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Promoter hypermethylation mediated downregulation of FBP1 in human hepatocellular carcinoma and colon cancer.

Chen M, Zhang J, Li N, Qian Z, Zhu M, Li Q, Zheng J, Wang X, Shi G - PLoS ONE (2011)

Bottom Line: Restoring FBP1 expression in low expressed cells significantly inhibited cell growth and colony formation ability through the induction of G2-M phase cell cycle arrest.Moreover, the observed effects coincided with an increase in reactive oxygen species (ROS) generation.In summary, epigenetic inactivation of FBP1 is also common in human liver and colon cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China.

ABSTRACT
FBP1, fructose-1,6-bisphosphatase-1, a gluconeogenesis regulatory enzyme, catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate. The mechanism that it functions to antagonize glycolysis and was epigenetically inactivated through NF-kappaB pathway in gastric cancer has been reported. However, its role in the liver carcinogenesis still remains unknown. Here, we investigated the expression and DNA methylation of FBP1 in primary HCC and colon tumor. FBP1 was lowly expressed in 80% (8/10) human hepatocellular carcinoma, 66.7% (6/9) liver cancer cell lines and 100% (6/6) colon cancer cell lines, but was higher in paired adjacent non-tumor tissues and immortalized normal cell lines, which was well correlated with its promoter methylation status. Methylation was further detected in primary HCCs, gastric and colon tumor tissues, but none or occasionally in paired adjacent non-tumor tissues. Detailed methylation analysis of 29 CpG sites at a 327-bp promoter region by bisulfite genomic sequencing confirmed its methylation. FBP1 silencing could be reversed by chemical demethylation treatment with 5-aza-2'-deoxycytidine (Aza), indicating direct epigenetic silencing. Restoring FBP1 expression in low expressed cells significantly inhibited cell growth and colony formation ability through the induction of G2-M phase cell cycle arrest. Moreover, the observed effects coincided with an increase in reactive oxygen species (ROS) generation. In summary, epigenetic inactivation of FBP1 is also common in human liver and colon cancer. FBP1 appears to be a functional tumor suppressor involved in the liver and colon carcinogenesis.

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Methylation of FBP1 promoter in human liver and colon cancer.(A) Schematic structure of the FBP1 CGI, with the exon 1 and MSP and BGS region indicated. Each short vertical line represents one CpG site. The position of MSP primers were marked as arrows. The methylation status of the FBP1 CGI was analyzed by MSP (B) and BGS (C). MSP = Methylation-specific PCR; USP = Unmethylation-specific PCR. For BGS in (C), each circle indicates one CpG site and circles filled in black represent methylated CpG sites. One row of circles represents a single colony.
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pone-0025564-g002: Methylation of FBP1 promoter in human liver and colon cancer.(A) Schematic structure of the FBP1 CGI, with the exon 1 and MSP and BGS region indicated. Each short vertical line represents one CpG site. The position of MSP primers were marked as arrows. The methylation status of the FBP1 CGI was analyzed by MSP (B) and BGS (C). MSP = Methylation-specific PCR; USP = Unmethylation-specific PCR. For BGS in (C), each circle indicates one CpG site and circles filled in black represent methylated CpG sites. One row of circles represents a single colony.

Mentions: Indeed one typical CpG Islands (CGI) were found around FBP1 exon 1 using the following criteria: GC content >55%, Obs CpG/Exp CpG >0.65, and length >500 bp (Figure 2A). The methylation status of this CGI in human liver and colon cancer cells was determined by MSP. As shown in Figure 2B, methylation of FBP1 promoter was readily detected in liver cancer cell lines HepG2, HuH7, HCC-LM3, BEL-7402, SMMC-7721, Sk-Hep1, MHCC-97H and MHCC-97L, and human colon cancer cell lines HT29, SW480, SW620, HCT116, LoVo and RKO. Besides, BGS also revealed that FBP1 promoter was heavily methylated in HepG2, Huh7, BEL-7402 cells and 8T tumor tissue, and no methylation was dedtected in 8N adjacent non-tumor tissue (Figure 2C).


