiation failed to protect the epidermis against UVA damage, whereas diluted in culture medium or in oily emulsion and applied to the epidermis reduced cellular death (cellular recovery values are, respectively, 24% and 21%). Since cosmetic or pharmaceutical formulations can be directly applied on the reconstructed epidermis as in vivo, this model in combination with a fluorescent viability assay appears to be a suitable approach for pharmaco-toxicological evaluations.

Human epidermis reconstructed on synthetic membrane: influence of experimental conditions on terminal differentiation.

Cell suspensions of human keratinocytes seeded onto cell culture inserts may undergo terminal differentiation in the absence of fibroblasts. Among the parameters that control these morphogenic events, exposure to air and the composition of the culture medium were investigated. In the latter case, three media were considered DMEM:Ham's F12, MCDB 153, and keratinocyte SFM medium at equivalent calcium (1.5 mM) and fetal calf serum (5%) concentrations. Immunochemical methods and transmission electron microscopy show that cells cultured in DMEM:Ham's F12 medium, and then raised at the air-liquid interface, form a basal layer plus suprabasal cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasal keratinocyte layers show morphologies that resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-filaggrin granules. When the cultures are kept submerged, the keratinocytes show occasional keratohyalin granules and are connected by fewer desmosomes. Additionally, no proper stratum corneum is formed. In keratinocyte SFM medium and MCDB 153, cultures raised at the air-liquid interface are not able to form an epithelium of normal architecture and do not express terminal differentiation markers. Differentiation is initiated, however, since desmosomes and bundles of keratin filaments appear; on the other hand, filaggrin is not expressed even after 28 d in culture. Membrane-bound transglutaminase is expressed throughout the entire suprabasal compartment in MCDB153 and DMEM:Ham's F12 media but never appears in keratinocyte SFM medium. These studies show the relative independence of epidermal differentiation program to the composition (including the calcium concentration) of the media contacting the dermis and filling the extracellular space.(ABSTRACT TRUNCATED AT 250 WORDS)

A new three-dimensional culture of human keratinocytes: optimization of differentiation.

Many attempts have been made to obtain reconstructed human epidermis comprised of keratinocytes and extracellular-matrix constituents (essentially collagen) in the presence or absence of fibroblasts. A simple model of cultured human keratinocytes, grown at the air-liquid interface of a noncoated artificial membrane, has been developed. This culture system offers many advantages: easy control of environmental factors and routine examination using optical or electronic microscopy, immunohistochemistry and indirect immunofluorescence techniques. This model enables the analysis of well-known differentiation markers and also integrins, a family of cell-surface molecules involved in cell-cell and cell-extracellular matrix interactions, whose receptors are expressed on all basal keratinocytes. In our culture system, the expression of the different integrin subunits (alpha 2, alpha 3, alpha 5, alpha 6, beta 1) was studied as a function of the differentiation state in two different media (K-SFM or DMEM/Ham's F12) supplemented with 5% fetal calf serum and adjusted to 1.5 mmol/L calcium. The most significant data are the preponderant expression of the alpha 2 and alpha 3 subunits in the basal and suprabasal layers, with membrane expression differing according to the culture medium; terminal differentiation was obtained in DMEM/Ham's F12. The use of membrane inserts represents a significant technological advance in culturing keratinocytes and is an easy-to-handle and valid model for determining the influence of physiological or pharmacological factors on cell proliferation or differentiation.

Influence of fibroblasts on epidermization by keratinocytes cultured on synthetic porous membrane (insert) at the air-liquid interface.

