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Components of heat-treated Helianthus annuus L. pectin inhibit tumor growth and promote immunity in a mouse CT26 tumor model
Journal of Functional Foods 48 (2018) 190–199
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Components of heat-treated Helianthus annuus L. pectin inhibit tumor growth and promote immunity in a mouse CT26 tumor model ⁎
Yuan Guan, Zhongyu Zhang, Xiyao Yu, Jingmin Yan, Yifa Zhou, Hairong Cheng , Guihua Tai
T ⁎
Jilin Province Key Laboratory on Chemistry and Biology of Changbai Mountain Natural Drugs, School of Life Sciences, Northeast Normal University, Changchun 130024, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Akt Anti-tumor Heat-treated pectin Promote immunity
Heat-treated pectin is known to have anti-tumor activity. The present study was designed to identify its active components and mechanism of action. Here, we investigated the anti-tumor effect of heat-treated Helianthus annuus L. pectin (HT-HAP), alkali-inactivated HT-HAP representing the pectin fragment fraction (HT-HAP-P), and heat-treated galacturonic acid representing the small molecule fraction (HT-HAP-S). Our results demonstrate that HT-HAP induces colon cancer CT26 cells apoptosis and inhibits CT26 tumor growth like 5-fluorouracil, whereas it shows no spleen and thymus toxicity in contrast to 5-fluorouracil. Mechanistically, HT-HAP attenuates Akt activation in tumors, and enhances it in the spleen and thymus. Interestingly, HT-HAP-S inhibited tumor growth, but promoted immunity mildly, whereas HT-HAP-P had little effect on tumor growth, but effectively promoted immunity. Overall, we attribute the anti-tumor effect from HT-HAP to the action of small molecules and the immune enhancement primarily to pectin fragments, with Akt activation being the underlying mechanism of action.
1. Introduction Colorectal cancer is the third most commonly diagnosed cancer and the fourth cause of cancer death worldwide (Ferlay et al., 2010; Siegel, DeSantis, & Jemal, 2014). Several epidemiological studies have proven the association of colon cancer with dietary habit, in particular the consumption of pectin in fruits and vegetables (Rose, DeMeo, Keshavarzian, & Hamaker, 2007; Slavin & Lloyd, 2012; Trock, Lanza, & Greenwald, 1990; Young, Hu, Le Leu, & Nyskohus, 2005). Pectin, a family of complex polysaccharides rich in galacturonic acid (GalA), has a complex structure composed of various elements and domains. The most abundant structure in pectin is homogalacturonan (HG), a linear chain of α1,4-linked-D-GalA with esterification or acetylation of some residues. The second most abundant structure, type I rhamnogalacturonan, has a backbone of repeating disaccharide units of [-α-D-GalA-1,2α-L-Rha-1,4-]n that can also be substituted with arabinan, galactan or arabinogalactan side chains. Pectin may also contain type II rhamnogalacturonan (RG-II), apigalacturonan, and/or xylogalacturonan. All of these domains have a galacturonan backbone (Caffall & Mohnen, 2009; Schols & Voragen, 1996; Yapo, 2011). As a dietary fiber, pectin is known to play a role in the prevention of colon cancer (Rose et al., 2007; Trock et al., 1990). Heat-modified pectin has been shown to possess additional activities. For example,
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heat-treated citrus pectin (HTCP) induces significant apoptosis in androgen-responsive and androgen-independent human prostate cancer cells and induces cell apoptosis and autophagy in HepG2 and A549 cells (Jackson et al., 2007; Leclere et al., 2015). Similarly, heat-modified ginseng pectin exhibits more significant anti-proliferative effects on human colon cancer cells compared to its un-modified counterparts (Cheng et al., 2011). Koyama et al. reported that heat-treatment of uronic acid or polysaccharides containing uronic acid residues generates a small bioactive molecule 4,5-dihydroxy-2-cyclopenten-1-one (DHCP) that promotes apoptosis of HL-60 cancer cells and is cytotoxic to other cancer cells (Koyama et al., 2000; Leclere et al., 2016). However, the function of DHCP has yet to be demonstrated in animal models. In addition to DHCP or DHCP-like small molecules, heattreatment also degrades pectin into fragments of various lengths. The effectiveness of these fragments remains unclear. Helianthus annuus L. (Sunflower) is a traditional industrial crop from which only the seeds, but not the heads, are utilized. To make use of the discarded head products and increase the economic value of this crop, we have extracted pectin from the Helianthus annuus L. heads that are rich of HG pectin (Peng et al., 2016). In this study, we heat-modified the pectin and investigated its anti-tumor efficacy on colon cancer cells and in a colon cancer model in mice.
Corresponding authors. E-mail addresses: [email protected] (H. Cheng), [email protected] (G. Tai).
https://doi.org/10.1016/j.jff.2018.07.001 Received 1 February 2018; Received in revised form 20 June 2018; Accepted 1 July 2018 1756-4646/ © 2018 Published by Elsevier Ltd.
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2. Materials and methods
well. Twenty-four hours later, media were removed and fresh media containing various concentrations of pectin were added. After 48 h, the MTT assay was used to assess cell viability.
2.1. Materials The heads of Helianthus annuus L. were collected from Changbai mountain area in Jilin Province, PR China. The enzyme Endo-PG was purchased from Sigma Aldrich (St Louis, MO, USA). RPMI1640 medium and fetal bovine serum were obtained from Gibco (Grand Island, NY, USA). An antibody to actin (Cat. No. 612657) was purchased from BD Biosciences (San Diego, CA). Antibodies to p38 (Cat. No. 9212), p-p38 (Cat. No. 9215), p-ERK (Cat. No. 9101), ERK (Cat. No. 9102), JNK (Cat. No. 9252), p-JNK (Cat. No. 9255), Akt (Cat. No. 4691), p-Akt (Cat. No. 9271) and Ki67 (Cat. No. 12202) were purchased from Cell Signaling Technology (Beverly, MA). Mouse interleukin-12 (IL-12, Cat. No. E-ELM0726c) and interferon-γ (IFN-γ, Cat. No. E-EL-M0048c) were purchased from Elabscience Biotechnology Co. Ltd (Wuhan, China). Other reagents were of analytical grade or better.
