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Grp78 promotes the invasion of hepatocellular carcinoma.

Su R, Li Z, Li H, Song H, Bao C, Wei J, Cheng L - BMC Cancer (2010)

Bottom Line: Our results also revealed that overexpression of Grp78 in SMMC7721 cells accelerated the process of cell spreading and promoted lamellipodia formation.Grp78 siRNA knockdown decreased FAK activation and activity.These data suggested that Grp78 inhibited RhoA activity by up-regulated phospho-p190RhoGAP level and Grp78 mediated p190RhoGAP phosphorylation is FAK dependent.

View Article: PubMed Central - HTML - PubMed

Affiliation: Central Laboratory, Liaoning Medical College, Jinzhou, PR China. rongjiansu@hotmail.com

ABSTRACT

Background: Glucose regulated protein 78 (Grp78) is involved in the invasion and metastasis in many human cancers including gastric cancer, breast cancer, prostate cancer. But the role of Grp78 in the invasion of human hepatocellular carcinoma has not been reported. In this article, we examined if Grp78 was associated with the invasion of hepatocellular carcinoma and explored the possible underlying mechanism.

Methods: The Grp78 and FAK expression levels in 44 patients with hepatocellular carcinoma were examined using immunohistochemistry. Grp78 overexpressing SMMC7721 cells were established by pcDNA3.1 (+)-Grp78 transfection and screened by G418. Grp78 and FAK levels in Grp78 overexpressing cells were down-regulated by siRNA transfection. The invasion status of tumor cells was evaluated by transwell assay in vitro, and chick embryo metastasis model in vivo. Cell spreading was determined by cell spreading assay, and quantitatively measured by Orisis software HUG. Grp78, pY397 FAK, pY576/577 FAK and FAK levels were detected by western blot. RhoA activity was detected by GST pulldown assay. The distribution of actin cytoskeleton was observed by fluorescent staining.

Results: Grp78 expression levels in 44 patients with hepatocellular carcinoma were negatively correlated with tumor grading, and positively correlated with portal invasion and intra-hepatic invasion. Overexpression of Grp78 in SMMC7721 cells promoted the invasion of cancer cells in vitro and in vivo, and this increase in tumor cell invasion was blocked by Grp78 siRNA knockdown. Our results also revealed that overexpression of Grp78 in SMMC7721 cells accelerated the process of cell spreading and promoted lamellipodia formation. Further analysis showed that overexpression of Grp78 in SMMC7721 cells increased pY397 and pY576/577 levels of FAK. Grp78 siRNA knockdown decreased FAK activation and activity. Our results also revealed that Grp78 overexpression in SMMC7721 cells decreased RhoA-GTP level, and Grp78 siRNA knockdown rescued RhoA-GTP level in Grp78 overexpressing cells, indicating Grp78 inhibited RhoA activity in hepatocellular carcinoma cells. Furthermore, overexpression of Grp78 in SMMC7721 cells increased phospho-p190RhoGAP level. FAK siRNA knockdown in Grp78 overexpressing cells reversed phospho-p190RhoGAP level. These data suggested that Grp78 inhibited RhoA activity by up-regulated phospho-p190RhoGAP level and Grp78 mediated p190RhoGAP phosphorylation is FAK dependent.

Conclusion: Grp78 promoted the invasion of hepatocellular carcinoma both in vitro and in vivo. Overexpression of Grp78 in hepatocellular carcinoma cells enhanced the activation and activity of FAK which negatively regulated Rock kinase activity by promoting the phosphorylation of p190RhoGAP.

