Hye-Eun Kim

Caffeoylserotonin Protects Human Keratinocyte HaCaT Cells against H2O2-Induced Oxidative Stress and Apoptosis through Upregulation of HO-1 Expression via Activation of the PI3K/Akt/Nrf2 Pathway

Caffeoylserotonin (CaS) has strong radical scavenging activity as well as antioxidant activities, protecting cells from lipid peroxidation, intracellular reactive oxygen species generation, DNA damage, and cell death. The molecular mechanism by which CaS protects against oxidative stress is not well understood. Here, we analyzed the cytoprotective activity of CaS in hydrogen peroxide (H2O2)‐treated keratinocyte HaCaT cells. H2O2 induced apoptosis in the cells through activation of pro‐apoptotic p21, Bax, and caspase‐3. Pretreatment with CaS inhibited apoptotic gene expression and activated the anti‐apoptotic gene, Bcl‐xL. Although CaS did not directly affect heme oxygenase‐1 (HO‐1) expression, pretreatment with CaS augmented HO‐1 expression through an increase in NF‐E2‐related factor (Nrf2) stability and stimulation of Nrf2 translocation to the nucleus upon H2O2 exposure. H2O2 also induced the phosphorylation and subsequent activation of ERK, p38 MAPK, and Akt. Analysis using specific inhibitors of p38 MAPK and Akt demonstrated that only Akt activation was involved in HO‐1 and Nrf2 expressions. In addition, PI3K and PKC inhibitors suppressed HO‐1/Nrf2 expression and Akt phosphorylation. These results demonstrate that CaS protects against oxidative stress‐induced keratinocyte cell death in part through the activation of Nrf2‐mediated HO‐1 induction via the PI3K/Akt and/or PKC pathways, but not MAPK signaling. Copyright

Induction of ATP synthase β by H2O2 induces melanogenesis by activating PAH and cAMP/CREB/MITF signaling in melanoma cells.

Hydrogen peroxide (H2O2) production due to oxidative stress is associated with apoptosis and melanogenesis in melanocytes. Here, we analyzed the effects of H2O2 on melanogenesis by measuring the melanin content and analyzing the expression of melanogenesis-related proteins, including cAMP-responsive element binding protein (CREB), microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), and phenylalanine hydroxylase (PAH). Treatment with 1mM H2O2 increased the cellular melanin content; the expression of PAH, TYR, and MITF; and the phosphorylation of CREB in B16F10 and SK-Mel-2 cells. In addition, H2O2 increased the expression of ATP synthase β (ATP5B), a mitochondrial F1 complex, and increased intracellular ATP levels. Studies using the ATP5B inhibitor oligomycin (OM) showed that the induction of cAMP resulted from an increase in ATP caused by the induction of ATP5B. OM treatment increased H2O2-mediated apoptosis via accelerated ATP depletion and apoptosis-related gene expressions. In summary, H2O2 may induce melanogenesis via the upregulation of PAH and activation of cAMP/p-CREB/MITF signaling by increasing intracellular cAMP levels through the induction of ATP5B.

Epigenetic Silencing of SPINT2 Promotes Cancer Cell Motility via HGF-MET Pathway Activation in Melanoma

Aberrant HGF-MET signaling activation via interactions with surrounding stromal cells in tumor microenvironment plays significant roles in malignant tumor progression. However, extracellular proteolytic regulation of HGF activation which is influenced by the tumor microenvironment and its consequential effects on melanoma malignancy remain uncharacterized. In this study we identified SPINT2: a proteolytic inhibitor of hepatocyte growth factor activator (HGFA), which plays a significant role in the suppression of the HGF-MET pathway and malignant melanoma progression. SPINT2 expression is significantly lower in metastatic melanoma tissues compared to those in early stage primary melanomas which also corresponded with DNA methylation levels isolated from tissue samples. Treatment with the DNA hypomethylating agent decitabine in cultured melanoma cells induced transcriptional reactivation of SPINT2, suggesting that this gene is epigenetically silenced in malignant melanomas. Furthermore, we show that ectopically expressed SPINT2 in melanoma cells inhibits HGF induced MET-AKT signaling pathway and decreases malignant phenotype potential such as cell motility, and invasive growth of melanoma cells. These results suggest that SPINT2 is associated with tumor suppressive functions in melanoma by inhibiting an extracellular signal regulator of HGF which is typically activated by tumor-stromal interactions. These findings indicate that epigenetic impairment of the tightly regulated cytokine-receptor communications in tumor microenvironment may contribute to malignant tumor progression.

