Biological pathways of hydralazine

The bioinformatics analysis showed the effects of hydralazine on both healthy and cancerous cells through the HIF-1 and VEGF pathways (Fig. 2). HIFs (Hypoxia-inducible factors) can regulate epithelial-mesenchymal transition, angiogenesis, and metabolic adaptations in lung and breast cancers. They consist of three isoforms, HIF-1, HIF-2, and HIF-3 [13]. This pathway suggests that hydralazine can increase tumor angiogenesis by increasing VEGF gene expression through its effects on HIF-1 and prolyl hydroxylase domain (PHD).

Fig. 2

The HIF-1 pathway in the KEGG database responses to cellular oxygen levels. In normoxic conditions, PHD enzymes use 2-oxoglutarate, ascorbate, iron, and oxygen to hydroxylate Pro402 and Pro564 residues in the oxygen-dependent degradation domain of the HIF-1α subunit. In hypoxic conditions, deficiency in co-factors or an increase in nitric oxide and reactive oxygen species leads to a reduction in the hydroxylation and degradation of HIF-1α. So, it will be stabilized and translocated to the nucleus, where it dimerizes with HIF-1β. This complex binds to hypoxia response elements in the promoter of the VEGF gene and causes angiogenesis. (2-oxoglutarate (2OG), ascorbate (Asc), iron (Fe2+), oxygen (O2), nitric oxide (NO), and reactive oxygen species (ROS))

Biological pathways of ATRA

The results of KEGG and Drugbank databases showed that ATRA can affect both healthy and cancerous cells through the regulation of the cell cycle, VEGF, and Wnt/β-catenin pathways. In the ATRA-RAR signaling pathway, ATRA binds to retinoic acid receptors (RARs) and retinoid X receptors (RXRs) in the cell nucleus, activating them. These activated complexes reduce VEGF levels, leading to less blood vessel formation, tumor growth, and cell proliferation. Additionally, ATRA lowers the expression of CCND1, a Wnt/β-catenin target, causing cell cycle arrest and reduced proliferation. It encourages pathway inhibitors to bind to Wnt ligands or Fz receptors, reducing pathway activation. It highlights the diverse roles of ATRA in controlling cell cycle progression, signaling pathways, and cellular differentiation (Fig. 3).

Fig. 3

Functional pathways of ATRA in the human body. (A) ATRA in the cell cycle pathway. (B) ATRA-RAR signaling pathway in breast cancer. (C) ATRA suppresses the expression of CCND1 as a Wnt/β-catenin target, leading to cell cycle arrest and reduced proliferation

ATRA halts the cell cycle in breast cancer cells by increasing the expression of cyclin-dependent kinase (CDK) inhibitors such as p21 and p27, which reduce CDK activity and prevent cell cycle progression in breast cancer [14] (Fig. 3 Section A).

In the ATRA-RAR signaling pathway, ATRA binds to RARs and RXRs in the cell nuclei, activating them. These activated complexes decrease VEGF expression, leading to reduced angiogenesis, tumor growth, and proliferation [15] (Fig. 3 Section B).

Besides, ATRA suppresses the expression of CCND1, a Wnt/β-catenin target, leading to cell cycle arrest and reduced proliferation. The dysregulation of the Wnt/β-catenin signaling pathway leads to tumor growth, invasion, and metastasis [16] (Fig. 3 Section C). This figure illustrates the multifaceted roles of ATRA in regulating cell cycle progression, signaling pathways, and cellular differentiation, highlighting its potential therapeutic effects in breast cancer treatment.

Drug targets

Molecular targets of the drug obtained from the way2drug database are listed in Table 2. The target classification chart from SwissTargetPrediction is shown in Fig. 4. The results indicate that these drugs have various targets in the body, so they can affect different molecules and pathways. Therefore, by combining these two drugs, different mechanisms impact cancer cells.

