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Clin Exp Metastasis (2017) 34:141–154 DOI 10.1007/s10585-017-9836-z

RESEARCH PAPER

Antimetastatic pectic polysaccharide from Decalepis hamiltonii; galectin-3 inhibition and immune-modulation

Sathisha U. Venkateshaiah1 · Mallikarjuna S. Eswaraiah1 · Harish Nayaka M. Annaiah1 · Shylaja M. Dharmesh1 

Received: 1 December 2015 / Accepted: 16 January 2017 / Published online: 3 February 2017 © Springer Science+Business Media Dordrecht 2017

effective anti-metastatic effect of SRPP due to both galec-tin-3 blockade and immunomodulation.

Keywords B16F10-mouse melanoma cells · Galectin-3 · Metastasis · Swallow root pectic polysaccharide

AbbreviationsSRPP Swallow root pectic polysaccharideGRPP Ginger pectic polysaccharideCPP Citrus pectic polysaccharideMMPs Matrixmetalloproteinase

Introduction

Melanoma is one of the most prevalent malignant tumors in humans [1], and its frequency has increased significantly in both men and women. The frequency of occurrence is related to change in sun exposure and environmental lev-els of UV light followed by its unique character of exhibit-ing resistance to chemo/radiation therapy. Comprehensive analysis of melanomas revealed that melanoma subgroups developed by different mechanisms depending on the pat-terns of sun exposure [2]. Notably, melanomas on the skin with intermittent sun induced damage have shown a ten-dency of hyperpigmentation due to UV-activated tyrosi-nase enzyme levels, which subsequently enhances melanin production [2] and Reactive Oxygen Species (ROS) [3]. Increased melanogenesis and its association with immuno-suppression further enhance the severity of melanomas [4].

Melanin is a naturally occurring photoprotective pig-ment of skin and plays a crucial role in protecting keratino-cytes from the mutagenic effects of ultraviolet radiation (UVR) through its ability to filter UVR and as a scaven-ger of ROS [3]. The mechanisms that mediate melanoma

Abstract Melanoma is a malignant neoplasm of major concern because of its high mortality rate and failure of chemotherapy. Previously we have shown that galectin-3, a galactose specific lectin, plays a pivotal role in the initiation of metastasis. It was hypothesized that blocking galectin-3 with galactose rich dietary pectic polymer would inhibit metastasis. The current study analyzes the preventive effect and mode of action of a pectic polymer from Swallow Root (Decalepis hamiltonii) in a preventative study of B16F10 cells lung colonization. Matrix metalloproteinase (MMPs) activity was assayed by zymography. Apoptotic/prolifera-tive markers and cytokines were analyzed by immunoas-say. Results indicated ~88% inhibition of lung coloniza-tion by SRPP as compared to 60% by CPP and only 7% by GRPP. Further molecular analysis revealed that galectin-3 blockade was associated with down regulation of MMPs and NFκB. Activation of caspases supported the apop-totic effect of SRPP. Infiltration of inflammatory cells into the lung was evidenced by presence of CD11b+ cells and release of the pro-inflammatory cytokine-IL-17, indicating inflammation during the cancer cell colonization process. SRPP enhanced the release of IL-12 that enables the reduc-tion of inflammation. Our data for the first time indicate the

Sathisha U. Venkateshaiah and Mallikarjuna S. Eswaraiah are equal first authors.

Electronic supplementary material The online version of this article (doi:10.1007/s10585-017-9836-z) contains supplementary material, which is available to authorized users.

* Shylaja M. Dharmesh shylaja@cftri.res.in; cancerbiolab@gmail.com

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

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progression appear to be due to enhanced conversion of l-tyrosine to melanin by tyrosinase [2]. Recent studies have also revealed the role of melanin in immunosuppression [4].

Recent studies have revealed the involvement of altered levels of several molecules including tumor suppressor genes—p16 and p53, transcription factors, cell adhesion molecules and matrix metalloproteinases (MMPs) in the initiation and progression of malignant melanoma [5]. It is also reported that galectin-3, a protein member of lectins group specifically bind to β-galactosides [6], was expressed in higher levels in melanocytes during tumor progression [7]. Increased levels of galectin-3 was previously reported from our group in B16F10 melanoma cell lines [8] suggest-ing that galectin-3 plays a key role in metastatic melanomas similar to that observed in other malignant metastatic can-cers. Raz and Lotan [9] and our group reported the effective blockade of galectin-3 mediated events by galectin-inhib-itors in vitro; events include galectin-3 mediated cell–cell interaction, induction of apoptosis and invasion including some associated molecular changes [8, 10]. SRPP had been shown to offer significant protection in in vitro metastatic cell models against metastatic markers [8, 11]. In the cur-rent study, we evaluated the in  vivo efficacy of SRPP in B16F10 melanoma cell induced mouse lung metastasis model.

Materials and methods

Cell culture

B16F10 (mouse melanoma) cells were obtained from National Centre for Cell Sciences (NCCS), Pune, India; cultured with DMEM-high glucose (4.5  g/L) with 4  mM glutamine, and supplemented with 1.5 g/L sodium bicarbo-nate, penicillin (100 units/mL), streptomycin (0.1 mg/mL) and 10% fetal bovine serum (Himedia, Mumbai, India) at 37 °C in a humidified chamber with 95% air and 5% CO2. Cultured cell growth and survival was monitored by MTT assay [12].

Purification and characterization of SRPP

Swallow Root Pectic polysaccharide (SRPP) was prepared and purified on DEAE cellulose column as described previously [8]. Briefly, SRPP (0.5  g) was dissolved in 20  mL water and loaded on to DEAE-cellulose column (65 × 2.5 cm); the elution was carried out with water, fol-lowed by increased gradients of ammonium carbonate (0.05–0.2  M) and subsequently with sodium hydroxide (0.1–0.2  M) at a flow rate of 1 mL/min. Eight mL frac-tions were collected and assayed for total sugar by phenol

sulfuric acid method [13]. Carbohydrate positive fractions were pooled, dialyzed and lyophilized. Ammonium car-bonate fraction of concentration 0.15 M showed maximum galectin-3 mediated agglutination inhibitory activity and was termed as the ‘active fraction’ and was considered for further studies.

