Annals of oncology : official journal of the European Society for Medical Oncology •
Previous clinical trials have not proved that adding epidermal growth factor receptor inhibitors to chemotherapy confers a survival benefit for patients with advanced biliary tract cancer (ABTC). Whether the KRAS mutation status of tumor cells confounded the results of past studies is unknown. ABTC patients stratified by KRAS status, Eastern Cooperative Oncology Group performance status, and primary tumor location were randomized 1 : 1 to receive GEMOX (800 mg/m(2) gemcitabine and 85 mg/m(2) oxaliplatin) or C-GEMOX (500 mg/m(2) cetuximab plus GEMOX) every 2 weeks. The primary end point was objective response rate (ORR). The study enrolled 122 patients between December 2010 and May 2012 (62 treated with C-GEMOX and 60 with GEMOX). Compared with GEMOX alone, C-GEMOX was associated with trend to better ORR (27% versus 15%; P = 0.12) and progression-free survival (PFS, 6.7 versus 4.1 months; P = 0.05), but not overall survival (OS, 10.6 versus 9.8 months; P = 0.91). KRAS mutations, which were detected in 36% of tumor samples, did not affect the trends of difference in ORR and PFS between C-GEMOX and GEMOX. The two treatment arms had similar adverse events, except that more patients had skin rashes, allergic reactions, and neutropenia in the C-GEMOX arm. Of patients with C-GEMOX, the presence of a grade 2 or 3 skin rash was associated with significantly better ORR, PFS, and OS. Addition of cetuximab did not significantly improve the ORR of GEMOX chemotherapy in ABTC, although a trend of PFS improvement was observed. The trend of improvement did not correlate with KRAS mutation status. This study is registered at ClinicalTrials.gov (NCT01267344). All patients gave written informed consent.
Cell-surface glycans are functional docking sites for tissue lectins such as the members of the galectin family. This interaction triggers a wide variety of responses; hence, there is a keen interest in defining its structural features. Toward this aim, we have used enzyme-linked lectinosorbent (ELLSA) and inhibition assays with the prototype rat galectin-5 and panels of free saccharides and glycoconjugates. Among 45 natural glycans tested for lectin binding, galectin-5 reacted best with glycoproteins (gps) presenting a high density of Galbeta1-3/4GlcNAc (I/II) and multiantennary N-glycans with II termini. Their reactivities, on a nanogram basis, were up to 4.3 x 10(2), 3.2 x 10(2), 2.5 x 10(2), and 1.7 x 10(4) times higher than monomeric Galbeta1-3/4GlcNAc (I/II), triantennary-II (Tri-II), and Gal, respectively. Galectin-5 also bound well to several blood group type B (Galalpha1-3Gal)- and A (GalNAcalpha1-3Gal)-containing gps. It reacted weakly or not at all with tumor-associated Tn (GalNAcalpha1-Ser/Thr) and sialylated gps. Among the mono-, di-, and oligosaccharides and mammalian glycoconjugates tested, blood group B-active II (Galalpha1-3Gal beta1-4GlcNAc), B-active IIbeta1-3L (Galalpha1-3Galbeta1-4GlcNAc beta1-3Galbeta1-4Glc), and Tri-II were the best. It is concluded that (1) Galbeta1-3/4GlcNAc and other Galbeta1-related oligosaccharides with alpha1-3 extensions are essential for binding, their polyvalent form in cellular glycoconjugates being a key recognition force for galectin-5; (2) the combining site of galectin-5 appears to be of a shallow-groove type sufficiently large to accommodate a substituted beta-galactoside, especially with alpha-anomeric extension at the non-reducing end (e.g., human blood group B-active II and B-active IIbeta1-3L); (3) the preference within beta-anomeric positioning is Galbeta1-4 > or = Galbeta1-3 > Galbeta1-6; and (4) hydrophobic interactions in the vicinity of the core galactose unit can enhance binding. These results are important for the systematic comparison of ligand selection in this family of adhesion/growth-regulatory effectors with potential for medical applications.