Promoter hypermethylation mediated downregulation of FBP1 in human hepatocellular carcinoma and colon cancer.

Chen M, Zhang J, Li N, Qian Z, Zhu M, Li Q, Zheng J, Wang X, Shi G - PLoS ONE (2011)

Methylation of FBP1 promoter in human liver and colon cancer.(A) Schematic structure of the FBP1 CGI, with the exon 1 and MSP and BGS region indicated. Each short vertical line represents one CpG site. The position of MSP primers were marked as arrows. The methylation status of the FBP1 CGI was analyzed by MSP (B) and BGS (C). MSP = Methylation-specific PCR; USP = Unmethylation-specific PCR. For BGS in (C), each circle indicates one CpG site and circles filled in black represent methylated CpG sites. One row of circles represents a single colony.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198434&req=5

pone-0025564-g002: Methylation of FBP1 promoter in human liver and colon cancer.(A) Schematic structure of the FBP1 CGI, with the exon 1 and MSP and BGS region indicated. Each short vertical line represents one CpG site. The position of MSP primers were marked as arrows. The methylation status of the FBP1 CGI was analyzed by MSP (B) and BGS (C). MSP = Methylation-specific PCR; USP = Unmethylation-specific PCR. For BGS in (C), each circle indicates one CpG site and circles filled in black represent methylated CpG sites. One row of circles represents a single colony.
Mentions: Indeed one typical CpG Islands (CGI) were found around FBP1 exon 1 using the following criteria: GC content >55%, Obs CpG/Exp CpG >0.65, and length >500 bp (Figure 2A). The methylation status of this CGI in human liver and colon cancer cells was determined by MSP. As shown in Figure 2B, methylation of FBP1 promoter was readily detected in liver cancer cell lines HepG2, HuH7, HCC-LM3, BEL-7402, SMMC-7721, Sk-Hep1, MHCC-97H and MHCC-97L, and human colon cancer cell lines HT29, SW480, SW620, HCT116, LoVo and RKO. Besides, BGS also revealed that FBP1 promoter was heavily methylated in HepG2, Huh7, BEL-7402 cells and 8T tumor tissue, and no methylation was dedtected in 8N adjacent non-tumor tissue (Figure 2C).

Bottom Line: Restoring FBP1 expression in low expressed cells significantly inhibited cell growth and colony formation ability through the induction of G2-M phase cell cycle arrest.Moreover, the observed effects coincided with an increase in reactive oxygen species (ROS) generation.In summary, epigenetic inactivation of FBP1 is also common in human liver and colon cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China.

ABSTRACT
FBP1, fructose-1,6-bisphosphatase-1, a gluconeogenesis regulatory enzyme, catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate. The mechanism that it functions to antagonize glycolysis and was epigenetically inactivated through NF-kappaB pathway in gastric cancer has been reported. However, its role in the liver carcinogenesis still remains unknown. Here, we investigated the expression and DNA methylation of FBP1 in primary HCC and colon tumor. FBP1 was lowly expressed in 80% (8/10) human hepatocellular carcinoma, 66.7% (6/9) liver cancer cell lines and 100% (6/6) colon cancer cell lines, but was higher in paired adjacent non-tumor tissues and immortalized normal cell lines, which was well correlated with its promoter methylation status. Methylation was further detected in primary HCCs, gastric and colon tumor tissues, but none or occasionally in paired adjacent non-tumor tissues. Detailed methylation analysis of 29 CpG sites at a 327-bp promoter region by bisulfite genomic sequencing confirmed its methylation. FBP1 silencing could be reversed by chemical demethylation treatment with 5-aza-2'-deoxycytidine (Aza), indicating direct epigenetic silencing. Restoring FBP1 expression in low expressed cells significantly inhibited cell growth and colony formation ability through the induction of G2-M phase cell cycle arrest. Moreover, the observed effects coincided with an increase in reactive oxygen species (ROS) generation. In summary, epigenetic inactivation of FBP1 is also common in human liver and colon cancer. FBP1 appears to be a functional tumor suppressor involved in the liver and colon carcinogenesis.

Show MeSH
Related in: MedlinePlus