Gel permeation chromatography was performed on Sepharose CL-4B column (1.6 × 92 cm). The active pec-tic polysaccharide fractions (10  mg) were dissolved in 1 mL distilled water (w/v), centrifuged at 6000g for 10 min at room temperature (28 ± 2 °C) and the supernatant was loaded (1 mL) onto the column. The elution was carried out by using NaCl (0.1  M) containing sodium azide (0.05%) at a constant flow rate of 16 mL/h. Fractions (3 mL) were collected and analyzed for the presence of total sugar and appropriate fractions were pooled. Dextran series standards (T-40, T-70, T-150, T-500, T-2000) were used to determine molecular weight and blue dextran was used to determine void volume. A calibration curve was prepared by plot-ting Ve/V0 versus log molecular weight, where, Ve = elu-tion volume, V0 = void volume. To ascertain the purity of the 0.15 M fraction, 10 mg of this was dissolved in deion-ized water (1 mL) and loaded on to an E-linear and E-1000 column (4.0 × 250  cm) connected to a Shimadzu LC-10A HPLC system (Shimadzu Corp. Kyoto, Japan). Elution was performed with milli Q water as a mobile phase at room temperature at a flow rate of 0.8 mL/min and emergence of peaks was monitored using RI detector.

Effect of selected dietary pectic polysaccharides against B16F10 induced melanoma metastasis mice model

Swiss albino mice weighing 28–36  g were maintained under standard conditions of temperature, humidity and light. The animals were provided with standard rodent pel-let diet (Amruth feeds, Bangalore, India) and water ad libi-tum. The study was approved by the institutional ethi-cal committee, which follows the guidelines of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals, Reg. No. 49, 1999), Govern-ment of India, New Delhi, India. SRPP, CPP and GRPP were used for in vivo studies. GRPP (Ginger pectic poly-scahides) was isolated in the laboratory by employing the same protocol, which was followed for the SRPP isolation [8]. Citrus pectic polysaccharides (CPP) were procured commercially. Samples were given to animals through oral intubation.

Animals were divided into eight groups with eight ani-mals in each group (n = 8). Group I–VIII include healthy control (Group I); Metastasis Induced (Group II) where animals were injected with B16F10 cells (2 × 106); Group III–V were treated with 200  mg/kg body weight SRPP

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(Group III), CPP (Group IV) and GRPP (Group V), respec-tively for 21 days prior to injection of B16F10 cells via oral gavage daily; Groups VI, VII and VIII served as respec-tive sample controls for SRPP, CPP and GRPP where ani-mals received samples at similar doses as that of the Group III–V (200 mg/kg b.w.). Sample treatment via oral gavage continued for 21 days. Lung metastasis was induced by injecting B16F10 (2 × 106) cells through tail vein [14, 15]. After another 21 days, animals were sacrificed by cervical dislocation; the lungs were excised and the metastatic nod-ules were quantitatively evaluated by two observers using the models described below. Growth/cell invasion index and % implantation of tumor were calculated and compared with set groups. Lung/liver tissue and blood samples were collected for further biochemical analysis. Tissue homoge-nates and serum samples were prepared as described pre-viously [16] for toxicity and biochemical studies. A small portion of the lung tissue was fixed in 10% formalin in PBS for histological analysis.

Evaluation of tumor growth index, implantation/invasion and histopathology

Macroscopic and microscopic analyses were carried out to evaluate the induction/protection of metastasis [17]. The area of the metastasis within each lobule and their maxi-mum and minimum diameters were obtained by captur-ing the image at varied magnifications and the following parameters were calculated [18]: (1) percentage of implan-tation (area of metastasis per lobule/total area × 100); (2) growth index (mean area of metastasis/total area) and (3) invasion index (area of metastasis per lobule/mean area of metastasis).

Histological changes in different portions of the tissue sections were examined by microscopic observations at 40× magnification. Evidence for tumors in the lung tissue was analyzed by infiltration, localization of the tumor mass, tissue damage and by accumulation of melanin pigments. Infiltration was monitored by specific CD11b, IL17, IL12; reactivity using respective monoclonal/polyclonal antibod-ies [19].

Determination of tumor markers and oxidant/antioxidant status in serum/lung homogenate

Galectin-3 levels in the serum samples were estimated employing the monoclonal anti-galectin-3 based ELISA as described previously [20]. Serum samples were analyzed for total protein, antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and antioxidant—glu-tathione (GSH) by employing the protocols as described in our earlier paper [16]. MMP activity was analyzed in lung

homogenates by zymographic method and monoclonal antibody based ELISA [20, 21].

Preparation of lung homogenate for western blot analysis

Lung tissues were collected from experimental animal groups, after dissecting the mice, washed with PBS and homogenized in ice-cold lysis buffer (50  mM Tris–HCl, 150 mM Nacl, 1 mM EGTA, 1 mM EDTA, 1 mM PMSF, 100  mM Na3VO4 and protease inhibitor cocktail (pH 7.4) using a homogenizer for 30  min and centrifuged at 10,000g  for 20  min at 4 °C. The supernatant was used to study expression levels of proteins (Galectin-3, Beta-actin) by western blot analysis. Briefly, aliquots of equal amounts of protein (50  μg) from lysates were subjected to 10% SDS–PAGE electrophoresis. Thereafter, proteins were elec-trophoretically transferred to nitrocellulose membranes and nonspecific sites were blocked with blocking buffer [5% non-fat dry milk and 1% tween 20 in 20  mM Tris buffer saline (TBS), pH 7.5] by incubating for 1 h at room tem-perature. The membranes were then probed overnight with the galectin-3 monoclonal primary antibody at 4 °C. After washing with TBS with 1% tween 20 (TBST), membranes were incubated with the horseradish peroxidase–conjugated goat-anti-mouse IgG secondary antibody at (1:5000 v/v) for 1 h at room temperature. The unbound secondary anti-bodies were removed by washing and protein expression was detected by enhanced chemiluminescence detection system as per manufacturer’s protocol (PIERCE Biotech, USA) and autoradiography with X ray film (Kodak).

Effect of SRPP on proliferation, apoptosis and adhesion of B16F10 cells

B16F10 cells (2 × 104 cells/mL) were cultured with the specified medium in a 96 well microplate and after 24, 48 and 72 h. 15 µL of MTT solution (5 mg/mL) was added and incubated at 37 °C for 2 h. 100 µL of DMSO was added to dissolve the dark blue crystals and absorption of formazan solution was measured at 570  nm in a microplate reader (Spectra Max-340, Molecular Devices, Germany). Apopto-sis assay was performed using ethidium bromide and acrid-ine orange dye method [22] as well as observing character-istic features of cells by microscopy. For adhesion assay, 24 well plates were coated with matrigel (5 mg/well) overnight at 4 °C. Wells were washed three times with PBS, blocked with 1% BSA (in PBS) for 2  h, and washed with PBS. B16F10 melanoma cells were resuspended in DMEM con-taining 0.1% BSA and added at 1 × 104 cells per well, pre-incubated with 50 and 100 μg/mL of SRPP/CPP, at 37 °C for 10 min. Pre-incubated cells were added to coated wells followed by incubation for 1 h at 37 °C. Un adhered cells

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were removed from wells by washing with PBS. Bound cells were detached from wells using trypsin–EDTA and counted [23]. MMP activity was analyzed in lung homoge-nates by enzymographic method [21].

