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. 2017 Aug 31;2(8):4632-4646.
doi: 10.1021/acsomega.7b00688. Epub 2017 Aug 17.

Potent Anticancer Activity With High Selectivity of a Chiral Palladium N-Heterocyclic Carbene Complex

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Free PMC article

Potent Anticancer Activity With High Selectivity of a Chiral Palladium N-Heterocyclic Carbene Complex

Anuj Kumar et al. ACS Omega. .
Free PMC article

Abstract

Five enantiomeric pairs of palladium complexes of 1,2,4-triazole-derived chiral N-heterocyclic carbene ligands were investigated to probe the influence of chirality on the compound's anticancer activity. Although no chirality-related influence was observed for any of the enantiomeric pair, strong anticancer activity was seen for a particular pair, (1S,2S,5R)-1c and (1R,2R,5S)-1c, which was significantly more active than the benchmark drug cisplatin for human breast cancer cells, MCF-7 (ca. 24-27-fold), and human cervical cancer cells, HeLa (ca. three- to fourfold). Broadening its scope of application, (1R,2R,5S)-1c also exhibited antiproliferative activity against lung cancer (A549), skin cancer (B16F10), and multidrug-resistant mammary tumor (EMT6/AR1) cell lines. Interestingly, (1R,2R,5S)-1c displayed 8- and 16-fold stronger antiproliferative activity toward B16F10 and MCF-7 relative to their respective noncancerous counterparts, L929 (fibroblast skin cells) and MCF10A (epithelial breast cells), thereby upholding the potential of these complexes for further development as anticancer agents. (1R,2R,5S)-1c inhibited tumor-cell proliferation by blocking the cells at the G2 phase. (1R,2R,5S)-1c caused DNA damage in MCF-7 cells, leading to mitochondrial reactive oxygen species production and subsequently cell death. We also present evidence indicating that (1R,2R,5S)-1c induced p53-dependent programmed cell death in MCF-7 cells.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Menthyl-derived chiral palladium complexes of 1,2,4-triazole-based NHC ligands.
Scheme 1
Scheme 1. Synthesis of the Menthyl-Derived Chiral 1,2,4-Triazole-Based NHC Complexes of Palladium
Figure 2
Figure 2
Oak Ridge thermal ellipsoid plot (ORTEP) of 1S,2S,5R-1c with thermal ellipsoids are shown at the 50% probability level. Selected bond lengths (angstrom) and angles (degrees): Pd(1)–C(1) 2.025(4), Pd(1)–C(15) 2.030(4), Pd(1)–O(1) 2.028(3), Pd(1)–O(4) 2.029(3), C(1)–Pd(1)–C(15) 178.65(17), O(1)–Pd(1)–O(4) 179.18(13), C(1)–Pd(1)–O(4) 87.09(14), and C(15)–Pd(1)–O(1) 87.95(14).
Figure 3
Figure 3
ORTEP of 1R,2R,5S-1c with thermal ellipsoids are shown at the 50% probability level. Selected bond lengths (angstrom) and angles (degrees): Pd(2)–C(3) 2.047(11), Pd(2)–C(4) 2.028(10), Pd(2)–O(7) 2.036(7), Pd(2)–O(5) 2.019(7), C(4)–Pd(2)–C(3) 179.0(5), O(7)–Pd(2)–O(5) 178.5(3), C(4)–Pd(2)–O(7) 92.2(3), and C(3)–Pd(2)–O(5) 92.3(4).
Figure 4
Figure 4
Graphical representation of the anticancer activity of the (NHC)2PdX2-type complexes, where X is Br or OCOCF3 against MCF-7 cells.
Figure 5
Figure 5
(a) Graphical representation of the comparison of the anticancer activity of the most potent Pd–NHC complexes (1S,2S,5R)-1c and (1R,2R,5S)-1c with cisplatin against MCF-7 and HeLa cell lines. (b) Graphical representation of the comparison of the anticancer activity of (1R,2R,5S)-1c against cancer cell lines, MCF-7 and B16F10, and their respective noncancerous counterparts, MCF10A and L929.
