Fukutaro Mizuhashi

Antitumor activities of IKP-104, a 4(1H)-pyrizinone derivative, on cultured and implanted tumors

Antitumor activities of IKP‐104, a 4(1H)‐pyrizinone derivative, were investigated with cultured tumor cell lines and implanted tumors in mice. IKP‐104 inhibited the growth of cultured murine tumor cell lines (L1210 leukemia, Lewis lung carcinoma and B16 melanoma) and human tumor cell lines (K562 leukemia and HeLa cervical carcinoma). It also had antitumor effects on implanted murine ascitic tumors (L1210 leukemia and sarcoma 180) and a murine solid tumor (Lewis lung carcinoma). IKP‐104 could be classified as a phase‐dependent cytostatic drug based on the mode of growth inhibition of cultured B16 melanoma cells compared with those of several other antitumor agents. The effect of IKP‐104 on the cell cycle traverse of cultured B16 melanoma cells was estimated by morphological and flow cytometric analyses. Cells accumulated in the mitotic phase, and abortive mitosis or polyploidy or multinucleation was induced from 6 h after exposure to IKP‐104. Based on these results, IKP‐104 is expected to be useful for the treatment of tumors, and its mode of action seemed to be similar to that of metaphase arrestants such as colchicine or vinca alkaloids.

Podophyllotoxin and Nocodazole Counter the Effect of IKP104 on Tubulin Decay

Tubulin, the subunit protein of microtubules, undergoes a time-dependent loss of functional properties known as decay. We have previously shown that the drug 2-(4-fluorophenyl)-1-(2-chloro-3,5-dimethoxyphenyl)-3-methyl-6-phenyl-4(1H)-pyridinone (IKP104) accelerates decay, but that in the presence of colchicine, IKP104 becomes a stabilizer of tubulin. To see if this is due to conformational effects specific to colchicine or simply to occupancy at the colchicine site, we examined the effects of nocodazole and podophyllotoxin, two well-known competitive inhibitors of colchicine for binding to tubulin, on IKP104’s acceleration of decay. We found that podophyllotoxin abolished IKP104’s accelerating effect and, like colchicine, turned it into a stabilizer of tubulin. Nocodazole’s effects were similar to those of podophyllotoxin and colchicine, in that it abolished IKP104-induced enhancement of decay; however, in the presence of nocodazole, IKP104 caused little or no stabilization of tubulin. Since colchicine, nocodazole, and podophyllotoxin have very different interactions with tubulin, but all inhibit the IKP104-induced enhancement of decay, our findings suggest that this inhibition arises from occupancy of the colchicine site rather than from a direct conformational effect of these two drugs.

Distinct and overlapping binding sites for IKP104 and vinblastine on tubulin

IKP104 is one of a group of tubulin-binding drugs whose interaction with tubulin suggests that it may bind to the protein at or close to the region where vinblastine binds. By itself IKP104 is a potent enhancer of tubulin decay as evidenced by the fact that it induces the exposure of the sulfhydryl groups and hydrophobic areas on tubulin. In this respect, IKP104 differs from vinblastine and other drugs such as phomopsin A, dolastatin 10, rhizoxin, and maytansine which are competitive or noncompetitive inhibitors of vinblastine binding. In contrast, however, in the presence of colchicine, IKP104 behaves differently and strongly stabilizes tubulin, to an extent much greater than does colchicine alone. IKP104 appears to have two classes of binding site on tubulin, differing in affinity; the acceleration of decay appears to be mediated by the low-affinity site (Chaudhuriet al., 1998,J. Protein Chem., in press). We investigated the relationship of the binding of IKP104 and vinblastine. We found that the high-affinity site or sites of IKP104 overlap with or interact with the vinblastine-binding sites, but that the low-affinity site is distinctly different.

