Korean J Gynecol Oncol.  2008 Mar;19(1):26-39. 10.3802/kjgo.2008.19.1.26.

Induction of apoptosis by the kinase inhibitor flavopiridol in human ovarian cancer cell lines

• Hur SY
• Lee JM

• KMID: 1979499
• DOI: http://doi.org/10.3802/kjgo.2008.19.1.26

Abstract

OBJECTIVE
Flavopiridol that inhibits cyclin-dependent kinase, can cause cell cycle arrest, induce apoptosis in human tumor cell lines. In the present study, we investigated apoptotic effects of flavopiridol and the underlying molecular mechanisms in human ovarian cancer cell lines.
METHODS
We used TOV-21G and TOV-112D cell lines. The cell viability was tested by MTT assay and apoptosis was assessed by TUNEL assay and annexin-V binding. Western blot was used to examine apoptosis related protein levels. MAP kinase activity was analyzed by non-radioactive MAP kinase assay kit.
RESULTS
Treatment of TOV-21G and TOV-112D cells with flavopiridol (50 nM to 1000 nM) led to a dose- and time-dependent inhibition of cell growth and survival. Dose-related induction of apoptosis was also observed in these cell lines. Flavopiridol (500 nM) induced striking decreases in the levels of the antiapoptic proteins Mcl-1, Bcl-X(L), and XIAP in both cell lines. In contrast, expression of Bax, Bcl-2, and AIF was not significantly influenced by flavopiridol. Although flavopiridol resulted in accumulation of p53 in both cells, flavopiridol mediated apoptosis was p53 independent because it occurred to the same degree in TOV-112D cells in which p53 was inactivated by mutation. Flavopiridol treatment resulted in enhanced cleavage of pro-caspase 9 and activation of caspase 3. Apoptosis was associated with suppression of ERK activity.
CONCLUSION
Although the precise mechanisms of flavopiridol mediated cytotoxicity have not been fully defined, these data suggest that flavopiridol has activity against ovarian cancers in vitro and is worthy of continued clinical development in the treatment of ovarian cancer.

Keyword

Flavopiridol; Ovarian cancer cell lines; Apoptosis

MeSH Terms

Apoptosis
Blotting, Western
Caspase 3
Cell Cycle Checkpoints
Cell Line
Cell Line, Tumor
Cell Survival
Flavonoids
Humans
In Situ Nick-End Labeling
Ovarian Neoplasms
Phosphotransferases
Piperidines
Proteins
Strikes, Employee
Caspase 3
Flavonoids
Phosphotransferases
Piperidines
Proteins

Figure

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  Fig. 1 Effect of flavopiridol on the viability of the ovarian cancer cell lines TOV-21G (A) and TOV-112D (B). Cells were treated with flavopiridol at the indicated concentrations 24 hours after plating. At 24, and 48 hours after treatment, cells were stained MTT, and the absorbance was read at 570 nm. Results were presented as percentage of control which was calculated using the equation: (mean absorbance of treated cells/mean absorbance of control cells) ×100. Data were expressed as mean±standard deviation (SD) from four independent experiments. *p<0.05 as compared to corresponding control cells.

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  Fig. 2 Ovarian cancer cell lines, TOV-21G (A) and TOV-112D (B) labelled by the TUNEL method. Cells were treated with 100 nM flavopiridol for 24 hours. Nuclear morphology was examined under a fluorescence microscopy. Nuclei of apoptotic cells were green. Live cells were totally blue without green spot. Original magnification, ×200.

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  Fig. 3 Induction of apoptosis by flavopiridol in ovarian cancer cell lines TOV-21G. Cells were treated with the indicated concentrations of flavopiridol (100, 300, and 500 nM) for 24 and 48 hours, respectively. (A) for apoptosis, the externalization of phosphatidylserine was assessed by measuring annexin-V-Fluos binding using propidium iodide as a counterstain. Quadrant rectangular dot grams from a representative of 3 independent experiments is shown. (B) DNA fragmentation were determined using terminal deoxynucleotidyl transferase for incorporation of fluorescein-12-dUTP at free 3'-OH DNA ends (TUNEL assay). The percentage of TUNEL-positive cells was counted for each condition at 40× magnification in five separate fields of at least 100 cells each. Data were expressed as the mean±standard deviation (SD) from three independent experiments.

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  Fig. 4 Induction of apoptosis by flavopiridol in ovarian cancer cell lines TOV-112D. Cells were treated with the indicated concentrations of flavopiridol (100, 300, and 500 nM) for 24 and 48 hours, respectively. (A) For apoptosis, the externalization of phosphatidylserine was assessed by measuring annexin V-Fluos binding using propidium iodide as a counterstain. Quadrant rectangular dot grams from a representative of 3 independent experiments is shown. (B) DNA fragmentation were determined using terminal deoxynucleotidyl transferase for incorporation of fluorescein-12-dUTP at free 3'-OH DNA ends (TUNEL assay). The percentage of TUNEL-positive cells was counted for each condition at 40× magnification in five separate fields of at least 100 cells each. Data were expressed as the mean±standard deviation (SD) from three independent experiments.

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  Fig. 5 Activation of caspase 3 by flavopiridol in ovarian cancer cell lines TOV-21G and TOV-112D. Cells were treated with 500 nM flavopiridol for the times indicated. Protein extracts were obtained from aliquots of cells and assayed for caspase 3 activity using the fluorogenic substrate DEVD-AMC.

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  Fig. 6 The effect of flavopiridol on the expression of apoptosis related proteins (AIF, XIAP, p53, Mcl-1, Bcl-XL, Bcl-2, and Bax) in ovarian cancer cell lines TOV-21G and TOV-112D. Cells were treated with flavopiridol (100, 300, and 500 nM) for 24 hours, respectively. Aliquots of cells were transferred and protein extracts were assayed for western blot analysis. The expression of actin were used as the loading control.

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  Fig. 7 Activation and processing of caspase 9 during flavopiridol induced apoptosis in ovarian cancer cell lines TOV-21G and TOV-112D. Cells were treated with flavopiridol (100, 300, and 500 nM) for 24 hours. Aliquots of cells were transferred and lysed in SDS sample buffer and lysates were subjected to western blot analysis with specific antibody, which recognized the pro-form and the active cleaved form of caspase 9, Mr 46 kDa and 34 kDa, respectively.

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  Fig. 8 Flavopiridol-induced suppression of the activity of ERK, MAP kinase in ovarian cancer cell lines TOV-21G and TOV-112D. Cells were treated with flavopiridol (100, and 500 nM) for 24 hours. Cell lysates were immunoprecipitated with anti-ERK antibodies before a further incubation with protein A-Sepharose beads. These immune complexes were reacted with myelin basic protein (MBP) as a substrate and then the phosphorylated substrate was analyzed by immunoblot analysis, probing with a monoclonal phospho-MBP antibody.

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