Stimulation of Melanogenesis by Nordihydroguaiaretic Acid in Human Melanoma Cells

Nordihydroguaiaretic acid (NDGA), a lignan found in vegetables, fruits and legumin, has been shown to possess antineoplastic, antiviral and antioxidant characteristics. In this study, we examined the effect of NDGA on melanogenesis in human melanoma cells (HMVII). In vitro, NDGA does not alter mushroom tyrosinase activity. However, in NDGA-treated HMVII cells, cellular tyrosinase activity increased in both a time- and dose-dependent manner. The concomitant increases in melanin content in NDGA-treated cells indicated an elevation of melanin synthesis by tyrosinase activation. In addition, after a 7-day incubation, melanin content in 20 μM NDGA-treated cells increased 5.02 fold. Tyrosinase protein also increased by treatment with NDGA. Nevertheless, tyrosinase mRNA was not altered in NDGA-treated cells. Our results suggest that NDGA can increase tyrosinase activity and de novo synthesis of melanin in human melanoma cells. We found that NDGA is a novel potent stimulator of melanogenesis in human melanoma cells.     

Specific phosphorylation and activation of ERK1c by MEK1b: a unique route in the ERK cascade

Extracellular signal-regulated kinases (ERKs) are key signaling molecules that regulate a large number of cellular processes, including mitosis. We showed previously that ERK1c, an alternatively spliced form of ERK1, facilitates mitotic Golgi fragmentation without the involvement of ERK1 and ERK2. Here we demonstrate that activation of ERK1c is mainly mediated by mitogen-activated protein kinase (MAPK)/ERK kinase 1b (MEK1b), which is an alternatively spliced form of MEK1 that was previously considered an inactive kinase. MEK1b phosphorylation and activity are preferentially stimulated by nocodazole, to induce its specific activity toward ERK1c. MEK1/2, on the other hand, preferentially target ERK1/2 in response to growth factors, such as EGF. As previously demonstrated for ERK1c, also MEK1b expression and activity are elevated during mitosis, and thereby enhance Golgi fragmentation and mitotic rate. MEK1 activity is also increased during mitosis, but this isoform facilitates mitotic progression without affecting the Golgi architecture. These results illustrate that the ERK cascade is divided into two routes: the classic MEK1/2–ERK1/2 and the splice-variant MEK1b–ERK1c, each of which regulates distinct cellular processes and thus extends the cascade specificity.     

Cellular and molecular features of mammalian pigmentation--tyrosinase and TRP

Melanin pigments are found in all biological kingdoms. At the cellular level, correct pigmentation depends not only on correct migration of pigment cells (melanocytes) and their precursors (melanoblasts), but also on specialized cytoplasmic organelles, melanosomes. Three pigment cell-specific enzymes inside melanosomes are involved in melanogenesis: tyrosinase, TRP-1 and TRP-2/DOPA-chrome tautomerase. A point mutation of tyrosinase is sufficient to give rise to a nonfunctional enzyme, leading to albinism and associated decreased visual acuity. Several regulatory elements have been identified within the promoters of the genes encoding these three enzymes. These promoter fragments have been used to target the expression of heterogeneous genes in pigment cells of transgenic mice.     

Valproic acid overcomes transforming growth factor-β-mediated sorafenib resistance in hepatocellular carcinoma

Sorafenib is a multi-kinase inhibitor approved for hepatocellular carcinoma, but rarely causes tumor regression in patients with chronic liver diseases. To investigate whether growth factor-mediated signaling is involved in sorafenib resistance, HepG2 and PLC/PRF/5 hepatoma cells were exposed to epidermal growth factor (EGF), hepatocyte growth factor (HGF) or transforming growth factor-β (TGF-β) prior to treatment with sorafenib. Furthermore, to identify an effective combination treatment with sorafenib, growth factor-sensitized cells were treated with sorafenib alone or in combination with celecoxib, lovastatin or valproic acid (VPA). Trypan blue staining and Annexin V assays showed that the cytotoxic effect of sorafenib was inhibited by 15-54% in cells sensitized to TGF-β (P<0.05). Western blotting analysis showed that TGF-β significantly activated extracellular signal-regulated kinase (ERK)-mediated AKT signaling, and sorafenib failed to suppress both ERK and AKT in TGF-β-sensitized cells. The decreased anti-tumor effect of sorafenib was rescued by chemical inhibition of ERK and AKT. When TGF-β-sensitized cells were treated with sorafenib plus VPA, the levels of phosphorylated ERK and AKT were considerably suppressed and the numbers of dead cells were increased by 3.7-5.7-fold compared with those exposed to sorafenib alone (P<0.05). Moreover, low dose sorafenib-induced cell migration was effectively suppressed by combination treatment with sorafenib and VPA. Collectively, TGF-β/ERK/AKT signaling might play a critical role in sorafenib resistance in hepatoma cells, and combination treatment with VPA may be effective against this drug resistance.     

