Stimulatory effect of estrogen on the growth of endometrial

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Mechanism of apicidin-induced cell cycle arrest and apoptosis in Ishikawa human endometrial cancer cells Mee Young Ahn a , Jaewon Lee a , Yong Jin Na b , Wahn Soo Choi c , Byung Mu Lee d ,Keon Wook Kang e , Hyung Sik Kim :

Mechanism of apicidin-induced cell cycle arrest and apoptosis in Ishikawa human endometrial cancer cells Mee Young Ahn a , Jaewon Lee a , Yong Jin Na b , Wahn Soo Choi c , Byung Mu Lee d ,Keon Wook Kang e , Hyung Sik Kim Chemico-Biological Interactions 179 (2009)

BACKGROUND:

BACKGROUND Histone deacetylase (HDAC) inhibitors are a promising new class of anticancer agents that act by inhibiting cell proliferation and inducing apoptosis in a variety of cancer cells. Although apicidin acts as a potent HDAC inhibitor. This study examined the anti-tumor effects of apicidin in Ishikawa cancer cells. The level of cell proliferation, the stage of the cell cycle, and apoptosis were measured after the apicidin treatment.

Deacetylation of histone:

Deacetylation of histone

AIM OF THE STUDY:

AIM OF THE STUDY Examined the effects of apicidin on the modulation of cell death, cell cycle arrest and apoptosis in human endometrial carcinoma cells.

Methodology Used:

Methodology Used

1. Cell culture:

1. Cell culture The Ishikawa cells were maintained in DMEM supplemented with 10% heat inactivated fetal bovine serum and 1% penicillin/streptomycin at 37 ◦C with 5% CO2 in a incubator. For the apicidin treatment, the cells were plated for 48 h in DMEM containing 10% FBS. The medium was then changed to DMEM containing 5% FBS with various concentrations of apicidin.

2. Cytotoxicity assay:

2. Cytotoxicity assay The Ishikawa cells were grown in 96 well. The cells were allowed to attach for 48 hrs ,and exposed to apicidin. Cytotoxicity by MTT Assay

3. Western blot analysis :

3 . Western blot analysis Westrn blot analysis of Ishikawa cell lysate and Probed sequentially with the antibodies against aceylated H3, P21, CDK4, PARP cleavage, cytochrome c, Bcl-2, Bax and β -actin antibody. Blots were developed using anenhanced chemiluminescence (ECL) kit

4. Reverse transcriptase- PCR:

4. Reverse transcriptase- PCR cDNA of (cyclin D1, cyclin E, CDK2, CDK4, p53, GAPDH) was synthesized using a reverse transcription method.

5. Quantitative real-time RT- PCR:

5. Quantitative real-time RT- PCR Amplification of cDNA of (cyclin D1, cyclin E, CDK2, CDK4, p53, GAPDH) by using Quantitative real-time RT-PCR Using light cycler DNA Master SYBER Green I kit.

6. Flow Cytometry analysis :

6. Flow Cytometry analysis The Ishikawa cells were treated with apicidin for 48 h. The total number of cells, both adherent and suspended, were collected, and stained with a propidium iodine (PI) solution.

7. Immunocytochemistry :

7. Immunocytochemistry The cells were grown on coverslips After the apicidin treatment, the cells were processed for immunostaining with anti-p21 andanti-p53 antibodies. Primary antibody was detected by FITC- conjugated goat anti-mouse IgG and nuclear staining was achieved by PI. The cells were observed using confocal laser scanning microscope.

8. Caspase activity assay :

8. Caspase activity assay The caspase-3, -8 and -9 activities in the cell lysate were determined using colorimetric assay kits. The substrates (DEVD-pNA for caspase- 3, IETD-pNA for caspase-8 and LEHD-pNA for caspase-9) are used and the chromophores were quantified spectrophotometrically at a wavelengthof 405 nm.

8. TUNEL assay:

8. TUNEL assay The level of apoptosis was determined by this method and PI staining . The cell were observed by confocal microscopy.

9. immunofluorescence:

9. immunofluorescence The cells were treated with the primary monoclonal antibodies against cytochrome c and visualized by secondary antibody goat anti-mouse immunoglobulin G.

Results and Discussion:

Results and Discussion

1. Apicidin inhibits the proliferation of Ishikawa endometrial cancer cells and induces morphological changes :

1. Apicidin inhibits the proliferation of Ishikawa endometrial cancer cells and induces morphological changes

1. Apicidin inhibits the proliferation of Ishikawa endometrialcancer cells and induces morphological changes:

1. Apicidin inhibits the proliferation of Ishikawa endometrialcancer cells and induces morphological changes

2. Expression levels of histone acetylation and cell cycle regulators :

2. Expression levels of histone acetylation and cell cycle regulators

2. Expression levels of histone acetylation and cell cycle regulators:

2. Expression levels of histone acetylation and cell cycle regulators

2. Expression levels of histone acetylation and cell cycle regulators:

2. Expression levels of histone acetylation and cell cycle regulators

2. Expression levels of histone acetylation and cell cycle regulators:

2. Expression levels of histone acetylation and cell cycle regulators

2. Expression levels of histone acetylation and cell cycle regulators:

2. Expression levels of histone acetylation and cell cycle regulators

2. Expression levels of histone acetylation and cell cycle regulators:

2. Expression levels of histone acetylation and cell cycle regulators

3. Apicidin increases the proportion of cells in the G1 phase:

3. Apicidin increases the proportion of cells in the G1 phase

4. Apicidin induces apoptotic cell death:

4. Apicidin induces apoptotic cell death

5. Apicidin induces caspases activity:

5. Apicidin induces caspases activity

6. Apicidin induces cytochrome c release and pro-apoptoticprotein expression:

6. Apicidin induces cytochrome c release and pro-apoptoticprotein expression

Conclusions:

Conclusions Apicidin had antiproliferative effects by inducing apoptosis in human endometrial cancer cells. Apicidin increasedthe proportion of cells in the G1 phase of the cell cycle, which is associated with a decrease in cyclin D1 and CDK4 expression and theinduction of p21. Apicidin-induced apoptosis might be related tocaspase-3 activation and the down-regulation of Bcl-2 expression. Overall, these results suggest that apicidin is a potential anti-tumoragent against human endometrial cancer cells

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