In recent years, researchers are actively exploring natural plants to discover drugs that are both non-toxic and effective against tumor cells [4]. Over the course of human history, essential oils have been extensively utilized in folk medicine due to their diverse range of low toxicity and pharmacological applications [15]. The anti-tumor effect of CEO has been documented in limited scientific literature, especially with regards to breast cancer. Recent studies by Tamimi and Mitoshi et al have demonstrated the cytotoxic effects of CEO on HT-29 and HCT116, respectively [16, 17]. An et al have reported the CEO significantly suppressed the proliferation and invasion of breast cancer cells through PI3K/AKT/mTOR pathway [18]. Zu et al observed that the cytotoxicity of CEO on prostate cancer cell was higher than on lung and breast cancer cells [19]. Alkhatib et al found that combination of CEO and chemotherapeutic agent mitomycin C penetrated the cell membrane and directly affected the nucleus, causing damage to Hela cells [13]. Ali et al evaluated the anticancer activity of CEO on the MCF-7 cells [20]. In this particular research, we observed that the CEO inhibited cell proliferation in breast cancer cells by inducing cell cycle arrest. Our network pharmacology analysis revealed that most of the drug-disease common targets were predominantly associated with various signaling pathways; the 4-Cyano-6-dicyanomethyl-2-methyl-2,5-diphenyl-1,2-dihydropyridine, 1,2-dihydropyrazol-3-one,4-(4-bromomethyl-4,5-dihydrothiazol-2-ylamino)-1,5-dimethyl-2-phenyl and tricyclo[4.2.1.1(2,5) decan-3-ol from CEO were an essential components of the CEO, exhibited the highest affinity when interacting with nine core targets. Though there some papers have reported CEO is potential in combating breast cancer, however, this is the initial instance we have documented the substantial restraint of CEO against breast cancer and evaluated its mechanism through network pharmacology analysis.
The signaling pathway known as neuroactive ligand receptor interaction primarily encompasses a collection of genes that encode neuroreceptors such as dopamine receptor and proto-oncogene, and it plays a important role in processing environmental information and mediating the interaction of signaling molecules [21]. The discovery of this pathway revealed its connection to specific neuropsychiatric disorders and congenital diseases [22]. Furthermore, multiple studies have demonstrated an overrepresentation of genes primarily linked to various types of cancer within the signaling pathway of neuroactive ligand receptor interaction [23, 24]. Based on KEGG analysis, the fifty-three identified targets play a role in the neuroactive ligand receptor interaction pathway including NR3C1 which presents unique affinity with 4-Cyano-6-dicyanomethyl-2-methyl-2,5-diphenyl-1,2-dihydropyridine from CEO.
The gene NR3C1, which encodes for a protein called glucocorticoid receptor, is intricately involved in multiple biological process including cell growth, cell death, migration and immune system control, and its crucial function influences the emergence, progression, therapy and prognosis of diverse tumor types [25]. The lower expression level of NR3C1 was observed in breast tumor tissue during recent research on NR3C1 and breast cancer; the agents such as naringenin, cortisol and sinomenine have been demonstrated to enhance the expression of NR3C1 in vivo and in vitro and have exhibited a significant anti-proliferative impact [26–28]. Furthermore, researches have shown that the use of essential oils in aromatherapy can enhance the quality of sleep and alleviate feelings of anxiety in patients diagnosed with cancer [29, 30]. Hence, We hypothesized that the CEO could regulate the expression level of NR3C1 through the pathway of interaction between neuroactive ligands and receptors, thereby exerting its effects on breast cancer cells.
The PI3K-AKT pathway has been found to play a crucial role in most breast cancers and has been associated with proliferation, apoptosis, survival, migration and invasion [31]. Presently, there is immense attention dedicated to the progress of inhibitors designed to target breast cancer within this specific pathway. Furthermore, activation of PI3K/AKT/mTOR pathway associated with reduced sensitivity to chemotherapy and radiotherapy, but suppressing this pathway effectively overcomes drug resistance in breast cancer cells [32]. KEGG analysis revealed that the thirty-four targets are associated with the PI3K-AKTpathway including PTK2, JAK1, JAK2, VGFR, EGFR, HSP90AA1 and PIK3CA. These targets have demonstrated strong binding affinity with 1,2-Dihydropyrazol-3-one,4-(4-bromomethyl-4,5-dihydrothiazol-2-ylamino)-1,5-dimethyl-2-phenyl and methyl octadeca-6,9,12-trienoate from CEO. The gene PTK2, which is also called focal adhesion kinase (FAK), shows a high level of expression in several cancer types such as hepatocellular carcinoma, lymphocytic leukemia and lung cancer, and may contribute to cell growth, survival, migration and invasion [33, 34]. A prior investigation demonstrated that PTK2 stimulates survival signaling pathway in breast cancer and has the potential to impede tumor development [35]. PTK2 expression levels in breast cancer tissues were found to be higher than normal breast tissues, making it a powerful prognostic indicator for breast cancer, as discovered by Chen and colleagues [36]. Lisiak et al discovered that oleanane triterpenoids down-regulated the expressions of PTK2 and phosphorylation status of respective proteins, thereby effectively suppressed the cell migratory and invasive abilities of MCF7, MDA-MB-468 and MDA-MB-231 cell lines [37]. Janus kinase (JAK) proteins in cancer cells are widely recognized as important part in cell proliferation, differentiation, apoptosis and migration [38]. Diverse inhibitors such as ruxolitinib have been developed to target the JAK family, specifically JAK1 and JAK2 [39]. Some authors observed that specific JAK2 inhibitors may be a better approach in treating