**1. Introduction**

European mistletoe (Loranthaceae) grows parasitically on trees, including oaks and apples, across Europe and Asia [1]. This semi-parasite has been used for generations as a medicinal plant in Germany and Switzerland to treat hypertension, arterial sclerosis, and cancer [2,3]. Several studies are proving its pharmacological effects. Various active components isolated from mistletoe have been demonstrated to possess anti-cancer effects in extensive detail [4,5]. The active ingredients responsible for these effects are lectin (a glycoprotein), viscotoxin (a protein component), and oleanolic acid (a triterpenoid). Scientific evidence suggests that these components not only possess anti-cancer properties but also play a vital role in enhancing the immune system's activity [2,6–9]. For example, among the pharmacological components of mistletoe, polysaccharides, oligosaccharides, amines, and alkaloids have a lesser direct killing effect on cancer cells than lectin, but they can activate macrophages to induce cell-mediated immunity [10]. In particular, a variant of European mistletoe, Korean mistletoe (*Viscum album* L. var. *coloratum*), has been reported to have superior immune cell stimulation activity compared to European mistletoe [11]. Korean mistletoe is a domestically native plant distinct from European mistletoe, and it has been used as a medicine for back pain, high blood pressure, and toothache in private and oriental medicine [12,13]. Recently, researchers have actively investigated the anti-cancer activity of Korean mistletoe extract. Most studies on chemotherapy with mistletoe use mistletoe water extract [14]. The anti-cancer activity of mistletoe extract is produced not

**Citation:** Lim, W.-T.; Hong, C.-E.; Lyu, S.-Y. Immuno-Modulatory Effects of Korean Mistletoe in MDA-MB-231 Breast Cancer Cells and THP-1 Macrophages. *Sci. Pharm.* **2023**, *91*, 48. https://doi.org/ 10.3390/scipharm91040048

Academic Editor: Barbara De Filippis

Received: 30 August 2023 Revised: 23 September 2023 Accepted: 4 October 2023 Published: 18 October 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

only by the direct cytotoxic effects of its constituents but also by inducing tumor-specific cell-mediated immune enhancement. This occurs due to the activation of immune cells (such as macrophages) against tumor cells [15].

Clinically diverse phenotypes characterize breast cancer, with different subtypes classified into progesterone receptor (PR), estrogen receptor (ER), and human epithelial growth factor receptor 2 (HER2) based on immunohistochemical staining expression [16]. Triple-negative breast cancer, a subtype in which PR, ER, and HER2 are not expressed, represents 10–20% of all breast cancer patients [17–19]. Therapeutic effects are noticeably lower in triple-negative breast cancers compared to other hormone receptors or treatments targeting HER2-positive subtypes. Consequently, treatment for triple-negative breast cancer still relies heavily on non-specific measures such as surgery, radiation therapy, and chemotherapy, despite their associated side effects [19,20]. Furthermore, triple-negative breast cancer aggressively metastasizes to major organs such as the bone, liver, lung, and brain throughout the course of the tumor. Doctors identify metastasis as a significant obstacle in effectively treating triple-negative breast cancer, which consequently results in a clinically poor prognosis [21–26].

Breast cancer encompasses an intricate microenvironment within the tumor, involving blood vessels, immune cells, fibroblasts, cytokines, and extracellular matrices. These components interact with each other, playing significant roles at every stage of metastasis [27]. Macrophages, integral to innate immunity, function differently within the tumor microenvironment compared to general macrophages. Specifically, in breast cancer microenvironments, they transform into tumor-associated macrophages (TAMs) upon activation [28]. TAMs constitute around 50% of the total cell population in this microenvironment, making them the most abundant component. They secrete stimulatory or inhibitory signaling molecules that manipulate growth during solid tumor progression [29–31].

The human immune response and inflammation lead to the secretion of inflammatory mediators from cells to protect against external stimuli like infections or tissue damage. However, this process can also promote chronic cancer cell death by inhibiting inflammatory mediators like interleukin (IL)-4, IL-6, interferon (IFN)-γ, and transforming growth factor (TGF)-β [13,32]. Tumor cells secrete matrix metalloproteases (MMPs) and proteolytic enzymes, along with inflammatory cytokines, that degrade the extracellular matrix forming the cellular scaffold and modulate the intra-tumoral environment [33,34].

Moreover, an inflamed microenvironment leads to the persistent activation of signal transducer and transcription (STAT) proteins within cells, triggering further inflammation along with metastasis and neovascular synthesis in cancerous cells, exacerbating the tumor [35]. Under normal conditions, STAT proteins play crucial roles in cell development, differentiation, and survival. However, excessive activation of these same proteins often underlies cancer development. An illustrative case of this phenomenon is the active pathway of STAT3, extensively studied due to its overexpression across various cancer cell types [36]. In an inflammatory microenvironment, tumor cells interact with IL-6 via IL-6 receptors, leading to their conversion into phospho-STAT3 (p-STAT3), an activated form [37]. P-STAT3 infiltrates the nucleus of tumor cells, upregulating metastasis and neovascularization-related genes [38]. While advancements in early diagnosis technology and anti-cancer drugs have led to a decrease in cancer mortality rates [39], cancer cell metastasis remains the primary cause of death in cancer patients [40]. Thus, the development of anti-cancer drugs inhibiting STAT3 activation is anticipated to offer therapeutic benefits by curbing cancer cell metastasis and new blood vessel synthesis.

