Animals ; Antineoplastic Agents, Phytogenic ; chemistry ; pharmacology ; Cell Division ; drug effects ; Colchicine ; analogs & derivatives ; chemistry ; pharmacology ; Humans ; Molecular Structure ; Structure-Activity Relationship ; Tubulin ; drug effects ; Tumor Cells, Cultured

This experimental research was aimed to establish an optimum system of enucleation, purification and identification for preparing the cytoplasts of suspension culture cells in order to undertake cell recombination. Human leukemia HL-60 cells in suspension culture were purified by 42% Percoll density gradient centrifugation and low-speed centrifugation at 1 500r/min, respectively. The purified HL-60 cells were treated with cytochalasin B (CB) alone or combined with colcchicine and enucleated by isopycnic gradient centrifugation on 50% Percoll at 25 degrees C and 34 degrees C, respectively. Cytoplasts made from HL-60 cells were purified through gradient centrifugation by 37%, 38% and 40% Percoll, respectively. The final cytoplasts were identified by Wright-Giemsa staining and 4,6-diamidino-2-phenylidole dihydrochloride (DAPI)/5, 6-carboxyflu-orescein diacetate succinimidyl ester (CFSE) double-staining. The phenotype and mitochondrial membrane potential of HL-60 cytoplasts were analyzed by flow cytometry. The results indicated that the enucleation ratio of HL-60 cells induced by CB combined with colcchicine was up to 91. 98% +/-4. 29%, which was significantly higher than that in CB alone group (74. 95% +/- 3. 02%)(P<0. 01). The rates of enucleation and cytoplast with diameter over 5min in 34 degrees C group were higher than those in 25 degrees C group (all P<0. 01). The cytoplast purities were (95.43 +/- 0. 59)% in 38% Percoll groups,which were higher than those of 40% Percoll (P<0. 05). Nucleus and caryoplasm could be clearly distinguished by DAPI and CFSE double labeling. The results further showed that the phenotype of HL-60 cytoplasts had no significant change, and the activity of the cytoplasts was above 80% within 12h. It is concluded that enucleation throuth density gradient centrifugation on 50% Percoll mediated by CB combined with colcchicine, 38%Percoll of purification followed by DAPI/CFSE double labeling and MMP detection is an optimum scheme for preparation and identification of cytoplast from suspension culture cells.
Cell Compartmentation ; Cell Nucleus ; Cell Separation ; Centrifugation, Density Gradient ; Colchicine ; analogs & derivatives ; pharmacology ; Cytochalasin B ; pharmacology ; Cytoplasm ; HL-60 Cells ; Humans

New colchicine analogs have been synthesized with the aim of developing stronger potential anticancer activities. Among the analogs, CT20126 has been previously reported to show immunosuppressive activities. Here, we report that CT20126 also shows potential anticancer effects via an unusual mechanism: the modulation of microtubule integrity and cell cycle arrest at the G2/M phase before apoptosis. When we treated COS-7 cells with CT20126 (5 muM), the normal thread-like microtubules were disrupted into tubulin dimers within 10 min and thereafter repolymerized into short, thick filaments. In contrast, cells treated with the same concentration of colchicine exhibited microtubule depolymerization after 20 min and never underwent repolymerization. Furthermore, optical density (OD) analysis (350 nm) with purified tubulin showed that CT20126 had a higher repolymerizing activity than that of Taxol, a potent microtubule-polymerizing agent. These results suggest that the effects of CT20126 on microtubule integrity differ from those of colchicine: the analog first destabilizes microtubules and then stabilizes the disrupted tubulins into short, thick polymers. Furthermore, CT20126 induced a greater level of apoptotic activity in Jurkat T cells than colchicine (assessed by G2/M arrest, caspase-3 activation and cell sorting). At 20 nM, CT20126 induced 47% apoptosis among Jurkat T cells, whereas colchicine induced only 33% apoptosis. Our results suggest that the colchicine analog CT20126 can potently induce apoptosis by disrupting microtubule integrity in a manner that differs from that of colchicine or Taxol.
Acetylation/drug effects ; Animals ; Apoptosis/*drug effects ; COS Cells ; Caspase 3/metabolism ; Cattle ; Cell Division/drug effects ; Cercopithecus aethiops ; Colchicine/*analogs & derivatives/chemistry/pharmacology ; Enzyme Activation/drug effects ; G2 Phase/drug effects ; Humans ; Jurkat Cells ; Microtubules/*metabolism ; Poly(ADP-ribose) Polymerases/metabolism ; Tubulin/metabolism ; Tubulin Modulators/chemistry/*pharmacology

