*2.4. Analysis for Apoptosis in BPL2-Treated Lung Cancer Cell Lines*

To evaluate whether treatment with BPL2 induced apoptosis, an Annexin V/PI staining assay was performed. After treatment with BPL2 at 20 μg/mL for 48 h, significant apoptosis induction was observed. The apoptosis rate increased to 17.1% (early: 0.1%, late: 17.0%) in A549, 9.5% (early: 1.5%, late: 7.0%) in H460, and 18.8% (early: 9.7%, late: 9.1%) in H1299 cells after treatment with BPL2. Necrotic cell death was observed at 2.7% in A549 and 0.5% in H460 and H1299 cells (Figure 7).

**Figure 7.** Analysis of apoptosis rates in BPL2-treated lung cancer cell lines. Lung cancer cells were incubated for 48 h with BPL2 and stained with Annexin V/PI for flow cytometric analysis. The upper left panel indicates necrotic cell death; the lower left panel indicates live cells; the upper right panel indicates late apoptosis, and the lower right panel indicates early apoptosis.

#### *2.5. Effect of Concomitant Treatment with Gefitinib and BPL2 on Cell Viability*

Concomitant treatment with gefitinib and BPL2 (10 μM and 10 μg/mL, respectively) showed a synergistic effect on the cancer cell lines compared with monotherapy. BPL2 or gefitinib monotherapy showed approximately 90% and 70% A549 cell viability, respectively, while concomitant therapy showed approximately 40% viability in the same cell line (Figure 8A). H460 cells treated with a combination of gefitinib-BPL2 did not show as much decrease in viability compared to A549 cells (Figure 8B). An increase in incubation time with the treatment agents led to a further 10% reduction in cell viability in both cell lines.

**Figure 8.** Concomitant treatment or monotherapy using gefitinib (10 μM) and BPL2 (10 μg/mL) on lung cancer cell lines. (**A**) A549, (**B**) H460. Con shows untreated cell lines. B+G shows cells treated with both agents.

#### **3. Discussion**

Similar to all other medicines, a lectin that is to be investigated for its therapeutic activity must be purified from its source [26]. Stepwise purification using two different affinity chromatographic techniques were successful in separating *Bryopsis plumosa* lectins. As previously reported [23,25], both GalNAc binding lectins (BPL1 and BPL3) were isolated by GalNAc affinity chromatography using a two-step elution method, and then the mannosebinding lectin (BPL2) was purified using D-mannose affinity chromatography [25]. The purity of the isolated lectins was sufficient to determine their effectiveness and mechanisms of action in cancer cell lines.

Several anticancer lectins from plants and animals have been reported in recent decades, such as galectin, C-type lectins, sialic acid binding, and Mistletoe lectin [14]. It is known that these lectins recognize carbohydrates on the cell surface and inhibit the survival of cancer cells by various mechanisms. A plant lectin has also been reported to affect apoptosis and autophagy by regulating a signal transduction pathway [14]. The specific binding of lectin to the cancer cell was well reported in targeting and imaging cancer cells. The alternation of cancer cell surface glycan was a well-known phenomenon and the carbohydrate recognition properties of lectin were often applied to cancer cell imaging [27]. Although the direct binding of BPL2 was not determined in this study, the binding of BPL2 on cancer cells could be assumed. The glycan binding specificity of BPL2 has been determined by hemagglutinating inhibition assay and it was specific to the α-methyl-D-mannose (Minimum inhibitory concentration, 3.9 mM), D-mannose (1.9 mM), L-fucose (7.8 mM), and D-glucose (125 mM) [25]. The abundance of high-mannose N-glycan or fucosylated on cancer cells has been reported [28,29]. Owing to the binding properties, lectins have been suggested as potential therapeutic agents that recognize the high-mannose N-glycans occurring at the membrane of various cancer cells [29]. Therefore, mannose-specific algal lectins such as Bryopsis lectin may have anti-cancer and anti-viral activity [29,30].

As expected, the D-mannose-specific lectin, BPL2, showed anticancer activity. However, GlcNAc- and GalNAc-binding lectins did not show any anticancer effects. It may be assumed that different lectins show specific cytotoxic effects against certain cancer cell

lines and that the latter two lectins could have anticancer activity in other cancer cells (e.g., cutaneous cancer). The specificity of lectins for distinct cancer cells is a well-known phenomenon. Tian et al. reported the binding affinity and specificity of 27 different lectins in four distinct colorectal cancer cell lines. In addition to the different interactions of 27 lectins with colorectal cancer cell lines, the same lectin displayed differences in four distinct cell lines [31].

