Normalization of the vascular nets in pathological circumstances by using a safe strategy has consistently been addressed due to the regular non-selectivity of conventional therapies so that they damage both normal and abnormal cells [34, 35]. However, membrane glycosylation has been pointed to as a useful aim for identification and medication of abnormalities like neoplastic lesions [21, 22]. Cancerous cells exhibit peculiar membrane glycosylation arrangements, which differ according to the category of cancer and the tumor phase. The most common glycosylation alterations include the obstruction synthesis and the neo-synthesis of sugars, modified branching, and the presence of novel structures, sialylations, fucosylation, and the manifestation of Lewis X/A arrangements in glycosphingolipids as a cancer antigen. Also, the elevated appearance of cell surface N-glycans, the aberrant genesis of mucin, and abnormal appearance of galectins also organize the main alterations related to glycosylation that distinguish the dissimilarity between tumor and normal cells. These variations are functionally associated with cell movement, invasion, escape of the immune response, and metastasis [7]. Accordingly, it has widely been suggested that plant lectins are promising therapeutic agents targeting specific carbohydrate structures [16]. Although little is known, emerging evidence demonstrates that utilization of these biological molecules can be adapted for an alternative anti-angiogenic platform as well as a glyco-targeting approach [27, 36]. In this respect, Park et al., have illustrated that the inhibitory effect of the galactose- and N-acetyl galactose amin-specific agglutinin, a 60 kDa-lectin isolated from Viscum album, on tumor growth and metastasis is related to the programmed cell death and angiogenesis [37]. Also, Bhutia et al. suggested that Abrus agglutinin (AGG) is a potent molecule against the proliferative and angiogenic properties of human breast tumors with minimal toxicity to normal cells, expressing cancer-selective properties. AGG has a high specificity towards [gal (b 1–3) gal NAc]-containing structures and it has been shown to detach HUVECs from the matrix via Insulin growth factor binding protein-2 pathway [36]. In more recent years, chitin-specific lectins have been introduced as putative molecular probes for diverse biological aims [19]. As for the issue under discussion here, Singh et al. have studied the anti-cancer and anti-angiogenic activities of two chitotriose-specific lectins, BhL and DiL9, which have the same function from different structural characteristics. They optimized the effective inhibitory doses of these dietary lectins which show the cancer-exclusive impacts on human pancreatic tumor cells by inducing apoptotic death, whereas these lectins did not threat the viability of normal cells. Also, both BhL (homodimer, 34 kDa) and DiL9 (monomer, 9 kDa) were shown to disturb the HUVECs-induced tubular architectures at non-toxic doses [27]. Similarly, we showed that UDA has variable toxicity on different cells (Fig. 1). Particularly, this lectin could not to a large extent sensitize the proliferative characteristic of normal cells even at a high dose (about 0.5 mg/ml) for a long time of exposure as tested on several normal cell lines from different tissues. It is interesting to note that our studied human and mouse cancer cells were much more sensitive to the cyto-toxic effect of UDA even at low doses in a short period. Surprisingly, cyto-toxic effect of this lectin was very low and we assumed it to be neglectable on HUVECs and MCF-10A cells even at the highest dose for a long time. The results demonstrate that our investigated normal cells: HUVEC, MCF-10A, and HDF (partially) from human as well as L-929 from mouse have a similar non-responsive proliferative behavior with respect to the UDA treatments, suggesting the possibility of the presence of the same glycosylated status on their membrane for UDA binding. As an opinion from glyco-science, the observations that UDA had an inhibitory effect on cancer cells can be discussed by the fact that the dynamic status of their glycome may be related to the functional differences of these cells (the tissue origin, differentiation, and stage of development and their metabolic activity) that may affect glycomics-based drug response in vitro, and thus, researches in vivo help us to achieve more real knowledge. According to our previous report, we demonstrated that UDA can affect the vascularization process and integrity of vascular nets in chick chorioallantoic membrane as an angiogenic model [50]. However, given that the response of a cell type to UDA reflects the abundance of GlcNAc in its pattern of membrane glycosylation [38, 39], the cells with an elevated and/or re-programmed property such as cancer cells may have the glycome in favor of the sugar specificity of UDA This suggests a new glycomic probe also towards cancer and endothelial cells for angiogenesis inhibition that is needed to be tested in the future.
