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Background Vitiligo is an acquired disease that involves low pigment variation in the skin. The use of cultured melanocytes for treatment of recalcitrant vitiligo has become a well-established treatment modality. In vitro cultured melanocytes present an effective autologous transplantation treatment modality for vitiligo. The present study investigated the in vitro culture of epidermal melanocytes sampled from multiple body parts, as well as the differences in total propagation time. Methods Skin specimens were collected from 30 participants (14 males and 16 females) who supplied normal colored skin sections from various regions of their body, including the face, chest, abdomen, buttock, and extremities. Subsequently, all of these specimens were treated with an identical melanocyte purification and culturing process. Results Melanocytes from the face demonstrated the most rapid growth and longest total propagation time. Melanocytes from the buttock, abdomen, and extremities displayed similar results to one another, and melanocytes from the chest and back had the slowest growth and shortest total propagation time. Conclusion Selecting the most favorable site to obtain epidermal melanocytes will reduce the required quantity of skin and culturing time, and maximize the growth and total propagation time of melanocytes. Therefore, care should be exercised when selecting the region of skin when culturing epidermal melanocytes.

Nickel compounds are associated with lung and skin cancer incidence increase and accumulation of nickel in the body contributes to carcinogenesis. Upregulation of certain integrins in the primary tumor is associated with cancer metastasis and poor prognosis. However, the molecular mechanisms of nickel-induced cancer metastasis are still unclear. The purpose of the present study was to investigate the effects of nickel chloride (NiCl2 ) on the progression of cancer during metastasis. The results of showed that NiCl2 induces the expression of integrin β3 mRNA and protein in a dose- and time-dependent manner. Inhibition of integrin αvβ3 activation by ITGB3 ligand mimetics and GR144053, as well as downregulation of ITGB3 by lentiviral shRNA gene silencing, diminished NiCl2 -induced secretion of vascular endothelial growth factor-a (VEGF-a). Furthermore, pretreatment with type I TGF-β receptor inhibitor, SB525334, suppressed the expression of ITGB3 at cell surface and secretion of VEGF-a in NiCl2 -treated cells. In conclusion, NiCl2 induces the expression of ITGB3 through TGF-β signaling activation, followed by increasing VEGF-a secretion, revealing a novel role for ITGB3 in nickel compound-induced cancer metastasis and tumor angiogenesis.

Background Cisplatin is a widely used antineoplastic drug. However, cisplatin-induced dyspepsia syndromes, including delayed gastric emptying, gastric distension, early satiety, nausea, and vomiting, often force patients to take doses lower than those prescribed or even refuse treatment. D-methionine has an appetite-enhancing effect and alleviates weight loss during cisplatin treatment. Methods This work established a model of anorexia and dyspepsia symptoms with intraperitoneal injection of cisplatin (5 mg/kg) once a week for three cycles. Presupplementation with or without D-methionine (300 mg/kg) was performed. Orexigenic and anorexigenic hormones (ghrelin, leptin, and glucagon-like peptide-1), tryptophan hydroxylase 1 (TPH1), 5-hydroxytryptamine receptors (5-HT2C and 5-HT3 ), and hypothalamic feeding-related peptides were measured by immunohistochemistry staining, enzyme-linked immunosorbent assay, and real-time PCR assay. Key results Cisplatin administration caused marked decrease in appetite and body weight, promoted adipose and fat tissue atrophy, and delayed gastric emptying and gastric distension, and D-methionine preadministration prior to cisplatin administration significantly ameliorated these side effects. Besides, cisplatin induced an evident increase in serum ghrelin level, TPH1 activity, and 5-HT3 receptor expression in the intestine and decreased plasma leptin levels and gastric ghrelin mRNA gene expression levels. D-methionine supplementation recovered these changes. The expression of orexigenic neuropeptide Y/agouti-related peptide and anorexigenic cocaine- and amphetamine-regulated transcript proopiomelanocortin neurons were altered by D-methionine supplementation in cisplatin-induced anorexia rats. Conclusions and inferences D-methionine supplementation prevents cisplatin-induced anorexia and dyspepsia syndrome possibly by attenuating intestinal tryptophan hydroxylase 1 activity and increasing plasma leptin concentration. Therefore, D-methionine can be used as an adjuvant therapy for treating cisplatin-induced adverse effects.

