Mechanism of action of colchicine. V. Neutrophil adherence and phagocytosis in patients with acute gout treated with colchicine.
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Arthritis↗
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[Individual variations of the content in colchicine, demthyl-3 colchicine and cholchicoside of the seeds of cultivated colchicum (author's transl)].
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Colchicine↗
Efficacy and safety assessment of 0.5% and 1% colchicine cream in the treatment of actinic keratoses.
BACKGROUND: Topical colchicine has been reported to be an effective treatment for actinic keratoses, but the optimal concentration has not been fully defined. OBJECTIVE: The aim of this study was to further support the beneficial effect of topical colchicine therapy for actinic keratoses, and to compare the efficacy and safety of two different concentrations of colchicine cream, 0.5% and 1%. METHODS: Sixteen patients with actinic keratoses were enrolled in this comparative randomized study. Eight patients applied 1% colchicine cream, twice daily on their lesions while the other eight were treated with a 0.5% colchicine cream for 10 days. Some patients were applied a second course of 10 days' therapy. Patients were examined before treatment and at 10 days, and followed up at 1, 2 and 6 months of treatment. Visible and palpable actinic keratoses lesions in each group were counted. Safety and efficacy were also assessed by clinical examination at each study visit. Routine laboratory tests were performed before and after treatment. RESULTS: Actinic keratoses lesions showed significant clinical improvement following treatment with 0.5% and 1% colchicine cream. Complete healing of actinic keratoses were observed in six of the eight patients in the 1% colchicine group, and in seven of the eight patients in the 0.5% colchicine group. The reduction rate in number of actinic keratoses at the end of treatment in the 1% colchicine group was 73.9% (48/65) (p < 0.001), and the reduction rate in the 0.5% colchicine group was 77.7% (52/67) in total (p < 0.001). The reduction in number of actinic keratoses (mean +/- SD) at the end of treatment was similar in the 1% colchicine group (0.7 +/- 1.3), and the 0.5% colchicine group (mean 0.6 +/- 1.7) (p > 0.05). Systemic side effects were not seen in either concentration. CONCLUSIONS: Topical colchicine is an effective and safe alternative agent. Cream containing 0.5% of colchicine is equally effective as 1% colchicine cream in the treatment of actinic keratoses.
Administration, Cutaneous↗
Fab-bound colchicine appears to adopt Fab fragment disposition in rats.
The disposition of colchicine-specific Fab fragments and the effect of Fab fragment administration on the disposition of colchicine were studied in anaesthetized bile duct-cannulated rats. One group of rats (n = 6) received a 125I-Fab dose of 38 mg kg-1 i.v. The plasma disposition was characterized by a volume of distribution of 179 +/- 48 mL kg-1, total body clearance of 1.02 +/- 0.07 mL min-1 kg-1, t1/2 alpha of 0.17 +/- 0.03 h and t1/2 beta of 1.3 +/- 0.3 h. Fab fragments were in part excreted by the renal route (15.6 +/- 6% of the Fab dose), while biliary excretion was a minor route (< 2% of the Fab dose). Two other groups of rats received 15 micrograms kg-1 colchicine (n = 6) or 15 micrograms kg-1 colchicine plus 38 mg kg-1 colchicine-specific Fab fragments (n = 6) by intravenous infusion. Pharmacokinetics of colchicine was markedly altered in the Fab-colchicine-treated rats. In this group, distribution volume and total body clearance of colchicine were decreased by factors of 22 and 10, respectively, compared with the values in the colchicine-treated group and were very similar to those of Fab fragments. An 80% reduction of cumulative biliary excretion of colchicine was observed in Fab-colchicine-treated rats (P < 0.01). The fraction of colchicine dose excreted by the urinary route was 38 +/- 6.9 and 9 +/- 0.7% respectively in Fab-colchicine- and colchicine-treated groups (P < 0.01). These data show that during Fab treatment, colchicine followed the elimination kinetics of Fab fragments.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Mechanism of colchicine-induced steroidogenesis in rat adrenocortical cells.
