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Cited by 688Natural product bioactivities and synthesisPhytochemical Studies and BioactivitiesPhytochemical compounds biological activities

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Betulin Is a Potent Anti-Tumor Agent that Is Enhanced by Cholesterol

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Mullauer, Franziska B., Kessler, Jan H., Medema, Jan Paul·2009·PLoS ONE
DOI 10.1371/journal.pone.0005361PMC PMC2671171
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Abstract

Betulinic Acid (BetA) and its derivatives have been extensively studied in the past for their anti-tumor effects, but relatively little is known about its precursor Betulin (BE). We found that BE induces apoptosis utilizing a similar mechanism as BetA and is prevented by cyclosporin A (CsA). BE induces cell death more rapidly as compared to BetA, but to achieve similar amounts of cell death a considerably higher concentration of BE is needed. Interestingly, we observed that cholesterol sensitized cells to BE-induced apoptosis, while there was no effect of cholesterol when combined with BetA. Despite the significantly enhanced cytotoxicity, the mode of cell death was not changed as CsA completely abrogated cell death. These results indicate that BE has potent anti-tumor activity especially in combination with cholesterol.

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Figures

Figure 1 Cholesterol strongly enhances cytotoxic effects of BE but not BetA. Jurkat cells were treated with the indicated concentrations of BetA (A), BetA in combination with 5 µM cholesterol (B), BE (C), BE in combination with 5 µM cholesterol (D) or various concentrations of cholesterol only (E). Cell death was monitored after 12, 24, 48 and 72 hours using PI exclusion. A549 lung cancer (F) and HeLa cervix carcinoma (G) cell lines were treated with 5 µM cholesterol (chol), 5 µg/ml BE (5 BE) or the combination of 5 µg/ml BE with 5 µM cholesterol (5 BE+chol) and after 24 hours cell death was analyzed via PI exclusion.
Figure 1 Cholesterol strongly enhances cytotoxic effects of BE but not BetA. Jurkat cells were treated with the indicated concentrations of BetA (A), BetA in combination with 5 µM cholesterol (B), BE (C), BE in combination with 5 µM cholesterol (D) or various concentrations of cholesterol only (E). Cell death was monitored after 12, 24, 48 and 72 hours using PI exclusion. A549 lung cancer (F) and HeLa cervix carcinoma (G) cell lines were treated with 5 µM cholesterol (chol), 5 µg/ml BE (5 BE) or the combination of 5 µg/ml BE with 5 µM cholesterol (5 BE+chol) and after 24 hours cell death was analyzed via PI exclusion.CC BY · PubMed Central
Figure 2 BE/cholesterol induces apoptosis in Jurkat cells. (A) Jurkat cells were pretreated with 20 µM zVAD.fmk for at least one hour prior to addition of either DMSO, 5 µM cholesterol (5 Chol), 5 µg/ml BE (5 BE) or 5 µg/ml BE in combination with 5 µM cholesterol (5 BE+chol). After 24 hours DNA fragmentation was assessed by FACS analysis of propidium iodide (PI) stained nuclei. (B) Jurkat cells were treated as described in (A) but after 24 hours PARP cleavage was assessed by immunoblotting. The protein kinase ERK was used as a loading control.
Figure 2 BE/cholesterol induces apoptosis in Jurkat cells. (A) Jurkat cells were pretreated with 20 µM zVAD.fmk for at least one hour prior to addition of either DMSO, 5 µM cholesterol (5 Chol), 5 µg/ml BE (5 BE) or 5 µg/ml BE in combination with 5 µM cholesterol (5 BE+chol). After 24 hours DNA fragmentation was assessed by FACS analysis of propidium iodide (PI) stained nuclei. (B) Jurkat cells were treated as described in (A) but after 24 hours PARP cleavage was assessed by immunoblotting. The protein kinase ERK was used as a loading control.CC BY · PubMed Central
Figure 3 The death receptor pathway is not involved in BE/cholesterol induced apoptosis. Jurkat control (JA3), FADD deficient (FADD ko) or caspase-8 deficient (Casp-8 ko) cells were treated with TRAIL (0.5 µg/ml plus 1 µg/ml anti-FLAG) (A) or with either 5 µM cholesterol (chol) or 5 µg/ml BE in combination with 5 µM cholesterol (5 BE+chol) and after 24 hours DNA fragmentation was analyzed.
Figure 3 The death receptor pathway is not involved in BE/cholesterol induced apoptosis. Jurkat control (JA3), FADD deficient (FADD ko) or caspase-8 deficient (Casp-8 ko) cells were treated with TRAIL (0.5 µg/ml plus 1 µg/ml anti-FLAG) (A) or with either 5 µM cholesterol (chol) or 5 µg/ml BE in combination with 5 µM cholesterol (5 BE+chol) and after 24 hours DNA fragmentation was analyzed.CC BY · PubMed Central
Figure 4 BE/cholesterol induced apoptosis is not affected by Bcl-2 over-expression but is inhibited in the presence of cyclosporin A. (A) Jurkat control (wt) or Bcl-2 over-expressing cells (Bcl-2) were treated as indicated (5BE = 5 µg/ml BE; chol = 5 µM cholesterol; CsA = 5 µg/ml cyclosporin A), after 24 hours intracellular staining for cytochrome c release was performed. (B, C, D) Jurkat control (wt) or Bcl-2 over-expressing cells were treated with 5 µg/ml BE/ 5 µM cholesterol either in the absence or presence of 5 µg/ml cyclosporin A. After 24 hours ROS (B), DNA fragmentation (C) and overall cell death (D) were assessed by FACS analysis. (E, F) Jurkat control (wt) or Bcl-2 over-expressing cells were treated with 5 µg/ml BE/ 5 µM cholesterol and PI exclusion (E) or DNA fragmentation (F) were measured after 0, 4, 8, 16 and 24 hours.
Figure 4 BE/cholesterol induced apoptosis is not affected by Bcl-2 over-expression but is inhibited in the presence of cyclosporin A. (A) Jurkat control (wt) or Bcl-2 over-expressing cells (Bcl-2) were treated as indicated (5BE = 5 µg/ml BE; chol = 5 µM cholesterol; CsA = 5 µg/ml cyclosporin A), after 24 hours intracellular staining for cytochrome c release was performed. (B, C, D) Jurkat control (wt) or Bcl-2 over-expressing cells were treated with 5 µg/ml BE/ 5 µM cholesterol either in the absence or presence of 5 µg/ml cyclosporin A. After 24 hours ROS (B), DNA fragmentation (C) and overall cell death (D) were assessed by FACS analysis. (E, F) Jurkat control (wt) or Bcl-2 over-expressing cells were treated with 5 µg/ml BE/ 5 µM cholesterol and PI exclusion (E) or DNA fragmentation (F) were measured after 0, 4, 8, 16 and 24 hours.CC BY · PubMed Central
Figure 5 BE/cholesterol induced apoptosis is independent of Bax/Bak. (A) Wildtype (wt) or Bax/Bak double knockout (DKO) mouse embryonic fibroblasts (MEFs) were treated as indicated and after 24 hours cell death was assessed by PI exclusion. Etoposide was included as a control for functionality of the cells. (B) Wt or DKO MEFs were treated as indicated and after 24 hours cells were subjected to immunoblotting to determine PARP processing. ERK was used as control for equal protein amounts. (C) Wt and DKO MEFs were treated as indicated for 24 hours before measuring cytochrome c release by intracellular FACS staining.
Figure 5 BE/cholesterol induced apoptosis is independent of Bax/Bak. (A) Wildtype (wt) or Bax/Bak double knockout (DKO) mouse embryonic fibroblasts (MEFs) were treated as indicated and after 24 hours cell death was assessed by PI exclusion. Etoposide was included as a control for functionality of the cells. (B) Wt or DKO MEFs were treated as indicated and after 24 hours cells were subjected to immunoblotting to determine PARP processing. ERK was used as control for equal protein amounts. (C) Wt and DKO MEFs were treated as indicated for 24 hours before measuring cytochrome c release by intracellular FACS staining.CC BY · PubMed Central