Fig. 4

Target categorization of hydralazine and ATRA

Table 2 Direct and indirect targets of hydralazine and ATRA, along with the probability of binding for each drugGene expression analysis

The results of gene expression analysis obtained from the GTEX and TNMplot databases show that these genes are suitable for the study (Fig. 5).

Fig. 5

Gene expression analysis in healthy and cancerous conditions. (A) Gene expression Heat map in normal tissues obtained from GTEX, which indicates that all three genes have high expression levels in breast tissue. (B) The gene expression graphs in three states of healthy, primary tumor, and metastatic, along with the p-value of the graphs indicating their significance

Patients survival

The survival rate of patients using the Kaplan-Meier plotter database showed that the aberrant expression of HIF1A, VEGFA, and CCND1 genes was associated with reduced survival in breast cancer patients (Fig. 6).

Fig. 6

Kaplan-Meier plots showing patients’ survival differences with gene expression changes due to mutations, including statistical analysis for survival time

Enrichment pathways

ShinyGO enrichment pathways analysis for the genes of interest is listed in Table 3. Results show their involvement in various types of cancers.

Table 3 Biological pathways of HIF1A, VEGFA, and CCND1 genes that are ordered from smallest to largest enrichment FDR. This analysis indicates the importance of these genes in various disease pathways and cancer typesMTT method

After the cell culture steps (Fig. 7), the MTT test results were obtained. The optical density read by the ELISA reader is shown in Fig. 8. The IC50 for ATRA in the MDA-MB-231 cells was about 130 µM, and in the MCF10 cells, it was 456 µM. The IC50 for hydralazine could not be calculated due to its stimulatory effect on cell growth; therefore, a concentration of 100 µM of this drug was considered for the final combined drug formulation. These results show decreased survival in ATRA-treated cells and increased survival in hydralazine-treated cells by dose. At similar concentrations, MCF10 cells’ survival in ATRA treatment was higher compared to MDA-MB-231 cells, indicating the higher cytotoxicity of this drug on malignant tumor cells. Hydralazine can stimulate breast cancer cell growth in both tumorigenic and non-tumorigenic states, but its effect on MCF10 cells was greater at similar concentrations.

Fig. 7

Morphology of breast cell lines. (A) Non-tumorigenic, island-like MCF10 cells, which are epithelial-like and tend to aggregate in the flask. (B) Tumorigenic MDA-MB-231 cells, which are spindle-shaped and more dispersed in the flask

Fig. 8

MTT test results show the cytotoxicity of ATRA and the growth stimulation of hydralazine in both cell lines with concentration increase. (A) MDA-MB-231 cells. (B) MCF10 cells

Isobologram test

The results obtained from the reduction of formazan crystals after 48 h are shown in Fig. 9. A constant concentration of 100 µM of hydralazine and a variable concentration of 200 µM of ATRA were used solely to ensure the reducing effect on cell viability in case of increased concentration.

Fig. 9

The results of the isobologram test in both cell lines show cell viability in treatments with selected constant concentration of 100 µM for hydralazine, and a variable concentration close to the IC50 of ATRA at 100 µM has a very strong inhibitory effect similar to the control drug doxorubicin. By increasing the concentration of ATRA in the combination, viability decreases. The different effects of this combined treatment on breast cells in MDA-MB-231 and MCF10 are significant (P-values < 0.05), indicating the specific impact of this combination on tumor cells (unlike doxorubicin). (A) Treatment in MDA-MB-231 cells. (B) Treatment in MCF10 cells

Wound healing test

Statistical analysis graphs for both cell lines are shown in Fig. 10. MDA-MB-231 control cells were not treated with any drug, and after 48 h, the gap was almost filled. In the doxorubicin treatment, the morphology of the cells changed, and cell migration was completely inhibited. Treatment with hydralazine alone showed that the cell migration was the same as the control cells. ATRA treatment changed cell morphology and decreased the invasion rate of cells compared to the control. The hydralazine and ATRA combination had a greater impact on preventing cell migration in this line compared to the individual drugs. This combination reduced the wound closure rate in 24 h, and after 48 h, in addition to the reduced rate, the cells were apoptosis, and the wound even expanded (Fig. 11).

