Swallow root (Decalepis hamiltonii) pectic oligosaccharide (SRO1) induces cancer cell death via modulation of galectin-3 and survivin

Carbohydrate Polymers 186 (2018) 402–410

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Swallow root (Decalepis hamiltonii) pectic oligosaccharide (SRO1) induces cancer cell death via modulation of galectin-3 and survivin S.E. Mallikarjuna, Shylaja M. Dharmesh

T

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Department of Biochemistry, CSIR-Central Food Technological Research Institute, Mysuru, 570 020, Karnataka, India

A R T I C L E I N F O

A B S T R A C T

Keywords: SRO1 SRPP B16F10 cells Galectin-3 Survivin Metastasis

Swallow root pectic oligosaccharide fraction (SRO1) from swallow root pectic polysaccharide (SRPP) possessed a molecular size of 831 Da. Structural analysis revealed that it is a rhamnogalacturonan I type, bearing arabinogalactan side chain with β-D-(1→4) galactose along with α-L-Araf (1→5)-α-L-Araf (1→3) structure on α-D-GalAOAc-(1→2)-α-L-Rha-(1→4)- linear backbone. β-D (1→4) linked galactose being the specific sugar for galectin-3, SRO1 had potentials in inhibiting galectin-3 mediated cancer progression. SRO1 inhibited galectin-3 mediated agglutination, in vitro, effectively with MIC of 1.08 μg/ mL and down regulated mRNA levels of galectin-3 (∼92%) along with its downstream key protein that inhibits apoptosis – survivin (∼78%) suggesting the capability of SRO1 in inhibiting galectin-3 mediated cancer promoting pathway. This is the first report, which highlights the inhibition of interplay of galectin-3 and survivin by a dietary pectic oligosaccharide.

1. Introduction Cancer remains as one of the major causes of mortality worldwide (Chen, Sun, Zhang, Liao, & Liao, 2017; Jin et al., 2013). Galectin-3, a carbohydrate binding protein (32 kDa) has been shown extensively both from our group and others, that it plays a significant role in accelerating the proliferation and metastasis along with inhibition of apoptosis, leading to “difficult-to-treat” condition in the affected patients (Balasubramanian et al., 2009; Takenaka, Fukumori & Raz, 2004). In addition to metastasis, chemoresistant nature of cancer cell is another important bull's eye that needs to be targeted for effective cancer management. Available literature suggests that survivin, a 16 kDa anti-apoptotic protein belonging to “Inhibitor of Apoptosis” (IAP) family, is a key molecule responsible for chemoresistance and is reported to be over expressed in tumour cells (Virrey, Guan, Li, Schönthal, Chen, & Hofman, 2008). Survivin functioning through TRAIL receptor (Tumour necrosis factor − related Apoptosis Inducing Ligand) is known to promote metastasis and angiogenesis; therefore, believed to encompass entire tumorigenesis process including proliferation, migration, invasion and thus collectively, facilitating metastasis (Garg, Suri, Gupta, Talwar & Dubey, 2016). One interesting observation by Cheong, Shin & Chun, (2010) revealed that the silencing of galectin-3, also silenced the activity of survivin. In fact it appear to be viable to target both galectin-3 and survivin as the signal transduction pathway leading to cancer with metastatic spread encompasses galectin-3 as a key initiator molecule,

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while survivin is in the downstream of the pathway (Cheong et al., 2010). In this perspective, we are addressing, in this paper, whether an inhibitor of galectin-3 can inhibit survivin and, if so, does it augment apoptosis. Our question is pertinent since several reports suggested that down regulation of galectin-3, increases cancel cell sensitivity to chemotherapeutic molecules suggesting that galectin-3 indeed can be a key target to overcome chemoresistance (Cheong et al., 2010; Lin et al., 2009; Niture & Refai, 2013). In this scenario, we utilized the previously identified galectin inhibitor from swallow root (Decalepis hamiltonii), a pectic polysaccharide (SRPP) which was the best, effective and safer with an MIC of 1.86 μg/ mL (Sathisha, Jayaram, Nayaka & Dharmesh, 2007) as opposed to standard galactose which had a MIC of 25 μg/ mL, for elucidation of its activity against survivin. Further, over a decades of research, we also established that low molecular weight oligosaccharides from intact pectic polysaccharides were more potent than their large molecular weight polysaccharides, probably due to the better bioavailability and also accessibility to galectin-3 molecule (Kapoor & Dharmesh, 2017). In the current study therefore, we envisage the 1. Isolation of a low molecular weight oligosaccharide (SRO1) from Swallow Root Pectic Polysaccharide (SRPP); 2 Comparative evaluation of galectin-3 inhibitory property of SRPP and swallow root oligosaccharide (SRO1); 3. Determination of sugar composition and structural backbone present in SRO1 to understand its structure-function relationship; 4. Effect of galectin-3 inhibitor on apoptosis and proliferation of cancer cells and; 5. Substantiation of mRNA levels as well as protein levels of galectin-3 and

Corresponding author. E-mail addresses: [email protected], [email protected] (S.M. Dharmesh).

https://doi.org/10.1016/j.carbpol.2018.01.053 Received 7 November 2017; Received in revised form 15 January 2018; Accepted 17 January 2018 Available online 31 January 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved.

