Article Figures & Data
Figures
- Fig. 1.
A, synthesis of 9-nitro-noscapine. 9-Nitro-nos was synthesized by the aromatic nitration of noscapine using silver nitrate in acetonitrile and TFAA at 25°C. B, 9-nitro-nos binds tubulin. B shows the fluorescence-quenching spectrum of tubulin for 9-nitro-nos. ▪, control; •, 10 μM; ▴, 20 μM; ▾, 50 μM; ♦, 75 μM; and ○, 100 μM 9-nitro-nos. C, the double reciprocal plot which gives a Kd value of 86 ± 6 μM for 9-nitro-nos binding to tubulin. D, 9-nitro-nos does not significantly change the polymerization rate and the steady-state polymer mass of purified tubulin in vitro. The assay was based on the light-scattering ability of tubulin polymer, reflected as the absorbance at 550 nm wavelength. An equivalent amount of the solvent dimethyl sulfoxide was used as a negative control. ▪, control; •, 25 μM 9-nitro-nos; ▴, 100 μM 9-nitro-nos. Results are representative of four independent experiments.
- Fig. 2.
9-Nitro-nos actively inhibits the proliferation of various human cancer cells, including those that are resistant to vinblastine, teniposide, and paclitaxel. CEM, CEM/VLB100, CEM/VM-1-5, 1A9, and 1A9/PTX22 cells were treated with 9-nitro-nos at gradient concentrations for 72 h. The IC50 value was then measured using standard in vitro proliferation assays. The MTS assay was used for nonadherent suspension cells (CEM, CEM/VLB100, and CEM/VM-1-5), and the sulforhodamine B (SRB) assay was performed for adherent cells (1A9 and 1A9/PTX22). Each value represents the average of two independent experiments with five replicates each.
- Fig. 3.
9-Nitro-nos arrests the cell cycle at G2/M phase in human cancer cells. A and B, the percentage of G2/M and sub-G1 cells as a function of time of treatment with 9-nitro-nos. C to G show an increase in the population of cells with degraded DNA (sub-G1 amount in the far left) in the three-dimensional FACS of DNA amounts. H, the effect of 9-nitro-nos on the cell cycle profile of normal primary fibroblast cells. Even concentrations as high as 100 μM did not alter the cell cycle progression profile of normal human cells. All cells were harvested for analysis at the indicated times, stained with propidium iodide, and analyzed by flow cytometry (FACS) using the CellQuest software. The x-axis shows intensity of PI fluorescence, which is indicative of the total DNA content of cells in different phases of the cell cycle. The y-axis represents the number of cells in each phase of the cell cycle, and the z-axis shows the time points 0, 24, 48, and 72 h. Results are representative of three experiments performed in triplicate.
- Fig. 4.
9-Nitro-nos kills cancer cells by inducing apoptosis. This is revealed by major changes in the nuclear morphology of these cells. A, top, untreated cells that display normal microtubule arrays. 9-Nitro-nos treatment for 72 h completely changes the morphologies, as seen by the fragmented condensed apoptotic bodies (A, bottom). Scale bar, 20 μM. B, the fragmented DNA in the terminal stages of apoptosis was measured using a flow cytometry-based quantitative TdT-mediated BrdUTP reaction (TUNEL assay) in a representative cell line (1A9/PTX22). Untreated control cells (bottom) and 50 μM 9-nitro-nos-treated 1A9/PTX22 cells (top) are shown. After 72 h of 9-nitro-nos treatment, cells were processed for apoptosis by a flow cytometry-based TUNEL assay. The fragmented DNA was also visualized using confocal microscopy as an abundance of TUNEL-positive cells (top right, open arrowhead). Untreated control cells appear red (bottom right, solid arrowhead). Scale bar, 20 μM. Data shown are from a representative experiment of two experiments performed. C, 9-nitronos treatment causes activation of caspase-3. The time-dependent increase in caspase-3 activity on 9-nitronos treatment in CEM, CEM/VLB100, CEM/VM-1-5, 1A9, and 1A9/PTX22 cells is shown. After drug incubation, caspase-3 activity was analyzed using the luminogenic substrate N-benzyloxycarbonyl-Asp-Glu-Val-Asp-aminoluciferin.
Tables
- TABLE 1
Effect of 9-nitro-nos on cell cycle progression of drug-resistant cell lines and their normal counterparts Cells were treated with 9-nitro-nos for the indicated times before being stained with propidium iodide for cell cycle analysis.
Cell Cycle Parameters CEM CEM/VLB100 CEM/VM-1-5 1A9 1A9/PTX22 0 h 24 h 48 h 72 h 0 h 24 h 48 h 72 h 0 h 24 h 48 h 72 h 0 h 24 h 48 h 72 h 0 h 24 h 48 h 72 h % G0/G1 45.5 24.8 19.7 13.9 46.9 25.5 23.7 8.7 36.0 25.4 13.7 10.5 50.0 30.6 28.6 16.7 57.0 27.3 5.9 14.1 S 25.0 10.0 8.1 8.9 17.6 17.7 9.3 4.5 19.4 13.0 11.0 8.7 21.8 14.3 10.6 9.6 8.8 4.7 3.4 5.7 G2/M 20.9 34.2 40.0 16.9 25.7 36.0 30.2 12.5 30.7 41.0 15.0 12.2 21.2 34.2 17.2 9.0 20.4 31.4 44.3 13.7 Sub-G1 0.25 6.5 23.8 44.3 3.6 14.8 30.7 56.1 3.4 18.8 58.0 63.0 0.7 12.0 38.0 56.3 3.5 30.0 44.6 59.0
Erratum
Correction to �Development of a Novel Nitro-Derivative of Noscapine for the Potential Treatment of Drug-Resistant Ovarian Cancer and T-Cell Lymphoma�
In the above article Aneja R, Vangapandu SN, Lopus M, Chandra R, Dulal P, and Joshi HC (2006) Mol Pharmacol 69:1801�1809, there was an error in Fig. 4 on page 1807. One of the published panels was from an experiment different from that described in the figure legend. The corrected version of Fig. 4 appears online only. The figure legend has not been changed.
The online version has been corrected in departure from the print version.
The authors regret this error and apologize for any confusion or inconvenience it may have caused.