Conflicting data for the effects of colchicine on cholesterol transport and steroidogenesis raise the question of the role of microtubules in cholesterol transport from the lipid droplet to mitochondria in steroidogenic cells. In this study, using corticosterone radioimmunoassay and immunofluorescence microscopy, we re-evaluated the effects of colchicine on hormone production and morphological changes of lipid droplets' and studied the signaling pathway involved in colchicine-induced steroidogenesis. Colchicine stimulated steroid production in a dose- and time-dependent manner. The structural integrity of both the microtubules and the lipid droplet capsule was destroyed by colchicine treatment. Disruption of the lipid droplet capsule occurred later than microtubule depolymerization. After cessation of colchicine treatment and a 3 h recovery in fresh medium, capsular protein relocated to the droplet surface before the cytoplasmic microtubule network was re-established. beta-lumicolchicine, an inactive analogue of colchicine, disrupted the capsule and increased hormone production without affecting microtubular structure. Thus, microtubule depolymerization is not required for the increase in steroid production and capsular disruption. To explore the signaling pathway involved in colchicine-induced steroidogenesis, we measured intracellular cAMP levels. Unlike ACTH, colchicine did not increase cAMP levels, suggesting that the cAMP-PKA system is not involved. Colchicine and ACTH had additive effects on corticosterone production, whereas colchicine and PMA did not, implying that part of the PKC signaling mechanism may be involved in colchicine-induced steroidogenesis. Cycloheximide, a protein synthesis inhibitor, completely inhibited colchicine-induced steroidogenesis and capsular disruption. These results demonstrate that the steroid production and lipid droplet capsule detachment induced by colchicine are both protein neosynthesis-dependent and microtubule-independent.
Adrenal Cortex↗
Persistence of colchicine resistance in Ehrlich-Lettré ascites tumors and cell strains.
The effect of colchicine on mitoses of mutant HD33 Ehrlich-Lettŕe ascites cells growing in vivo and in vitro was studied. HD33 mouse ascites tumors are colchicine-resistant. The LD50 of colchicine in mice bearing HD33 ascites tumors was 1.4 mg/kg body weight (b.w.), but a single dose of 3.33 mg colchicine/kg b.w. failed to suppress the anaphase of HD33 tumor mitoses for 24 h. No change in the level of colchicine resistance was observed after 269 weekly transplantations of HD33 ascites tumors without colchicine. In suspension culture, growth of HD33 ascites cells ceased at 1.5 x 10(-6) M colchicine. 10(-5) M colchicine suppressed the anaphase of HD33 mitoses and produced typical C-mitoses within one hour. The same effects on mitoses of colchicine sensitive Ehrlich ascites cells in vitro were achieved with 10(-6) M colchicine. In HD33 ascites cell cultures grown without colchicine, only a slight increase in colchicine sensitivity was registered after 5 years. Parallel cultures were propagated for the same period in the presence of 10(-7) M colchicine (HD33C ascites cells) without detectable growth alterations; the resistance level increased slightly. The limit of 10(-6) M colchicine was tolerated by the ascites cells in permanent culture without growth reduction (HD33CS ascites cells). 3H-colchicine binding studies suggest a permeability barrier of the plasma membrane as a mechanism of genetically fixed resistance.
Animals↗
Colchicine inhibits GABA(A) receptors independently of microtubule depolymerization.
Colchicine is a microtubule depolymerizing agent used extensively in the study of cytoskeleton-dependent cell functions. In studying the possible functional interaction between the GABA(A) receptor and the cytoskeleton, we found that colchicine inhibits GABA(A) receptor function by mechanisms independent of microtubule depolymerization. Human GABA(A) receptor alpha1beta2gamma2L subunits were co-expressed in Xenopus oocytes and the effects of colchicine on GABA(A) receptor function was assessed using the two-electrode voltage-clamp technique. Co-application of GABA (10 microM) with colchicine (100 microM) resulted in a 59.9% inhibition of GABA-gated chloride currents. This effect was instantaneous in onset with no pre-incubation required and reversed within seconds. Other depolymerizing agents, such as nocodazole (20 microM) and vinblastine (200 microM), did not affect GABA(A) receptor function using the same co-application protocol used with colchicine. The polymerizing agent taxol (10-50 microM) did not affect colchicine inhibition of the GABA responses and did not itself alter GABA-gated chloride currents. The inhibitory effect of colchicine was present under conditions in which the oocyte microtubules had been depolymerized by cold temperature. These results indicate that colchicine inhibits the GABA(A) receptor via mechanisms unrelated to microtubule depolymerization. To further examine the inhibitory effect of colchicine on the GABA response, GABA (10-3000 microM) concentration-response curves were performed in the absence or presence of various concentrations of colchicine (30-300 microM). In the presence of colchicine, the GABA concentration-response curve was shifted to the right in a parallel fashion. A Schild plot of this data yielded a linear slope indicating that colchicine acts as a competitive antagonist at the GABA binding site. We conclude that colchicine is a competitive antagonist at the GABA(A) receptor and that studies using colchicine to examine the functional interaction between GABA(A) receptors and microtubules should be interpreted with caution.