The effects of Caffeoylserotonin on inhibition of melanogenesis through the downregulation of MITF via the reduction of intracellular cAMP and acceleration of ERK activation in B16 murine melanoma cells

In this study, we evaluated the anti-melanogenesis effects of Caffeoylserotonin (CaS) in B16 melanoma cells. Treatment with CaS reduced the melanin content and tyrosinase (TYR) activity in B16 melanoma cells in a dose-dependent manner. CaS inhibited the expression of melanogenesis-related proteins, including microphthalmia-associated transcription factor (MITF), TYR, and tyrosinase-related protein-1 (TRP-1), but not TRP-2. α-MSH is known to interact with melanocortin 1 receptor (MC1R) thus activating adenylyl cyclase and increasing intracellular cyclic AMP (cAMP) levels. Furthermore, cAMP activates extracellular signal-regulated kinase 2 (ERK2) via phosphorylation, which phosphorylates MITF, thereby targeting the transcription factor to proteasomes for degradation. The CaS reduced intracellular cAMP levels to unstimulated levels and activated ERK phosphorylation within 30 min. The ERK inhibitor PD98059 abrogated the suppressive effect of CaS on α-MSH-induced melanogenesis. Based on this study, the inhibitory effects of CaS on melanogenesis are derived from the downregulation of MITF signaling via the inhibition of intracellular cAMP levels, as well as acceleration of ERK activation. [BMB Reports 2012; 45(12): 724-729]

Diagnostic value of serum glutathione peroxidase 3 levels in patients with lung cancer

We selected glutathione peroxidase 3 (GPx3) as a specific candidate that is down regulated in patients with lung cancer. In this study, we examined the diagnostic value of serum GPx3, which is an extracellular protein and readily detectable in blood.

Caffeoylserotonin suppresses THP-1 monocyte adhesion and migration via inhibition of the integrin β1/FAK/Akt signalling pathway.

We investigated the effect of caffeoylserotonin (CaS) on THP-1 monocyte migration and adhesion to fibronectin in response to MCP-1. CaS decreased monocyte adhesion and migration induced by MCP-1, together with CCR2 expression and α5β1 integrin, and activated β1 integrin expression on the cell surface. CaS also inhibited FAK and Akt phosphorylation. We found that CaS had anti-inflammatory activity based on inhibition of adhesion and migration via inhibition of the integrin β1/FAK/Akt signalling pathway. Thus, the inhibitory effects of CaS on monocyte function may support the future development of this compound as a potential treatment for inflammation-dependent diseases.

Association between cancer stem cell-like properties and epithelial-to-mesenchymal transition in primary and secondary cancer cells

One of the theories on cancer stem cells (CSCs) states that these cells initiate most tumors and give rise to more-or-less differentiated tumor cells. Genetic signatures of CSCs are thought to predict tumor recurrence and metastases, thus, supporting the notion that CSCs may be metastatic precursors and induce epithelial-to-mesenchymal transition (EMT). In this study, we tried to examine the association between CSCs and EMT (using specific markers) in the mucoepidermoid carcinoma cell line YD15 and its derivative cell line YD15M (lymph node metastasis). Relative protein expression levels were analyzed by western blotting, flow cytometry, and immunofluorescence assays. In addition, cell cycle assay and aldehyde dehydrogenase (ALDH) activity assay were carried out. Under growth conditions, YD15M cells formed irregular spherical colonies consistent with a stem cell phenotype. YD15M cells demonstrated the low expression of E-cadherin and β-catenin but high expression of vimentin than that in YD15 cells. In the metastatic cells (YD15M), the coexpression of vimentin and CD133 was detected. Weak proliferation based on cell cycle analysis and decreased PCNA expression was also observed. In addition, expression levels of ALDHA1, OCT4, and NANOG (CSC-like properties) were significantly increased in YD15M cells. Taken together, these findings should help to elucidate the interplay between EMT and CSC-like properties during metastasis and may provide useful information for the development of a novel classification system and therapeutic strategies against head and neck cancer.

