Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a
naphthoquinone isolated from the roots of Plumbaginaceae plants, has potential antiproliferative activity against several
tumor types. We have examined the effects of
plumbagin on cellular microtubules ex vivo as well as its binding with purified
tubulin and microtubules in vitro. Cell viability experiments using human non-small lung epithelium
carcinoma cells (A549) indicated that the IC 50 value for
plumbagin is 14.6 microM. Immunofluorescence studies using an antitubulin
FITC conjugated antibody showed a significant perturbation of the interphase microtubule network in a dose dependent manner. In vitro polymerization of purified
tubulin into microtubules is inhibited by
plumbagin with an IC 50 value of 38 +/- 0.5 microM. Its binding to
tubulin quenches
protein tryptophan fluorescence in a time and concentration dependent manner. Binding of
plumbagin to
tubulin is slow, taking 60 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 235.12 +/- 36 M (-1) s (-1) and 11.63 +/- 11 M (-1) s (-1) at 25 degrees C respectively. The stoichiometry of
plumbagin binding to
tubulin is 1:1 (mole:mole) with a dissociation constant of 0.936 +/- 0.71 microM at 25 degrees C.
Plumbagin competes for the
colchicine binding site with a K i of 7.5 microM as determined from a modified Dixon plot. Based on these data we conclude that
plumbagin recognizes the
colchicine binding site to
tubulin. Further study is necessary to locate the pharmacophoric point of attachment of the inhibitor to the
colchicine binding site of
tubulin.