Interaction of a fluorescent analog of N -deacetyl-N -methyl-colchicine (colcemid) with liver alcohol dehydrogenase

The evidence for specific binding of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-colcemid (NBD-colcemid), a fluorescent analog of colcemid (N-deacetyl-N-methyl-colchicine), to liver alcohol dehydrogenase is presented. Alcohol dehydrogenase bound NBD-colcemid in a time-dependent manner, enhanced the fluorescence intensity, and caused a large blue shift of the emission maximum of the free drug. The specificity of binding was determined for both the colchicine nucleus and the NBD moiety. The binding was not affected by the presence of alcohol or NAD in the reaction mixture. Preincubation of horse liver alcohol dehydrogenase with colcemid inhibited the binding to a considerable extent. NBD-colcemid inhibited the enzymic activity of alcohol dehydrogenase in a mixed-type noncompetitive mode with a Ki value of 32 μM, whereas colcemid showed noncompetitive inhibition with a Ki of 100 μM. The association rate constant of NBD-colcemid binding with liver alcohol dehydrogenase was 587 M-1 s-1 at 25 °C. The stoichiometry and dissociation constant of the binding reaction were 0.62/dimer and 12 μM, respectively. Donor quenching experiments showed that both tryptophans of alcohol dehydrogenase transferred energy to the bound NBD-colcemid. Thus, this study reports the binding of a colchicine analog to a protein other than tubulin with high affinity. It is concluded that NBD-colcemid binding to dehydrogenases is a general phenomenon, but the common structural element(s) that is responsible for the binding activity, and which exists among tubulin and dehydrogenases, has yet to be determined

N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)colcemid, a probe for different classes of colchincine-binding site on tubulin

The nature of binding of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-colcemid (NBD-colcemid), an environment-sensitive fluorescent analogue of colchicine, to tubulin was tested. This article reports the first fluorometric study where two types of binding site of colchincine analogue on tubulin were detected. Binding of NBD-colcemid to one of these sites equilibrates slsowly. NBD-colcemid competes with colchicine for this site. Binding of NBD-colcemid to this site also causes inhibition of tubulin self-assembly. In contrast, NBD-colcemid binding to the other site is characterised by rapid equilibration and lack of competition with colchicine. Nevertheless, binding to this site is highly specific for the cholchicine nucleus, as alkyl-NBD analogues have no significant binding activity. Fast-reaction-kinetic studies gave 1.76 × 105 M-1 s-1 for the association and 0.79 s-1 for the dissociation rate constants for the binding of NBD-colcemid to the fast site of tubulin. The association rate constants for the two phases of the slow site are 0.016 × 10-4 M-1 s-1 and 3.5 × 10-4 M-1 respectively. These two sites may be related to the two sites of colchicine reported earlier, with binding characteristics altered by the increased hydrophobic nature of NBD-colcemid

α-Fodrin in Cytoskeletal Organization and the Activity of Certain Key Microtubule Kinesins

Cortical cytoskeletal proteins are significant in controlling various cellular mechanisms such as migration, cell adhesion, intercellular attachment, cellular signaling, exo- and endocytosis and plasma membrane integrity, stability and flexibility. Our earlier studies involving in vitro and ex vivo approaches led us to identify certain undiscovered characteristics of α-fodrin, a prominent cortical protein. The conventional functions attributed to this protein mainly support the plasma membrane. In the present study, we utilized a global protein expression analysis approach to detect underexplored functions of this protein. We report that downregulation of α-fodrin in glioblastoma cells, U-251 MG, results in upregulation of genes affecting the regulation of the cytoskeleton, cell cycle and apoptosis. Interestingly, certain key microtubule kinesins such as KIF23, KIF2B and KIF3C are downregulated upon α-fodrin depletion, as validated by real-time PCR studies

6-Shogaol Inhibits Breast Cancer Cells and Stem Cell-Like Spheroids by Modulation of Notch Signaling Pathway and Induction of Autophagic Cell Death