The viability of lung cancer cell lines was greatly decreased after exposure to BPL2. The IC50 value was approximately 50 μg/mL for A549 and H1299 cells. This is quite a low concentration compared with that of the red alga *Kappaphycus striatus* lectin, KSL, which was reported to have an IC50 value in the range of 0.80–1.94 μM (0.22–0.54 mg/mL) [32]. Owing to the cytotoxicity of BPL2 against non-cancerous cell lines, the minimum concentration of BPL2 (<20 μg/mL) for other experiments was determined based on the cell viability results that did not show cell toxicity.

Although the mechanisms underlying the anticancer activity of BPL2 were unclear, the molecule clearly showed effective inhibition of cell growth in a colony-forming assay at a low concentration (20 μg/mL). The reduction in cell growth may have been mediated by binding to the surface carbohydrates of cancer cells and inducing cytotoxicity. Mannosebinding plant lectin from *Remusatia vivipara* exhibits a strong glycan-mediated cytotoxic effect and inhibits the growth and motility of human breast cancer cells [33]. Cancer cells often exhibit alterations in the cell surface of polysaccharides that act as tumor-associated antigens. Lectin recognizes altered cell surface carbohydrates and inhibits cell growth through several mechanisms, such as the reactive oxygen species-dependent pathway [34] and an apoptosis-inducing mechanism [35]. Owing to this, BPL2 may have mechanisms that are similar to those of other plant lectins.

Inhibition of cancer invasion and migration is a priority in cancer therapy because most cancer deaths are caused by metastasis [36]. BPL2 clearly inhibited the invasion and movement of cancer cells in all the tested lung cancer cell lines. In general, cancer cell invasion and migration are affected by several mechanisms. For example, a lectin from *Bandeiraea simplicifolia* seeds (BS-I) inhibited cancer cells, hepatocellular carcinoma, invasion, and migration, mediated by inhibiting the activation of the AKT/GSK-3β/β-catenin pathway [37]. AKT/GSK3β/β-catenin signaling contributes to cell migration and the EMT pathway [38], which affects EMT gene expression patterns. EMT is a program of cells that are vital for embryonic development, wound healing, and the malignant progression of cancer [39]. Three of the EMT marker genes, viz. zinc-finger E-box binding protein 1 (ZEB1), vimentin, and Twist, among the reported genes (i.e., ZEB1, Snail, and Twist) for the EMT marker [40] were selected, and the gene expression patterns after or without exposure to BPL2 were determined. All the analyzed genes in the three tested lung cancer cell lines were downregulated following treatment with BPL2, which corresponds to cell migration and invasion experiments. The protein expression at the same conditions correlated significantly with the corresponding gene expression level, although, for vimentin, the trend was unclear. Therefore, the gene expression could be assumed to reflect the corresponding protein levels.

ZEB1 is a well-known transcription factor that is upregulated in various tumor cell lines and is related to the invasion and migration of cells in patients with lung cancer [41]. It is also a critical regulator of cell plasticity, DNA damage, cancer cell differentiation, and metastasis [42]. BPL2 suppresses ZEB1 gene regulation in lung cancer cell lines and induces cell death. Signal transduction and activation of ZEB1 in EMT plays an important role in embryonic development and malignant progression. It is also associated with resistance to cancer therapies [43]. Suppression of ZEB1 gene expression decreases cancer angiogenesis while eliciting continuous cancer vascular normalization [44]. BPL2 diminished ZEB1 expression, and it could be assumed that it inhibits cancer cell migration via the same mechanisms.

Twist and snail, key transcription factors, are involved in the EMT pathway and play an essential role in cell migration, invasion, and metastasis [45,46]. Although a slight difference was observed among the tested cancer cell lines, downregulation of the Twist and snail genes was clearly defined. Therefore, we assumed that the anticancer ability of BPL2 was related to the inhibition of the EMT pathway.

