Progress in the development of early diagnosis and a drug with unique pharmacology to improve cancer therapy.

Cancer continues to be one of the major health and socio-economic problems worldwide, despite considerable efforts to improve its early diagnosis and treatment. The identification of new constituents as biomarkers for early diagnosis of neoplastic cells and the discovery of new type of drugs with their mechanistic actions are crucial to improve cancer therapy. New drugs have entered the market, thanks to industrial and legislative efforts ensuring continuity of pharmaceutical development. New targets have been identified, but cancer therapy and the anti-cancer drug market still partly depend on anti-mitotic agents. The objective of this paper is to show the effects of KAR-2, a potent anti-mitotic compound, and TPPP/p25, a new unstructured protein, on the structural and functional characteristics of the microtubule system. Understanding the actions of these two potential effectors on the microtubule system could be the clue for early diagnosis and improvement of cancer therapy.

New anti-mitotic drugs with distinct anti-calmodulin activity.

Bisindole Vinca alkaloids target microtubule system causing anti-mitotic activity. The problem of their clinical application is the lack of selectivity resulting in toxic side effects. In this paper we review the late history of new bisindole derivatives focusing on KARs recognized as potent anti-cancer drugs with low side effect. KARs, just as other bisindoles, impede microtubule assembly of mitotic spindle, however, they display no anti-calmodulin activity. This new drug family appears to be less potent than vinblastine in vitro systems, but it shows high antitumor efficacy with considerably higher doses being well tolerated in the animal tumor models. 3D data of calmodulin complexed with KAR-2 explain the specificity and unique pharmacology of KAR derivatives.

TPPP/p25: from unfolded protein to misfolding disease: prediction and experiments.

TPPP/p25, the first representative of a new protein family, identified as a brain-specific unfolded protein induces aberrant microtubule assemblies in vitro, suppresses mitosis in Drosophila embryo and is accumulated in inclusion bodies of human pathological brain tissues. In this paper, we present prediction and additional experimental data that validate TPPP/p25 to be a new member of the "intrinsically unstructured" protein family. The comparison of these characteristics with that of alpha-synuclein and tau, involved also in neurodegenerative diseases, suggested that although the primary sequences of these proteins are entirely different, there are similarities in their well-defined unstructured segments interrupted by "stabilization centres", phosphorylation and tubulin binding motives. SK-N-MC neuroblastoma cells were transfected with pEGFP-TPPP/p25 construct and a stable clone denoted K4 was selected and used to establish the effect of this unstructured protein on the energy state/metabolism of the cells. Our data by analyzing the mitochondrial membrane polarization by fluorescence microscopy revealed that the high-energy phosphate production in K4 clone is not damaged by the TPPP/p25 expression. Biochemical analysis with cell homogenates provided quantitative data that the ATP level increased 1.5-fold and the activities of hexokinase, glucosephosphate isomerase, phosphofructokinase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase were 1.2 to 2.0-fold higher in K4 as compared to the control. Our modelling using these data and rate equations of the individual enzymes suggests that the TPPP/p25 expression stimulates glucose metabolism. At pathological conditions TPPP/p25 is localized in inclusion bodies in multiple system atrophy, it tightly co-localizes with alpha-synuclein, partially with tubulin and not with vimentin. The previous and the present studies obtained with immunohistochemistry with pathological human brain tissues rendered it possible to classify among pathological inclusions on the basis of immunolabelling of TPPP/p25, and suggest this protein to be a potential linkage between Parkinson's and Alzheimer's diseases.

Tubulin and microtubule are potential targets for brain hexokinase binding.

The metabolite-modulated association of a fraction of hexokinase to mitochondria in brain is well documented, however, the involvement of other non-mitochondrial components in the binding of the hexokinase is controversial. Now we present evidence that the hexokinase binds both tubulin and microtubules in brain in vitro systems. The interaction of tubulin with purified bovine brain hexokinase was characterized by displacement enzyme-linked immunosorbent assay using specific anti-brain hexokinase serum (IC(50)=4.0+/-1.4 microM). This value virtually was not affected by specific ligands such as ATP or glucose 6-phosphate. Microtubule-bound hexokinase obtained in reconstituted systems using microtubule and purified hexokinase or brain extract was visualized by transmission and immunoelectron microscopy on the surface of tubules. The association of purified bovine brain hexokinase with either tubulin or microtubules caused about 30% increase in the activity of the enzyme. This activation was also observed in brain, but not in muscle cell-free extract. The possible physiological relevance of the multiple heteroassociation of brain hexokinase is discussed.

Combined enhancement of microtubule assembly and glucose metabolism in neuronal systems in vitro: decreased sensitivity to copper toxicity.