β-catenin is important in development of lung cancer. In our previous study, GMI, a fungal immunomodulatory protein, inhibits lung cancer cell survival. The aim of this study is to evaluate the effect of GMI on β-catenin inhibition and apoptosis induction. GMI induced apoptosis in lung cancer cells bearing wild-type and mutated EGFR. GMI did not reduce the β-catenin mRNA expression but suppressed the protein expressions of β-catenin that resulted in the transcriptional downregulation of its target genes: survivin and cyclin-D1. The transcriptional activation activity of β-catenin was demonstrated by TOPFLASH/FOPFLASH luciferase reporter assay. Inhibition of GSK-3β and proteasome blocked the inhibiting effect of GMI on β-catenin and its target genes. β-catenin silencing increased activation of apoptosis in GMI-treated H1355 cells. This is the first study to reveal the novel function of GMI in inducing apoptosis via β-catenin inhibition. These results provide a new potential of GMI in against lung cancer.

Objective DDX3 has diverse biological functions in translation control, cell growth regulation, and tumor progression. Oral squamous cell carcinoma (OSCC) is a common malignant tumor worldwide with a poor clinical prognosis. The impact of DDX3 expression in OSCC is seldom discussed. Materials and Methods Tumor tissues and adjacent normal tissues were obtained from 324 patients with OSCC. In this study, we used immunohistochemical staining methods to investigate the associations between DDX3 expression and the clinicopathological characteristics of OSCC. Results Low/negative DDX3 expression in tumor cells was significantly associated OSCC patient characteristics including male gender (P

BACKGROUND AND PURPOSE Autophagic cell death is considered a self-destructive process that results from large amounts of autophagic flux. In our previous study, GMI, a recombinant fungal immunomodulatory protein cloned from Ganoderma microsporum, induced autophagic cell death in lung cancer cells. The aim of this study was to examine the role of autophagosome accumulation in GMI-mediated cell death. EXPERIMENTAL APPROACH Western blot analysis, flow cytometry and confocal microscopy were used to evaluate the effects of different treatments, including silencing of ATP6V0A1 by use of short hairpin RNAi, on GMI-mediated cell death, lung cancer cell viability and autophagosome accumulation in vitro. KEY RESULTS Lysosome inhibitors bafilomycin-A1 and chloroquine increased GMI-mediated autophagic cell death. GMI and bafilomycin-A1 co-treatment induced the accumulation of large amounts of autophagosomes, but did not significantly induce apoptosis. GMI elicited autophagy through the PKB (Akt)/mammalian target of rapamycin signalling pathway. Silencing of ATP6V0A1, one subunit of vesicular H+-ATPases (V-ATPases) that mediates lysosome acidification, spontaneously induced autophagosome accumulation, but did not affect lysosome acidity. GMI-mediated autophagosome accumulation and cytotoxicity was increased in shATP6V0A1 lung cancer cells. Furthermore, ATP6V0A1 silencing decreased autophagosome and lysosome fusion in GMI-treated CaLu-1/GFP-LC3 lung cancer cells. CONCLUSION AND IMPLICATIONS We demonstrated that autophagosome accumulation induces autophagic cell death in a GMI treatment model, and ATP6V0A1 plays an important role in mediating autophagosome-lysosome fusion. Our findings provide new insights into the mechanisms involved in the induction of autophagic cell death.

