Targeting mitochondria is emerging as a successful therapeutic approach for cancer because of the involvement of mitochondria in redox signaling, cellular metabolism, and cellular homeostasis [1]. The mitochondria are the site of adenosine triphosphate (ATP) synthesis from glucose and fatty acids through a series of biochemical reactions known as oxidative phosphorylation. The essential requirement for oxidative phosphorylation is the electron transport chain (ETC) that is associated with the production of reactive oxygen species (ROS), as well as adenosine triphosphate (ATP) synthesis [2]. Thus, an efficient redox balance during cellular ATP synthesis is necessary for cellular proliferation and viability. However, dysfunction of mitochondria has a significant negative impact on overall ROS generation and cellular energy metabolism, which ultimately adversely affects the fate of cells. Nevertheless, in cancer cells, the shifting of energy metabolism from the usual oxidative phosphorylation to active glycolysis impedes mitochondrial activity, leading to increased levels of ROS generation, which can ultimately be linked to numerous oncogenic signals [2].

Mitochondrial membrane potential (abbreviated to $\mathrm{\Delta }$$\mathrm{\Psi }$m) is a global indicator of mitochondrial function. The electrochemical gradient resulting from the imbalance of H+ outside and inside the inter-membrane space causes the development of mitochondrial membrane potential [3]. In a normal cell, mitochondrial membrane potential ranges between –108 and –159 mV, while optimum membrane potential for maximum ATP production fluctuates between –130 and –140 mV in almost all living creatures. Alteration by approximately 10% in the mitochondria membranes potentials results in, approximately, a 90% depletion in ATP synthesis and an increase in the generation of potentially toxic ROS by a similar magnitude [4]. Cancer cells are $>$50% more hyperpolarized across the inner mitochondrial membrane ($\mathrm{\Psi }$IM) than normal cells [5]. Furthermore, the invasiveness of cancer is associated with the increased hyperpolarization of $\mathrm{\Psi }$IM [6, 7, 8]. In cancerous cells, an increased mitochondrial membrane potential compared to cytosol also makes mitochondria a potential selective target as mitochondria allow selective agents to pass across the double membrane. The outer membrane of the mitochondria is associated with signals related to cell survival and death [9]. The porosity of the mitochondrial membrane increases during the activation of proapoptotic proteins in the state of oxidative stress, enabling the extensive escape of cytochrome c to the cytosol and thereby leading to cellular damage [10].

Many recent reports showed that herbal extracts and active isolated compounds can cause the disruption of mitochondrial membrane potential in different cancer cell lines [11, 12, 13]. Solanum nigrum (black nightshade) is a commonly used medicinal herb in alternative systems of herbal medicine. The crude extract from Solanum nigrum has shown anticancer potential in different types of cancer including breast cancer, cervical cancer, endometrial cancer, colorectal cancer, and human melanoma [14, 15, 16, 17]. The Solanum nigrum water extract from leaves is significantly cytotoxic in breast cancer cell lines due to apoptosis and epithelial-mesenchymal transition [18]. The leaf extract of Solanum nigrum possesses antioxidant [19] and anti-inflammatory [20] properties that can efficiently cope with oxidative stress and inflammation, the primary hallmarks of cancer. Furthermore, the leaf extract of Solanum nigrum also proved capable of promoting mitochondrial fission and, hence, reducing the normal function of mitochondria in breast cancer cell lines [18]. These reports suggest that Solanum nigrum works mechanistically by targeting mitochondria membrane potential and hampering ATP generation in breast cancer. We, therefore, attempted to investigate the effect of Solanum nigrum on mitochondria membrane potential and ATP generation in breast cancer cells.

