The serum/PDGF-dependent "melanogenic" role of the minute level of the oncogenic kinase PAK1 in melanoma cells proven by the highly sensitive kinase assay.

We previously demonstrated that the oncogenic kinase PAK4, which both melanomas and normal melanocytes express at a very high level, is essential for their melanogenesis. In the present study, using the highly sensitive "Macaroni-Western" (IP-ATP-Glo) kinase assay, we investigated the melanogenic potential of another oncogenic kinase PAK1, which melanoma (B16F10) cells express only at a very minute level. After transfecting melanoma cells with PAK1-shRNA for silencing PAK1 gene, melanin content, tyrosinase activity, and kinase activity of PAK1 were compared between the wild-type and transfectants. We found that (i) PAK1 is significantly activated by melanogenic hormones such as IBMX (3-isobutyl-1-methyl xanthine) and α-MSH (melanocyte-stimulating hormone), (ii) silencing the endogenous PAK1 gene in melanoma cells through PAK1-specific shRNA reduces both melanin content and tyrosinase activity in the presence of both serum and melanogenic hormones to the basal level, (iii) the exogenously added wild-type PAK1 in the melanoma cells boosts the α-MSH-inducible melanin level by several folds without affecting the basal, and (iv) α-MSH/IBMX-induced melanogenesis hardly takes place in the absence of either serum or PAK1, clearly indicating that PAK1 is essential mainly for serum- and α-MSH/IBMX-dependent melanogenesis, but not the basal, in melanoma cells. The outcome of this study might provide the first scientific basis for explaining why a wide variety of herbal PAK1-blockers such as CAPE (caffeic acid phenethyl ester), curcumin and shikonin in cosmetics are useful for skin-whitening.

Effect of Okinawa Propolis on PAK1 Activity, Caenorhabditis elegans Longevity, Melanogenesis, and Growth of Cancer Cells.

Propolis from different areas has been reported to inhibit oncogenic/aging kinase PAK1, which is responsible for a variety of conditions, including cancer, longevity, and melanogenesis. Here, a crude extract of Okinawa propolis (OP) was tested against PAK1 activity, Caenorhabditis elegans (C. elegans) longevity, melanogenesis, and growth of cancer cells. We found that OP blocks PAK1 and exhibits anticancer activity in the A549 cell (human lung cancer cell) line with IC50 values of 6 µg/mL and 12 µg/mL, respectively. Most interestingly, OP (1 µg/mL) significantly reduces reproduction and prolongs the lifespan of C. elegans by activating the HSP-16.2 gene, as shown in the PAK1-deficient strain. Furthermore, OP inhibits melanogenesis in a melanoma cell line (B16F10) by downregulating intracellular tyrosinase activity with an IC50 of 30 µg/mL. Our results suggest that OP demonstrated a life span extending effect, C. elegans, anticancer, and antimelanogenic effects via PAK1 inactivation; therefore, this can be a potent natural medicinal supplement against PAK1-dependent diseases.

Combination of immunoprecipitation (IP)-ATP_Glo kinase assay and melanogenesis for the assessment of potent and safe PAK1-blockers in cell culture.

Cucurbitacin I (CBI) is a triterpene from a bitter melon called Goya grown in Okinawa, Japan, and directly inhibits both the Tyr-kinase JAK2 and the G protein RAC, leading to the inactivation of PAK1 (RAC/CDC42-activated kinase 1). Bio 30, a propolis produced in New Zealand, contains CAPE (caffeic acid phenethyl ester) as the major anti-cancer ingredient which directly down-regulates RAC, leading to the inactivation of PAK1. Since PAK1 is essential for the growth of RAS cancer cells such as A549 cell line which carry an oncogenic K-RAS mutant, and the melanogenesis in skin cells, here using these PAK1-blockers as model compounds, we introduce a new approach to the quick assessment of PAK1-blockers in cell culture. First, combining the immuno-precipitation (IP) of PAK1 from cell lysate and the in vitro ATP_Glo kinase assay kit (called "Macaroni-Western" assay), we confirmed that both CBI and Bio 30 inactivate PAK1 in A549 lung cancer cells in 24 h, and inhibit their PAK1-dependent growth in 72 h. Furthermore, we verified that CBI inhibits the PAK1/PAK4-dependent melanogenesis in melanoma cells by far more than 50%, while Bio 30 inhibits the melanogenesis only by 50%, with only a merginal effect on their growth per se. Since the "Macaroni-Western" kinase assay and melanogenesis are both rather simple and quick, the combination of these two cell culture assays would be highly useful for selecting both "potent" (highly cell-permeable) and "safe" (non-toxic) natural or synthetic PAK1-blockers.

Hispidin and related herbal compounds from Alpinia zerumbet inhibit both PAK1-dependent melanogenesis in melanocytes and reactive oxygen species (ROS) production in adipocytes.

Recently several compounds from Okinawa plants including Alpinia zerumbet (alpinia) were shown to inhibit directly the oncogenic/ageing kinase PAK1 (p21-activated kinase 1). Furthermore, it was recently revealed that both PAK1 and PAK4 (p21-activated kinase 4) are equally essential for the melanogenesis in melanoma cells. Thus, in this study, we tested if several alpinia compounds inhibit the melanogenesis in melanoma (B16F10) cells, as well as the PAK1-dependent up-regulation of both reactive oxygen species (ROS) and nitric oxide (NO) in cultured adipocytes (3T3-L1) without any cytotoxicity. The effect of alpinia compounds on the melanogenesis was measured by both the melanin content and intracellular tyrosinase activity in melanoma cells treated with 3-isobutyl-1-methylxanthine (IBMX), a melanogenesis stimulating hormone. We found that (1E,3E,5E)-6-methoxyhexa-1,3,5-trien-1-yl)-2,5-dihydrofuran (MTD), 5,6-dehydrokawain (DK), labdadiene, hispidin and dihydro-5,6-dehydrokawain (DDK) at 50 µg/mL reduced the melanin content by 63-79%. The MTD, DK and hispidin, at 50 µg/mL, inhibited tyrosinase activity by 70-83% in melanoma cells. Among these compounds, labdadiene, MTD, (E)-2,2,3,3-Tetramethyl8-methylene-7-(oct-6-en-1-yl)octahydro-1H-quinolizine (TMOQ) and hispidin strongly inhibited the ROS production. Hispidin, labdadiene and MTD at 20 µg/mL inhibited NO production by over 70%. These findings altogether suggest that some of these alpinia compounds could be potentially useful for the prevention or treatment of hyperpigmentation and obesity.