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Cited by 183Telomeres, Telomerase, and SenescenceHistone Deacetylase Inhibitors ResearchCell death mechanisms and regulation

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A Novel Indication for Panobinostat as a Senolytic Drug in NSCLC and HNSCC

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Samaraweera, Leleesha, Adomako, Alfred, Rodriguez-Gabin, Alicia, McDaid, Hayley M.·2017·Scientific Reports
DOI 10.1038/s41598-017-01964-1PMC PMC5432488
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Abstract

Panobinostat (pano) is an FDA-approved histone deacetylase inhibitor. There is interest in evaluating alternate dosing schedules and novel combinations of pano for the treatment of upper aerodigestive and lung malignancies; thus we evaluated it in combination with Taxol, a chemotherapeutic with activity in both diseases. Dose-dependent synergy was observed in Non-Small Cell Lung Cancer (NSCLC) and Head and Neck Squamous Cell Carcinoma (HNSCC) cell lines and was due to senescence rather than potentiation of cell death. Senescence occurred following cisplatin- or Taxol-treatment in cell lines from both cancer types and was associated with decreased histone 3 (H3) acetylation and increased Bcl-xL expression: the latter a biomarker of senescence and target of anti-senescence therapeutics, or senolytics. Since H3 acetylation and Bcl-xL expression were altered in senescence, we subsequently evaluated pano as a senolytic in chemotherapy-treated cancer cells enriched for senescent cells. Pano caused cell death at significantly higher rates compared to repeat dosing with chemotherapy. This was associated with decreased expression of Bcl-xL and increased acetylated H3, reversing the expression patterns observed in senescence. These data support evaluating pano as a post-chemotherapy senolytic with the potential to kill persistent senescent cells that accumulate during standard chemotherapy in NSCLC and HNSCC.