Modulation of cancer specific markers by SRPP

Melanin content in lung

Melanin content was estimated using the modified method of Hosoi et  al. [24]. Briefly, to 100  μL of lung tissue homogenate, 200 μL of 1 N NaOH containing 10% DMSO was added, heated at 80 °C for 1 h and cooled; the amount of melanin was determined spectrophotometrically by recording the absorbance at 475 nm.

Caspase‑3, NFκB and p53

Comparative evaluation of levels of caspases (enzymes required for apoptotic cell death), NFκB (a transcriptional factor) and p53 (a tumor suppressor gene product) was carried out in lung homogenates. Monoclonal antibodies (MAbs) specific to caspase-3, NFκB-p65 and p53 were used for the analysis by ELISA [8]. Primary antibodies at 1:1000 (v/v) dilution and secondary antibody (alkaline phosphatase conjugated rabbit anti mouse IgG) at 1:5000 (v/v) were used followed by p-nitrophenyl phosphate-PNPP (1 mg/mL) as substrate. The absorbance was measured at 405 nm in a microplate ELISA reader (Molecular Devices, Spectramax 340, and Germany).

Statistical analysis

All results were expressed as mean ± standard deviation (n = 8). The analyses utilized student’s t test to test each treatment group mean against control mean. A value of p < 0.05, p < 0.01, p < 0.001 was considered significant, very significant, and highly significant, respectively.

Results

Purification and characterization of dietary pectic polysaccharides

The yield of swallow root and ginger pectins were around 4 and 0.6%, respectively on dry weight basis. Since SRPP showed potent activity, it was further fractionated on DEAE cellulose ion exchange chromatography. SRPP was fractionated into four fractions namely, 0.05, 0.10, 0.15 and 0.20  M ammonium carbonate eluted fractions (Fig.  1a). Active fraction of SRPP was eluted by 0.15  M ammo-nium carbonate on DEAE cellulose column, had sugar

composition of rhamnose (7): arabinose (66): galactose (27) (w/w) [8]. GRPP [25] did not show activity even at >600  µg/mL. Commercially available citrus pectin (CPP) gave moderate activity (MIC value-25  µg/mL). The per-centage yield of 0.15 M fraction was higher (62%) followed by neutral (17%), 0.10 M (15%) and 0.05 M (4%) fractions. Approximately 15-fold higher yield and sixfold higher activity was observed in 0.15 M fraction when compared to that of 0.05 M fraction [8]. Molecular weight of the ‘active pectic polysaccharide’ eluted with 0.15 M ammonium car-bonate was found to be 250 kDa (Fig.  1d). Since 0.15 M fraction showed better activity compared to other fractions, the purity of this fraction was examined. Figure 1b, c shows homogeneity of active SRPP fraction.

SRPP inhibited lung metastasis in experimental animal model effectively than CPP; GRPP had no significant protection

Injection of B16F10 cells through the lateral tail vein resulted in the formation of metastatic nodules in the lungs (Fig.  2a, b). Significant reduction in tumor colonies and their growth were observed in the lung of SRPP and CPP (Fig. 2a, c, d) treated mice. On the contrary, GRPP treated animals did not show any significant change (Fig.  2a, e), when compared with metastasis induced (MI) group. Lungs of SRPP, CPP and GRPP (Group-VI–VIII) control groups of animals were without any lesions and were similar to that of healthy controls.

Figure 3a shows superficial metastatic nodules. The MI group, with only B16F10 cells, showed metastatic nod-ules between 176 and 438, which was randomly distrib-uted over the lung surface with a mean of 328.17 ± 86.04. Group III, treated with SRPP showed a significant reduc-tion (p < 0.001) of ~88% in the formation of superficial metastatic nodules with a mean value of 40.67 ± 6.05 as compared to that of metastasis induced group (MI). Group IV treated with CPP showed a mean of 108.17 ± 7.44 indicating significant (p < 0.01) reduction of ~67% com-pared to MI group. However, Group V treated with GRPP showed no significant (p > 0.05) difference in the superfi-cial metastatic nodule (315.17 ± 91.12) formation sug-gesting the inability of GRPP to offer protection. The results shown in Fig.  3b indicated the implantation per-centage; MI Group showed an implantation percentage of the lung parenchyma in the range of 16.08 and 34.98%, with a mean of 24.54 ± 7.08% and high growth and inva-sion index. The SRPP, CPP and GRPP treated group showed implantation percentage of 3.07 ± 1.37 (p < 0.001), 4.99 ± 1.17, (p < 0.001) and (22.8 ± 4.7), respectively. The SRPP treated animals showed the greatest reduction in invasion as compared to other groups with statistical sig-nificance of p < 0.001. The invasion index results revealed

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that metastasis control group (MI) showed a mean inva-sion index of 136.06 ± 10.40, while SRPP and CPP group showed 14.65 ± 3.13 and 32.72 ± 2.75, respectively. A significant (p < 0.001) reduction in invasive index of ten and fivefolds was seen in SRPP and CPP treated groups (Fig. 3c). GRPP showed no significant effect in the invasion index (126.45 ± 8.44), indicating the increased invasion of B16F10 melanoma cells to the lungs as that of metastasis induced group (136.06 ± 10.40). This increased invasion of B16F10 melanoma cells to the lungs was effectively pro-tected by SRPP treatment. The reduction in invasion was proportional to the reduction in growth of melanoma cells demonstrating the ability of the compound to inhibit the early process of tumor cell growth. Growth index of vari-ous groups of mice is depicted in Fig. 3d. MI group showed a growth index of 0.0025 ± 0.0007 while, SRPP and CPP treated group showed a growth index of 0.0009 ± 0.0004 (p < 0.01) and 0.001 ± 0.0003 (p < 0.01), respectively. Sta-tistically significant reduction of two and twofold was seen

in SRPP (p < 0.01) and CPP (p < 0.01) group, respectively, suggesting the reduction in growth of melanoma cells upon treatment with SRPP and CPP. In GRPP treated group (group V), there was a marginal reduction in the growth index (0.0023 ± 0.0004) which was statistically non signifi-cant (p > 0.05).

