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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 promotes the spreading and cell polarity formation of hepatocellular carcinoma cells. (A), SMMC7721 cells that stably transfected with Grp78 or mock transfected were allowed to spread on Fibronectin (10 μg/ml) pre-coated surface for 2 hours. The extent of cell spreading was photographed under phase-contrast microscopy at 1 hour and 2 hours. This experiment was repeated for three times in triplicate. (Scale bar = 20 μm) (B), Overexpression of Grp78 in SMMC7721 cells promoted cell spreading which is represented as the mean area of cells (pixels). (C), Overexpression of Grp78 in SMMC7721 cells promotes cell polarity formation which is depicted as the ratio of long axis and short axis. (D) SMMC7721 cells that stably transfected with Grp78 form obvious thin, transparent lamellipodia at the wound edge compared with mock-transfected cells.
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Figure 3: Overexpression of Grp78 promotes the spreading and cell polarity formation of hepatocellular carcinoma cells. (A), SMMC7721 cells that stably transfected with Grp78 or mock transfected were allowed to spread on Fibronectin (10 μg/ml) pre-coated surface for 2 hours. The extent of cell spreading was photographed under phase-contrast microscopy at 1 hour and 2 hours. This experiment was repeated for three times in triplicate. (Scale bar = 20 μm) (B), Overexpression of Grp78 in SMMC7721 cells promoted cell spreading which is represented as the mean area of cells (pixels). (C), Overexpression of Grp78 in SMMC7721 cells promotes cell polarity formation which is depicted as the ratio of long axis and short axis. (D) SMMC7721 cells that stably transfected with Grp78 form obvious thin, transparent lamellipodia at the wound edge compared with mock-transfected cells.

Mentions: To determine if Grp78 affected the spreading of tumor cells, we observed the spreading status of Grp78 overexpressing cells for 2 h after the cells were replated on fibronectin-coated coverslips (10 μg/ml). We found that Grp78 overexpressing cells exhibited an accelerated spreading as compared with mock-transfected cells. Most Grp78 overexpressing cells exhibited a polar, elongated, morphology after 2 h of replating. However mock-transfected cells presented a round symmetric morphology without obvious polarity formation (fig 3A).


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 promotes the spreading and cell polarity formation of hepatocellular carcinoma cells. (A), SMMC7721 cells that stably transfected with Grp78 or mock transfected were allowed to spread on Fibronectin (10 μg/ml) pre-coated surface for 2 hours. The extent of cell spreading was photographed under phase-contrast microscopy at 1 hour and 2 hours. This experiment was repeated for three times in triplicate. (Scale bar = 20 μm) (B), Overexpression of Grp78 in SMMC7721 cells promoted cell spreading which is represented as the mean area of cells (pixels). (C), Overexpression of Grp78 in SMMC7721 cells promotes cell polarity formation which is depicted as the ratio of long axis and short axis. (D) SMMC7721 cells that stably transfected with Grp78 form obvious thin, transparent lamellipodia at the wound edge compared with mock-transfected cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2821360&req=5

Figure 3: Overexpression of Grp78 promotes the spreading and cell polarity formation of hepatocellular carcinoma cells. (A), SMMC7721 cells that stably transfected with Grp78 or mock transfected were allowed to spread on Fibronectin (10 μg/ml) pre-coated surface for 2 hours. The extent of cell spreading was photographed under phase-contrast microscopy at 1 hour and 2 hours. This experiment was repeated for three times in triplicate. (Scale bar = 20 μm) (B), Overexpression of Grp78 in SMMC7721 cells promoted cell spreading which is represented as the mean area of cells (pixels). (C), Overexpression of Grp78 in SMMC7721 cells promotes cell polarity formation which is depicted as the ratio of long axis and short axis. (D) SMMC7721 cells that stably transfected with Grp78 form obvious thin, transparent lamellipodia at the wound edge compared with mock-transfected cells.
Mentions: To determine if Grp78 affected the spreading of tumor cells, we observed the spreading status of Grp78 overexpressing cells for 2 h after the cells were replated on fibronectin-coated coverslips (10 μg/ml). We found that Grp78 overexpressing cells exhibited an accelerated spreading as compared with mock-transfected cells. Most Grp78 overexpressing cells exhibited a polar, elongated, morphology after 2 h of replating. However mock-transfected cells presented a round symmetric morphology without obvious polarity formation (fig 3A).

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