Caffeic Acid Phenethyl Ester Inhibits Alpha-Melanocyte Stimulating Hormone-Induced Melanin Synthesis through Suppressing Transactivation Activity of Microphthalmia-Associated Transcription Factor

Caffeic acid phenethyl ester (<b>1</b>), a natural compound found in various plants and propolis, is a well-known anti-inflammatory, immunomodulatory, and cytotoxic agent. The present study aimed to investigate the molecular events underlying the antimelanogenic activity of <b>1</b> in alpha-melanocyte stimulating hormone (α-MSH)-stimulated B16-F10 melanoma cells. In this investigation, <b>1</b> effectively reduced α-MSH-stimulated melanin synthesis by suppressing expression of melanogenic enzymes such as tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2), although this compound did not directly inhibit tyrosinase enzyme activity. On the other hand, the expression and nuclear translocation of microphthalmia-associated transcription factor (MITF) as a key transcription factor for tyrosinase expression regulating melanogenesis were not affected by treatment with <b>1</b>. The upstream signaling pathways including cAMP response element-binding protein (CREB), glycogen synthase kinase-3β (GSK-3β), and Akt for activation and expression of MITF were also not influenced by <b>1</b>. Interestingly, <b>1</b> inhibited transcriptional activity of a tyrosinase promoter by suppressing the interaction of MITF protein with an M-box containing a CATGTG motif on the tyrosinase promoter. Given the important role of MITF in melanogenesis, suppression of <b>1</b> on the function of MITF to transactivate tyrosinase promoter may present a novel therapeutic approach to treat hyperpigmentation disorders

Suppression of apoptotic signal events by GM3 RNA interference in CDDP-exposed cells.

<p>HCT116 cells were transfected with three siRNA targeting GM3 and negative control siRNA. Cells were then cultured for 12(30 µg/mL). Total RNA and protein lysates from the cells were prepared as described in Materials and Methods. (A) The levels of GM3 synthase mRNA in total RNA obtained from each cell was detected by RT-PCR. (B) Total protein was prepared from the cells and analyzed by western blotting using the indicated antibodies.</p

HCT116 cell apoptosis induced by CDDP via a ROS-dependent pathway.

<p>(A) After treatment of CDDP at the indicated concentrations for 24 h, genomic DNA was isolated and separated by electrophoresis on a 2% agarose gel. The DNA was stained with ethidium bromide and visualized under UV light. (B) Total protein from HCT116 cells was isolated, and immunoblotting analysis was performed with the indicated antibodies. (C) HCT116 cells were treated with or without 30 µg/mL CDDP after pre-treatment with 10 nM NAC, followed by the replacement of the culture medium with freshly prepared medium containing 10 µM DCFH-DA. After 30 min of incubation at 37°C, fluorescence intensity was measured by flow cytometry. Results represent the fold increase from their respective controls (cells not treated with CDDP), considered as 1. Data represent the mean ± SD of 3 independent measurements. ^***P<0.001 vs. the CDDP-treated control. (D) HCT116 cells were treated with or without CDDP (30 µg/mL) after pre-treatment of NAC (10 nM). Protein was isolated from the cells and analyzed by immunoblotting using indicated antibodies. GAPDH was included as an internal control.</p

Regulation of 12-LOX activity by CDDP-induced ganglioside GM3 in HCT116 cells.

<p>(A) HCT116 cells were transfected with siRNA targeting GM3 and negative control siRNA. The cells were then cultured for 12 h in the presence or absence of CDDP (30 µg/mL). The culture medium was replaced with freshly prepared medium containing 10 µM DCFH-DA. After 30 min of incubation at 37°C, fluorescence intensity was measured by flow cytometry. Data represent the mean ± SD of 3 independent measurements. ^**P<0.01 and ^***P<0.001 vs. the CDDP-treated control. ns: no significance. For RT-PCR, total RNA and protein lysates from the cells were prepared as described in Materials and Methods. The levels of GM3 synthase mRNA in total RNA obtained from each cell were detected by RT-PCR. (B) HCT116 cells were treated with or without 30 µg/mL CDDP after pre-treatment of 10 nM NAC or 1 µM Baicalein, and then culture medium was replaced with freshly prepared medium containing 10 µM DCFH-DA. After 30 min of incubation at 37°C, fluorescence intensity was measured by flow cytometry. Data represent the mean ± SD of 3 independent measurements. ^***P<0.001 vs. the CDDP-treated control. For RT-PCR, total RNA and protein lysates from the cells were prepared as described in Materials and Methods. The levels of GM3 synthase mRNA in total RNA obtained from each cell were detected by RT-PCR.</p

Enhanced expression of GM3 synthase and its product GM3 in HCT116 cells after CDDP treatment.