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Figures

Figure 1 Synergy Between Taxol and Pano is Associated with Suppression of Proliferation in NSCLC and HNSCC. Dose response curves for Taxol, or pano and the concurrent combination in (A) A549 (NSCLC) and (B) FaDu (HNSCC) cells. Combination index (CI) versus Fraction Affect (FA) plots comparing concurrent and sequencing regimens, in (C) A549 and (D) FaDu. Each data point represents mean ± SEM (n = 6). (E) Immunoblot analysis for proliferation (PCNA) and cell death (p89-PARP) to evaluate the effect of low dose, concurrent Taxol and pano treatment. Doses used were: Taxol - 6.25 nM (A549), 2.5 nM (FaDu) and pano- 25 nM (both cell lines) for 72 hrs. (F) Caspase 3/7 activity using the same conditions outlined in E. Activity is indicated as % relative to vehicle treatment. Each bar represents mean ± SEM (n = 3).
Figure 1 Synergy Between Taxol and Pano is Associated with Suppression of Proliferation in NSCLC and HNSCC. Dose response curves for Taxol, or pano and the concurrent combination in (A) A549 (NSCLC) and (B) FaDu (HNSCC) cells. Combination index (CI) versus Fraction Affect (FA) plots comparing concurrent and sequencing regimens, in (C) A549 and (D) FaDu. Each data point represents mean ± SEM (n = 6). (E) Immunoblot analysis for proliferation (PCNA) and cell death (p89-PARP) to evaluate the effect of low dose, concurrent Taxol and pano treatment. Doses used were: Taxol - 6.25 nM (A549), 2.5 nM (FaDu) and pano- 25 nM (both cell lines) for 72 hrs. (F) Caspase 3/7 activity using the same conditions outlined in E. Activity is indicated as % relative to vehicle treatment. Each bar represents mean ± SEM (n = 3).CC BY · PubMed Central
Figure 2 The Combination of Taxol and Pano is Synergistic due to Senescence Induction. Representative proliferation assays for (A) A549 and (B) FaDu showing sustained proliferative arrest in combination-treated cells. Cells treated with single agents exhibited transient proliferative arrest. The statistical significance (unpaired t-test) for combination-treated cells relative to single agents on day 12 is indicated. Transcriptome analysis (qRT-PCR) of SASP-associated genes (IL6, IL8, CXCL1, PAI-1) and proliferation (cyclin A2) in 6 day drug-treated (C) A549 and (D) FaDu cells. Increased SASP expression and absence of proliferation are typical of senescence. Bar represents mean ± SD (n = 3). (E) Representative images of SA-β-gal-stained A549 cells after 6 days treatment with the indicated doses of Taxol, pano or combo. SA-β-gal quantitation (bottom left) are indicated (mean ± SD). Magnification 5X.
Figure 2 The Combination of Taxol and Pano is Synergistic due to Senescence Induction. Representative proliferation assays for (A) A549 and (B) FaDu showing sustained proliferative arrest in combination-treated cells. Cells treated with single agents exhibited transient proliferative arrest. The statistical significance (unpaired t-test) for combination-treated cells relative to single agents on day 12 is indicated. Transcriptome analysis (qRT-PCR) of SASP-associated genes (IL6, IL8, CXCL1, PAI-1) and proliferation (cyclin A2) in 6 day drug-treated (C) A549 and (D) FaDu cells. Increased SASP expression and absence of proliferation are typical of senescence. Bar represents mean ± SD (n = 3). (E) Representative images of SA-β-gal-stained A549 cells after 6 days treatment with the indicated doses of Taxol, pano or combo. SA-β-gal quantitation (bottom left) are indicated (mean ± SD). Magnification 5X.CC BY · PubMed Central
Figure 3 Senescence is a Common Outcome of Chemotherapy in NSCLC and HNSCC and is Associated with Altered Histone Acetylation and Bcl-xL Expression. Chemotherapy-Induced Senescent (CIS) populations were obtained by treating NSCLC (A549, H460) and HNSCC (UMSCC47, FaDu) cells with either cisplatin (CDDP) or Taxol for 10 days. The doses indicated were chosen to induce maximal cell death and residual, surviving cells typically become senescent. (A) Representative phase images of SA-β-gal-stained CIS populations (Magnification 10X), (B) Transcriptome analysis (qRT-PCR) of SASP-related gene expression and proliferation in 10 day old CIS populations. The dotted line represents baseline expression in vehicle-only treated asynchronous cells. Bars represent mean ± SD (n = 3) (C) Representative immunoblot showing decreased H3 acetylation and increased expression of Bcl-xL in CIS populations [C] compared to asynchronous [A] NSCLC and HNSCC cells. Cisplatin (CDDP) was used as the senescent inducer and doses are denoted in 3A.
Figure 3 Senescence is a Common Outcome of Chemotherapy in NSCLC and HNSCC and is Associated with Altered Histone Acetylation and Bcl-xL Expression. Chemotherapy-Induced Senescent (CIS) populations were obtained by treating NSCLC (A549, H460) and HNSCC (UMSCC47, FaDu) cells with either cisplatin (CDDP) or Taxol for 10 days. The doses indicated were chosen to induce maximal cell death and residual, surviving cells typically become senescent. (A) Representative phase images of SA-β-gal-stained CIS populations (Magnification 10X), (B) Transcriptome analysis (qRT-PCR) of SASP-related gene expression and proliferation in 10 day old CIS populations. The dotted line represents baseline expression in vehicle-only treated asynchronous cells. Bars represent mean ± SD (n = 3) (C) Representative immunoblot showing decreased H3 acetylation and increased expression of Bcl-xL in CIS populations [C] compared to asynchronous [A] NSCLC and HNSCC cells. Cisplatin (CDDP) was used as the senescent inducer and doses are denoted in 3A.CC BY · PubMed Central
Figure 4 Pano has Senolytic Activity in Chemotherapy-Induced Senescent NSCLC. (A) Schema of experimental design. CIS populations of A549 obtained after treatment with either CDDP or Taxol (cycle 1), were subsequently treated with either (i) cycle 2 of chemotherapy (at the same dose as cycle 1), or (ii) pano (200 nM). Cells were assayed for survival at 13 and 17 days post-treatment initiation, corresponding to 3 and 7 days post-cycle 2. Representative SRB-stained images indicate dramatic cell loss following pano-treatment in (B) CIS (CDDP - cisplatin), or (C) CIS (Taxol) populations (Magnification 5X). Quantitative analysis of cell loss in (D) CIS (CDDP) and (E) CIS (Taxol) senescent populations following cycle 2 treatment (mean ± SEM, n = 6). Data indicate significant loss (P < 0.0001: unpaired t-test) for pano-treated cells versus chemotherapy (either CDDP or Taxol) by day 17. Vehicle controls reflect treatment of CIS populations for either duration. (F) Increased caspase 3/7 activity in pano-treated (3 days) CIS populations of A549. Bar represents mean ± SEM (n = 3) and statistical significance (unpaired t-test) is indicated. (G) Representative immunoblot showing increased PARP cleavage, decreased Bcl-xL and increased acetylated H3 in both CIS populations of A549 cells following 1- and 3-day treatment with pano (200 nM).
Figure 4 Pano has Senolytic Activity in Chemotherapy-Induced Senescent NSCLC. (A) Schema of experimental design. CIS populations of A549 obtained after treatment with either CDDP or Taxol (cycle 1), were subsequently treated with either (i) cycle 2 of chemotherapy (at the same dose as cycle 1), or (ii) pano (200 nM). Cells were assayed for survival at 13 and 17 days post-treatment initiation, corresponding to 3 and 7 days post-cycle 2. Representative SRB-stained images indicate dramatic cell loss following pano-treatment in (B) CIS (CDDP - cisplatin), or (C) CIS (Taxol) populations (Magnification 5X). Quantitative analysis of cell loss in (D) CIS (CDDP) and (E) CIS (Taxol) senescent populations following cycle 2 treatment (mean ± SEM, n = 6). Data indicate significant loss (P < 0.0001: unpaired t-test) for pano-treated cells versus chemotherapy (either CDDP or Taxol) by day 17. Vehicle controls reflect treatment of CIS populations for either duration. (F) Increased caspase 3/7 activity in pano-treated (3 days) CIS populations of A549. Bar represents mean ± SEM (n = 3) and statistical significance (unpaired t-test) is indicated. (G) Representative immunoblot showing increased PARP cleavage, decreased Bcl-xL and increased acetylated H3 in both CIS populations of A549 cells following 1- and 3-day treatment with pano (200 nM).CC BY · PubMed Central
Figure 5 Pano has Senolytic Activity in Other Chemotherapy-Induced Senescent NSCLC and HNSCC Cells. Quantitative analysis of SRB-stained adherent CIS cells at 13 and 17 days post treatment indicating significant pano-mediated cell loss, relative to chemotherapy, in (A) H460 CIS (CDDP) (B) H460 CIS (Taxol) (C) UMSCC47 CIS (CDDP) (D) UMSCC47 CIS (Taxol) (E) FaDu CIS (CDDP) and (F) FaDu CIS (Taxol) cells. The same experimental design outlined in schema Fig. 4A was utilized. Bar represents mean ± SEM (n = 6). An unpaired t-test of pano versus chemotherapy at day 17 indicate a statistically significant difference in (A–D) (P < 0.0001); and (P < 0.001) in FaDu, (E and F). (G) Increased caspase 3/7 activity in pano-treated (3 days) CIS populations of H460 cells. Bar represents mean ± SEM (n = 3). P = <0.05: unpaired t-test of pano versus vehicle for both CIS (CDDP and Taxol populations).
Figure 5 Pano has Senolytic Activity in Other Chemotherapy-Induced Senescent NSCLC and HNSCC Cells. Quantitative analysis of SRB-stained adherent CIS cells at 13 and 17 days post treatment indicating significant pano-mediated cell loss, relative to chemotherapy, in (A) H460 CIS (CDDP) (B) H460 CIS (Taxol) (C) UMSCC47 CIS (CDDP) (D) UMSCC47 CIS (Taxol) (E) FaDu CIS (CDDP) and (F) FaDu CIS (Taxol) cells. The same experimental design outlined in schema Fig. 4A was utilized. Bar represents mean ± SEM (n = 6). An unpaired t-test of pano versus chemotherapy at day 17 indicate a statistically significant difference in (A–D) (P < 0.0001); and (P < 0.001) in FaDu, (E and F). (G) Increased caspase 3/7 activity in pano-treated (3 days) CIS populations of H460 cells. Bar represents mean ± SEM (n = 3). P = <0.05: unpaired t-test of pano versus vehicle for both CIS (CDDP and Taxol populations).CC BY · PubMed Central