2.6. Flow cytometry assay Cells treated with different concentrations of pectin for 48 h were stained with the Annexin V-FITC/PI kit (Keygen Biotechnology, Jiangsu, China) according to the manufacturer’s instructions. 2.7. In vivo assays Specific-pathogen-free female Balb/c mice, age 6–8 weeks and weighing 16–18 g, were obtained from Beijing HFK Bioscience Co. Ltd (Beijing, China). All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals. Mice were housed under pathogen-free conditions and allowed access to food and water ad libitum. Animals were randomly divided into 10 groups (n = 6 per group). Then 1 × 105 of CT26 cells were injected subcutaneously into the right axilla of the mice. On day 2 following injection, HAP, HT-HAP, HTHAP-P or HT-HAP-S was administered intragastrically daily for 20 days at the dosages of 150, 300, 110 or 220 mg/kg body weight (BW), as indicated. 5, 10, 15 mg/kg BW of 5-FU was applied by intraperitoneal injection every other day. The control group was administered distilled water. After 20 days, the mice were sacrificed, and the tumor, spleen and thymus were dissected, weighed and lysed for western blotting analyses or subjected to immunohistochemistry sectioning, respectively. The tumor inhibition rate and spleen and thymus index were calculated as follows:
2.2. Preparation of HAP, HT-HAP, HT-HAP-S and HT-HAP-P HAP was prepared according to the method described by our group, with some modifications (Peng et al., 2016). Briefly, the heads of Helianthus annuus L. were extracted with 0.2% oxalic acid (solid: liquid ratio 1:20, w/v) at 100 °C for 1 h and precipitated with 60% aqueous ethanol. After centrifugation, the pectin was re-dissolved in distilled water and obtained by spray drying. HT-HAP was prepared as previously reported with some modifications (Hao et al., 2013; Jackson et al., 2007). Briefly, the HAP was dissolved in distilled water at the concentration of 5 mg/mL and treated with Endo-PG at 37 °C for 12 h. Then the solution was heated at 121 °C for 1 h for 4 times. The product was lyophilized and stored at −20 °C. HT-HAP-S was prepared from galacturonic acid (GalA, purchased from Sigma Aldrich, St Louis, MO, USA) by treatment of GalA solution (5 mg/mL in distilled water) at 121 °C for 1 h for 4 times as described above. HT-HAP-P was prepared as previously reported with minor modifications (Jackson et al., 2007). HT-HAP was dissolved in distilled water at the concentration of 10 mg/mL, and the pH was adjusted to 12.0 with ammonia, then 7.0 with acetic acid. The solution was then evaporated and lyophilized to give HT-HAP-P.
Tumor inhibition rate(%) = (Wc−Wt)/Wc × 100% where Wc is the tumor weight of the control group, and Wt is the tumor weight of the test group. Spleen or Thymus Index (mg/g) = Weight of spleen or thymus/ Body weight. Frozen tumor sections (8 μm) were subjected to immunostaining using Ki67 antibody and Hoechst 33342 as described previously (Chen, Huang, Huang, Tseng, & Tseng, 2006). Macrophages and NK cells were obtained from the peritoneal cavity and spleen of the tumor-bearing mice, respectively. Macrophage phagocytosis and NK cell cytotoxicity assays were performed as reported previously (Sheng et al., 2017).
2.3. Physicochemical properties of HAP, HT-HAP, HT-HAP-S and HTHAP-P
2.8. Western blotting
Monosaccharide composition was determined by using high performance liquid chromatography (HPLC) as described by Zhang et al. (2009). Molecular weight was measured using a TSK-gel G-3000PWxL column (7.8 × 300 mm, TOSOH, Japan) connected to a Shimadzu HPLC system, as described by Zhang et al. (2009). For detection of the active compound (DHCP), samples dissolved in elution buffer (5% methanol and 0.05% trifluoroacetic acid) were applied onto a Kromasil C18 column (4.6 × 250 mm) connected to a Shimadzu HPLC system. Material was eluted at a flow rate of 0.6 mL/ min and monitored by UV at 235 nm (Aoyagi, Ishii, Ugwu, & Tanaka, 2008).
Cells treated with HAP, HT-HAP, HT-HAP-P and HT-HAP-S were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.5% Triton X-100, 150 mM sodium chloride, 0.1 mM PMSF, and Roche incomplete protease inhibitor cocktail). Tumor, spleen or thymus proteins were lysed by grinding the tissue in lysis buffer. Equal amounts of protein were separated by using 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membranes, blotted with specific antibodies (dilution of 1:5000 for the actin antibody and 1:1000 for all other antibodies), and detected following incubation with enhanced chemiluminescence (ECL) reagent using a chemiluminescence detection system (MiniChemi, Beijing Sage Creation Science Co., Ltd). The bands were quantified by densitometry using Lane 1D software (MiniChemi, Beijing Sage Creation Science Co., Ltd).
2.4. Cell culture The mouse colon cancer cell line CT26 was purchased from ATCC and cultured in RPMI 1640, supplemented with 10% fetal bovine serum, 100 U/mL of penicillin and 100 μg/mL of streptomycin. Cells were cultured at 37 °C in a 5% CO2 incubator.
2.9. ELISA assay Tumors of the mice were ground and lysed in lysis buffer. IL-12 and IFN-γ from the tumors of the mice in each group were analyzed by commercially available ELISA kits according to the manufacturer’s
2.5. Cell viability assay CT26 cells were plated in 96-well plates (Costar) at 1 × 104 cells/ 191
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Fig. 1. Effect of HT-HAP on tumor growth and immune organ toxicity in tumor bearing mice. Mice treated with HAP, HT-HAP or 5-FU were sacrificed. The tumor weight (A), body weight (D), spleen and thymus index (E, F) were analyzed. Ki67 expression in tumors was tested with immunostaining (B). Bar, 50 μm. For Ki67 quantification (C), five sections from each of the three tumors per group were analyzed, with the value representing the mean ± SD, n = 15. Data in A, D, E and F were in a normal distribution, and each value represents mean ± SD of six animals in each group. The data of tumor weight, spleen and thymus index were analyzed by using the one-way ANOVA, followed by LSD's test. The data of body weight was analyzed by using the one-way ANOVA followed by Dunnett’s T3. Data in C was in non-normal distribution, and was analyzed by Kruskal–Wallis test, followed by the Mann-Whitney U test. *P < 0.05, **P < 0.01 compared to each control group.