Animals↗
P-glycoprotein is more efficient at limiting uptake than inducing efflux of colchicine and vinblastine in HL-60 cells.
PURPOSE: To investigate the role of the P-glycoprotein (P-gp) drug efflux pump in the intracellular disposition of colchicine and vinblastine. METHODS: Uptake and efflux kinetics were studied in vitro in human lymphocytes and in HL-60 cells with or without the P-gp modulator, verapamil. RESULTS: In human lymphocytes, colchicine was slowly taken up (uptake half-life was 18.9+/-1.1 hr.) and verapamil increased colchicine uptake by 37%, whereas it did not modify colchicine efflux from cells. In HL-60 cells, colchicine uptake was non-linear and slower than that of vinblastine, the colchicine uptake half-life (11.1+/-0.5 hr.) being 25-fold longer than that of vinblastine at 25 nM. Verapamil did not significantly modify colchicine uptake half-life, but increased its intracellular accumulation by 23% and that of vinblastine by 81%. Immuno-flow cytometry showed that P-gp expression in HL-60 cells increased significantly from 24 hr. following colchicine or vinblastine exposure. The significant increase in colchicine uptake induced by verapamil at 24 hr. was correlated with this enhanced P-gp expression. The drug efflux half-life was 11.5-fold higher for colchicine (23+/-0.9 hr) than vinblastine, indicating a much slower elimination of colchicine from cells that could be related to its longer dissociation half-life from the tubulin receptor. Verapamil treatment did not modulate either colchicine or vinblastine efflux kinetics, suggesting that the intracellular drugs are not available to the transmembrane P-gp binding sites. CONCLUSIONS: P-gp may not be the main reason for the slowness of colchicine uptake. It may be more efficient at controlling entry of colchicine and vinblastine through the plasma membrane than at mediating their efflux from HL-60 cells.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Colchicine binding to bovine anterior pituitary slices and inhibition of growth-hormone release.
The uptake of [ring C-methoxyl-3H]colchicine into bovine anterior pituitary slices was studied. The data suggest that more than one site exists for the binding of colchicine. At low concentrations colchicine binds to saturable trypsin-sensitive site(s), with a dissociation constant of 3.1 +/- 0.69 mug. The binding capacity of these sites is 8.58 +/- 0.60 pmol of colchicine/mg of wet pituitary. At higher colchicine concentrations binding occurs predominantly to sites which exhibit non-saturation kinetics. Subcellular fractionation of colchicine-labelled slices shows that 90% of the saturable sites are present in the fraction containing cytosol, where the binding protein has a molecular weight of about 11.9 x 10(4) and constitutes 0.7% of the protein present. The nuclear fraction contains 10% of the saturable sites, and the mitochondria and granule fraction contain only non-saturable sites. The rate of colchicine uptake was studied at 0.84 mm- and 2mum-colchicine. At both concentrations the colchicine space exceeded the total tissue water within 10 min. Equilibration with the saturable binding sites was complete in 120 min at 2mum-colchicine. A concentration of colchicine (13.4 mum) which would give 81% maximum binding was found to decrease the length of observable microtubules in tissue fixed at 37 degrees C in glutaraldehyde by 83 +/- 4%. The colchicine-binding protein could be partially purified by using a standard procedure for isolation of brain tubulin. Colchicine inhibits the release of growth hormone in the presence of 3-isobutyl-1-methylxanthine (0.1 mm), but does not alter basal release. The concentration-dependence of colchicine inhibition is similar to that of colchicine binding, but maximum inhibition is only 35%.
Animals↗
Colchicine and Longitudinal Dynamics of Clonal Hematopoiesis: An Exploratory Substudy of the LoDoCo2 Trial.
