Griseofulvin stabilizes microtubule dynamics, activates p53 and inhibits the proliferation of MCF-7 cells synergistically with vinblastine.
BACKGROUND
Griseofulvin, an antifungal drug, has recently been shown to inhibit proliferation of various types of cancer cells and to inhibit tumor growth in athymic mice. Due to its low toxicity, griseofulvin has drawn considerable attention for its potential use in cancer chemotherapy. This work aims to understand how griseofulvin suppresses microtubule dynamics in living cells and sought to elucidate the antimitotic and antiproliferative action of the drug.


METHODS
The effects of griseofulvin on the dynamics of individual microtubules in live MCF-7 cells were measured by confocal microscopy. Immunofluorescence microscopy, western blotting and flow cytometry were used to analyze the effects of griseofulvin on spindle microtubule organization, cell cycle progression and apoptosis. Further, interactions of purified tubulin with griseofulvin were studied in vitro by spectrophotometry and spectrofluorimetry. Docking analysis was performed using autodock4 and LigandFit module of Discovery Studio 2.1.


RESULTS
Griseofulvin strongly suppressed the dynamic instability of individual microtubules in live MCF-7 cells by reducing the rate and extent of the growing and shortening phases. At or near half-maximal proliferation inhibitory concentration, griseofulvin dampened the dynamicity of microtubules in MCF-7 cells without significantly disrupting the microtubule network. Griseofulvin-induced mitotic arrest was associated with several mitotic abnormalities like misaligned chromosomes, multipolar spindles, misegregated chromosomes resulting in cells containing fragmented nuclei. These fragmented nuclei were found to contain increased concentration of p53. Using both computational and experimental approaches, we provided evidence suggesting that griseofulvin binds to tubulin in two different sites; one site overlaps with the paclitaxel binding site while the second site is located at the alphabeta intra-dimer interface. In combination studies, griseofulvin and vinblastine were found to exert synergistic effects against MCF-7 cell proliferation.


CONCLUSIONS
The study provided evidence suggesting that griseofulvin shares its binding site in tubulin with paclitaxel and kinetically suppresses microtubule dynamics in a similar manner. The results revealed the antimitotic mechanism of action of griseofulvin and provided evidence suggesting that griseofulvin alone and/or in combination with vinblastine may have promising role in breast cancer chemotherapy.
Rathinasamy
Krishnan
K
Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai - 400076, Maharashtra India.
Jindal
Bhavya
B
Asthana
Jayant
J
Singh
Parminder
P
Balaji
Petety V
PV
Panda
Dulal
D
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001665:Binding Sites; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D020090:Chromosome Segregation / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006118:Griseofulvin / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D018386:Kinetochores / Q000187:drug effects / Q000378:metabolism; D018613:Microscopy, Confocal; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013997:Time Factors; D014162:Transfection; D014404:Tubulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050257:Tubulin Modulators / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D014747:Vinblastine / Q000494:pharmacology
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; C495901:TP53 protein, human; D014404:Tubulin; D050257:Tubulin Modulators; D016159:Tumor Suppressor Protein p53; D006118:Griseofulvin; D014747:Vinblastine