Incidentally Found Primary Cerebral Malignant Melanoma Associated with Ota Nevus—Wide Dissemination after an Initial Phase of Slow Growth

Primary cerebral malignant melanoma accounts for 1% of all melanomas and for 0.7% of all primary tumours of the central nervous system (CNS). We report an incidentally found primary malignant melanoma in the right temporal lobe of a 76-year-old woman with an Ota nevus on her right eyelid and sclera. The lesion was initially characterised by slow growth, followed by tumoural bleeding and wide leptomeningeal dissemination. Magnetic resonance imaging (MRI) during brain checkup demonstrated an 8-mm mass in the right uncus with high intensity on T1-weighted images. The mass grew slowly over the next two years, but asymptomatic tumoural haemorrhage eventually developed. The patient underwent tumour removal via right frontotemporal craniotomy. Extensive subarachnoid dissemination from the right temporal tumour was found; the pathologic diagnosis was malignant melanoma. Despite adjuvant therapy comprising whole-brain radiation and nivolumab, the patient died of severe leptomeningeal dissemination at 4.5 months after the operation (33 months after the initial MRI study). Our literature review found 46 cases of primary CNS malignant melanoma, predominantly in middle-aged to elderly individuals. The most frequent symptom was headache, followed by visual disturbance, nausea/vomiting, and hemiparesis. Only one other case of primary cerebral malignant melanoma had been found incidentally; 11 previously reported patients manifested congenital nevi (Ota nevus, n = 5; other nevi, n = 6). Six patients suffered tumoural haemorrhage, 4 experienced leptomeningeal dissemination, and 5 developed extracranial metastases. The median survival time of the patients was 31 months

Induction of p53-dependent p21 limits proliferative activity of rat hepatocytes in the presence of hepatocyte growth factor.

BACKGROUND: Hepatocyte growth factor (HGF), a potent mitogen for hepatocytes, enhances hepatocyte function without stimulating proliferation, depending on the physiological conditions. p53, a transcription factor, suppresses the cell proliferation by expressing p21(WAF1/CIP1) in various tissues. AIM: To investigate the mechanism through which the hepatocytes maintain mitotically quiescent even in the presence of HGF. METHODS: We studied the relationship between p53 and p21 expression and the effect of p53-p21 axis on hepatocyte proliferation in primary cultured rat hepatocytes stimulated by HGF. Hepatic p21 levels are determined serially after partial hepatectomy or sham operation in rats. RESULTS: DNA synthesis was markedly increased by HGF addition in rat hepatocytes cultured at low density but not at high density. Cellular p53 levels increased in the hepatocytes cultured at both the densities. p21 levels were increased and correlated with cellular p53 levels in hepatocytes cultured at high density but not at low density. When the activity of p53 was suppressed by a chemical inhibitor for p53, cellular p21 levels were reduced, and DNA synthesis was increased. Similarly, p21 antisense oligonucleotide increased the DNA synthesis. In rats after partial hepatectomy, transient elevation of hepatic p21 levels was observed. In contrast, in sham-operated rats, hepatic p21 levels were increased on sustained time scales. CONCLUSION: p53-related induction of p21 may suppress hepatocyte proliferation in the presence of HGF in the setting that mitogenic activity of HGF is not elicitable

DNA Methyltransferase Inhibitor Zebularine Inhibits Human Hepatic Carcinoma Cells Proliferation and Induces Apoptosis

<div><p>Hepatocellular carcinoma is one of the most common cancers worldwide. During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Zebularine (1-(β-_D-ribofuranosyl)-1,2-dihydropyrimidin-2-one) acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both <em>in vitro</em> and <em>in vivo</em>. In this study, we explore the effect and possible mechanism of action of zebularine on hepatocellular carcinoma cell line HepG2. We demonstrate that zebularine exhibits antitumor activity on HepG2 cells by inhibiting cell proliferation and inducing apoptosis, however, it has little effect on DNA methylation in HepG2 cells. On the other hand, zebularine treatment downregulated CDK2 and the phosphorylation of retinoblastoma protein (Rb), and upregulated p21^WAF/CIP1 and p53. We also found that zebularine treatment upregulated the phosphorylation of p44/42 mitogen-activated protein kinase (MAPK). These results suggest that the p44/42 MAPK pathway plays a role in zebularine-induced cell-cycle arrest by regulating the activity of p21^WAF/CIP1 and Rb. Furthermore, although the proapoptotic protein Bax levels were not affected, the antiapoptotic protein Bcl-2 level was downregulated with zebularine treatment. In addition, the data in the present study indicate that inhibition of the double-stranded RNA-dependent protein kinase (PKR) is involved in inducing apoptosis with zebularine. These results suggest a novel mechanism of zebularine-induced cell growth arrest and apoptosis via a DNA methylation-independent pathway in hepatocellular carcinoma.</p> </div