Fig. 10

Wound healing graphs show a decrease in migration rate in the combined drug treatment (P-values < 0.05 are significant). (A) Results in MDA-MB-231 cells. (B) Results in MCF10 cells. After 48 h following treatment with doxorubicin, the cells were so damaged that the scratch completely disappeared, and calculation with the ImageJ software was not possible; however, to identify the result significance, a value of 200 was assumed

Fig. 11

Results of the wound healing test in MDA-MB-231 cells, showing the effectiveness of the ATRA/hydralazine combination on the cells’ migration

MCF10 control cells were not treated with any drug, and after 48 h, the gap was almost closed. Doxorubicin Treatment showed complete inhibition of cell migration, apoptosis, and morphological changes in these non-tumor cells in a way that analysis with ImageJ software was not possible in the images taken 48 h later. The treatment with hydralazine alone showed cell migration as the control cells. Treatment with ATRA indicated complete inhibition of cell migration, degradation, destruction, and morphological changes in this cell line. The hydralazine and ATRA combination did not show significant effects on cell migration compared to the individual drugs, indicating the non-cytotoxicity and lack of inhibition of this combination on these healthy breast tissue cells (Fig. 12).

Fig. 12

Results of the wound healing test in MCF10 cells show the combination’s lesser impact compared to doxorubicin

Gene expression analysis by real-time PCR

According to Fig. 13, the expression of each gene was directly examined in both cell lines. Using the Tukey test, the significance comparison of the graphs for each comparison was measured. All of the p-values were < 0.0001.

Fig. 13

Comparative analysis of RT-PCR. The combination of ATRA and hydralazine can be selected as the most effective treatment with the greatest effect on MDA-MB-231 cells and the least effect on the MCF10 cells. This effect appears on key genes related to cell cycle regulation (CCND1), angiogenesis (VEGFA, VEGFA2), and hypoxia adaptation (HIF1A, HIF1A-AS). The reduction rate is generally higher in MDA-MB-231 cells, indicating a potentially greater therapeutic efficacy of this compound in breast cancer cells compared to non-tumor cells

In the MDA-MB-231 cell line, all three treatments (Hydralazine, ATRA, and hydralazine/ATRA) significantly reduce the expression of CCND1, with ATRA showing the greatest reduction. In MCF10 cells, CCND1 expression is also reduced by all three treatments. Hydralazine significantly reduces the expression of this gene in these cells, which can have negative effects on the body. The reduction in MCF10 is much less affected compared to MDA-MB-231.

Hydralazine significantly increased the expression of VEGFA in MDA-MB-231 cells, but it decreased in MCF10 cells. Treatment with ATRA has shown little effect on this gene, and few changes were observed in the combined treatment. The harmful effects of hydralazine are deactivated when it is combined with ATRA, so angiogenesis is inhibited.

The VEGFA2 gene expression is similar in all three treatments. In all treatments, MDA-MB-231 shows a significant increase compared to the control sample, while a slight increase is observed in the MCF10 cell line. The increased expression of this gene is more significant in MDA-MB-231 cells compared to MCF10 cells.

In MDA-MB-231 cells, HIF1A expression is reduced by the combination drug and slightly by ATRA, while hydralazine did not affect these cells alone. In MCF10 cells, hydralazine reduced HIF1A expression, affecting normal body cells under hypoxic conditions. ATRA and the combination treatment were ineffective on this gene’s expression in these cells. The negative effects of hydralazine were eliminated when combined with ATRA.

In MDA-MB-231 cells, ATRA and hydralazine significantly reduced the expression of HIF1A-AS. However, in the combined treatment, the expression of this gene slightly increased. The negative effects of hydralazine were eliminated in combination with ATRA. In MCF10 cells, none of the treatments showed any significant effect on HIF1A-AS expression.