Ricin (RCA60) is a potent cytotoxic protein with lectin domains, contained in the seeds of the castor bean Ricinus communis. It is a potential biohazard. To corroborate the biological properties of ricin, it is essential to understand the recognition factors involved in the ricin-glycotope interaction. In previous reports, knowledge of the binding properties of ricin was limited to oligosugars and glycopeptides with different specificities. Here, recognition factors of the lectin domains in ricin were examined by enzyme-linked lectinosorbent (ELLSA) and inhibition assays, using mammalian Gal/GalNAc structural units and corresponding polyvalent forms. Except for blood group GalNAcalpha1-3Gal (A) active and Forssman (GalNAcalpha1-3GalNAc, F) disaccharides, ricin has a broad range of affinity for mammalian disaccharide structural units-Galbeta1-4Glcbeta1-(Lbeta), Galbeta1-4GlcNAc (II), Galbeta1-3GlcNAc (I), Galbeta1-3GalNAcalpha1-(Talpha), Galbeta1-3GalNAcbeta1-(Tbeta), Galalpha1-3Gal (B), Galalpha1-4Gal (E), GalNAcbeta1-3Gal (P), GalNAcalpha1-Ser/Thr (Tn) and GalNAcbeta1-4Gal (S). Among the polyvalent glycotopes tested, ricin reacted best with type II-containing glycoproteins (gps). It also reacted well with several T (Thomsen-Friedenreich), tumor-associated Tn and blood group Sd. (a+)-containing gps. Except for bird nest and Tamm-Horsfall gps (THGP), this lectin reacted weakly or not at all with ABH-blood type and sialylated gps. From the present and previous results, it can be concluded that: (i) the combining sites of these lectin domains should be a shallow-groove type, recognizing Galbeta1-4Glcbeta1- and Galbeta1-3(4)GlcNAcbeta- as the major binding site; (ii) its size may be as large as a tetrasaccharide and most complementary to lacto-N-tetraose (Galbeta1-3GlcNAc beta1-3Galbeta1-4Glc) and lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc); (iii) the polyvalency of glycotopes, in general, enhances binding; (iv) a hydrophobic interaction in the vicinity of the binding site for sugar accommodation, increases the affinity for Galbeta-. This study should assist in understanding the glyco-recognition factors involved in carbohydrate-toxin interactions in biological processes. The effect of the polyvalent P/S glycotopes on ricin binding should be examined. However, this is hampered by the lack of availability of suitable reagents.
Artocarpus integrifolia agglutinin (Jacalin) from the seeds of jack fruits has attracted considerable attention for its diverse biological activities and has been recognized as a Galbeta1-->3GalNAc (T) specific lectin. In previous studies, the information of its binding was limited to the inhibition results of monosaccharides and several T related disaccharides, but its interaction with other carbohydrate structural units occurring in natural glycans has not been characterized. For this reason, the binding profile of this lectin was studied by enzyme linked lectinosorbent assay (ELLSA) with our glycan/ligand collection. Among glycoproteins (gps) tested for binding, high density of multi-Galbeta1-->3GalNAcalpha1--> (mT(alpha)) and GalNAcalpha1-->Ser/Thr (mTn) containing gps reacted most avidly with Jacalin. As inhibitors expressed as nanograms yielding 50% inhibition, these mT(alpha) and mTn containing glycans were about 7.1 x 10(3), 4.0 x 10(5), and 7.8 x 10(5) times more potent than monomeric T(alpha), GalNAc, and Gal. Of the sugars tested and expressed as nanomoles for 50% inhibition, Tn containing peptides, T(alpha), and the human P blood group active disaccharide (P(alpha), GalNAcbeta1-->3Galalpha1-->) were the best and about 283 times more active than Gal. We conclude that the most potent ligands for this lectin are mTn, mT, and possibly P(alpha) glycotopes, while GalNAcbeta1-->4Galbeta1-->, GalNAcalpha1-->3Gal, GalNAcalpha1-->3GalNAc, and Galalpha1-->3Gal determinants were poor inhibitors. Thus, the overall binding profile of Jacalin can be defined in decreasing order as high density of mTn, and mT(alpha) >>> simple Tn cluster > monomeric T(alpha) > monomeric P(alpha) > monomeric Tn > monomeric T > GalNAc > Gal > Methylalpha1-->Man z.Gt; Man and Glc (inactive). Our finding should aid in the selection of this lectin for biological applications.