SRPP inhibited metastatic cell growth in vitro and induction of apoptosis

Significant inhibition of cell viability by SRPP-60% (p < 0.01) and CPP-48% (p < 0.05) at 100 µg/mL (Fig. 4a) was observed when compared with control. This was confirmed by monolayer changes in the B16F10 cells by SRPP (Fig.  4d, b) and CPP. A typical necrotic type cell lysis and apoptosis were observed in SRPP (Fig. 4d, e) and CPP treated cells. Further, number of cells lysed as per microscopic observation, ethidium bromide and

Fig. 1 Purification and characterization of SRPP. a DEAE cellulose column chromatography profile of SRPP; b purity of active SRPP fraction—0.15  M by gel permeation chromatography on sepharose

CL-4B column; c HPLC analysis and d calibration curve for molecu-lar weight determination

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acridine orange stained cells were counted. SRPP and CPP showed 48 and 42% apoptosis (Fig. 4c) in B16F10 cells.

Inhibition of B16F10 melanoma cell adhesion to extracellular matrix proteins (ECM)

Whether SRPP is capable of inhibiting tumor metastasis by directly preventing tumor cell adherence was investi-gated by in vitro matrigel assay. SRPP significantly inhib-ited B16F10 cell adhesion to matrigel in a concentration dependent manner (Fig.  4b). SRPP showed significant reduction in cell adhersion by 37% (p < 0.05) and 52%, (p < 0.01) at concentration of 50 and 100  µg/mL, respec-tively. Wheareas, CPP showed 28% (p < 0.05) and 42% (p < 0.01) reduction in cell adhersion at 50 and 100 µg/mL concentration, respectively.

Modulation of galectin-3, antioxidant and antioxidant enzymes

High levels of galectin-3 were observed in MI groups (71%) when compared to healthy group animals. Around 40, 60 and 13.9% reduction was observed in SRPP, CPP and GRPP treated groups, respectively when compared to MI (Table  1). Total protein did not vary significantly between control, CPP and GRPP when compared to MI group while, 1.71 fold increase was seen in SRPP group. MI group showed 1.21 fold depletion in GSH levels whereas; SRPP showed no significant difference in GSH levels when compared to healthy controls. CPP and GRPP showed no effect on restoring GSH levels when compared to MI group. Two fold depletion of SOD level in MI group was normalized in treated groups. SRPP and CPP treated group showed around twofold increase in SOD activ-ity compared to MI group (p < 0.01) Although significant

Fig. 2 In vivo efficacy of SRPP in prevention of metastatic lung tumors: A macroscopic observation of pulmonary metastatic nodules. a Healthy, b metastatic induced, c SRPP, d CPP, e GRPP-pretreated followed by MI induction. B Histopathological observations of lung tissue from healthy, metastasis induced and treated groups. B 1–5 represents sections observed at 10× while B 6–10 reveals the same

at 40×. 1 and 6 normal lung, 2 and 7 metastatic lung, 3 and 8 SRPP treated, 4 and 9 CPP treated, 5 and 10 GRPP treated. Arrows in B 2 indicate inflammatory infiltrates (a), actively proliferative hyperchro-matic cells reveal increased mitotic potency (b), disrupted and dyspla-sic (c), altered glandular architecture of lungs and in B7 (d) indicates-cells with typical metastatic characteristics were observed

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reduction in CAT activity was observed in MI group when compared to that of healthy controls, twofold increase in the activity was seen in SRPP (p < 0.001) treated groups, which may be due to antioxidant potentials present in the sample. CPP and GRPP showed significant changes in CAT levels compared to MI (Table 1).

Biochemical parameters in the lung homogenate

Melanin and galectin‑3 levels

Figure  3e reveals 4 and 2.5 folds reduction in melanin content in SRPP (p < 0.01) and CPP (p < 0.01) treated groups, respectively. Only slight reduction was seen

in GRPP treated groups, which could be attributed to marginally reduced growth rate of melanoma cells, as depicted in Fig.  2. Similar changes were observed in galectin-3 levels also (Fig.  5a). Western blot analysis revealed that, SRPP treatment group resulted in reduced expression of galectin-3. SRPP was more potent than CPP. GRPP however did not show any change in the expression of galectin-3 (Fig.  5e). Data substantiates with ELISA data (Fig.  5a). A good correlation between galectin-3 and tumor index in treated and untreated ani-mals suggests the role of galectin-3 in active metastasis (MI) group, and down regulation of the same by SRPP/CPP treatment. Further proportional changes in melanin (R ~ 1) content reveals the reduction in tumor formation.

Fig. 3 Analysis of area of the metastases within each lobule: (*p < 0.05 versus MI, **p < 0.01 versus MI, ***p < 0.001 versus MI, ns non significant). a Frequency of the pulmonary metastatic nodules of experimental animal groups, b implantation percentage, c invasion index and d growth index of pulmonary metastasis in experimentally induced metastasis (MI) and sample (SRPP) pretreated followed by mestastasis induction. As indicated under “Materials and methods” section, total No. of B16F10 cells harbored in the lung per total lung area has been considered. Determination of biochemical parameters in the lung homogenate of metastasis induced (MI) and samples

pretreated followed by metastasis induction: e melanin content was measured and the average of melanin content found in animals of MI group was considered as 100% and melanin content in sample treated groups were expressed as relative % melanin content. f Matrix metal-loproteinases was measured in the lung homogenate of healthy (H), MI and SRPP treated groups by enzymographic analysis; bands indi-cated by arrow corresponds MMP-2. g Matrix metalloproteinasese-2 measured in the lung homogenate of healthy (H), MI and SRPP treated groups by ELISA analysis

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Fig. 4 Antiproliferative and apoptotic effect of SRPP on B16F10 cells in  vitro (*p < 0.05 versus control, **p < 0.01 versus control, ***p < 0.001 versus control). a Effect of SRPP and CPP on the growth of B16F10 cell lines, cell growth inhibition was assessed by MTT assay. b Inhibition of B16F10 melanoma cell adhesion to ECM proteins by SRPP and CPP. c Measurement of apoptosis in metastatic B16F10 cells. Number of cells lysed as per microscopic observation

and ethidium bromide and acridine orange stained cells were counted and percent apoptosis was calculated. d Morphological changes and induction of apoptosis in metastatic B16F10 cells; number of cells lysed as per microscopic observation (a, b) and ethidium bromide and acridine orange (c, d) stained cells were counted and percent apopto-sis was calculated

Table 1 Serum levels of galectin-3, total protein, antioxidant and antioxidant enzymes

Serum was collected from different animals under healthy (H), metastastis induced (MI), SRPP,CPP and GRPP treated groups was subjected to quantitative estimations of galectin-3, total protein, GSH and SOD/CAT. Galectin-3 and total protein content were measured by ELISA and colorimetric methods respectively and the values are represented as μg/dL; GSH is expressed as mg/mg protein. SOD/CAT activities are rep-resented as U/mg protein. Values are expressed as mean ± SD (n = 8) *p < 0.05, **p < 0.01, ***p < 0.001, ns non-significant, represent significant changes relative to metasta-stis induced group (MI)

