Human IL12p80 Promotes Murine Oligodendrocyte Differentiation to Repair Nerve Injury.

Nerve injury of the central nervous system and the peripheral nervous system still poses a major challenge in modern clinics. Understanding the roles of neurotrophic factors and their molecular mechanisms on neuro-regeneration will not only benefit patients with neural damage but could potentially treat neurodegenerative disorders, such as amyotrophic lateral sclerosis. In this study, we showed that human IL12 p40-p40 homodimer (hIL12p80) within PLA and PLGA conduits improved sciatic nerve regeneration in mice. As such, the group of conduits with NSCs and hIL12p80 (CNI) showed the best recovery among the groups in the sciatic functional index (SFI), compound muscle action potential (CMAP), and Rotarod performance analyses. In addition, the CNI group had a faster recovery and outperformed the other groups in SFI and Rotarod performance tests beginning in the fourth week post-surgery. Immunohistochemistry showed that the CNI group increased the diameter of the newly regenerated nerve by two-fold (p < 0.01). In vitro studies showed that hIL12p80 stimulated differentiation of mouse NSCs to oligodendrocyte lineages through phosphorylation of Stat3 at Y705 and S727. Furthermore, implantation using PLGA conduits (C2.0 and C2.1) showed better recovery in the Rotarod test and CMAP than using PLA conduits in FVB mice. In B6 mice, the group with C2.1 + NSCs + hIL12p80 (C2.1NI) not only promoted sciatic functional recovery but also reduced the rate of experimental autotomy. These results suggested that hIL12p80, combined with NSCs, enhanced the functional recovery and accelerated the regeneration of damaged nerves in the sciatic nerve injury mice. Our findings could further shed light on IL12's application not only in damaged nerves but also in rectifying the oligodendrocytes' defects in neurodegenerative diseases, such as amyotrophic lateral sclerosis and multiple sclerosis.

Inhibition of Epstein-Barr virus reactivation by the flavonoid apigenin.

BACKGROUND: Lytic reactivation of EBV has been reported to play an important role in human diseases, including NPC carcinogenesis. Inhibition of EBV reactivation is considered to be of great benefit in the treatment of virus-associated diseases. For this purpose, we screened for inhibitory compounds and found that apigenin, a flavonoid, seemed to have the ability to inhibit EBV reactivation. METHODS: We performed western blotting, immunofluorescence and luciferase analyses to determine whether apigenin has anti-EBV activity. RESULTS: Apigenin inhibited expression of the EBV lytic proteins, Zta, Rta, EAD and DNase in epithelial and B cells. It also reduced the number of EBV-reactivating cells detectable by immunofluorescence analysis. In addition, apigenin has been found to reduce dramatically the production of EBV virions. Luciferase reporter analysis was performed to determine the mechanism by which apigenin inhibits EBV reactivation: apigenin suppressed the activity of the immediate-early (IE) gene Zta and Rta promoters, suggesting it can block initiation of the EBV lytic cycle. CONCLUSION: Taken together, apigenin inhibits EBV reactivation by suppressing the promoter activities of two viral IE genes, suggesting apigenin is a potential dietary compound for prevention of EBV reactivation.

Luteolin inhibits Epstein-Barr virus lytic reactivation by repressing the promoter activities of immediate-early genes.

The lytic reactivation of Epstein-Barr virus (EBV) has been reported to be strongly associated with several human diseases, including nasopharyngeal carcinoma (NPC). Inhibition of the EBV lytic cycle has been shown to be of great benefit in the treatment of EBV-associated diseases. The administration of dietary compounds is safer and more convenient than other approaches to preventing EBV reactivation. We screened several dietary compounds for their ability to inhibit EBV reactivation in NPC cells. Among them, the flavonoid luteolin showed significant inhibition of EBV reactivation. Luteolin inhibited protein expression from EBV lytic genes in EBV-positive epithelial and B cell lines. It also reduced the numbers of EBV-reactivating cells detected by immunofluorescence analysis and reduced the production of virion. Furthermore, luteolin reduced the activities of the promoters of the immediate-early genes Zta (Zp) and Rta (Rp) and also inhibited Sp1-luc activity, suggesting that disruption of Sp1 binding is involved in the inhibitory mechanism. CHIP analysis revealed that luteolin suppressed the activities of Zp and Rp by deregulating Sp1 binding. Taken together, luteolin inhibits EBV reactivation by repressing the promoter activities of Zp and Rp, suggesting luteolin is a potential dietary compound for prevention of virus infection.

EBV reactivation as a target of luteolin to repress NPC tumorigenesis.