Culture of keratinocytes on a noncoated porous synthetic membrane maintained at the air-liquid interface allows the establishment of a fibroblast/keratinocyte co-culture, without direct cell-cell contact between the two cellular layers. The influence of fibroblasts (proliferating, confluent or blocked by mitomycin C) on epidermization (i.e., expression of integrins and markers of epidermal differentiation) was studied by immunohistochemistry in two culture media. In the medium supplemented with FCS or Ultroser G and in the absence of fibroblasts, alpha 2, alpha 3, alpha 5 and alpha 6 subunits of integrins are expressed by the basal keratinocytes, except alpha 5 which does not appear with the medium supplemented with Ultroser G. During stratification, the alpha 3 subunit is the only one to persist on suprabasal cells and all the markers of epidermal differentiation studied (filaggrin, involucrin, transglutaminase, keratins K1/K10) are expressed at the 14th day of emerged culture. The presence of fibroblasts modifies the expression profile of integrins: when they are proliferative, the expression of alpha 2 and alpha 6 chains is delayed in the medium supplemented with FCS, and the alpha 6 chain is absent in the medium supplemented with Ultroser G; when they are confluent or blocked by mitomycin C, greater changes are observed only in the medium supplemented with Ultroser G and lead to inhibition or delay of the expression of alpha 2 and alpha 6.(ABSTRACT TRUNCATED AT 250 WORDS)

Activity of Vittel water on proliferation of human fibroblasts, proliferation and differentiation of human keratinocytes.

Synopsis The effects of Vittel water on the proliferation of fibroblasts, proliferation and differentiation of keratinocytes from human origin were studied. To determine the relative importance of calcium and other elements of the mineral water, cultures were raised in a low-calcium medium (low-Ca medium), in a medium prepared with Vittel water (Vittel medium) and in a medium containing an identical calcium concentration to that of Vittel medium (Ca medium). The fibroblasts and keratinocytes were cultured in immersion for proliferation assays and on a reconstructed epidermis at the air-liquid interface to evaluate keratinocyte differentiation. Vittel medium decreased proliferation of keratinocytes when compared to low-Ca medium. The effect was similar to that of Ca medium at the beginning of the experiment, but significantly higher at day 7. A stratified epithelium appeared with the three types of media when keratinocytes were incubated at the air-liquid interface; however the number of sheets was more regular and greater in Vittel medium and Ca medium than in low-Ca medium. Filaggrin and transglutaminase expression appeared earlier with Vittel medium than with the other media. After 2 weeks, expression of markers was similar in the three media. After 3 weeks culture in Vittel medium, there was a greater expression of filaggrin. Proliferation of young fibroblasts was significantly higher in Vittel medium than in Ca medium. It was lower in low- Ca medium. With old fibroblasts the degree of proliferation was lower than with young fibroblasts. The augmentation of proliferation happen earlier in Vittel medium than in low-Ca medium and Ca medium. Vittel medium regulated the growth rate of old fibroblasts, rendering it identical to that of young fibroblasts in low-Ca medium. The effects of Vittel water were not linked to the sole presence of calcium since, with medium at an equimolecular concentration in calcium, the medium containing Vittel water had a better activity. One explanation of these effects of Vittel water might be the presence of magnesium.

Free-radical damage by UV or hypoxanthine-xanthine oxidase in cultured human skin fibroblasts: Protective effects of two human plasma fractions.

Two in vitro methods to investigate free radical damage to cultured human skin fibroblasts have been used: irradiation with UVA or UVB, producing intracellular free radicals and DNA damage, or free radical production by the enzymatic system hypoxanthine-xanthine oxidase, releasing free radicals into the culture medium. These methods differ not only in the location of the free radicals generated but also in their nature and kinetics. The antioxidant properties of two human plasma extracts A and B (derived from Cohn's fraction IV and Cohn's fraction I + III) were investigated before, during and/or after the oxidative stress. Protection was observed when the fractions were added concomitantly with the enzymatic system (at 5 g/litre, fractions A and B exhibited, respectively, 77 and 50% activity) or during UVB irradiation (37 and 68% activity for fractions A and B, respectively at 5 g/litre). A small degree of protection was observed against UVA damage. No preventive or restorative effect was observed with the UVB system. The two fractions prevented UVA damage (at 2.5 g/litre, fraction A and B elicited 22 and 23% activity, respectively) but only fraction A also exhibited a restorative effect (at 5 g/litre, activity was 26%). One of the protective mechanisms could be the enhancement of intracellular antioxidant enzyme activity by incubation of cells with fractions A and B (after 24 hr of contact with fraction B, total glutathione peroxidase and glutathione peroxidase Se-dependent activities were significantly increased by 60 and 42%, respectively, compared with control values).

