Korean J Gynecol Oncol.
2005 Dec;16(4):333-346.
Differential protein expression of etoposide-treated CaSki cervical carcinoma cells
- Affiliations
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- 1Department of Medical Bioscience, The Catholic University of Korea College of Medicine, Seoul, Korea.
- 2Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, The Catholic University of Korea College of Medicine, Seoul, Korea. jspark@catholic.ac.kr
- 3Department of Bioscience and Biotechnology, Sejong University, Seoul, Korea.
Abstract
OBJECTIVE
This study was designed to examine the pharmaco-dynamic pattern of proteomic expression in cervical carcinoma cells (CaSki cell line; HPV-16 positive) after in vitro treatment by the etoposide.
METHODS
We analyzed proteomic profiling in cervical carcinoma cells after etoposide treatment using two-dimensional gel electrophoresis (2-DE) with MALDI-TOF-MS used for protein identification. Then, we tested the several experimental methods for verification and functional identification, including MTT assay, PI staining, DNA fragmentation assay, FDA, FACS and Western blot analysis.
RESULTS
Etoposide inhibited the CaSki cervical cancer cell growth in a dose-dependent manner and the optimal concentration of etoposide is 2micrometer(IC50) in the CaSki cervical cancer cells. The etoposide induced apoptosis, as determined by DNA fragmentation assay, FACS, and Western blot. The etoposide increased the protein expression of Fas (Apo-1/CD95), p53, pRb and caspase-3, but decreased the level of Bcl-2 and caspase-3 precursor and subsequently triggered the mitochondrial apoptotic pathway (release of cytochrome c and activation of caspase-9). To this end, we analyzed CaSki cancer cells using 2-DE. Eight proteins (XAP-5, HXC-36, serine/threonine protein phosphatase 2B catalytic subunit, G2/mitotic-specific cyclin B1, T-box transcription factor TBX20, diacylglycerol kinase, amiloride-sensitive amine oxidase, HEF-like protein, ras-related protein Rab-20) were down-regulated and nine proteins (RNA 3'-terminal phosphate cyclase-like protein, late endosomal/lysosomal Mp1 interacting protein, glia maturation factor, replication protein A 14 kDa subunit, mago sashi protein homolog, 14 kDa phosphohistidine phosphatase, protein C14 or f48, cyclin-dependent kinase 4 inhibitior A, retinoic acid-binding protein II) were up-regulated in etoposide-treated CaSki cells when compared with non-treated cells.
CONCLUSION
Our results clearly indicate that etoposide induced cell death by apoptosis. These findings may provide insights into the mechanisms underlying the apparent anti-tumoral effects of etoposide.
Keyword
MeSH Terms
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Apoptosis
Blotting, Western
Calcineurin
Caspase 3
Catalytic Domain
Cell Death
Cell Line
Cyclin B1
Cyclin-Dependent Kinase 4
Cytochromes c
Diacylglycerol Kinase
DNA Fragmentation
Electrophoresis, Gel, Two-Dimensional
Etoposide
Glia Maturation Factor
Human papillomavirus 16
Oxidoreductases
Proteomics
Replication Protein A
Transcription Factors
Uterine Cervical Neoplasms
Calcineurin
Caspase 3
Cyclin B1
Cyclin-Dependent Kinase 4
Cytochromes c
Diacylglycerol Kinase
Etoposide
Glia Maturation Factor
Oxidoreductases
Replication Protein A
Transcription Factors
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