Effect of zebularine on the DNMTs expression and DNA methylation in HepG2 cells.

<p>(A) The protein level of DNMT1, DNMT3a, and DNMT3b after zebularine treatment for 72 h at different concentrations. After treatment, the cells were harvested and western blot analysis was performed to detect the protein level of DNMT1, DNMT3a, and DNMT3b. GAPDH was used as a loading control. Data are the means ± SEM of results from at least three independent experiments. *<i>p</i><0.05, compared to 0 μM. (B) Scatter plot of the average beta values at 485,415 CpG sites for zebularine-treated (y-axis) and control (x-axis) HepG2 cells (n = 3 for each group). Dots for CpG sites whose delta-beta value is >0.1 or <−0.1 are shown in green (35 [0.0072%] hypermethylated and 162 [0.033%] hypomethylated CpG sites).</p

Effects of zebularine on the protein expression of cell-cycle regulator.

<p>The protein level of CDK2, p-Rb, and Rb after zebularine treatment for 24 h at different concentrations. After treatment, the cells were harvested and western blot analysis was performed to detect the protein level of CDK2, p-Rb, and Rb. GAPDH was used as a loading control. Data are the means ± SEM of results from at least three independent experiments. *<i>p</i><0.05, compared to 0 μM.</p

Cellular p53 and p21 levels in hepatocytes treated with HGF.

<p>Rat hepatocytes were cultured in WE containing 10% FCS and various concentrations of HGF for 18 hours. Open circles denote hepatocytes cultured in the absence of HGF. Dotted open circles denote hepatocytes cultured with 2.5 ng/mL HGF. Dotted closed circles denote hepatocytes cultured with 5 ng/mL HGF. Closed circles denote hepatocytes cultured with 10 ng/mL HGF. (A) Hepatocytes cultured at high density. (B) Hepatocytes cultured at low density.</p

Changes in DNA synthesis of hepatocytes after HGF treatment.

<p>Rat hepatocytes were cultured in WE containing 10% FCS, 10 ng/mL HGF and 0.1 mmol/L BrdU, and were harvested serially. Closed circles denote hepatocytes cultured at high density. Open circles denote hepatocytes cultured at low density. Data are mean ± SEM of eight dishes. *p<0.01 compared with the values cultured for 0 hours.</p

Effects of zebularine on phosphorylation of PKR.

<p>(A) The phosphorylation and expression of PKR after zebularine treatment for 72 h at different concentrations. After treatment, the cells were harvested and western blot analysis was performed to detect the phosphorylated and total PKR protein level. GAPDH was used as a loading control. *<i>p</i><0.05, compared to 0 μM. (B) Effect of the overexpression of PKR in zebularine-induced cell death. The forward transfection of the empty vector (Halo Tag control vector) as the control or the plasmid-containing PKR cDNA sequence (pFN21A-hPKR) was performed, and the cells were then treated with different concentrations of zebularine for 72 h. *<i>p</i><0.05, compared to control. Data are the means ± SEM of results from at least three independent experiments.</p

The effect of pifithrin-α on DNA synthesis of hepatocytes in the presence of HGF.

<p>Rat hepatocytes were cultured at high density in the same medium as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078346#pone-0078346-g006" target="_blank">Figure 6</a>, except that the medium contained 1 mmol/L BrdU, for 24 hours. An open bar denotes hepatocytes cultured in the absence of HGF. Closed bars denote hepatocytes cultured with pifithrin-α in the presence of 10 ng/mL HGF. A dotted bar denotes hepatocytes cultured with DMSO in the presence of 10 ng/mL HGF. Data are mean ± SEM of eight dishes. *p<0.01 compared with the values treated only with HGF or values treated with HGF and DMSO.</p