Effect of biophysical changes on propidium iodide access to DNA during oxidative stress of cultured human skin cells.

The sensitive single-cell analytical techniques of flow cytometry and propidium iodide-binding have been used to examine the molecular effect of oxidative stress on cultured human skin fibroblasts. Cells synchronized by limited time attachment were exposed to a hypoxanthine-xanthine oxidase (HX/XO) system at different intervals after subculture. The characteristic feature of the treated population was a variation of the amount of nuclear DNA propidium iodide (PI)-fluorescence staining. Increased fluorescence intensity was observed with a shift of the G1/G0 and G2/M peak, which is dependent on both cell cycle stage and treatment level. When scavenger molecules (catalase, silybin) were added to the oxidative reaction, the nuclear DNA histogram of HX/XO-treated cells was similar to that obtained from untreated cells. In parallel, UV absorbance studies in vitro have shown that PI is capable of binding extensively to DNA when isolated from HX/XO-exposed cells, compared with control cells or HX/XO-exposed cells in the presence of scavengers. These results indicate that free radicals are responsible for the increase in fluorescence intensity in the HX/XO-exposed cells. This change in DNA stainability would be due to an opening of the DNA strands in situ, leading to an unmasking of new PI-binding sites on DNA. The strand separation may facilitate access on the fluorochrome to DNA, thereby enhancing dye binding. This flow cytometric assay based on DNA biophysical changes caused by free radicals is a useful means of measuring pro- and/or anti-oxidant potential.

Screening tests for free radical scavengers on cutaneous cultured cells.

Synopsis Numerous studies discuss the important development of free radicals in human skin after exposure to UV irradiation. They suggest that these reactive molecules are responsible for sun-accelerated cutaneous aging. We reproduced two types of radical agression on cutaneous human cell cultures. The first brings into action oxygen radicals generated by hypoxanthine-xanthine oxidase system and the second involves the use of UV radiation. These complementary models may be used as screening methods for antioxidant molecule research because they allow the distinction between molecules with filtering properties and free radical scavengers. The possibility of introducing molecules at different stages (before, during and after exposure to agression) permits the study of the molecular mecanism. These models were tested on silymarin, catechin, ascorbic acid and alpha tocopherol.

In vitro cytotoxic effects of enzymatically induced oxygen radicals in human fibroblasts: Experimental procedures and protection by radical scavengers.

Introduction of hypoxanthine and xanthine oxidase into human fibroblast cultures induces a dose-dependent cytotoxicity as a result of free-radical formation. The influence of medium, cell density and the power of recovery after free-radical attack were investigated. It appears that toxicity is higher in physiological Dulbecco phosphate buffer or Hanks' balanced salt solution than in modified Eagle medium, is inversely proportional to cell density and that damage is most often irreversible. Using this model, we studied the protective effects of a hydrosoluble flavonoid, silybin, and of a well known antioxidant, BHT (butylated hydroxytoluene). These molecules were administered before and during free-radical attack. With BHT significant protection was observed when it was added before free-radical attack (24% protection at a concentration of 10(-4)m) and before and during exposure (20% protection at a concentration of 10(-5)m). When silybin is applied during radical attack maximal activity is recorded at a concentration of 8 x 10(-4)m (45%), but the most interesting results are observed when 1 x 10(-4) and 8 x 10(-4)m are used, respectively, before and during radical exposure (63% of activity).