Antitumor activity of DABA-containing carboxylate Zn complexes on breast cancer cell line MCF-7

doi:10.21203/rs.3.rs-2437557/v1

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Antitumor activity of DABA-containing carboxylate Zn complexes on breast cancer cell line MCF-7

https://doi.org/10.21203/rs.3.rs-2437557/v1

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The coordination polymer [Zn₂(DABA)₃(CH₃OH)] (DABA=4-dimethylaminobenzoic acid) (ZP) was obtained by hydrothermal synthesis using DABA and Zn(NO₃)₂·6H₂O as substrates. The exact structure of ZP was confirmed by thermogravimetric analysis (TGA), Fourier transforms infrared spectroscopy (FTIR)， and single-crystal X-ray diffraction. The Cell Counting Kit-8 (CCK-8) assay was used to detect the cytotoxicity of ZP on the following cell types (A549, HepG2, Huh-7, MCF-7, LO2 cells), and the results showed that the complex was significantly toxic to MCF-7 cell line and less toxic to normal hepatocytes LO2. In addition, the cell cycle and mitochondrial membrane potential of the MCF-7 cell line were further measured by flow cytometry, and the experimental results indicated that different concentrations of the drug blocked the cell cycle in the S phase and significantly reduced the mitochondrial membrane potential. Western Blot experiments illustrated that ZP was able to upregulate the expression levels of cleaved PARP, cleaved caspase and bax protein and downregulate the expression level of bcl-2. These results suggest that ZP inhibits tumor cell apoptosis through a mitochondria-mediated pathway, accompanied by the regulation of bcl-2 and caspase family proteins.

Zinc-based

Coordination polymer

Anti-tumor activity

MCF-7

Bcl-2

MOFs with specific killing of cancer cells and low toxicity to normal cells were prepared.

Breast cancer has replaced lung cancer as the most commonly diagnosed cancer in the world, according to statistics released by the International Agency for Research on Cancer (IARC) as of December 2020 [1]. Breast cancer is divided into three subtypes based on the expression of estrogen or progesterone receptors and the amplification of the ERBB2 gene [2]. At present, endocrine therapy, monoclonal antibody therapy, and chemotherapy are used to treat different subtypes of breast cancer. However, there are some disadvantages in these treatments, such as drug resistance, side effects, or a long course of treatment [2–3]. Recently, metal-based complexes have attracted widespread interest due to the successful clinical use of cisplatin, which offers unique advantages owing to their ability to improve drug efficacy, reduce drug side effects and decrease drug resistance [4–5].

Nowadays, many metal ions, such as ruthenium, cobalt, nickel, copper, and zinc ions, have been used to study the anti-tumor effects. Chen et al. demonstrated that novel complexes of 40-methoxy-5,7-dihydroxy-isoflavone ligands with several transition metal ions (zinc, manganese, copper, cobalt, and nickel) exhibited superior inhibitory effects on the growth of five human cancer cell lines [6]. Zhu et al. reported that the quinolone Ru (II)-aryl complex induced apoptosis in T-24 bladder cancer cells by producing ROS metabolites, triggering increased caspase-9 and loss of mitochondrial membrane potential [7]. Rao et al. found that C-N cyclopentolate 2H-indazole Ru(II) and Ir(III) complexes promoted apoptosis through nuclear condensation, cell membrane hemorrhage, and caspase 3/7 activation [8]. Among those metals, zinc ions stand out due to the important advantages of safety and non-toxicity [9]. For example, Zana et al. found that complexes of zinc with S-alkyl derivatives of thiosalicylic acid were able to bind to calf thymus DNA and exhibited good antitumor activity against human B-lymphocyte leukemia (JVM-13) and human breast cancer cells (MDA-MB-468) [10]. Qi et al. designed Zn(II) -thiosemicarbonate complexes, which could shorten the G2 phase of the MCF-7 cell cycle and promoted cancer cell apoptosis [11]. Qin et al. demonstrated that curcumin-cryptoterpene zinc(II) complex could increase intracellular Ca²⁺ and reactive oxygen species (ROS) levels, impair mitochondrial membrane potential, and inhibit the growth of T-24 bladder cancer cells in vivo [12]. Tan et al. studied the activity of quercetin zinc (II) complexes and found that it could be inserted into the stacked base pairs of DNA, showing significant cytotoxicity to three tumor cell lines (HepG2, SMMC7721, and A549) [13]. Therefore, it would have stupendous importance of choosing zinc ion as a potential antitumor metal.