Glycoproteins (gps) contain many carbohydrate epitopes or crypto-glycotopes for Gal and GalNAc reactive lectins. They are present on the cell surface and function as receptors in various life processes. Many exist in soluble or gel form and serve as biological lubricants or as barriers against microbial invasion. During the past two decades, eleven mammalian structural units have been used to express the binding domain of applied lectins. They are: F, GalNAcalpha1-->3GalNAc; A, GalNAcalpha1-->3Gal; T, Galbeta1-->3GalNAc; I, Galbeta1-->3GlcNAc; II, Galbeta1--> 4GlcNAc; B, Galalpha1-->3Gal; E, Galalpha1-->4Gal; L, Galbeta1--> 4Glc; P, GalNAcbeta1-->3Gal; S, GalNAcbeta1-->4Gal and TN, GalNAcalpha1-->Ser(Thr). Except L and P, all of the units can be found in glycoproteins. TN, which is an important marker for breast/colon cancer and vaccine development, exists only in O-glycans. Natural TN gp, the simplest mammalian O-glycan, is exclusively expressed in the armadillo salivary gland. Antifreeze gp is composed of repeating units of T. Pneumococcus type XIV capsular polysaccharide has uniform II disaccharide as carbohydrate side chains. Asialo human alpha(1)-acid gp and asialo fetuin provide multi-antennary II structures. Human ovarian cyst gps, which belong to the complex type of glycoform, comprise most of the structural units. To facilitate the selection of lectins that could serve as structural probes, the carbohydrate binding properties of Gal/GalNAc reactive lectins have been classified according to their highest affinity for structural units and their binding profiles are expressed in decreasing order of reactivity. Hence, the binding relationship between glycoproteins and Gal/GalNAc specific lectins can be explored.
Expression of the binding properties of Gal/GalNAc specific lectins, based on the affinity of decreasing order of mammalian glycotopes (determinants) rather than monosaccharide inhibition pattern, is probably one of the best ways to express carbohydrate specifity and should facilitate the selection of lectins as structural probes for studying mammalian glycobiology. Eleven mammalian structural units have been selected to express the binding domain of applied lectins. They are: 1. F, GalNAcalpha1 --> 3GalNAc; 2. A, GalNAcalpha1 --> 3Gal; 3. T, Galbeta1 --> 3GalNAc; 4. I, Galbeta 1 --> 3GlcNAc; 5. II, Galbeta1 --> 4GlcNAc; 6. B, Galalpha1 --> 3Gal; 7. E, Galalpha1--> 4Gal; 8. L, Galbeta1 --> 4Glc; 9. P, GalNAcbeta1 --> 3Gal; 10. S, GalNAcbeta1 --> 4Gal and 11. Tn, GalNAcalpha1 --> 4Ser (Thr) of the peptide chain. Thus, the carbohydrate specificity of Gal/GalNAc reactive lectins can be divided into classes according to their highest affinity for the above disaccharides and/or Tn residue. Examples of the binding properties of these lectins can be demonstrated by Ricimus communis agglutinin (RCA1), grouped as II specific lectin and its binding property is II > I > B > T; Ahrus precatorius agglutinin (APA), classified as T and its carbohydrate specificity is T > I/II > E > B > Tn; Artocarpus integrifolia (jacalin, AIL), as T/Tn specific and its binding reactivity is T > Tn >> I (II) and Geodia cydonium (GCL), as F/A specific, and with affinity for F > Ah [GalNAcalpha1-->43(L(Fuc)alpha1-->2)Gal] >> I > L. Due to the multiple reactivity of lectins toward mammalian glycotopes, the possible existence of different combining sites or subsites in the same molecule has to be examined, and the differential binding properties of these combining sites (if any) have to be characterized. To establish the relationship among the amino acid sequences of the combining sites of plant lectins and mammalian glycotopes should be an important direction to be addressed in lectinology.
