Appl Biochem Biotechnol DOI 10.

1007/s12010-012-9805-6

Animal Lectins: Potential Antitumor Therapeutic Targets in Apoptosis

Zhe Liu & Qian Zhang & Hao Peng & Wen-zhi Zhang

Received: 11 December 2011 / Accepted: 10 July 2012 # Springer Science+Business Media, LLC 2012

Abstract Lectins, a group of carbohydrate-binding proteins ubiquitously distributed into plants and animals, are well-known to have astonishing numerous links to human cancers. In this review, we present a brief outline of the representative animal lectins such as galectins, Ctype lectins, and annexins by targeting programmed cell death (or apoptosis) pathways, and also summarize these representative lectins as possible anti-cancer drug targets. Taken together, these inspiring findings would provide a comprehensive perspective for further elucidating the multifaceted roles of animal lectins in apoptosis pathways of cancer, which, in turn, may ultimately help us to exploit lectins for their therapeutic purposes in future drug discovery. Keywords Lectin . Cancer . Apoptosis . Galectin . C-type lectin . Annexin

Introduction Lectins, a group of highly diverse non-immune origin proteins ubiquitously distributed in plants, animals, and fungi, contain at least one non-catalytic domain which enables them to selectively recognize and reversibly bind to specific free sugars or glycans present on glycoproteins and glycolipids without altering the structure of carbohydrate [1]. Animal lectins (endogenous) have been drawing a rising attention for their multifaceted roles implicated in the extrinsic and intrinsic apoptotic pathways in cancer; therefore, they would be further used not only as biomarkers but as potential targets for cancer therapeutics [2]. Among these, galectin-1 (Fig. 1a), P-selectin (Fig. 1b), and annexin-1 (Fig. 1c) are most
Z. Liu : W.-z. Zhang (*) Department of Hepatobiliary Surgery, General Hospital of PLA, Beijing 100853, China e-mail: sn3064@163.com Q. Zhang School of Life Sciences, Sichuan University, Chengdu 610064, China H. Peng (*) BeiGene (Beijing) Co., Ltd, Beijing 102206, China e-mail: zhaoxu1986sc@163.com

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Fig. 1 The structures of three representative animal lectins. a Molecular structure of galectin-1; b molecular structure of P-selectin; c molecular structure of annexin-1

closely associated with some core apoptosis-related pathways as potential anti-cancer drug targets [3]. In this review, we present a brief outline of the representative animal lectins (e.g., galectins, C-type lectins, and annexins) as potential targets implicated in the apoptotic pathways in cancer. To sum up, these findings may provide a better understanding of the roles of animal lectins in apoptosis of human cancers, thus, helping us exploit diverse origin lectins as potential targets for future preclinical and clinical therapies of cancer (Table 1).

Galectins Galectins, a family of galactose-binding lectins known as Ca2+-independent S-type animal lectins, are widely distributed in many types of mammalian cells [4]. To date, there have been 15 kinds of galectins identified to possess a carbohydrate recognition domain (CRD) and bind to N-acetyllactos probably via recognizing the -gal unit. According to the number and arrangement of the CRDs, galectins are classified into three main types. First is the proto-type galectins that contain two non-covalent homodimers of CRDs which can crosslink ligands on cell surfaces and extracellular matrix [5], including galectins-1, -2,-5, -7, -10, -11, -13, and -14 [6]. Another is the chimera-type galectin that possesses a combined structure including a C-terminal CRD and tyrosine-rich N-terminal domain which help to construct higher order oligomers [7]. As far as we know, only galectins-3 has been classified into this group [8]. The tandem repeat galectins have two distinct CRDs and galectins-4, -6, 8, -9, and -12 [9] are in this group. Galectins bind -galactosides through evolutionarily conserved sequence elements of the carbohydrate recognition domain (CRD). Each galectin has an individual carbohydrate-binding preference in cytoplasm and nucleus, respectively. In the recent years, several reports have indicated that the correlations between galectins and tumor progression are remarkable, indicating that galectins can be used as potential biomarkers and even potential targets for cancer diagnosis and therapy [10]. Of note, galectins, located in the cytoplasm and nucleus, can secrete outside the cells and function extracellularly. Some galectins have been reported to bind to several cell surface antigens and receptors in a carbohydrate-dependent style. Moreover, galectins have been demonstrated to function inside cells in response to its carbohydrate-binding activities. As mentioned above, galectins may regulate cell signal transduction by binding intracellular ligands and participating in several apoptotic pathways in cancer.