BACKGROUND: Clonal hematopoiesis (CH) is an aging-related hematologic condition associated with increased risk for cardiovascular events. Larger CH clones associate more strongly with cardiovascular risk. Preclinical data indicate that inflammatory signaling drives expansion of CH clones and CH-associated cardiovascular disease. However, the effect of anti-inflammatory therapies on CH clonal dynamics in humans is unclear. OBJECTIVES: The goal of this study was to test the association of randomization to colchicine vs placebo with CH growth in participants with chronic coronary artery disease. It also assessed the association of colchicine use with change in inflammatory biomarkers over time according to CH status. METHODS: In this exploratory substudy of the LoDoCo2 (Low-Dose Colchicine 2) trial, high-coverage targeted sequencing was used to detect CH driver mutations and to quantify variant allele frequency at 4 timepoints: baseline, after a 30-day open-label colchicine run-in phase (0.5 mg daily), 1 year postrandomization to colchicine or placebo, and at end of study (median follow-up of 25.0 months). Clonal dynamics were assessed by using a generalized linear mixed model. High-sensitivity C-reactive protein and interleukin-6 were additionally measured at baseline, randomization, and 1 year postrandomization. RESULTS: In total, 854 participants contributed 2,047 observations across 4 timepoints, including before and after the prerandomization colchicine run-in period. Randomization to placebo was associated with a 14.9% annual increase in CH clone size (βtime = 0.14; 95% CI: 0.08 to 0.21) vs a nonsignificant 6.3% increase with colchicine (βtime on colchicine: 0.06; 95% CI: -0.01 to 0.14), although this difference between treatment arms was not statistically significant (Pinteraction = 0.13). Compared with placebo, colchicine was associated with attenuated clonal growth in TET2 CH (βtime on colchicine: 0.09 [95% CI: -0.04 to 0.22]; βtime placebo: 0.27 [95% CI: 0.16 to 0.37]; Pinteraction= 0.04). Among individuals with non-DNMT3A CH, interleukin-6 levels increased to a lesser extent in those receiving colchicine vs placebo over 1 year (30.0% vs 98.1% increase, respectively; Pinteraction = 0.01). CONCLUSIONS: In this exploratory analysis, treatment with low-dose colchicine was associated with attenuated clonal expansion in TET2 CH. These findings suggest the potential for colchicine to curb the proliferative advantage of key CH driver mutations and to mitigate their associated risk of cardiovascular disease. Further validation in prospective studies is warranted.
Humans↗
Uptake of [3H]colchicine into brain and liver of mouse, rat, and chick.
The uptake of [ring A-4-3H] colchicine and [ring C-methoxy-3H]colchicine has been compared in mice from 1 to 24 hr after administration. Less radioactivity was found in brain after administration of ring-labeled colchicine than after administration of the methoxy-labeled colchicine. Three hr after administration of ring-labeled colchicine, 5% of the label was in liver and about 0.01% of the label was present in brain. Forty percent of the brain radioactivity was bound to tubulin as determined by vinblastine precipitation. After 3 hr, an average of 8% of the radioactivity from methoxy-labeled colchicine was found in the liver and 0.16% in brain. However, less than 5% of the activity in brain was precipitated by vinblastine, and the colchicine equivalent was comparable to that found after administration of the ring-labeled colchicine. The amount of colchicine entering mouse brain after subcutaneous injection is comparable to the minimum behaviorally effective dose when administered to the caudate. The metabolism of [ring C-methoxy-3H] and [ring A-3H]colchicine was also studied in rats. The general pattern was similar to mice; less radioactivity was found in brain after administration of the ring-labeled alkaloid than after administration of methoxy-labeled colchicine. Again, 40-50% of ring-labeled colchicine was precipitated by vinblastine. A much smaller percentage of the methoxy-labeled drug was precipitated by vinblastine than of the ring A-labeled colchicine. These experiments, together with behavioral experiments [7], support the hypotheses that structural alterations in synapses by recently synthesized proteins which are transported down the axons and dendrites may be an essential process for long-term memory formation.
Animals↗
Colchicine reduces proteinuria in passive Heymann nephritis.