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of these side effects with HT-HAP (Fig. 1D–F). On the other hand, the administration of HT-HAP at 300 mg/kg BW did significantly increase the thymus index to the level observed in the non-tumor (normal) group. Thus, the use of HT-HAP appears to be better than 5-FU in terms of their effects on the spleen and thymus in tumor bearing mice.
instructions. 2.10. Statistical analysis All experiments were performed at least three times, and SPSS 21.0 (IBM Corporation, NY, USA) statistical software program was used for all data analyses. The normal distribution of the data was analyzed by using the Shapiro-Wilk test (Stephens, 1965). Results were expressed as the mean ± SD. Single factor analysis of variance was performed using ANOVA, followed by a post hoc LSD's test when the data were normally distributed and exhibited a homogeneous variance. For data that did not show a homogeneous variance, Dunnett’s T3 analysis (Dunnett, 1980) was used as the post hoc test. Non-normally distributed data were analyzed by using Kruskal–Wallis (Kruskal & Wallis, 1952) followed by Mann-Whitney U test (Mann & Whitney, 1947). Statistical significance was defined as p < 0.05 (*), or p < 0.01(**).
3.2. Heat generated small molecule(s) accounted for the anti-tumor activity both in in vitro and in vivo Heat treatment is known to degrade HG into fragments, oligosaccharides and monosaccharides (GalA), which we refer to here as the ‘pectin fragment fraction’. The resulting GalA can be further modified into UV 235 nm-positive small molecules (e.g. DHCP) (Aoyagi et al., 2008; Cheng et al., 2011; Koyama et al., 2000; Leclere et al., 2016) that we refer to as the ‘small molecule fraction’. In order to identify the effective component(s) in HT-HAP, we prepared two samples: HT-HAPP prepared by alkaline treatment of HT-HAP to disrupt the UV 235 nmpositive small molecule(s) (Jackson et al., 2007) that represents the pectin fragment fraction, and HT-HAP-S, prepared by heat treatment of GalA, that represents the small molecule fraction. Size distribution of these samples determined by HPGPC (High-Performance Gel Permeation Chromatography) is shown in Fig. 2A. Consistent with the degradative effect of endo-PG and heat treatment, HT-HAP and HT-HAP-P displayed a major peak at the elution position of GalA, and peaks between 12 min and 16 min correspond to fragments and oligosaccharides. The 235 nm-positive small molecules were assessed by using HPLC with a C18 column. As shown in Fig. 2B, HT-HAP and HT-HAP-S displayed an obvious peak of DHCP at 6.7 min, whereas HAP and HT-HAPP showed no such peak. Monosaccharide composition analyses showed that HAP, HT-HAP, HT-HAP-P, and HT-HAP-S were all primarily composed of GalA (79.9%, 71.5%, 72.1% and 96.5%, respectively), with HAP, HT-HAP, and HT-HAP-P also containing small amounts of rhamnose, glucose, arabinose, mannose, xylose and glucuronic acid residues (Table 1). The MTT assay showed that HT-HAP and HT-HAP-S significantly suppressed cell viability in a concentration-dependent manner, whereas HAP and HT-HAP-P had no effect (Fig. 3A). The cell viability curves of
3. Results 3.1. HT-HAP inhibits tumor growth with no spleen and thymus toxicity Mice implanted with CT26 tumors were used to study the antitumor activities of HAP (untreated pectin) and HT-HAP (heat-treated HAP) in vivo. 5-FU, a chemotherapeutic usually given to colon cancer patients, was used as the positive control. As shown in Fig. 1A, HT-HAP inhibited tumor growth by 22.0% and 42.2%, whereas HAP had negligible effect (9.1% and 4.7%) at 150 and 300 mg/kg BW, respectively. The anti-tumor effect from HT-HAP at 300 mg/kg BW was similar to that from 10 mg/kg BW of 5-FU (43.4%) (Fig. 1A). The expression of Ki67, an indication of cell proliferation, was also similar in the two groups, with both being remarkably less than control or HAP groups (Fig. 1B–C). Thus, HT-HAP exerts an inhibitory effect on tumor growth in vivo. Side effects are an important issue associated with the use of chemotherapeutic drugs. We observed a significant decrease in body weight (Fig. 1D), spleen index (Fig. 1E), and thymus index (Fig. 1F), with the administration of 10 mg/kg BW 5-FU. In contrast, we saw none
Fig. 2. Assessment of HAP, HT-HAP, HT-HAP-P and HT-HAP-S. (A) elution profiles on a TSK-gel G-3000PWxL column with RI detection. Vo, void volume. GalA, the elution position of GalA. *The elution position of NaCl added to the sample. (B) elution profiles on Kromasil C18 column with UV detection at 235 nm. The molecular formula and elution peak of DHCP are indicated. 193
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HT-HAP and HT-HAP-S were identical when plotted against the contents of DHCP in each sample (Fig. 3B). Flow cytometry demonstrated that HT-HAP and HT-HAP-S induced apoptosis, whereas HAP and HTHAP-P did not (Fig. 3C). The observed apoptosis was caspase-independent, because it could not be abrogated by using the pan-caspase inhibitor z-VAD-fmk (Fig. 3C). The caspase-independent nature of DHCP-induced apoptosis was also reported previously (Leclere et al., 2015). Quantification of these data from three independent experiments showed similar results (Fig. 3 D), suggesting that heat-generated small molecule(s) accounted for the anti-tumor effect in vitro. Similarly, our in vivo studies showed that HT-HAP-S inhibited tumor
Table 1 Monosaccharide composition of HAP, HT-HAP, HT-HAP-P and HT-HAP-S. Sample
HAP HT-HAP HT-HAP-P HT-HAP-S
Monosaccharide composition (mol %) GalA
Rha
Glc
Ara
Gal
Man
Xyl
GlcA
Fuc
79.9 71.5 72.1 96.5
4.8 6.7 8.0 –
4.1 4.6 4.2 –
3.1 4.0 4.1 –
3.6 4.4 3.5 1.1
1.7 2.6 3.0 –
2.3 2.5 2.6 2.3
0.4 2.1 1.7 –
– 1.7 0.8 –
Fig. 3. Analyses of the effective anti-tumor components of HT-HAP in vitro. (A–B) cell viability as determined by the MTT assay. CT26 cells were treated with HAP, HT-HAP, HT-HAP-S or HT-HAP-P at the indicated concentrations (A) or DHCP contents (B). (C) cell apoptosis was determined by using flow cytometry. CT26 cells were pretreated with or without z-VAD-fmk and then treated with indicated concentrations of HAP, HT-HAP, HT-HAP-S or HT-HAP-P. (D) statistic data of C. Each value represents the mean ± SD of three independent experiments. **P < 0.01 compared to the 0 mg/mL group (one-way ANOVA followed by LSD's test).