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Overexpression of Grp78 induces hepatocellular carcinoma cells invasion. (A), Western blot of protein lysates from mock- and Grp78-transfected SMMC7721 cells, indicating Grp78 expression level. The lower blot was used as loading control. (B), A transwell assay using mcok- and Grp78-transfected cells was performed on inserts pre-coated with ECM gel. Images are representative of the invasion of Grp78 overexpressing cells in three separate experiments performed in triplicate. (C), A chick embryo metastasis assay was performed using mock- and Grp78-transfected cells suspended in PBS. The extent of tumor cell invasion was evaluated by human specific Alu PCR using DNA extracts from chick embryonic lungs and livers. Negative control: DNA extracts from untreated 7-day chick embryonic lungs and livers. Positive control: DNA extracts from human colorectal caricinoma. (D), Western blot analysis showing Grp78 level in Grp78 siRNA and control siRNA transfected Grp78 overexpressing cells. (E) Representative image of a transwell assay using Grp78 siRNA knockdown Grp78 overexpressing cells and control siRNA transfected Grp78 overexpressing cells for further elucidating the role of Grp78 in tumor cells invasion. The experiments were repeated for three times in triplicate.
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Figure 2: Overexpression of Grp78 induces hepatocellular carcinoma cells invasion. (A), Western blot of protein lysates from mock- and Grp78-transfected SMMC7721 cells, indicating Grp78 expression level. The lower blot was used as loading control. (B), A transwell assay using mcok- and Grp78-transfected cells was performed on inserts pre-coated with ECM gel. Images are representative of the invasion of Grp78 overexpressing cells in three separate experiments performed in triplicate. (C), A chick embryo metastasis assay was performed using mock- and Grp78-transfected cells suspended in PBS. The extent of tumor cell invasion was evaluated by human specific Alu PCR using DNA extracts from chick embryonic lungs and livers. Negative control: DNA extracts from untreated 7-day chick embryonic lungs and livers. Positive control: DNA extracts from human colorectal caricinoma. (D), Western blot analysis showing Grp78 level in Grp78 siRNA and control siRNA transfected Grp78 overexpressing cells. (E) Representative image of a transwell assay using Grp78 siRNA knockdown Grp78 overexpressing cells and control siRNA transfected Grp78 overexpressing cells for further elucidating the role of Grp78 in tumor cells invasion. The experiments were repeated for three times in triplicate.

Mentions: To confirm our previous results, we transfected SMMC7721 cells with pcDNA 3.1(+)-Grp78 recombinant by Fugene HD followed by G418 selection. The transfection efficiency was assessed by western blot, indicating a significant increase (= 2.5 fold) as compared with mock-transfected cells (Fig.2A).


Grp78 promotes the invasion of hepatocellular carcinoma.

Su R, Li Z, Li H, Song H, Bao C, Wei J, Cheng L - BMC Cancer (2010)

Overexpression of Grp78 induces hepatocellular carcinoma cells invasion. (A), Western blot of protein lysates from mock- and Grp78-transfected SMMC7721 cells, indicating Grp78 expression level. The lower blot was used as loading control. (B), A transwell assay using mcok- and Grp78-transfected cells was performed on inserts pre-coated with ECM gel. Images are representative of the invasion of Grp78 overexpressing cells in three separate experiments performed in triplicate. (C), A chick embryo metastasis assay was performed using mock- and Grp78-transfected cells suspended in PBS. The extent of tumor cell invasion was evaluated by human specific Alu PCR using DNA extracts from chick embryonic lungs and livers. Negative control: DNA extracts from untreated 7-day chick embryonic lungs and livers. Positive control: DNA extracts from human colorectal caricinoma. (D), Western blot analysis showing Grp78 level in Grp78 siRNA and control siRNA transfected Grp78 overexpressing cells. (E) Representative image of a transwell assay using Grp78 siRNA knockdown Grp78 overexpressing cells and control siRNA transfected Grp78 overexpressing cells for further elucidating the role of Grp78 in tumor cells invasion. The experiments were repeated for three times in triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Overexpression of Grp78 induces hepatocellular carcinoma cells invasion. (A), Western blot of protein lysates from mock- and Grp78-transfected SMMC7721 cells, indicating Grp78 expression level. The lower blot was used as loading control. (B), A transwell assay using mcok- and Grp78-transfected cells was performed on inserts pre-coated with ECM gel. Images are representative of the invasion of Grp78 overexpressing cells in three separate experiments performed in triplicate. (C), A chick embryo metastasis assay was performed using mock- and Grp78-transfected cells suspended in PBS. The extent of tumor cell invasion was evaluated by human specific Alu PCR using DNA extracts from chick embryonic lungs and livers. Negative control: DNA extracts from untreated 7-day chick embryonic lungs and livers. Positive control: DNA extracts from human colorectal caricinoma. (D), Western blot analysis showing Grp78 level in Grp78 siRNA and control siRNA transfected Grp78 overexpressing cells. (E) Representative image of a transwell assay using Grp78 siRNA knockdown Grp78 overexpressing cells and control siRNA transfected Grp78 overexpressing cells for further elucidating the role of Grp78 in tumor cells invasion. The experiments were repeated for three times in triplicate.
Mentions: To confirm our previous results, we transfected SMMC7721 cells with pcDNA 3.1(+)-Grp78 recombinant by Fugene HD followed by G418 selection. The transfection efficiency was assessed by western blot, indicating a significant increase (= 2.5 fold) as compared with mock-transfected cells (Fig.2A).

