Highly Promising Antitumor Agent of a Novel Platinum(II) Complex Bearing a Tetradentate Chelating Ligand

Abstract

A new mononuclear cationic platinum(II) coordination compound with 6,6′-bis(NH-benzimidazol-2-yl)-2,2′-bipyridine (L) ligand having N₄-tetradentate binding pocket [Pt(L)]Cl₂·2H₂O (Complex 1) was synthesized and characterized by FT-IR(ATR), UV–vis, ¹H NMR, APCI and MALDI MS, and CHN analysis. The antigrowth effect of Complex 1 was tested in breast cancer (MDA-MB-231), lung cancer (A549), colorectal cancer (HCT-116), prostate cancer (PC-3) cell lines, and bronchial epithelial cell line (BEAS-2B) by the SRB and ATP cell viability assays. Apoptosis was detected with Annexin V, mitopotential, BCL-2 inactivation, and γH2AX assays by flow cytometry. Complex 1 was found to have cytotoxic activity of MDA-MB-231, A549, HCT-116, and PC-3 cancer cell lines in a dose-dependent manner for 48 h. Complex 1 has been found to cause cell death through different mechanisms depending on the type of cancer. The findings indicated that complex induced intrinsic apoptosis with the increased mitochondrial membrane depolarization level, Bcl-2 inactivation, and DNA damage in PC-3 and A549 cell lines.

Since the discovery of cisplatin as a clinically approved anticancer drug, many researchers have focused on the synthesis of new analogous platinum(II) coordination compounds to developed more potent drugs with low side effects.¹ Carboplatin and Oxaliplatin were developed to overcome the resistance observed in some cancer types.² The progress in the new design of platinum(II) drugs has been recently reviewed.³ Chi-Ming Che and co-workers have reported that the Fe(II)-polypyridine complexes which are stable under physiological conditions show higher cytotoxicity toward a series of human carcinoma cell lines than cisplatin and excellent cytotoxic effect on taxol-resistant hepatocellular carcinoma cell line.⁴ The observed high cytotoxic activity of the iron(II) complexes can be ascribed by the stable chelate formation of the polypyridine ligands under physiological conditions. It has been suggested that in order to obtain a high cytotoxic Pt drug, the corresponding Pt(II) complex is supposed to be cationic and planar to increase the interaction with DNA and also should have a favorable chemical structure bearing significant functional groups, such as a NH moiety for H-bonding interactions toward DNA.⁵⁻⁷ In addition, the compound incorporating a large interacting aromatic moiety shows better interactions with DNA.⁸ As previously mentioned, they successfully showed that the calculated binding constants, K_B, increase in the order [Pt(bipy)(py)₂]²⁺ < [Pt-(terpy)(py)]²⁺ < [Pt(quaterpy)]²⁺, upon increasing the aromatic planar surface extension as expected.⁸ It is very well-known that minor changes in the chemical structure can lead to pronounced differences in the biological activity of the organometallic coordination compounds.⁹ This is confirmed by the Pt(bpy)Cl₂ and analogous coordination compounds with 2,2′-biyridine (bpy) moieties which exhibit lower cytotoxic activity than cisplatin, but the cis-platinum(II) complex with 2′-pyridyl(benzimidazole) shows the notable cytotoxicity toward certain tumor cell lines. Yau et al. have shown that the binding of the platinum(II) complex [Pt(QP)](CF₃SO₃)₂ (QP = quaterpyridine, N₄-donor ligand) to DNA is comparatively stronger that Ru(II)–cyclam complex in which the N4-donor ligand cyclam does not contain planar aromatic rings.¹⁰ The present study offers a new approach for developing a promising anticancer drug based on platinum(II) coordination compound. The new complex is a dicationic Pt(II) complex showing square-planar geometry and is very stable in the solid state and in solution. The ligand has a tetradentate binding pocket which makes the complex essentially planar. The extension of the aromatic surface was achieved by introducing the benzimidazole moieties at 6,6′-positions of 2,2′-bipyridine chromophore. Here we present the cytotoxic studies of the mononuclear square-planar Pt(II) Complex 1 which provides an extended aromatic surface with NH functionalities on the benzimidazole groups (Scheme 1).

Scheme 1. Schematic Presentation of the Synthesis of Complex 1.

