Neokestose suppresses the growth of human melanoma A2058 cells via inhibition of the nuclear factor­κB signaling pathway.

Neokestose has superior prebiotic effects compared with the commercial fructooligosaccharides (FOS). In addition, the branched structure of neokestose, a type of neo­FOS, confers improved chemical stability compared with conventional FOS; therefore, the investigation of the branched structure by the present study may be of high biomedical value. The present study aimed to determine whether neokestose may suppress growth of the A2058 melanoma cell line. The cells were initially treated with neokestose; subsequently, in vitro cytotoxicity was assessed using MTT, and cell cycle progression and apoptosis were detected using flow cytometry. The protein expression levels of cyclin D1, phosphorylated (p)­inhibitor of κB (IκB) and nuclear factor­κB (NF­κB) were determined using western blotting. Treatment with neokestose led to a dose­dependent inhibition of cell viability. Flow cytometry data indicated that neokestose increased the sub­G1 cell population, and induced early and late apoptosis. Western blot analysis revealed that neokestose treatment reduced the expression levels of p­IκB and cyclin D1. These findings suggest that neokestose treatment may induce suppression of A2058 melanoma cell viability via inhibition of the NF­κB pathway. The present findings support the requirement for further investigation into the potential use of neokestose as an additional or chemopreventive therapeutic agent for the treatment of melanoma.

Antineoplastic effect of a novel chemopreventive agent, neokestose, on the Caco-2 cell line via inhibition of expression of nuclear factor-κB and cyclooxygenase-2.

Neokestose is a 6G-fructooligosaccharide (FOS) and an important prebiotic. When FOS are ingested by patients with colorectal cancer, they may come into contact with cancer cells prior to being fermented by bifidobacteria in the colon. In the present study, the effects of neokestose on cell proliferation, cell cycle and apoptosis of the colorectal cancer cell line Caco-2 were investigated to evaluate its anti-cancer effect. An MTT assay showed that neokestose-treated Caco-2 cells exhibited a significant and dose-dependent loss of viability. Flow cytometric analysis indicated that the sub-G1 population of Caco-2 cells was significantly increased following treatment with neokestose, and the percentage of Caco-2 cells in the stage of late apoptosis was also significantly increased in a dose-dependent manner. Western blot analysis showed that the overexpression of nuclear factor-κB, a central molecule responsible for the transition from inflammation to cancer, and cyclooxygenase-2, an important enzyme in colorectal tumorigenesis, in colorectal carcinoma cells was inhibited by neokestose. Accordingly, the present study provided in vitro evidence that neokestose may be used as a dietary chemopreventive agent, whose application is more rational than that of COX-2 inhibitors or aspirin for preventing colorectal cancer.

Continuous production of high-purity fructooligosaccharides and ethanol by immobilized Aspergillus japonicus and Pichia heimii.

High-purity fructooligosaccharides (FOS) were produced from sucrose by an innovative process incorporating immobilized Aspergillus japonicus and Pichia heimii cells. Intracellular FTase of A. japonicus converted sucrose into FOS and glucose, and P. heimii fermented glucose mainly into ethanol. The continuous production of FOS was carried out using a tanks-in-series bioreactor consisting of three stirred tanks. When a solution composed of 1 g L(-1) yeast extract and 300 g L(-1) sucrose was fed continuously to the bioreactor at a dilution rate of 0.1 h(-1), FOS at a purity of up to 98.2 % could be achieved and the value-added byproduct ethanol at 79.6 g L(-1) was also obtained. One gram of sucrose yielded 0.62 g FOS and 0.27 g ethanol. This immobilized dual-cell system was effective for continuous production of high-purity FOS and ethanol for as long as 10 days.

Production of high-purity neofructooligosaccharides by culture of Xanthophyllomyces dendrorhous.

Neofructooligosaccharides (neo-FOS) were produced in submerged cultures of Xanthophyllomyces dendrorhous. Among the various strains of X. dendrorhous that have intracellular (6)G-fructofuranosidase ((6)G-FFase), BCRC 21346 with high enzyme activity (3.60 U/mL) and BCRC 22367 with low enzyme activity (0.59 U/mL) were investigated in this work. Neo-FOS were generated in a 5-L jar fermenter at 20°C, 100rpm and 2vvm with the pH controlled at 6.9±0.1, using 250g/L of sucrose as the substrate. Through the catalytic action of X. dendrorhous(6)G-FFase on sucrose, monosaccharides as well as neo-FOS were produced. A portion of these monosaccharides was consumed by the yeast cells. However, the production of monosaccharides was low in concentration in culture with low (6)G-FFase activity, indicating they might be used up concurrently during the fermentation. Consequently, neo-FOS at a purity of up to 87.4% could be obtained.

Correlation between protein expression and epigenetic and mutation changes of Wnt pathway-related genes in oral cancer.

The components of the Wnt-signaling pathway are reported to be mutated in human cancer cells, but the relationship between the components and oral squamous carcinoma (SCC) is still unknown. In this study, we analyzed the epigenetic changes and expression patterns of four member proteins of the Wnt-signaling pathway and analyzed the mutations of beta-catenin and AXIN 1 genes, in order to explore the roles of the pathway in the development of oral cancer. The results showed that there are no beta-catenin and AXIN 1 gene mutations and no methylation of the CpG island of beta-catenin, AXIN I and GSK3beta genes in oral cancer cells; methylation of the CpG island of APC occurs in the precancerous stage and it is a dynamic change; the aberrant expressions or abnormal localization of the Wnt-signaling pathway proteins have no relationship with methylation status or mutation. From our results, we suggest that the Wnt pathway related genes play a very limited role in the development of oral SCC.

