Soledad Chazarra

Kojic Acid, a Cosmetic Skin Whitening Agent, is a Slow-binding Inhibitor of Catecholase Activity of Tyrosinase

Abstract— It was found that kojic acid, which is used in cosmetics for its excellent whitening effect, inhibits catecholase activity of tyrosinase in a non‐classical manner. A decrease in the initial velocity to a steady‐state inhibited velocity can be observed over a few minutes. This time‐dependence, which is unaltered by prior incubation of the enzyme with the inhibitor, is consistent with a first‐order transition. The kinetic data obtained correspond to those for a postulated mechanism that involves the rapid formation of an enzyme inhibitor complex that subsequently undergoes a relatively slow reversible reaction. Kinetic parameters characterizing this type of inhibition were evaluated by means of nonlinear regression of product accumulation curves.

Synthesis and biological activity of a 3,4,5-trimethoxybenzoyl ester analogue of epicatechin-3-gallate.

Despite presenting bioavailability problems, tea catechins have emerged as promising chemopreventive agents because of their observed efficacy in various animal models. To improve the stability and cellular absorption of tea polyphenols, we developed a new catechin-derived compound, 3- O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG), which has shown significant antiproliferative activity against several cancer cell lines, especially melanoma. The presence of methoxy groups in its ester-bound gallyl moiety drastically decreased its antioxidant and prooxidant properties without affecting its cell-antiproliferative effects, and the data indicated that the 3-gallyl moiety was essential for its biological activity. As regards its action mechanism, we demonstrated that TMECG binds efficiently to human dihydrofolate reductase and down-regulates folate cycle gene expression in melanoma cells. Disruption of the folate cycle by TMECG is a plausible explanation for its observed biological effects and suggests that, like other antifolate compounds, TMECG could be of clinical value in cancer therapy.

Kinetic study of the suicide inactivation of latent polyphenoloxidase from iceberg lettuce (Lactuca sativa) induced by 4-tert-butylcatechol in the presence of SDS

In plants, polyphenoloxidase undergoes an irreversible inactivation during the oxidation of o-diphenol to o-quinones. In the present paper, using latent polyphenoloxidase from iceberg lettuce (Lactuca sativa), in the presence of an activating agent, SDS, the kinetic parameters that characterize the enzyme during its action on the suicide substrate 4-tert-butylcatechol have been determined. The effect of pH has also been considered. It was seen that the presence of SDS in the reaction medium changed the kinetic parameters of the enzyme during suicide inactivation, this phenomenon depended on the SDS concentration up to saturating concentrations. Variations were also observed in the kinetic parameters at pH values below 5 where SDS provoked inactivation of the enzyme. This differing kinetic behaviour during suicide inactivation in the presence of SDS may be caused by the conformational changes provoked by SDS in the enzyme in latent state. Thus, polyphenoloxidases showing suicide inactivation would present an enzymatic activity resulting from the balance between the activation process and suicide inactivation.

Binding of natural and synthetic polyphenols to human dihydrofolate reductase.

Dihydrofolate reductase (DHFR) is the subject of intensive investigation since it appears to be the primary target enzyme for antifolate drugs. Fluorescence quenching experiments show that the ester bond-containing tea polyphenols (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) are potent inhibitors of DHFR with dissociation constants (KD)of 0.9 and 1.8 μM, respectively, while polyphenols lacking the ester bound gallate moiety [e.g., (-)-epigallocatechin (EGC) and (-)-epicatechin (EC)] did not bind to this enzyme. To avoid stability and bioavailability problems associated with tea catechins we synthesized a methylated derivative of ECG (3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin; TMECG), which effectively binds to DHFR (KD = 2.1 μM). In alkaline solution, TMECG generates a stable quinone methide product that strongly binds to the enzyme with a KD of 8.2 nM. Quercetin glucuronides also bind to DHFR but its effective binding was highly dependent of the sugar residue, with quercetin-3-xyloside being the stronger inhibitor of the enzyme with a KD of 0.6 μM. The finding that natural polyphenols are good inhibitors of human DHFR could explain the epidemiological data on their prophylactic effects for certain forms of cancer and open a possibility for the use of natural and synthetic polyphenols in cancer chemotherapy.