Brain cell-free extract greatly stimulates the polymerization rate of purified tubulin with a reduction of the nucleation period and without a significant alteration of the final assembly state. This effect is mimicked by neuroblastoma extract at 10-fold lower extract concentration, but not by excess muscle extract. Copper inhibits microtubule assembly in vitro but in the presence of brain extract the copper effect is suspended. Electron microscopic images showed that intact microtubules are formed and decorated by cytosolic proteins in the absence and presence of copper, while the copper alone induces the formation of S-shaped sheets and oligomeric threads. The flux of triosephosphate formation from glucose is enhanced by microtubules in brain extract, but not in muscle extract. Copper inhibits the glycolytic flux; however, the presence of microtubules not only suspends the inhibition by copper but the activation of glycolysis by microtubules is also preserved. We conclude that the organization of neuronal proteins modifies both the rates of microtubule assembly and glycolysis, and reduces their sensitivities against the inhibition caused by copper.

New semisynthetic vinca alkaloids: chemical, biochemical and cellular studies.

A new semisynthetic anti-tumour bis-indol compound, KAR-2 [3'-(beta-chloroethyl)-2',4'-dioxo-3,5'-spiro-oxazolidino-4-dea cetoxy-vinblastine] with lower toxicity than vinca alkaloids used in chemotherapy binds to calmodulin but, in contrast to vinblastine, does not exhibit anti-calmodulin activity. To investigate whether the modest chemical modification of bis-indol structure is responsible for the lack of anti-calmodulin potency and for the different pharmacological effects, new derivatives have been synthesized for comparative studies. The synthesis of the KAR derivatives are presented. The comparative studies showed that the spiro-oxazolidino ring and the substitution of a formyl group to a methyl one were responsible for the lack of anti-calmodulin activities. The new derivatives, similar to the mother compounds, inhibited the tubulin assembly in polymerization tests in vitro, however their inhibitory effect was highly dependent on the organization state of microtubules; bundled microtubules appeared to be resistant against the drugs. The maximal cytotoxic activities of KAR derivatives in in vivo mice hosting leukaemia P388 or Ehrlich ascites tumour cells appeared similar to that of vinblastine or vincristine, however significant prolongation of life span could be reached with KAR derivatives only after the administration of a single dose. These studies plus data obtained using a cultured human neuroblastoma cell line showed that KAR compounds displayed their cytotoxic activities at significantly higher concentrations than the mother compounds, although their antimicrotubular activities were similar in vitro. These data suggest that vinblastine/vincristine damage additional crucial cell functions, one of which could be related to calmodulin-mediated processes.

Interaction of a new bis-indol derivative, KAR-2 with tubulin and its antimitotic activity.

1. KAR-2 (3"-(beta-chloroethyl)-2",4"-dioxo-3,5"-spiro-oxazolidino- 4-deacetoxy-vinblastine), is a bis-indol derivative; catharantine is coupled with the vindoline moiety which contains a substituted oxazolidino group. Our binding studies showed that KAR-2 exhibited high affinity for bovine purified brain tubulin (Kd-3 microM) and it inhibited microtubule assembly at a concentration of 10 nM. 2. Anti-microtubular activity of KAR-2 was highly dependent on the ultrastructure of microtubules: while the single tubules were sensitive, the tubules cross-linked by phosphofructokinase (ATP: D-fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11) exhibited significant resistance against KAR-2. 3. The cytoplasmic microtubules of Chinese hamster ovary mammalian and Sf9 insect cells were damaged by 1 microgram ml-1 KAR-2, as observed by indirect immunofluorescence and transmission electron microscopy. Scanning electron microscopy revealed intensive surface blebbing on both types of cells in the presence of KAR-2. 4. KAR-2 was effective in the mouse leukaemia P338 test in vivo without significant toxicity. Studies on a primary cerebro-cortical culture of rat brain and differentiated PC12 cells indicated that the toxicity of KAR-2 was significantly lower than that of vinblastine. The additional property of KAR-2 that distinguishes it from bis-indol derivatives is the lack of anti-calmodulin activity.

The interaction of a new anti-tumour drug, KAR-2 with calmodulin.

