New 1,2,3-triazole linked ciprofloxacin-chalcones induce DNA damage by inhibiting human topoisomerase I& II and tubulin polymerization

Abstract

A series of novel 1,2,3-triazole-linked ciprofloxacin-chalcones 4a-j were synthesised as potential anticancer agents. Hybrids 4a-j exhibited remarkable anti-proliferative activity against colon cancer cells. Compounds 4a-j displayed IC₅₀s ranged from 2.53-8.67 µM, 8.67-62.47 µM, and 4.19-24.37 µM for HCT116, HT29, and Caco-2 cells; respectively, whereas the doxorubicin, showed IC₅₀ values of 1.22, 0.88, and 4.15 µM. Compounds 4a, 4b, 4e, 4i, and 4j were the most potent against HCT116 with IC₅₀ values of 3.57, 4.81, 4.32, 4.87, and 2.53 µM, respectively, compared to doxorubicin (IC₅₀ = 1.22 µM). Also, hybrids 4a, 4b, 4e, 4i, and 4j exhibited remarkable inhibitory activities against topoisomerase I, II, and tubulin polymerisation. They increased the protein expression level of γH2AX, indicating DNA damage, and arrested HCT116 in G2/M phase, possibly through the ATR/CHK1/Cdc25C pathway. Thus, the novel ciprofloxacin hybrids could be exploited as potential leads for further investigation as novel anticancer medicines to fight colorectal carcinoma.

Keywords: Click synthesis; DNA damage; HCT116 cells; chalcone; ciprofloxacin; cytotoxicity; topoisomerase I and II inhibitors; tubulin polymerisation inhibition.

Graphical abstract

Figure 1.

Chemical structures of several examples of some reported N-4 piperazinyl ciprofloxacin derivatives with anti-proliferative activities.

Figure 2.

Potential anticancer drugs based on 1,2,3-triazole nucleus in active clinical trials.

Figure 3.

Some recently reported anti-proliferative 1,2,3-triazole/chalcone hybrids.

Figure 4.

Pharmacophore of target compounds 4a-j.

Scheme 1.

Reagents and conditions: (a) Conc. H₂SO₄, NaNO₂, NaN₃, 0–5 °C; (b) Proper aromatic aldehydes, 60% sodium hydroxide, ethanol, stirred overnight at 0–5 °C; (c) Propargyl bromide, NaHCO₃, DMF, stirred overnight at 80 °C; (d) CuSO₄·5H₂O, Sodium ascorbate, DMF, CH₂Cl₂, MeOH, H₂O (3:2:1:1) rt stirring 24 h.

Figure 5.

Cytotoxic effects of CP hybrids (4a, 4b, 4e, 4i, and 4j) and doxorubicin against non-cancerous cells (HEK293) after 72 h incubation period. (A) Concentration-cell viability curves of CP hybrids and doxorubicin treated HEK293 cells using different concentrations (0.01–100 µM, n = 3) and 72 h incubation period. (B) IC₅₀ values were estimated by fitting the cytotoxicity data into a linear regression model using GraphPad Prism software. Data were statistically compared by one-way ANOVA followed by Tuckey’s multiple comparisons test (****p < 0.0001, ns = not significant). (C and D). Microscopic images of HEK293 cells treated with different concentrations of CP hybrids (4a, 4b, 4e, 4i, and 4j) and doxorubicin for 72 h (20× objective lens, total magnification = 200×). Scale bar = 400 µM.

Figure 6.

The effect of IC₅₀ values of CP hybrids 4a, 4b, 4e, 4i, 4j and camptothecin (CPT, positive control in Topo-I assay) on the activity of human topoisomerase I. (A) DNA relaxation assay. (B) in-vitro enzyme assay. Data are plotted as means ± SEM of 3 experiments. ^∗∗ p < 0.01 and ^∗∗∗ p < 0.001 refer to the statistically significant differences in comparison to DMSO (vehicle, negative control treatment).

Figure 7.

The effect of ciprofloxacin hybrids 4a, 4b, 4e, 4i, 4j, and etoposide (ETO, positive control in Topo-II assay) on the activity of human topoisomerase II. (A). Agarose gel electrophoresis of human topoisomerase II assay. (B). Topo-II inhibitory activity of ciprofloxacin hybrids.

Figure 8.

Effect of ciprofloxacin derivatives on tubulin polymerisation. HCT116 cells were untreated (control) and treated with IC₅₀ values of compounds 4a, 4b, 4e, 4i, 4j, paclitaxel or nocodazole for 36 h. (A) Morphological changes of HCT116 cells visualised by immunofluorescence microscopy. Tubulin were shown in green and the nuclei were in blue. (B) Western blotting analysis.

Figure 9.

CP hybrids induce DNA damage. (A) γH2AX staining in HCT116 cells treated with IC₅₀ values of CP hybrids 4a, 4b, 4e, 4i, 4j, or DMSO (negative control). (B) γH2AX-positive nuclei %. (C) Western blot revealed that levels of γH2AX were increased in HCT116 cells treated with CP hybrids 4a, 4b, 4e, 4i, 4j, DMSO (negative control), and GAPDH (loading control). (D) The relative protein level of γ-H2AX, in comparison to GAPDH was measured by ImageJ. Data are plotted as means ± SEM of 3 experiments. ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001 refer to the statistically significant differences in comparison to DMSO.

Figure 10.

Analysis of cell cycle. HCT116 cells were treated with different concentrations of ciprofloxacin hybrids 4a, 4b, 4e, 4i, and 4j for 24 h. Doxorubicin, camptothecin, and etoposide were used as assay controls. The X-axis displays the fluorescence intensity corresponding to the DNA content per HCT116 cell. DNA histograms were analysed by ModFit LT software.

Figure 11.

Ciprofloxacin hybrids 4a, 4b, 4e, 4i, and 4j induce G2/M cell cycle arrest possibly via ATR/CHK1/Cdc25C pathway A. Western blot revealed that expressions of p-ATR, p-CHK1, and p-Cdc25C were increased in HCT116 cells treated with Ciprofloxacin hybrids 4a, 4b, 4e, 4i, 4j, DMSO (negative control), and GAPDH (loading control) B. The relative protein levels of p-ATR, p-CHK1, and p-Cdc25C, compared to their total form ATR, CHK1, and Cdc25C, respectively, were measured by ImageJ. Data are plotted as means ± SEM of 3 experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 refer to the statistically significant differences in comparison to DMSO.

Figure 12.

Binding mode and H-bonds interactions of compound 4a (left) and camptothecin (right) within ‎1T8I ‎active site: (a) 3 D structure and (b) 2D interactions.

Figure 13.

Binding mode and H-bonds interactions of compound 4a (left) and etoposide (right) within ‎6ZY7 ‎active site: (a) 3 D structure and (b) 2 D interactions.