*3.6. Inhibition of MMP-2 and -9 Expression*

MMP-2 and MMP-9 are enzymes released by the metastatic cancer cell line MDA-MB-231 that tear down the extracellular matrix, a barrier surrounding the cell that allows tumor cells to migrate to neighboring organs [53]. We confirmed the suppression of MMP-2 or MMP-9 expression through Western blot and immunofluorescence assays after treating MDA-MB-231 cells and MDA-MB-231/THP-1 co-cultured cells for 48 h with 0.1 and 2 μg/mL of mistletoe extract (Figure 10a,b). As a result, when MDA-MB-231 and MDA-MB-231/THP-1 co-culture groups were treated with 0.1 g/mL mistletoe extract, no significant difference in MMP-2 protein expression was found. However, when 2 μg/mL mistletoe was treated, MMP-1 protein was expressed 0.9 ± 0.1 times and 0.4 ± 0.2 times less in the MDA-MB-231 and MDA-MB-231/THP-1 co-culture group, respectively, showing a significant difference in inhibiting the MMP-1 protein expression (Figure 10a). Similarly, when MDA-MB-231 and MDA-MB-231/THP-1 co-culture groups were treated with 0.1 g/mL mistletoe extract, no significant difference in MMP-9 protein expression was found. On the other hand, when 2 μg/mL mistletoe was treated, MMP-1 protein was expressed 0.7 ± 0.1 times and 0.4 ± 0.1 times less in the MDA-MB-231 and MDA-MB-231/THP-1 co-culture group, respectively, showing a significant difference in inhibiting the MMP-9 protein expression (Figure 10b). In addition, immunofluorescence revealed a significant inhibition of expression in the MBA-MB-231/THP-1 co-culture group upon treatment with 2 μg/mL mistletoe water extract (Figure 11).

**Figure 10.** Inhibitory effects of mistletoe water extract on MMP-2 and MMP-9 expression in (**a**) MDA-MB-231 and (**b**) MDA-MB-231/THP-1 co-cultured cells. Both groups were treated with mistletoe water extracts (0, 0.1, and 2 μg/mL) for 48 h, and their relative protein expression levels were quantified and presented using a bar graph. Each bar represents the mean ± standard deviation (S.D.) of experiments performed in triplicates. The *p*-value indicates significant differences between MDA-MB-231 and MDA-MB-231/THP-1 co-cultured cells (\*\*\* *p* < 0.001, \* *p* < 0.001, ns: not significant).

**Figure 11.** Representative images from immunofluorescence staining of (**a**) MMP-2 and (**b**) their quantitative bar graph. MDA-MB-231 and MDA-MB-231/THP-1 co-cultured cells were treated with various concentrations of Korean mistletoe water extracts (0, 0.1, and 2 μg/mL) for 48 h. The expressed proteins were labeled with Alexa Fluor™ 488 (green), while nuclear DNA was counterstained with DAPI (blue) then observed using a fluorescence microscope (magnification ×200). Fluorescence intensity was quantified using Image J software. Scale bars: 20 μm. The results are represented as the mean ± standard deviation (S.D.). The *p*-value indicates significant differences between MDA-MB-231 and MDA-MB-231/THP-1 co-cultured cells (\* *p* < 0.001, ns: not significant).

#### *3.7. Effect on EMT Marker Expression*

Through the regulation of related proteins, epithelial–mesenchymal transition (EMT) converts epithelial cells into an invasive and metastatic state. E-cadherin and N-cadherin are recognized as the markers for this transformation, where E-cadherin is a suppressor and N-cadherin an activator [54]. To evaluate whether mistletoe could inhibit the metastasis of breast cancer cells by modulating the expression of cadherin protein, 0.1 and 2 μg/mL of mistletoe extract was added to MDA-MB-231 cells and MDA-MB-231/THP-1 co-cultured cells and incubated for 48 h. As a result, E-cadherin, an EMT-inhibiting protein, was expressed 1.7 ± 0.2 times more than the control in MDA-MB-231/THP-1 co-cultured cell when treated with 0.1 μg/mL mistletoe extract; however, there was no significant difference in MDA-MB-231 cells. When 2 μg/mL of mistletoe was treated, E-cadherin protein was expressed 1.4 ± 0.1 times and 2.5 ± 0.3 times more than the control in the MDA-MB-231 and MDA-MB-231/THP-1 co-culture group, respectively, showing a significant difference between the two groups (Figure 12).

