Apoptosis Induction by New Coumarin Derivatives in a Mice Model of Breast Cancer

1. Introduction

Breast cancer (BC) remains the most common malignancy diagnosed in females and the second leading cause of cancer-related death in women worldwide ( 1 ). Breast cancer is a complex disease that may involve numerous signaling pathways and molecules, leading to different treatment options depending on tumor type and molecular profile ( 2 , 3 ). Currently, classical BC treatment strategies, including chemotherapy, immunotherapy, and radiotherapy, are invasive, expensive, and ineffective. Therefore, the identification of novel therapeutic approaches applied alone or in combination with other methods would be of great benefit in improving the clinical care of BC patients ( 2 ). Natural products, such as phytoconstituents or their relative structures, still account for 50% of drugs prescribed for cancer chemotherapy. In this context, previous studies have demonstrated the anticancer effects of new agents, such as doxorubicin-G2 FA ( 4 ), ICD-85 (venom-derived peptides) ( 5 ), on BC cells, as well as caffeic acid phenethyl ester and Matricaria chamomilla essential oil on non-small cell lung cancer cells A549 ( 6 ). Moreover, in recent decades, scientists have identified and developed a variety of phytochemicals that not only have inhibitory activity against various types of cancers but also have fewer side effects ( 7 ). Among several phytochemotherapeutic agents, chromenes (benzopyrans) and their derivatives have been studied in recent years because of their interesting biological properties. In these heterocyclic rings, a pyran ring is combined with a benzene ring. Tocopherols, anthocyanins, alkaloids, flavonoids, and tannins are examples of benzopyrans that form the backbone of different types of polyphenols ( 8 ). Natural compounds derived from coumarin (1,2-benzopyrones or 2H-chromen-2-one or phenylpropanoids 1) exhibit various biological, pharmacological, and biochemical activities against cancer ( 9 ). Some coumarins and their active metabolite, 7-hydroxycoumarin analogs, have shown effects against BC through a variety of mechanisms, the most notable of which were the inhibition of aromatase and sulfatase. Selective estrogen receptor modulators (SERMs) based on coumarin and coumarin-estrogen conjugates may also have a potential role in BC treatment ( 10 ). In the present study, the effect of a series of novel synthetic coumarin derivatives on the induction of programmed cell death in BC was investigated in the animal model of BALB/c mice using quantitative molecular assays and pathological studies.

2. Materials and Methods

2.1. Chemistry and preparation of coumarin derivatives

At the Department of Organic Chemistry and Biochemistry, University of Tabriz, Tabriz, Iran, we synthesized two coumarin compounds (Figure 1), namely 2-amino-4-(4-(2-hydroxyethoxy)-3-methoxyphenyl)-5-oxo-4H,5H-pyrano[3,2-c]coumarin-3-carbonitrile and 2-amino-4-(4-hydroxyphenyl)-5-oxo-4H,5H-pyrano[3,2-c]coumarin-3-carbonitrile (Figure 1A), designated as A and B, respectively. Stock solutions at a concentration of 10 mg/ml of each of the compounds were also prepared and stored at room temperature in the laboratory for future studies.

Figure 1.a: Structure of the present synthetic coumarin derivatives. b: Cell viability was evaluated using MTT assay, which was dose-dependent and decreased at higher doses (P<0.05, mean ± SD, n=3)

2.2. Cell culture

Murine metastatic mammary tumor cell line 4T1 was obtained from the National Cell Bank of Iran (Pasteur Institute, Iran), cultured in Roswell Park Memorial Institute 1640 medium (GIBCO, USA), and supplemented with 10% fetal bovine serum (GIBCO, USA), 100 units/ml penicillin, and 100μg/ml streptomycin. The flasks were incubated at 37°C with 95% humidity and 5% CO₂.

2.3. Animals and ethical approval

Female BALB/C mice (n=60) in good health (25-35 g, 7-9 weeks) were purchased from the Pasteur Institute in Karaj, Iran. They were kept under standard conditions (22°C, 12/12 h light/dark cycle, and 40-60% humidity) and given food and water at all times. Ethical approval was included on the “cover”.

