Cytotoxic, analgesic and anti-inflammatory activity of colchicine and its C-10 sulfur containing derivatives

Abstract

10-Alkylthiocolchicines have been obtained and characterized by spectroscopic methods and their biological activities as: cytotoxic, anti-inflammatory and analgesic activities have been tested. Cytotoxic activity against SKOV-3 ovarian cell line for 10-alkylthiocolchicine analogues was reported and tested compounds showed to be more active than commonly used doxorubicin. Some of tested C-10 alkylthiolated colchicines have been found to exhibit cytotoxicity at levels comparable to that of the natural product-colchicine. 10-Methylthiocolchicine has IC₅₀ = 8 nM and 10-ethylthiocolchicine has IC₅₀ = 47 nM in comparison to colchicine IC₅₀ = 37 nM. Moreover for 10-alkylthioderivatives apoptosis test, cyclin B1 and cell cycle tests were performed. 10-n-Butylthiocolchicine was tested for anti-inflammatory and analgesic activities it showed to produce analgesic rather than anti-inflammatory effect.

Figure 1

Colchicine 1 and its derivatives 2–6.

Figure 2

Effect on α-tubulin polymerization in SKOV-3 cells incubated with tested compounds for 24 h. (A, B) control, (C,D) 1 0,1 μM and 1.0 μM respectively; (E,F) compound 2 0,1 μM and 1.0 μM respectively; (G,H) compound 6 0.1 μM and 1.0 μM (bar, 10 μm; FITC-conjugated primary antibody).

Figure 3

Effect of tested compounds on cell cycle distribution in SKOV-3 ovarian cancer cells. Cells were treated with tested compounds: colchicine 1, 2 and 6 at concentrations 0.1 μM, 1.0 μM, and 1.0 μM for 24 h (A) and 72 h (B). After incubation cells were stained with propidium iodide and analyzed using flow cytometry. Statistical significances are marked with asterisks (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.

Figure 4

Apoptosis induction in SKOV-3 treated with compounds: 1, 2 and 6 at concentrations 0.1 μM, 1.0 μM, and 1.0 μM for 24 h (A) and 72 h (B). After incubation cells were stained with stained Hoechst 33,258 and propidium iodide and evaluated using fluorescence microscopy. Statistical significances are marked with asterisks (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.

Figure 5

Impact of tested compounds on Cyclin B1 expression in SKOV-3 cells. Cells were treated with tested compounds: colchicine, 2 and 6 at concentrations 0.1 μM, 10.0 μM for 24 h. All tested groups were significantly different from control group, p < 0.001 (A) Western blot, (B) corresponding results of densitometric analysis.

Figure 6

Impact of tested compounds: colchicine, 2 and 6 on Caspase 3/7 activation in SKOV-3 cells. Cells were treated with tested compounds at concentrations 0.1 μM, and 10.0 μM for 24 h. All tested groups were significantly different from control group, p < 0.001, except group treated with compound 6 at concentration 0.1 µM, p < 0.01.

Figure 7

Effect of tested compounds: colchicine, 2 and 6 on apoptosis (white bars) and necrosis (grey bars) assayed using ELISA in SKOV-3 cells after 24 h of incubation. Statistical significances are marked with asterisks (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.

Figure 8

Impact of verapamil (ABCB₁ inhibitor) on cytotoxic activity of tested compounds: colchicine, 2 and 6. Statistical significances between groups treated without verapamil are marked with asterisks (*) p < 0.05; (**) p < 0.01; (***) p < 0.001. Statistical significances between groups treated with verapamil are marked with asterisks (*) p < 0.05; (**) p < 0.01; (***) p < 0.001. Statistical significances between groups treated with the same concentration of tested compound but with or without verapamil are marked with asterisks (*) p < 0.05; (**) p < 0.01; (***) p < 0.001. (ANOVA F(3, 33) = 16.5; p = 0.0000).

Figure 9

Influence of 6 and indomethacine on anti-inflammatory activity in rats. Values are mean ± SEM, ΔG—value expressing change in paw’s thickness against baseline (before inflammation) after 3 h, 6 (6–1.0) a dose of 1.0 mg/kg, 6 (6–10) a dose of 10.0 mg/kg, **, *—significant difference vs Control group; p < 0.05 or p < 0.1, respectively.

Figure 10

Influence of 6 and morphine on analgesic activity using tail-flick test. Values are mean ± SEM, 6 (6–1.0) a dose of 1.0 mg/kg, 6 (6–10) a dose of 10.0 mg/kg, **, * significant difference vs Control group; p < 0.05 or p < 0.1, respectively.