On the other hand, carboxylic acid also played an excellent anti-tumor effect as complexes ligand. Yang et al. prepared 2-pyridine carboxaldehyde 2-pyridine carboxylic acid copper complexes and found that these complexes could lead to S phase arrest in cancer cells [14]. Liu et al. designed two novels Ru (II) polypyridyl complexes with hydroxybenzoic acid groups, which could be capable of inducing apoptosis in cancer cells via reactive oxygen species (ROS)-a mediated pathway and enhancing the tumor-targeting ability of photosensitizers [15]. In addition,4-dimethylaminobenzoic acid (DABA), as a typical benzoic acid derivative, showed unique advantages as a pharmacophore because the substitution on its scaffold provided a large compound library, which could play various roles such as anti-cancer and anti-drug resistance. PrasherP et al reported that o-aminobenzoic acid derivatives exhibited interesting antibacterial, antiviral and insecticidal properties, while derivatives based on the o-aminobenzoic acid diamide scaffold were applied as P-glycoprotein inhibitors for the management of drug resistance in cancer cells [16].

Therefore, in this study, ZP was first synthesized by hydrothermal synthesis, and their structural characterization was detected using FTIR, TGA and XRD. Secondly, the cytotoxicity of the Zn complex was examined by CCK-8, and the regulation of Zn complex on apoptosis, mitochondrial membrane potential, cell cycle progression was studied. Finally, the expression levels of Bcl-2 and caspases family proteins were also investigated.

2.1 Materials and methods

All reagents are purchased from commercial suppliers and are of analytical purity grade, so no further purification is required. The required solvents are purified in accordance with standard operations. Zinc nitrate hexahydrate (Zn (NO₃)₂·6H₂O), 4-dimethylaminobenzoic acid (DABA) and trypsin were purchased from Shanghai Macklin Biochemical Co. Ltd. DMEM and DMSO were obtained from Beijing Solarbio Science & Technology Co. Ltd. Cell Counting Kit-8 (CCK-8 Kit) was purchased from Dojindo. RIPA protein extraction reagent (Beyotime Biotechnology, Shanghai, China), β-actin, Apoptosis Antibody Sampler Kit, Bcl-2 and Bax Antibody (1:1000 dilutions, Cell Signaling Technology, USA), fgoat anti-rabbit IgG H&L (Alexa Fluor 488) (Santa Cruz Biotechnology, USA). Cells lines of A549, HepG2, Huh-7, MCF-7and normal cell LO2 were purchased from the American Type Culture Collection. Microanalysis (C, H, and N) was performed with the CHNS-932LECO elemental analyzer (Isomass Scientific Inc., Calgary, Canada). FTIR measurements had been acquired by an FT-IR-5700 Spectrometer (Nicolet, USA). ESI-MS was measured by high-resolution mass spectrometry (Bruker, micro TOF-Q II, Germany).

2.2 Synthesis and characterization

The mixture of DABA (3 mmol, 0.594 g) and Zn (NO₃)₂·6H₂O (2 mmol, 0.495 g) in the ratio of DMF: CH₃OH (4 ml: 6 ml) was used as the solvent, and the reaction was carried out under magnetic agitation for 1 hour. After a period of sufficient reaction, the reaction liquid was transferred to the High-Temperature Reactor, the corresponding mark was made, the temperature was raised to 100°C, kept for 24 hours, then lowered to room temperature and kept for 48 hours. After the crystallization, the crystal solution was repeatedly washed with methanol until the solution became clear and transparent, filtered, and dried at room temperature.

Element analysis (C, H, N, and O) was performed on an analyzer called Perkin-Elmer 2400chn. Complexes III was used as a reagent for the determination of zinc complexes and Eriocchrome Black T was used as an indicator. Calc for C₂₈H₃₃N₃O₇Zn₂:C, 55.23%; H, 4.65%; N, 5.92%; O, 15.78%. Found: C,55.12%; H, 4.55%; N, 5.72%; O, 15.69%.

2.3 Cytotoxicity in vitro

The cytotoxicity of the complexes to cancer cells was detected by cell counting kit-8kit [18]. Cells were grown in 96-well plates at a density of 5 × 10³ cells / 100 µL and incubated at 37°C with 5% CO₂ for 24h. After the cells adhered to the wall stably, the cells were incubated for 48 hours with different concentrations (10µM, 50µM, 100µM) of complexes. The absorbance of the microplate reader was adjusted to 450nm and the OD of the 96-well plate was measured, with each plate being averaged at least three times. Cell viability was calculated according to the formula: [(As-Ab) / (Ac-Ab)] × 100%. As: absorbance of experimental wells (with cells, medium, CCK-8 solution and drug solution); Ac: absorbance of control wells (with cells, medium, CCK-8 solution, without drug); Ab: absorbance of blank wells (with medium, CCK-8 solution, without cells and drug). Four groups of cancer cells and one group of normal cells were subjected to the experiment: A549, HepG2, Huh-7, MCF-7, LO2 cells.

