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  • br Since lactose is known to interact with galectin


    Since lactose is known to interact with galectin-3 and results have shown that 3CSF also bind to galectin-3, the effects of 3CSF in Kainic acid pre-viously treated with lactose were investigated to determine the exten-sion of galectin-3-mediated effects induced by 3CSF. As shown in Fig. 3B, the treatment with 3CSF in HCT116 cells previously exposed to lactose did not increase the inhibitory proliferative effect. On the other hand, 3CSF and pre-treated cells with lactose plus the additional inclu-sion of 3CSF treatment had a strong inhibitory effect on the proliferation
    Fig. 2. Inhibition of hemagglutination assay. The negative control contains just the erythrocytes; positive control contains the erythrocytes plus galectin-3; and lac (lactose) 4 μg was the minimum quantity that inhibits the hemagglutination. 1CSF: chelate-soluble fraction extracted from papaya with 1 day after harvest; 2CSF: chelate-soluble fraction extracted from papaya with 2 days after harvest; 3CSF: chelate -soluble fraction extracted from papaya with 3 days after harvest; 4CSF: chelate -soluble fraction extracted from papaya with 4 days after harvest; 5CSF: chelate -soluble fraction extracted from papaya with 5 days after harvest.
    of HT29 cells when compared with only lactose, indicating that this CSF fraction also induces galectin-3-independent effects. The differences be-tween the effects of 3CSF in HCT116 and HT29 cells seem to be related to different kinds of mutations in these colon cancer cell lines, as previ-ously reported [22,38]. Besides the differences in mutation types, HT29 has a mutated APC and wild-type β-catenin and HCT116 ex-presses wild-type APC and a mutant β-catenin [39]. Galectin-3 is a β-catenin important regulator of tumor metastasis [40] and the differ-ences in results presented herein could be derived of these distinct mu-tations and interaction with galectin-3 and β-catenin. Pectin inhibits β-catenin expression in the colon [41] possibly due to inhibition of galectin-3-β-catenin complex formation [40]. Somehow, in HT29 cell line the 3CSF probably could also interfere in β-catenin expression and reduce the cell viability. Further studies are needed to confirm these mechanisms.
    3.3. Structural characterization of CSF fractions indicates that 3CSF has lower molecular weight and more ramifications
    Fig. 4A shows that CSF fractions have relatively high molecular weight (between 710 and 1800 kDa – Void volume); however, the mo-lecular weight decreases along with ripening. Notably, 3CSF had an ad-ditional peak between 80 and 410 kDa, indicating an enrichment of lower molecular weight polysaccharides. To visualize changes in size, 3CSF was analyzed through AFM, and results were compared to that of 1 CSF, which is the CSF with the highest molecular weight. As shown in Fig. 4B, 3CSF was smaller than 1CSF.
    Compositional analysis revealed that CSF fractions were composed mainly of GalA, confirming that CSF are HG-rich fractions, with minor amounts of neutral sugars (Fig. 4C). The degree of esterification revealed that 1CSF had a smaller degree of esterification (33%) compared to 2-5CSF, which had a degree of esterification around 50% (Fig. 5D).
    FT-IR spectroscopy was used to calculate the degree of esterification and to identify specific wavenumbers to discriminate different polysac-charides functional groups. Polysaccharides are normally presented in 
    900 cm−1 being indicated by different types of neutral sugars [43]. A more detailed analysis of 3CSF through 1D 1H NMR and 2D 13 C\\1H HSQC and HMBC clarified its preponderant structure. 1D 1H spectrum (Fig. 5A) showed several anomeric signals. Attempts to iden-tify the different spin systems using 1H - 1H COSY and TOCSY spectra failed due to the high complexity of the spectra. We then employed 1H/13C HSQC (Fig. 5B), which showed a well-defined set of signals, eas-ily assigned by comparison with the chemical shifts reported for a