triple-negative breast cancer [40]. Over the last few years, many researches have revealed that malignant cells exploit the expression of EGFR and VGFR to enhance active targeting [41]. VGFR, which is also known as vascular permeability factor, plays a important role in the formation of blood vessels through vasculogenesis and angiogenesis [42]. Elevation of VGFR levels in cells resulted in high rate of metastasis accompanied by migration and mitosis of endothelial cells [43]. EGFR is highly abundant in numerous cancer cell types and functions as a receptor embedded within the cell membrane. The EGFR receptor has the ability to impact a range of cellular pathways, including RAF/MEK/ERK, JAK/STAT, and P13K/AKT extra [44]. The frequent mutation at the L858R gene has been related to the activation of EGFR in various cancer cells including lung, colon and breast cancer [45, 46]. Various monoclonal antibodies and formulations including cetuximab and nimotuzumab, have mainly been employed for inhibiting angiogenesis and gaining clinical approval in order to target VGFR and EGFR [47, 48]. Heat shock protein 90-alpha (HSP90AA1), a crucial molecular chaperone, is released into the extracellular environment and migrate to the nucleus, where it can induce the generation of immune memory and participate in the evolutionary process of tumor formation [49]. HSP90AA1 has a major function in controlling DNA damage, regulating the cell cycle and gene expression, and thereby stimulating cell proliferation, metastasis, survival and invasion [50]. Furthermore, many reports suggested that HSP90AA1 shows promise as a powerful target for the cancer therapy [51]. The gene PIK3CA, which codifies for the PI3K, is a crucial component in a signaling pathway that enhances cell viability, growth and metabolic processes [52]. A study indicated that PIK3CA can serve as an unfavorable prognostic biomarker in patients with triple negative breast cancer who experience a poor consequence, suggesting its potential in targeted therapy [53]. Furthermore, we found cyclin-dependent kinase 2 (CDK2), a identified targets, also participated in PI3K-AKT pathway in our KEGG analysis. CDK2 is a vital molecule involved in controlling the progression of the cell cycle and frequently exhibits increased expression levels in cancerous cells; the inhibition of CDK2 has been proven to effectively treat various types of cancers like breast carcinomas, leukemia, and lymphomas [54]. Additionally, our findings suggest that the CEO could potentially have an impact on the expression of CDK2 through the PI3K-AKT signaling pathway, despite CDK2 not being identified as a core target in PPI netwok.
Calcium signaling pathway is another important signaling pathway in which the thirty-five identified targets were involved including VGFR and EGFR. Ionic Ca2+, a vital component of breast milk, controls a multiple intracellular signalling pathways integral to gene transcription, differentiation, proliferation, apoptosis, cell cycle arrest, and cell migration [55]. Makena et al hypothesized that subtype-specific targeting of calcium signaling could be another technique of precision medicine in the triple negative breast cancer treatment [56].
MAPK pathway is one of the suggested pathway by our research in which twenty-six identified targets were involved including MAPK14, MAPK11 and VGFR [57]. MAPK signaling pathway is involved in the development of several types of cancer and is being targeted for therapeutic interventions [58]. JNK1 (MAPK8) and ERK1/2 (MAPK3/1) has been implicated in regulating the maintenance of breast cancer stem cells, leading to the development of chemo-resistance and the promotion of epithelial-mesenchymal transition (EMT) process [59]. The MAPK signaling including p38α (MAPK14), p38β (MAPK11), p38γ (MAPK12) and p38δ (MAPK13), among them p38α and p38β are the predominant form [60]. Previous research have shown that MAPK14 and MAPK11 play different role in breast tumorigenesis and EMT in breast cancer cells by cooperating with TGF-β [61]. Several studies have indicated that inhibiting MAPK14 can reduce the tumorigenic capability in animal models of breast cancer [62]. However, the overexpression of MAPK11 is linked to the up-regulation of monocyte chemotactic protein-1 (MCP-1), resulting in the differentiation of osteoclasts and the advancement of bone metastasis in patients with breast cancer [63]. In addition, a few studies have indicated new functions of MAPK11 in the progression of tumors controlled by TGF-1β and VEGF growth factors, even without the involvement of the proapoptotic MAPK14 [64].
The twenty-eight identified targets were involved in the cyclic AMP (cAMP) signaling pathway including PIK3CA. The signaling molecule cAMP controls cellular metabolism through the activation of protein kinase A (PKA) and the targeting of exchange protein directly activated by cAMP (EPAC) [65]. The suppression of cell proliferation, migration and cytoskeleton remodeling by stimulating EPAC indicates its potential to act as a target for treatment of cancer [66].
Taken together, CEO might regulate the target genes through those signaling pathways we described above, and exhibits anti-breast cancer efficacy. Despite the many agents have been in clinical use for decades, the new classes of anti-breast cancer drugs should have been developed and our findings will provide new evidence for usage of CEO in the breast cancer treatment. However, there are many shortcomings presented in our study. Due to the limitation of financial resources, the detailed bioactive compounds in CEO have not been validate yet; chamomile flower is expensive and sufficient amount of CEO hard to collect for animals treatment, hence, in vivo antitumor experiments are yet to be discovered; core targets and involving pathways needs to analysis. Although additional investigation is required to uncover the fundamental processes involved, these discoveries present a fresh concept in utilizing CEO as a possible innovative medication for the breast cancer therapy.