Breast cancer (BC) is a multifactorial disease involving many pathways and target molecules. Multi-target therapy through multi-compound herbal medicines is an alternative strategy to treat BC. In the present study, we elucidate the molecular mechanism of Ocimum basilicum (OB) as an anticancer agent using system bioinformatic approach and investigate its cytotoxic effect against MCF-7 cells. We performed network pharmacology (NP) and molecular docking studies to provide scientific information regarding the underlying anti-BC mechanism of OB. Based on topology parameters obtained from protein-protein interaction (PPI), we identified six potential targets that play a significant role in the network including SRC, PI3KCA, EGFR, ESR1, AKT1, and MAPK1. Furthermore, consensus docking suggested rutin, quercetin-3-O-diglucoside, and kaempferol-3-O-β-D-rutinoside as the potential compounds of OB. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that the cytotoxic effect of OB might be related to the modulation of several pathways such as PI3K-Akt, VEGF, and HIF-1, breast cancer, and estrogen signaling pathways. The in vitro assay revealed that various extracts of OB demonstrated cytotoxic effects against MCF-7 with IC₅₀ = 231 µg/mL (OB ethanolic extract), 408 µg/mL (OB methanolic extract), 479 µg/mL (OB ethyl acetate extract), 1887 µg/mL (OB n-hexanoic extract) and 767 µg/mL (OB butanolic extract) respectively.

Ocimum basilicum, MCF-7 cells, Cytotoxicity, Network Pharmacology, Molecular Docking

Breast cancer (BC) is one of the most common cancers diagnosed in women worldwide, and its occurrence exceeded lung cancer for the first time in 2020 (Zhang et al. 2022). The latest statistics from the International Agency for Research on Cancer (IARC) estimated that there were 2.26 million new BC cases in 2020 and the incidence of BC was predicted to increase to more than 3 million new cases and 1 million deaths in 2040 (Arnold et al. 2022).

The underlying molecular mechanisms of BC are complex involving dysregulation of various signaling pathways with their respective target molecules (Feng et al. 2018). As a consequence, the treatment of BC and other cancers requires a multi-target approach rather than a single-target to optimize the efficacy in certain cases (e.g. resistance) (Giordano and Petrelli 2008). This multi-target approach can be achieved by applying a combination of drugs (drug cocktail), or a single drug with multiple targets (Bolognesi and Rossi 2020; Makhoba et al. 2020). Moreover, multi-compound herbal medicine also offers multi-target effects, thus it can be an alternative therapy for cancer treatment. It has been suggested that combining herbal medicine with conventional cancer drugs might improve bioavailability and decrease adverse effects or toxicity by reducing the dose of standard medicines (Baktiar Laskar et al. 2020).

Based on current data, of 250,000 plant species found in the kingdom Plantae, only 10% of species have been explored for their anticancer activity (Baktiar Laskar et al. 2020). This indicates there is still a great opportunity to find anticancer agents from plants. Ocimum basilicum (OB) belongs to Lamiaceae (Labiatae) family and is a medicinal plant with several pharmacological activities including bronchodilatory effect, antioxidant, anti-inflammatory, and anticancer activity (Aminian et al. 2022). Methanol extract OB has been reported to show anti-BC activity but the molecular mechanism was not determined (Al-Ali et al. 2013). The molecular mechanism study of OB decoct preparation as anti-BC had been conducted previously by manually selecting targets without considering the chemical contribution of OB (Torres et al. 2018).

As a multifactorial disease, molecular mechanism elucidation of BC is challenging at the cellular level, particularly when using multi-compound substances (e.g. extract) since the biological activity is an accumulation of multi-target and multi-compound interactions. Therefore, it is important to consider all possible targets modulated by the compounds. To address this, network pharmacology (NP) offers a systematic and comprehensive approach to analyzing the molecular mechanism of extract materials (Yang et al. 2022). To our knowledge, there were no studies to investigate the molecular mechanism of OB through NP. Therefore, in this study, the underlying molecular mechanisms of OB as an anticancer were elucidated through NP. Molecular docking was conducted for preliminary validation that active compounds of OB can bind to the potential targets suggested by NP. Furthermore, we also explored the cytotoxic activity of various extracts of OB leaves against the MCF-7 cells. The current study provides basic knowledge before performing future experimental studies in developing OB as an anti-BC agent.

OB is a spicy plant commonly used in traditional medicine for treating several conditions such as dysentery, flatulence, colds, and nausea (Eid et al. 2023). Through network pharmacology and molecular docking studies, we hypothesized that the cytotoxic effect of OB on MCF7 is achieved by inhibiting six main targets including SRC, PI3KCA, EGFR, ESR1, AKT1, and MAPK1. Interestingly, these targets are closely related to each other. SRC is a partner for EGFR in its signal transduction (de Diesbach et al. 2010). SRC:EGFR interaction induces activation of PIK3/AKT and MAPK pathways (Irwin et al. 2011). Both pathways in turn induce ligand-independent activation of ESR1 (Eanes and Patel 2016). It has been known that this relationship is involved in the mechanism of resistance to endocrine therapy in ERPBC (Xu and Sun 2010). Therefore, the extract of OB is promising for further development as an alternative treatment for endocrine-resistant ERPBC.

Our docking simulation revealed that almost all compounds were able to bind to these main targets simultaneously, indicating OB has a multitarget and synergic effect, thus increasing their anticancer activity. Based on the consensus score (Table 5), quercetin-3-O-diglucoside was the most potent compound against three targets: PI3KCA, ESR1, and AKT1.

