Apoptotic effect of novel Schiff based CdCl₂(C₁₄H₂₁N₃O₂) complex is mediated via activation of the mitochondrial pathway in colon cancer cells

Abstract

The development of metal-based agents has had a tremendous role in the present progress in cancer chemotherapy. One well-known example of metal-based agents is Schiff based metal complexes, which hold great promise for cancer therapy. Based on the potential of Schiff based complexes for the induction of apoptosis, this study aimed to examine the cytotoxic and apoptotic activity of a CdCl2(C14H21N3O2) complex on HT-29 cells. The complex exerted a potent suppressive effect on HT-29 cells with an IC50 value of 2.57 ± 0.39 after 72 h of treatment. The collapse of the mitochondrial membrane potential and the elevated release of cytochrome c from the mitochondria to the cytosol indicate the involvement of the intrinsic pathway in the induction of apoptosis. The role of the mitochondria-dependent apoptotic pathway was further proved by the significant activation of the initiator caspase-9 and the executioner caspases-3 and -7. In addition, the activation of caspase-8, which is associated with the suppression of NF-κB translocation to the nucleus, also revealed the involvement of the extrinsic pathway in the induced apoptosis. The results suggest that the CdCl2(C14H21N3O2) complex is able to induce the apoptosis of colon cancer cells and is a potential candidate for future cancer studies.

Figure 1. Lactate dehydrogenase (LDH) assay, demonstrated the cytotoxicity of CdCl₂(C₁₄H₂₁N₃O₂) complex against HT-29 cells.

The result showed significant cytotoxicity at concentration of 3 μg/mL. The data represent the means ± SD of three independent experiments. *P < 0.05 compared with the no-treatment group.

Figure 2. Activity of CdCl₂(C₁₄H₂₁N₃O₂) complex on cell cycle arrest in the S/M phase.

(A) After incubation with DMSO (negative control) or Schiff based compound (3 μg/mL) for 24 h, HT-29 cells were collected, stained with BrdU (representing the S phase) and Phospho-Histone H3 (representing the M phase), and subjected to cell cycle analysis using a Cellomics ArrayScan HCS reader. (B) Representative bar charts showing that treatment with CdCl₂(C₁₄H₂₁N₃O₂) complex caused no significant changes in the BrdU and Phospho-Histone H3 fluorescence intensities, suggesting that the cells do not arrest at the S/M phases. The data represented the means ± SD of the fluorescence intensity readings from three independent experiments. *P < 0.05 compared with the no-treatment group.

Figure 3. Effect of CdCl₂(C₁₄H₂₁N₃O₂) complex on cell cycle progression in HT-29 cells.

This effect was assessed by flow cytometry. After incubation with the Schiff based compound for 24 and 48 h, significant cell cycle arrest at the G₁ phase was observed. All of the data are expressed as the means ± standard error of triplicate measurements. *P < 0.05 compared with the no-treatment group.

Figure 4. Fluorescent micrographs of acridine orange and propidium iodide double-stained HT-29 cells.

(A) Untreated HT-29 cells were healthy after 72 h. In addition, early apoptotic features, including blebbing and chromatin condensation, were observed after (B) 24 and (C) 48 h. (D) Late apoptosis events were observed after 72 h of treatment with 3.0 μg/ml CdCl₂(C₁₄H₂₁N₃O₂) complex (magnification: 200×).VC: Viable cells; BL: Blebbing of cell membrane; CC: Chromatin condensation; LA: Late apoptosis.

Figure 5. ROS generation in the presence of CdCl₂(C₁₄H₂₁N₃O₂) complex.

At concentration of 3 μg/mL, the Schiff based compound caused significant ROS formation in HT-29 cells. All of the data are expressed as the means ± standard error of triplicate measurements. *P < .05 compared with the no-treatment group.

Figure 6. Effects of CdCl₂(C₁₄H₂₁N₃O₂) complex on nuclear morphology, membrane permeability, mitochondrial membrane potential (MMP) and Cytochrome c release.

(A) Representative images of HT-29 cells stained with Hoechst 33342, cytochrome c, membrane permeability, and MMP dyes after treatment with 3 μg/mL CdCl₂(C₁₄H₂₁N₃O₂) complex (magnification: 20×). (B) Representative bar charts indicating the dose-dependent reduction in MMP, the increased cell permeability, and the cytochrome c release in treated HT-29 cells. All of the data are expressed as the means ± standard error of triplicate measurements. *P < 0.05 compared with the no-treatment group.

Figure 7. Effect of various concentrations of CdCl₂(C₁₄H₂₁N₃O₂) complex on caspase 3/7, 8, and 9 activation in HT-29 cells after 24 h of treatment.

The results revealed significant activation of caspases-3/7, -8, and -9. All of the data are expressed as the means ± standard error of triplicate measurements. *P < 0.05 compared with the no-treatment group.

Figure 8. NF-κB Translocation.

(A) Images and (B) representative bar chart of HT-29 cells after treatment with various concentrations of CdCl₂(C₁₄H₂₁N₃O₂) complex for 3 h and subsequent exposure to TNF-α (1 ng/mL) as an NF-κB activator for 30 min. The results did not reveal any significant translocation of NF-κB from the cytoplasm to the nucleus. All of the data are expressed as the means ± standard error of triplicate measurements. *P < 0.05 compared with the no-treatment group.

Figure 9. Western blot analysis of CdCl₂(C₁₄H₂₁N₃O₂)-treated HT-29 cells.

Western blot analysis revealed the expression levels of cleaved caspase-3 and truncated Bid in CdCl₂(C₁₄H₂₁N₃O₂)-treated HT29 and CCD841 cells. β-actin served as a loading control.

Figure 10. Apoptosis evaluation of HT-29 cells treated with Calcium cheator BAPTA/AM piror to CDCL2(C14H21N3O2) compound.

(A) Represents the untreated cells as the control, (B) 24 h treatment of HT-29 cell with compound, (C) 24 h treatment of HT-29 cell with BAPTA/AM and CDCL2(C14H21N3O2) compound.

Figure 11. HT-29 cells were treated with DMSO or different concentrations of CdCl₂(C₁₄H₂₁N₃O₂) complex for 12 hours.

RNAs were isolated and converted to cDNA. Quantitative real-time PCR was performed to determine expression level of Bcl-2, Bcl-xl and Bax genes. GAPDH was used as a housekeeping gene.

Figure 12. Chemical structure of CdCl₂(C₁₄H₂₁N₃O₂).