**Figure 12.** Effects of Korean mistletoe water extracts on regulating EMT markers in (**a**) MDA-MB-231 and (**b**) MDA-MB-231/THP-1 co-cultured cells using Western blot analysis. Both cell groups were treated with mistletoe water extracts (0, 0.1, and 2 μg/mL) for 48 h, and their relative protein expression levels were quantified and presented using a bar graph. The results are represented as the mean ± standard deviation (S.D.). The *p*-value indicates significant differences between MDA-MB-231 and MDA-MB-231/THP-1 co-cultured cells (\*\*\* *p* < 0.001, \*\* *p* < 0.01, \* *p* < 0.001).

In addition, the expression of N-cadherin, a protein that induces EMT, was confirmed via Western blot. When 0.1 μg/mL of mistletoe was treated, N-cadherin protein was expressed 1.3 ± 0.2 times and 0.9 ± 0.1 times more compared to the control in the MDA-MB-231 and MDA-MB-231/THP-1 co-culture group, respectively, showing a significant difference between the two groups. In the case of 2 μg/mL mistletoe treatment, N-cadherin protein was expressed 1.2 ± 0.1 times and 0.4 ± 0.1 times more compared to the control in the MDA-MB-231 and MDA-MB-231/THP-1 co-culture group, respectively, showing a significant difference between the two groups (Figure 12).

#### **4. Discussion**

Breast cancer is a major problem for women worldwide, accounting for around onequarter of all female cancer cases [39,55,56]. Breast cancer treatment often entails targeting particular receptors such as ER, PR, and HER2, inducing pharmacological reactions that result in cell death. Developing medications that successfully attack triple-negative breast cancer, on the other hand, provides a distinct difficulty. This subtype, which affects around 10% to 20% of patients, lacks the typical receptors ER, PR, and HER2 [16]. The only therapy presently approved by the Food and Medication Administration (FDA) in the United States is a medication that inhibits the expression of programmed death-ligand 1 (PD-L1) in triple-negative breast cancer [48]. To make things worse, triple-negative breast cancer is notorious for its high recurrence rates and disease spread to distant organs, both of which contribute considerably to death rates [40]. To prevent any further complications, the primary treatment for triple-negative breast cancer involves surgical surgery, radiation therapy, and non-specific chemotherapy [20]; however, these chemotherapeutic procedures—utilizing highly cytotoxic drugs—are associated with a wide range of side effects. These adverse reactions in turn diminish the quality of life among patients suffering from triple-negative breast cancer [18]. Therefore, discovering medications that precisely suppress triple-negative breast cancer without causing adverse effects is emerging.

Mistletoe, a medicinal plant widely distributed throughout Europe and Asia, serves as a complementary agent for anti-cancer drugs that increase apoptosis in cancer cells, decrease cancer mortality, and lessen drug adverse effects [1,3]. Recent studies have shown that mistletoe water extract promotes cell-mediated immunity to tumor cells by protecting monocytes DNA and stimulating immune cells such as macrophages as well as direct cytotoxicity to cancer cells [57]. The bone marrow produces monocytes, which then differentiate into macrophages; these macrophages undergo further differentiation, dependent on various environmental factors, which enable them to perform dual functions within the tumor microenvironment. In general, macrophages use cell-mediated immune responses to kill cancer cells, mediate phagocytosis, and cause vascular damage and necrosis of tumors; however, in a malignly established tumor microenvironment, cancer cells survive, proliferate angiogenesis, and their immune avoidance contributes to cancer progression and metastasis [58]. In recent immuno-cancer studies using macrophages, methods of manipulating macrophages to enhance anti-cancer activity or blocking the access of macrophages to tumors have been mainly proposed. This study confirms that mistletoe water extract holds potential as an immuno-cancer drug for treating triplenegative breast cancer by manipulating the function of macrophages.