In addition, the activation of Bcl-2 family proteins such as Bax and Bcl-2 triggers the direct apoptosis of tumor cells [41]. These activated Bax protein oligomers bind to the mitochondrial outer membrane, inducing mitochondrial outer membrane permeabilization (MOMP) [42,43]. This process allows apoptosome leakage from the mitochondria, subsequently activating cysteine-aspartic proteases (caspases) [44]. Among these, caspase-3 acts as an effector caspase, inducing apoptosis by deactivating poly ADP-ribose polymerase (PARP) in the nucleus of tumor cells, thereby hindering DNA repair [45]. Apoptosis is

genetically regulated by infection and DNA damage, setting it apart from necrosis. In the study of anti-cancer drugs, inducing apoptosis is crucial; unlike necrosis, it avoids triggering additional inflammatory reactions. Consequently, only tumor cells are selectively eliminated without causing harm to normal tissues [46].

Furthermore, cancer cell metastasis is a sequential process, progressing from primary tumors to the development of new tumors in distant organs. Epithelial–mesenchymal transition (EMT) is a theory closely associated with the early stages of this metastasis process [47]. The initiation phase occurs when MMP-2 and MMP-9 are secreted by the primary tumor, enabling the infiltration of local tissues and blood vessels [48]. Consequently, these infiltrated cancer cells gradually undergo a phenotypic change from epithelial to mesenchymal cells, characterized by a decrease in the expression of the cell adhesion molecule E-cadherin and an increase in the expression of N-cadherin, which weakens cell adhesion [49,50]. EMT transforms solid tumor cells into mesenchymal cells, endowing these newly transformed cells with enhanced mobility. This mobility allows detached cancerous epithelial cells to enter circulatory systems like blood vessels and metastasize to other organs. A notable feature of EMT is the conversion of cadherins, in particular an increase in N-cadherin accompanied by a loss of E-cadherin [51].

Recent studies have actively pursued immuno-cancer research, with a focus on investigating anti-cancer activity by meticulously examining and enhancing tumor microenvironments. However, no research has reported on how mistletoe water extract activates macrophages to improve the tumor microenvironment. Therefore, in this experiment, we aimed to explore the potential of Korean mistletoe extract as an immune enhancer. We sought to compare and investigate whether activated macrophages induce apoptosis more effectively in triple-negative breast cancer cells while concurrently inhibiting EMT and neovascular synthesis.

#### **2. Materials and Methods**

#### *2.1. Reagents and Antibodies*

The antibodies used in this study were obtained from multiple providers: Cell Signaling Technology (CST, Danvers, MA, USA), Thermo Fisher Scientific (Waltham, MA, USA), and BD Biosciences (Franklin Lakes, NJ, USA). The biotin anti-human IL-6 detection antibody used for the enzyme-linked immunosorbent assay (ELISA) was procured from BD Biosciences. For Western blot experiments, primary antibodies were supplied by CST, including anti-mouse Bcl-2 (1:1000, Cat#15071), anti-rabbit Bax (1:1000, Cat#5023), anti-rabbit caspase-3 (1:1000, Cat#9664), anti-rabbit cleaved caspase-3 (1:1000, Cat#9664), anti-rabbit PARP (poly ADP-ribose polymerase, Cat#9532) (1:1000), anti-rabbit cleaved PARP (1:1000, Cat#5625), anti-mouse STAT3 (Tyr705, 1:1000, Cat#9138), anti-mouse p-STAT3 (1:1000, Cat#9145), anti-rabbit MMP-2 (1:1000, Cat#40994), anti-rabbit MMP-9 (1:1000, Cat#13667), anti-rabbit E-cadherin (1:1000, Cat#3195), and anti-rabbit N-cadherin (1:1000, Cat#13116). Cleaved caspase-3 (1:1000, Cat#700182) and β-actin antibodies (Cat#MA1-744) were obtained from Thermo Fisher Scientific. For Western blot experiments, secondary antibodies including anti-mouse IgG and horseradish peroxidase (HRP)-linked antibodies (Cat#7056) as well as anti-rabbit IgG and HRP-linked antibodies (Cat#7074), were procured from CST. In the immunofluorescence experiment, Alexa Fluor™ 488 goat anti-rabbit IgG (H+L) served as the secondary antibody and was acquired from Thermo Fisher Scientific.

#### *2.2. Manufacturing Mistletoe Water Extract*

Korean mistletoe was harvested from oak trees in Kangwon-do, Korea. The botanical verification was conducted by Professor Jon-Suk Lee from Seoul Women's University, Korea, and a voucher specimen (VCA101) was deposited at the College of Pharmacy, Sunchon National University, Korea. Initially, 100 g of crushed samples containing dried mistletoe leaves, stems, and branches (EV-MC6000, Everyhome Co., Busan, Republic of Korea) were combined with distilled water in a proportion of four times the weight of the dried sample. This mixture was gently agitated for a day within a shaking incubator operating at

120 rpm and maintained at a temperature of 4 ◦C. Subsequently, the resulting supernatant was separated via filtration using cotton material. The remaining residue underwent two additional extraction cycles using the same quantity of distilled water, followed by shaking. The obtained supernatant was then subjected to centrifugation at 4 ◦C for 20 min at 4500 rpm utilizing a centrifuge (VS-550, Vision Co., Daejeon, Republic of Korea), and then filtered using filter paper (No. 2, Advantec Toyo Roshi Kaisha, Ltd., Tokyo, Japan). The filtered supernatant was freeze-dried and stored at −20 ◦C until it was ready for use.

#### *2.3. Cell Culture*

Human breast cancer cells (MDA-MB-231) and human mononuclear cells (THP-1) were procured from the Korean Cell Line Bank (Seoul, Republic of Korea). Both cell lines were cultivated in a RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco Co., Grand Island, NY, USA), 2.05 mM L-glutamine, and 1% antibiotics (100 U/mL penicillin-100 μg/mL streptomycin). The cells were maintained in a controlled environment at 37 ◦C in a 5% CO2 incubator (Sanyo, Osaka, Japan).