Vorinostat (suberoylanilide hydroxamic acid), a class of histone deacetylase inhibitors, represents an emerging class of anticancer agents currently progressing in clinical trials. It causes cell growth inhibition, differentiation, and apoptosis of many tumor types in vitro and in vivo. Recently, it was reported that hTERT is one of the targets for cancer therapy in cancer cells. Telomerase repeat amplification protocol assay was used to analyze the expression of hTERT after vorinostat treatment in the A549 lung cancer cells. Vorinostat inhibited telomerase activity by reducing the expression of human telomerase reverse transcriptase (hTERT) in A549 human lung cancer cells. The epigenetic regulation mechanism is responsible for the repression of hTERT by vorinostat, analyzed through the methylation-specific PCR and bisulfite sequencing of the hTERT promoter. Vorinostat induced the demethylation of site-specific CpGs on the promoter region of hTERT, which was caused by the down-regulation of DNA methyltransferases. DNA methyltransferases (DNMT1 and DNMT3b) were also decreased in vorinostat-treated A549 cancer cells. Furthermore, chromatin immunoprecipitation analysis of the hTERT promoter revealed that vorinostat decreased the level of inactive chromatin markers dimethyl-H3K9, and the declined binding of DNMT1 and DNMT3b were associated. The novel insights showed that vorinostat down-regulated telomerase via epigenetic alteration in lung cancer to vorinostat-mediated cancer-specific therapies.

BACKGROUND: Glutathione S-transferases M2 (GST-M2) is a detoxifying enzyme. Low expression levels of GST-M2 have been detected in lung cancer cells. However, little is known about the regulation of GST-M2 in lung cancer cells. In this study, the authors investigated the epigenetic regulatory mechanisms of GST-M2 in lung cancer cells. METHODS: The authors evaluated the promoter methylation of GST-M2 in lung cancer cells after treatment with the DNA methyltransferase (DNMT) inhibitor 5′-aza-2′-deoxycytidine (5′-aza-dC). Reporter activity assays, chromatin immunoprecipitation (ChIP), electrophoretic mobility-shift assays, and small interfering RNA (siRNA) assays were used to determine whether the methylation of specificity protein 1 (Sp1) affected binding to the GST-M2 promoter or regulated GST-M2 transcription. Real-time polymerase chain reaction was used to determine GST-M2 and DNMT-3b messenger RNA levels in 73 nonsmall cell lung cancer (NSCLC) tissues. RESULTS: GST-M2 expression was restored after treatment with 5′-aza-dC in lung cancer cells. GST-M2 exhibited high frequency of promoter hypermethylation in lung cancer cells and NSCLC tumor tissues. CpG hypermethylation abated Sp1 binding to the GST-M2 promoter in lung cancer. Knockdown of Sp1 in normal lung cells reduced GST-M2 expression, and silencing of DNMT-3b increased GST-M2 expression in lung cancer cells. In addition, DNMT-3b expression was significantly higher in lung tumors with low levels of GST-M2 expression than in lung tumors with high levels of GST-M2 expression, especially among women and among patients who had stage I disease. CONCLUSIONS: Epigenetic silencing of GST-M2 was distinguished from Sp1-mediated GST-M2 transcriptional expression. The authors concluded that this represents a mechanism that leads to decreased expression of GST-M2 in lung cancer cells. Cancer 2011. © 2011 American Cancer Society.