2.4 Determination of mitochondrial membrane potential

The JC-1 kit was utilized to determine the mitochondrial membrane potential values of cells. Cells were diluted to a density of 1 × 10⁵ cells / mL with DMEM containing 10% FBS and then inoculated into 6-well plates. After 24 hours, MCF-7 cells were treated with ZP of different concentrations and incubation continued for 24h. Then, according to the instructions in advance configuration JC-1 dyeing working solution and dyeing buffer. Take out the culture plate, wash them twice with PBS, add 1ml of JC-1 dyeing working solution to each well, mix well, and incubate at 37°C for 20min. After incubation, wash twice with precooled JC-1 buffer, then add 2 ml culture solution to each hole and place under the Fluorescence microscope (Leica TCS SP8, Germany).

2.5 Flow cytometry for cell cycle analysis

Cells were diluted to a density of 1×10⁶ cells / mL with DMEM containing 10% FBS and then inoculated into 6-well plates. After treatment with different concentrations of ZP, incubation was continued for 24h. Subsequently, RNase: PI was prepared in advance in a staining working solution at a ratio of 1:9, cells were washed in PBS and collected by centrifugation for 5min at a speed of 2000r. The single-cell suspension was collected, the supernatant was removed, 500ul of cold ethanol at a volume fraction of 70% was added for fixation and stored at 4°C. The fixative was washed off with PBS, pre-configured PI/RNase staining solution was added, and the cells were protected from light at room temperature for 60 min. Detection was performed by flow cytometry(BD FACSCanto II) and red fluorescence was recorded. The data is processed with FlowJo.7.6 software.

2.6 Detection of apoptosis by Annexin V-FITC/PI

The test was performed according to the following standard protocol.MCF-7 was seeded in 6-well plates at a density of 1 x 10⁵ and incubated for 24 h. After treatment with different concentrations of ZP, incubation was continued in the incubator for 24 h. After digestion with trypsin, the cells were collected and washed twice with PBS.

Add Binding Buffer to suspend the cells, then add 5 µL of Annexin V-FITC and mix, followed by 5 µL of Propidium Iodide and mix. The reaction was carried out for 10 min at room temperature and protected from light and was rapidly detected by flow cytometry.

2.7 Western blot analysis

Cells were diluted to a density of 1×10⁶ cells/mL with DMEM containing 10% FBS and treated with different concentrations of ZCP for 24 hours. The lysate was prepared in advance according to Ripa: enzyme inhibitor: PMSF = 98:1:1. The cells were washed twice with precooled PBS, and 100µ of cell lysate was added to each pore, blowing until the bubbles formed and covered the entire surface. After lysis on ice for 30min, the proteins were collected with a cell scraper, transferred to a 1.5ml centrifuge tube using a pipette, and centrifuged at 12000r/min at 4°C for 15min. The supernatant was dispensed and the protein concentration was quantified by means of a BCA protein assay kit. The proteins were separated on a pre-prepared 12% SDS-PAGE and transferred onto methanol-activated PVDF membranes. The membrane was placed in PBST containing 5% skimmed milk powder to seal the blank protein sites on the surface of the membrane. The membranes were washed three times with PBST and after the membranes were washed, they were placed in a hybridization cassette containing the primary antibody, labeled with the name of the antibody and placed on a shaker and incubated overnight at 4°C. After washing in TBST, the imprinting was incubated at room temperature for 1 hour with a Horseradish peroxidase bound goat antibody to rabbit immunoglobulin G.Western imprinted protein bands were obtained by visualizing the immune response bands using enhanced chemiluminescence.

2.8 Statistical analysis

All data were obtained from at least three independent, parallel experiments, expressed as an average ± standard deviation.SPSS (version 19.0; SPSS, IBM, Armonk, NY, USA) is used for statistical analysis of data. P < 0.05 was considered to indicate a statistically significant difference.

3.1 Synthesis and characterization

For the specific fine structure of the complexes, single-crystal X-ray diffraction analysis showed crystallization in the single-crystal P 1 21/n 1 space group, which belonged to the monoclinic crystal system. As shown in Scheme 1, Zn²⁺was hexavalent bounded to the oxygen atom of methanol and the carboxyl group of DABA. The basic unit of the whole structure consisted of three DABA ligands, two zinc ions, and one methanol molecule. The two zinc ions were in different crystallographic coordination environments, where Zn1 was dimeric and linked to two oxygen atoms, and Zn2 was trimeric and linked to three oxygen atoms. In addition, the complexes maintain structural stability through DABA benzene ring π-π bond interactions, and the independent crystallographic unit connected ligand with zinc ions through Zn-O bond, showing a 1D chain-like structure in space (Scheme 2).