Healthy control Metastatic SRPP CPP GRPP

Galectin-3 (μg/dL) 2.5 ± 0.56*** 8.6 ± 1.2 5.2 ± 0.89*** 3.5 ± 1.15*** 7.4 ± 0.861ns

Total protein (mg/mL) 13.96 ± 4.14ns 12.11 ± 1.35 21.48 ± 2.18*** 15.2 ± 3.49ns 13.45 ± 1.64ns

SOD (U/mg protein) 3.32 ± 0.129*** 5.86 ± 0.33 2.73 ± 0.38** 2.40 ± 0.23* 5.01 ± 0.40ns

CAT (U/mg protein) 16.35 ± 3.64*** 3.72 ± 4.89 32.78 ± 2.72*** 10.87 ± 3.46** 7.78 ± 2.52*GSH (mg/mg protein) 2.20 ± 0.16* 1.81 ± 0.23 2.23 ± 0.26* 1.73 ± 0.55ns 1.60 ± 0.49ns

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Analysis of matrix metalloproteinases

Although galectin-3 has been depicted in metastatic spread, invasion is promoted by matrix metalloprotein-ases activity that hydrolyzes the basement membrane [26]. As indicated in Fig. 3f, g, a substantial increase in levels of MMP-2 was observed upon induction of metas-tasis in MI group when compared to that of healthy con-trol groups. Significant inhibition of this was observed in SRPP treated groups. Results thus, suggest that SRPP may inhibit cell invasion by virtue of its MMP-2 inhibit-ing property.

Caspase-3, NFκB and p53 levels

Reduction in melanoma cell growth in treated groups could be due to cell death/apoptosis. Caspases are a family of proteins and identified as main executors of the apoptotic process. To confirm the possible mechanism of apoptosis in response to treatment with SRPP, CPP and GRPP, we evaluated caspase-3 levels by ELISA. Regression in tumor colonies could also be predicted due to either the activa-tion of tumor suppressor component—p53 or reciprocal down regulation of proliferative favoring component—NFκB. Substantial activation of Caspases (Fig. 5d) and p53

Fig. 5 Determination of biochemical parameters in the lung homoge-nate (*p < 0.05 versus MI, **p < 0.01 versus MI, ***p < 0.001 versus MI, ns non significant). a Galectin-3, b p53, c NF-κB and d cas-pases-3 levels were measured by respective specific monoclonal anti-body based ELISAs. Galectin-3 levels were quantitated using stand-

ard human galectin-3 and values are expressed as μg of galectin-3/g of lung tissue. p53, NF-κB and caspase-3 results are expressed as relative percentage by considering the average of MI group as 100%. galectin-3 (e) levels were also substantiated by western blot analysis

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(Fig.  5b) and reduction in NFκB (Fig.  5c) were observed in SRPP (p < 0.01) and CPP (p < 0.05) treated groups when compared to that of MI group. However, GRPP had no sig-nificant reducing effect on levels of caspase-3, NFκB and p53 when compared to MI (p > 0.05).

SRPP treatment reduced IL17 and IL10 while augmenting IL12

Lungs of animals infused with B16F10 cells showed har-boring of inflammatory cells as evidenced by intense CD11b staining. CD11b stained area also showed staining for IL-17 (Fig.  6). SRPP treated tissues however showed reduction in IL-17, while increased levels of IL-12 (Fig. 6).

Toxicity studies

Results of toxicity studies indicated a gradual increase in the body weight of mice in sample control groups IV, VI and VIII, similar to healthy animals (Table 2). In addition, higher amounts of total protein (non-significant) compared to the healthy group (I) were also observed. Liver func-tion enzyme—SGPT in all the three groups (VI–VIII) was

almost similar to that of healthy animals. No significant change in SGOT levels were (p > 0.05) observed. Data thus suggest no adverse effects on major organs. Animals, after the experimental schedule, remained healthy as that of control animals with normal food and water intake, body weight gain and behavior (Table 2).

Discussion

Melanoma has been known as one of the most frequently metastasizing malignant neoplasias [1] causing ~80% of skin cancer related deaths. No remedial measures are avail-able due to resistance rendered by malignant melanoma cells to chemo/radiation therapies [27]. Thus data warrants immediate attention for development of alternative treat-ments for melanoma patients. Dietary/herbal sources have been traditionally known to have potential preventive and curative property against chronic disease such as cancer [28, 29]. Modified citrus pectin (MCP) is thought to be useful in the prevention and treatment of metastatic can-cer, especially in solid tumors like melanoma and cancers of the prostate, colon, and breast [30]. Number of studies

Fig. 6 Immunohistological analysis analysis of lung tissues of healthy, metastasis induced and sample (SRPP and GRPP) pretreated animals. Metastasis induced group showed intense staining for CD11b, IL-17 while, showed reduced staining for IL-12, which induces tumor regression. Tissues from SRPP treatment showed reduced staining for IL-17 and increased levels of IL-12. GRPP treated tissues showed almost similar staining pattern as that of the metastasis group indicating no effect or least effect on tumor reduction

GRPP pretreated

Healthy

MetastasisInduced

SRPPPretreated

CD11b IL-12 IL-17

151Clin Exp Metastasis (2017) 34:141–154

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in humans and animals have reported that MCP interferes with cancer cells interactions with other cells by acting as a galectin-3 antagonist [31, 32]. MCP thus appears to disrupt the processes that allow cancer cells to communicate with one another. Further it is reported that when MCP mole-cules bind to galectin-3 at the surface of cancer cell, they block galectin-3 and other molecules from penetrating into nearby healthy tissue to create a new tumor and to estab-lish the tumor’s blood supply (angiogenesis). In this way, MCP seems to play a role in preventing cancerous tumors from metastasizing and spreading to other organs—one of the main causes of death from cancer [32, 33]. Since the citrus pectin (CPP) had the effect of metastasis inhibition, we have taken CPP as positive control for SRPP. Previous reports from other laboratories [29, 34] and our laboratory [8] have shown Swallow root pectic polysaccharide (SRPP) as one of the best galectin-3 inhibitor.