Nasopharyngeal carcinoma (NPC) is a malignancy derived from the epithelial cells of the nasopharynx. Although a combination of radiotherapy with chemotherapy is effective for therapy, relapse and metastasis after remission remain major causes of mortality. Epstein-Barr virus (EBV) is believed to be one of causes of NPC development. We demonstrated previously that EBV reactivation is important for the carcinogenesis of NPC. We sought, therefore, to determine whether EBV reactivation can be a target for retardation of relapse of NPC. After screening, we found luteolin is able to inhibit EBV reactivation. It inhibited EBV lytic protein expression and repressed the promoter activities of two major immediate-early genes, Zta and Rta. Furthermore, luteolin was shown to reduce genomic instability induced by recurrent EBV reactivation in NPC cells. EBV reactivation-induced NPC cell proliferation and migration, as well as matrigel invasiveness, were also repressed by luteolin treatment. Tumorigenicity in mice, induced by EBV reactivation, was decreased profoundly following luteolin administration. Together, these results suggest that inhibition of EBV reactivation is a novel approach to prevent the relapse of NPC.

EGCG inhibits proliferation, invasiveness and tumor growth by up-regulation of adhesion molecules, suppression of gelatinases activity, and induction of apoptosis in nasopharyngeal carcinoma cells.

(-)-Epigallocatechin-3-gallate (EGCG), a major green tea polyphenol, has been shown to inhibit the proliferation of a variety of tumor cells. Epidemiological studies have shown that drinking green tea can reduce the incidence of nasopharyngeal carcinoma (NPC), yet the underlying mechanism is not well understood. In this study, the inhibitory effect of EGCG was tested on a set of Epstein Barr virus-negative and -positive NPC cell lines. Treatment with EGCG inhibited the proliferation of NPC cells but did not affect the growth of a non-malignant nasopharyngeal cell line, NP460hTert. Moreover, EGCG treated cells had reduced migration and invasive properties. The expression of the cell adhesion molecules E-cadherin and ß-catenin was found to be up-regulated by EGCG treatment, while the down-regulation of matrix metalloproteinases (MMP)-2 and MMP-9 were found to be mediated by suppression of extracellular signal-regulated kinase (ERK) phosphorylation and AP-1 and Sp1 transactivation. Spheroid formation by NPC cells in suspension was significantly inhibited by EGCG. Oral administration of EGCG was capable of suppressing tumor growth in xenografted mice bearing NPC tumors. Treatment with EGCG was found to elevate the expression of p53 and p21, and eventually led to apoptosis of NPC cells via caspase 3 activation. The nuclear translocation of NF-κB and ß-catenin was also suppressed by EGCG treatment. These results indicate that EGCG can inhibit the proliferation and invasiveness, and induce apoptosis, of NPC cells, making it a promising agent for chemoprevention or adjuvant therapy of NPC.

Epstein-Barr virus BALF3 mediates genomic instability and progressive malignancy in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a head and neck cancer prevalent throughout Southern China and Southeast Asia. Patient death following relapse after primary treatment remains all too common but the cause of NPC relapse is unclear. Clinical and epidemiological studies have revealed the high correlation among NPC development, Epstein-Barr virus (EBV) reactivation and host genomic instability. Previously, recurrent EBV reactivation was shown to cause massive genetic alterations and enhancement of tumor progression in NPC cells and these may be required for NPC relapse. Here, EBV BALF3 has the ability to induce micronuclei and DNA strand breaks. After recurrent expression of BALF3 in NPC cells, genomic copy number aberrations, determined by array-based comparative genomic hybridization, had accumulated to a significant extent and tumorigenic features, such as cell migration, cell invasion and spheroid formation, increased with the rounds of induction. In parallel experiments, cells after highly recurrent induction developed into larger tumor nodules than control cells when inoculated into NOD/SCID mice. Furthermore, RNA microarrays showed that differential expression of multiple cancer capability-related genes and oncogenes increased with recurrent BALF3 expression and these changes correlated with genetic aberrations. Therefore, EBV BALF3 is a potential factor that mediates the impact of EBV on NPC relapse.

The synergistic effect of chemical carcinogens enhances Epstein-Barr virus reactivation and tumor progression of nasopharyngeal carcinoma cells.