The bond distances and bond angles of the crystals were calculated by referring to the reported literature [17]. The bond lengths of Zn2-O1, Zn2-O4, and Zn2-O6 centered on Zn2 are 1.9272(19) Å, 1.943(2) Å, and 1.929(2) Å, indicating that the carboxylate bone length tends to be uniform. The bond angles of O4-Zn2-O1, O6-Zn2-O1, and O6-Zn2-O4 centered on Zn2 are 106.27˚,107.82˚, and 124.50˚, showing the consistency of the six coordination of zinc. Moreover, the crystallographic data and structural refinement of ZP are shown in Table 1, and the bond lengths and bond angles of ZP crystal units are listed in Table 2.

SEM image shows that the complex has a cubic crystal structure frame (see in Fig. 1). The single XRD pattern results showed that the diffraction peak positions of the experimental model are in general agreement with the prepared complex (see in Fig. 2), proving that the complex is a crystal of high purity.

Figure 3 plots the results of the TGA thermal stability experiment. The TGA showed three weight loss phases, and the first stage occurred between the temperature interval of 22 and 360°C with a weight loss of 5.0% (calculated value 4.92%), corresponding to the loss of methanol molecules. The second stage occurred between 360°C and 545°C, which was probably due to the elimination of DABA ligands (calculated value 70.37%; found 70.45%). The third stage of 26.36% (545°C to 700°C) weight loss was associated with the production of zinc oxide compounds (calculated value 25.46%).

Figure 4 shows the FITR curves of DABA and ZP. The characteristic peaks 1196 cm^− 1, 1580 cm^− 1 and 1650 cm^− 1 were detected in the FITR curves of DABA (see in Fig. 4A). The peak at 1196 cm^− 1was belonged to be the C-N bending vibration of dimethylamine, the peak at 1580 cm^− 1 was ascribed to be the C = C backbone stretching vibration of the benzene ring. The peak at 1650 cm^− 1 was assigned to the C = O stretching vibration of the carboxyl group [18]. Similarly, in the FITR characteristic curve of ZP, the above characteristic peaks of 1196 cm^− 1, 1580 cm^− 1, and 1650 cm^− 1 were also were detected (see Figure in 4B), indicating that DABA was successfully connected with Zn²⁺.

3.2 Cytotoxicity in vitro

The cytotoxicity of the complex against a range of cancer cells including A549 cells, HepG2 cells, Huh-7 cells, MCF-7 cells, and LO2 (human normal cells) was assayed by CCK8, and the cisplatin-treated group was served as a positive control group (see in Fig. 5). As seen from Fig. 5, different concentrations of ZP showed good inhibitory effects on selected groups of cancer cells. Particularly, ZP with a concentration of 100 µM possessed the best effect towards MCF-7 cells, which was close to the positive drug cisplatin group. In addition, ZP was less toxic to normal cells compared to the positive group. These results illustrated that ZP might be a very promising antitumor drug.

3.3 Determination of mitochondrial membrane potential

JC-1 is an ideal fluorescent probe that is widely used to detect mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix and forms a polymer, at which point red fluorescence could be detected. However, at lower levels, it is a monomer and green fluorescence is trapped. A decrease in mitochondrial membrane potential is a sign of early apoptosis [19]. Figure 6 depicts the mitochondrial membrane potential fluorescence intensity as well as the fluorescence intensity ratio of MCF-7 cells. The results showed that the percentage of green fluorescent cells increased more significantly, and the intensity of red fluorescence decreased sequentially after MCF-7 cells were treated with different concentrations of ZP for 24 h (see in Fig. 6A). Furthermore, the ratios of red and green fluorescence between the control group and the drug treatment group were 0.88, 0.67, 0.57, 0.46, respectively (see in Fig. 6B). These results suggested that the mitochondrial membrane potential of cancer cells decreased after drug treatment, implying that ZP could induce apoptosis by triggering the mitochondrial membrane potential.

3.4 Flow cytometry for cell cycle analysis

DNA damage has a significant impact on the process of cell mitosis and the distribution of the cell cycle [20]. To investigate the mechanism of ZP inhibition of cell proliferation, flow cytometry was employed to detect the effect of the drug on the cancer cell cycle. As shown in Fig. 7, a very significant change in the proportion of cells in the G1 phase was observed. The percentage of cells in the G1 phase in the control group was 84.34%, while the cell cycle percentage of MCF-7 cells treated with different concentrations (10 µM, 50 µM, and 100 µM) of ZP were 77.59%, 70%, and 60.59%, respectively. Meanwhile, the proportion of cell populations in the S phase was significantly increased to 15.66%, 22.41%, 30%, and 39.41% in the control and drug-treated groups, respectively. The above results indicated that the cell cycle of MCF-7 cells was blocked by ZP in the S phase.