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Table 1 Animal lectins as possible therapeutic antitumor targets Animal lectin Galectin-1 Cancer type Breast carcinoma, colon carcinoma, ovary cancer, bladder carcinomas, gliomas, endometrial adenocarcinoma, head, and neck cancers, prostate carcinoma Thyroid, central nervous system, head and neck squamous cell carcinoma, pancreas, bladder, stomach, and renal carcinoma, hepatocellular, ovary, breast, endometrium, skin, colon, prostate, salivary glands cancer Thyroid tumors, colon carcinoma Mechanism Contribute to cell migration, adhesion, angiogenesis, tumor immune escape, induce apoptosis Reference [18, 19]

Galectin-3

Involve in cell transformation and growth, inhibition of apoptosis, affect tumor invasion and metastasis

[20, 21]

Galectin-7

Correlate with keratinocyte differentiation, p53 stabilization, and apoptosis induction. increases lymphoma metastasis Inhibit cell adhesion, suppresses migration, modulate cell surface receptors, induce apoptosis Promote the proliferation, induce apoptosis

[22]

Galectin-8

Colon cancer

[23]

P-seletion

Colon carcinoma, breast cancer

[24]

NK-receptor

Renal cell carcinoma

Induce tumor apoptosis through TRAIL and Fas ligands (pre-clinical) An angiogenic factor that induces vascular endothelial growth factor production through the HIF-1 pathway Acts as tumor suppressor by negative regulating the RasRafMAPK signaling pathway

[25]

Annexin A3

Colorectal, prostate cancer

[26]

Annexin A6

Squamous epithelial carcinoma, malignant melanomas

[27]

Galectin-1 and galectin-3 Galectin-1 contains two non-covalent homodimers of CRDs cross-linking ligands on cell surfaces and extracellular matrix, and its crystal structure of human galectin-1 consists of a six-stranded and a five-stranded -sheet in an antiparallel arrangement [11]. Galectin-1 may favor tumor growth and lead to tumor progression and metastasis by influencing various biological processes such as cell migration, adhesion, angiogenesis, and apoptosis [12] since it can conjugate to H-Ras and promote its location to the plasma membrane which is a key procedure of tumor transformation [13], and thus resulting in sustained activation of Raf-1 and MAPK-1 that play the key promoters in apoptosis [14]. Moreover, galectin-1 expression has been examined in several malignant tumors including breast cancer, neuroblastoma, oral

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squamous cell carcinoma, and lung adenocarcinoma [1517]. Other examples of the roles of galectin-1 in tumor biology include modulation of tumor invasion in hepatocellular carcinoma cell lines [18]. Some reports have also shown that galectin-1 can promote proliferation and invasion of pancreatic cancer cells [19]. Galectin-3, the sole member of the chimera-type galectin which contains an N-terminal region composed of short amine acid segments and a C-terminal CRD, lacks a signal sequence required for secretion through some classical secretary pathways. Recent reports have shown that its translocation to the cytosolic side of the plasma membrane plays a key role in galectin-3 translocation [20], and galectin-3 binds to multivalent carbohybrate ligands on the cell surface, thus initiating various cellular signaling events and affecting various cellular functions [21]. In contrast to the extracellular cell death signal triggered by galectin1, the anti-apoptotic activity of galectin-3 might result from an intracellular function of this chimeric protein [22]. Moreover, numerous cytosolic molecules have been identified as galectin-3 ligands. The first molecular detected as a galectin-3 ligand was Bcl-2, a molecule involved in apoptosis [23], suggesting that Bcl-2 binds to galectin-3 through its CRD. Also, other apoptosis-related molecules have been reported to be novel galectin-3 binding ligands. In addition, CD95 (APO-1/Fas), a member of the death receptor family, was reported to interact with galectin-3 by co-immunoprecipitation and confocal microscopic analysis [24]. Nucling, a protein involved in apoptosis regulation, was identified as a novel galectin-3 binding molecule, and Alix/Aip1 was identified as a galectin-3-binding partner in jurkat cells [25]. Galectin-3 can affect K-Ras protein and Akt protein, suggesting that cytosolic galectin-3 is involved in regulation of cell proliferation, differentiation, survival, and apoptosis [26], while synexin was detected to interact with galectin-3 in human epithelial cells [27]. These findings reveal that mitochondria are new localization sites of galectin-3, indicating the intracellular function of galectin-3 in apoptosis. Moreover, galectin-3 can mediate endocytosis of 1 integrin (CD29) via a caveolae-like signaling pathway as well as endocytosis of AGE products. Galectin-3 can also bind to CD98 on the membrane and subsequently induce cell apoptosis through Ca2+ influx pathway [28] (Fig. 2). Additionally, a recent report has shown that Gal-3 is consistently overexpressed in pancreatic cancer as compared to both chronic pancreatitis and normal pancreas, and the overexpression of galectin-3 enhanced the resistance to apoptosis [29]. Other Galectins Galectin-7, a one-CRD galectin mainly found in stratified squamous epithelium, is an early transcriptional target of the tumor-suppressor protein p53 [30]. A recent report has shown that galectin-7 expression is upregulated after ultraviolet B irradiation of epidermal [31], resulting in p53 stabilization and apoptosis induction, which indicates that galectin-7 may participate in the pro-apoptotic effects of p53 on keratinocytes. Moreover, galectin-7 can promote JNK activation and mitochondrial cytochrome c release, a mitochondrial PCD pathway [32]. Moreover, recombinant galectin-8 can inhibit adhesion of human carcinoma 1,299 cells to plates coated with integrin ligands and thus inducing cancer cell apoptosis [33] (Fig. 2). Furthermore, by a cellular model of rheumatoid arthritis, some studies have shown that galectin-8 can induce apoptosis in synovial fluid cells, possibly by interacting specifically with the CD44vRA isoform of CD44 [34]. Galectin-9 is a two-CRD galectin with its three isoforms differing in the lengths having been identified. Recombinant galectin-9 induces apoptosis in thymocytes, in a dose-dependent and lactose-inhibitable manner [35]. The apoptosis component p38 is also an important mediator of galectin-9-induced dendritic cell maturation [36].

