Mitsuko Kawano

Antimelanogenesis effect of Tunisian herb Thymelaea hirsuta extract on B16 murine melanoma cells

Abstract:u2002 Skin pigmentation is the result of melanogenesis that occurs in melanocytes and/or melanoma cells. Although melanogenesis is necessary for the prevention of DNA damage and cancer caused by UV irradiation, excessive accumulation of melanin can also cause melanoma. Thus, we focused on the antimelanogenesis effect of an extract from Thymelaea hirsuta, a Tunisian herb. Murine melanoma B16 cells were treated with T. hirsuta extract, and then cell viability and synthesized melanin content were measured. We found that the T. hirsuta extract decreased the synthesized melanin content in B16 cells without cytotoxicity. Tyrosinase is a key enzyme of melanogenesis and extracellular signal‐regulated kinase (ERK)‐1/2 phosphorylation is known to be related to melanogenesis inhibition. To clarify its mechanism, we also determined ERK1/2 phosphorylation and tyrosinase expression level. ERK1/2 was immediately phosphorylated in cells just after treatment with the extract. The tyrosinase expression was inhibited after 24u2003h of stimulation with the extract. The T. hirsuta extract was fractionated, and we found that one fraction considerably decreased the melanin synthesis in B16 cells and that this fraction contains daphnanes as the main component. This indicates that our findings might be attributable to daphnanes.

Stress response of mammalian cells incubated with landfill leachate

Environmental contamination from landfill leachate has been linked to disturbances in human health. Often, however, only global parameters, such as dissolved organic content, chemical oxygen demand, and 5-d biological oxygen demand, are used to evaluate wastewater quality. In the present study, we determined leachate cytotoxicity and stress response of leachate-treated mammalian cells using in vitro bioassays and other molecular techniques. The modified E-screen assay using human breast cancer MCF-7 cells was used to determine the estrogenic potential and/or cytotoxicity of water samples from two solid-waste landfills in Tunisia. The cytotoxicity mechanism of the leachate was determined by DNA fragmentation and lactate dehydrogenase assays. The stress response of heat shock protein (HSP) 47-positive Chinese hamster ovary cells treated with leachate also was determined. Proteomics analyses and real-time reverse transcription-polymerase chain reaction were used to determine and confirm the enhanced expressions of certain stress-related proteins. Results showed that the leachate samples generally did not have estrogenic activity. Instead, they were cytotoxic toward MCF-7 cells, and the cytotoxicity was by necrosis during the early stages of incubation. Leachate also enhanced the expression of HSP and various stress-related proteins, such as heterogeneous nuclear ribonucleoprotein E1, phosphoglycerate mutase, and nuclear matrix protein 200, in MCF-7 cells. These can be considered as survival mechanisms against leachate-induced cytotoxicity.

Hair growth regulation by the extract of aromatic plant Erica multiflora

Hair growth problems can affect human physical and mental health and are of particular relevance during the aging process. In an effort to resolve such problems, we attempted to find plants having hair growth regulation activity and thus collected plant extracts from Tunisia for bioprospecting purposes. Among them, we investigated the Erica multiflora extract to evaluate the hair growth promotion activity by using the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay and cell cycle assay on human dermal papilla cells in vitro and an administration assay on mouse dorsal skin in vivo. The results showed that the Erica multiflora extract promotes dermal papilla cell growth and cell cycle with high activity, and induced hair growth in vivo by induction of anagen phase from telogen phase.

pH‐dependent reversible inhibition of violaxanthin de‐epoxidase by pepstatin related to protonation‐induced structural change of the enzyme

The redox enzyme violaxanthin de‐epoxidase (VDE) was found to be sensitive to pepstatin, a specific inhibitor of aspartic protease. The inhibition was similar to that of aspartic protease in that it was reversible and accompanied by the protonation of the enzyme. Of the two peaks of VDE appearing on anion exchange chromatography, VDE‐I predominated at pH 7.2. On lowering the pH of the chromatography, VDE‐I decreased and VDE‐II increased. Furthermore, re‐chromatography of either peak yielded both peaks. These results suggest that VDE‐I and VDE‐II are interconvertible depending on pH, and thus, they represent the de‐protonated and protonated forms of the enzyme, respectively. Presumably the protonation‐induced structural change of the enzyme is responsible for the interaction with pepstatin, and also with substrate.

Effect of Tunisian Aromatic Plant Extracts on Melanogenesis

Melanin plays an important role in the prevention of DNA damage and protection of skin tissues from UV irradiation. However, excessive accumulation of melanin can also cause freckle and melanoma. Thus, we focused on the melanogenesis regulation effect of Tunisian aromatic plants. The melanin synthesis system is known as melanogenesis, and tyrosinase is a key enzyme of melanogenesis (Fang and Setaluri, 1999; Kameyama et al., 1995). Currently, many research efforts have focused on the specific mechanism involved in melanogenesis. It has been reported that ERK1/2 phosphorylation is related to the inhibition of melanogenesis (Kim et al., 2006). Murine melanoma B16 cells were treated with the extract of Tunisian aromatic plants, namely, Capparis spinosa, Erica multiflora and Thymelaea hirsuta, and then cell viability and synthesized melanin content were measured. The C. spinosa and E. multiflora extract increased the synthesized melanin content in B16 cells without cytotoxity. The tyrosinase expression was clearly increased in cells treated with the extracts. The T. hirsuta extract significantly decreased the synthesized melanin content in B16 cells without cytotoxicity. ERK1/2 was immediately phosphorylated in cells just after treatment with the extract. The tyrosinase expression was inhibited in cells treated with the extracts.

Hair Growth Regulation by an Aromatic Plant Extract

Recently, people are experiencing more stress and some have problems regarding hair growth as a part of the aging process. Resolving this hair growth problem is important for the aging society in the future. This is because not only bodily health but also mental health is important for a healthy life. Thus, we attempted to find plants having hair growth regulation activity.

Effect of Tunisian Plant Extract on Melanogenesis

In this study, we determined whether Tunisian aromatic plants can induce melanogenesis in the cultured mouse melanoma cell line, B16. The cells were cultured with or without Tunisian aromatic plant extracts. We found that melanogenesis was enhanced by Tunisian aromatic plant extract without affecting cell growth and cell shape. This denotes that Tunisian aromatic plant can induce melanogenesis in B16 cells without causing transformation. In succeeding experiments, we found that Tunisian aromatic plant promoted melanogenesis without affecting tyrosinase protein expression. Moreover, we did not detect the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), which are related to inhibition of melanogenesis.