Colchicine was given to rats in the heterologous phase of passive Heymann nephritis to see whether this drug could reduce proteinuria. Treatment with 0.06 mg/day for 14 days caused significant reductions in proteinuria and albuminuria. Administration of dimethyl sulfoxide (DMSO) alone or in combination with colchicine also reduced protein and albumin excretion. In a long-term experiment, rats treated with colchicine had significantly less proteinuria. After stopping therapy, urine protein excretion was similar to controls. No differences in glomerular C3 and IgG deposition were found between treated and control rats 24 h, 3,7 and 14 days after immunization. Depressed serum C3 levels were measured at 24 h in colchicine-treated rats. No difference in serum-circulating immune complexes was detected between the two groups. Concurrent administration of indomethacin and colchicine to rats with passive Heymann nephritis (PHN) partially reversed the reduction in proteinuria and albuminuria seen in rats treated with colchicine alone. The G.F.R, however, was significantly reduced in colchicine-treated rats as well as in rats treated with colchicine and indomethacin. Serum cholesterol and triglyceride levels were significantly lower in colchicine-treated rats than in controls. Serum cholesterol concentrations in rats given both colchicine and indomethacin were similar to control values. These findings suggest that colchicine reduces urine protein and albumin excretion, and hyperlipidemia in PHN. The finding that indomethacin partially blocks the effects of colchicine suggests that renal prostaglandin stimulation by colchicine may have been involved in the reduction in proteinuria.
Animals↗
Colchicine in experimental alkaline burns of the rat esophagus: an old drug, a new indication?
AIM: An experimental study was performed to investigate the efficacy of colchicine in the prevention of fibrosis in the alkaline burn of the esophagus in rats. METHOD: A standard esophageal alkaline burn was produced by the application of 37.5% NaOH for a period of 90 s to the distal esophagus followed by a water rinse. The experiments were conducted twice with two different dosages of colchicine. In the first experiment, colchicine 1 mg/kg (i.p.) was applied once, on the first day. Group A (n:8), the sham laparotomy group; group B (n:8), the untreated group (positive control group); Group C (n:16), where the standard esophageal burn was induced and colchicine applied at a dose of 1 mg/kg i.p. in 1 ml volume, and group D (n:14), where the rats did not have any operation, but were treated with colchicine (1 mg/kg, i.p.) as in group C. In the second experiment, colchicine was applied at repeated doses of 100 microg/kg (i.p.) on the first, 7th, 14th, and 21st days. Twenty-five rats were divided into groups. Group AA (n:8), the sham laparotomy group; group BB (n:9), the untreated group (positive control group); and group CC (n:8), where the standard esophageal burn was induced and colchicine was applied at repeated doses. All the rats were killed on the 28th day and evaluated for esophageal tissue hydroxyproline content and histopathologic damage score. RESULTS: Colchicine treatment at a dose of 1 mg/kg significantly reduced the quantity of hydroxyproline in the esophageal tissue in groups C and D compared with the positive control group B (P < 0.05). However, it is associated with a mortality rate of 60%. Colchicine at repeated doses of 100 microg/kg showed no significant effect in group CC compared to the untreated group BB and in the quantity tissue of hydroxyproline content (P > 0.05). CONCLUSION: At non-toxic doses, colchicine was not effective in the treatment of alkaline esophageal burn in rats. Colchicine-like molecules with less adverse effects or colchicine itself in titrated doses may be hopeful in preventing the development of fibrosis in the alkaline burns of the esophagus.
Analysis of Variance↗
Effect of colchicine on hepatobiliary function in CCl4 treated rats.
A number of toxic chemicals affect the biliary excretory function of liver. Organochlorines and halomethanes are known to enhance bile flow. Despite the demonstration that a diversity of agents modify biliary function, the mechanism by which these chemicals manifest this effect is not fully understood. This study was designed to assess the effect of colchicine (0.1, 1.0, or 2.5 mg/kg, i.p., in saline) administration on biliary excretory function 6 and 24 hr later. Additionally, the effect of colchicine (1 mg/kg, i.p. in saline) pretreatment in rats 2 hr prior to the administration of a single low dose of CCl4 (100 microL/kg, i.p., in corn oil) or corn oil alone (1 mL/kg, i.p.) on hepatic biliary excretory function was also assessed at 6 and 24 hr after the last treatment. The hepatotoxicity was evaluated by serum enzymes, alanine and aspartate aminotransferases, and histopathological alterations of the liver. Biliary excretion of intravenously administered phenolphthalein glucuronide (PG) was assessed in bile duct cannulated anesthetized rats. Only the highest dose of colchicine (2.5 mg/kg) resulted in detectable liver injury as revealed by elevations of serum transaminases. While the lowest dose of colchicine (0.1 mg/kg) did not influence bile secretion, the two higher doses caused a slight choleretic effect at 24 hr. The highest dose caused a transient inhibition of bile flow, but this effect was no longer evident at 6 hr. Biliary excretion of PG was inhibited significantly by colchicine within 6 hr after administration, an effect that was also persistent at 24 hr. Colchicine at a 1 mg/kg dose did not cause any adverse effect on hepatobiliary function. Therefore, for the interactive toxicity study with CCl4, 1 mg colchicine/kg was chosen as a moderate dose which did not cause any significant adverse effect on hepatobiliary function. Biliary excretion of PG was significantly lower in rats at 6 and 24 hr after the combination treatment with colchicine + CCl4 than in rats receiving either CCl4 or colchicine alone. In contrast, rats receiving CCl4 alone or colchicine + CCl4 showed a significant increase in cumulative bile flow at 6 hr, whereas, at 24 hr, the bile flow was increased significantly in rats receiving colchicine regardless of CCl4 treatment. The data suggest that colchicine pretreatment leads to significant inhibition of hepatobiliary excretion in CCl4 treated rats. Serum alanine transaminase and aspartate transaminase levels were elevated significantly after the colchicine + CCl4 combination, indicating hepatic injury.(ABSTRACT TRUNCATED AT 400 WORDS)
Alanine Transaminase↗
Role of P-glycoprotein in colchicine and vinblastine cellular kinetics in an immortalized rat brain microvessel endothelial cell line.