Aplysia gonad lectin (AGL), which has been shown to stimulate mitogenesis in human peripheral lymphocytes, to suppress tumor cells, and to induce neurite outgrowth and improve cell viability in cultured Aplysia neurons, exhibits a peculiar galacturonic acid/galactose specificity. The carbohydrate binding site of this lectin was characterized by enzyme-linked lectino-sorbent assay and by inhibition of AGL-glycan interactions. Examination of the lectin binding with 34 glycans revealed that it reacted strongly with the following glycoforms: most human blood group precursor (equivalent) glycoproteins (gps), two Galalpha1-->4Gal-containing gps, and two d-galacturonic acid (GalUA)-containing polysaccharides (pectins from apple and citrus fruits), but poorly with most human blood group A and H active and sialylated gps. Among the GalUA and mammalian saccharides tested for inhibition of AGL-glycan binding, GalUA mono- to trisaccharides were the most potent ones. They were 8.5 x 10(4) times more active than Gal and about 1.5 x 10(3) more active than the human blood group P(k) active disaccharide (E, Galalpha1-->4Gal). This disaccharide was 6, 28, and 120 times more efficient than Galbeta1-->3GlcNAc(I), Galbeta1-->3GalNAc(T), and Galbeta1--> 4GlcNAc (II), respectively, and 35 and 80 times more active than melibiose (Galalpha1-->6Glc) and human blood group B active disaccharide (Galalpha1-->3Gal), respectively, showing that the decreasing order of the lectin affinity toward alpha-anomers of Gal is alpha1-->4 > alpha1-->6 > alpha1-->3. From the data provided, the carbohydrate specificity of AGL can be defined as GalUAalpha1-->4 trisaccharides to mono GalUA > branched or cluster forms of E, I, and II monomeric E, I, and II, whereas GalNAc is inactive.
Bandeiraea (Griffonia) simplicifolia lectin-I, isolectin A(4)(GS I-A(4)), which is cytotoxic to the human colon cancer cell lines, is one of two lectin families derived from its seed extract. It contains only a homo-oligomer of subunit A, and is most specific for GalNAcalpha1-->. In order to elucidate the GS I-A(4)-glycoconjugate interactions in greater detail, the combining site of this lectin was further characterized by enzyme linked lectino-sorbent assay (ELLSA) and by inhibition of lectin-glycoprotein interactions. This study has demonstrated that the Tn-containing glycoproteins tested, consisting of mammalian salivary glycoproteins (armadillo, asialo-hamster sublingual, asialo-ovine, -bovine, and -porcine submandibular), are bound strongly by GS I-A(4.)Among monovalent inhibitors so far tested, p-NO2-phenylalphaGalNAc is the most potent, suggesting that hydrophobic forces are important in the interaction of this lectin. GS I-A(4)is able to accommodate the monosaccharide GalNAc at the nonreducing end of oligosaccharides. This suggests that the combining site of the lectin is a shallow cavity. Among oligosaccharides and monosaccharides tested as inhibitors of the binding of GS I-A(4), the hierarchy of potencies are: GalNAcalpha1-->3GalNAcbeta1-->3Galalpha1-->4Galbeta 1-->4Glc (Forssman pentasaccharide) > GalNAcalpha1-->3(LFucalpha1-->2)Gal (blood group A)()> GalNAc > Galalpha1-->4Gal > Galalpha1-->3Gal (blood group B-like)> Gal.