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Fig. 2 Animal lectins are involved in the apoptotic pathways. In this context of cancer, to inhibit apoptosis, galectin-1 can combine with H-Ras to activate Raf-1/MEK-1/ERK signaling pathway. Galectin-3 have three ways to activate caspase-3 to induce apoptosis including binding to Bcl-1 to activate caspase-9, binding to CD95/Fas to enhance caspase-8 and combining with CD98 under Ca2+ influx. Galectin-3 can also bind to KRas to enhance PI3K/Akt pathway to inhibit apoptosis. Moreover, galectin-7 as well as Alix/AIP1 can combine with JNK to promote caspase-9 and then caspase-3 to activate apoptosis. P-selectin either works on K-Ras or PI3K to inhibit apoptosis. In addition, Annexin can regulate several signaling pathways to inhibit apoptosis. Annexin A1 can activate IKK and then NF-B. Annexin A3 can promote HIF-1and Annexin A6 can activate Raf-1/MEK-1/ERK pathway

C-Type Lectins C-type lectin s, a superfamily of mammalian lectins mainly including selectin, DC-SIGN, and NK-receptors have been linked to various biological processes such as immune responses modulation, adhesion, proliferation, and apoptosis [37]. Selectin is the most important member of C-type lectin, including P-selectin, L-selectin, and E-selectin. Among them, P-selectin was reported to promote the proliferation of colon carcinoma through p38 and PI3K signaling pathways, suggesting that a complex made up of activated PI3K and p38 can result in activation of integrin dependent on P-selectin and then happen with P-selectinmediated cell spreading. Integrins have been shown to have a close relationship with cytoskeleton, which might play important roles as signal-transducting receptors, activating different biochemical pathways such as MAPK cascade, and thus regulating cell activation, proliferation, and apoptosis [38]. Additionally, other studies also revealed that PI3K and p38 signaling pathways activated by various stimuli played either a direct or an indirect role in tumor evolution. For instance, insulin-like growth factor could activate PI3K and p38 pathways in breast cancer migration [39]. Within this context, heregulin-mediated-PI3K and -p38 activation could facilitate the activation of the matrix metalloproteinase (MMP)-2 [33] and MMP-9 [37], as significant proteins in tumor invasion [38]. After its adhesion to colon carcinoma cells, E-selectin could promote tyrosine phosphorylation of various proteins including c-Src and p38 MAPK [39, 40].

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It is well-known that special location dooms DC-SIGN to involve in immune responses and modulate signaling pathways with counter-ligand. Recent studies have revealed that restraint of DC-SIGN by specific antibodies can contribute to ERK1/2 and Akt phosphorylation without concomitant p38 activation [41]. Furthermore, DC-SIGN co-precipitates with the tyrosine kinases Lyn and Syk, indicating that DC-SIGN acts as intracellular signaling molecules triggering activation of ERK1/2, Lyn, Syk, etc. [42]. Glycosylated ICAM-2 can recognize and bind to DC-SIGN, forming a DC-SIGN-ICAM-2 complex to initiate the maturation of DCs which can induce specific cytotoxic T lymphocyte-modulated antitumor immune response. Besides, PI3K activation mediates NF-B-induced dendritic cell maturation [43]. Of note, NK cells can directly induce tumor apoptosis via perforin granzyme pathway or death receptors such as TRAIL and Fas ligands which are produced on tumor cell surfaces [44]. Therefore, the sensitization of tumor target cells to NK cellmediated apoptosis can present a novel drug target for cancer therapy (Fig. 2).