Carbohydrate Polymers 186 (2018) 402–410

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system. SRO was resolved into 3 components – SRO1, SRO2 and SRO3. Active fraction SRO1 was re-chormatrographed on the same column under similar conditions and the fraction was found to be 99.5% pure. Molecular weight of SRO1 was estimated according to Kapoor & Dharmesh (2017) using ESI–MS (Alliance, Waters 2695) in a positive mode (100 V). All our further studies were performed with SRO1.

survivin in presence and absence of the active fraction of SRO – SRO1. Results of the study highlights the modulation of both galectin-3 and survivin in cells by galectin inhibitor and appear to be due to the structural motif present in SRO1. Study thus impinges on the signal transducing effect of galectin inhibitor on the key initiator molecule – galectin-3 and the downstream molecule survivin. Other studies in our laboratory have shown the effect of galectin- 3 inhibitor on apoptosis activating molecule such as caspases also (Jayaram, Kapoor & Dharmesh, 2015; Venkateshaiah, Eswaraiah, Annaiah & Dharmesh, 2017).

2.2.3. Estimation of total sugar and uronic acid Total sugar content of SRO1 was estimated by phenol-sulphuric acid method (Rao & Pattabiraman, 1989). To various aliquot of test molecules, 0.3 mL of 5% phenol and 2 mL of concentrated sulfuric acid was added. Absorbance was measured against a blank at 490 nm. Glucose was used as a reference standard. Uronic acid estimation was carried as described previously (Bitter & Muir, 1962). To different aliquot of test molecules, 3 mL of concentrated sulphuric acid was added. The mixture was boiled for 20 min and brought down to 25°C. Carbazol (0.1%) was added and the mixture was incubated in dark for 2 h. The absorbance was measured at 530 nm. The concentration of uronic acid in samples was estimated using galactouronic acid as reference standard.

2. Material and methods 2.1. Chemicals Dulbecco’s modified eagle’s medium (DMEM) (AL007A), antibiotic solution (A018), FBS (fetal bovine serum) (RM9955), skimmed milk powder (GRM-1254), MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl 2-H-tetrazolium bromide) (TC-191), were procured from Himedia, India (Cell Culture grade). 6-diaminidino-2-phenylindole (DAPI) (D9542), mercaptoethanol, protein molecular weight marker (D1992), trypsin, HPLC grade carbohydrate standards such as rhamnose, arabinose, xylose, mannose, galactose and glucose, protease, termamylase, glucoamylase etc., were purchased from Sigma Chemical Co, St. Louis, MO, USA. HRP conjugated-goat anti-mouse IgG secondary antibody (ab97023), anti-survivin mouse monoclonal (ab93274) and anti-galectin-3 monoclonal (ab2785) antibodies were procured from Abcam, UK. The other chemicals such as Triton X 100, SDS, Tween 20, TEMED, EDTA (ethylene diamine tetra acetic acid), hexane, ammonium oxalate, iodine solution, sodium phosphate buffer, glutaraldehyde, glycine, sodium chloride, amberlite IR 120H+ resin, sulphuric acid and solvents used were of the analytical grade purchased from Sisco Research Laboratories, Mumbai, India.

2.2.4. Sugar composition analysis by gas liquid chromatography (GLC) GLC was performed to analyse neutral sugar composition of SRO1 according to Thejaswini et al. (2013) and Sathisha et al. (2007). To 10 mg of sample, 10% sulphuric acid was added and subjected to acid hydrolysis by refluxing for 8–12 h. Neutralization and deionization of the hydrolysed sample was achieved by treating with solid barium carbonate and amberlite IR 120H+ resin, respectively. Alditol acetates were prepared and subjected for sugar composition analysis on RTX-1 column using a Shimadzu GLC (Kyoto, Japan) The flow rate was 40 mL/ min; the column temperature was maintained at 200°C and the injector temperature was 250°C (Sathisha et al., 2007). 2.2.5. FTIR and NMR analysis SRO1 was subjected to IR spectral study using a Perkin-Elmer spectrum 2000 spectrometer. Spectra was followed from 4000 to 400 cm−1 in an absorbance mode at a resolution of 4 cm−1. 10 mg of SRO1 dissolved in 600 μL deuterated water was subjected to 1H and 13C NMR analysis for its characterisation. The spectrum was recorded using Bruker AQS 400 MHz NMR spectrophotometer. 1H NMR spectrum was recorded at 500 MHz of 10,330 Hz spectral width containing water presaturation pulse program zgpr. 13C spectrum was recorded at 125 MHz with a spectral width of 28,985 Hz. Based on the signals of FTIR, NMR and sugar composition, tentative structure of SRO1 has been proposed. Molecular weight was calculated using Expasy glycan mass analysis software and compared with the mass obtained by ESI–MS.