Solanum nigrum is used in various medicinal systems due to its numerous valuable pharmacological properties [25, 26, 27, 28]. Our results showed that Solanum nigrum water extract (SNWE) inhibited the growth of both estrogen receptor-positive as well as estrogen receptor-negative breast cancer cell lines (MCF-7 and MDA-MB-231, respectively) in a concentration-dependent manner (Fig. 2). Several studies showed the cytotoxic effect of Solanum nigrum water extract in breast cancer cell line AU565; cervical cancer cell line U14; hepatoma cell line HepG2; endometrial cancer cell lines HEC1A, HEC1B, and KLE; and colorectal cancer cell lines DLD-1 and HT-29 [14, 15, 16, 29, 30]. Furthermore, SNWE induces apoptosis and autophagy in cancer cells [31, 32]. Similarly, lower concentrations of SNWE caused autophagy and no apoptosis, whereas high concentrations inhibited p-Akt and resulted in apoptosis- and autophagy-induced cellular damage [16]. Moreover, adjuvant therapy with SNWE can also augment the cellular toxicity of known cancer drugs such as docetaxel, doxorubicin, and cisplatin [32, 33, 34].

In our study, we found that SNWE significantly diminished intracellular ATP levels in a dose-dependent manner (Fig. 3). The intracellular ATP levels are markers for cell viability and can be used to evaluate the efficacy of anticancer drugs in cancer cells. Various concentrations of SNWE significantly reduced the ATP levels in the breast cancer cells in a dose-dependent manner, while the untreated control cells showed higher ATP levels. These results reveal the failure of the mitochondria to produce ATP for cellular proliferation in both MCF-7 and MDA-MB-231 breast cancer cells. Similarly, Hernández et al. [35] observed that Petiveria alliacea L leaf extracts reduced the cellular ATP concentrations in breast cancer cells and breast cancer murine models.

Our results show that SNWE treatment significantly disrupts mitochondrial membrane potential (MMP) in breast cancer cells (Fig. 5). Mitochondria are the central regulators of the intrinsic apoptosis pathway as they maintain the balance between pro-apoptotic and anti-apoptotic events while controlling programmed cell death [36]. The recent literature proposes targeting mitochondria as an attractive strategy to combat cancer [37]. The various concentrations of SNWE caused the dose-dependent reduction in oxidative phosphorylation and electron transport through the disruption of MMP, which leads to the induction of caspase-dependent apoptosis. Rhodamine-123 is a fluorescent chemical probe widely used for its ability to accumulate in the mitochondria and thus acts as an indicator for mitochondria membrane potential [38]. High retention of rhodamine-123 fluorescence was detected in both untreated MCF-7 and MDA-MB-231 cells (Fig. 4). Further, the fluorescence was significantly reduced after SNWE treatment in both types of breast cancer cells. This mechanism of action suggests that SNWE induces mitochondrial damage and thereby leads to ATP depletion that, in turn, causes cell death. Several other evidence-based studies have shown that a significant number of naturally occurring agents and herbs, including withanone from Withania somniferum, can induce mitochondria-mediated cancer cell death by elevating oxidative stress, disturbing the cellular redox system, reducing ATP levels, and causing loss of mitochondrial membrane potential [39, 40]. The concept of mitochondria-targeted anticancer agents is attracting increased attention and, in the literature, such agents have been termed “mitocans”, mitochondria-targeting anticancer drugs, with various modes of action [41]. Naturally occurring products act as mitocans by targeting the mitochondrial membrane potential [42].