<p>(A) Total RNA from HCT116 cells was isolated following treatment with the indicated concentrations of CDDP treatment for 12 h and GM3 synthase mRNA was detected by RT-PCR. (B) HCT116 cells were transiently transfected with GM3 synthase gene promoter (pGL3-1600) and CREB mutation of GM3 synthase promoter (pGL3-1600 CREB Mu), and then cultured with or without CDDP for 12 h. Luciferase activity was determined from cell lysates as described in the Materials and Methods. The results shown are means ± SD of three independent experiments with triplicate measurement. ^***P<0.001 vs. the CDDP-treated control. (C) HCT116 cells were cultured and then incubated with indicated concentration of CDDP treatment for 12 h. Immunoreactivity against ganglioside GM3 was detected using a fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM without or with the M2590 antibody. (D) The gangliosides isolated from the HCT116 cells treated with CDDP (0, 10, and 30 µg/mL) were analyzed by HPTLC.</p

Schematic representation of the mechanism underlying the <i>P</i>. <i>lactiflora</i> induced LIF expression in the endometrium and its relevance.

<p><i>P</i>. <i>lactiflora</i> induces LIF expression through the activation of p38 and MEK/ERK pathways, leading to an increase in the trophoblast adhesion to the endometrium. Inhibition of LIF expression blocks the expression of adhesion molecules, such as integrin β3 and β5, and adhesion of the trophoblast to the endometrium.</p

Expression of adhesion molecules in PL-PP treated Ishikawa cells.

<p><b>(A)</b> Ishikawa cells were treated with or without PL-PP (50 μg/mL) for 24 h, and total RNA was extracted. The expression levels of <i>LIF</i>, <i>ITGAV</i>, <i>ITGB1</i>, <i>ITGB3</i>, <i>ITGB4</i>, <i>ITGB5</i>, <i>ICAM-1</i>, <i>L-selectin</i>, <i>E-cadherin</i>, and <i>CD44</i> mRNA were analysed by RT-PCR. β-actin was used as an internal control. <b>(B)</b> The pLKO.1 –or shLIF—transfected Ishikawa cells were treated with PL-PP (50 μg/mL) for 24 h. Total RNA was extracted and <i>ITGB3</i> and <i>ITGB5</i> mRNA expression levels were measured by RT-PCR. β-actin was used as an internal control.</p

The effect of PL-PP on LIF expression and adhesion of JAr spheroids to Ishikawa cells.

<p><b>(A, B)</b> Total RNA and protein were isolated after treatment with the indicated concentrations of PL-PP in serum—free medium for 24 h. Relative levels of LIF mRNA and protein were examined by RT-PCR and western blot analysis, respectively. The intensity of the band of interest was estimated by densitometric analysis and calculated as the mean ± SD of three independent experiments (* <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001 compared to the control group). <b>(C)</b> Ishikawa cells were cultured in 24-well plates and treated with or without PL-PP (50 μg/mL) for 48 h. Twenty JAr spheroids were added onto the Ishikawa cell monolayer. The number of JAr spheroids bound to confluent Ishikawa cells was manually counted and calculated as the mean ± SD of three independent experiments (* <i>P</i> < 0.05 compared to each group).</p

<i>Paeonia lactiflora</i> Enhances the Adhesion of Trophoblast to the Endometrium via Induction of Leukemia Inhibitory Factor Expression

<div><p>In the present study, we investigated the role of <i>Paeonia lactiflora Pall</i>. extract on embryo implantation <i>in vitro</i> and <i>in vivo</i>. A polysaccharides depleted-water extract of <i>P</i>. <i>lactiflora</i> (PL-PP) increased LIF expression in human endometrial Ishikawa cells at non-cytotoxic doses. PL-PP significantly increased the adhesion of the human trophectoderm-derived JAr spheroids to endometrial Ishikawa cells. PL-PP-induced LIF expression was decreased in the presence of a p38 kinase inhibitor SB203580 and an MEK/ERK inhibitor U0126. Furthermore, endometrial LIF knockdown by shRNA reduced the expression of integrins β3 and β5 and adhesion of JAr spheroids to Ishikawa cells. <i>In vivo</i> administration of PL-PP restored the implantation of mouse blastocysts in a mifepristone-induced implantation failure mice model. Our results demonstrate that PL-PP increases LIF expression via the p38 and MEK/ERK pathways and favors trophoblast adhesion to endometrial cells.</p></div

Identification of signaling pathways involved in PL-PP-induced LIF expression.

<p>Ishikawa cells were pre-treated with SP600125 (10 μM), LY294002 (15 μM), SB203580 (10 μM), and/or U0126 (0.5 μM) for 1 h. After treatment of each signaling inhibitor, the cells were stimulated with PL-PP (50 μg/mL) for 24 h. Total RNA and protein were isolated from each sample and LIF expression was evaluated by RT-PCR <b>(A)</b> and western blot analysis <b>(B)</b>. The intensity of the band of interest was estimated by densitometric analysis and calculated as means ± SD of two independent experiments (^<b>#</b> <i>P</i> < 0.01 vs. the untreated control. * <i>P</i> < 0.05 and ** <i>P</i> < 0.01 vs. the PL-PP-treated control). Ishikawa cells were treated with PL-PP for the indicated times (0, 0.5, 1, 2, and 4 h). Total protein was isolated from each sample and p38 and ERk phosphorylations were evaluated by western blot analysis <b>(C)</b>. The intensity of the band of interest was estimated by densitometric analysis and calculated as means ± SD of three independent experiments (* <i>P</i> < 0.05 compared to each control).</p