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Fig. 4. Analyses of the effective components of HT-HAP in vivo. Mice were treated with HT-HAP, HT-HAP-P or HT-HAP-S as indicated. The tumor weight (A), spleen index (B), thymus index (C), NK cell cytotoxicity (D) and phagocytic rate of macrophage (E) were determined. In A–C, each value represents the mean ± SD of six animals in each group. In D and E, each value represents the mean ± SD of triplicate. Data in C were in a non-normal distribution and analyzed by using the Kruskal–Wallis followed by Mann-Whitney U test. Other data were in normal distributions and analyzed by using the one-way ANOVA followed by LSD's test. * P < 0.05, **P < 0.01 compared to control group.
cytotoxity and macrophage activation by HT-HAP-P was greater than that elicited by HT-HAP-S. These data indicate that both the pectin fragments and small molecules can promote an immune response, with the former being more effective than the later.
growth by 21.8% and 46.8% at dosages of 110 and 220 mg/kg BW (where the DHCP content was equivalent to that of 150 and 300 mg/kg BW HT-HAP), respectively, whereas HT-HAP-P exerted only a 14.6% and 11.9% inhibition at doses of 150 and 300 mg/kg BW, respectively (Fig. 4A). This confirmed our conclusion that heat-generated small molecule(s) accounted for the anti-tumor effect.
3.4. Opposing effects of HT-HAP on Akt activation in tumors and immune organs
3.3. Pectin fragments promote immunity more than small molecules To further understand the effects of HT-HAP on tumor growth and immunity, we investigated some signaling molecules frequently involved in cell proliferation and/or apoptosis (Kong, Yu, Chen, Mandlekar, & Primiano, 2000; Wu, 2007; Zhang et al., 2013). The in vitro assay showed that HT-HAP and HT-HAP-S, both of which had antitumor activity, down-regulated Akt, but up-regulated JNK, ERK and p38 phosphorylation in CT26 cells in a time-dependent manner (Fig. 5). In contrast, HAP and HT-HAP-P, both of which exhibited no anti-tumor activity, showed no obvious effects (Fig. 5). It appears that Akt
HT-HAP, HT-HAP-P (representing the pectin fragment fraction) and HT-HAP-S (representing the small molecule fraction) were also compared for their effects on the immunity in tumor-bearing mice. Our results show that all three samples had no significant effect on the spleen index compared to control group (Fig. 4B). However, all of these samples significantly increased the thymus index and NK cytotoxity (Fig. 4C and D). HT-HAP and HT-HAP-P also significantly increased macrophage activation (Fig. 4E). Moreover, the increase in NK 195
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Fig. 5. Akt and MAPK activation in CT26 cells. CT26 cells were treated with HAP, HT-HAP, HT-HAP-P or HT-HAP-S for the indicated times. Whole-cell extracts were prepared and subjected to western blotting using specific antibodies. (A) representative immunoblots. (B) densitometric analysis of the ratio of p-Akt/Akt, p-ERK/ ERK, p-JNK/JNK and p-p38/p38. Each value represents the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 compared to each 0 h group (oneway ANOVA followed by LSD's test).
activity) produced a robust decrease in Akt phosphorylation in CT26 tumors, whereas HT-HAP-P (no anti-tumor activity) exhibited no obvious changes in Akt phosphorylation (Fig. 7A–B). Collectively, both in vitro and in vivo data indicate that inhibition of Akt activation is a mechanism for the inhibition effect of HT-HAP on tumors. The spleen and thymus of tumor-bearing mice were lysed and subjected to western blot analysis. In the HT-HAP group, Akt activation was increased in both spleen and thymus (Fig. 7C–F), in contrast to the decrease of Akt activation in tumors. Furthermore, HT-HAP-P and HTHAP-S, both of which showed immune-enhancing activity, also promoted Akt activation in the spleen and thymus, although to different extents (Fig. 7C–F). Thus, the increase of Akt activation is a mechanism
inactivation and MAPK activation are related to the anti-tumor effect. Consistent with these in vitro data, Akt activation in CT26 tumors in mice was inhibited by HT-HAP (with anti-tumor activity), but not by HAP (without anti-tumor activity) (Fig. 6). However, the involvement of MAPKs was excluded by this in vivo study (Fig. 6) for two reasons: (1) phosphorylation of ERK in tumors was up-regulated by both HAP (without anti-tumor activity) and HT-HAP (with anti-tumor activity), albeit to different levels, and (2) p38 phosphorylation was largely unchanged, while JNK phosphorylation was decreased in tumors in contrast to results from our in vitro cell studies. Thus, the inhibition of Akt activation could be a mechanism for HT-HAP’s anti-tumor action. Supporting this proposal, we found that HT-HAP-S (with anti-tumor 196
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Fig. 6. Akt and MAPK activation in CT26 tumors. Tumors of mice treated with HAP or HT-HAP were dissected, lysed and subjected to western blotting. (A) representative immunoblots. (B) densitometric analysis of the ratio of p-Akt/Akt, p-ERK/ERK, p-JNK/JNK and p-p38/p38. Each value represents the mean ± SD of four tumors in each group. *P < 0.05, **P < 0.01 compared to each control group (one-way ANOVA followed by LSD's test).
pectin also showed no correlation between immune-enhancement and anti-tumor activity in a mouse sarcoma 180 tumor model (Hao et al., 2013). However, these studies did not exclude the possibility that, in a long term, enhanced immunity may help the body combat cancer.