Melanoma coordinates general and cell-specific mechanisms to promote methotrexate resistance.

Melanoma, the most aggressive form of skin cancer, is notoriously resistant to all current modalities of cancer therapy, including to the drug methotrexate. Melanosomal sequestration and cellular exportation of methotrexate have been proposed to be important melanoma-specific mechanisms that contribute to the resistance of melanoma to methotrexate. In addition, other mechanisms of resistance that are present in most epithelial cancer cells are also operative in melanoma. This report elucidates how melanoma orchestrates these mechanisms to become extremely resistant to methotrexate, where both E2F1 and checkpoint kinase 1 (Chk1), two molecules with dual roles in survival/apoptosis, play prominent roles. The results indicated that MTX induced the depletion of dihydrofolate in melanoma cells, which stimulated the transcriptional activity of E2F1. The elevate expression of dihydrofolate reductase and thymidylate synthase, two E2F1-target genes involved in folate metabolism and required for G(1) progression, favored dTTP accumulation, which promoted DNA single strand breaks and the subsequent activation of Chk1. Under these conditions, melanoma cells are protected from apoptosis by arresting their cell cycle in S phase. Excess of dTTP could also inhibit E2F1-mediated apoptosis in melanoma cells.

Comparison of a Pair of Synthetic Tea-Catechin-Derived Epimers: Synthesis, Antifolate Activity, and Tyrosinase-Mediated Activation in Melanoma

Despite bioavailability issues, tea catechins have emerged as promising chemopreventive agents because of their efficacy in various animal models. We synthesized two catechin‐derived compounds, 3‐O‐(3,4,5‐trimethoxybenzoyl)‐(−)‐catechin (TMCG) and 3‐O‐(3,4,5‐trimethoxybenzoyl)‐(−)‐epicatechin (TMECG), in an attempt to improve the stability and cellular absorption of tea polyphenols. The antiproliferative and pro‐apoptotic activities of both compounds were analyzed with various cancer cell systems, and TMCG, which was easily synthesized in excellent yield, was more active than TMECG in both melanoma and non‐melanoma cell lines. TMCG was also a better inhibitor of dihydrofolate reductase and was more efficiently oxidized by tyrosinase, potentially explaining the difference in activity between these epimers.

Characterization of Patatin Esterase Activity in AOT-Isooctane Reverse Micelles

Patatin is a family of glycoproteins that accounts for 30–40% of the total soluble protein in potato ( Solanum tuberosum L.) tubers. This protein has been reported not only to serve as a storage protein but also to exhibit lipid acyl hydrolase (LAH) activity. In this study patatin is characterized in AOT‐isooctane reverse micelles. The influence on the enzymatic activity of characteristic parameters of reverse micelles, wo (= H2O/AOT), and the percentage of H2O, θ, were investigated. The results obtained show that patatin esterase activity varies with wo but remains constant throughout the range of θ values studied. The variation with wo showed that the activity follows an S‐shaped behavior pattern, reaching a maximum at about wo = 20 for 2% H2O. Patatin esterase activity was compared with p‐nitrophenyl (PNP) fatty acid esters of different chain lengths. The activity was much higher for PNP‐caprylate. The pH optimum was 6.0, different from the value obtained when patatin esterase activity was measured in mixed micelle systems. The optimal temperature was 35 °C, above which the activity decreased to almost zero. The kinetic parameters were also evaluated ( Km = 10 mM, Vm = 158 μM/min, Vm/ Km = 15.8 × 10−3 min−1). This paper shows the suitability of reverse micelles for measuring patatin esterase activity, since it allows the study of the enzyme in similar conditions to that prevailing in vivo.