Uptake and efflux of colchicine and vinblastine, whose effects are related to their high-affinity binding to tubulin, were studied in the immortalized rat brain microvessel endothelial cell line RBE4. At 10 nM extracellular drug concentration, uptake equilibrium was approached at 45 hr for colchicine, but at only 3.5 hr for vinblastine. After 1 hr preincubation with 200 nM colchicine or vinblastine, drug efflux fitted biexponential kinetics with an initial fast phase (half-life = 2.2 min and 9.6 min, respectively) and a later slow phase (half-life = 3.6 hr and 1.8 hr, respectively). After 6 hr preincubation with 200 nM colchicine, only the slow phase (half-life = 3.6 hr) could be observed. The colchicine and vinblastine uptake rate was increased by cyclosporin A, an inhibitor of the drug efflux pump P-glycoprotein, which is expressed at the blood-brain barrier. Whereas cyclosporin A decreased vinblastine efflux, its effect on colchicine efflux was apparent after only 13 hr washout and was associated with the re-uptake by cells of colchicine molecules. Differences in uptake kinetics of colchicine and vinblastine could be related to differences in their lipid solubility, and mainly in their binding affinities to tubulin. Differences in efflux kinetics could in addition be explained by the involvement of P-glycoprotein in the efflux of vinblastine, whereas efflux of colchicine was not influenced by this pump. Indeed, binding of colchicine to tubulin would imply that most intracellular colchicine may be inaccessible to P-glycoprotein. In the case of a cytotoxic drug such as colchicine, which is tightly bound to intracellular receptors, the role of P-glycoprotein within the blood-brain barrier would be more to protect the brain against entry of this drug than to detoxify the brain by its extraction.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
A vulnerable period of colchicine toxicity during goldfish optic nerve regeneration.
The effects of intraocular (i.o.) administration of the alkaloid colchicine on visual recovery following axotomy of the goldfish optic nerve were investigated. Under the experimental conditions used, control goldfish recovered vision, measured behaviorally, within 5-7 weeks of retro-orbital optic nerve crush. Fish injected i. o. with 0.1 microg of colchicine within 3 days of optic nerve crush (post-crush; PC) recovered vision after some delay relative to control fish, while injection with colchicine between 7 and 14 days PC produced a much more profound inhibition of recovery of vision, in most cases a complete block for the duration of the study (98 days). Further evidence for a delayed susceptibility of the regenerating optic nerve to colchicine following crush was reflected in a suppression of neurite outgrowth normally seen in explanted retinal tissue taken from PC goldfish. In addition, retrograde transport of the fluorescent dye 4-(4-didecylaminostyryl)-N-methylpyridinium iodide from the optic tectum to the retina as a measure of axonal continuity revealed substantially less labeling following i.o. administration of colchicine 1 week PC when compared to retinas from fish receiving colchicine at the time of optic nerve crush. Histological sections of the retina showed no evidence of residual retinal damage resulting from the colchicine injections or from interactions of axotomy and the drug administration. These results indicate a period of increased vulnerability of the regenerating visual system to the toxic effects of i.o. administered colchicine, beginning 3-5 days PC, and remaining until regenerating optic nerve fibers have begun to reach the tectum. While colchicine has many known effects on nerve function, it is proposed that the delayed susceptibility to disruption of regeneration observed in these experiments is largely, if not entirely, attributable to a colchicine-induced accumulation of tubulin heterodimers, which are known to block microtubule assembly and to participate in a feedback inhibition of tubulin synthesis. Thus, it is during the maximal induction of tubulin synthesis and of microtubule formation which normally occurs several days following axotomy that colchicine has its greatest effect. The results suggest that colchicine may be especially neurotoxic during neural development and regeneration.