Mutational, epigenetic and expressional analyses of caveolin-1 gene in cervical cancers.

Caveolin-1 (CAV-1) protein, an integral membrane protein of caveolae membranes, is highly expressed in terminally differentiated cells and down-regulated in cells transformed by human papilloma virus infection. It may also be involved in the tumorigenesis of cervical cancer. CAV-1 gene is regarded as a candidate for the tumor suppressor gene and it can be inactivated in several ways, including point mutations, chromosomal deletions and promoter methylation. We used direct sequencing, methylation specific PCR, and immunohistochemical staining methods to explore the role of CAV-1 gene in the development of cervical cancer. Our results showed that 4 of 72 cases (6%) had methylated CpG-island on the CAV-1 promoter, 17 of 72 cases (26.1%) having no methylation on the promoter showed no expression of CAV-1 protein, and 2 of 72 cases had a GAC right curved arrow GAT transition polymorphism at codon 82. Three types of CAV-1 expression patterns were observed in cervical cancer tissues, and the expression pattern had no relationship with mutation status. From these results, we suggest that CAV-1 gene can be inactivated through mutations, and does not play a role, through methylation of promoter, or an unknown mechanism which may play a role, in the development of cervical cancer.

Epigenetic changes of tumor suppressor genes, P15, P16, VHL and P53 in oral cancer.

We performed methylation specific PCR to explore the mechanism of inactivation of tumor suppressor genes P15, P16, P53 and VHL in 48 oral SCC. The frequencies of aberrant methylation on the promoter of the P15, the P16, the P53 and the VHL genes were 0.27 (13/48), 0.42 (20/48), 0.04 (2/48) and none, respectively. Altogether, over 50% of the samples showed the CpG-island methylation modification in at least one of the three tumor suppressor genes, indicating that the frequent inactivation of these genes may be an important step during oral cancer development, and the methylation inactivation of P15 or P16 may occur at pre-cancerous stage.

The relationship among the polymorphisms of SULT1A1, 1A2 and different types of cancers in Taiwanese.

Sulfotransferase (SULT) enzymes play an important role in the detoxification, metabolism and bioactivation of numerous xenobiotics, many dietary and environmental mutagens, drugs, neurotransmitters and hormones. The genes for SULT1A1 and SULT1A2 contain common genetic polymorphisms that are associated with individual variations in the level of enzyme activities as well as variations of biochemical and physical properties. We developed a PCR-RFLP method to analyze the frequencies of SULT1A1 and SULT1A2 alleles among cancerous patients and normal controls in Taiwan. The results showed that SULT1A1*1 and SULT1A2*1 were in positive linkage disequilibrium. Neither SULT1A1*3 nor SULT1A2*3 were found in this study. The frequencies of SULT1A1*2 and SULT1A2*2 for hepatic, colon, lung, oral, gastric, renal and cervical cancerous patients were 3.95, 5.56, 4.92, 3.84, 2.70, 7.41 and 4.50%, respectively. No statistical significance was found for these cancer patients after comparison with normal controls (4.0%) for the allelic frequencies of SULT1A1*2 and SULT1A2*2.

The correlation between CpG methylation and protein expression of P16 in oral squamous cell carcinomas.

We examined the P16 expression by immunohistochemical stain and detected the methylation by methylation specific polymerase chain reaction (MSP) in 48 primary oral squamous cell carcinoma (SCC) tissues. The results showed that 20/48 (41.7%) of cancerous tissues had CpG methylation around the promoter region, but 8/48 (17%) of the nearby non-cancerous tissues also had CpG methylation, around the promoter region. The results from immunohistochemical studies showed that reduced and heterogeneous expression of P16 were found in the tissues, which had CpG methylation around the promoter region. In conclusion, the methylation of P16 in oral SCC occurs in pre-cancerous and cancerous stage, which results in decreasing or abolishing the P16 expression, which is heterogeneous in the cancer cells.

The correlation between CpG methylation on promoter and protein expression of E-cadherin in oral squamous cell carcinoma.

BACKGROUND: Reduction of E-cadherin in most common epithelial tumors relates to metastasis, which results from the silence of E-cadherin by CpG methylation. MATERIALS AND METHODS: We examined the E-cadherin expression by immunohistochemical staining and detected methylation by methylation-specific polymerase chain reaction (MSP) in 48 primary oral SCC tissues. RESULTS: The results showed that 41 out of 48 (85.4%) cancerous tissues and 16 out of 48 (33.3%) nearby non-cancerous tissues had CpG methylation on the promoter region of E-cadherin. In these non-cancerous tissues, 2 out of 16 (12.5%) had no methylation change in their paired cancerous part. Immunohistochemical study showed that a decreased expression pattern was found in the tissue which had CpG methylation on the promoter region, but an over expression island or aberrant expression was also frequently found in these cases. CONCLUSION: The methylation of E-cadherin in oral SCC may occur in the precancerous stage and the process is dynamic, which has no relationship with the aberrant expression of E-cadherin protein.