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Metadata

Authors
Samaraweera, L., Adomako, A., Rodriguez-Gabin, A., & McDaid, H. M.
Affiliations
Albert Einstein College of Medicine (US)
Corresponding author
McDaid, Hayley M.
ORCID
McDaid, Hayley M.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2017-05-15
Volume
7
Issue
1
Pages
1900-1900
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-017-01964-1
PMC
PMC5432488
OpenAlex
W2612435930
Cited by
183
References
44
Citations by year
2026: 13 · 2025: 23 · 2024: 17 · 2023: 21 · 2022: 28 · 2021: 26 · 2020: 26 · 2019: 16 · 2018: 11 · 2017: 2
FWCI
11.13
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Breast Cancer Research Foundation
  • Albert Einstein Cancer Center (P30CA013330)
  • National Institutes of Health (5k12ca132783-12)
  • National Cancer Institute (K12-CA132783, R01CA077263, CA 077263, CA132783, P30CA013330)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Telomeres, Telomerase, and Senescence
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Histone Deacetylase Inhibitors Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cell death mechanisms and regulation
Keywords
panobinostat, senolytics, histone deacetylase inhibitors, non-small cell lung cancer, head and neck squamous cell carcinoma, paclitaxel, cisplatin, cellular senescence, Bcl-xL, histone H3 acetylation, drug synergy
MeSH terms
Squamous Cell Carcinoma of Head and Neck, Panobinostat, Antineoplastic Agents, Carcinoma, Non-Small-Cell Lung, Cell Survival, Dose-Response Relationship, Drug, Drug Synergism, Humans, Lung Neoplasms, Cellular Senescence, Apoptosis, Paclitaxel, Cell Line, Tumor, Histone Deacetylase Inhibitors

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