Aloe-emodin (AE), extracted from the rhizome of Rheum palmatum, has an anti-proliferative effect on different human cancer cell lines. Nonetheless, the underlying mechanism by which AE inhibits nasopharyngeal carcinoma (NPC) cell invasion is still unclear. The results of this study show that treatment of NPC cells with growth suppressive concentrations of AE caused cell cycle arrest at the S-G(2)/M phase. Coimmunoprecipitation and small interfering RNA (siRNA) studies demonstrated that AE-induced cell cycle arrest in NPC cells was associated with increasing levels of cyclin B1 bound to cyclin-dependent kinase 1. The inhibition of NPC cell invasion by AE was evidenced through the suppression of matrix metalloproteinases-2 (MMP-2) expression. MMP-2 promoter activity and cell invasion were inhibited by p38 mitogen-activated protein kinase (MAPK) siRNA, inhibitor 4-(4-Fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-5-(4-pyridyl)-1H-imidazole (SB203580), and AE, but not by JNK siRNA and inhibitor 1,9-pyrazoloanthrone. Treatment with AE, SB203580, NF-kappaB inhibitors N-p-tosyl-(L)-phenylalanine chloromethyl ketone (TPCK) and pyrrolidine dithiocarbamate (PDTC) or transfection with p38 MAPK siRNA significantly inhibited NF-kappaB transcriptional activity. In addition, TPCK and PDTC treatment inhibited the expression and promoter activity of MMP-2 and thereby significantly inhibited cell invasion activity. The involvement of p38 MAPK activity in NF-kappaB-mediated MMP-2 function was further confirmed through the attenuation of p38 MAPK by SB203580 and NF-kappaB ectopic expression. Collectively, our results indicate that AE inhibits invasion of NPC cells by suppressing the expression of MMP-2 via the p38 MAPK-NF-kappaB signaling pathway.

Background and Objectives The role of human nonmetastatic clone 23 type 1 (nm23-H1), a metastasis-associated gene, is less clear-cut in cancer of uterine cervix; therefore, we investigate its expression in cancer tissues and its correlation with clinicopathologic variables and survival of patients. Methods Thirty cervical cancer tissues and their normal counterparts were collected to evaluate quantitative nm23-H1 mRNA expression. From them, 16 cancer and 16 normal tissues were collected and added with another 64 cancer tissues to construct a 96-tissue core microarray for immunohistochemical study. We evaluated the relationships among nm23-H1 immunostaining using semi-quantitative H scores, clinicopathologic parameters, recurrence and survival in cervical cancer patients. Results Nm23-H1 mRNA expression and H score (median H scores: 2.0 vs. 0.3, P = 0.001) were higher in cervical cancer tissues than normal counterparts, respectively. Nm23-H1 expression was significantly associated with depth of stromal invasion (P = 0.003), tumor diameter (P = 0.044) and cell differentiation (P = 0.025). Other than stage II, poor cell differentiation as well as positive parametrium invasion and lymph node metastasis, positive nm23-H1 expression was significantly correlated with poor overall survival. Conclusion High nm23-H1 expression is predictive of worse overall survival for cervical cancer patients. J. Surg. Oncol. © 2008 Wiley-Liss, Inc.

Telomerase expression is the hallmark of tumor cells, and activation of this ribonucleoprotein complex may be a rate-limiting or critical step in cellular immortalization and oncogenesis. Fungal immunomodulatory protein, FIP-gts, has been isolated from Ganoderma tsugae. In the present study, we expressed and purified the recombinant fungal immunomodulatory protein reFIP-gts in E. coli. We found that reFIP-gts significantly and selectively inhibits the growth of A549 cancer cells while not affecting the growth of normal MRC-5 fibroblasts. The reFIP-gts suppression of telomerase activity is concentration-dependent, due to the downregulation of the telomerase catalytic subunit (hTERT). It also happens at the mRNA level. These results were confirmed by transient transfections of A549 cells with pGL3-Basic plasmid constructs containing the functional hTERT promoter and its E-box-deleted sequences cloned upstream of a luciferase reporter gene. With electrophoretic mobility shift assays and Western blotting, we demonstrated that in response to reFIP-gts, binding of c-myc transcriptional factor to the E-box sequence on the hTERT promoter is inhibited. These results show that reFIP-gts suppresses telomerase activity and inhibits transcriptional regulation of hTERT via a c-myc-responsive element-dependent mechanism. Our findings provide new insight into both the anticancer function of reFIP-gts and the regulation of hTERT/telomerase expression, which may be valuable in the development of a promising chemopreventive agent.