To apply UDA for carbohydrate-mediated targeting in angiogenesis-related therapeutics, we also mechanistically compared the growth inhibition of two types of cancer cells with the different expression levels of EGFR including U87 Glioblastoma and A431 carcinoma cells and HUVECs in the UDA exposure. The U87 is an EGFR-negative cell line [40] whereas A431 cells highly express this receptor [25] and endothelial cells intrinsically respond to EGF, an important pro-angiogenic mediator [41]. The UDA was shown to impede the growth of these cell types diversely. Remarkably different from HUVECs, both the EGFR-positive and EGFR-negative cancer cells were sensitive to UDA (A431: highly, U87: moderately, and HUVECs: non-sensitive). As a result, the anti-proliferative activity of UDA may not be contributed to the amount of EGF receptor, at least, on our cells. Based on former literature on the anti-cancer activity of UDA (the active constituent of the water extract from U. dioica rhizomes), this lectin has been reported to exert fifty percent of growth inhibition on A431 epidermoid carcinoma cells at 21 µg/ml by preventing the EGF from binding to its cognate receptor and such inhibitory point on human cervical epithelial cancer cells has been also calculated at 5 µg/ml of UDA by affecting the attachment of EGF/bFGF to HeLa cell line. Moreover, this interaction has been proposed to interpret the therapeutic role of UDA against the benign hyperplastic lesions in prostate tissue [25, 42, 43]. Besides, UDA has been described to be able to induce cyto-toxic and apoptotic impacts on human gastric adeno-carcinoma (AGS) cells at 20 µg/ml after 24 hours [38]. Generally, plant lectins have been shown to possess variable tumor-suppressive activities [36] and a new model for induction of programmed cell death by these lectins has suggested that UDA may trigger an apoptotic cascade via blocking EGFER [26]. More precisely, the exact interaction mode of UDA and GlcNAc-oligomers in crystal structures revealed that this chitin-binding lectin has two identical carbohydrate-recognition domains with different tendencies to bind GlcNAc residues in an individual chito-oligomer, A: the stronger and B: the weaker ligand-binding site, as illustrated by Saul et al [21]. This differential binding manner or dual binding affinity of UDA to its target molecule can also be contributable to the observed irregularities (dose and time-independencies) in the UDA-treated cells. On the other hand, carbohydrate-binding profiling of UDA has shown that this chitin-specific protein can recognize cell surface N-glycans containing oligo-mannose structures or high mannose-type N-glycans [19]. Also, EGFR test previously showed that UDA at 0.5 µg/ml was able to inhibit this receptor while other herbal lectins such as Concanavalin A (Con A), a mannose (Man)-specific lectin, and WGA, a dimeric tandem repeat-type lectin from Hevein family, did not exhibit this interaction with EGFR [25]. It is noteworthy that EGFR has a mannose-oligomeric side-chain conjugated to the amino acid at position 337 of its extra-cellular region, Immunoglobulin-like domain 3 involved in ligand binding [44, 45]. As a straightforward effect of glycosylation, this glyco-conjugated residue that may provide a regulatory structural feature conformationally affecting ligand binding and activation of EGFR probably makes this N-glycan suitable for serving as a candidate receptor for UDA. Consequently, the presence of the deregulated EGFR in normal cells like MCF-10A [46] may be the most probable reason that our studied normal cells were not vulnerable to UDA, and conversely, the up-regulated EGFR in malignant cells like A431 [25] may execute the high vulnerability of abnormal cells to this lectin. Amazingly, the sensitivity of the EGFR-negative brain tumor (U87) cells to UDA might be associated with other glycans bearing carbohydrates similar to UDA targets. However, UDA-EGFR interaction may not be the only main mechanism for the biological role of this herbal lectin. Yet, it remains unclear whether the state of low inhibitory effect on the proliferation process in normal cells is ubiquitously manifested by UDA or even other chitin-binding lectins, implying the potential safety for their applications. Although the previously reported chitin-binding lectins have been shown to display anti-proliferative activities on HUVECs and L-929 cells, GI-50 and GI-90 after 48 h > 130 µg/ml for BhL and 520 µg/ml for Dil9 (27), UDA was found to show different activities on these cells (Fig. 1). In Comparison to BhL and Dil9, UDA was non-toxic on HUVECs even at 480 µg/ml GI < 10 %) after 48 hours. Therefore, UDA can be supposed to be exclusively applicable against cancer in an optimized dose administration with restricted side effects. For instance, since the mouse 4T1 cells that mimic stage IV human breast tumor cells [47] were highly sensitive to UDA, contrary to the human normal breast (MCF-10A) cells, an investigation on UDA-treated breast tumors is now underway. According to these findings, it simply can be deduced that the cellular physiological and pathological actions, especially cancer progression and/or any step of angiogenesis, sensitive to a chitin-binding lectin, like UDA differentially present a particular glycosylation pattern as well as a kind of distribution of cell surface components conjugated with chito-oligomers or sugar arrangements favorable for chitin-binding proteins. Interestingly, Con A has been shown to have the GI-50 value at 25 µg/ml by inducing apoptosis in HUVECs [27]. These may send out the presence of notable structure-function distinctions of these lectins in behaving HUVECs. Furthermore, the results regarding the lack of such toxicity on the human endothelial cells obtained from this effort motivated our enthusiasm more to further assess the possible preventive influences of UDA on the other events of vascularization using in vitro models, providing additional information to support the utilization of chitin-binding lectins, like UDA, in a safe glycomics-based strategy against angiogenesis.