of action for the HT-HAP-mediated enhancement of immunity in mice. 4. Discussion Chemotherapeutic drugs are widely used in the treatment of various cancers; however their therapeutic effectiveness is usually accompanied by adverse side effects (Chabner & Roberts, 2005; Dezern et al., 2013; Verstappen, Heimans, Hoekman, & Postma, 2003). Chemotherapeutic drugs, particularly when used in high-doses and for long periods of time, can therefore lead to a decline in body weight, splenocyte proliferation, organ index, macrophage phagocytosis and natural killer (NK) cell activity (Dezern et al., 2013; Hayashi, Nakano, Hashimoto, Kanekiyo, & Hayashi, 2008; Zhang, Nie, Huang, Li, & Xie, 2013). Thus, it is important for patients to avoid the damage that occurs with the use of immunosuppressive drugs. Nutritional supplements and dietary changes are deemed crucial to regulating immune responses (Keusch, 2003; Maslowski & Mackay, 2011). Here we investigated the effects of HT-HAP on both tumor growth and immunity using a xenograft CT26 tumor model in normal Balb/c mice that are widely used in tumor and immunological studies (Anderson, & Grey, 1974; Besedovsky, & Sorkin, 1974; Hori, Nomura, & Sakaguchi, 2003; Song, Baik, Hong, & Sung, 2016; Yuan, Song, Li, Li, & Dai, 2006; Zhang et al., 2012). We report that HT-HAP inhibits tumor growth, yet promotes immunity, in tumor-bearing mice. Furthermore, we identified the effective components and a signaling molecule related to these activities. Even though the anti-tumor activity of HT-HAP may result from immune-enhancement, we believe that this is unlikely, primarily because HT-HAP-P can enhance immunity and not anti-tumor activity, whereas HT-HAP-S exhibited less immune stimulating activity and better anti-tumor activity than HT-HAP-P. As further confirmation, we investigated cytokine expression in tumors. ELISA results show that both HAP and HT-HAP have little effect on IL-12 and IFN-γ expression (Supplementary Fig. 1). Our previous study with heat-modified citrus
5. Conclusion Our results demonstrate that HT-HAP plays an inhibitory role on CT26 tumor growth and an enhanced effect on immunity. The inhibitory effect on tumor was exerted by small molecules in HT-HAP while the enhancement effect on immunity was primarily exerted by pectin fragments in HT-HAP. The effects of HT-HAP on tumor and immunity are correlated with the Akt activation, which might be the underlying mechanism of action. The protective effect of HT-HAP on immunity in tumor-bearing mice prompts us to propose that ingestion of heat-modified pectin in the daily diet may be beneficial to cancer patients being treated with immunosuppressive drugs. Our study also suggests that the unfractionated sample may have an advantage over individual components, because the mixture exerts multiple actions. 6. Conflict of interest The authors declare no conflict of interest. 7. Ethics statements The research did not include any human subjects. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals. Acknowledgment This work was supported by the University S&T Innovation Platform 197
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Fig. 7. Akt activation in tumors, spleen and thymus. The tumor, spleen and thymus from mice treated with HT-HAP, HT-HAP-P or HT-HAP-S were lysed and subjected to western blotting. (A, C and E) representative immunoblots. (B, D and F) densitometric analysis of the ratio of p-Akt/Akt. Each value represents the mean ± SD of four animals in each group. *P < 0.05, **P < 0.01 compared to each control group (one-way ANOVA followed by LSD's test).
of Jilin Province for Economic Fungi (#2014B-1), the Fundamental Research Funds for the Central Universities (No.: 2412016KJ044), the National Natural Science Foundation of China (Nos. 31370805 and 31470798). We are grateful to Prof. Kevin Mayo for critical reading and editing of this manuscript.
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Components of heat-treated Helianthus annuus L. pectin inhibit tumor growth and promote immunity in a mouse CT26 tumor model ⁎
Yuan Guan, Zhongyu Zhang, Xiyao Yu, Jingmin Yan, Yifa Zhou, Hairong Cheng , Guihua Tai
T ⁎
Jilin Province Key Laboratory on Chemistry and Biology of Changbai Mountain Natural Drugs, School of Life Sciences, Northeast Normal University, Changchun 130024, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Akt Anti-tumor Heat-treated pectin Promote immunity
Heat-treated pectin is known to have anti-tumor activity. The present study was designed to identify its active components and mechanism of action. Here, we investigated the anti-tumor effect of heat-treated Helianthus annuus L. pectin (HT-HAP), alkali-inactivated HT-HAP representing the pectin fragment fraction (HT-HAP-P), and heat-treated galacturonic acid representing the small molecule fraction (HT-HAP-S). Our results demonstrate that HT-HAP induces colon cancer CT26 cells apoptosis and inhibits CT26 tumor growth like 5-fluorouracil, whereas it shows no spleen and thymus toxicity in contrast to 5-fluorouracil. Mechanistically, HT-HAP attenuates Akt activation in tumors, and enhances it in the spleen and thymus. Interestingly, HT-HAP-S inhibited tumor growth, but promoted immunity mildly, whereas HT-HAP-P had little effect on tumor growth, but effectively promoted immunity. Overall, we attribute the anti-tumor effect from HT-HAP to the action of small molecules and the immune enhancement primarily to pectin fragments, with Akt activation being the underlying mechanism of action.