Tubulin stability and decay: Mediation by two distinct classes of IKP104-binding sites

IKP104, a novel antimitotic drug, has two classes of binding sites on bovine brain tubulin with different affinities. IKP104, by itself, enhances the decay of tubulin, but in the presence of colchicine or podophyllotoxin, it stabilizes tubulin instead of opening up the hydrophobic areas [Luduena et al. (1995), Biochemistry34, 15751–15759], Here, we have dissected these two apparently contradictory effects of IKP104 by cleaving the C-terminal ends of both α and β subunits of tubulin with subtilisin. We have found that the selective removal of the C-terminal ends from both the α and β subunits of αβ tubulin lowers the sulfhydryl titer by approximately 1.5 mol/mol of dimer. Interestingly, IKP104 does not increase either the sulfhydryl liter or the exposure of hydrophobic areas of this subtilisin-treated tubulin (αsβs). Moreover, IKP104 lowers the sulfhydryl titer of αsβs tubulin approximately by 1 mol/mol and appears to inhibit completely the time-dependent decay of αsβs tubulin. The cleavage at the C-terminal ends of both α and β modulates the effect of IKP104 on the β subunit, but not on the α subunit. Fluorometric binding data analysis suggests that IKP104 binds to the αsβs tubulin only at the high-affinity site; the low-affinity site(s) disappear almost completely. The sulfhydryl titer data for α and β and the fluoromelric data therefore suggest that the interaction of IKP104 at the high-affinity site on tubulin is not regulated by the C-terminal domains of α and β and the effect of the high-affinity site is restricted largely to the α subunit, while the low-affinity-site binding is modulated by the C-terminal domain of β. It also appears that the stabilization and the acceleration of the decay of tubulin are mediated by distinct interactions of IKP104 with its high- and low-affinity sites on tubulin, respectively.

Interaction of the Tumor Inhibitor IKP-104, a 4(1H)-Pyridinone Derivative, with Microtubule Proteins

The effects of a mitotic arrestant, IKP‐104, which has an antitumor activity, on the in vitro., polymerization and depolymerization of rat brain microtubules were investigated. IKP‐104 inhibited microtubule polymerization at concentrations greater than 0.71 × 10‐6M.,a nd its IC50 value was determined to be 1.31 × 10‐6 M by probit analysis. Fifty‐two percent of pre‐polymerized microtubules depolymerized at 1.31 × 10‐6M IKP‐104. Electron micrographs of microtubules taken immediately after treatment with 1 × 10‐3M IKP‐104 revealed a fraying of microtubule ends into elongated coil‐like filaments, which were composed of 2 or 3 protofilaments. When microtubule protein treated with 1 × 10‐3M IKP‐104 was cleaved by trypsin, fragments of 41,36, 34, 23,21,19 and 16 kilodaltons (kDa) derived from a‐tubulin were produced. In particular, the 19, 23 and 34 kDa fragments were characteristically observed in the trypsin cleavage of microtubules treated with IKP‐104, and these fragments were not observed with untreated microtubules. The effects of IKP‐104 on microtubule protein mentioned above were mostly similar to those of vinblastine (VLB) and we suggest that IKP‐104 bound to the site or sites near “VLB‐binding site or sites” of α‐tubulin subunit, resulting in induction of conformational changes.

Effects of the tumor inhibitor IKP-104, a 4(1H)-pyridinone derivative, on cytoskeletal microtubules of cultured tumor cells

The effects of IKP‐104, a 4(lH)‐pyridinone derivative, on the mitotic profile and cytoskeletal microtubule dynamics of cultured B16 melanoma cells were examined in order to investigate the mechanism of its antitumor activity. The exposure to IKP‐104 caused accumulation of cells in abnormal metaphase with chromosomes scattered within the cytoplasm and induced polyploid and multinucleate cells as detected by differential staining microscopy with brilliant blue R and safranin O. An immunofluorcsceiicc study with monoclonal anti‐α‐tubulin antibody revealed that IKP‐104 diminished cytoskeletal microtubules of both interphase and mitotic cells, resulting in induction of a few fragments resembling “microtubular bundles” induced by vinblastine (VLB). These results indicated that IKP‐104 arrests cells in the mitotic phase by inhibition of polymerization and induction of depolymerization of cytoskeletal microtubules, similarly to VLB.