Tier 4 — mechanistic

Fisetin Induces Apoptosis Through p53-Mediated Up-Regulation of DR5 Expression in Human Renal Carcinoma Caki Cells

Min KJ, Nam JO, Kwon TK
Molecules 2017 Volume 22, issue 8, article 1285

Bibliography

PubMed
PMID 28767099
PubMed Central
PMC6151974
DOI
10.3390/molecules22081285
Funding
National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIP) (2014R1A5A2010008).
Competing interests
The authors declare no conflict of interest.

Study snapshot

DesignIn vitro mechanistic study using flow cytometry, western blotting, siRNA knockdown, DAPI/DNA fragmentation assays and promoter-luciferase reporter assays to dissect the apoptosis pathway induced by fisetin.
ModelHuman renal carcinoma (Caki) cells; comparator human hepatoma (SK-Hep1) cells and normal mouse kidney (TCMK-1) cells.
SampleExperiments performed in triplicate (three independent samples).
InterventionFisetin 50-200 µM for 24 hours; pan-caspase inhibitor z-VAD-fmk (20 µM) co-treatment; DR5, p53 and CHOP siRNA knockdown; cycloheximide (20 µg/mL) protein-stability chase; ROS scavengers N-acetylcysteine (5 mM) and glutathione ethyl ester (2 mM).
Duration24-hour treatment for most endpoints; time-course analysis of DR5 induction over 0-24 hours.
EndpointsSub-G1 apoptotic population (flow cytometry); PARP cleavage and caspase-3 (DEVDase) activity; Death receptor 5 (DR5) and DR4 mRNA/protein expression; p53 protein expression and stability; CHOP/ATF4 endoplasmic reticulum stress markers; Reactive oxygen species production (H2DCF-DA)

What the study showed, in plain terms

This South Korean lab study asked how fisetin kills kidney cancer cells at the molecular level, using human renal carcinoma (Caki) cells grown in a dish.

Fisetin triggered clear apoptosis (programmed cell death) in the cancer cells, and this was blocked when the researchers used a drug that shuts down caspases, the enzymes that execute apoptosis — confirming a classic caspase-dependent death pathway.

The key discovery was that fisetin turns up a 'death receptor' on the cell surface called DR5, and this was the trigger for apoptosis: when the researchers silenced the DR5 gene, fisetin's cancer-killing effect was significantly blunted. They traced the chain of events further back and found fisetin stabilises the well-known tumour-suppressor protein p53, which in turn switches on DR5.

Interestingly, fisetin also increased cell stress signals (CHOP, a marker of endoplasmic reticulum stress, and reactive oxygen species) but these turned out to be side effects, not the actual cause of the cell death — silencing CHOP or scavenging the free radicals did not stop fisetin's apoptotic effect. This kind of careful pathway dissection is valuable mechanistic science, though it stays entirely at the level of cells in a dish.

Key findings

  • Fisetin (50-200 µM, 24h) dose-dependently induced apoptosis in Caki renal carcinoma cells (sub-G1 population, PARP cleavage, chromosomal condensation/fragmentation, DNA fragmentation), and normal TCMK-1 kidney cells were less sensitive than the cancer cells.
  • The pan-caspase inhibitor z-VAD-fmk completely blocked fisetin-induced apoptosis, confirming a caspase-dependent mechanism; caspase-3 (DEVDase) activity rose in a dose-dependent manner.
  • Fisetin markedly up-regulated death receptor 5 (DR5) at both mRNA and cell-surface protein level within 6 hours, and DR5 siRNA knockdown significantly reduced fisetin-induced apoptosis and PARP cleavage.
  • Fisetin increased p53 protein levels via enhanced protein stability (not increased mRNA); p53 siRNA knockdown markedly inhibited fisetin-induced DR5 up-regulation, establishing a p53-to-DR5 signalling axis.
  • Fisetin also induced CHOP/ATF4 endoplasmic reticulum stress markers and reactive oxygen species production, but neither CHOP knockdown nor ROS scavengers (NAC, GEE) affected fisetin-induced apoptosis, indicating these are parallel but non-causal effects in this cell type.
  • Fisetin similarly induced apoptosis in human hepatoma SK-Hep1 cells, suggesting the pro-apoptotic effect is not unique to renal carcinoma cells.

What this study can and cannot tell us

This study used only cultured cell lines; there is no animal tumour model or human data to confirm this mechanism operates, or that fisetin has anti-tumour efficacy, in a living organism.

Fisetin concentrations of 50-200 µM used throughout are well above the low micromolar-or-lower plasma concentrations typically achievable with oral fisetin supplementation in humans, given fisetin's poor bioavailability.

The p53-DR5 apoptosis mechanism identified here contrasts with other fisetin cancer papers (e.g., in gastric cancer) where p53 induction did not drive fisetin-induced apoptosis, indicating the pathway is cell-type dependent and cannot be generalised across all cancers.

Editorial review

Reviewed by the Biohack Blueprint research team

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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