3.5 Detection of apoptosis by Annexin V-FITC/PI

To investigate the role of the complex ZP in the induction of apoptosis, Annexin V-FITC/PI staining was performed on MCF-7 cells to determine the rate of apoptosis [21]. As shown in Fig. 8, the percentage of apoptotic cells was significantly increased in the ZP-treated group compared to the control group. In addition, the percentage of apoptosis in Annexin V-FITC/PI staining after treating MCF-7 cells with different concentrations (10 µM, 50 µM and 100 µM) of ZP, was 13.4%, 16.2%, and 19.3%, respectively. These results suggested that the proliferation inhibition of ZP cells was achieved by inducing apoptosis.

3.6 Western blot analysis

Caspase-3 and bax are pro-apoptotic proteins, and bcl-2 is an anti-apoptotic protein. PARP is an important protein for activating the mitochondrial pathway in MCF-7 cells. As shown in Fig. 9, the expression of cleaved PARP, bax, and cleaved caspase-3 increased in MCF-7 cells after ZP treatment. Specifically, cleaved PARP increased approximately 8.3-fold, cleaved caspase-3 increased 2.7-fold, and Bax increased 5.1-fold at a ZP concentration of 50 µM compared to the control group. However, the expression of Bcl-2 decreased with increasing ZP concentration, and the lowest expression of bcl-2 was observed when ZP was 100 µM. These results suggested that ZP-induced apoptosis is related to the mitochondria-mediated apoptotic pathway.

In conclusion, a new zinc metal complex ZP was synthesized in this study, and its antitumor activity was investigated. In vitro cytotoxicity assays have confirmed its effective ability to inhibit tumor cell growth, with the advantage of being less toxic to normal cells. Apoptosis assays showed that the rate of apoptosis also increased significantly with increasing ZP concentration. In addition, cell cycle data showed that ZP blocked the cell cycle of cancer cells in the S phase. Western blotting data showed that the complex was able to upregulate caspases and downregulate Bcl-2, thereby inducing apoptosis in cancer cells. All of the above scientific data suggested that the complex could induce apoptosis through a mitochondria-mediated pathway, acting as an anti-cancer agent. This work would contribute to the design and synthesis of novel antitumor drugs, which have a better prospect in the field of nano antitumor.

Acknowledgments

The authors would like to thank the grants from the National Natural Science Foundation of Guangdong Province (No.2021B1515140020 and No. 2019A1515110017), Science and Technology Foundation of Chinese Medicine Department of Guangdong Province (No.20212100), Dongguan Sci-tech Commissioner Program No.20201800500062, Science and Technology Foundation of Guangdong Medical University (No.GDMUZ2020006), Medical Scientific Research Foundation of Guangdong Province (No.B2021056), Dongguan social development science and technology project (No.20211800905542), and Discipline construction project of Guangdong Medical University (No.4SG21229GDGFY01 and 4SG22006G).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author, [Z.C], upon reasonable request.

Conflict of interest statement

The authors declare that there are no conflicts of interest，we do not have any possible conflicts of interest.

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Schemes 1 and 2 are available in the Supplementary Files section

No competing interests reported.

Graphicalabstract.docx
Scheme1.png
Scheme 1 Structure of complex ZP
Scheme2.png
Scheme 2 One-dimensional chain structure of ZP

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Antitumor activity of DABA-containing carboxylate Zn complexes on breast cancer cell line MCF-7

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Version 1

Abstract

Figures

Highlights

1. Introduction

2. Materials And Methods

2.1 Materials and methods

2.2 Synthesis and characterization

2.3 Cytotoxicity in vitro

2.4 Determination of mitochondrial membrane potential

2.5 Flow cytometry for cell cycle analysis

2.6 Detection of apoptosis by Annexin V-FITC/PI

2.7 Western blot analysis

2.8 Statistical analysis

3. Results And Discussion

3.1 Synthesis and characterization

3.2 Cytotoxicity in vitro

3.3 Determination of mitochondrial membrane potential

3.4 Flow cytometry for cell cycle analysis

3.5 Detection of apoptosis by Annexin V-FITC/PI

3.6 Western blot analysis

4. Conclusions

Declarations

References

Scheme

Additional Declarations

Supplementary Files

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