Anticancer Function and ROS-Mediated Multi-Targeting Anticancer Mechanisms of Copper (II) 2-hydroxy-1-naphthaldehyde Complexes

Abstract

Multi-targeting of oncoproteins by a single molecule represents an effectual, rational, and an alternative approach to target therapy. We carried out a systematic study to reveal the mechanisms of action of newly synthesized Cu²⁺ compounds of 2-naphthalenol and 1-(((2-pyridinylmethyl)imino)methyl)- (C1 and C2). The antiproliferative activity of the as-synthesized complexes in three human cancer cell lines indicates their potential as multi-targeted antitumor agents. Relatively, C1 and C2 showed better efficacy in vitro relative to Cisplatin and presented promising levels of toxicity against A-549 cells. On the whole, the Cu²⁺ complexes exhibited chemotherapeutic effects by generating reactive oxygen species (ROS) and arresting the cell cycle in the G₀/G₁ phase by competent regulation of cyclin and cyclin-dependent kinases. Fascinatingly, the Cu²⁺ complexes were shown to activate the apoptotic and autophagic pathways in A-549 cells. These complexes effectively induced endoplasmic reticulum stress-mediated apoptosis, inhibited topoisomerase-1, and damaged cancer DNA through a ROS-mediated mechanism. The synthesized Cu²⁺ complexes established ROS-mediated targeting of multiple cell signaling pathways as a fabulous route for the inhibition of cancer cell growth.

Keywords: 2-hydroxy-1-naphthaldehyde; Cu(II) complex; anticancer mechanism; cytotoxicity.

Figure 1

(A) Chemical structures of Cu²⁺ compounds. (B) Synthetic routes of Cu²⁺ compounds (C1 and C2).

Figure 2

(A) Effect of ROS levels of A-549 cells treated with C1 (1.4 μM) and C2 (1.4 μM) for 24 h compared with untreated cells, quantification of the flow cytometric results. (B) Cu and Pt contents (%) in A-549 cells after treatment with C1 (1.4 μM) and C2 (1.4 μM) and analyzed by ICP-MS. (C) Western blot analysis of the expression level of LC3-II, Beclin-1, and P62 in A-549 cells treated with C1 (1.4 µM) and C2 (1.4 µM) relative to control. (D) The percentage expression level of the LC3-II, Beclin-1, and P62. Mean ± SD, n = 3, * p < 0.05, ** p < 0.01.

Figure 3

Tumor spheroid analysis of A-549 cells after being treated with C1 and C2 with specified concentrations for 7 days.

Figure 4

(A) Effect of the cell cycle of A-549 cells treated with C1 (1.4 µM) and C2 (1.4 µM) compared with untreated cells. (B) Western blot analysis of CDK2 and cyclin E in A-549 cells treated with C1 (1.4 μM) and C2 (1.4 μM). (C) Percentage expression levels of CDK2 and cyclin E. The percentage values are those relative to the control. Results are the mean ± SD, n = 3, * p < 0.05, ** p < 0.01.

Figure 5

(A) Effect of cell apoptosis of A-549 cells treated with C1 (1.4 µM) and C2 (1.4 µM) comparing with untreated cells. (B) Western blot analysis of Bax, Bcl-2, Bcl-xl, caspase 3 (cleaved), Caspase 9 (cleaved), and Cytochrome-c in A-549 cells treated with C1 (1.4 µM) and C2 (1.4 µM). (C) The percentage expression level of Casp-3 (cleaved), Casp-9 (cleaved), and cytochrome-c relative to control. (D) The percentage expression level of Bax, Bcl-2, and Bcl-xl relative to its control value. Results are the mean ± SD, n = 3, * p < 0.05, ** p < 0.01.

Figure 6

Assay of the A-549 cells’ mitochondrial membrane potential with JC-1 fluorescence probe staining compared with untreated cells.

Figure 7

(A) A-549 cells treated with C1 and C2 and analysis of Ca²⁺ concentration. (B) Western blot analysis of PERK, eIF2α, and CHOP in A-549 cells. (C) Graphical presentation of the expression levels of PERK, eIF2α, and CHOP. Mean ± SD, (n = 3), * p < 0.05, **p < 0.01.

Figure 8

Generation of ROS by using 2′,7′-dichlorofluorescein (DCF) and analysis of ER-tracker red by binding to sulfonylurea receptors in the ER.

Figure 9

(A) Western blot analysis of Akt1/2, P38, JNKs, Erk-1/2 in A-549 cells treated with C1 (1.4 µM) and C2 (1.4 µM). (B) Percentage expression levels of Akt1/2, P38, JNKs, Erk-1/2 relative to their control value. Mean ± SD, n = 3, * p < 0.05, ** p < 0.01.

Figure 10

(A) Cleavage of pBR322 plasmid DNA by C1 and C2, using agarose gel electrophoresis. (B) Cleavage of pBR322 plasmid DNA by C1 and C2 in the presence of H₂O₂ using agarose gel electrophoresis; lane 1: Cleavage of pBR322 plasmid DNA by C1 and C2, using agarose gel electrophoresis. (B) Cleavage of pBR322 plasmid DNA by C1 and C2 in the presence of H₂O₂ using agarose gel electrophoresis; lane 1: Plasmid DNA + H₂O₂; lane 2: Plasmid DNA + H₂O₂ + CuCl₂; lane 3: Plasmid DNA + H₂O₂ + C1 (0.7 µM); lane 4: Plasmid DNA + H₂O₂ + C1 (1.4 µM); lane 5: Plasmid DNA + H₂O₂ + C2 (0.7 µM); lane 6: Plasmid DNA + H₂O₂ + C2 (1.4 µM). C. Inhibition of topoisomerase-I activity; lane 1: Plasmid DNA; lane 2: Plasmid DNA + 1U topoisomerase I; lane 3: Plasmid DNA + 1U topoisomerase-I + C1 (0.7 µM); lane 4: Plasmid + 1U topoisomerase-I + C (1.4 µM); lane 5: Plasmid DNA + 1U topoisomerase-I + C2 (0.7 µM); lane 6: Plasmid DNA + topoisomerase-I + C2 (1.4 µM).

Figure 11

(A) UV-Vis titration of 5 µM C1 with ct-DNA in 10 mM Tris-HCl (7.4). (B) UV-Vis titration of 5 µM C2 with ct-DNA in 10 mM Tris-HCl (7.4). (C) Effect of C1 on the emission intensity of EB (8 µM) bound to ctDNA (10 µM) in pH 7.2 Tri-HCl buffer. (D) Effect of C2 on the emission intensity of EB (8 µM) bound to ctDNA (10 µM) in pH 7.2 Tri-HCl buffer.