2.2. Isolation and physicochemical characterization of SRO 2.2.1. Isolation of swallow root oligosaccharide (SRO) from swallow root polysaccharide (SRPP) SRPP was isolated according to Phatak, Chang & Brown (1988) using ammonium oxalate extraction method. Defatted samples (100 g) were subjected to protease, termamylase and glucoamylase treatment to degrade proteins, amylose and amylopectin, respectively by providing optimum reaction conditions for enzymes. The deproteinzed and destarched residue was subjected to extraction with 200 mL of 0.5% (w/v), ammonium oxalate solution, pH 3.5; boiled for 3 h at 70°C with occasional agitation. The content was filtered and the supernatant was subjected to precipitation with four volumes (v/v) of absolute ethanol at 4°C. After 3 h, the pellet was separated by centrifugation at 5000 g at room temperature for 20 min and was washed twice with 50 mL of ethanol. The pellet was resuspended in 10 mL of water and lyophilised to obtain Swallow Root Pectic Polysaccharide (SRPP). Oligosaccharides from SRPP- SRO was obtained using the protocol described previously from our laboratory (Kapoor & Dharmesh, 2016). Briefly, to 100 mg of SRPP, 10 mL of 2 M acetic acid was added and kept in a boiling water bath to hydrolyze the polysaccharide. Three volumes of ethanol (v/v) were added after 10 h of hydrolysis and stirred for 30 min at 4°C. The content was centrifuged at 4000g for 15 min and excess acid in the supernatant was removed by co-distilling with water. The content was dried completely; the residue obtained was resuspended in water and lyophilised to get Swallow Root pectic Oligosaccharides (SRO).

2.3. Evaluation of galectin-3 inhibitory, antiproliferative and apoptotic activity of SRO1 and SRPP 2.3.1. Galectin-3 inhibition assay The test molecules were evaluated for its galectin-3 inhibitory activity based on hemagglutination assay, according to the protocol described by Sathisha et al. (2007). Briefly, human erythrocytes were prepared from heparinised blood and were washed four times with five volumes of 0.15 M NaCl. A 4% erythrocyte suspension in 0.02 M PBS, pH 7.4 containing 1 mg/ mL trypsin was incubated for 1 h at 37°C. The trypsinized cells were washed with five volumes of 0.15 M NaCl and fixed in five volumes of 0.02 M PBS, pH 7.4 containing 1% glutaraldehyde for 1 h at room temperature. Termination of glutaraldehyde fixation was achieved by the addition of five volumes of 0.1 M glycine in PBS, pH 7.4 at 4°C. The fixed erythrocytes were employed for the hemagglutination assay. The assay was performed in a microtitre agglutination assay plate. The reaction mixture contained 150 μL of 4% erythrocyte suspension with or without serially diluted SRO1 and SRPP (50 μg). Minimum Inhibitory Concentration (MIC) of the test molecules required to inhibit the galectin mediated agglutination of RBCs was

2.2.2. HPLC analysis; determination of purity and molecular weight The purity of SRO was analysed using HPLC. 25 μL of 1 mg/ mL (in deionised water) was injected to ultra-hydrogel E-linear column (Waters 2695). Sample was eluted using deionised water at a flow rate of 1 mL/ min and elution was monitored using ELSD 2424 detector 403

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determined and was compared with standard galectin specific sugars − galactose and lactose to analyse the inhibitory potency of test molecules.

AACGCAAACA-3′;Galectin-3-R-5′-CTCTCAAAGGGGAAGGCTGA-3′;βactin-F-CCTTCCAGCAGATGTGG ATCA-3′; β-actin −R-AACGTAGCTTAG TAACAGTTC-3′] and isolated c-DNA (10 ng) as template in 20 μL reaction volume. The amplification parameters were as follows: initial denaturation at 95°C for 5 min prior to 44 thermal cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s, with final extension of 65°C for 5 min.

2.3.2. Cell culture B16F10 cells (mouse melanoma cells) were obtained from the National Center for Cell Sciences, Pune, India. The cells were maintained on Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS at 37°C in a CO2 incubator (5% CO2, 95% humidity) (Eppendoff, model – Galaxy 170 S).