AIMTo search for colchicine derivatives which have high efficacy and low toxicity.

METHODSColchicine was firstly converted into thiocolchicine, and then it was hydrolyzed to get 7-(N-deacetylthiocolchicine). At last, 7-(N-deacetylthiocolchicine) was amidated to get the target compounds. The chemical structure of these new derivatives was confirmed with 1H NMR, IR, MS, and HR-MS. The cytotoxicity of the compounds was tested by MTT assay. Their in vivo antitumor activity was evaluated against mice tumor H22 and U14.

RESULTSTwelve thiocolchicine derivatives are new compounds.

CONCLUSIONIn vitro antitumor activity has showed that some of these thiocolchicines possessed cytotoxic activity superior to colchicine. However, in vivo antitumor activity indicated that these derivatives have poor efficacy in mice.

Animals ; Antineoplastic Agents, Phytogenic ; chemical synthesis ; chemistry ; pharmacology ; Cell Line, Tumor ; Cell Survival ; drug effects ; Colchicine ; analogs & derivatives ; chemical synthesis ; chemistry ; pharmacology ; Humans ; Inhibitory Concentration 50 ; Liver Neoplasms, Experimental ; pathology ; prevention & control ; Male ; Mice ; Mice, Inbred ICR ; Models, Chemical ; Molecular Structure ; Neoplasm Transplantation ; Prostatic Neoplasms ; pathology ; prevention & control ; Structure-Activity Relationship

Colchicine has been shown to regulate the expression of inflammatory gene, but this compound possesses much weaker anti-inflammatory activity. In this study, we synthesized a new colchicine derivative CT20126 and examined its immunomodulatory property. CT20126 was found to have immunosuppressive effects by inhibiting lymphocyte proliferation without cytotoxicity and effectively inhibit the transcriptional expression of the inflammatory genes, iNOS, TNF-alpha, and IL-1beta, in macrophages stimulated by LPS. This effect was nearly comparable to that of cyclosporine A. This compound also significantly suppressed the production of nitric oxide and Th1-related pro-inflammatory cytokines, IL-1beta, TNF-alpha, and IL-2, with minimal suppression of Th2-related anti-inflammatory cytokines IL-4 and IL-10 in the sponge matrix allograft model. Moreover, administration of CT20126 prolonged the survival of allograft skins from BALB/c mice (H-2d) to the dorsum of C57BL/6 (H-2b) mice. The in vivo immune suppressive effects of CT20126 were similar to that of cyclosporine A. These results indicate that this compound may have potential therapeutic value for transplantation rejection and other inflammatory diseases.
Animals ; Cell Line ; Colchicine/*analogs & derivatives/chemistry/*pharmacology ; Cytokines/*biosynthesis ; Female ; Gene Expression Regulation/drug effects ; Graft Survival/*drug effects ; Immunosuppression ; Interleukin-1beta/genetics/metabolism ; Lipopolysaccharides/pharmacology ; Lymphocyte Culture Test, Mixed ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Nitric Oxide/biosynthesis ; Nitric Oxide Synthase Type II/genetics/metabolism ; Skin Transplantation/*immunology ; Th1 Cells/*drug effects/immunology/metabolism ; Th2 Cells/*drug effects/immunology/metabolism ; Transplantation, Homologous ; Tumor Necrosis Factor-alpha/genetics/metabolism