2.4. MTT assay

To determine the viability of the cells and the appropriate dose of coumarin derivatives, the MTT (3‐ (4, 5‐dimethylthiazol‐2‐yl) ‐2, 5‐diphenyltetrazolium bromide) assay was performed. Briefly, cells (1.5×10³) were seeded in 96-well plates and incubated at 37C for 24 h. After treatment with 8 ascending concentrations (0.0001, 0.001, 0.01, 0.1, 1, 10,100, and 1,000 µmol) of the test compounds (including A and B), and 0.1 ml of dimethyl sulfoxide (DMSO) as control, the cells were incubated for another 24 h at 37°C. Afterward, 50 μl of the prepared MTT solution (Sigma-Aldrich, Germany) was added to each well and incubated for an additional 4 h. Following that, the medium in each well was removed, 200 μl of DMSO (Sigma‐Aldrich) was added to the wells to dissolve MTT formazan crystals, and incubated for another 30  min. Finally, the absorbance values of each well were read at 570 nm using a Sunrise enzyme-linked immunosorbent assay (ELISA) reader (Tecan, Switzerland). The experiment was performed three times.

2.5. Breast cancer induction, treatment, and sampling

To demonstrate the inhibitory effects of the selected coumarins on BC growth, 60 healthy BALB/c mice were randomly divided into six groups of 10 mice each (Table 1), and examined daily for their general health. A suspension of 4T1 cells with a concentration of 1×10⁶/ml was obtained after trypsinization and centrifugation of the cells cultured in complete media. Subsequently, 0.2 ml of the suspension was injected subcutaneously into the left flank of mice under ketamine and xylazine anesthesia (10 mg/kg BW IP) ( 11 ). Tumors appeared 2 weeks after the cell injection, and the main treatment was started with 0.4 ml of equal injection volumes, according to table 1. At the end of the experiment (after 5 weeks), all animals were euthanized, and the internal organs, including the liver, lungs, and tumor mass, were removed for further examinations (quantitative real-time polymerase chain reaction [qRT-PCR] and histopathology).

2.6. RNA extraction and quantitative reverse transcription-polymerase chain reaction

The RT-qPCR method was used to measure fold changes in the expression of some genes associated with apoptosis/survival, including c-Myc, BCL-2, caspase-9, and COX-2. Total RNA from tissues of all tumor-induced mice in each group was isolated using TRIzol reagent (RiboEx Kit, GeneAll, South Korea) according to the manufacturer's instructions and quantified by measuring absorbance at 260 nm and 280 nm using NanoDrop (Thermo Scientific, USA). Complementary DNA (cDNA) was synthesized using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher) according to the manufacturer’s protocol. The RT-qPCR technique was performed using 2X SYBR Green pre-mix (Amplicon, UK) in a StepOnePlus^TM RT-PCR System (Applied Biosystems, US). Mean expression in each treated group was presented as an increase/decrease in parity, compared to mean expression in the control group, which was assigned the desired value of 1. In addition, the glyceraldehyde-3-phosphate dehydrogenase gene was used to normalize expression data. The primer sequences are listed in Table 2.

2.7. Histopathological examinations

First, tissue samples were immediately fixed in a 10% formalin buffer solution for at least 48 h. Subsequently, the formalin-fixed tissue samples were routinely passaged, embedded in kerosene, sectioned, and stained with conventional hematoxylin and eosin. The tissue sections were examined under a light microscope (Olympus, Japan) for tumor proliferation, invasion, and distant metastasis associated with the probable histopathological changes, such as vascular congestion, hemorrhage, necrosis, and infiltration of inflammatory cell with grades of normal (N), very mild (+1), mild +2), moderate (+3), severe (+4), and very severe (+5).

2.8. Statistical analysis

Data analysis was performed in Sigma Stat software (SPSS, USA) using ANOVA. Data were reported as mean ± SD, and p-values of less than 0.05 were considered statistically significant. In addition, the nonparametric tests (Kruskal-Wallis H and Mann-Whitney U) were employed for statistical analysis of pathological lesions (semiquantitative data) between different groups, and a p < 0.05 was considered significant.

3. Results

3.1. MTT assay

The MTT assay was used to determine the cell viability and response of 4T1 cells to two coumarin compounds at different concentrations and the effective dose. Based on the data shown in figure 1B, eight different concentrations of the coumarin compounds (A and B) were tested, and the most effective dose with more than 80% cell viability was selected for further analysis.

3.2. Tumor inhibitory results by qRT-PCR and histopathology

The effect of coumarin derivatives on the gene expressions of caspase-9, BCL-2, c-Myc, and COX-2 was demonstrated by qRT-PCR and melting curve analyses. According to the qRT-PCR results illustrated in Figure 2, the expression of caspase-9 was increased by coumarin derivatives and doxorubicin in all treated groups. On the other hand, the expression of BCL-2, c-Myc, and COX-2 was decreased in all groups undergoing treatment with coumarin derivatives and doxorubicin, which was significant compared to the cancer control group (P<0.05). It is noteworthy that most changes in caspase-9, BCL-2, c-Myc, and COX-2 were observed in A2 (P<0.05), B1 (P<0.05), B1 (P<0.01), and A1 (P<0.01) groups after treatments with coumarin derivatives.