Annexins The annexins are a superfamily of Ca2+-regulated phospholipid-binding proteins of 70 amino acid residues [45]. The annexins are composed of two main structural domains: the variable amino-terminus mediating interactions with protein ligands, as well as the conserved carboxyl terminus containing the calcium-binding and membrane-binding sites [46]. They have a diverse range of cellular functions including apoptosis, inflammation, and growth regulation [47]. Current changes in the expression of individual annexins, except for annexin A9 and annexin A13, have been observed in different types of cancer, implicating the changes in annexin expression and/or their subcellular localizations in tumor development and progression, especially tumor invasion and metastasis, as well as angiogenesis and drug resistance. Annexin A1 is a major substrate for epidermal growth factor receptor kinases and serine/ threonine kinases, and it is involved in a range of cellular signal transduction pathways, such as inflammation, cell differentiation, cell proliferation, and tumor invasion [48]. The increased expression of annexin A1 can correlate with the increasing tumor stage as well as the presence of metastases and poor survival [49], indicating its diagnostic value in certain types of cancer, such as hairy cell leukemia [50]. Moreover, annexin A1 has also been used as a potential antitumor drug by inhibiting the NF-B signal transduction pathway [51]. Cell surface annexin A2, a receptor for both proteases and extracellular matrix proteins, could overexpress in several types of cancer, indicating a role in suppressing tumor invasion and metastasis [52]. Paradoxically, it is also a receptor for angiostatin by blocking annexin A2dependent plasmin production, suggesting a possible way to develop new anti-angiogenic therapies [53, 54]. Accordingly, annexins can play multiple roles in tumor development and progression, which are relevant to tumor invasion and metastasis, as well as angiogenesis and apoptosis (Fig. 2).

Conclusions Hitherto, a bulk of updated data demonstrates that lectins from animals have been widely used as remarkable biomarkers and potential novel drug targets, respectively, in diagnosis, prognosis, and especially cancer therapy. Galectins, the most well-known animal lectins, have been gradually utilized not only as the key biomarkers for recognition of tumor

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initiation and progression from different stages, but as potential new targets that may play the pivotal roles for regulation of cancer-related signaling pathways for anti-neoplastic drug discovery. In addition, C-type lectin receptors (CLRs), known as pattern recognition receptors implicated in cancer, have been found that their complex mechanisms which glycosylation can be changed during inflammation, cancer progression, or apoptosis thus creating new opportunities of therapeutic intervention in the immune system. Thus, researching in galectins and C-type lectins might contribute significantly to the understanding of the causes and mechanisms of carcinogenesis; hence, the thrust in research ought to be focused on elucidating molecular mechanisms of actions of galectins and C-type lectins implicated in their interactions with other genes or proteins, focusing on the aim of providing leads to develop more new sophisticated tools, as well as to improve the currently available means of cancer detection and treatment. Our understanding of the multifaceted roles of lectins in cancer has benefited from the availability of all the above-mentioned data; however, there are significant disadvantages inherent in the complex molecular mechanisms of lectins, as well as a pressing need for more additional key information. Thus, further discoveries are being driven by an abundance of structural information on the potential drug targets; thereby, X-ray crystallography and nuclear magnetic resonance would be invaluable in the efforts to harness these lectins for cancer drug discovery. Therefore, the novel methods would help provide more novel insights into how these animal lectins could play the key roles as potential antitumor drugs or targets in cancer treatment. In summary, we have demonstrated a brief outline of representative animal lectins as potential targets in cancer therapy. Therefore, these inspiring findings would provide a comprehensive perspective for the key roles of lectins from animals in cancer, which in turn may ultimately help cancer biologists and clinicians to exploit them for their therapeutic purposes in future drug discovery.
Acknowledgments We are grateful to Miss Qian Liu (National University of Singapore) for her critical review on this manuscript. We also thank Miss Xin Wen (Sichuan University) for her good suggestion on this work. Additionally, this work was supported by the grants from the Eleventh Five-year Plan military special fund (no. 08BJ01), the Young teacher's fund of Sichuan University (no. 2010SCU11066) and the Science Foundation for Post Doctorate Research of China (no. 20110491725).

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