When we treated mistletoe extract on MDA-MB-231 cells, a triple-negative breast cancer cell, for 48 h, the 50% inhibitory concentration (IC50) was 20 μg/mL. According to the plant screening system of the National Cancer Institute (NCI) in the United States, the crude extract of plants is considered to be cytotoxic in vitro when IC50 is less than 20 μg/mL after 24–72 h culture [59]. Our results are similar to those of Goda et al., who conducted prior studies on the cytotoxicity of mistletoe extracts in MDA-MB-231 cells [60]. Therefore, we considered mistletoe water extract as a potential cytotoxic drug for triple-negative breast cancer. Furthermore, when mistletoe extracts were treated on THP-1 cells (human mononuclear cell lines), our findings revealed a survival rate of more than 80% at dosages less than 2 μg/mL. Mishra et al. confirmed that the mistletoe water extract for THP-1 cells showed a survival rate of about 60% at 2 g/mL [61], which differed slightly from our findings. However, this might be because Mishra et al. employed *Viscum articulatum* and did not differentiate THP-1 cells into macrophages, which differs from our work.

Cancer cells secrete inflammatory mediators; these are a significant factor in tumor progression [32]. For instance, in the case of triple-negative breast cancer-secreting cytokines such as IL-4, IL-6, IFN-, and TGF-β, it is these that cause chronic inflammation within this specific microenvironment. The over-expression of such inflammatory cytokines results in accelerated metastasis, alongside neovascularization and cancer cell infiltration [62]. The inflammatory cytokine, IL-6, which plays a pivotal role in cancer progression through

the activation of the JAK2/STAT3 signaling pathway [48], has been found to associate STAT3-activated cancer cells with survival and differentiation, metastasis, and infiltration, as well as an increase in EMT [18]. This study's results revealed that when THP-1 cells (differentiated into macrophages) and MDA-MB-231/THP-1 co-cultured cells were treated with 0.1 and 2 μg/mL Korean mistletoe water extract, there was a significant reduction in IL-6 secretion compared to that seen in MDA-MB-231 cells. Additionally, we studied the JAK2/STAT3 signaling pathway using Western blotting. When we treated the MDA-MB-231/THP-1 co-cultured cells with mistletoe extract at a concentration of 2 μg/mL, it significantly reduced p-STAT3 protein expression. Choi et al. reported through their studies on complex plant extracts that they inhibit STAT3 activation within MDA-MB-231 cells; such inhibition further suppresses IL-6 production in cancer cells [17]. Based on this report, we can assume that Korean mistletoe extract actively reduces the secretion of IL-6 in MDA-MB231 cells by inhibiting the activation of STAT3 between tumor cells and macrophages. Faggioli et al. [63] observed an increase in IL-6 expression within MDA-MB231 cells via nuclear factor kappa (NF-kB)-light-chain enhancer pathway in parallel studies [63], and Suarez-Crevo et al. [64] suggested that the increase in p38, which is a mitogen-activated protein kinase (MAPK) signaling subgroup, and extracellular signal-regulated kinases (ERK1/2) mediated IL-6 expression in MDA-MB-231 cells [64]. However, since this study cannot confirm whether mistletoe water extract affects IL-6 expression in MDA-MB-231 cells by targeting molecules other than STAT3 in MDA-MB-231 cells, it is necessary to study other signaling proteins closely related to inflammatory cytokines. In addition, expressions of IL-4, TGF-β, and IFN-γ were qualitatively analyzed using the Multi-Analyte ELISArray kit (Qiagen) to find out what immune regulation Korean mistletoe extract is involved in between MDA-MB-231 and macrophages. As a result, the expression of the antiinflammatory cytokines IL-4 and TGF-β increased in the MDA-MB-231/THP-1 co-culture group compared to the MBA-MB-231 cell culture group. It was reported that TGF-β reacts with TGF-β receptor 1 to activate the formation of reactive oxygen specifications (ROS) in cells through the TGF-β/Smad pathway, which activates the suppressor of mothers again (Smad) protein, resulting in the activation of mitochondria, cytochrome c, caspase-9, and finally caspase-3 [65,66]. Since ELISArray confirmed that TGF-β increased expression in the MDA-MB-231/THP-1 co-culture group compared to the MDA-MB-231 cell culture group, we expected that apoptosis targeting mitochondria in triple-negative breast cancer cells would increase in the co-culture group. Also, it was reported that IFN-γ is involved when M0 macrophages are differentiated into M1 macrophages [67]. Based on these facts, we can assume that Korean mistletoe extract can differentiate M0 macrophages into M1 via initiating IFN-γ expression. However, a follow-up study is needed.