2.3.6. Analysis of galectin-3 and survivin protein levels by western blot For Western blot analysis, exponentially growing (60–90% confluence) B16F10 cells were treated with SRO1, SRPP and dacarbazine (50 μg/ ml) for 48 h. Cells were harvested, washed with PBS and resuspended in 200 μL RIPA buffer containing protease inhibitor cocktail (Sigma Aldrich, USA). Cells were homogenized and then centrifuged at 14000g for 10 min at 4°C. The clear supernatant was used for the immuno blot analysis. The protein samples (50 μg) were subjected to SDS polyacrylamide gel (10%) separation according to Laemmli, (1970). The separated bands were transferred to PVDF membrane (pore size 0.22 μ, Pall life sciences) at 100 V for 2 h and thereafter blocked using skim milk (5%). Overnight primary antibody (1:1000 v/v) (Abcam, UK) treatment was done at 4°C against galectin-3 and survivin independently and β- actin was used as a loading control. The blots were washed with TBST to remove unbound primary antibody and incubated with the respective HRP-conjugated secondary antibody (1:2500 v/v) for 2 h at 25°C. After washing with TBST (to remove unbound secondary antibody) the blots were developed with enhanced chemiluminescence method by clarity western substrate from Biorad using Syngene gel doc system (GiBOX Chemi XT4).

2.3.3. Antiproliferative assay Antiproliferative activity of SRO1 and SRPP, on B16F10 cells, was monitored using MTT assay as described previously by Sathisha et al. (2007) with slight modification. Cells were seeded onto 96 well plate at a density of 2 × 104 cells/ well and was incubated at 37°C in a CO2 incubator (5% CO2, 95% humidity). After 12 h, growth media was replaced with media containing varying concentrations (20, 40 and 80 μg/ mL) of SRO1, SRPP and dacarbazine (positive control). The cells were incubated for another 48 h. After incubation, 15 μL of freshly prepared 5 mg/ mL MTT solution (5-3-(4, 5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) was added. After 4 h of incubation, media was removed without disturbing the formazan crystals. DMSO (100 μL) was added to each well and tapped gently to solubilise the blue formazan crystals, formed as a reduced product of MTT due to the activity of NADH oxidoreductase in living cells. Absorbance was recorded using the microtiter plate reader (model: Spectromax- SPX340, Shimadzu, Japan), after 20 min, at 570 nm. Results were expressed as percent cell viability.

2.4. Statistical analysis Data are expressed as the mean ± standard deviation (SD) from three independent experiments. All experimental analyses were compared with one-way analyses of variance (ANOVAs) using Graphpad prism software. Differences were considered significant at p < 0.05.

2.3.4. Apoptosis assay The ability of the pectic oligo/polysaccharides to induce apoptosis was evaluated by ethidium bromide and acridine orange dye method (Jayaram et al., 2015) along with observing characteristic features of cell under the microscope. B16F10 cells were seeded onto 96 well plate at density of 2 × 104 cells/well. After 12 h, cells were treated with 20, 40 and 80 μg/ mL concentrations of SRO1, SRPP and dacarbazine. After incubating for 48 h, media was removed and to the plate, 10 μL of dye (100 μg/ mL each ethidium bromide and acridine orange) was added to each well. The cells were observed under inverted microscope at 20X magnification and the picture was captured using CCD camera attached to the microscope. Dead and live cells were counted and percent death has been calculated and represented. DAPI (4, 6-diaminidino-2-phenylindole) was also performed to evaluate the apoptotic propriety of SRO1, SRPP and dacarbazine. DAPI staining was performed employing the protocol followed by Jayaram et al. (2015). Briefly, B16F10 cells were seeded onto 60 mm-dish (2 × 105cells/dish). After 12 h, the cells were treated with 80 μg/ mL of SRO1, SRPP and dacarbazine and incubated for another 48 h. Cells were harvested by subjecting to trypsin treatment. The harvested cells were washed thrice with PBS, pH 7.4 and stained with DAPI for 30 min at 37°C in the dark. Cells were observed using a fluorescence microscope with ultraviolet (UV) excitation at 300–500 nm.

3. Results 3.1. Isolation and physicochemical characterization of SRO1 3.1.1. Isolation of swallow root oligosaccharide (SRO1) using swallow root polysaccharide (SRPP) Extraction of the polysaccharide with hot water followed by deproteinization, destarchification and precipitation resulted in an enriched pectic polysaccharide fraction. The yield of the SRPP was found to be 6% and it was in accordance with the previous report by Sathisha et al. (2007). This fraction was further subjected to controlled hydrolysis to produce lower molecular weight oligosaccharides (SRO). The yield of SRO was 15% of the native polysaccharide (SRPP). Total Sugar content of SRO was analysed using phenol-sulphuric acid method and was found to be 910 mg/ g. HPLC analysis indicated the resolving of SRO into SRO1, SRO2 and SRO3 (Fig. 1A). However, SRO1 was the major peak and it constituted up to 99.52%. The molecular weight of SRO1 was found to be ∼ 831 Da (Fig. 1B). Since only SRO1 showed galectin-3 inhibitory activity, for all our future structure and functional studies, SRO1 was considered.

