Cancer Res Treat.  2006 Apr;38(2):112-117.

Augmentation of Sodium Butyrate-induced Apoptosis by Phosphatidylinositol 3-kinase Inhibition in the Human Cervical Cancer Cell-line

• Park JK
• Cho CH
• Ramachandran S
• Shin SJ
• Kwon SH
• Kwon SY
• Cha SD

Abstract

PURPOSE: Sodium butyrate (NaBT) is principally a histone deacetylase (HDAC) inhibitor, and it has the potential to arrest HPV-positive carcinoma cells at the G1 to S phase transition of the cell cycle. The aim of study was to determine whether phosphatidylinositol 3-kinase (PI3K) inhibition can enhance the inhibitory effect of NaBT on a human cervical cancer cell line (HeLa).
MATERIALS AND METHODS
Cervical cancer cells (HeLa) were treated with NaBT alone or in combination with the PI3K inhibitors wortmannin or LY294002. Cell viability analysis and FACS analysis were carried out. The expressions of the cell cycle related proteins were evaluated by Western-blot analysis.
RESULTS
Inhibition of PI3K enhanced NaBT-mediated apoptosis and this decreased the HeLa cell viability. Either wortmannin or LY294002, combined with NaBT, enhanced the activation of caspase 3 and caspase 9, and this enhanced the subsequent cleavage of poly (ADP-ribose) polymerase (PARP). Cervical cancer cells were arrested in the subG1 and G2/M phase, as was detected by FACS analysis. NaBT treatment in combination with PI3K inhibitors showed the increased expression of the CDK inhibitors p21(Cip1/Waf1) and p2(7Kip1), in a p53 dependent manner, and also the increased dephosphorylation of Rb whereas there was a reduction in the expression levels of cyclin A, cyclin D1 and cyclin B1.
CONCLUSION
The results demonstrate that inhibition of PI3K enhances NaBT-mediated cervical cancer cell apoptosis through the activation of the caspase pathway. Moreover, these findings will support future investigation using the PI3K inhibitors in combination with adjuvant treatment for treating carcinoma of the cervix.

Keyword

Cervical cancer; Sodium butyrate; Phosphatidylinositol 3-kinase; Apoptosis

MeSH Terms

Apoptosis*
Butyric Acid
Caspase 3
Caspase 9
Cell Cycle
Cell Line
Cell Survival
Cervix Uteri
Cyclin A
Cyclin B1
Cyclin D1
Female
HeLa Cells
Histone Deacetylases
Humans*
Phosphatidylinositol 3-Kinase*
Phosphatidylinositols*
S Phase
Sodium*
Uterine Cervical Neoplasms*
Butyric Acid
Caspase 3
Caspase 9
Cyclin A
Cyclin B1
Cyclin D1
Histone Deacetylases
Phosphatidylinositol 3-Kinase
Phosphatidylinositols
Sodium

Figure

• 

  Fig. 1 Growth inhibition in the HeLa cells treated for 24 h with a combined concentration of NaBT with the PI3K inhibitor (wortmannin). Cell viability was measured using the Cell Titer Cell Proliferation Assay and the results are expressed as a % of the control culture conditions. *p<0.05, †p<0.01.

• 

  Fig. 2 Effect of a treatment with NaBT alone or NaBT with wortmannin or LY294002 treatment on the cell cycle profile. After treatment for 24 h with 5 mM/L NaBT alone or DMSO only or 5 mM/L NaBT with 400 nM/L wortmannin or 20µM/L LY294002, the HeLa cells were collected, fixed, stained with propidium iodide and analyzed by flow cytometry. The values represent the number of cells in a phase of the cell cycle as a percentage of the total cells.

• 

  Fig. 3 Effects of PI3K inhibitors and NaBT on the cell cycle-related genes. HeLa cells were treated for 24 h with 5 mM/L NaBT alone or DMSO only or with 5 mM/L NaBT with 400 nM/L wortmannin or 20µM/L LY294002. The whole cell lysates were prepared and they were used for the detection of each protein expression using antibodies against p21Cip1/Waf1, p27Kip1, pRb and p53 by Western blot analysis. β-tubulin was used as an internal control.

• 

  Fig. 4 Effects of PI3K inhibitors and NaBT on cyclins and cdks. The whole cell lysates were prepared and used for the detection of each protein expression with using antibodies against CDK2, CDK4, cyclin A, cyclin B1, cyclin D1 and cyclin E by Western blot analysis. β-tubulin was used as an internal control.

• 

  Fig. 5 Effects of PI3K inhibitors and NaBT on caspase activation and PARP cleavage. The whole cell lysates were prepared and used for the detection of each protein expression with using antibodies against caspase 3, caspase 9 and PARP by Western blot analysis. β-tubulin was used as an internal control.

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