Aside from direct apoptosis, the mechanism of tumor cell death produced by the component of mistletoe water extract is linked to immune cell activation, namely natural killer cells (NK cells), lymphocytes, and macrophages [67–69]. Clinical investigations have indicated that immune cells triggered by mistletoe water extracts react directly or indirectly to tumor cells, inhibiting tumor cell proliferation and improving patient survival rates [12,70]. Therefore, we investigated whether the activation of human macrophage THP-1 by Korean mistletoe water extract could induce apoptosis in MDA-MB-231 cells through cell-mediated immunity. MDA-MB-231 cells and the MDA-MB-231/THP-1 coculture group were each exposed to Korean mistletoe water extracts at concentrations of 0.1 and 2 μg/mL for a duration of 48 h. It was observed that in the MDA-MB-231/THP-1 co-culture group at a concentration of 2 μg/mL, there was a significant augmentation in the Bax/Bcl-2 protein expression ratio compared to the MDA-MB-231 cell culture group, indicating a significant increase in the initiation of apoptosis.

Additionally, we hypothesized that mistletoe could release apoptosomes into the mitochondria's cytoplasm, through Bcl-2 family regulation, which leads to caspase-3 activation. Typically, caspase-3 exists in the cytoplasm in an inactive form, and when cleaved by caspase-9 or other proteases, it is activated when the active site is exposed [71]. To compare and analyze the degree of activation of caspase-3 in this study, the ratio of cleaved

caspase-3 was confirmed. When 2 μg/mL mistletoe extract was treated, the expression of cleaved caspase-3 increased significantly in the MDA-MB-231/THP-1 co-culture group. From this result, it can be said that mistletoe can regulate Bax and Bcl-2 proteins leading to an increase in MOMP, resulting in caspase-3 activation.

Apoptosis is caused when cleaved caspase-3 cuts and inactivates PARP, which aids in the repair of damaged DNA in tumor cells' nuclei. Therefore, increasing cleaved PARP induces apoptosis in tumor cells [72].

In this study, cleaved PARP expression increased in both the 0.1 and 2 μg/mL mistletoetreated MDA-MB-231/THP co-culture group. However, the expression of cleaved caspase-3 increased only in the 2 μg/mL mistletoe-treated MDA-MB-231/THP-1 co-culture group. There was a correlation between the increase in cleaved caspase-3 and cleaved PARP, but it was not proportional. This could be explained by a previous study indicating that cleaved caspase-3 enters the nucleus through active transport in the nuclear pore complex rather than through simple diffusion. Furthermore, active nuclear transport of cleaved caspase-3 is dependent on morphological changes in the nucleus caused by various apoptotic triggers and transport proteins that carry out active nuclear transport [73]. Also, Cui et al. reported that the cutting of PARP during apoptosis was performed by calpain, a calcium-dependent protease, rather than caspase-3, by inducing p53 genes and caspase-9 in breast cancer cell lines [74]. Based on the findings, it is possible to explain why there was an increase in cleaved PARP expression even though there was no significant rise in cleaved caspase-3 when mistletoe water extract at 0.1 g/mL was treated. The mechanism by which mistletoe extract induces apoptosis in MDA-MB-231 cells is likely to cleave PARP and trigger apoptosis through pathways other than caspase-3.