2.3.5. Evaluation of mRNA levels of galectin-3 and survivin using RT- PCR Exponentially growing (60–90% confluence) B16F10 cells were treated with SRO1, SRPP, and dacarbazine (50 μg/ ml) for 48 h. Cells were harvested, washed with PBS and re-suspended in 1 mL of Trizol (Sigma Aldrich, USA). RNA was extracted according to manufacturer’s protocol and quantified using a Nanodrop spectrophotometer (Thermo scientific, US). One μg of total RNA was converted to cDNA using verso select cDNA synthesis kit (Thermo scientific, US) according to manufacturer’s protocol. Real time PCR was performed in BIO-RAD CFX96 system (BioRad, USA) by using universal SYBR green master mix (BioRad, USA) and forward and reverse primers [Survivin-F-5′-TGCCCCGACGTTGCC-3′; Survivin-R5′-CAGTTCTTGAATGTAGAGATGCGGT-3′;Galectin-3-F-5′-AGTGAAACCC

3.1.2. Sugar composition of SRO1 The neutral sugar composition analysis, by gas chromatography, revealed that SRO1 is composed of rhamnose (21%), arabinose (53%), xylose (1%), Mannose (1%) and galactose (24%) (Table 1). Arabinose was the predominant neutral sugar followed by galactose and rhamnose. Increased rhamnose content in the SRO1 followed by higher levels of arabinose and galactose suggested the presence of hairy region of pectin. Xylose and mannose, which are present in smaller quantities, may have originated from cellulose and hemicelluloses. The uronic acid content was found to be 171 mg/ g in SRO1 (Table-1) suggesting ∼17%. 404

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Fig. 1. (A). HPLC chromatogram of SRO1. (B). Molecular weight of SRO1.

were assigned to H-2, H-3, H-4 and H-5 of α-(1,4)-linked D-GalA. The signals at 3.5 ppm and 1.8 ppm corresponded to H-4 and H-6 of rhamnopyranosyl units. These assignments suggested the presence of a type I arabinogalactan (AG-I) with unsubstituted rhamnose residues. Based on the signals of FTIR, NMR and molecular weight, characteristic structural features of SRO1 has been suggested to be a disaccharide unit of α-D-GalA-OAc (O-Acetyl) (a) −(1 → 2)-α-L-Rha- (1 → 4)-(b) and linear Gal residue of β1 → 4 (c) with α-L-Ara (1 → 3) (d) & α-L-Ara (1 → 5) (e) and depicted in Fig. 2D. The molecular mass of the proposed structure corresponds to ∼ 809 Da. The fact that we have observed 831 Da in ESI–MS, it is possible that remaining 22 Da has been contributed from interfering Na+ ion, which is quite common in mass spectral analysis.

Table 1 Sugar composition analysis and galectin-3 inhibitory activity of SRO1. Parameters

Content(s)/Activity

Total sugar (%) Uronic acid (mg/ g total sugar) Neutral sugars (Relative%) Rhamnose Arabinose Xylose Mannose Galactose Galectin-3 Inhibition Activity SRO1(MIC μg/mL) Galectin−3 Inhibitory Activity of SRPP (MIC μg/mL)a

91 171

a

21 53 1 1 24 1.08 1.86

Result of SRPP is already published (Sathisha et al., 2007).

3.2. Evaluation of galectin-3 inhibitory, antiproliferative, apoptotic activity of SRO1 and SRPP

3.1.3. Physicochemical characterization of SRO1 The structure of SRO1 was characterized using FTIR and NMR studies. The infrared spectra of extracted sample showed characteristic peaks of pectic oligosaccharide (Fig. 2A). The spectra revealed an intense peak in the region 3320 cm−1 indicating the presence of hydroxyl group. In case of pectin, absorption in the OeH region was due to inter and intra molecular H-bonding of galactouronic acid backbone. The absorption at 1636 cm−1 indeed provided evidence for the presence of uronic acid. The wavelengths- 1077 and 928.0 cm−1 represent the rhamnogalacturonan/ arabinogalacturonan and glycosidic linkage, respectively. The assignment of NMR signals for the SRO1 is summarized in Table 2. Respective 13C and 1H NMR chemical shifts are shown in Fig. 2B & C. 13C NMR spectrum indicated signals at 107.5, 81.3, 78.01, 85.4 and 68.2 ppm, which represents the alpha L-arabinose residue and signals at 99.05, 77.2, 74.2, 72.3, 71.9 and 175.4 could be attributed to C-1, C-4, C-5, C-3, C-2 and C-6, respectively of (1 → 4)-linked-D-galactopyranosyl units. Signal at 70–72 ppm attributed O-Ac group at the galactouronic acid. Proton signals at 3.69, 3.96, 4.70 and 4.74 ppm

SRO1 was tested for its anticancer potentials in comparison with its parent molecule SRPP and commercially available anticancer drug dacarbazine (DC). 3.2.1. Galectin- 3 inhibitory potentials Galectin-3 inhibitory activity of isolated SRPP and its controlled hydrolysed product, SRO1, was found to have MIC values of 1.86 μg/ mL (Sathisha et al., 2007) and 1.08 μg/ mL, respectively. SRO1 showed ∼ 42% better inhibition on galectin-3 mediated agglutination of red blood cells, compared to SRPP (Table 1) suggesting that low molecular weight oligosaccharide may display better galectin-3 inhibitory property than the large molecular weight polysaccharide as highlighted in our previous report (Kapoor & Dharmesh, 2017). 3.2.2. Antiproliferative assay Cells were treated with varying concentrations of SRPP and SRO1 to 405

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Fig. 2. (A) FTIR spectra of SRO1(B).