1. Introduction Colorectal cancer is the third most commonly diagnosed cancer and the fourth cause of cancer death worldwide (Ferlay et al., 2010; Siegel, DeSantis, & Jemal, 2014). Several epidemiological studies have proven the association of colon cancer with dietary habit, in particular the consumption of pectin in fruits and vegetables (Rose, DeMeo, Keshavarzian, & Hamaker, 2007; Slavin & Lloyd, 2012; Trock, Lanza, & Greenwald, 1990; Young, Hu, Le Leu, & Nyskohus, 2005). Pectin, a family of complex polysaccharides rich in galacturonic acid (GalA), has a complex structure composed of various elements and domains. The most abundant structure in pectin is homogalacturonan (HG), a linear chain of α1,4-linked-D-GalA with esterification or acetylation of some residues. The second most abundant structure, type I rhamnogalacturonan, has a backbone of repeating disaccharide units of [-α-D-GalA-1,2α-L-Rha-1,4-]n that can also be substituted with arabinan, galactan or arabinogalactan side chains. Pectin may also contain type II rhamnogalacturonan (RG-II), apigalacturonan, and/or xylogalacturonan. All of these domains have a galacturonan backbone (Caffall & Mohnen, 2009; Schols & Voragen, 1996; Yapo, 2011). As a dietary fiber, pectin is known to play a role in the prevention of colon cancer (Rose et al., 2007; Trock et al., 1990). Heat-modified pectin has been shown to possess additional activities. For example,
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heat-treated citrus pectin (HTCP) induces significant apoptosis in androgen-responsive and androgen-independent human prostate cancer cells and induces cell apoptosis and autophagy in HepG2 and A549 cells (Jackson et al., 2007; Leclere et al., 2015). Similarly, heat-modified ginseng pectin exhibits more significant anti-proliferative effects on human colon cancer cells compared to its un-modified counterparts (Cheng et al., 2011). Koyama et al. reported that heat-treatment of uronic acid or polysaccharides containing uronic acid residues generates a small bioactive molecule 4,5-dihydroxy-2-cyclopenten-1-one (DHCP) that promotes apoptosis of HL-60 cancer cells and is cytotoxic to other cancer cells (Koyama et al., 2000; Leclere et al., 2016). However, the function of DHCP has yet to be demonstrated in animal models. In addition to DHCP or DHCP-like small molecules, heattreatment also degrades pectin into fragments of various lengths. The effectiveness of these fragments remains unclear. Helianthus annuus L. (Sunflower) is a traditional industrial crop from which only the seeds, but not the heads, are utilized. To make use of the discarded head products and increase the economic value of this crop, we have extracted pectin from the Helianthus annuus L. heads that are rich of HG pectin (Peng et al., 2016). In this study, we heat-modified the pectin and investigated its anti-tumor efficacy on colon cancer cells and in a colon cancer model in mice.
Corresponding authors. E-mail addresses: [email protected] (H. Cheng), [email protected] (G. Tai).
https://doi.org/10.1016/j.jff.2018.07.001 Received 1 February 2018; Received in revised form 20 June 2018; Accepted 1 July 2018 1756-4646/ © 2018 Published by Elsevier Ltd.
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2. Materials and methods
well. Twenty-four hours later, media were removed and fresh media containing various concentrations of pectin were added. After 48 h, the MTT assay was used to assess cell viability.
2.1. Materials The heads of Helianthus annuus L. were collected from Changbai mountain area in Jilin Province, PR China. The enzyme Endo-PG was purchased from Sigma Aldrich (St Louis, MO, USA). RPMI1640 medium and fetal bovine serum were obtained from Gibco (Grand Island, NY, USA). An antibody to actin (Cat. No. 612657) was purchased from BD Biosciences (San Diego, CA). Antibodies to p38 (Cat. No. 9212), p-p38 (Cat. No. 9215), p-ERK (Cat. No. 9101), ERK (Cat. No. 9102), JNK (Cat. No. 9252), p-JNK (Cat. No. 9255), Akt (Cat. No. 4691), p-Akt (Cat. No. 9271) and Ki67 (Cat. No. 12202) were purchased from Cell Signaling Technology (Beverly, MA). Mouse interleukin-12 (IL-12, Cat. No. E-ELM0726c) and interferon-γ (IFN-γ, Cat. No. E-EL-M0048c) were purchased from Elabscience Biotechnology Co. Ltd (Wuhan, China). Other reagents were of analytical grade or better.
2.6. Flow cytometry assay Cells treated with different concentrations of pectin for 48 h were stained with the Annexin V-FITC/PI kit (Keygen Biotechnology, Jiangsu, China) according to the manufacturer’s instructions. 2.7. In vivo assays Specific-pathogen-free female Balb/c mice, age 6–8 weeks and weighing 16–18 g, were obtained from Beijing HFK Bioscience Co. Ltd (Beijing, China). All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals. Mice were housed under pathogen-free conditions and allowed access to food and water ad libitum. Animals were randomly divided into 10 groups (n = 6 per group). Then 1 × 105 of CT26 cells were injected subcutaneously into the right axilla of the mice. On day 2 following injection, HAP, HT-HAP, HTHAP-P or HT-HAP-S was administered intragastrically daily for 20 days at the dosages of 150, 300, 110 or 220 mg/kg body weight (BW), as indicated. 5, 10, 15 mg/kg BW of 5-FU was applied by intraperitoneal injection every other day. The control group was administered distilled water. After 20 days, the mice were sacrificed, and the tumor, spleen and thymus were dissected, weighed and lysed for western blotting analyses or subjected to immunohistochemistry sectioning, respectively. The tumor inhibition rate and spleen and thymus index were calculated as follows:
2.2. Preparation of HAP, HT-HAP, HT-HAP-S and HT-HAP-P HAP was prepared according to the method described by our group, with some modifications (Peng et al., 2016). Briefly, the heads of Helianthus annuus L. were extracted with 0.2% oxalic acid (solid: liquid ratio 1:20, w/v) at 100 °C for 1 h and precipitated with 60% aqueous ethanol. After centrifugation, the pectin was re-dissolved in distilled water and obtained by spray drying. HT-HAP was prepared as previously reported with some modifications (Hao et al., 2013; Jackson et al., 2007). Briefly, the HAP was dissolved in distilled water at the concentration of 5 mg/mL and treated with Endo-PG at 37 °C for 12 h. Then the solution was heated at 121 °C for 1 h for 4 times. The product was lyophilized and stored at −20 °C. HT-HAP-S was prepared from galacturonic acid (GalA, purchased from Sigma Aldrich, St Louis, MO, USA) by treatment of GalA solution (5 mg/mL in distilled water) at 121 °C for 1 h for 4 times as described above. HT-HAP-P was prepared as previously reported with minor modifications (Jackson et al., 2007). HT-HAP was dissolved in distilled water at the concentration of 10 mg/mL, and the pH was adjusted to 12.0 with ammonia, then 7.0 with acetic acid. The solution was then evaporated and lyophilized to give HT-HAP-P.
Tumor inhibition rate(%) = (Wc−Wt)/Wc × 100% where Wc is the tumor weight of the control group, and Wt is the tumor weight of the test group. Spleen or Thymus Index (mg/g) = Weight of spleen or thymus/ Body weight. Frozen tumor sections (8 μm) were subjected to immunostaining using Ki67 antibody and Hoechst 33342 as described previously (Chen, Huang, Huang, Tseng, & Tseng, 2006). Macrophages and NK cells were obtained from the peritoneal cavity and spleen of the tumor-bearing mice, respectively. Macrophage phagocytosis and NK cell cytotoxicity assays were performed as reported previously (Sheng et al., 2017).
