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

Affiliations
  • 1Department of Obstetrics and Gynecology, Keimyung University School of Medicine, Daegu, Korea. sdcha@dsmc.or.kr
  • 2Department of Pathology, Keimyung University School of Medicine, Daegu, Korea.

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.


Reference

1. Dent P, Yacoub A, Contessa J, Caron R, Amorino G, Valerie K, et al. Stress and radiation-induced activation of multiple intracellular signaling pathways. Radiat Res. 2003; 159:283–300. PMID: 12600231.
2. Ma BB, Bristow RG, Kim J, Siu LL. Combine-modality treatment of solid tumors using radiotherapy and molecular targeted agents. J Clin Oncol. 2003; 21:2760–2776. PMID: 12860956.
3. Roche S, Koegl M, Courtneidge SA. The phosphatidylinositol 3-kinase α is required for DNA synthesis induced by some, but not all, growth factors. Proc Natl Acad Sci USA. 1994; 91:9185–9189. PMID: 8090789.
4. Philpott KL, McCarthy MJ, Klippel A, Rubin LL. Activated phosphatidylinositol 3-kinase and Akt kinase promote survival of superior cervical neurons. J Cell Biol. 1997; 139:809–815. PMID: 9348296.
Article
5. Davidson HW. Wortmannin causes mistargeting of procathepsin D. Evidence for the involvement of a phosphatidylinositol 3-kinase in vesicular transport to lysosomes. J Cell Biol. 1995; 130:797–805. PMID: 7642698.
Article
6. Leevers SJ, Vanhaesebroeck B, Waterfield MD. Signalling through phosphoinositide 3-kinases: the lipids take centre stage. Curr Opin Cell Biol. 1999; 11:219–225. PMID: 10209156.
Article
7. Arbeit JM, Howley PM, Hanahan D. Chronic estrogen-induced cervical and vaginal squamous carcinogenesis in human papillomavirus type 16 transgenic mice. Proc Natl Acad Sci USA. 1996; 93:2930–2935. PMID: 8610145.
Article
8. Cummings JH. Short-chain fatty acids in the human colon. Gut. 1981; 22:763–779. PMID: 7028579.
9. Finzer P, Kuntzen C, Soto U, zur Hausen H, Rosl F. Inhibitors of histone deacetylase arrest cell cycle and induce apoptosis in cervical carcinoma cells circumventing human papillomavirus oncogene expression. Oncogene. 2001; 20:4768–4776. PMID: 11521189.
Article
10. Finzer P, Ventz R, Kuntzen C, Seibert N, Soto U, Rosl F. Growth arrest of HPV-positive cells after histone deacetylase inhibition is dependent of E6/E7 oncogene expression. Virology. 2002; 304:265–273. PMID: 12504567.
11. Mandal M, Kumar R. Bcl-2 expression regulates sodium butyrate-induced apoptosis in human MCF-7 breast cancer cells. Cell Growth Differ. 1996; 7:311–318. PMID: 8838861.
12. Coradini D, Pellizzaro C, Marimpietri D, Abolafio G, Daidone MG. Sodium butyrate modulates cell cycle-related proteins in HT29 human colonic adenocarcinoma cells. Cell Prolif. 2000; 33:139–146. PMID: 10959623.
Article
13. Newmark HL, Young CW. Butyrate and phenylacetate as differentiating agents: practical problems and opportunities. J Cell Biochem Suppl. 1995; 22(suppl):247–253. PMID: 8538206.
Article
14. Alonio LV, Picconi MA, Dalbert D, Mural J, Bartt O, Bazan G, et al. Ha-ras oncogene mutation associated to progression of papillomavirus induced lesions of uterine cervix. J Clin Virol. 2003; 27:263–269. PMID: 12878090.
Article
15. Vanhaesebroeck B, Leevers SJ, Ahmadi K, Timms J, Katso R, Driscoll PC, et al. Synthesis and function of 3-phosphorylated inositol lipids. Annu Rev Biochem. 2001; 70:535–602. PMID: 11395417.
Article
16. Benistant C, Chapuis H, Roche S. A specific function for phosphatidylinositol 3-kinase α(p85α-p110α) in cell survival and for phosphatidylinositol 3-kinase β(p85α-p110β) in de novo DNA synthesis of human colon carcinoma cells. Oncogene. 2000; 19:5083–5090. PMID: 11042696.
17. Kermorgant S, Aparicio T, Dessirier V, Lewin MJ, Lehy T. Hepatocyte growth factor induces colonic cancer cell invasiveness via enhanced motility and protease overproduction. Evidence for PI3 kinase and PKC involvement. Carcinogenesis. 2001; 22:1035–1042. PMID: 11408346.
Article
18. Ma YY, Wei SJ, Lin YC, Lung JC, Chang TC, Whang-Peng J, et al. PIK3CA as an oncogene in cervical cancer. Oncogene. 2000; 25:2739–2744. PMID: 10851074.
Article
19. Janson W, Brandner G, Siegel J. Butyrate modulates DNA-damage-induced p53 response by induction of p53-independent differentation and apoptosis. Oncogene. 1997; 15:1395–1406. PMID: 9333015.
20. Yih LH, Lee TC. Arsenite induces p53 accumulation through an ATM-dependent pathway in human fibroblasts. Cancer Res. 2000; 60:6346–6352. PMID: 11103796.
21. Wymann MP, Bulgarelli-Leva G, Zvelebil MJ, Pirola L, Vanhaesebroeck B, Waterfield MD, et al. Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction. Mol Cell Bio. 1996; 16:1722–1733. PMID: 8657148.
Article
22. Kennedy SG, Wagner AJ, Conzen SD, Jordan J, Bellacosa A, Tsichlis PN, et al. The PI 3-kinase/Akt signaling pathway delivers an antiapoptotic signal. Genes Dev. 1997; 11:701–713. PMID: 9087425.
Article
23. Boulton S, Kyle S, Yalcintepe L, Durkacz BW. Wortmannin is a potent inhibitor of DNA double strand break but not single strand break repair in Chinese hamster ovary cells. Carcinogenesis. 1996; 17:2285–2290. PMID: 8968039.
Article
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