Currently, inhibition of endothelial cell adhesion and migration, and interference with ECM are the purposes of anti-angiogenic strategies [48]. According to the wound repair model, the movement of HUVECs was efficiently declined even at low doses of UDA. Thus, this lectin may inhibit an angiogenic event by affecting the migratory capacity of endothelial cells. This valuable non-toxic anti-migratory activity of UDA may discover a new potency of UDA for its antagonistic effect on cancer metastasis or other angiogenesis-related patho-physiological conditions. UDA was also demonstrated to prevent the motility of human EGFR-negative brain cancer (U87) cells, suggesting the EGFR-independent anti-migratory effect of UDA on these cells. Furthermore, fifty percent of the motility of endothelial cells was inhibited by UDA at 30 µg/ml (Fig. 2). As we experimented with the UDA concentrations on the HUVECs-generated tube-like structures in a three-dimensional cell culture model, this lectin was also shown to prevent the tube formation process in endothelial cells (Fig. 4). The results showed that this anti-migratory dose (30 µg/ml) of UDA was completely preventive for vessel sprouting. Meaningfully, this concentration affected the migration of U87 cells (Fig. 3). As a consequence, an optimized dose of UDA can be applied against cancer metastatic events, especially for the brain far from the limitations of the brain-blood barrier. In Comparison to UDA, other chitin-binding lectins also have anti-tubulogenesis activities as the HUVECs exposed to these lectins detached from the matrix, BhL at 8 µg/ml and Dil9 at 142 µg/ml [27]. Therefore, these lectins can prevent angiogenesis in a different range of doses. Also, the partial inhibition in migration of HUVECs at 7.5 µg/ml of UDA (Fig. 2) accompanied with partial anti-tubulogenesis at this concentration (Fig. 4) was the overlapping data denoting the presence of a migration-associated mechanism. The capability of cells to move during angiogenesis or chemotaxis is facilitated by the generation of the filopodia and lamellipodia at their leading edge. In general, integrins, Collagen receptors, and their related molecular pathways are involved in these structures [49]. Expression of integrin αVβ3, αVβ5, and α2β1 in HUVECs has been implicated in angiogenesis. The modulating role of integrin α2β1 in this process observed in vitro illustrates its involvement in supporting VEGF signaling and HUVEC migration. Studies from other researchers support the concept that integrin α2β1 contributes to the regulation of VEGF signaling. This integrin complex is closely associated with VEGFR-2 and EGFR, modulating the activation of these receptors during angiogenesis. Since these integrins perhaps reveal novel pharmacological targets, their inhibitors that simultaneously affect a growth factor signaling in a cross-talk can be used in combination therapy. For example, this inhibitory capacity can be seen in lectins such as C-type lectins. The mRNA expression level of integrin α2 is highly regulated in an angiogenic cross-talk related to VEGF. Also, VEGF-A is the major angiogenesis regulatory ligand for VEGF receptors, especially VEGFR-2, and induces neovascularization via interaction with endothelial cells [15]. Concentrating on this molecular mechanistic point of view, the quantitative expression analyses in this study showed that UDA could down-regulate the VEGF-A and integrin α2 mRNA levels, suggesting its anti-angiogenic role in balancing the regulatory loop between integrin α2 and VEGF likely through binding integrin α2-containing complexes. However, many cell surface N-glycans may have several binding sites for UDA targets. Therefore, because a decreasing trend in VEGFR-2 expression was also seen in UDA-treated HUVECs, UDA interactions with growth factor (co-) receptors such as VEGFR-2, which is involved in angiogenesis, are other possible mechanisms for its action against this process. It should not be forgotten that galectins are important glyco-modulators for growth or death factor receptors [10] and exogenous lectins, like UDA, may antagonize the regulatory role of galectins via competition for binding sugar residues on cell receptors. The details of such interactions and integrin α2 and VEGFR-2 putative binding sites for UDA, due to their unknown glycosylated structures, are still in their infancy and need further investigations. Taken together, UDA was reported to possibly hold promise for safe glyco-targeting of the processes related to angiogenesis due to its non-toxicity on endothelial and other normal cells.
As a common feature of plant lectins, UDA also perfectly prevented the proliferation and migration of cancer cells. This lectin can inhibit the migratory and tubulogenesis capacity of endothelial cells. Moreover, it could be concluded that this small lectin may have therapeutic potencies with a preventive manner towards membrane N-glycans expressed on both human endothelial and tumor cells. This is because cellular receptors like TCR and EGFR have been previously suggested to be putative targets for UDA. And, it is better to say that UDA prefers cell surface glyco-conjugates containing its favorite carbohydrate structures such as GlcNAc and/or, even with more affinity, Mannose-oligomers. The underlying anti-angiogenic mechanism for UDA may be through the downregulation of VEGF- integrin cross-talks engaged in a wide range of steps during endothelial tubulogenesis. Our results from the reliable experiments in vitro provide additional pharmacological data of the therapeutic efficacy of UDA, and it would be regarded as a new empowering insight to develop a novel anti-angiogenic drug by engineering chitin-binding lectins, like UDA. Hence, the selective and safe elimination of the abnormal cells without interfering with the integrity of the normal cells will be the fast track for success to cross out the risky strategies, for example, against the failures in the brain and eyes by using a glyco-targeting approach.