Tier 4 — mechanistic

Dietary flavonoid fisetin binds to β-tubulin and disrupts microtubule dynamics in prostate cancer cells

Mukhtar E, Adhami VM, Sechi M, Mukhtar H
Cancer Letters 2015 Volume 367, issue 2, pages 173-183

Bibliography

PubMed
PMID 26235140
PubMed Central
PMC4570246
DOI
10.1016/j.canlet.2015.07.030
Funding
United States Public Health Service Grants RO1 CA 160867 and RO1 CA 160867 S1 (to H. Mukhtar); Regione Autonoma della Sardegna grant CRP-25920 (to M. Sechi).
Competing interests
The authors declared no conflicts of interest, financial or otherwise.

Study snapshot

DesignIn vitro tubulin polymerisation assay, surface plasmon resonance (SPR) binding assay, computational molecular docking, and cell-based assays (proliferation, migration, invasion, cell cycle, western blot) in prostate cancer cell lines.
ModelHuman prostate cancer cell lines PC-3 and DU145, and the P-glycoprotein-overexpressing multidrug-resistant cell line NCI/ADR-RES; purified porcine tubulin protein for in vitro polymerisation and SPR assays.
SampleIn vitro/cell-based experiments; no animal or human subjects.
InterventionFisetin at 10 µM (tubulin polymerisation, SPR) and 10-80 µM (cell-based proliferation, migration, invasion, cell cycle and western blot assays), compared against paclitaxel as an active-comparator microtubule-stabilising agent.
DurationAcute in vitro exposures from 60 minutes (polymerisation assay) to 72 hours (cell viability, invasion, colony formation).
EndpointsIn vitro microtubule polymerisation rate; Surface plasmon resonance binding affinity (KD) to β-tubulin; Computational docking free binding energy to the paclitaxel binding pocket; Acetylated α-tubulin, MAP-2, MAP-4 and NudC protein expression; Cell cycle distribution (G2/M arrest); Proliferation, migration and invasion in prostate cancer cells; Viability and colony formation in P-glycoprotein-overexpressing NCI/ADR-RES cells

What the study showed, in plain terms

This is a laboratory (in vitro) study from the same University of Wisconsin group behind several other fisetin-prostate-cancer papers in this Data Center. It asks a narrower, more mechanistic question than most: does fisetin directly interact with the cell's internal scaffolding — the microtubule network — the way established chemotherapy drugs like paclitaxel do?

Using purified tubulin protein, the researchers found that fisetin makes microtubules assemble faster and more efficiently than paclitaxel does under the same test conditions, and a biophysical binding assay (surface plasmon resonance) showed fisetin binds directly to β-tubulin with an affinity in the same range as paclitaxel. Computer modelling suggested fisetin fits into the same binding pocket on tubulin that paclitaxel uses, with a stronger predicted binding energy.

In prostate cancer cells, fisetin treatment increased markers of stable, well-organised microtubules, arrested more cells in the G2/M phase of division (a hallmark of microtubule-targeting chemotherapy drugs), and reduced cell proliferation, migration and invasion. It also reduced viability and colony formation in a cancer cell line engineered to pump out chemotherapy drugs (a model of multidrug resistance), and lowered the level of the resistance-conferring P-glycoprotein pump itself.

Taken together, this paper proposes fisetin as a completely different kind of anti-cancer mechanism from the PI3K/Akt/mTOR-pathway story told in other fisetin-prostate-cancer papers in this Data Center: a microtubule-stabilising agent similar in principle to paclitaxel, but from a dietary source. It is entirely a cell-culture study — there is no whole-animal or human evidence here that fisetin acts this way in a living body.

Key findings

  • In a cell-free tubulin polymerisation assay, fisetin enhanced microtubule assembly faster and more efficiently than paclitaxel at the same 10 µM concentration (Vmax 65 vs 12 mOD/min).
  • Fisetin-treated PC-3 cells resisted cold-induced microtubule depolymerisation far longer than untreated cells, indicating fisetin stabilises the microtubule network in living cells, not just in the test tube.
  • Surface plasmon resonance showed fisetin binds directly to β-tubulin with a KD of 1.59 µM, comparable to paclitaxel's KD of 2.26 µM; computational docking placed fisetin in the same binding pocket as paclitaxel with a more favourable calculated binding energy (~-16 kcal/mol vs ~-6 kcal/mol for paclitaxel).
  • Fisetin treatment (20-80 µM, 24h) dose-dependently increased acetylated α-tubulin and the microtubule-stabilising proteins MAP-2 and MAP-4 in PC-3 and DU145 cells, and downregulated the motor-complex regulator NudC.
  • Fisetin increased the proportion of cells arrested in G2/M phase (31% to 46% at 80 µM) and significantly inhibited PC-3 cell proliferation (p=0.04), invasion (p=0.05) and migration in a dose- and time-dependent manner.
  • In the P-glycoprotein-overexpressing multidrug-resistant NCI/ADR-RES cell line, fisetin decreased cell viability and colony formation and downregulated P-glycoprotein protein expression, suggesting a possible route past chemotherapy resistance.

What this study can and cannot tell us

This entire study is in vitro — purified protein assays and cultured prostate cancer cell lines. There is no animal or human evidence that fisetin acts as a microtubule-stabilising agent in a living organism, and the concentrations used (10-80 µM) are well above achievable free plasma fisetin levels after an oral dose in people.

The proposed binding site was inferred by computational docking into a published crystal structure, using assumptions about a shared paclitaxel/epothilone binding pocket; the authors explicitly state that fisetin's precise binding site on β-tubulin 'remains to be established' by direct structural methods.

As with other in vitro fisetin-cancer papers, this is mechanistic proof-of-concept work from a single laboratory group, not a therapeutic efficacy study, and does not establish a clinical dose, safety profile, or comparative effectiveness against approved microtubule-targeting chemotherapy.

Related papers in the corpus

Tier 4
Dietary flavonoid fisetin: a novel dual inhibitor of PI3K/Akt and mTOR for prostate cancer management
Adhami VM et al. · Biochemical Pharmacology · 2012
Tier 4
Fisetin: A Dietary Antioxidant for Health Promotion
Khan N et al. · Antioxidants & Redox Signaling · 2013
Tier 4
Anti-cancer effects of fisetin on mammary carcinoma cells via regulation of the PI3K/Akt/mTOR pathway: In vitro and in vivo studies
Sun X et al. · International Journal of Molecular Medicine · 2018

Editorial review

Reviewed by the Biohack Blueprint research team

Last verified

Dr. Khaled Mahmoud, MBBChCritical Care Medicine, Longevity Science & Editorial Direction Dr. Amr Yasser, PhDCardiometabolic, Musculoskeletal, Gut & Bioavailability Dr. Abdulla Mostafa, PhDCellular Senescence, Neurological, Mitochondrial & Brain-Ageing Biology