Immunotherapy is designed to help the immune sys-tem to identify and attack cancer cells. The U.S. Food and Drug Administration has approved the checkpoint inhibitor drugs—ipilimumab (Yervoy®), pembrolizumab (Keytruda®) and nivolumab (Opdivo®) to treat melanoma. Ipilimumab targets the CTLA-4 cell receptor; pembroli-zumab and nivolumab target the PD-1 cell receptor. Check-point inhibitors work by targeting signaling proteins that allow cancer cells to disguise themselves as healthy cells. These protein receptors help to regulate the immune system by suppressing the activity of T-cells. Cytokines are mole-cules that regulates immune activity. The drugs alpha-inter-feron (IFN-alpha) and interleukin-2 (IL-2) are cytokines that may be used to treat some cases of advanced mela-noma. These drugs stimulate the rapid growth and activity of immune cells so that they quickly attack the cancer cells [35]. Immunotherapy however, may not be recommended for all patients since immunotherapy treatments have been known to also cause side effects including skin rashes and gastrointestinal problems.

In the present study, we have shown the in vivo efficacy of a dietary pectic polysaccharide—SRPP and probable

mode of sequential action of as per the Scheme  1. Thus, while unraveling the mode of action of SRPP, we addressed the effect of SRPP on crucial steps of pathogenicity in a systematic manner, such as (1) galectin-3 inhibition, (2) inhibition of MMP activation, (3) reciprocal regulation of p53 and NFκB, (4) induction of apoptosis, (5) modulation of molecules required for the execution of these events and (6) blocking of inflammatory cytokines, which otherwise may lead to inflammation that can aggravate tumors.

Results of our study indicated that SRPP has inhibited metastasis by controlling/blocking galectin-3 action dur-ing cancer spread. Our conclusion on this is based on the fallowing research findings: (a) SRPP could inhibit galec-tin-3 mediated agglutination very effectively with MIC of 1.85 µg/mL. Indeed SRPP has shown as a better inhibitor of galectin-3 than the known galectin inhibitor—CPP (MIC-25 µg/mL), which is reported in our paper [8], (b) western blot analysis of lung tissue from animals of healthy, metas-tasis induced, SRPP/CPP treated groups revealed signifi-cant down regulation of galectin-3 when compared to that of metastasis induced group, (c) for galectin-3 inhibition, galectin specific sugar residue, that is galactose appear to be important. Our compositional analysis had revealed the presence of 32 and 19% galactose in SRPP and CPP, respectively, confirming that SRPP and CPP may bind to galectin-3 particularly to carbohydrate binding domain which has the affinity for galactose residue [10], (d) galec-tin-3 inhibition by galactose containing pectic polysaccha-ride has been reported by other group of researchers [8, 10, 11, 30, 31, 34]. Interestingly gene array analysis (un published data) of cancer induced tissue v/s galactose con-taining pectic polysaccharide/oligosaccharide from dietary sources carried out recently from our laboratory clearly revealed normalization of galectin-3 levels further strength-ening our predication that pectic polysaccharides possess the ability to block the action galectin-3.

Our view that galectin-3 inhibitor may block galec-tin-3 gene expression is substantiated by observation of similar results provided by Zhang et  al. [36], where they

Table 2 Toxicity studies

Effect of swallow root components on liver function enzymes (SGPT, SGOT, ALP, TBARS), serum protein, weight gain in sample control groups in comparison with those of healthy controls. Values are expressed as mean ± SD (n = 8)ns non-significant changes compared to healthy control

Healthy control SRPP (without MI) CPP (without MI) GRPP (without MI)

Total protein (mg/mL) 13.96 ± 4.14ns 19.78 ± 2.67ns 18.4 ± 3.04ns 15.69 ± 0.89ns

SGPT (U/mg protein) 262.78 ± 8.01ns 261.56 ± 4.76ns 240 ± 11.40ns 236 ± 8.29ns

SGOT (U/mg protein) 349.78 ± 3.12ns 297.33 ± 25.80ns 297.22 ± 23.91ns 308.44 ± 5.22ns

ALP (mg/mg protein) 32.30 ± 0.16ns 29.23 ± 0.26ns 27.73 ± 0.55ns 26.60 ± 0.49ns

TBARS (nmoles of MDA/mg protein)

0.125 ± 0.094ns 0.196 ± 0.067ns 0.185 ± 0.087ns 0.149 ± 0.10ns

Body weight (gms) 31.56 ± 2.75ns 34.26 ± 1.47ns 34.44 ± 1.55ns 34.23 ± 2.65ns

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have clearly shown how galectin-3 binding polysaccha-ride would inhibit galectin-3 gene expression. Further, our observations are also supported by research findings of several investigators; Menachem et  al. [37], who reports that MCP, a modified citrus pectin, which is also known as galectin-3 binding inhibitor has down regulated galec-tin-3 expression in anaplastic thyroid carcinoma cells [37]. Pienta et  al. [38]; Platt and Raz [31] have reported that MCP interacts directly with Gal-3 [39, 40] to inhibit vari-ous cellular processes in cancer [41, 42].

Further, the present study indicated drastic increase in MMP-2 level in MI group of animals and significant down regulation of the same by SRPP group suggesting

that, galectin-3-inhibitors, by virtue of its binding abil-ity to galectin-3 may block the conformational changes of galectin-3, which otherwise is required for the activa-tion of MMPs [5]. It is intriguing to note that galectin-3 is a potential substrate for MMPs, the action of which is crucial for invasion of cancer cells into newer areas, hence being responsible for cancer spread. Down regulation of MMPs (key component of cell invasion) thus may result in inhibition of cell invasion. Reduction in number of lung melanoma cells as evidenced by macroscopic and histo-pathologic observations followed by reduction in melanin content confirms the lack of cell survival ability of B16F10 melanoma cells during SRPP/CPP treatment.

B16F10 cells

Lung Metastasis

SRPP

Inhibition of Lung Metastasis

+

Increased; Cell survival, Tumor mass,Melanin, Galectin-3, MMP,NF-kB, IL-17 & CD11b

Decreased; p53 & IL-12

a

b

c

d

Decreased; Cell survival, Tumor mass,Melanin, Galectin-3, MMP,NF-kB, IL-17 & CD11b

Increased; p53 & IL-12

Scheme  1 Mechanism of the metastasis inhibition by SRPP. Cul-tured melanoma cells (a) upon inocultion to tail vein of expermen-tal mice resulted in lung metastasis (b). Lung metastasis was con-firmed by increseased cell survival, melanin, tumor mass, NFkB,

MMP, galectin-3, Bcl-2 (anti apoptotic protein), IL-17, and CD11b and decreased p53 (tumor suppressor protein) and IL-12. Treatment with SRPP (c) resulted in inhibiton of lung metastasis (d) followed by reciprocal regulation of tumor and immunological markers

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The reduction of cancer cell growth may essentially due to induction of apoptosis. Our results are supported by increase in caspase-3 which helps in pushing cancer cells towards their fragmentation and autophagy. Blockade of galectin-3 mediated lung metastasis hence appears to be due to inhibition of signaling cascades that are induced during metastasis. Down regulation of nuclear factor kB, a transcriptional regulator and the activation of p53, a well known tumor suppressor gene product by SRPP adds to the intriguing role of SRPP galectin-3 inhibitors in signaling cascade.

