Proteomic analysis of anti-cancer effects by paclitaxel treatment in cervical cancer cells.
OBJECTIVE
Paclitaxel (Taxol), a potent drug of natural origin isolated from the bark of the Pacific yew, is widely used in the treatment of ovarian, lung and breast cancer. At present, there is little information regarding the anti-cancer mechanism of paclitaxel against cervical carcinoma cells. We thus tried to show the anti-cancer effect of paclitaxel on cervical carcinoma cell line carrying HPV by using a proteomic analysis and to investigate the mechanism of actions.


METHODS
We treated paclitaxel to cervical carcinoma cells and then carried out MTT assay to observe the anti-proliferate activity. Using proteomics analysis including two-dimensional (2-DE) gel electrophoresis and MALDI-TOF-MS, we tried to find the anti-proliferate activity-related proteins. Among them, paclitaxel treatment suppressed the expression of the mitotic checkpoint protein BUB3. Functional proteomic analysis by small interfering RNA (siRNA) targeting was tried to illuminate a role of mitotic checkpoint protein BUB3 in cell cycle progression.


RESULTS
The cytotoxicity effects of paclitaxel were determined in HPV-16 positive CaSki, HPV-18 positive HeLa and HPV-negative C33A cervical carcinoma cell lines. Using efficient proteomics methods including 2-DE/MALDI-TOF-MS, we identified several cellular proteins that are responsive to paclitaxel treatment in HeLa cells. Paclitaxel treatment elevated mainly apoptosis-related, immune response-related and cell cycle check point-related proteins. On the other hand, paclitaxel treatment diminished growth factor/oncogene-related proteins and transcription regulation-related proteins. Paclitaxel showed anti-proliferate activity through the membrane death receptor (DR)-mediated apoptotic pathway involving activation of caspase-8 with a TRAIL-dependent fashion as well as the mitochondrial-mediated pathway involving down-regulation of bcl-2 by cytochrome c release. Furthermore, we found siRNA-induced BUB3 knock down on cell cycle progression blocked by cell cycle arrest after paclitaxel treatment.


CONCLUSIONS
The proteome profiling technique provided a broad-base and effective approach for the identification of protein changes induced by paclitaxel and showed anti-proliferate activity through the membrane death receptor-mediated apoptotic pathway, the mitochondrial-mediated pathway. This study shows the power of proteomic profiling with functional analysis using RNAi technology for the discovery of novel molecular targets and a better understanding of the actions of paclitaxel at the molecular level in cervical carcinoma cells.
Lee
Keun-Ho
KH
Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Kangnam St. Mary's Hospital, Seoul 137-040, Korea.
Yim
Eun-Kyoung
EK
Kim
Chan-Joo
CJ
Namkoong
Sung-Eun
SE
Um
Soo-Jong
SJ
Park
Jong-Sup
JS
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D018797:Cell Cycle Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006367:HeLa Cells; D006801:Humans; D017239:Paclitaxel / Q000494:pharmacology*; D000075223:Poly-ADP-Ribose Binding Proteins; D040901:Proteomics; D034741:RNA, Small Interfering / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013778:Tetrazolium Salts; D013844:Thiazoles; D014162:Transfection; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism* / Q000473:pathology
D000972:Antineoplastic Agents, Phytogenic; C489444:BUB3 protein, human; D018797:Cell Cycle Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D034741:RNA, Small Interfering; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue; D017239:Paclitaxel