Figure 2. Evaluation of gene expression levels in tumor mass by qRT-PCR. The expression levels of caspase-9 were increased in all treated groups compared to the cancer control group, particularly in the A2 and B2 (P<0.05) groups. The expressions of BCL-2, c-Myc, and COX-2 decreased in all treated groups compared to the control group (mean±SD, n=10), especially in groups B1, B1, and A1 (P<0.05). A1 (A/low dose group): 10µm; A2 (A/high dose group): 1 mM; B1 (B/low dose group): 10µm; B2 (A/high dose group: 1 mM); C: tumor control group treated with normal saline; D: tumor group treated with doxorubicin 5 mg/kg BW

Histopathological evaluation is briefly illustrated in Figure 3. After tumor induction and treatment with normal saline, severe malignancy was observed in the control group. Specifically, a high nuclear/cytoplasmic ratio, the presence of bizarre neoplastic cells, severe necrosis associated with hemorrhage, and inflammation in the tumor mass were noted. Similar lesions were observed in the lung and liver, along with extensive metastases. As expected, all pathologic lesions were significantly reduced after the application of doxorubicin (P<0.05). Histologic changes and tumor malignancy decreased significantly in the low and high coumarin-recipient dose groups (P<0.05), especially in the high dose groups, which was more pronounced in the A2 group. It should be noted that microscopic metastases in the liver and lung decreased significantly in all four treated groups.

Figure 3. Evaluation of breast cancer inhibitory effects of the present synthetic coumarin derivatives by histopathological examination, mice (n=10). C: tumor control group treated with normal saline; D: tumor group treated with doxorubicin 5 mg/kg BW; A1 (A/low dose group): 10µm; A2 (A/high dose group): 1 mM; B1 (B/low dose group): 10µm; B2 (A/high dose group: 1 mM). Severe malignancy was detected in the control group. Similar lesions with extensive metastases were observed in the lung and liver. The pathological lesions decreased significantly after the application of doxorubicin (P<0.05). Histological changes and tumor malignancy were significantly lower in the low- and high-coumarin-dose groups, especially in the high-dose groups. Microscopic metastases in the liver and lungs were significantly reduced in all four treated groups, although this was more pronounced in the A groups. M: metastases, N: necrosis. Hematoxylin and eosin staining

4. Discussion

Our data showed that the administration of coumarin compounds could significantly increase apoptosis and reduce microscopic metastases in BC in a mouse model, which was confirmed by histopathological and qRT-PCR studies. The current results suggested that the present coumarin compounds were able to inhibit tumor growth. However, it seemed that 2-amino-4-(4-(2-hydroxyethoxy)-3-methoxyphenyl)-5-oxo-4H,5H-pyrano[3,2-c]coumarin-3-carbonitrile was more successful than 2-amino-4-(4-hydroxyphenyl)-5-oxo-4H,5H-pyrano[3,2-c]coumarin-3-carbonitrile. The expression levels of the essential apoptotic genes, including caspase-9, BCL-2, c-Myc, and COX-2, were significantly altered in the treated groups. In parallel with apoptosis induction, the pathological lesions and microscopic metastases were significantly decreased by the coumarin compound treatment of BC. On the other hand, as expected, the present coumarin derivatives had no toxic effects on the liver and kidney, which was confirmed by normal histological and biochemical indicators and body weight.

Coumarins are a naturally occurring compound found in numerous plants, some spices, and herbs ( 12 ) and can be extracted from plants or synthesized from phenol, ortho-Cresol, and salicylaldehyde for commercial purposes; they are highly soluble in chloroform, ethanol, and oils and slightly soluble in water ( 13 ). Moreover, they have great anticancer potential with low adverse effects due to their original structure and functional groups. They can affect various cellular pathways, such as the inhibition of angiogenesis, cell proliferation, and metastasis, as well as the suppression of enzymes that mainly contribute to cancer pathophysiology ( 14 , 15 ).

Although chemotherapy is currently the most common treatment for BC, most chemotherapeutic anticancer drugs have high toxicity and low specificity ( 16 ). Therefore, the development of new anticancer drugs for the treatment of BC is needed.