Severity of metastasis has been found to be augmented by inflammation. Inflammation although is a response from the host to eject tumors in the localized tissue, infil-tration is known to trigger the aggravation of tumors [43, 44]. Thus, an attempt has been made in the current study to understand the role of cytokines in untreated and treated tumor bearing mice. CD11b staining depicts the harboring of inflammatory cells in the tumor area and the release of pro-inflammatory cytokine IL-17 justifies the severity of tumor aggressiveness. Upon treatment with SRPP, IL-12 levels were increased. Results of the study clearly depicts that SRPP could inhibit the tumor aggressiveness, prob-ably by inhibiting the infiltration of inflammatory cells. Based on similar type of results Tugues et  al. [45] have highlighted the promising strategies of the use of IL-12 as tumor suppressing agent with limiting adverse effects. Differential role of IL-12 could be due to the immunologi-cal status and the stage of the disease [46]. Since SRPP—a potent inhibitor of metastasis augmented IL-12, while GRPP could neither inhibit metastasis nor augment IL-12 may suggest that SRPP may be functioning as anticancer component via regulation of IL-12 also. Further, it is to be emphasized that cytokines produced by M1 form can kill tumor cells; while that from M2 form can favor cancer cell growth. Hypoxic condition has been implicated in such conversions. The inhibition of tumor metastasis upon SRPP treatment could be both by galectin-3 blockade as well as modulation of inflammatory cytokines. The inability of GRPP to inhibit melanoma cell growth in vivo, when com-pared to that of SRPP, could be due to the lack of GRPP to block galectin-3.

The decrease in antioxidant (GSH) and antioxidant enzyme (catalase and superoxide dismutase) levels in met-astatic conditions were normalized upon treatment with SRPP as well as CPP. Toxicity studies indicated no signifi-cant damage to the vital organs, such as liver as evidenced maintenance of liver function enzymes (SGPT, SGOT and ALP). Also increased TBARS in MI group was normalized in all treated groups. There was a normal weight gain in the sample treated groups as compared to healthy groups that were fed with standard mice diet suggesting no effects of the selected test components. Additionally since galectin-3

inhibitors show specific affinity to galectin-3, they are tar-geted only to high galectin-3 expressing B16F10 cells that are harbored in the lung and not normal lung cells per se.

In conclusion, Results have shown that SRPP act as an antimetastatic compound which can alter the adhesion of B16F10 melanoma cells, inhibit their proliferation and induce apoptosis. The role of dietary components in cancer progression and metastasis can thus be a promising reme-dial agent as effective therapeutics to explore against com-plex metastatic melanomas. The key molecular signaling and immunomodulatory pathway through, which the pectin inhibits melanoma cell proliferation and apoptosis induc-tion, needs further studies.

Acknowledgements The authors wish to thank the Director, CSIR-Central Food Technological Research Institute, for his keen interest in the work and encouragement. SMD is thankful to Department of Sci-ence and Technology and Department of Biotechnology, New Delhi for the financial assistance through projects. Dr. Mahadeva, Profes-sor of English, University of Mysore, Mysore has been acknowledged for his kind support in English correction. UVS, SEM and HMA gracefully acknowledge the Senior Fellowships provided by Indian Council of Medical Research and Council of Scientific and Industrial Research, New Delhi, India, respectively.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interest.

References

1. Goldsmith LA, Askin FB, Chang AE, Cohen C, Dutcher JP, Gilgor R, Swanson NA (1992) Diagnosis and treatment of early melanoma: NIH Consensus Development Panel on Early Mela-noma. JAMA 268:1314–1319

2. Chamelli J, Frances P, Noonan, Merlino G (2003) Ultravio-let radiation and cutaneous malignant melanoma. Oncogene 22:3099–3112

3. Friedmann PS, Gilchrest BA (1987) Ultraviolet radiation directly induces pigment production by cultured human melanocytes. J Cell Physiol 133:88–94

4. Kanavy HE, Gerstenblith MR (2011) Ultraviolet radiation and melanoma. Semin Cutan Med Surg 30:222–228

5. Hofmann UB, Westphal JR, Waas ET (1999) Matrix metallopro-teinases in human melanoma cell lines and xenografts: increased expression of activated matrix metalloproteinase-2 (MMP-2) correlates with melanoma progression. Br J Cancer 81:774–782

6. Herrmann J, Truck CW, Atchison RE (1993) Primary structure of the soluble lactose binding lectin L-29 from rat and dog and interaction of its non-collagenous proline, glycine, tyrosine rich sequence with bacterial and tissue collagenase. J Biol Chem 208:26704–26711

7. Victor G, Prieto A, Alexandra (2006) Galectin-3 expression is associated with tumor progression and pattern of sun exposure in melanoma. Clin Cancer Res 12:6709–6715

8. Sathisha UV, Smitha J, Harish Nayaka MA (2007) Inhibition of galectin-3 mediated cellular interactions by pectic polysaccha-rides from dietary sources. Glycoconj J 24:497–507

154 Clin Exp Metastasis (2017) 34:141–154

1 3

9. Raz A, Lotan R (1987) Endogenous galactoside-binding lectins: a new class of functional tumor cell surface molecules related to metastasis. Cancer Metastasis Rev 6:433–452

10. Gao X, Zhi Y, Sun L, Peng X, Zhang T, Xue H, Tai G, Zhou Y (2013) The Inhibitory effects of a rhamnogalacturonan (RG-I) domain from ginseng pectin on galectin-3 and its structure-activ-ity relationship. J Biol Chem 288:33953–33965

11. Inohara R, Raz A (1987) Effects of natural complex carbohy-drate (citrus pectin) on murine melanoma cell properties related to galectin-3 functions. Glycoconj J 11:527–532

12. Hansen MB, Nielsen SE, Berg K (1989) Reexamination and fur-ther development of a precise and rapid dye method for measur-ing cell growth/cell kill. J Immunol Methods 119:203–210

13. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related sub-stances. Anal Chem 28:350–356

14. Cristina MC, Vicente VO, Josefa YM (2005) Treatment of metastastic melanoma B16F10 by the flavonoids tangeretin, rutin and diosmin. J Agric Food Chem 53:6791–6797

15. Smitha Jayaram, Sabeeta Kapoor, Shylaja M Dharmesh (2015) Pectic polysaccharide from corn (Zea mays L.) effectively inhib-ited multistep mediated cancer cell growth and metastasis. Chem Biol Interact 235:63–75