2.3. Physicochemical properties of HAP, HT-HAP, HT-HAP-S and HTHAP-P
2.8. Western blotting
Monosaccharide composition was determined by using high performance liquid chromatography (HPLC) as described by Zhang et al. (2009). Molecular weight was measured using a TSK-gel G-3000PWxL column (7.8 × 300 mm, TOSOH, Japan) connected to a Shimadzu HPLC system, as described by Zhang et al. (2009). For detection of the active compound (DHCP), samples dissolved in elution buffer (5% methanol and 0.05% trifluoroacetic acid) were applied onto a Kromasil C18 column (4.6 × 250 mm) connected to a Shimadzu HPLC system. Material was eluted at a flow rate of 0.6 mL/ min and monitored by UV at 235 nm (Aoyagi, Ishii, Ugwu, & Tanaka, 2008).
Cells treated with HAP, HT-HAP, HT-HAP-P and HT-HAP-S were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.5% Triton X-100, 150 mM sodium chloride, 0.1 mM PMSF, and Roche incomplete protease inhibitor cocktail). Tumor, spleen or thymus proteins were lysed by grinding the tissue in lysis buffer. Equal amounts of protein were separated by using 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membranes, blotted with specific antibodies (dilution of 1:5000 for the actin antibody and 1:1000 for all other antibodies), and detected following incubation with enhanced chemiluminescence (ECL) reagent using a chemiluminescence detection system (MiniChemi, Beijing Sage Creation Science Co., Ltd). The bands were quantified by densitometry using Lane 1D software (MiniChemi, Beijing Sage Creation Science Co., Ltd).
2.4. Cell culture The mouse colon cancer cell line CT26 was purchased from ATCC and cultured in RPMI 1640, supplemented with 10% fetal bovine serum, 100 U/mL of penicillin and 100 μg/mL of streptomycin. Cells were cultured at 37 °C in a 5% CO2 incubator.
2.9. ELISA assay Tumors of the mice were ground and lysed in lysis buffer. IL-12 and IFN-γ from the tumors of the mice in each group were analyzed by commercially available ELISA kits according to the manufacturer’s
2.5. Cell viability assay CT26 cells were plated in 96-well plates (Costar) at 1 × 104 cells/ 191
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Fig. 1. Effect of HT-HAP on tumor growth and immune organ toxicity in tumor bearing mice. Mice treated with HAP, HT-HAP or 5-FU were sacrificed. The tumor weight (A), body weight (D), spleen and thymus index (E, F) were analyzed. Ki67 expression in tumors was tested with immunostaining (B). Bar, 50 μm. For Ki67 quantification (C), five sections from each of the three tumors per group were analyzed, with the value representing the mean ± SD, n = 15. Data in A, D, E and F were in a normal distribution, and each value represents mean ± SD of six animals in each group. The data of tumor weight, spleen and thymus index were analyzed by using the one-way ANOVA, followed by LSD's test. The data of body weight was analyzed by using the one-way ANOVA followed by Dunnett’s T3. Data in C was in non-normal distribution, and was analyzed by Kruskal–Wallis test, followed by the Mann-Whitney U test. *P < 0.05, **P < 0.01 compared to each control group.
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of these side effects with HT-HAP (Fig. 1D–F). On the other hand, the administration of HT-HAP at 300 mg/kg BW did significantly increase the thymus index to the level observed in the non-tumor (normal) group. Thus, the use of HT-HAP appears to be better than 5-FU in terms of their effects on the spleen and thymus in tumor bearing mice.
instructions. 2.10. Statistical analysis All experiments were performed at least three times, and SPSS 21.0 (IBM Corporation, NY, USA) statistical software program was used for all data analyses. The normal distribution of the data was analyzed by using the Shapiro-Wilk test (Stephens, 1965). Results were expressed as the mean ± SD. Single factor analysis of variance was performed using ANOVA, followed by a post hoc LSD's test when the data were normally distributed and exhibited a homogeneous variance. For data that did not show a homogeneous variance, Dunnett’s T3 analysis (Dunnett, 1980) was used as the post hoc test. Non-normally distributed data were analyzed by using Kruskal–Wallis (Kruskal & Wallis, 1952) followed by Mann-Whitney U test (Mann & Whitney, 1947). Statistical significance was defined as p < 0.05 (*), or p < 0.01(**).
3.2. Heat generated small molecule(s) accounted for the anti-tumor activity both in in vitro and in vivo Heat treatment is known to degrade HG into fragments, oligosaccharides and monosaccharides (GalA), which we refer to here as the ‘pectin fragment fraction’. The resulting GalA can be further modified into UV 235 nm-positive small molecules (e.g. DHCP) (Aoyagi et al., 2008; Cheng et al., 2011; Koyama et al., 2000; Leclere et al., 2016) that we refer to as the ‘small molecule fraction’. In order to identify the effective component(s) in HT-HAP, we prepared two samples: HT-HAPP prepared by alkaline treatment of HT-HAP to disrupt the UV 235 nmpositive small molecule(s) (Jackson et al., 2007) that represents the pectin fragment fraction, and HT-HAP-S, prepared by heat treatment of GalA, that represents the small molecule fraction. Size distribution of these samples determined by HPGPC (High-Performance Gel Permeation Chromatography) is shown in Fig. 2A. Consistent with the degradative effect of endo-PG and heat treatment, HT-HAP and HT-HAP-P displayed a major peak at the elution position of GalA, and peaks between 12 min and 16 min correspond to fragments and oligosaccharides. The 235 nm-positive small molecules were assessed by using HPLC with a C18 column. As shown in Fig. 2B, HT-HAP and HT-HAP-S displayed an obvious peak of DHCP at 6.7 min, whereas HAP and HT-HAPP showed no such peak. Monosaccharide composition analyses showed that HAP, HT-HAP, HT-HAP-P, and HT-HAP-S were all primarily composed of GalA (79.9%, 71.5%, 72.1% and 96.5%, respectively), with HAP, HT-HAP, and HT-HAP-P also containing small amounts of rhamnose, glucose, arabinose, mannose, xylose and glucuronic acid residues (Table 1). The MTT assay showed that HT-HAP and HT-HAP-S significantly suppressed cell viability in a concentration-dependent manner, whereas HAP and HT-HAP-P had no effect (Fig. 3A). The cell viability curves of