Vimentin expression is affected by the downregulation of ZEB1, in turn constraining tumor migration [47,48]. The regulation of Twist is also associated with the expression of membrane proteins (N-cadherin, fibronectin, and vimentin) involved in cell adhesion in cancer cells [49]. Because of the downregulation of transcription factors ZEB1 and Twist, a reduction in vimentin expression after treatment with BPL2 was expected. The expression of vimentin was reduced in the tested cell lines, although different expression levels were observed in each tested cell line. The membrane protein vimentin is widely distributed in the fibroblasts, white blood cells, and vascular endothelial cells. It supports cell membranes and organelles, and a lack of vimentin induces cell migration. BPL2 appears to affect the transcription factor of EMT, disturbs vimentin expression, and ultimately inhibits cancer cell growth, invasion, and migration. Therefore, BPL2 appears to be a candidate inhibitor of the EMT pathway. However, the mechanisms of BPL2 in cancer cell lines are still unclear, whether it is directly or indirectly related; therefore, further comprehensive studies are required to understand the inhibition mechanisms.

The regulation of N-cadherin and E-cadherin is switched during EMT signaling by a complex network of signaling pathways and transcription factors. Downregulation of E-cadherin is often observed in malignant epithelial cancers and is accepted as a tumor suppressor. In contrast to E-cadherin, N-cadherin is downregulated in tumor cells [50]. Similar to the regulation of N-cadherin and E-cadherin in the inhibition of tumor cell lines, treatment with BPL2 led to the upregulation of E-cadherin and downregulation of N-cadherin. The results of the EMT pathway involving marker gene regulation following treatment with BPL2 were well aligned with the suppression of the EMT pathway in tumor cell lines.

Cell surface glycan alteration during the EMT process has been observed in various cancer models. It has been reported that modification of the glycan on the cell surface plays a pivotal role in metastasis [51].

The mannan-binding lectin in the reduction of EMT has been reported to be related to the calcium entry machinery [52]. BPL2 does not require a divalent ion for its activity [25]; therefore, it could be assumed that BPL2 is not associated with calcium channels. There are few reports on the involvement of lectin in the EMT pathway. Although BPL2 has not been confirmed to directly contribute to the suppression of the EMT pathway, it could be assumed to inhibit the migration of cancer cells by recognizing the cell surface glycan alternations (high-mannose N-glycan) on cancer cells with metastatic ability.

Gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), is a well-known drug used for the treatment of non-small cell lung cancer [53]. Concurrent treatment with anticancer agents to attain therapeutic success is accepted as a common regimen. Simultaneous treatment with BPL2 and gefitinib resulted in synergistic effects. We confirmed that the level of total EGFR expression decreased following BPL2 treatment. BPL2 decreased the activation of ERK and AKT in A549 and H460 cells, followed by the downregulation of cellular EGFR levels. Similarly, a study reported decreased expression of EGFR by lectin protein from *Pseudomonas fluorescens* in gastric cancer cells [54,55]. Consistently, BPL2 significantly reduced the expression of EGFR along with the activation of ERK and AKT, downstream of the EGFR signaling pathway in lung cancer cells. Similarly, the synergistic effect of a combination treatment of gefitinib and docetaxel in EGFR-TKI-sensitive cells has been reported [56].

*Polygonatum odoratum* lectin elicits apoptosis and autophagy in cancer cells. Apoptosis is induced by the Akt-NF-κB pathway in lung cancer cells [57], and the EGFR-mediated Ras-Raf-MEK-ERK pathway in breast cancer cells [55]. Similar to *Polygonatum odoratum* lectin, BPL2 treatment resulted in differential expression of EGFR and EMT pathway-related proteins. Based on the results, it could be concluded that BPL2 could induce apoptosis by similar mechanisms.

The main role of gefitinib is to inhibit tyrosine kinase, involved in cellular proliferation [58] and promotes apoptosis [59]. Based on the results of the Annexin V/PI staining assay, BPL2 was found to induce apoptosis rather than necrosis in lung cancer cell lines. Like BPL2, induction of apoptosis by lectin has been reported, like mistletoe lectin [14,60], a lectin from *Dioclea lasiocarpa* [61], and lectin from *Sophora flavescens* [62]. The synergetic effect may have led to the induction of apoptosis. The combination of mistletoe lectin with other compounds showed a synergistic anti-cancer effect in breast cancer cells [63].

The anticancer activity of BPL2 was determined in this study, and it was related to the inhibition of the EMT pathway and induction of apoptosis. Furthermore, concurrent treatment with another anticancer agent, gefitinib, showed a synergistic effect in two lung cancer cell lines (A549 and H460). Therefore, the mannose-binding lectin, BPL2, could be a good candidate for drug development in anticancer therapeutics.