Effects of ghrelin on protein expression of antioxidative enzymes and iNOS in the rat liver

Introduction

We investigated the effects of ghrelin on protein expression of the liver antioxidant enzymes superoxide dismutases (SODs), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR), nuclear factor κB (NFκB) and inducible nitric oxide synthase (iNOS). Furthermore, we aimed to investigate whether extracellular regulated protein kinase (ERK1/2) and protein kinase B (Akt) are involved in ghrelin-regulated liver antioxidant enzymes and iNOS protein expression.

Material and methods

Male Wistar rats were treated with ghrelin (0.3 nmol/5 µl) injected into the lateral cerebral ventricle every 24 h for 5 days, and 2 h after the last treatment the animals were sacrificed and the liver excised. The Western blot method was used to determine expression of antioxidant enzymes, iNOS, phosphorylation of Akt, ERK1/2 and nuclear factor κB (NFκB) subunits 50 and 65.

Results

There was significantly higher protein expression of CuZnSOD (p < 0.001), MnSOD (p < 0.001), CAT (p < 0.001), GPx, (p < 0.001), and GR (p < 0.01) in the liver isolated from ghrelin-treated animals compared with control animals. In contrast, ghrelin significantly (p < 0.01) reduced protein expression of iNOS. In addition, phosphorylation of NFκB subunits p65 and p50 was significantly (p < 0.001 for p65; p < 0.05 for p50) reduced by ghrelin when compared with controls. Phosphorylation of ERK1/2 and of Akt was significantly higher in ghrelin-treated than in control animals (p < 0.05 for ERK1/2; p < 0.01 for Akt).

Conclusions

The results show that activation of Akt and ERK1/2 is involved in ghrelin-mediated regulation of protein expression of antioxidant enzymes and iNOS in the rat liver.     

CK2β Is Expressed in Endometrial Carcinoma and Has a Role in Apoptosis Resistance and Cell Proliferation

Protein kinase CK2 (CK2) is a serine/threonine kinase that participates in important cellular processes. We have recently demonstrated that CK2 plays a role in resistance to TRAIL/Fas-induced apoptosis in endometrial carcinoma (EC) by regulating FLIP. Here, we assessed the immunohistochemical expression of CK2β in EC and checked its role in cell proliferation and anchorage-independent cell growth. CK2β immunostaining was assessed in two tissue microarrays, one constructed from paraffin-embedded blocks of 95 ECs and another from 70 samples of normal endometrium. CK2β expression was correlated with histological type; grade and stage; cell proliferation (Ki-67) and apoptotic index; immunostaining for cyclin D1, PTEN, AKT, β-catenin, and FLIP. Moreover, the Ishikawa EC cell line was subjected to down-regulation of CK2 by shRNA. CK2β expression was frequent in EC (nuclear, 100%; cytoplasmic, 87.5%). The staining was more intense in EC than in normal endometrium (P = 0.000), and statistically correlated with AKT, PTEN, β-catenin, and FLIP. In EC, CK2β expression correlated with cell proliferation. Knock-down of CK2β blocked colony formation of EC in soft agar, and also resulted in decreased expression of cyclin D1 and ERK phosphorylation. The results confirm that CK2β is widely expressed in EC, and suggest a role in cell proliferation and anchorage-independent cell growth.     

Methionine Adenosyltransferase 2B–GIT1 Complex Serves as a Scaffold to Regulate Ras/Raf/MEK1/2 Activity in Human Liver and Colon Cancer Cells