At each stage of metastasis, breast cancer epithelial cells are polarized into endothelial cell phenotypes through the progress of EMT, increasing mobility, and a loss of E-cadherin and an increase in N-cadherin are observed at the molecular level [75,76]. In addition, MMPs are involved in the process of cancer cells breaking down and invading the extracellular matrix. Among various types of MMPs, MMP-2 and MMP-9, with the activity of breaking down gelatin, are secreted to the cell surface and locally decompose the extracellular matrix that binds to the cell membrane, making the cell motile [77]. Because the findings imply that the mobility of cancer cells is connected to the level of MMP expression, investigations on cancer metastasis inhibition targeting MMPs are being conducted [78–80]. Therefore, in this study, when mistletoe water extract was treated on MDA-MB-231 cells and MDA-MB-231/THP-1 co-cultured cells, the difference in mobility inhibition between the two groups was compared via wound healing and Transwell migration assay. Also, to validate the suppression of metastasis of triple-negative breast cancer cells, the regulation of E-cadherin and N-cadherin, and the inhibition of MMP-2 and MMP-9 expression, were confirmed. Firstly, the MDA-MB-231 cell culture group and the MDA-MB-231/THP-1 coculture group were attached to a plate or membrane insert, and then the mobility inhibition of MDA-MB-231 cells was observed after they were treated with mistletoe extract. It was confirmed that the co-culture group significantly inhibited the movement to the wound area compared to the MDA-MB-231 cell culture group. The mobility of MDA-MB-231 attached to the insert membrane was inhibited by mistletoe extract, dose-dependently.

To compare the inhibition of metastasis between the two groups due to the regulation of cell movement-related protein expression at the molecular level, an EMT marker that regulates the mobility of cancer cells within the tumor microenvironment was observed through Western blot analysis. First, when the expression of E-cadherin, an EMT inhibitor, was observed, there was a significant increase in expression in the MDA-MB-231/THP-1 co-culture group compared to the MDA-MB-231 cell culture group. In addition, expression of the EMT product, N-cadherin, was considerably reduced in the co-culture group compared to the single culture group. Therefore, it was confirmed that mistletoe water extract can inhibit the EMT of MDA-MB-231 cells at the molecular level by activating THP-1 macrophages. Furthermore, the comparison of Western blot results between the two groups revealed a significant inhibition in the expression of MMP-2 and MMP-9. Following 48 h of

treatment with mistletoe water extract at a dosage of 2 μg/mL, both MMP-2 and MMP-9 were considerably suppressed in the MDA-MB-231/THP-1 co-culture group. Therefore, it was confirmed at the molecular level that mistletoe water extract can inhibit metastasis by inhibiting the stage of local infiltration of tissue by activating THP-1 macrophages and inhibiting the expression of MMP-2 and MMP-9 in MDA-MB-231 cells through cell-mediated immunity. The observed effects on EMT markers and MMPs provide valuable insights into the potential mechanisms underlying mistletoe water extract's anti-metastatic properties. These findings align with previous research indicating the anti-cancer properties of mistletoe extracts and highlight its potential as a therapeutic agent in the field of metastatic breast cancer [81].

According to the findings of this research, Korean mistletoe water extract inhibits triple-negative breast cancer cells by enhancing the human immune system via activating macrophages. In particular, we could confirm apoptosis which induced MOMP in triplenegative breast cancer cells through cell-mediated immunity of activated macrophages. In addition, we looked into the STAT3 pathway and also the regulation of cancer cell mobility by controlling numerous inflammation-related cytokines. However, further studies are needed to determine whether mistletoe can influence the immunity of breast cancer subtypes other than triple-negative breast cancer cells. We also need to determine how macrophages were activated and which component in mistletoe extract contributes to this activation. Lastly, the subject of whether mistletoe influences other immune cells in the human body, such as natural killer (NK) cells, remains unanswered, thus further study is needed.

**Author Contributions:** Conceptualization: C.-E.H. and S.-Y.L.; funding acquisition: S.-Y.L.; investigation: W.-T.L., C.-E.H. and S.-Y.L.; methodology: W.-T.L. and C.-E.H.; resources: W.-T.L. and C.-E.H.; supervision: S.-Y.L.; writing—original draft: W.-T.L.; writing—review and editing: S.-Y.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