Figure 6
Figure 6
Graphical representation of the anticancer activity of the most potent Pd–NHC complex (1R,2R,5S)-1c against various cancer cell lines, namely, MCF-7, HeLa, B16F10, A549, and EMT6/AR1.
Figure 7
Figure 7
(1R,2R,5S)-1c cleaves PARP and causes apoptosis in MCF-7 cells. (a) (1R,2R,5S)-1c treatment leads to cell death in MCF-7 cells. MCF-7 cells were incubated without and with 4 and 8 μM (1R,2R,5S)-1c for 48 h and analyzed by flow cytometry. Representative images from three independent sets of experiments are shown. (b) (1R,2R,5S)-1c cleaves PARP in MCF-7 cells, indicating apoptosis. MCF-7 cells were treated without and with 4 and 8 μM (1R,2R,5S)-1c for 48 h. PARP cleavage was determined by western blot using anti-PARP immunoglobulin G (IgG). Actin was used as a loading control. The experiment was performed three times. Representative blot is shown.
Figure 8
Figure 8
(1R,2R,5S)-1c causes DNA damage in MCF-7 cells. (a) MCF-7 cells were treated without and with 4 and 8 μM (1R,2R,5S)-1c for 36 h and immunostaining was performed using anti-γ-H2AX IgG (pink). DNA was stained using Hoechst 33258 (blue). The scale bar is 20 μm. (b) γ-H2AX intensity was calculated using ImageJ software. The experiment was performed three times, and in each case, 500 cells were scored. *p < 0.05 and **p < 0.01 show statistical significance of the data.
Figure 9
Figure 9
(1R,2R,5S)-1c generates reactive oxygen species in MCF-7 cells. (a) Flow cytogram showing intracellular ROS generation in MCF-7 cells untreated (negative control) and treated (positive control) with 30 μM H2O2 and 4 and 8 μM (1R,2R,5S)-1c for 24 h. (b) Percentage of DCFH-DA-positive cells were quantified and plotted. **p < 0.01 shows statistical significance of the data.
Figure 10
Figure 10
(1R,2R,5S)-1c arrested MCF-7 cells at the G2 phase of the cell cycle. (a) (1R,2R,5S)-1c blocked cells at the G2/M phase of the cell cycle. DNA distribution profiles in different phases of the cell cycle after 36 h treatment with media alone (control) or 2 and 4 μM of (1R,2R,5S)-1c were determined by flow cytometry. Data analysis was done using FlowJo software. (b) (1R,2R,5S)-1c did not arrest cells at mitosis. MCF-7 cells were incubated without and with 2 and 4 μM (1R,2R,5S)-1c for 36 h. Mitotic cells were visualized on the basis of DNA morphology after staining the cells with Hoechst 33258. The scale bar is 20 μm. (c) Mitotic index was calculated from the microscopy data and plotted. The experiment was performed three times, and 1000 cells were scored in each case. *p < 0.05 shows statistical significance of the data.
Figure 11
Figure 11
(1R,2R,5S)-1c activates p53 pathway and its downstream target p21. (a) (1R,2R,5S)-1c treatment leads to nuclear accumulation of p53. MCF-7 cells were treated without and with 4 and 8 μM (1R,2R,5S)-1c for 36 h, fixed, and processed for immunostaining using anti-p53 IgG. Left panels show DNA stained with Hoechst 33258 (blue), middle panels show the corresponding cells stained with anti-p53 IgG (red), and right panels show merged images. The scale bar is 10 μm. (b) (1R,2R,5S)-1c activates the p21. MCF-7 cells were processed as mentioned above and stained using anti-p21 IgG. Left panels show DNA stained with Hoechst 33258 (blue), middle panels show the corresponding cells stained with anti-p21 IgG (red), and right panels show merged images. The scale bar is 10 μm.

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