Methionine adenosyltransferase 2B (MAT2B) encodes for variant proteins V1 and V2 that interact with GIT1 to increase ERK activity and growth in human liver and colon cancer cells. MAT2B or GIT1 overexpression activates MEK. This study explores the mechanism for MEK activation. We examined protein-protein interactions by co-immunoprecipitation and verified by confocal microscopy and pull-down assay using recombinant or in vitro translated proteins. Results were confirmed in an orthotopic liver cancer model. We found that MAT2B and GIT1-mediated MEK1/2 activation was not mediated by PAK1 or Src in HepG2 or RKO cells. Instead, MAT2B and GIT1 interact with B-Raf and c-Raf and enhance recruitment of Raf proteins to MEK1/2. MAT2B-GIT1 activates c-Raf, which is the key mediator for MEK/12 activation, because this still occurred in RKO cells that express constitutively active B-Raf mutant. The mechanism lies with the ability of MAT2B-GIT1 to activate Ras and promote B-Raf/c-Raf heterodimerization. Interestingly, MAT2B but not GIT1 can directly interact with Ras, which increases protein stability. Finally, increased Ras-Raf-MEK signaling occurred in phenotypically more aggressive liver cancers overexpressing MAT2B variants and GIT1. In conclusion, interaction between MAT2B and GIT1 serves as a scaffold and facilitates signaling in multiple steps of the Ras/Raf/MEK/ERK pathway, further emphasizing the importance of MAT2B/GIT1 interaction in cancer growth.Methionine adenosyltransferase (MAT) is an essential enzyme expressed in all mammalian cells that catalyzes the formation of S-adenosylmethionine (SAMe), the principal biological methyl donor.¹ There are three mammalian MAT genes. MAT1A and MAT2A encode for the catalytic subunit (α1 and α2) of the different MAT isoforms, and MAT2B encodes for a regulatory subunit (β) that modulates the activity of the MAT2A-encoded isoenzyme (MATII).¹ MAT1A is predominantly expressed in normal hepatocytes, whereas MAT2A is expressed in all extrahepatic tissues.¹ MAT2B shares a similar expression pattern as MAT2A.² There are two major variant proteins encoded by MAT2B, MAT2BV1 (or V1) and MAT2BV2 (or V2), and both variants can increase MATII efficiency by lowering the Michaelis constant of MATII for its substrates methionine and ATP.^2,3 In addition to regulating the kinetic properties of MATII, MAT2B is overexpressed in hepatocellular carcinoma (HCC) and colon cancer and offers the cancer cell a growth advantage.^2,4A key mechanism for MAT2B to enhance growth is ERK1/2 activation.^2,4 Our previous work found that increased ERK1/2 activation occurs only when both MAT2B variants are present in addition to GIT1, a scaffold protein that facilitates c-Src–dependent mitogen-activated protein kinase (MAPK) activation.⁴ We found that both MAT2B variants directly interact with GIT1, and when these proteins are overexpressed, there is enhanced recruitment of ERK2 to MEK1 and the activity of both ERK1/2 and MEK1 increased.⁴ This finding proved to be important in tumorigenesis because overexpression of either V1 or V2 with GIT1 enhanced growth and lung metastasis in an orthotopic HCC model.⁴ Conversely, knockdown of endogenous V1, V2, or GIT1 lowered MEK1 and ERK1/2 activity.⁴ Thus, our previous work established MAT2B-GIT1 as a scaffold that facilitates MEK-ERK signaling.⁴ However, we did not examine how MAT2B-GIT1 complex activates MEK. Our current work examined the signaling pathways that can lead to MEK activation and identified MAT2B-GIT1 as a scaffold that acts on multiple levels of the Ras-Raf-MEK-ERK signaling cascade to facilitate their activation in human liver and colon cancer cells.     

RAF1-MEK1-ERK/AKT axis may confer NSCLC cell lines resistance to erlotinib

The fact that advanced NSCLC patients with wild type (wt) EGFR can benefit from erlotinib therapy makes it critical to find out biomarkers for effective selection of patients and improving the therapy effects. In present study, 3 NSCLC cell lines (U1752, Calu-6 and NCI-H292) with wt EGFR and different sensitivities to erlotinib were used for microarray analysis. The differential basal gene expression between 2 NSCLC cell lines was analyzed, about 353 genes were expression-altered with higher than 2-fold changes between Calu-6 and U1752. And Ingenuity Pathway Analysis (IPA) showed that these genes were mainly enriched in regulation of epithelial–mesenchymal transition (EMT) pathway, Wnt-β catenin signaling, Tec kinase signaling and some types of cancer-related signaling. More interestingly, RAF1 (c-raf), MAP2K1 (MEK1), SNAI and downstream signaling molecules ERK and AKT were predicted to be activated in erlotinib-resistant cell line by IPA. Subsequent immunoblotting experiments showed that the phosphorylation of ERK and AKT were exactly increased stepwise from erlotinib sensitive cell line to erlotinib resistant cell lines. Collectively, activation of RAF1-MEK1-ERK/AKT axis may determine the resistance of NSCLC cell lines bearing wt EGFR to erlotinib. Our work provides potential biomarkers and therapeutic targets for NSCLC patients harboring wt EGFR.