Bottom Line: Our results also revealed that overexpression of Grp78 in SMMC7721 cells accelerated the process of cell spreading and promoted lamellipodia formation.Grp78 siRNA knockdown decreased FAK activation and activity.These data suggested that Grp78 inhibited RhoA activity by up-regulated phospho-p190RhoGAP level and Grp78 mediated p190RhoGAP phosphorylation is FAK dependent.

View Article: PubMed Central - HTML - PubMed

Affiliation: Central Laboratory, Liaoning Medical College, Jinzhou, PR China. rongjiansu@hotmail.com

ABSTRACT

Background: Glucose regulated protein 78 (Grp78) is involved in the invasion and metastasis in many human cancers including gastric cancer, breast cancer, prostate cancer. But the role of Grp78 in the invasion of human hepatocellular carcinoma has not been reported. In this article, we examined if Grp78 was associated with the invasion of hepatocellular carcinoma and explored the possible underlying mechanism.

Methods: The Grp78 and FAK expression levels in 44 patients with hepatocellular carcinoma were examined using immunohistochemistry. Grp78 overexpressing SMMC7721 cells were established by pcDNA3.1 (+)-Grp78 transfection and screened by G418. Grp78 and FAK levels in Grp78 overexpressing cells were down-regulated by siRNA transfection. The invasion status of tumor cells was evaluated by transwell assay in vitro, and chick embryo metastasis model in vivo. Cell spreading was determined by cell spreading assay, and quantitatively measured by Orisis software HUG. Grp78, pY397 FAK, pY576/577 FAK and FAK levels were detected by western blot. RhoA activity was detected by GST pulldown assay. The distribution of actin cytoskeleton was observed by fluorescent staining.

Results: Grp78 expression levels in 44 patients with hepatocellular carcinoma were negatively correlated with tumor grading, and positively correlated with portal invasion and intra-hepatic invasion. Overexpression of Grp78 in SMMC7721 cells promoted the invasion of cancer cells in vitro and in vivo, and this increase in tumor cell invasion was blocked by Grp78 siRNA knockdown. Our results also revealed that overexpression of Grp78 in SMMC7721 cells accelerated the process of cell spreading and promoted lamellipodia formation. Further analysis showed that overexpression of Grp78 in SMMC7721 cells increased pY397 and pY576/577 levels of FAK. Grp78 siRNA knockdown decreased FAK activation and activity. Our results also revealed that Grp78 overexpression in SMMC7721 cells decreased RhoA-GTP level, and Grp78 siRNA knockdown rescued RhoA-GTP level in Grp78 overexpressing cells, indicating Grp78 inhibited RhoA activity in hepatocellular carcinoma cells. Furthermore, overexpression of Grp78 in SMMC7721 cells increased phospho-p190RhoGAP level. FAK siRNA knockdown in Grp78 overexpressing cells reversed phospho-p190RhoGAP level. These data suggested that Grp78 inhibited RhoA activity by up-regulated phospho-p190RhoGAP level and Grp78 mediated p190RhoGAP phosphorylation is FAK dependent.

Conclusion: Grp78 promoted the invasion of hepatocellular carcinoma both in vitro and in vivo. Overexpression of Grp78 in hepatocellular carcinoma cells enhanced the activation and activity of FAK which negatively regulated Rock kinase activity by promoting the phosphorylation of p190RhoGAP.

Show MeSH
Related in: MedlinePlus