Mechanism of Dihydrofolate Reductase Downregulation in Melanoma by 3-O-(3,4,5-Trimethoxybenzoyl)-(-)- Epicatechin

In our search to improve the stability and cellular absorption of tea polyphenols, we synthesized 3‐O‐(3,4,5‐trimethoxybenzoyl)‐(−)‐epicatechin (TMECG), which showed high antiproliferative activity against melanoma. TMECG downregulates dihydrofolate reductase (DHFR) expression in melanoma cells and we detail the sequential mechanisms that result from this even. TMECG is specifically activated in melanoma cells to form a stable quinone methide (TMECG‐QM). TMECG‐QM has a dual action on these cells. First, it acts as a potent antifolate compound, disrupting folate metabolism and increasing intracellular oxidized folate coenzymes, such as dihydrofolate, which is a non‐competitive inhibitor of dihydropterine reductase, an enzyme essential for tetrahydrobiopterin (H4B) recycling. Such inhibition results in H4B deficiency, endothelial nitric oxide synthase (eNOS) uncoupling and superoxide production. Second, TMECG‐QM acts as an efficient superoxide scavenger and promotes intra‐cellular H2O2 accumulation. Here, we present evidence that TMECG markedly reduces melanoma H4B and NO bioavailability and that TMECG action is abolished by the eNOS inhibitor Nω‐nitro‐L‐arginine methyl ester or the H2O2 scavenger catalase, which strongly suggests H2O2‐dependent DHFR downregulation. In addition, the data presented here indicate that the simultaneous targeting of important pathways for melanoma survival, such as the folate cycle, H4B recycling, and the eNOS reaction, could represent an attractive strategy for fighting this malignant skin pathology. J. Cell. Biochem. 110: 1399–1409, 2010.

Cinnamate of inulin as a vehicle for delivery of colonic drugs

Colon diseases are difficult to treat because oral administrated drugs are absorbed at the stomach and intestine levels and they do not reach colon; in addition, intravenous administrated drugs are eliminated from the body before reaching colon. Inulin is a naturally occurring polysaccharide found in many plants. It consists of β 2-1 linked D-fructose molecules having a glucosyl unit at the reducing end. Various inulin and dextran hydrogels have been developed that serve as potential carrier for introduction of drugs into the colon. Because inulin is not absorbed in the stomach or in the small intestine, and inulin is degraded by colonic bacteria, drugs encapsulated in inulin-coated vesicles could be specifically liberated in the colon. Therefore, the use of inulin-coated vesicles could represent an advance for the treatment of colon diseases. Here, we study the use of a cinnamoylated derivative of chicory inulin as a vehicle for the controlled delivery of colonic drugs. The encapsulation of methotrexate in inulin vesicles and its release and activity was studied in colon cancer cells in cultures.

Purification and Kinetic Properties of Human Recombinant Dihydrofolate Reductase Produced in Bombyx mori Chrysalides

Recent reports describe the inhibition of human dihydrofolate reductase (hDHFR) by natural tea polyphenols. This finding could explain the epidemiologic data on their prophylactic effects for certain forms of cancer, and it raises the possibility that natural and synthetic polyphenols could be used in cancer chemotherapy. In order to obtain larger quantities of hDHFR to support structural studies, we established and validated a baculovirus system for the expression of this protein in Bombyx mori chrysalides (pupae of the silkworm enclosed in a cocoon). To isolate the expressed protein, whole infected pupae were homogenized, and the expressed protein was purified by affinity chromatography. Here, we demonstrate the efficient expression of recombinant hDHFR in this model and report that this newly expressed protein has high enzymatic activity and kinetic properties similar to those previously reported for recombinant hDHFR expressed in Escherichia coli. The purified protein showed dissociation constants for the binding of natural polyphenols similar to that expressed in E. coli, which ensures its usage as a new tool for further structural studies. Although the hDHFR yield per individual was found to be lower in the chrysalides than in the larvae of B. mori, the former system was optimized as a model for the scaled-up production of recombinant proteins. Expression of proteins in chrysalides (instead of larvae) could offer important advantages from both economic and biosecurity aspects.