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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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FBP1 overexpression reduced liver cancer cell colony formation abilities.(A) FBP1 expression level in the transfected SW480 and SMMC-7721 cells was confirmed by Real-Time RT-PCR. (B) The effect of ectopic FBP1 expression on liver cancer cell growth was investigated by the monolayer colony formation assay. The scanned colony formation in six-well plates was shown in the panel.. (C) The growth of liver and colon cancer cell lines (SW480 and SMMC-7721) with and without FBP1 expression was determined with MTS cell growth assay. Stable expression of FBP1 suppressed the growth of liver and colon cancer cells. The results were shown as values of mean ± SD. P values were calculated using Student's t-test (* p<0.05).
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pone-0025564-g004: FBP1 overexpression reduced liver cancer cell colony formation abilities.(A) FBP1 expression level in the transfected SW480 and SMMC-7721 cells was confirmed by Real-Time RT-PCR. (B) The effect of ectopic FBP1 expression on liver cancer cell growth was investigated by the monolayer colony formation assay. The scanned colony formation in six-well plates was shown in the panel.. (C) The growth of liver and colon cancer cell lines (SW480 and SMMC-7721) with and without FBP1 expression was determined with MTS cell growth assay. Stable expression of FBP1 suppressed the growth of liver and colon cancer cells. The results were shown as values of mean ± SD. P values were calculated using Student's t-test (* p<0.05).

Mentions: The effect of exogenous FBP1 expression on the growth of human liver cancer cells was investigated by a monolayer colony formation assay. To further investigate the potential role of FBP1 in tumor suppression, liver cancer cells SMMC-7721 and colon cancer SW480 was transfected with pcDNA-FBP1 expressing vector and the cell colony formation ability was examined under the selection of G418. Compared with control cells transfected with empty vector pcDNA3.1, cancer cells transfected with pcDNA-FBP1 expressing vector showed decreased colony formation ability (Figure 4B). These data suggest that FBP1 may play a role in tumor suppression. Besides, FBP1 stable expression levels in those cells including SW480 and SMMC-7721 was also confirmed by Real-Time RT-PCR as shown in Figure 4A.


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)

FBP1 overexpression reduced liver cancer cell colony formation abilities.(A) FBP1 expression level in the transfected SW480 and SMMC-7721 cells was confirmed by Real-Time RT-PCR. (B) The effect of ectopic FBP1 expression on liver cancer cell growth was investigated by the monolayer colony formation assay. The scanned colony formation in six-well plates was shown in the panel.. (C) The growth of liver and colon cancer cell lines (SW480 and SMMC-7721) with and without FBP1 expression was determined with MTS cell growth assay. Stable expression of FBP1 suppressed the growth of liver and colon cancer cells. The results were shown as values of mean ± SD. P values were calculated using Student's t-test (* p<0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0025564-g004: FBP1 overexpression reduced liver cancer cell colony formation abilities.(A) FBP1 expression level in the transfected SW480 and SMMC-7721 cells was confirmed by Real-Time RT-PCR. (B) The effect of ectopic FBP1 expression on liver cancer cell growth was investigated by the monolayer colony formation assay. The scanned colony formation in six-well plates was shown in the panel.. (C) The growth of liver and colon cancer cell lines (SW480 and SMMC-7721) with and without FBP1 expression was determined with MTS cell growth assay. Stable expression of FBP1 suppressed the growth of liver and colon cancer cells. The results were shown as values of mean ± SD. P values were calculated using Student's t-test (* p<0.05).
Mentions: The effect of exogenous FBP1 expression on the growth of human liver cancer cells was investigated by a monolayer colony formation assay. To further investigate the potential role of FBP1 in tumor suppression, liver cancer cells SMMC-7721 and colon cancer SW480 was transfected with pcDNA-FBP1 expressing vector and the cell colony formation ability was examined under the selection of G418. Compared with control cells transfected with empty vector pcDNA3.1, cancer cells transfected with pcDNA-FBP1 expressing vector showed decreased colony formation ability (Figure 4B). These data suggest that FBP1 may play a role in tumor suppression. Besides, FBP1 stable expression levels in those cells including SW480 and SMMC-7721 was also confirmed by Real-Time RT-PCR as shown in Figure 4A.

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