Cancer Letters

Cancer Letters

Volume 367, Issue 2, 28 October 2015, Pages 173-183
Cancer Letters

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

https://doi.org/10.1016/j.canlet.2015.07.030Get rights and content

Highlights

  • Fisetin enhances tubulin polymerization and increases α-tubulin acetylation and MAP-2 and MAP-4 expression.

  • Fisetin renders cells resistant to cold-induced microtubule depolymerization.

  • Fisetin arrests cells in the G2/M phase of cell cycle.

  • Fisetin inhibits cell proliferation, invasion and migration.

  • Fisetin inhibits the viability, colony formation and decreases the expression of P-gp in multidrug-resistant cancer cell line NCI/ADR-RES.

Abstract

Microtubule targeting based therapies have revolutionized cancer treatment; however, resistance and side effects remain a major limitation. Therefore, novel strategies that can overcome these limitations are urgently needed. We made a novel discovery that fisetin, a hydroxyflavone, is a microtubule stabilizing agent. Fisetin binds to tubulin and stabilizes microtubules with binding characteristics far superior than paclitaxel. Surface plasmon resonance and computational docking studies suggested that fisetin binds to β-tubulin with superior affinity compared to paclitaxel. Fisetin treatment of human prostate cancer cells resulted in robust up-regulation of microtubule associated proteins (MAP)-2 and -4. In addition, fisetin treated cells were enriched in α-tubulin acetylation, an indication of stabilization of microtubules. Fisetin significantly inhibited PCa cell proliferation, migration, and invasion. Nudc, a protein associated with microtubule motor dynein/dynactin complex that regulates microtubule dynamics, was inhibited with fisetin treatment. Further, fisetin treatment of a P-glycoprotein overexpressing multidrug-resistant cancer cell line NCI/ADR-RES inhibited the viability and colony formation. Our results offer in vitro proof-of-concept for fisetin as a microtubule targeting agent. We suggest that fisetin could be developed as an adjuvant for treatment of prostate and other cancer types.

Introduction

Microtubules play an essential role in the intracellular cytoskeletal framework. They are polymers made of repeating αβ-tubulin heterodimers that are involved in cell division, migration, signaling, and intracellular trafficking which also makes them important in cancer cell proliferation and metastasis [1]. The dynamics of microtubule polymerization are central to their biological function. Polymerization dynamics allows microtubules to adapt to spatial arrangements that can change rapidly in response to cellular needs and to perform mechanical work. Correspondingly, microtubule dynamics as well as associated signaling pathways are important targets for novel anti-cancer and anti-neurodegenerative therapies. Thus, microtubule targeting agents (MTAs) have become a very successful class of cancer drugs that have proven their potency and efficacy against many cancer types including prostate cancer (PCa). Despite their success, resistance and severe side effects restrict their clinical use [2].

One of the major problems in chemotherapy is multidrug resistance (MDR) against anti-cancer drugs. ATP-binding cassette (ABC) transporters are a family of proteins that mediate MDR via ATP-dependent drug efflux pumps. Although many MDR inhibitors have been identified, none of them have proven clinically useful without side effects. Therefore, novel approaches are required to identify non-toxic MDR inhibitors that can inhibit or circumvent MDR. Many MTA are substrates of P-glycoprotein (P-gp), a 170 kDa protein encoded by human ABCB1 gene. This gene belongs to a broad spectrum ATP-dependent efflux pump that reduces drug efficacy and higher doses of these drugs are required to achieve adequate intracellular concentration in cancer cells [3], [4], [5]. Therefore, inhibition of P-gp function represents a logical approach to overcome MDR in cancer chemotherapy.

There has been substantial research effort to investigate the use of natural products, with good safety profile, to inhibit cancer and many other diseases [6], [7]. Fisetin (3,3′,4′,7-tetrahydroxy flavone) is a flavonol present in the diet at concentrations ranging from 2 to 160 µg/g, and at much higher concentrations as an ingredient in nutritional supplements. Fisetin has anti-proliferative, apoptotic and antioxidant activities, and is under study for its chemopreventive/chemotherapeutic effects against several cancers, as well as for its neuroprotective properties [8], [9], [10]. We report here that fisetin binds to β-tubulin and disrupts microtubule dynamics and is able to overcome drug resistance in the NCI/ADR-RES cell line.

Section snippets

Materials and methods

Fisetin and 4′,6-diamidino-2-phenyindole (DAPI), and paclitaxel were purchased from Sigma (St. Louis, MO); PC-3, DU-145 cells and dimethyl sulfoxide (DMSO) from ATCC (Manassas, VA). NCI/ADR-RES cell line was obtained from the DTP Human Tumor Cell Line Screen (Developmental Therapeutics Program, NCI, Frederick, MD). RPMI-1640 media, fetal bovine serum, penicillin, and streptomycin from Mediatech, Inc (Manassas, VA); α-tubulin and acetylated α-tubulin from Santa Cruz (Dallas, TX); FITC-conjugated

Fisetin stabilizes microtubule assembly in vitro

Using an in vitro tubulin polymerization assay, we discovered that fisetin enhanced microtubule polymerization to a greater extent than paclitaxel under conditions that required an enhancer for microtubules to polymerize efficiently (Fig. 1A). Paclitaxel stabilizes microtubule polymer and prevents it from disassembling [11]. Our observation suggests that fisetin at similar dose has the same effect but is faster and much efficient (fisetin Vmax = 65 m OD/min vs. paclitaxel Vmax = 12 m OD/min)

Discussion

Natural agents that target microtubules and disrupt the normal function of the mitotic spindle have an excellent selectivity against cancer cells and have been proven to be the key components of chemotherapeutic regimens for various solid tumors. Thus, development and discovery of new drugs, and exploring new treatment strategies that reduce side effects and circumvent drug resistance could provide more effective therapeutic options for patients with cancer. A better approach is to identify

Conclusion

Drugs that affect microtubule dynamics are among the most effective anticancer agents in routine clinical use. Although the vast majority of known MT-stabilizing agents are structurally complex, the data reported here provide the first evidence that fisetin is a microtubule stabilizing agent far superior than paclitaxel. In terms of therapy, almost all cytotoxic chemotherapeutic drugs arrest dividing cells by causing DNA damage or targeting products of proliferation-signature genes, as we

Conflict of interest

The authors declare no conflicts of interest, financial or otherwise.

Acknowledgements

This work was supported by the United States Public Health Service Grants RO1 CA 160867 and RO1 CA 160867 S1 (to H. Mukhtar). M.S. gratefully acknowledges the Regione Autonoma della Sardegna for financial support grant CRP-25920 (annualità 2010) within the frame of “Legge regionale n° 7/2007”, and also thanks the Precision Antibody (Columbia, MD, USA) for their helpful technical assistance. We would like to thank the Biological Testing Branch of National Cancer Institute for providing the

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