Animals↗
Roles of colchicine rings B and C in the binding process to tubulin.
The interactions of tubulin with colchicine analogues in which the tropolone methyl ether ring had been transformed into a p-carbomethoxybenzene have been characterized. The analogues were allocolchicine (ALLO) and 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB), the first being transformed colchicine and the second transformed colchicine with ring B eliminated. The binding of both analogues has been shown to be specific for the colchicine binding site on tubulin by competition with colchicine and podophyllotoxin. Both analogues bind reversibly to tubulin with the generation of ligand fluorescence. The binding of ALLO is slow, the fluorescence reaching a steady state in the same time span as colchicine; that of TCB is rapid. The displacement of ALLO by podophyllotoxin proceeds with a half-life of ca. 40 min. Binding isotherms generated from gel filtration and fluorescence measurements have shown that both analogues bind to tubulin with a stoichiometry of 1 mol of analogue/mol of alpha-beta tubulin. The equilibrium binding constants at 25 degrees C have been found to be (9.2 +/- 2.5) x 10(5) M-1 for ALLO and (1.0 +/- 0.2) X 10(5) M-1 for TCB. Binding of both analogues was accompanied by quenching of protein fluorescence, perturbation of the far-ultraviolet circular dichroism of tubulin, and induction of the tubulin GTPase activity, similarly to colchicine binding. Both inhibited microtubule assembly in vitro, ALLO substoichiometrically, and both induced the abnormal cooperative polymerization of tubulin, which is characteristic of the tubulin-colchicine complex. Analysis in terms of the simple bifunctional ligand binding mechanism developed for colchicine [Andreu, J.M., & Timasheff, S.N. (1982) Biochemistry 21, 534-543] and comparison with the binding of the colchicine two-ring analogue, 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one [Andreu, J. M., Gorbunoff, M. J., Lee, J. C., & Timasheff, S. N. (1984) Biochemistry 23, 1742-1752], have shown that transformation of the tropolone methyl ether part of colchicine into p-carbomethoxybenzene weakens the standard free energy of binding to tubulin by 1.4 +/- 0.1 kcal/mol, while elimination of ring B weakens it by 1.0 +/- 0.1 kcal/mol. The roles of rings C and B of colchicine in the thermodynamic and kinetic mechanisms of binding to tubulin were analyzed in terms of these findings.
Animals↗
Changes in the circular dichroic spectrum of colchicine associated with its binding to tubulin.
Circular dichroism has been used to study the interaction of colchicine with the tubulin alpha beta dimer at 26 degrees C. Tubulin purified from bovine brain microtubule protein exhibits negligible circular dichroism at wavelengths above 310 nm. Free colchicine exhibits a negative circular dichroic band at 340 nm characterized by an extremum in molar ellipticity [theta] of -3.35 (+/- 0.27) x 10(4) deg.cm2/dmol. This negative band either vanished or was greatly reduced, i.e., [theta] = 0 (+/- 0.57) x 10(4) deg.cm2/dmol, when colchicine was bound to tubulin. This was demonstrated by circular dichroic studies on stable [3H]colchicine-tubulin complexes which were separated from all unbound colchicine by means of gel filtration. Also, tubulin was titrated with colchicine, and at low colchicine concentrations, the observed ellipticity at 340 nm could be extrapolated to zero at 0 M colchicine; saturation occurred at a molar ratio of colchicine to tubulin of about 1:1. The association constant characterizing the drug-protein interaction was estimated to be about 0.9 micro M-1. As controls, three other acidic proteins were studied at a molar ratio of colchicine to protein of 2:1, and their presence had no effect on the circular dichroic properties of colchicine. These results are consistent with the idea that a conformational change in colchicine accompanies its binding to tubulin. The spectrum of the complex between 250 and 300 nm was quite similar to that expected from simple additivity of the spectra of drug and protein except between 255 and 265 nm. The technique described herein should be applicable to other protein-drug systems.
Animals↗