16. Srikanta BM, Siddaraju MN, Shylaja M Dharmesh (2007) A novel phenol-bound pectic polysaccharide from Decalepis ham‑iltonii with multi-step ulcer preventive activity. World J Gastro-enterol 13:5196–5207

17. Martinez C, Vicente V, Yanez M, Garcia JM, Canteras M, Alcaraz M (2005) Experimental model of pulmonary metastasis treatment with IFN alpha. Cancer Lett 225:75–83

18. Lentini A, Autuoria F, Mattiolia P, Caragliab M, Abbruzzeseb A, Beninati S (2000) Evaluation of the efficacy of potential antineo-plastic drugs on tumour metastasis by a computer-assisted image analysis. Eur J Cancer 36:1572–1577

19. Brown ER, Doig T, Anderson N, Brenn T, Doherty V, Xu Y, Bartlett JM, Smyth JF, Melton DW (2012) Association of galec-tin-3 expression with melanoma progression and prognosis. Eur J Cancer 48:865–874

20. Balasubramanian K, Vasudevamurthy R, Sathisha UV (2009) Galectin-3 in urine of cancer patients: stage and tissue specific-ity. J Cancer Res Clin Oncol 135:355–363

21. Shiu-Chen H, Chi-Tang H, Shoei-Yn L (2005) Carnosol inhibits the invasion of B16F10 mouse melanoma cells by suppressing metalloproteinase-9 through down-regulating nuclear factor-kappa B and c-Jun. Biochem Pharma 69:221–232

22. Powell AA, LaRue JM Martinez JD (2001) Bile acid hydropho-bicity is correlated with induction of apoptosis and/or growth arrest in HT 116 cells. Biochem J 356:481–486

23. Hibino S, Shibuya M, Engbring JA, Mochizuki M, Nomizu M, Kleinman HK (2004) Identification of an active site on the laminin alpha5 chain globular domain that binds to CD44 and inhibits malignancy. Cancer Res 64:4810–4816

24. Hosoi J, Abe E, Suda T (1985) Regulation of melanin synthesis of B16 mouse melanoma cells by 1 alpha, 25-dihydroxyvitamin D3 and retinoic acid. Cancer Res 45:1474–1478

25. Siddaraju MN, Dharmesh SM (2006) A bioactive fraction from zingiber officinale and a process for the preparation thereof. US patent, US8158608

26. Ochieng J, Fridman R, Nangia-Makker P (1994) Galectin-3 is a novel substrate for human matrix metalloproteinases-2 and -9. BioChemistry 33:14109–14114

27. Takenaka Y, Fukumori T, Yoshii T (2004) Nuclear export of phosphorylated galectin-3 regulates its anti-apoptotic

activity in response to chemotherapeutic drugs. Mol Cell Biol 24:4395–4406

28. Shathish K, Guruvayoorappan C (2014) Decalepis hamilto-nii inhibits tumor progression and metastasis by regulating the inflammatory mediators and nuclear factor κB subunits. Integr Cancer Ther 13:141–151

29. Watson E (2013) Natural galectin-3 inhibitors from pectins are the next big thing in heart-healthy foods, says start-up. Breaking News on Food and Beverage Development, North America, 22 Mar 2013

30. Azemar M, Hildenbrand B, Haering B, Heim ME, Unger C (2007) Clinical benefit in patients with advanced solid tumors treated with modified citrus pectin: a prospective pilot study. Clin Med Oncol 1:73–80

31. Pienta KJ, Naik H, Akhtar A et al (1995) Inhibition of spontane-ous metastasis in a rat prostate cancer model by oral administra-tion of modified citrus pectin. J Natl Cancer Inst 87:348–353

32. Nangia-Makker P, Hogan V, Honjo Y et al (2002) Inhibition of human cancer cell growth and metastasis in nude mice by oral intake of modified citrus pectin. J Natl Cancer Inst 94:1854–1862

33. Takenaka Y, Fukumori T, Raz A (2004) Galectin-3 and metasta-sis. Glycoconj J 19:543–549

34. Maxwell EG, Belshaw NJ, Waldron KW, Morris VJ (2012) Pec-tin-an emerging new bioactive food polysaccharide. Trends Food Sci Tech 24:64–73

35. Redman JM, Gibney GT, Atkins MB (2016) Advances in immu-notherapy for melanoma. BMC Med 14(1):1

36. Zhang L, Wang P, Qin Y, Cong Q, Shao C, Du Z, Ni X, Li P, Ding K (2016) RN1, a novel galectin-3 inhibitor, inhibits pan-creatic cancer cell growth in vitro and in vivo via blocking galec-tin-3 associated signaling pathways. Oncogene. doi:10.1038/onc.2016.306

37. Menachem A, Bodner O, Pastor J, Raz A, Kloog Y (2015) Inhi-bition of malignant thyroid carcinoma cell proliferation by Ras and galectin-3 inhibitors. Cell Death Discov 1:15047

38. Platt D, Raz A (1992) Modulation of the lung colonization of B16-F1 melanoma cells by citrus pectin. J Nat Cancer Inst 84:438–442

39. Zhang T, Zheng Y, Zhao D, Yan J, Sun C, Zhou Y, Tai G (2016) Multiple approaches to assess pectin binding to galectin-3. Int J Biol Macromol 91:994–1001

40. Zhang T, Lan Y, Zheng Y, Liu F, Zhao D, Mayo KH, Zhou Y, Tai G (2016) Identification of the bioactive components from pH-modified citrus pectin and their inhibitory effects on galec-tin-3 function. Food Hydrocoll 58:113–119

41. Liu FT, Rabinovich GA (2005) Galectins as modulators of tumour progression. Nat Rev Cancer 5:29–41

42. Kim K, Mayer EP, Nachtigal M (2003) Galectin-3 expression in macrophages is signaled by Ras/MAP kinase pathway and up-regulated by modified lipoproteins. Biochim Biophys Acta (BBA) 1641(1):13–23

43. Bugelski PJ, Kirsh RL, Sowinski JM, Poste G (1985) Changes in the macrophage content of lung metastases at different stages in tumor growth. Am J Pathol 118(3):419

44. Murray PJ, Wynn TA (2011) Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 11(11):723–737

45. Tugues S, Burkhard SH, Ohs I, Vrohlings M, Nussbaum K, Vom Berg J, Becher B (2015) New insights into IL-12-mediated tumor suppression. Cell Death Differ 22(2):237–246

46. Seder RA, Kelsall BL, Jankovic D (1996) Differential roles for IL-12 in the maintenance of immune responses in infectious ver-sus autoimmune disease. J Immunol 157(7):2745–2748