Phenylbutyrate-induced apoptosis is associated with inactivation of NF-κB in HT-29 colon cancer cells

Rena Feinman, Kevin O. Clarke, Lawrence Harrison

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Purpose: Cytotoxic chemotherapy has been used to treat patients with metastatic colorectal cancer with limited success. Therefore novel chemotherapeutic approaches are needed. Based on encouraging preclinical data, there has been an interest in developing derivatives of butyrate as clinically applicable agents. The purpose of this study was to investigate the effects of phenylbutyrate (PB), a butyrate analogue, on the cell growth and apoptosis in a colon cancer cell model. Methods: Growth curves, flow cytometric studies, Western blotting, DNA binding assays and transient transfection experiments were performed in vitro using the colon cancer cell line HT-29 after exposure to PB. Results: Exposure of HT-29 colon cancer cells to PB resulted in growth inhibition and induction of apoptosis as measured by annexin V staining. This increase in apoptosis was associated with a decrease in mitochondrial membrane potential, an increase in caspase-3 activity and a decrease in intact PARP protein levels. Since NF-κB plays a pivotal role in the regulation of apoptosis, we explored the effects of PB on the DNA binding and transcriptional activity of this transcription factor. After PB treatment, NF-κB-DNA binding was markedly decreased and specifically, this decreased DNA binding was observed in the p50:p65 heterodimer. The decreased NF-κB DNA binding was observed as early as 3 h after PB treatment, while no apparent changes in annexin V binding were detected until 12 h after PB treatment. Untreated HT-29 cells transfected with a κB-luciferase reporter plasmid demonstrated significant constitutive activity of the κB binding site, which was markedly decreased after treating the cells with PB. Conclusion: These results suggest that PB-induced apoptosis may be partly regulated through the inactivation of NF-κB. PB, an oral butyrate analogue, may have therapeutic potential in colon cancer.

Original languageEnglish (US)
Pages (from-to)27-34
Number of pages8
JournalCancer Chemotherapy and Pharmacology
Volume49
Issue number1
DOIs
StatePublished - Jan 18 2002
Externally publishedYes

Fingerprint

Phenylbutyrates
Colonic Neoplasms
Cells
Apoptosis
Butyrates
Annexin A5
DNA
Growth
B-Form DNA
HT29 Cells
Chemotherapy
Mitochondrial Membrane Potential
Cell growth
Therapeutics
Luciferases
Caspase 3
Transfection
Colorectal Neoplasms
Assays
Plasmids

All Science Journal Classification (ASJC) codes

  • Oncology
  • Toxicology
  • Pharmacology
  • Cancer Research
  • Pharmacology (medical)

Cite this

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abstract = "Purpose: Cytotoxic chemotherapy has been used to treat patients with metastatic colorectal cancer with limited success. Therefore novel chemotherapeutic approaches are needed. Based on encouraging preclinical data, there has been an interest in developing derivatives of butyrate as clinically applicable agents. The purpose of this study was to investigate the effects of phenylbutyrate (PB), a butyrate analogue, on the cell growth and apoptosis in a colon cancer cell model. Methods: Growth curves, flow cytometric studies, Western blotting, DNA binding assays and transient transfection experiments were performed in vitro using the colon cancer cell line HT-29 after exposure to PB. Results: Exposure of HT-29 colon cancer cells to PB resulted in growth inhibition and induction of apoptosis as measured by annexin V staining. This increase in apoptosis was associated with a decrease in mitochondrial membrane potential, an increase in caspase-3 activity and a decrease in intact PARP protein levels. Since NF-κB plays a pivotal role in the regulation of apoptosis, we explored the effects of PB on the DNA binding and transcriptional activity of this transcription factor. After PB treatment, NF-κB-DNA binding was markedly decreased and specifically, this decreased DNA binding was observed in the p50:p65 heterodimer. The decreased NF-κB DNA binding was observed as early as 3 h after PB treatment, while no apparent changes in annexin V binding were detected until 12 h after PB treatment. Untreated HT-29 cells transfected with a κB-luciferase reporter plasmid demonstrated significant constitutive activity of the κB binding site, which was markedly decreased after treating the cells with PB. Conclusion: These results suggest that PB-induced apoptosis may be partly regulated through the inactivation of NF-κB. PB, an oral butyrate analogue, may have therapeutic potential in colon cancer.",
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Phenylbutyrate-induced apoptosis is associated with inactivation of NF-κB in HT-29 colon cancer cells. / Feinman, Rena; Clarke, Kevin O.; Harrison, Lawrence.

