Figure 1.
NMK-TD-100- Structure and absorption spectrum.
(A) Structure of NMK-TD-100 and colchicine (B) The absorption spectrum of NMK-TD-100 (100 µM).
Figure 2.
Cytotoxicity of NMK-TD-100 in HeLa and PBMC cells.
(A) HeLa cells were cultured for 48 h with various concentrations (0-100 µM) of NMK-TD-100. Cell viability was assessed by MTT assay and is expressed as a percentage of control. Data are represented as the mean±SD. [*p<0.05 vs. control, where n=4]. (B) Percentage of cell death in freshly isolated PBMC after treatment with NMK-TD-100 for 48 h. Data are represented as the mean±SD [*p<0.05 vs. control, where n=3]. (C, D) Colony formation assay. Cultured HeLa cells were seeded in six-well plates at a density of 1,000 cells per well and cells were treated with NMK-TD-100 (0-10 µM) for 48 h. NMK-TD-100 containing media was replaced with fresh media and subsequently cells were cultured for 10 days. At the end, cells were fixed and stained with crystal violet, images were taken. Colony formation was quantified by dissolving stained cells in Sorenson’s buffer for colorimetric reading of OD at 550 nm. Data are represented as the mean±SD [*p<0.05 vs. control, where n=3].
Figure 3.
NMK-TD-100 induced mitochondrial pathway mediated apoptosis in HeLa cells.
(A) Annexin V-FITC /PI assay for showing that NMK-TD-100 induced apoptosis in HeLa cells. Cultured HeLa cells were treated with 0-10 µM NMK-TD-100 for 36 h, cells were harvested and stained with annexin V-FITC and PI. The percentage of early apoptotic cells in the lower right quadrant (annexin V-FITC positive/PI negative cells), as well as late apoptotic cells located in the upper right quadrant (annexin V-FITC positive/PI positive cells). (B) NMK-TD-100 induced collapse of mitochondrial membrane potential in HeLa cells. Cells were treated with 0-10 µM NMK-TD-100 for 36 h. Then treated-cells were stained with JC1 and analyzed using flow cytometer. Red fluorescence emitted from the cells with normal mitochondria (red population in figure) gradually decreases with concomitant increase in green fluorescence emitted from that containing declined mitochondrial membrane potential (green population in figure). (C) Western Blot analysis of change in expression of pro and anti-apoptotic proteins (p53, bax, bcl2 and procaspase 3) of 36 h ligand-treated HeLa cells. Probing of α- tubulin was used as a loading control. The results represent the best of data collected from three experiments with similar results.
Figure 4.
Cell cycle progression analysis and determination of mitotic index in NMK-TD-100 treated HeLa cells.
(A) Effect of NMK-TD-100 on cell cycle progression of HeLa cells. Cells were treated with ligand for 24 h. After incubation, cells were processed with RNase A and stained with PI for detection through flow cytometer. The numbers 1, 2 and 3 written on figure represent G 0/G1, S and G 2/M phases of the cell cycle in HeLa cells, respectively. Data are representative of three identical experiments. (B-D) Effect of NMK-TD-100 on chromosomes of HeLa cells. Cultured HeLa cells were fixed and stained with DAPI (1 µg/mL) for the observation of chromosomes in different stages of mitosis; also, normal and abnormal interphase nuclei are observed in the absence (control cells) and in the presence of NMK-TD-100 (2.5-5 µM) for 24 h. Arrow indicates mitotic cell. (E) Effect of NMK-TD-100 on mitosis in HeLa cells. Cultured HeLa cells were treated with 0-5 µM NMK-TD-100 for 24 h. Cells were then fixed and stained with DAPI (1 µg/mL). Mitotic indices were determined by counting interphase and mitotic cells at 40X magnification using a confocal microscope. At least 1000 cells per data point were counted. Data are represented as the mean ±SD [*p<0.05 vs. control, where n=3].
Figure 5.
Effect of NMK-TD-100 on interphase and spindle microtubules in HeLa cells.
(A-I) NMK-TD-100 perturbed the organization of interphase microtubule in HeLa cells. Cells were incubated in various concentration of NMK-TD-100 (0-5 µM) for 24 h, fixed and stained using antibody against α-tubulin (red) and nucleus with DAPI (blue). (J-O) NMK-TD-100 depolymerized the spindle microtubule with formation of multipolar spindles in HeLa cells. Microtubules were stained with antibody against α-tubulin (red) and nucleus with DAPI (blue). Details of the experiments are described in the ‘Materials and methods’ section.
Figure 6.
Effect of NMK-TD-100 on reassembly of cold depolymerized microtubule in HeLa cells.