Seroepidemiological studies imply a correlation between Epstein-Barr virus (EBV) reactivation and the development of nasopharyngeal carcinoma (NPC). N-nitroso compounds, phorbols, and butyrates are chemicals found in food and herb samples collected from NPC high-risk areas. These chemicals have been reported to be risk factors contributing to the development of NPC, however, the underlying mechanism is not fully understood. We have demonstrated previously that low dose N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.1 µg/ml) had a synergistic effect with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate (SB) in enhancing EBV reactivation and genome instability in NPC cells harboring EBV. Considering that residents in NPC high-risk areas may contact regularly with these chemical carcinogens, it is vital to elucidate the relation between chemicals and EBV and their contributions to the carcinogenesis of NPC. In this study, we constructed a cell culture model to show that genome instability, alterations of cancer hallmark gene expression, and tumorigenicity were increased after recurrent EBV reactivation in NPC cells following combined treatment of TPA/SB and MNNG. NPC cells latently infected with EBV, NA, and the corresponding EBV-negative cell, NPC-TW01, were periodically treated with MNNG, TPA/SB, or TPA/SB combined with MNNG. With chemically-induced recurrent reactivation of EBV, the degree of genome instability was significantly enhanced in NA cells treated with a combination of TPA/SB and MNNG than those treated individually. The Matrigel invasiveness, as well as the tumorigenicity in mouse, was also enhanced in NA cells after recurrent EBV reactivation. Expression profile analysis by microarray indicates that many carcinogenesis-related genes were altered after recurrent EBV reactivation, and several aberrations observed in cell lines correspond to alterations in NPC lesions. These results indicate that cooperation between chemical carcinogens can enhance the reactivation of EBV and, over recurrent reactivations, lead to alteration of cancer hallmark gene expression with resultant enhancement of tumorigenesis in NPC.

Reversed mutation rates of KRAS and EGFR genes in adenocarcinoma of the lung in Taiwan and their implications.

BACKGROUND: In western countries, the Kirsten ras oncogene homolog gene (KRAS) mutation rate is high in patients with nonsmall cell lung cancer (NSCLC), especially in those with adenocarcinoma (30%-50%), but the epidermal growth factor receptor gene (EGFR) mutation rate is very low (3%-8%). In addition, KRAS mutations reportedly were associated with EGFR tyrosine kinase inhibitor (EGFR-TKI) resistance. In Taiwan, high EGFR mutation rates associated with high EGFR-TKI response rates in patients with NSCLC have been reported; however, KRAS mutation data are limited and have not been correlated with TKI response. METHODS: KRAS mutation analysis was performed on 237 NSCLC specimens, and the results were correlated with clinicopathologic features. All but 2 tumors also underwent EGFR mutation analysis. RESULTS: KRAS mutations were identified in only 9 of 237 patients (3.80%). Five patients were women who were nonsmokers, and 4 patients were men who were ever-smokers. The mutation rate was 5.03% in patients with adenocarcinoma (8 of 159 patients) and 1.56% in patients with squamous cell carcinoma (1 of 64 patients). Four mutations were G12V, 3 mutations were G12D, 1 mutation was L19F, and 1 was the duplication insertion mutation dupT50_M72. In contrast, EGFR mutations were detected in 96 of 235 patients (40.8%) and in 90 of 157 adenocarcinomas (57.3%). None of the KRAS mutations coexisted with EGFR mutations. KRAS mutations were not associated significantly with any clinicopathologic characteristics, including smoking status. Among the 53 patients who had received TKI monotreatment, only 1 patient had a KRAS mutation and had progressive disease. CONCLUSIONS: The KRAS mutation rate was too low to play a significant role in TKI resistance or tumorigenesis among Taiwanese patients with NSCLC, which was the complete reverse of the results reported in western countries.

Increased epidermal growth factor receptor (EGFR) gene copy number is strongly associated with EGFR mutations and adenocarcinoma in non-small cell lung cancers: a chromogenic in situ hybridization study of 182 patients.

SUMMARY: To evaluate the association of epidermal growth factor receptor (EGFR) gene copy number with EGFR and k-ras mutation status and tyrosine kinase inhibitor (TKI) sensitivity in non-small cell lung cancer (NSCLC), EGFR gene copy number of 182 NSCLC tumor specimens were analyzed by chromogenic in situ hybridization (CISH). EGFR and k-ras mutation analyses were also performed for, respectively, 176 and 157 of the 182 patients. Additionally, 36 patients in this study had received TKI monotherapy. The tumor was considered to be CISH positive if the gene copy number was >or=5 signals per nucleus in >or=40% of tumor cells. CISH-positive tumors were strongly associated with adenocarcinoma (56.8%) compared with squamous cell carcinoma (15.9%) (p<0.0001). The CISH-positive tumors were also strongly associated with EGFR mutations (78%) compared with wild type (20.2%) (p<0.0001). Only six tumors had k-ras mutations. None had EGFR mutation and only one was CISH positive. In the patients treated with TKI, EGFR mutation was strongly associated with TKI responsiveness (22/25 responders) (p<0.0001), but the CISH-positive tumors were only marginally significant (18/25 responders) (p=0.0665). Patients with EGFR mutations or CISH-positive tumors were all associated with longer median survival, although not statistically significant. Our results suggest Increased EGFR copy number was highly correlated with EGFR mutation in adenocarcinoma. Although it is less correlated with TKI responsiveness when compared with EGFR mutations, it still could be a good alternative molecular predictive marker for TKI responsiveness, since CISH can be done on paraffin section and is much quicker than DNA sequencing.