1. KAR-2 (3"-(beta-chloroethyl)-2",4"-dioxo-3,5" -spiro-oxazolidino-4-deacetoxy-vinblastine) is a semisynthetic bis-indol derivative, with high anti-microtubular and anti-tumour activities but with low toxicity. KAR-2, in contrast to other biologically active bis-indols (e.g. vinblastine) did not show anti-calmodulin activity in vitro (enzyme kinetic, fluorescence anisotropy and immunological tests). 2. Direct binding studies (fluorescence resonance energy transfer, circular dichroism) provided evidence for the binding of KAR-2 to calmodulin. The binding affinity of KAR-2 to calmodulin (dissociation constant was about 5 microM) in the presence of Ca2+ was comparable to that of vinblastine. 3. KAR-2 was able to interact with apo-calmodulin as well; in the absence of Ca2+ the binding was of cooperative nature. 4. The effect of drugs on Ca2+ homeostasis in human neutrophil cells was investigated by means of a specific fluorescent probe. Trifluoperazine extensively inhibited the elevation of intracellular Ca2+ level, vinblastine did not appreciably affect it, KAR-2 stimulated the Ca2+ influx and after a transient enhancement the Ca2+ concentration reached a new steady-state level. 5. Comparison of the data obtained with KAR-2 and bis-indols used in chemotherapy suggests that the lack of anti-calmodulin potency resides on the spiro-oxazolidino portion of KAR-2. This character of KAR-2 manifested itself in various systems and might result in its low in vivo toxicity, established in an anti-tumour test.

Characterization of microtubule-phosphofructokinase complex: specific effects of MgATP and vinblastine.

Phosphofructokinase interacts with both microtubules and microtubules containing microtubule-associated proteins to produce bundling and periodical cross-bridging of tubules. Immunoelectron microscopy using anti-phosphofructokinase antibodies provided direct evidence that the kinase molecules are responsible for the cross-bridging of microtubules. Limited proteolysis by subtilisin, a procedure that cleaves the N-terminal segment of the free enzyme as well as the C-terminal "tails" of tubulin subunits exposed on microtubules, showed that while phosphofructokinase becomes resistant, tubulin retains sensitivity against proteolysis within the heterologous complex. These data suggest that the N-terminal segment of the enzyme, but not the C-terminal "tail" of tubulin subunits, is involved in the interaction between the microtubule and the kinase. The phosphorylation of phosphofructokinase or microtubules containing microtubule-associated proteins by the cAMP-dependent protein kinase did not interfere with the heterologous complex formation. MgATP prevents phosphofructokinase binding to the microtubules, and it can displace the enzyme from the single microtubules. However, the bundled microtubules are apparently resistant to the MgATP dissociation effect. Modelling of the assembly process suggests that the tubulin-kinase complex is able to polymerize as the free tubulin. Vinblastine, an anti-mitotic agent, inhibits tubulin assembly; however, its inhibitory effect is partially suppressed in the presence of phosphofructokinase. Fluorescence anisotropy measurements indicated that kinase and vinblastine compete for tubulin binding with no evidence for ternary complex formation. This competitive mechanism and the ability of the tubulin-enzyme complex to polymerize into microtubules may result in the resistance of the tubulin-enzyme complex against the inhibition of assembly induced by vinblastine. Microtubules formed in the presence of vinblastine plus phosphofructokinase can be visualized by electron microscopy. A molecular model is suggested that summarizes the effects of MgATP and vinblastine on the multiple equilibria in the tubulin/microtubules/phosphofructokinase system.

Anti-calmodulin potency of indol alkaloids in in vitro systems.

We have demonstrated that bis-indol Vinca alkaloids of anti-mitotic activities (vinblastine, vincristine, and navelbine) bind to calmodulin in a Ca(2+)-dependent manner. We designed direct binding tests (fluorescence energy transfer and circular dichroism measurements) to quantify the interactions of bis-indol derivatives with calmodulin. The dissociation constants of calmodulin-navelbine and calmodulin-vinblastine complexes with 1:1 stoichiometry are 0.5 microM and 3 microM, respectively. These values indicate that the binding affinities of these Vinca alkaloids to calmodulin and tubulin are comparable. Immunological, enzyme kinetic and fluorescence anisotropy measurements showed that bis-indol alkaloids inhibit the interactions of calmodulin with target proteins. The results of indirect enzyme-linked immunosorbent assay showed that bis-indol alkaloids effectively antagonize with anti-calmodulin antibody for calmodulin binding (IC50 = 90 microM, 400 microM, and 430 microM for navelbine, vincristine and vinblastine, respectively). According to the fluorescence anisotropy and enzyme kinetic measurements, vinblastine, vincristine and vinblastine, similarly to trifluoperazine, the classic calmodulin antagonist, compete with target enzyme [phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11)] for an inhibitory effect either on immunocomplex formation or on calmodulin-enzyme interaction. Navelbine appeared in our tests as the most potent drug in inhibiting the association of calmodulin to target proteins in comparison to other bis-indol derivatives. Since navelbine and vinblastine possess identical vindoline moiety, although they differ in the catharantine part, the difference in anti-calmodulin potencies is suggested to reside predominantly on this portion of the molecules. These findings might establish the pharmacological importance of these activities in the specificity and toxicity of the drugs.