The ligand (L), 6,6′-bis(NH-benzimidazol-2-yl)-2,2′-bipyridine was synthesized according to the literature method.¹¹ The ¹H and ¹³C NMR spectra (Figure S1 and S2, respectively) which are consistent with the literature data clearly confirm the structure of the ligand. The observed peak at m/z 411.1 in the ESI-MS (Figure S3) can be assigned to [L + Na]⁺. The [Pt(L)]Cl₂·2H₂O complex was obtained in good yield from the reaction of Pt(COD)Cl₂ and the ligand in DMF as shown in Scheme 1. The ¹H NMR spectrum of Complex 1 shows five well-resolved signals in the aromatic region (Figure S4), corresponding to the pyridyl and the benzimidazole protons as akin to that of corresponding Ru(II) complex.¹² The benzimidazole rings which are magnetically equivalent indicate that the molecule holds C₂ symmetry on the L ligand. The broad singlet at 12.90 ppm corresponding to the benzimidazole NH protons indicates that L coordinates to the metal center through the pyridyl and the benzimidzole tertiary N atoms to afford a dicationic complex ion, [Pt(L)]²⁺, in solution. The lowest field doublet centered at δ 9.13 ppm is assigned to the H3,3′ protons of the 2,2′-bipyridine. The doublet at δ 8.47 is assigned to the H5,5′ protons and that at δ 8.30 to the H4,4’ protons of the 2,2′-bipyridine moiety. The multiplets at between δ 7.74 and 7.30 ppm are due to the benzimidazole protons. It is interesting to note that the H3,3′-protons of the 2,2′-bipyridine undergo deshielding in comparison to those of the free ligand. This may be due to van der Waals forces between the adjacent protons as a result of the distortion of the chelate ligand from the planarity upon coordination to the metal center. The APCI MS confirms the structure of the complex in solution. In the MS spectrum (Figure S5), the major peak m/z 389.1 is assigned to the ligand. The peak at m/z 583.2 is assigned to the [Pt(L)]⁺ (calcd for C₂₄H₁₆N₆Pt 583.10). A small peak observed at m/z 690.1 can be assigned to the [Pt(L)]Cl₂·2H₂O (calcd for C₂₄H₂₀N₆O₂Cl₂Pt m/z 691.069). The softer mass analysis method, MALDI-TOF MS, shows the molecular ion peak at m/z 582.098 (Figure S6a) which evidently confirms the structure very well as the theoretical and experimental isotopic distribution are identical (Figure S6b). It is further confirmed by the CHN analysis that the title compound is hydrated in the solid state as well. Further support was provided by the FT-IR spectrum which shows a broad band at 3375 cm^–1 due to the ν(OH) water molecules (Figure S7). This very broad band masks the stretching band of the NH that appears in the same region of the spectrum. The complex exhibits characteristic ring stretching modes between 1630 and 1455 cm^–1 as observed for the related compounds.¹³ The present Complex 1 resembles the Pt(II) complex with quaterpyridine (QP), [Pt(QP)]²⁺, in which the chromophore is essentially planar with only slight twisting of the ligand frame.^14,15

PC-3, A549, MDA-MB-231, HCT-116, and BEAS-2B cells were treated with varying concentrations (1.25–40 μM) of Complex 1 for 48 h to evaluate the antigrowth/cytotoxic effect via SRB cell viability assay and confirmed by ATP assay. It was shown in previous studies that ATP assay is more sensitive than other cell viability tests.^16,17 As depicted in Figure 1, it was found that Complex 1 decreased cell viability in a dose-dependent manner in all five cell lines as a result of the SRB assay. Especially PC-3 prostate cancer and MDA-MB-231 breast cancer cell lines were found more sensitive than HCT-116 and A549 cell lines, according to ATP assay. On the other hand, A549 lung cancer cells exhibited resistance to Complex 1 up to 5 μM concentration. Although it was detected by ATP assay that it has no cytotoxic effect on BEAS-2B epithelial cell line. The SRB assay showed that Complex 1 has cytotoxicity in BEAS-2B cells (Figure 1A and B). This difference is due to the two assays’ principle of the method. In addition, phase contrast microscopic images were taken for each dose to evaluate morphological alteration in response to Complex1 (Figure S7).

Figure 1.

Cell viability analysis of Complex 1 for 48 h against A549, PC-3, MDA-MB-231, HCT-116, and BEAS-2B cells measured by (A) SRB assay and (B) ATP assay. Cytotoxicity of ligand alone for 48 h against five cell lines detected by (C) SRB assay. *** denotes statistically significant differences in comparison with control (p < 0.001).

