Exploration of Antiproliferative Activity and Apoptosis Induction of New Nickel(II) Complexes Encompassing Carbazole Ligands

Abstract

To attest the effectiveness of nickel complexes as anticancer drug candidates with minimum side effects, the present investigation describes the facile synthesis and anticancer activities of nickel(II) complexes enriched with three derivatives of carbazolone-based benzhydrazone ligands(L) having a [Ni(L)₂] composition. Analytical and spectral techniques were used to characterize the synthesized Ni(II) complexes. The single-crystal X-ray diffraction performed for complex 4 confirmed the square planar geometry with a [Ni(κ²-N,O-L)₂] arrangement. The MTT assay was carried out for the complexes to determine in vitro cytotoxicity against cancerous human-cervical carcinoma, human-colon carcinoma, and non-cancerous L929 (fibroblast) cells. All three complexes exhibited good toxicity against the cancer cells with a low IC₅₀ concentration. Complex 4, containing -OCH₃ fragment, exhibits high lipophilicity and revealed exceptional cytotoxicity against cancer cells. AO-EB fluorescent staining indicated apoptosis-associated cell morphological changes after exposure to complex 4. The apoptosis induction was further confirmed by a HOECHST-33342 fluorescent staining technique via chromosomal condensation and nuclear fragmentation. Further, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) mechanistic studies revealed that complex 4 can raise ROS levels and reduce MMP and promote mitochondrial dysfunction-mediated apoptotic cell death. Further, stimulation of late apoptosis by complex 4 in cervical cancer cells was quantitatively differentiated through the staining of phosphatidylserine externalization by flow cytometry. Furthermore, the ELISA analysis confirmed that complex 4 induced apoptosis through caspase activation.

Figure 1

Previous reports on biological activity of Ni(II) square planar complexes.

Scheme 1. Synthesis of Ni(II) Carbazolone Benzhydrazone Complexes

Figure 2

Newly synthesized Ni(II) square planar complexes.

Figure 3

ORTEP diagram of complex 4 with 30% probability. All the hydrogen atoms were omitted for clarity. The bond angles (deg.) around the Ni(1) ion: O(1)–Ni(1)–N(1) = 83.00(6), O(1)–Ni(1)–O(1) = 180.0, and N(1)–Ni(1)–N(1) = 180.0. Bond lengths (Å): Ni(1)–N(1) = 1.9446(14) and Ni (1)–O(1) = 1.8264(12).

Figure 4

AO-EB dual staining of HeLa cancer cells. (A) Control; (B) cells administered with complex 4 (IC₅₀ concentration) for 24 h; and (C) percentage of live and apoptotic cells.

Figure 5

HOECHST staining of HeLa cancer cells. (A) Control; (B) cells administered with complex 4 (IC₅₀ concentration) for 24 h; and (C) percentage of nonfragmented and fragmented cells.

Figure 6

ROS detection of HeLa cancer cells. (A) Control; (B) cells administered with complex 4 (IC₅₀ concentration) for 24 h; and (C) fluorescent intensities based on ROS generation.

Figure 7

JC-1 staining of HeLa cancer cells. (A) Control and (B) cells administered with complex 4 (IC₅₀ concentration) for 24 h.

Figure 8

Double staining method of HeLa cancer cells (A) control and (B) cells administered with complex 4 (IC₅₀ concentration) for 24 h.

Figure 9

Effect of complex 4 on caspase—3, 8, 9 in HeLa cells by the ELISA assay for 12 and 24 h.