<div><p>Cancer stem cells (CSCs) pose a serious obstacle to cancer therapy as they can be responsible for poor prognosis and tumour relapse. In this study, we have investigated inhibitory activity of the ginger-derived compound 6-shogaol against breast cancer cells both in monolayer and in cancer-stem cell-like spheroid culture. The spheroids were generated from adherent breast cancer cells. 6-shogaol was effective in killing both breast cancer monolayer cells and spheroids at doses that were not toxic to noncancerous cells. The percentages of CD44⁺CD24^-/^low cells and the secondary sphere content were reduced drastically upon treatment with 6-shogaol confirming its action on CSCs. Treatment with 6-shogaol caused cytoplasmic vacuole formation and cleavage of microtubule associated protein Light Chain3 (LC3) in both monolayer and spheroid culture indicating that it induced autophagy. Kinetic analysis of the LC3 expression and a combination treatment with chloroquine revealed that the autophagic flux instigated cell death in 6-shogaol treated breast cancer cells in contrast to the autophagy inhibitor chloroquine. Furthermore, 6-shogaol-induced cell death got suppressed in the presence of chloroquine and a very low level of apoptosis was exhibited even after prolonged treatment of the compound, suggesting that autophagy is the major mode of cell death induced by 6-shogaol in breast cancer cells. 6-shogaol reduced the expression levels of Cleaved Notch1 and its target proteins Hes1 and Cyclin D1 in spheroids, and the reduction was further pronounced in the presence of a γ-secretase inhibitor. Secondary sphere formation in the presence of the inhibitor was also further reduced by 6-shogaol. Together, these results indicate that the inhibitory action of 6-shogaol on spheroid growth and sustainability is conferred through γ-secretase mediated down-regulation of Notch signaling. The efficacy of 6-shogaol in monolayer and cancer stem cell-like spheroids raise hope for its therapeutic benefit in breast cancer treatment.</p></div

Effect of 6-shogaol on cell cycle of MCF-7 cells / spheroids.

<p>(A): Cell cycle analysis of MCF-7 cells treated with 16 μM 6-shogaol (2×IC₅₀) for different time points. (B): Cell cycle analysis of MCF-7 spheroid cells with different concentrations of 6-shogaol for 48 hours. The histogram is a representative of three independent experiments for both monolayer and spheroid cells. Bar graph represents percentage of cells in different phases of cell cycle. Error bars represent standard error of mean and have been calculated from three different experiments. *** denotes p ≤ 0.001; ** denotes p ≤ 0.005 and * denotes p ≤ 0.05.</p

Confirmation of the action of 6-shogaol on breast cancer spheroids.

<p>(A): Flow cytometric analysis of CD44⁺ /CD24^- expression in untreated spheres (right upper panel) and spheres treated with 40 μM 6-shogaol for 18 hours (right lower panel). (B): Effect of different concentrations of 6-shogaol on primary and secondary spheres. 5000 cells per well were seeded in quadruplicates with or without 6-shogaol. After 7 days, number of spheres were counted and then dispersed. From these, 5000 cells were again seeded and kept to regrow in fresh media without 6-shogaol. The error bars represent the standard error of mean from three different experiments. *** refers p ≤ 0.001.</p

6-shogaol induces autophagic flux and cell death in breast cancer cells.

<p>(A): MCF7 cells were treated with 6-shogaol or chloroquine for the indicated time points, stained with LC3A/B antibody and imaged as described in the Methods section. Green punctae are indicative of LC3II expression. Cell nuclei are labelled in blue. (B): MCF7 cells were treated for 48 hours with the indicated concentrations of 6-shogaol or chloroquine or their combination. Western blot was conducted with 50 μg of protein. Fold change in the expression of LC3II was calculated with respect to β-actin level of the control. A representative blot has been shown from three independent experiments. (C): MCF-7 cells were treated with chloroquine (CQ), 6-shogaol or combination of CQ and 6-shogaol in the indicated concentrations. After 48 hours, cell viability was checked by MTT assay. The error bars represent the standard deviation from three different experiments. ** denotes p ≤ 0.005. (D): Western blotting was conducted with cell lysates of MCF-7 spheroids after 40 μM 6-shogaol treatment as detailed in the Methods section. Cleavage of LC3I to LC3II was observed in the MCF-7 spheroid cells. Housekeeping gene β-actin was used as loading control. LC3II expression was quantitated and fold increase with respect to the control has been indicated.</p

Cytotoxic activity of 6-shogaol in breast cancer cells and spheroids and in noncancerous cells.

<p>Different concentrations of drugs were added one day and three days after seeding for monolayer and spheroids respectively. IC₅₀ was determined from MTT assay results after 48 hours using the nonlinear regression programme of Origin. Standard deviations from three different experiments are shown.</p