Korean J Physiol Pharmacol.  2012 Oct;16(5):361-365. 10.4196/kjpp.2012.16.5.361.

Gecko Proteins Exert Anti-Tumor Effect against Cervical Cancer Cells Via PI3-Kinase/Akt Pathway

• Jeong AJ
• Chung CN
• Kim HJ
• Bae KS
• Choi S
• Jun WJ
• Shim SI
• Kang TH
• Leem SH
• Chung JW

• KMID: 1493971
• DOI: http://doi.org/10.4196/kjpp.2012.16.5.361

Abstract

Anti-tumor activity of the proteins from Gecko (GP) on cervical cancer cells, and its signaling mechanisms were assessed by viable cell counting, propidium iodide (PI) staining, and Western blot analysis. GP induced the cell death of HeLa cells in a dose-dependent manner while it did not affect the viability of normal cells. Western blot analysis showed that GP decreased the activation of Akt, and co-administration of GP and Akt inhibitors synergistically exerted anti-tumor activities on HeLa cells, suggesting the involvement of PI3-kinase/Akt pathway in GP-induced cell death of the cancer cells. Indeed, the cytotoxic effect of GP against HeLa cells was inhibited by overexpression of constituvely active form of Akt in HeLa cells. The candidates of the functional proteins in GP were analyzed by Mass-spectrum. Taken together, our results suggest that GP elicits anti-tumor activity against HeLa cells by inhibition of PI3-kinase/Akt pathway.

Keyword

Cervical cancer; Gecko; Lizard; PI3-kinase; Tumor

MeSH Terms

Blotting, Western
Cell Count
Cell Death
HeLa Cells
Humans
Lizards
Phosphatidylinositol 3-Kinases
Propidium
Proteins
Uterine Cervical Neoplasms
Phosphatidylinositol 3-Kinases
Propidium
Proteins

Figure

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  Fig. 1 Effect of GP on survival of HeLa cells. HeLa cells were cultured with designated concentrations of GP for 48 hours. Viable cells were observed with phase contrast microscope (A), and dead or dying cells were analyzed by PI staining (B). Data are representatives of three independent experiments.

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  Fig. 2 Specific anti-tumor effect of GP against HeLa cells. (A) HeLa cell were treated with raw GP (left panel) or heat-inactivated GP (right panel) for 48 hours, and the viable cell numbers were counted. (B) Various types of normal cells were treated with raw GP for 48 hours and viable cell numbers were counted (data are averages of three independent experiments).

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  Fig. 3 Activation of MAP kinases. (A) HeLa cells were treated with designated concentration (0, 100, 200 µg/ml) of GP for 30 minutes, and the activation of Akt was analyzed by Western blot analysis. (B) HeLa cells were treated with wortmannin (Wort) and/or GP for 48 hours, and the viable cell numbers were counted. DMSO was included as a vehicle control.

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  Fig. 4 Confirmation of involvement of PI3-kinase/Akt in GP induced cell death of HeLa cells. (A) Control HeLa cells (pUSEamp) and the active Akt-overexpressing HeLa cells (CA-Akt) were treated with or without GP (200 µg/ml) for 48 hours, and the viable cell numbers were counted. Data are averages of three independent experiments. (B) After treatment of GP (200 µg/ml), the cells were stained with PI and the viability of the cells was investigated by microscopic analysis. Data are representative of three independent experiments.

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  Fig. 5 Protein separations of total GP on 2D gels, and categorization of the identified proteins by mass spectrometry. (A) After isoelectric focusing in pH gradient of the electric field, the electrophoretic separation is used in polyacrylamide gel (SDS-PAGE). Proteins were visualized after silver staining. (B) Identified proteins in Table 1 were categorized according to their cellular functions.

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