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Metadata

Authors
Mullauer, F. B., Kessler, J. H., & Medema, J. P.
Affiliations
  • Amsterdam UMC Location University of Amsterdam (NL)
  • Leiden University Medical Center (NL)
Corresponding author
Medema, Jan Paul
ORCID
Medema, Jan Paul
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2009-04-28
Volume
4
Issue
4
Pages
e1
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0005361
PMC
PMC2671171
OpenAlex
W2168735820
Cited by
688
References
39
Citations by year
2026: 7 · 2025: 23 · 2024: 25 · 2023: 30 · 2022: 29 · 2021: 55 · 2020: 35 · 2019: 29 · 2018: 37 · 2017: 34 · 2016: 45 · 2015: 39 · 2014: 43 · 2013: 55 · 2012: 55
FWCI
26.95
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Data Archiving and Networked Services (DANS) (submittedVersion)
  • UvA-DARE (University of Amsterdam) (submittedVersion, other-oa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Pure Amsterdam UMC (submittedVersion)
  • Data Archiving and Networked Services (DANS) (submittedVersion, other-oa)
Funders
Nationaal Fonds tegen Kanker
Topics
  • Physical Sciences › Chemistry › Organic Chemistry › Natural product bioactivities and synthesis
  • Physical Sciences › Chemistry › Organic Chemistry › Phytochemical Studies and Bioactivities
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Drug Discovery › Phytochemical compounds biological activities
Keywords
betulin, betulinic acid, cholesterol, apoptosis, anticancer activity, cyclosporine, cytotoxicity, combination therapy, cell death
MeSH terms
Betulinic Acid, Animals, Antineoplastic Agents, Phytogenic, Cell Line, Cells, Cultured, Cholesterol, Drug Synergism, HeLa Cells, Humans, Triterpenes, Gene Expression, Cyclosporine, Apoptosis, Reactive Oxygen Species, Mice, Knockout, Jurkat Cells, Genes, bcl-2, bcl-2 Homologous Antagonist-Killer Protein, bcl-2-Associated X Protein, Mice, Fas-Associated Death Domain Protein, Pentacyclic Triterpenes

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