Red light-emitting diode irradiation regulates oxidative stress and inflammation through SPHK1/NF-κB activation in human keratinocytes

Oxidative stress, in which the amount of oxidants exceeds the capacity of antioxidant defense system, is a well-accepted pathogenesis of several human diseases. Light-emitting diode irradiation (LEDI) is an efficient strategy to counteract this condition. The biological effect of phototherapy, using visible light, has attracted recent attention especially in dermatological practice. However, little is known about the molecular mechanism of the anti-oxidant and anti-inflammatory effects of red light irradiation. We evaluated these effects of LEDI in HaCaT human keratinocyte cells under phorbol-12-myristate-13-acetate (PMA) induced reactive oxygen species (ROS). Microarray analysis revealed changes in 309 genes after LEDI. LEDI at 625 nm produced ROS scavenging and anti-inflammatory effects. One of the most important genes identified by microarray analysis was sphingosine kinase-1 (SPHK1), which is a key molecule in sphingolipid metabolism. SPHK1 knock-down drastically reduced ROS scavenging efficiency as well as expression levels of inflammation-related proteins in PMA-treated HaCaT cells. These results not only indicate the potential for the clinical application of 625-nm LEDI in treating skin disorders via ROS and/or inflammation, but also suggest SPHK1 as a potential therapeutic target in phototherapy.

Cell proliferation and migration mechanism of caffeoylserotonin and serotonin via serotonin 2B receptor in human keratinocyte HaCaT cells

Caffeoylserotonin (CaS), one derivative of serotonin (5-HT), is a secondary metabolite produced in pepper fruits with strong antioxidant activities. In this study, we investigated the effect of CaS on proliferation and migration of human keratinocyte HaCaT cells compared to that of 5-HT. CaS enhanced keratinocyte proliferation even under serum deficient condition. This effect of CaS was mediated by serotonin 2B receptor (5-HT2BR) related to the cell proliferation effect of 5-HT. We also confirmed that both CaS and 5-HT induced G1 progression via 5-HT2BR/ERK pathway in HaCaT cells. However, Akt pathway was additionally involved in upregulated expression levels of cyclin D1 and cyclin E induced by CaS by activating 5-HT2BR. Moreover, CaS and 5-HT induced cell migration in HaCaT cells via 5-HT2BR. However, 5-HT regulated cell migration only through ERK/AP-1/MMP9 pathway while additional Akt/NF-κB/MMP9 pathway was involved in the cell migration effect of CaS. These results suggest that CaS can enhance keratinocyte proliferation and migration. It might have potential as a reagent beneficial for wound closing and cell regeneration.

Development of Novel Photosensitizer Using the Buddleja officinalis Extract for Head and Neck Cancer

Photodynamic therapy (PDT) is generally safer and less invasive than conventional strategies for head and neck cancer treatment. However, currently available photosensitizers have low selectivity for tumor cells, and the burden and side effects are so great that research is needed to develop safe photosensitizers. In this study, it was confirmed that the Buddleja officinalis (BO) extract, used in the treatment of inflammation and vascular diseases, shows fluorescence when activated by LED light, and, based on this, we aimed to develop a new photosensitive agent suitable for PDT. MTT, Diff-Quick® staining, and DCF-DA were performed to measure the effects of treating head and neck cancer cells with BO extract and 625 nm LED light (BO-PDT). Cell cycle, TUNEL, and western blot assays, as well as acridine orange staining, were performed to explore the mechanism of BO-PDT-induced cell death. We found that when the BO extract was irradiated with 625 nm LED light, it showed sufficient fluorescence and stronger intracellular toxicity and ROS effect than the currently commercially available hematoporphyrin. BO-PDT resulted in a decrease of mTOR activity that was correlated with an increase in the levels of ATG5, beclin-1, and LC3-II, which interfere with the formation of autophagosomes. In addition, BO-PDT induced the activation of PARP and led to an increase in the expression of proapoptotic protein Bax and a decrease in the expression of the antiapoptotic protein Bcl-2. Moreover, BO-PDT has been shown to induce the autophagy pathway 4 h after treatment, while apoptosis was induced 16 h after treatment. Finally, we confirmed that BO-PDT caused cell death of head and neck cancer cells via the intrinsic pathway. Therefore, we suggest that BO extract can be used as a new photosensitizer in PDT of head and neck cancer.