Panel A shows normal interphase microtubule in HeLa cells before incubating the cells at 4°C whereas panel B shows microtubule network after 3 h of cold depolymerization. Cold media was replaced by warm media in the presence of 0 (Panel C), 2.5 (Panel D) and 5 µM (Panel E) of NMK-TD-100 and incubated for 3 h at 37°C. Microtubules were tagged by anti- α-tubulin antibody, subsequently TRITC conjugated secondary antibody (red) and nucleus were probed by DAPI (blue). Further details of the experimental procedures are given in the ‘Materials and Methods’ section.
Figure 7.
Effect of NMK-TD-100 on tubulin polymerization in cell-free system.
(A) Tubulin was polymerized in the presence of various concentration of NMK-TD-100 (0-30 µM) and the kinetics of polymerization was monitored by increase using light scattering at 350 nm. (B) Transmission electron microscopic studies of products of tubulin polymerization in the absence and presence of 25 µM of NMK-TD-100. Scale bar is 500 nm. (C) Effect of NMK-TD-100 on depolymerization of preformed microtubule as monitored by decrease in light scattering at 350 nm. The end point absorbance at 350 nm was plotted against the concentration of NMK-TD-100. Data are representative of average of three similar experiments with SD.
Figure 8.
Binding of NMK-TD-100 to tubulin as assessed by enhancement of ligand fluorescence.
(A) Fluorescence emission spectra of NMK-TD-100 (2 µM) in buffer (curve a) and in the presence of 2 (curve b), 5 (curve c), 7.5 (curve d), 10 (curve e) and 15 (curve f) µM of tubulin. (B) Fluorescence emission spectra of NMK-TD-100 (20 µM) in the presence of aqueous buffer (PEM buffer) and methanol. (C) Scatchard Plot of NMK-TD-100 binding to tubulin. (D) Job Plot for binding of NMK-TD-100 to tubulin. The concentrations of NMK-TD-100 and tubulin were varied continuously whereas the total concentration of tubulin and NMK-TD-100 was kept fixed at 5 µM. The corrected fluorescence value at 450 nm was plotted against the mole fraction of NMK-TD-100. The excitation and emission wavelengths were 340 and 450nm respectively for all the above panels. Data are representative of three similar experiments. Details of all the experiments are described in the ‘Materials and methods’ section.
Figure 9.
Characterization of binding of NMK-TD-100 to tubulin as assessed by quenching of fluorescence of tryptophan residues of tubulin.
(A) NMK-TD-100 reduced the intrinsic tryptophan fluorescence of tubulin (1 µM) in a concentration-dependent manner. (B) Double reciprocal plot of NMK-TD-100 binding to tubulin. Fmax was determined from the plot of 1/(F0-F) versus 1/[NMK-TD-100]. (C) Linear plot of NMK-TD-100 binding to tubulin. The excitation and emission wavelengths were 295 and 335 nm, respectively for all the four panels. Data are representative of three identical experiments. (D) The semi-logarithmic plot of ln(Qmax -Qt) versus time.
Figure 10.
Characterization of binding site for NMK-TD-100.
(A) Time-dependent binding of NMK-TD-100 to preformed tubulin-colchicine complex as monitored by the fluorescence enhancement of NMK-TD-100. The excitation and emission wavelengths were 390 and 450 nm, respectively. (B) Characterization of concentration-dependent binding of NMK-TD-100 to preformed tubulin-colchicine complex. Tubulin (2 µM) was incubated with varied concentration of colchicine (0-100 µM) to form tubulin-colchicine complexes. 5 µM of NMK-TD-100 was added to each of preformed tubulin-colchicine complex and incubated for 30 mins. The enhancement of NMK-TD-100 fluorescence (at 450 nm) was measured for each sample after excitation at 390 nm. Curves 1, 2, 3, 4, 5 and 6 represent fluorescence emission spectra of NMK-TD-100 in presence of 0, 5,10,25,50 and 100 µM colchicine complexed with tubulin. (C) The emission maxima of NMK-TD-100 at 450 nm in presence of tubulin-colchicine complexes were plotted against different concentrations of colchicine. (D) Vinblastine did not inhibit the binding of NMK-TD-100 to tubulin. The excitation and emission wavelengths were 340 and 450 nm, respectively. Data are representative average of three identical experiments along with SD.
Figure 11.
Docking of NMK-TD-100 to tubulin.
(A) In silico binding of NMK-TD-100 (yellow) on tubulin heterodimers. In the ribbon diagram grey represents α-tubulin monomer and blue represents β-tubulin monomer. (B) 3D diagram representing binding site of NMK-TD-100 on tubulin heterodimers. Red is showing the polar domains, blue stands for non-polar domains and white stands for neutral domains on protein surface. (C) Ball and stick figure showing positions of hydrogen bonds which stabilizes NMK-TD-100 molecule on protein surface. (D) The crystal structure of αβ heterodimer of tubulin depicting the proximity of the binding sites for colchicine (yellow) and NMK-TD-100 (red).