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Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS
Published on: August 10, 2017
Combined proteasome and histone deacetylase inhibition in non-small cell lung cancer
Chadrick E Denlinger1, Michael D Keller, Marty W Mayo
1Department of Surgery, University of Virginia School of Medicine, Charlottesville, 22908, USA.
Objective:
Inhibitors of histone deacetylases are potent inducers of cell-cycle arrest and apoptosis in certain malignancies. We have previously demonstrated that chemotherapy activates the antiapoptotic transcription factor nuclear factor kappa B in non-small cell lung cancer and fails to induce significant levels of apoptosis. We hypothesize that nuclear factor kappa B inhibition with the proteasome inhibitor bortezomib (formerly known as PS-341) will sensitize non-small cell lung cancer cells to histone deacetylase inhibitor-mediated apoptosis.
Methods:
Tumorigenic non-small cell lung cancer cells (A549, H358, and H460) were treated with bortezomib, followed by the histone deactylase inhibitor sodium butyrate. After treatment, nuclear factor kappa B transcriptional activity was measured by using a luciferase reporter assay and transcription of the nuclear factor kappa B-dependent gene IL8. Apoptosis was determined on the basis of caspase-3 activation and DNA fragmentation. Western blot analyses for the cell-cycle regulatory proteins p21 and p53 were performed, and cell-cycle alterations were determined by means of FACS analysis. Experiments were performed in triplicate, and statistical significance was determined by using unpaired t tests.
Results:
Butyrate increased nuclear factor kappa B transcriptional activity 4-fold relative to that seen in control cells (P =.05) in all non-small cell lung cancer cell lines. Treatment with bortezomib reduced butyrate-induced activation of nuclear factor kappa B to baseline levels. The proteins p21 and p53 were stabilized after treatment with bortezomib, correlating with a G(2)/M cell-cycle arrest. Treatment with butyrate alone resulted in minimal apoptosis, but combined histone deacetylase and proteasome inhibition increased apoptosis 3- to 4-fold (P =.02).
Conclusions:
Combined molecular targeting of histone deacteylases and proteasomes synergistically induced apoptosis in non-small cell lung cancer. Pharmacologic nuclear factor kappa B suppression through proteasome inhibition, followed by treatment with histone deacetylase inhibitors, might represent a novel treatment strategy for patients with non-small cell lung cancer.
Insights
Combining proteasome inhibitors with histone deacetylase inhibitors synergistically enhances apoptosis in non-small cell lung cancer by suppressing nuclear factor kappa B activation. This novel strategy may improve treatment outcomes for lung cancer patients.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Therapeutics
Background:
- Histone deacetylase inhibitors (HDACi) induce cell-cycle arrest and apoptosis in malignancies.
- Chemotherapy activates antiapoptotic nuclear factor kappa B (NF-κB) in non-small cell lung cancer (NSCLC), limiting apoptosis.
- We hypothesize that inhibiting NF-κB with bortezomib sensitizes NSCLC to HDACi-induced apoptosis.
Purpose of the Study:
- To investigate the synergistic effect of bortezomib and sodium butyrate on apoptosis in NSCLC cells.
- To determine the impact of combined treatment on NF-κB activity, cell-cycle proteins, and apoptosis.
Main Methods:
- NSCLC cell lines (A549, H358, H460) were treated with bortezomib followed by sodium butyrate.
- NF-κB transcriptional activity was assessed using a luciferase reporter assay and IL8 gene transcription.
- Apoptosis was measured by caspase-3 activation and DNA fragmentation; cell-cycle proteins (p21, p53) and cell-cycle distribution were analyzed.
Main Results:
- Sodium butyrate increased NF-κB activity 4-fold; bortezomib reduced this to baseline.
- Bortezomib stabilized p21 and p53, inducing G2/M cell-cycle arrest.
- Combined HDAC and proteasome inhibition increased apoptosis 3- to 4-fold compared to butyrate alone.
Conclusions:
- Combined targeting of HDACs and proteasomes synergistically induces apoptosis in NSCLC.
- Pharmacologic NF-κB suppression via proteasome inhibition followed by HDAC inhibitors offers a novel NSCLC treatment strategy.
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