On the basis of the ATP assay results, the IC₅₀ and IC₉₀ values (50% and 90% inhibition of cell growth) of Complex 1 were calculated (Table 1). The IC₅₀ values were 2.1 μM for PC-3, 9.3 μM for A549, 4.8 μM for HCT-116, and <1.25 μM for MDA-MB-231 cells. However, the IC₅₀ value of Complex 1 for BEAS-2B could not be calculated because it was more than 40 μM according to the ATP assay result. Two different cell viability assays were done in this study. The SRB assay, as an initial assay that is also used by National Cancer Institute (USA), reflects the amount of protein mass of alive cells. However, ATP assay is the most sensitive cell viability assay. Therefore, ATP assay was done to confirm the results of the SRB assay. These two assays are basically biologically different assays and match each other very well. In the previous study, our group has shown the IC₅₀ values of well-known and broadly used chemotherapeutic drug cisplatin for PC-3, A549, and BEAS-2B as 27 μM, 16.6 μM, and 24.6 μM, respectively.¹⁸ Also, Yin et al. determined the IC₅₀ value of cisplatin on MDA-MB-231 cells as 7.8 μM.¹⁹Complex 1 showed greater cytotoxic activity on cancer cells and lower toxicity on BEAS-2B epithelial cells compared to cisplatin. Also, the IC₉₀ values were determined as 32.6 μM for PC-3, 33.5 μM for A549, 18.4 μM for HCT-116, 30.6 μM for MDA-MB-231, and 40 μM for BEAS-2B cells, in order to use in following experiments.

Table 1. IC₅₀ and IC₉₀ Values for Complex 1 Calculated on the Basis of the Results of ATP Assay with Cells Treated for 48 h.

Complex 1	IC50 Dose (μM) ± SD	IC90 Dose (μM) ± SD
PC-3	2.1 ± 1.4	32.6 ± 2.3
MDA-MB-231	<1.25	30.6 ± 1.6
A549	9.3 ± 1.9	33.5 ± 0.9
HCT-116	4.8 ± 0.9	18.3 ± 1.2
BEAS-2B	5 ± 1.2	>40

On the other hand, the cytotoxic activity of N4-donor ligand, that is a component of Complex 1, was investigated by using SRB assay, whether cell death is caused by complex or ligand itself. As a result of the SRB assay, the ligand was not caused by cytotoxicity alone (Figure 1C).

In the next experiment, the Muse Cell analyzer determined whether Complex 1 caused apoptosis and what the cell death mechanism was in PC-3, HCT-116, MDA-MB-231, A549, and BEAS-2B. Annexin V assay can detect viable, early apoptotic, late apoptotic, and necrotic cells separately through binding to expose phosphatidylserine to the outer membrane.²⁰ Therefore, annexin-V positivity was investigated after treatment with IC₉₀ values of Complex 1 for 24 and 48 h in five cell lines. Complex 1 increased apoptosis 21.6% and 18.2% in A549 cells, 31.8% and 44.7% in MDA-MB-231 cells, 13.8% and 20.1% in PC-3 cells, 18.55% and 28.75% in HCT-116 cells, and 21.7% and 46.7% in BEAS-2B cells for 24 and 48 h, respectively (Figure 2). Notably, Complex 1 was found very effective against all cancer cells, especially on MDA-MB-231 breast cancer cells. Interestingly, although A549 cells exhibited resistance to Complex 1-induced apoptosis, necrosis was detected after 48 h treatment with 32% population.

Figure 2.

Determination of apoptosis in (A) A549, (B) MDA-MB-231, (C) PC-3, (D) HCT-116, and (E) BEAS-2B cell lines treated with Complex 1 (IC₉₀ doses) for 24 and 48 h assessed by Annexin-V positivity.