In: Cancer Chemotherapy and Pharmacology, Vol. 49, No. 1, 18.01.2002, p. 27-34.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Phenylbutyrate-induced apoptosis is associated with inactivation of NF-κB in HT-29 colon cancer cells

AU - Feinman, Rena

AU - Clarke, Kevin O.

AU - Harrison, Lawrence

PY - 2002/1/18

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N2 - Purpose: Cytotoxic chemotherapy has been used to treat patients with metastatic colorectal cancer with limited success. Therefore novel chemotherapeutic approaches are needed. Based on encouraging preclinical data, there has been an interest in developing derivatives of butyrate as clinically applicable agents. The purpose of this study was to investigate the effects of phenylbutyrate (PB), a butyrate analogue, on the cell growth and apoptosis in a colon cancer cell model. Methods: Growth curves, flow cytometric studies, Western blotting, DNA binding assays and transient transfection experiments were performed in vitro using the colon cancer cell line HT-29 after exposure to PB. Results: Exposure of HT-29 colon cancer cells to PB resulted in growth inhibition and induction of apoptosis as measured by annexin V staining. This increase in apoptosis was associated with a decrease in mitochondrial membrane potential, an increase in caspase-3 activity and a decrease in intact PARP protein levels. Since NF-κB plays a pivotal role in the regulation of apoptosis, we explored the effects of PB on the DNA binding and transcriptional activity of this transcription factor. After PB treatment, NF-κB-DNA binding was markedly decreased and specifically, this decreased DNA binding was observed in the p50:p65 heterodimer. The decreased NF-κB DNA binding was observed as early as 3 h after PB treatment, while no apparent changes in annexin V binding were detected until 12 h after PB treatment. Untreated HT-29 cells transfected with a κB-luciferase reporter plasmid demonstrated significant constitutive activity of the κB binding site, which was markedly decreased after treating the cells with PB. Conclusion: These results suggest that PB-induced apoptosis may be partly regulated through the inactivation of NF-κB. PB, an oral butyrate analogue, may have therapeutic potential in colon cancer.

AB - Purpose: Cytotoxic chemotherapy has been used to treat patients with metastatic colorectal cancer with limited success. Therefore novel chemotherapeutic approaches are needed. Based on encouraging preclinical data, there has been an interest in developing derivatives of butyrate as clinically applicable agents. The purpose of this study was to investigate the effects of phenylbutyrate (PB), a butyrate analogue, on the cell growth and apoptosis in a colon cancer cell model. Methods: Growth curves, flow cytometric studies, Western blotting, DNA binding assays and transient transfection experiments were performed in vitro using the colon cancer cell line HT-29 after exposure to PB. Results: Exposure of HT-29 colon cancer cells to PB resulted in growth inhibition and induction of apoptosis as measured by annexin V staining. This increase in apoptosis was associated with a decrease in mitochondrial membrane potential, an increase in caspase-3 activity and a decrease in intact PARP protein levels. Since NF-κB plays a pivotal role in the regulation of apoptosis, we explored the effects of PB on the DNA binding and transcriptional activity of this transcription factor. After PB treatment, NF-κB-DNA binding was markedly decreased and specifically, this decreased DNA binding was observed in the p50:p65 heterodimer. The decreased NF-κB DNA binding was observed as early as 3 h after PB treatment, while no apparent changes in annexin V binding were detected until 12 h after PB treatment. Untreated HT-29 cells transfected with a κB-luciferase reporter plasmid demonstrated significant constitutive activity of the κB binding site, which was markedly decreased after treating the cells with PB. Conclusion: These results suggest that PB-induced apoptosis may be partly regulated through the inactivation of NF-κB. PB, an oral butyrate analogue, may have therapeutic potential in colon cancer.

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