13C

NMR spectra of SRO1, (C). 1H NMR spectra of SRO1, (D). Tentative structure of SRO1.

Table 2 NMR assignments to SRO1. Residues

C1H1

C2H2

C3H3

C4H4

C5H5

C6H6

OAc

-4)-α-D-GalpA-(1- (a) 2)-α-L-Rhap-(1 (b) 4)-β-D-Galp-(1 (c) α-L-Araf-(1 (d)

99.05/4.74 – – 107.5/–

70.3/3.6 76.6/4.0 71.9/3.5 81.3/4.7

70.8/3.9 68.9/3.8 74.2/3.8 78.01/4.0

77.4 71.4/3.1 69.9/3.9 85.4/4.0

74.2/4.7 69.6/3.7 77.2/3.6 68.2/3.8

175.4 1.1 61.3/3.7 –

173.5 – – –

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the double stranded DNA. Acridine orange stains the nuclei red when ethidium bromide is bound whereas, it stains the nuclei green in absence of ethidium bromide. Therefore, the nuclei of dead cells appear red while that of viable cells appear as green. There was an evident decrease in green coloured cells, when the cells were treated with 80 μg/mL of SRPP, SRO1 and dacarbazine, independently. SRO1 was found to be better than SRPP. However, dacarbazine was more effective in causing cell death and preventing its proliferation (Fig. 4A & B). Cell disruption was evident in SRPP, SRO1 and dacarbazine treatment. Results are quantitated and depicted in Fig. 4B. The cause for reduction in the proliferation of cells as evidenced by MTT upon test samples treatment was also studied by DAPI staining. DAPI stained cells were visualized and photographed using fluorescence microscope equipped with camera. Cells without sample treatment had intact homologous nuclei and therefore, DAPI did not enter the nuclei. In cells treated with SRO1, SRPP and dacarbazine showed blue fluorescence of DAPI indicating that loss of intact nuclei. As seen in Fig. 4C, SRO1/ SRPP treated cells showed condensed nuclei and apoptotic bodies similar to that of dacarbazine.

Fig. 3. Antiproliferative activity of SRO1 and SRPP in comparison with dacarbazine in B16F10 cells.

analyse the antiproliferative property. As seen in Fig. 3 both SRO1 and SRPP showed antiproliferative activity in a dose dependent manner. SRO1 showed 34%, 50% and 62% inhibition on proliferation at 20 μg/ mL, 40 μg/ mL and 80 μg/ mL, respectively, while at similar concentration range, SRPP showed 12%, 25%, 30% (∼2 folds lesser), suggesting an increase in the antiproliferative activity of SRO1 than SRPP. The known anticancer drug, dacarbazine showed activity similar to that of SRO1 at similar doses (40%, 55% and 65% antiproliferative activity). Data thus revealed that SRO1 was significant (p < 0.05) in displaying antiproliferative activity compared to SRPP.

3.2.4. Expression levels of galectin-3 and survivin by RT-PCR Effect of SRPP, SRO1 and dacarbazine on galectin-3 and survivin expressions at gene level was studied in order to analyse antimetastatic effect and apoptosis inducing ability of the test molecules. In real time PCR experiments, it was observed that untreated cells showed higher expression of galectin-3 and survivin. Treatment of the cells with SRO1 and SRPP, showed a significant reduction (p < 0.05; 92% and 59%, respectively) in mRNA levels of galectin-3. SRO1 was found to be 36% better than dacarbazine (Fig. 5A). Survivin mRNA level was reduced by 78%, 52% and 55% in SRO1, SRPP and dacarbazine treated cells, respectively. SRO1 was better than positive control, dacarbazine, (23%) in down regulating survivin gene transcription (Fig. 5B). However, SRPP and dacarbazine were on par with each other (p < 0.05) in down regulating mRNA expression levels of both galectin-3 and survivin.

3.2.3. Effect of SRO1 and SRPP on apoptosis inducing ability Cell treated with samples were stained with ethidium bromide and acridine orange to qualitatively analyse the effect of samples on cell death. Morphologically, programmed cell death or apoptosis is generally characterized by a series of modifications such as chromatin and cytoplasm condensation. Ethidium may enter the nucleus upon nuclear membrane disruption, characteristic feature of apoptosis, and bind to

Fig. 4. (A). Acridine – orange and EtBr stained images of SRO1, SRPP and dacarbazine (DC) treated cells, (B). Calculated percent cell death, (C) DAPI stained images of SRO1, SRPP and dacarbazine (DC) treated cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5. Effect of SRO1, SRPP and dacarbazine on m-RNA and protein expression levels of galectin-3 and survivin. (A) m-RNA levels of galectin-3, (B) m-RNA levels of survivin,(C) Protein levels of galectin-3 and (D) Protein levels of survivin. *p < 0.05 compared to control. Bars that do not have a common superscript show p < 0.05. Bars with common superscript show p > 0.05. Shown is the representative image from three independent experiments.