To detect the mitochondrial membrane potential (ΔΨm) and Bcl-2 inactivation, that is crucial for the intrinsic apoptotic pathway, a Bcl-2 Activation Dual Detection and Mitopotential assay was performed by using flow cytometry after cells were treated with Complex 1 (with IC₉₀ doses) for 24 h. It was found that Complex 1 caused mitochondrial membrane potential loss 25.9% and 73.85% in A549 cells, 17.28% and 1.52% in MDA-MB-231 cells, 26.85% and 69.2% in PC-3 cells, 12.03% and 24.1% in HCT-116 cells, and 31.57% and 35.94% in BEAS-2B cells for 24 and 48 h, respectively (Figure 3). Also, flow cytometric Bcl-2 analysis showed that Complex 1 increased the percentage of inactivated Bcl-2 in a time-dependent manner in A549 and PC-3 cell lines, from 40.9% to 71.1% and from 22% to 49.1%, respectively. Bcl-2 inactivation after Complex 1 treatment was determined as 2.6% and 0.6% in MDA-MB-231 cells, 1% and 2% in HCT-116 cells, and 20.3% and 9.8% in BEAS-2B cells for 24 and 48 h, respectively (Figure 4). Surprisingly, Complex 1 had no significant effect on Bcl-2 inactivation while altering mitochondrial membrane potential in HCT-116 cancer cells. Nevertheless, Bcl-2 inactivation via phosphorylation is an important indicator for intrinsic apoptosis, it is not the only mechanism underlying. As discussed in Shamas-Din et al.’s review, BH3-only proteins could also promote apoptosis by binding to antiapoptotic proteins such as Bcl-2 and Bcl-XL and leading to release of pro-apoptotic proteins such as Bax and Bak from antiapoptotic proteins. Furthermore, free Bax and/or Bak form oligomer on the mitochondrial outer membrane to form a pore.²¹ Mitochondrial membrane depolarization was detected after treatment with Complex 1 after 24 h but not for 48 h in MDA-MB-231 cells.

Figure 3.

Mitochondrial membrane depolarization was detected by the flow cytometer in (A) A549, (B) MDA-MB-231, (C) PC-3, (D) HCT-116, and (E) BEAS-2B cell lines after treatment with Complex 1 (IC₉₀ doses) for 24 and 48 h.

Figure 4.

Bcl-2 inactivation was detected by flow cytometer in (A) A549, (B) MDA-MB-231, (C) PC-3, (D) HCT-116, and (E) BEAS-2B cell lines after treatment with Complex 1 (IC₉₀ doses) for 24 and 48 h.

Complex 1-induced DNA damage was detected by H2AX Activation Dual Detection via flow cytometer. Flow cytometer results showed that Complex 1 caused DNA damage 23.6% and 36.7% in A549, 3.2% and 15.1% in MDA-MB-231, 16.6% and 44.64% in PC-3, 9.4% and 4.1% in HCT-116, and 39.6% and 29.2% in BEAS-2B cells for respectively 24 and 48 h (Figure 5). It has been shown that Complex 1 increased DNA damage in a time-dependent manner in A549 and PC-3 cell lines.

Figure 5.

DNA damage was detected via H2AX assay by a flow cytometer in (A) A549, (B) MDA-MB-231, (C) PC-3, (D) HCT-116, and (E) BEAS-2B cell lines after treatment with Complex 1 (IC₉₀ doses) for 24 and 48 h.

Taken together, these findings indicate that Complex 1 induced intrinsic apoptosis with the increased mitochondrial membrane depolarization level, Bcl-2 inactivation, and DNA damage in PC-3 prostate cancer and A549 lung cancer cell lines. While annexin-V positivity was detected in MDA-MB-231 cells for 24 and especially 48 h treatment with Complex 1, neither mitochondrial membrane depolarization nor Bcl-2 activation was detected. However, it has been detected that 45.45% and 49.85% of the population was dead by necrosis in 48 h treated MDA-MB-231 and HCT-116 cells, respectively. The platinum(II) complex Complex 1 induced mitochondrial membrane depolarization in a time-dependent manner in BEAS-2B. Also, it increased Bcl-2 inactivation and DNA damage in 24 h.

In conclusion, the in vitro analysis showed that the cationic square-planar platinum(II) coordination compound with N₄-donor ligand, [Pt(L)]²⁺ (Complex 1), induces apoptosis with different mechanisms in cell lines. Also, platinum-based Complex 1 caused cytotoxicity with less concentration to compare to clinically used platinum-based drugs such as cisplatin. All these results of this study further supported the potential use of Complex 1 as a drug in cancer treatment.

Glossary

Abbreviations

APCI MS

atmospheric pressure chemical ionization mass spectrometer

FT-IR

Fourier-transform infrared spectroscopy

QP

quaterpyridine

MALDI-TOF

matrix assisted laser desorption/ionization – time of flight

SRB

sulforhodamine B

IC₅₀

the 50% maximal inhibitory concentration

IC₉₀

the 90% maximal inhibitory concentration

SD

standard deviation

H2AX

histone 2 A.X

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00676.

• Experimental procedures for the ligand and the Complex 1 synthesis and characterization, materials and methods, biological assays, and phase-contrast microscopic images (PDF)

Author Contributions

^# I.Y. and O.R.A. contributed equally to this work.

The authors declare no competing financial interest.