The MDM2 gene is overexpressed in several human tumors and its product may be processed into various isoforms. Recently, alternative splicing forms of MDM2 mRNA have been detected in various types of tumors. In this study, lung tissue from human non small cell lung cancers was examined for MDM2 mRNA splicing variants by nested RT-PCR. Of the 117 lung cancer tissue samples analyzed, a total of 31 (26.5%) had splice variants for the MDM2 gene, while 59 (50.4%) had undetectable levels of MDM2 transcript. Further analysis indicated that the predominant variant for 26 of the 31 samples with alternative MDM2 splicing products was MDM2-657, a splice variant lacking exons 3-11. Significant associations were found between the frequency of alternative splicing and the gender and smoking habits of the patients. Approximately 36% of male patients had alternative splicing of MDM2 compared with only 9.5% of female patients (P = 0.008); 44.2% of the smoker patients had alternative MDM2 splice forms versus 16.2% of nonsmokers (P = 0.003). Furthermore, most normal lung cell lines examined possessed only full-length MDM2 mRNA, while among several lung cancer cell lines, only H1355 and CaLu-1 cells lacked alternatively spliced MDM2 transcripts. When H1355 cells were treated in vitro with the cigarette smoke carcinogen benzo[a]pyrene (B[a]P) or the B[a]P metabolite benzo[a]pyrene diolepoxide (BPDE), three MDM2 splicing products were detected by nested RT-PCR. Finally, with the use of several specific inhibitors, we found that BPDE-induced MDM2 mRNA alternative splicing in H1355 cells may occur through the PI3K or MAPK pathway. Overall, our results suggest that carcinogens present in cigarette smoke increase the risk of alternative MDM2 splicing, which is highly associated with lung cancer.

MDM2 is one of the downstream target genes for transcriptional activation by the product of the p53 tumor-suppressor gene. Transactivation of MDM2 gene expression is represented by the presence of a functional p53 protein. We hypothesized that MDM2 mRNA expression may be a more suitable prognostic factor than p53 or MDM2 protein expression and p53 gene mutations. In this study, expression of MDM2 mRNA, p53 protein, and MDM2 protein and mutations of the p53 gene were assessed in 81 lung tumor tissue specimens using RT-PCR, immunohistochemistry, and direct sequencing among exons 5-8, respectively. By immunohistochemistry, 33 and 42 of 81 patients with p53 (40.7%) and MDM2 (51.5%) protein expression were found in lung tumor specimens, respectively. The p53 direct sequencing data indicated that 13 of 81 patients (16.0%) had p53 mutations. However, Kaplan-Meier analysis showed that p53 protein and MDM2 protein expression and p53 mutation were not useful as prognostic factors. Interestingly, the survival of patients with MDM2 mRNA expression was longer than that of patients without MDM2 mRNA expression, though MDM2 mRNA expression was not associated with clinicopathological parameters, including tumor grade, tumor stage, tumor type, and TNM values. Moreover, Cox regression analysis showed that MDM2 mRNA expression was a significantly independent favorable prognostic factor in non-small-cell lung cancer (NSCLC) patients. Thus, measuring MDM2 mRNA expression using RT-PCR may be a simple, useful approach for predicting the survival of NSCLC patients.

A new fungal immunomodulatory protein (FIP-fve) has been isolated and purified from the edible golden needle mushroom (Flammulina velutipes). The apparent molecular mass of FIP-fve determined by SDS/PAGE agrees well with the value of 12704 Da calculated from its amino acid composition and sequence. The complete amino acid sequence of FIP-fve was elucidated by protein sequencing techniques. FIP-fve consists of 114 amino acid residues with an acetylated amino end, and lacks methionine, half-cystine and histidine residues. FIP-fve was able to hemagglutinate human red blood cells. The immunomodulatory activity of FIP-fve was demonstrated by its stimulatory activity toward human peripheral blood lymphocytes, and its suppression of systemic anaphylaxis reactions and local swelling of mouse footpads. FIP-fve was found to enhance the transcriptional expression of interleukin-2 and interferon-gamma.