Mechanistically, several bioactive ingredients found in Solanum nigrum, including uttroside B, solanine, solamargine, and physalins, were tested for their anticancer effects in both in-vitro and in-vivo cancer models [43]. $\alpha$Solanine is reported to be involved in the regulation of oncogenic miRNA-21 and tumor suppressor miRNA-138 in prostate cancer [43]. The active components of Solanum nigrum also produced inhibitory actions on various cellular pathways, such as matrix metalloproteinases (MMPs), VEGF/VEGFR, JAK-STAT, and PI3K/AKT in various cancer types [43]. $\alpha$-Solanine was shown to suppress cancer metastasis via the expression of MMPs and inhibition of epithelial-mesenchymal transition [44]. It can also down-regulate the expression of vascular endothelial growth factor (VEGF) through the ERK1/2-HIF-1$\alpha$ and STAT3 signaling pathways in cancer cells [45]. Molecular docking analysis showed that phytoconstituents of Solanum nigrum, including solanidine, solasodine, and solamargine, have positive drug-likeness values, suggesting the therapeutic potential of these compounds as future anticancer drugs [46]. Moreover, the glycoalkaloids and saponins found in Solanum nigrum did not show any general toxicity, tumorigenicity, or irritant effects [46]. These findings suggest that Solanum nigrum is a non-toxic medicinal herb that contains numerous bioactive compounds with pharmacological potentials. Moyo et al. [47] studied the impact of boiling and in-vitro human digestion of predominant compounds of S. nigrum including, myricetin, quercetin-3-O-arabinoside, 3,4-dicaffeoylquinic acid, 3-caffeoylquinic acid, and rutin. The study showed improvement in phenolics after boiling but reduction following in-vitro digestion, without affecting their bioactivity, particularly in preventing oxidative stress in Caco-2 cells [47]. Thus, the heating steps used for the preparation of SNWE in our study would have no detrimental effect on the stability of bioactive compounds, particularly, those with antioxidant activity.

Reprogramming of energy metabolism is one of the emerging hallmarks of cancers because uncontrolled cell division demands an increase in fuel and biosynthetic precursors by adjusting energy metabolism in cancerous cells. Our findings showed that SNWE significantly interferes with energy metabolism in cancer cells by disrupting mitochondrial membrane potential that results in the depletion of cellular ATP levels. However, the involvement of other potential targets behind the anticancer effects of SNWE may not be ruled out. For instance, solanine, the main ingredient of Solanum nigrum, showed antitumor ability against different tumors by targeting different proteins [48].

Thus, the use of SNWE alone or as an adjuvant with low doses of known cancer drugs could serve as an economical therapeutic modality with fewer side effects.

The SNWE treatment induced the loss of mitochondrial membrane potential, which resulted in reduced cellular ATP levels and subsequent apoptotic cell death in the MCF-7 and MDA-MB-231 breast cancer cells. Since this study was conducted in breast cancer cells, further studies are required to confirm the implications of our findings for the treatment of human breast cancer. In the early stages, breast cancer is treatable with high success rates. Pharmacotherapies based on traditional medicine are not only cost-effective but mostly free from side effects. Further studies are warranted to explore the therapeutic and prophylactic anticancer effects of Solanum nigrum using animal models of cancer.

Datasets used and/or analyzed for this study are available from the corresponding author upon appropriate request.

Conceptualization, HAK; methodology, HAK, NRP, AAA, SHA, BSA, AAKH; formal analysis, NRP, AAA, BSA, AAKH; investigation, HAK, NRP, AAA, SHA; resources, HAK, SHA, NRP; data curation, HAK, NRP, AAA, BSA, AAKH; original draft preparation, HAK, AAA; writing, review and editing, HAK, AAA, NRP; supervision, HAK, AAA; project administration, HAK; funding acquisition, HAK. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work to take public responsibility for appropriate portions of the content and agreed to be accountable for all aspects of the work in ensuring that questions related to its accuracy or integrity.

The study protocol was approved by the Institutional Review Board of King Saud University, Riyadh, Saudi Arabia (Approval No. KSU-SE-22-18, dated 24/03/2022). This study was conducted in rats and did not involve human subjects.

We acknowledge the technical support from the laboratory staff.

This research was funded by the National Plan for Science, Technology, and Innovation (MAARIFAH), King Abdulaziz City for Science and Technology, Kingdom of Saudi Arabia, Award Number (2-17-03-001-0044).

The authors declare no conflict of interest. HAK is serving as one of the Guest Editors and Editorial Board Members of this journal. We declare that HAK had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to SN.