Nangia-Makker (Nangia-Makker, Conklin, Hogan, & Raz, 2002), followed by our laboratory observations (Sathisha et al., 2007; Venkateshaiah et al., 2017). Data suggested that a dietary polysaccharide such as citrus pectin and swallow root pectic polysaccharide could inhibit metastasis effectively and this empowered the role of nutraceuticals in the arena of treatment for complicated disease like cancer. The supremacy of the dietary polysaccharide lies in its specific property to bind to galectin-3, which is a key molecule for triggering cascades of phenomenon that favour metastasis such as inhibition of apoptosis, angiogenesis, invasion and metastasis. Obviously binding of pectic component to galectin-3 inhibited the action of all these by silencing or inactivating the action of galectin-3, evidently by inhibiting the interaction of galectin-3 of tumour cells with their affinity ligands on normal cells or normal tissue epithelium (Nangia-Makker et al., 2002). Interestingly, galectin-3 mediated interactions between tumour cells and normal cells were found to be via carbohydrate binding domain of galectin-3 (Ahmed, Guha, Kaptan, & Bandyopadhyaya, 2011). The pectins being natural mimetics of galectin-3 ligand, via galactose residue, makes it as an eco-friendly binder to galectin-3, thereby inhibiting the action of galectin-3. In order to support this concept, it is mandatory to provide evidence for the structure, which can potentially bind to carbohydrate domain of galectin-3. In this perspective, structural analysis was undertaken and based on the characterization studies, a structural motif of SRO1 is presented.

Results have been validated by protein expression studies using western blot analysis. 3.2.5. Western blot analysis of galectin-3 and survivin Western blots for galectin-3/survivin protein expression corroborated with mRNA expression results. SRO1, SRPP and dacarbazine down regulated both galectin-3 (Fig. 5C) and survivin (Fig. 5D) levels by 62% / 56%; 42% / 58% and 69% / 60%, respectively when loaded at equal protein concentration of differentially treated cells extract. SRO1 was significantly better than SRPP (20%) (p < 0.05) and was on par with the dacarbazine in reducing galectin-3 levels in B16F10 cells. However, all the three test molecules exhibited similar level of effect on survivin levels (p < 0.05). Differential antiproliferative and apoptotic activity of dacarbazine could be attributed to its bioavailability, which needs to be elucidated. 4. Discussion Dietary pectic polysaccharides are known to have inhibitory potentials against several types of tumours and therefore, the utilization of these molecules in management of cancer is gaining great importance (Glinsky & Raz, 2009; Niture & Refai, 2013; Zhang, Xu & Zhang, 2015). The role of carbohydrates in inhibiting cancer cell growth in vitro (Sathisha et al., 2007) and tumours in vivo (Jayaram et al., 2015; Venkateshaiah et al., 2017) gained momentum for the first time by 408

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Scheme 1. Mechanism of modulation of Galectin-3 and Survivin by SRO1. A. Over expression of galectin-3 in cancer cell mediates phosphorylation of AKT (1), which prevents the cleavage of Bid (2) leading to blockade of loss of membrane potential (3) which in turn results in the loss of caspases activity. Over expression of galectin-3 resulted in overexpression of survivin also and upregulated survivin is known to inhibit caspases (4) and hence apoptosis (5). B. SRO1 treatment (6) resulted in down regulation of galectin-3 followed by survivin (8) and hence enhanced apoptosis of cancer cells (9).

of 0.25 μg/ mL than SRO1, which has an MIC of 1.08 μg/ mL. The increased activity of SrTPO1 could be due to the presence of additional galactose residues, which are specifically recognized by the carbohydrate binding domain of galectin-3. Careful attention must therefore be paid to the precise structure − function analysis between galectin-3 inhibitory oligosaccharide and galectin-3 inhibitory activity, particularly when we contemplate that low molecular weight pectic oligosaccharides function as better galectin-3 inhibitor than the large molecular weight pectins. Current observation has bearing on this concept and substantiates that modified pectins, which yields low molecular weight pectic oligosaccharide, display better functional ability (Sathisha et al., 2007; Harsha, Prakash & Dharmesh, 2016) than the large molecular weight pectins, although SrTPO1 with molecular size 1500 Da showed four fold (MIC 0.25 μg/mL) more activity than SRO1, which is 831 Da. Studies thus suggest that within the low molecular weight pectic oligosaccharide, galectin-3 inhibitory effect may vary, which in turn may have bearing on the precise sugar composition. Further exploration of additional bioefficacy and bioavailability of these oligosaccharide would enable to arrive at selection of required molecule of choice for effective management of cancer metastasis. Current study thus provides a hint to the lead structure for galectin-3 inhibitory domain, which has pertinence in its application. Further SRO1 exhibiting downregulation of survivin (Chemoresistance factor) boosts the efficacy of SRO1 in overcoming the survivin induced chemoresistance in addition to galectin-3 inhibitory property that necessitates metastasis inhibition. SRO1 and SRPP significantly reduced the proliferation of highly metastatic B16F10 cells and induced apoptosis and it is emphasized that it is via targeting galectin-3 that play a crucial role in enhanced proliferation and decreased apoptosis in cancer cells (Ferreira Cardoso, de Sousa Andrade, Bustos & Chammas, 2016). In this predicament, our observation that inhibition of galectin-3 function by SRO1 in the current paper and varieties of dietary polysaccharides in earlier investigations propel the role of these on the downstream molecule − survivin that dictates caspases activation to enable apoptosis (Sathisha et al., 2007). Survivin has been specifically selected since it has

