Tier 4 — mechanistic

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, Ma X, Li Q, Yang Y, Xu X, Sun J, Yu M, Cao K, Yang L, Yang G, Zhang G, Wang X
International Journal of Molecular Medicine 2018 Volume 42, issue 2, pages 811-820

Bibliography

PubMed
PMID 29749427
PubMed Central
PMC6034928
DOI
10.3892/ijmm.2018.3654
Video explainer
Watch on YouTube Biohack Blueprint's video walkthrough of this study.
Funding
Beijing Natural Science Foundation (grant nos. 7162084 and 7174312), National Natural Science Foundation of China (grant nos. 81373815 and 81673924), Beijing Municipal Education Commission (grant no. KM201510025025), and the Specialized Research Fund for the Doctoral Program of Higher Education of China (grant no. 20131107110014).
Competing interests
The authors declared that they have no competing interests.

Study snapshot

DesignIn vitro cell-line study (MTT viability assay, electrical cell-substrate impedance sensing for proliferation/migration/invasion, flow cytometry apoptosis assay, western blotting) plus an in vivo murine orthotopic mammary tumour model.
ModelMouse mammary carcinoma 4T1 and 4T1-luc2 cells, human breast cancer cell lines MCF-7 and MDA-MB-231, HUV-EC-C human umbilical vein endothelial cells; 30 female BALB/c mice (6-8 weeks) bearing 4T1 orthotopic mammary tumours.
SampleIn vitro experiments performed in triplicate; in vivo, 30 female BALB/c mice divided into vehicle, fisetin and control groups (n=10/group).
InterventionIn vitro fisetin at 0, 20, 40 and 80 µM (proliferation/apoptosis/western blot) or 0-40 µM (migration/invasion assays); in vivo intraperitoneal fisetin 223 mg/kg or vehicle (DMSO:polyethylene glycol 200, 1:4) daily for 3 successive weeks.
DurationIn vitro exposures of 24-72 hours; in vivo dosing for 21 days with tumour follow-up to day 34.
EndpointsBreast cancer cell viability (MTT assay); Proliferation, migration and invasion (ECIS platform); Apoptosis (Annexin V/PI flow cytometry, TUNEL); PI3K/Akt/mTOR pathway protein expression (western blot); Orthotopic tumour volume and weight in vivo; Liver and kidney biochemical function (ALT, AST, BUN, creatinine)

What the study showed, in plain terms

This Chinese oncology group tested fisetin against mouse and human breast cancer cells in the dish, then against real tumours grown in mice, to see whether the same PI3K/Akt/mTOR pathway that fisetin blocks in prostate and lung cancer also matters in breast cancer.

Fisetin reduced the number of viable 4T1, MCF-7 and MDA-MB-231 breast cancer cells, slowed their migration and invasion, and pushed more of the mouse 4T1 cells into apoptosis. In mice with real orthotopic breast tumours, daily fisetin injections for three weeks shrank tumour volume and weight compared with untreated controls.

The mechanistic story matches other fisetin cancer papers in this Data Center: fisetin lowered active (phosphorylated) PI3K, Akt and mTOR signalling in the tumour cells, alongside more of the pro-apoptotic protein Bax and less of the protective Bcl-xL.

The important caveat for readers is dosing and route. The mouse dose (223 mg/kg by injection, daily for three weeks) is far higher and far more sustained than any oral fisetin supplement dose in people, and it raised liver enzymes (ALT and AST) in the treated mice — a reminder that fisetin is not free of dose-dependent liver stress at high exposure, even though it showed no effect on kidney markers.

Key findings

  • Fisetin significantly reduced viability of 4T1, MCF-7 and MDA-MB-231 breast cancer cells in a concentration- and time-dependent manner (20-80 µM, 24-48 hours).
  • Using real-time impedance sensing, fisetin inhibited 4T1 cell proliferation, slowed wound-healing migration, and reduced the cells' ability to invade through a simulated vascular endothelial barrier.
  • Fisetin induced apoptosis of 4T1 cells, with apoptotic rates rising from about 1% in controls to 10-24% across the 20-80 µM range.
  • Western blotting showed fisetin reduced Akt, P70 and mTOR expression and their phosphorylated (active) forms, upregulated the pro-apoptotic protein Bax, and downregulated the anti-apoptotic Bcl-xL.
  • In an orthotopic 4T1 mouse mammary tumour model, daily intraperitoneal fisetin (223 mg/kg) for 21 days significantly reduced tumour volume and tumour weight compared with control and vehicle-only groups, and increased tumour cell apoptosis on TUNEL staining.
  • Fisetin-treated mice had significantly higher serum ALT and AST than controls, indicating a degree of liver stress at this high, sustained injected dose, while kidney markers (BUN, creatinine) were unaffected.

What this study can and cannot tell us

This is an in vitro and murine in vivo study only; there is no human data on fisetin and breast cancer. The in vivo fisetin dose (223 mg/kg/day, intraperitoneal, for three weeks) is far above any achievable or studied oral supplement dose in people and cannot be extrapolated to human dosing.

The authors themselves note that fisetin's poor solubility and low bioavailability limited the study: the three-week treatment window was too short to observe lung metastasis in this aggressive 4T1 model, and two mice in the fisetin group reached the humane endpoint (>10% body weight loss) during treatment, forcing early study termination before metastasis could be assessed.

The rise in ALT and AST in the fisetin-treated mice is a genuine safety signal at this dose and route, even though the authors could not fully separate the contribution of the DMSO/PEG-200 vehicle from fisetin itself.

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
Dietary flavonoid fisetin binds to β-tubulin and disrupts microtubule dynamics in prostate cancer cells
Mukhtar E et al. · Cancer Letters · 2015

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