On PI3KCA, the score of this compound was better than the co-crystal ligand. It formed H-bonds with Ser773, Ser774, and Asp933. Asp933 was reported as the residue that most frequently forms an H-bond with many inhibitors (Sabbah et al. 2010). This residue belongs to the conserved DFG motif in many kinase proteins that orient the γ-phosphate of ATP for transfer by binding to Mg²⁺ thus the catalytic process occurs (Maherwari et al. 2017). Meanwhile, an H-bond with Ser774 might be used to increase selectivity (Sabbah et al. 2010) and design mutant-specific inhibitors of PI3KCA (Sabbah et al. 2012). On ESR1, this compound occupies a slightly different position compared to the co-crystal (Fig. 4A). However, it can form an H-bond with Asp351, an essential residue in stabilizing the active conformation of ESR1. By interacting with this residue, an inhibitor will disturb the stability of the active conformation and inhibit ESR1 activity (Kim et al. 2005). On AKT1, this compound formed H-bonds with Lys179, Thr195, Glu198, and Asp292. A molecular dynamic simulation demonstrated that the complex of AKT1 with IN00145, the most active compound according to enzymatic assay, was stabilized by forming H-bonds with two residues included in our docking study, i.e. Lys179 and Asp292 (Mahajan et al. 2020).

Kaempferol-3-O-β-D-rutinoside, on the other hand, was the best-scored compound on EGFR and MAPK1. This compound binds to EGFR by forming H-bonds with Asp766 and Met769, while on MAPK1, it formed H-bonds with Met106, Lys115, and Ser151. Meanwhile, rutin showed the most negative score on SRC and formed H-bonds with Ala293, Lys295, Met341, and Asn391. Met769, Met106, and Met341 are the key residues at the hinge region of EGFR, MAPK1, and SRC, respectively. These targets are members of kinase protein, and it has been reported that H-bonds interaction with the kinase hinge is usually indispensable for potent inhibition (Xing et al. 2015).

In this study, we also investigated the potential anticancer activity of some extracts of OB on MCF-7 and found that they have cytotoxic effects with IC₅₀ = 231 µg/mL (ethanolic extract), 408 µg/mL (methanolic extract), 479 µg/mL (ethyl acetate extract), 1887 µg/mL (n-hexanoic extract) and 767 µg/mL (buthanolic extract). MCF-7 is one of BC cell cultures with estrogen receptors. Ethanolic extract provides better cytotoxic activity compared to other extracts. It seems that the compounds responsible for providing cytotoxic activity are absorbed in the ethanol solvent.

Several studies have tested the cytotoxic effect of OB. However, they used different parts (aerial part, seed, essential oil) of OB and different solvents. Moreover, the molecular mechanisms were not characterized (Arshad Qamar et al. 2010; Eid et al. 2023). So far, we found a study that determined the mechanism of aqueous leaf extracts of OB from decoction. This extract decreased MCF-7 cell growth by 80% at 1000 µg/mL. Here, Akt, mTOR, and AMPK were suggested as the target molecules of OB (Torres et al. 2018). Another study used machine learning-based screening to collect the potential active compounds of the essential oil of OB. BRCA1 and BRCA2 were selected as the target molecules for investigation through molecular docking. However, this study cannot represent comprehensively the total activity of the essential oil of OB against MCF-7 (Nguyen et al. 2022).

According to National Cancer Institute (NCI) and Geran protocol, the cytotoxic effect of a substance is categorized as strong (IC₅₀< 20 µg/mL), moderate (IC₅₀ = 21–200 µg/mL), weak (IC₅₀ = 201–500 µg/mL), and non-cytotoxic (IC₅₀ >501 µg/mL) (Youssef et al. 2022). Based on this, all extracts were classified as weak cytotoxic agents. However, by considering the potential anticancer activity suggested by NP and molecular docking described above, future development of the crude extract of OB is still needed. Several studies have shown that after fractionating, the cytotoxic effect of medicinal plants increases (Charles-Okhe et al. 2022). Thus, the fractionating step focuses on the presence of three potential compounds (rutin, quercetin-3-O-diglucoside, and kaempferol-3-O-β-D-rutinoside) which can be considered for future studies.

Although OB has a weak cytotoxic effect on MCF-7, it showed a good potential for development as an alternative anticancer agent after optimization, such as through the fractionation process. NP studies suggested that OB has cytotoxic activity possibly by modulating several pathways including breast cancer, estrogen, PI3K-Akt, VEGF, and HIF-1 signaling pathways, and targeting six main targets: SRC, PI3KCA, EGFR, ESR1, AKT1, and MAPK1. Meanwhile, three compounds: rutin, quercetin-3-O-diglucoside, and kaempferol-3-O-β-D-rutinoside were considered as the potential compounds of OB. Thus, the cytotoxic activity of OB might partly be due to thebiological activity of these compounds. For the next studies, an in vitro assay against the six main targets is needed to validate this hypothesized molecular mechanism of OB. Moreover, fractionating the crude extract by focusing on the potential compounds might be a strategy to improve the cytotoxic activity.

There is no conflict of interest between any of the authors. The manuscript hasn’t been released yet, nor has it been offered for publication anywhere. The manuscript’s publishing has the unanimous approval of all authors.

This work was funding by Research Grant 2023 from Health Research Organization, National Research and Innovation Agency (BRIN), Indonesia.