Structure revealed that it is a short oligosaccharide with molecular weight of ∼ 831 Da with rhamnogalacturonan I structure with alternative units of L-rhamnose and O-acetylated derivative of D-galacturonic acid with α-1→2 linkage. The side chains initiating with β1→4 D galactose are attached to rhamnose residues. Arabinose branch appear on the galactose residue as a core of α-1→3 arabinosyl (a pentose in the furanose ring form) residues suggesting the presence of arabinogalactan type oligosaccharide. Besides, there were also other signals in the structural studies, suggesting the presence of α-1→5 arabinosyl – arabinose indicating the presence of more than 1 arabinose residue and is supported by sugar composition analysis. The core structure intriguingly is reported in citrus pectin, which is also one of the potent sources of galectin-3 inhibitor (Zhang, Lan et al., 2016a; Zhang, Zheng et al., 2016b). A pertinent question to debate here is, despite the same core structure, why the activity of SRO1 is ∼24 times better than citrus pectin. It is to be notified here that the reported citrus pectin has additional structures attached to it such as rhamnogalacturonan I type with not only arabinogalactan, but also linear galactan and arabinan stuructures (Yapo, 2011). This infact may have diluted the galectin-3 inhibitory property. Results were further substantiated by extensive work carried out in our laboratory, where citrus pectin was subjected to size reduction process via acid and or enzymatic treatment. Obtained fragments were isolated and evaluated for galectin-3 inhibitory potentials (Dharmesh, Patil, Mann & Kapoor, 2017). Few oligosaccharides were more potent than the parent molecule suggesting that probably, the cleavage of additional domains such as galactans and arabinans on the rhamnogalcturonan I type citrus pectin must have yielded the potency. (Dharmesh et al., 2017; Zhang, Lan et al., 2016a; Zhang, Zheng et al., 2016b) Further one of the recently reported galectin-3 inhibitory oligosaccharide (SrTPO1) from tomato (Kapoor & Dharmesh, 2017) also revealed the presence of similar domains that are found in SRO1, but with extended galactose residues at the branch and extended rhamnose − galacturonic residues also with the molecular weight of 1500 Da as opposed to 831 Da in SRO1. It is interesting to note that SrTPO1 was ∼4 fold better in galectin-3 inhibitory property with MIC 409

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monarch strength in dictating cell survival that further leads to chemoresistance in varieties of cancers including melanoma. Results are supported by our previous observations that inhibition of galectin-3 expression collated with down regulation of transcription factors such as NFķB and upregulation of caspases (Jayaram et al., 2015; Kapoor & Dharmesh, 2017; Venkateshaiah et al., 2017). Survivin is the smallest member of the Inhibitor of apoptosis (IAP) family of proteins, involved in inhibition of apoptosis and regulation of cell cycle. These functional attributes make survivin a unique protein exhibiting divergent functions i.e. regulating cell proliferation and cell death. Expression pattern of survivin is also distinctive; it is prominently expressed during embryonal development, absent in most normal, terminally differentiated tissues but upregulated in a variety of human cancers. Expression of survivin in tumours correlates with not only inhibition of apoptosis and a decreased rate of cell death, but also resistance to chemotherapy and aggressiveness of tumours (Garg et al., 2016). Cancer drug discovery scheme infact explored this prime molecular target; several inhibitors were designed. However, since survivin functions through multiple mechanisms using varieties of partners, earlier studies hinted that blocking of one pathway may not be sufficient to block the complete survivin signalling. Since galectin3–survivin interplay dictates the action of both survivin and galectin-3, a common inhibitor was contemplated. Results of the current study thus indicate the exemplary role of SRO1 as a double edged sword in inhibiting both galectin-3 as well as survivin as evidenced by gene expression analysis and depicted in Scheme 1. Nevertheless, it is imperative to address the question, whether the inhibition of galectin-3 by SRO1 is resulting in down regulation survivin or does it also execute direct effect on survivin, or both; this needs to be elucidated.

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