Recent evidence demonstrates that coumarin compounds modulate apoptosis, proliferation, and invasion of various cancer cells, which are mediated by different signal transduction cascades. Based on the present results, the growth-inhibitory potential of coumarin compounds, when it leads to fruition, might be dependent on the nature and position of the functional group, which has already been suggested by other researchers. For instance, the presence and position of the hydroxyl groups and a catecholic acid group may be associated with the different inhibitory values. In this regard, there is increasing evidence demonstrating that nitration of 7-hydroxycoumarin suppresses the human malignant melanocytes ( 17 ). In addition, previous studies have reported that both coumarin and 7-hydroxycoumarin can reduce the proliferation of a number of human malignant cell lines ( 18 ) and various types of animal tumors in vitro ( 19 ). In addition, it was previously found that N-aryl carboxamide and phenyl substitution at the C-3 position and 1, 2, 3-triazolyl, trihydroxystilbene, and amino substitution at the C-4 position of the coumarin nucleus may be the most effective in combating lung cancer ( 20 ). some coumarins and their active metabolites the 7-hydroxycoumarin analogs are of particular interest for BC chemotherapy because they exhibit sulfatase- and aromatase-suppressive properties. As described previously, coumarin-estrogen conjugates and SERMs have also been proposed as potential agents for suppressing BC ( 10 , 21 ). Coumarin-estrogen conjugates showed growth-inhibitory effects in NCI-7- human BC cell lines ( 21 ). Similarly, fluorinated coumarin derivatives demonstrated anticancer effects in MCF-7 cancer cells ( 22 ).

A series of novel 4-substituted coumarin derivatives have shown antimitotic activity in human BC cell lines (MCF-7 and MCF-7/ADR) ( 23 ). On the other hand, coumarin derivatives induce programmed cell death in BC cell lines through BCL-2 and caspase-9 cascades ( 20 ). The results of a similar study indicated that 7,8-Dihydroxy-4-methylcoumarin can downregulate p53, Bax, p21, and COX-2 and upregulate c-Myc protein, which induces apoptosis ( 20 ). Our results were consistent with these findings. In the present coumarin structures, OH and OMe substitutions appeared to act as electron donor groups in para and meta positions, making the compounds more active and reactive.

The induction of programmed cell death is central to the tumor suppressive effects of some molecular targets that promote the expression of a number of genes that contribute to apoptosis ( 24 , 25 ). It was found that caspase-9 was the initiator caspase of the mitochondrial apoptotic pathway, and its function was essential for apoptosis. Activated caspase-9 cleaves downstream caspases, such as caspase-3, initiating the caspase cascade ( 26 , 27 ). BCL-2 is one of the major genes involved in the intrinsic pathway of apoptosis. In this regard, a previous study reported an alteration in the expression level of t-BCL-2 by coumarin compounds in cancer cells, which was in line with our results ( 20 ). c-Myc is an important proto-oncogene that is upregulated in numerous human malignancies ( 28 ). Up-regulation of COX-2 increases cancer cell proliferation and metastatic potential and inhibits apoptosis ( 29 ). In this case, all four treatments decreased the expression of COX-2 and c-Myc, which was more pronounced in groups A (especially A1) and B (especially B1). At the same time, all four treatments increased the expression of caspase-9, which might have affected apoptosis induction. It seems that the present coumarin compounds might have influenced the expression of c-Myc, which plays an important role in the development and progression of BC. Pathological changes were significantly reduced after coumarin treatment, with no significant differences between treated groups.

It can be concluded that the present coumarin compounds, namely 2-amino-4-(4-(2-hydroxyethoxy)-3-methoxyphenyl)-5-oxo-4H,5H-pyrano[3,2-c]coumarin-3-carbonitrile and 2-amino-4-(4-hydroxyphenyl)-5-oxo-4H,5H-pyrano[3,2-c]coumarin-3-carbonitrile, may facilitate BC cell death by altering some key apoptotic pathways through targeting caspase-9, BCL-2, c-Myc, and COX-2, which may play a chemotherapeutic role in BC therapy in the future. However, further extensive and complementary studies are needed.

Authors' Contribution

Conceptualisation: MK, MM. Methodology: MK, MM, DS, FJA, FEV, RTM, ST, MSA, YP. Writing/preparation of original draft: MK, MM, FJA, YP. Writing, review and editing: MK, MM. Supervision and funding acquisition: MM. All authors have read and approved the final version of the manuscript.

Ethics

The Animal Research Ethics Committee of Tabriz University of Medical Sciences approved and carried out all animal studies (IR.TBZMED.VCR.REC.1398.407).

Conflict of Interest

The authors declare no conflict of interest.

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