3. Results 3.1. HT-HAP inhibits tumor growth with no spleen and thymus toxicity Mice implanted with CT26 tumors were used to study the antitumor activities of HAP (untreated pectin) and HT-HAP (heat-treated HAP) in vivo. 5-FU, a chemotherapeutic usually given to colon cancer patients, was used as the positive control. As shown in Fig. 1A, HT-HAP inhibited tumor growth by 22.0% and 42.2%, whereas HAP had negligible effect (9.1% and 4.7%) at 150 and 300 mg/kg BW, respectively. The anti-tumor effect from HT-HAP at 300 mg/kg BW was similar to that from 10 mg/kg BW of 5-FU (43.4%) (Fig. 1A). The expression of Ki67, an indication of cell proliferation, was also similar in the two groups, with both being remarkably less than control or HAP groups (Fig. 1B–C). Thus, HT-HAP exerts an inhibitory effect on tumor growth in vivo. Side effects are an important issue associated with the use of chemotherapeutic drugs. We observed a significant decrease in body weight (Fig. 1D), spleen index (Fig. 1E), and thymus index (Fig. 1F), with the administration of 10 mg/kg BW 5-FU. In contrast, we saw none
Fig. 2. Assessment of HAP, HT-HAP, HT-HAP-P and HT-HAP-S. (A) elution profiles on a TSK-gel G-3000PWxL column with RI detection. Vo, void volume. GalA, the elution position of GalA. *The elution position of NaCl added to the sample. (B) elution profiles on Kromasil C18 column with UV detection at 235 nm. The molecular formula and elution peak of DHCP are indicated. 193
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HT-HAP and HT-HAP-S were identical when plotted against the contents of DHCP in each sample (Fig. 3B). Flow cytometry demonstrated that HT-HAP and HT-HAP-S induced apoptosis, whereas HAP and HTHAP-P did not (Fig. 3C). The observed apoptosis was caspase-independent, because it could not be abrogated by using the pan-caspase inhibitor z-VAD-fmk (Fig. 3C). The caspase-independent nature of DHCP-induced apoptosis was also reported previously (Leclere et al., 2015). Quantification of these data from three independent experiments showed similar results (Fig. 3 D), suggesting that heat-generated small molecule(s) accounted for the anti-tumor effect in vitro. Similarly, our in vivo studies showed that HT-HAP-S inhibited tumor
Table 1 Monosaccharide composition of HAP, HT-HAP, HT-HAP-P and HT-HAP-S. Sample
HAP HT-HAP HT-HAP-P HT-HAP-S
Monosaccharide composition (mol %) GalA
Rha
Glc
Ara
Gal
Man
Xyl
GlcA
Fuc
79.9 71.5 72.1 96.5
4.8 6.7 8.0 –
4.1 4.6 4.2 –
3.1 4.0 4.1 –
3.6 4.4 3.5 1.1
1.7 2.6 3.0 –
2.3 2.5 2.6 2.3
0.4 2.1 1.7 –
– 1.7 0.8 –
Fig. 3. Analyses of the effective anti-tumor components of HT-HAP in vitro. (A–B) cell viability as determined by the MTT assay. CT26 cells were treated with HAP, HT-HAP, HT-HAP-S or HT-HAP-P at the indicated concentrations (A) or DHCP contents (B). (C) cell apoptosis was determined by using flow cytometry. CT26 cells were pretreated with or without z-VAD-fmk and then treated with indicated concentrations of HAP, HT-HAP, HT-HAP-S or HT-HAP-P. (D) statistic data of C. Each value represents the mean ± SD of three independent experiments. **P < 0.01 compared to the 0 mg/mL group (one-way ANOVA followed by LSD's test).
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Fig. 4. Analyses of the effective components of HT-HAP in vivo. Mice were treated with HT-HAP, HT-HAP-P or HT-HAP-S as indicated. The tumor weight (A), spleen index (B), thymus index (C), NK cell cytotoxicity (D) and phagocytic rate of macrophage (E) were determined. In A–C, each value represents the mean ± SD of six animals in each group. In D and E, each value represents the mean ± SD of triplicate. Data in C were in a non-normal distribution and analyzed by using the Kruskal–Wallis followed by Mann-Whitney U test. Other data were in normal distributions and analyzed by using the one-way ANOVA followed by LSD's test. * P < 0.05, **P < 0.01 compared to control group.
cytotoxity and macrophage activation by HT-HAP-P was greater than that elicited by HT-HAP-S. These data indicate that both the pectin fragments and small molecules can promote an immune response, with the former being more effective than the later.
growth by 21.8% and 46.8% at dosages of 110 and 220 mg/kg BW (where the DHCP content was equivalent to that of 150 and 300 mg/kg BW HT-HAP), respectively, whereas HT-HAP-P exerted only a 14.6% and 11.9% inhibition at doses of 150 and 300 mg/kg BW, respectively (Fig. 4A). This confirmed our conclusion that heat-generated small molecule(s) accounted for the anti-tumor effect.
3.4. Opposing effects of HT-HAP on Akt activation in tumors and immune organs
3.3. Pectin fragments promote immunity more than small molecules To further understand the effects of HT-HAP on tumor growth and immunity, we investigated some signaling molecules frequently involved in cell proliferation and/or apoptosis (Kong, Yu, Chen, Mandlekar, & Primiano, 2000; Wu, 2007; Zhang et al., 2013). The in vitro assay showed that HT-HAP and HT-HAP-S, both of which had antitumor activity, down-regulated Akt, but up-regulated JNK, ERK and p38 phosphorylation in CT26 cells in a time-dependent manner (Fig. 5). In contrast, HAP and HT-HAP-P, both of which exhibited no anti-tumor activity, showed no obvious effects (Fig. 5). It appears that Akt
HT-HAP, HT-HAP-P (representing the pectin fragment fraction) and HT-HAP-S (representing the small molecule fraction) were also compared for their effects on the immunity in tumor-bearing mice. Our results show that all three samples had no significant effect on the spleen index compared to control group (Fig. 4B). However, all of these samples significantly increased the thymus index and NK cytotoxity (Fig. 4C and D). HT-HAP and HT-HAP-P also significantly increased macrophage activation (Fig. 4E). Moreover, the increase in NK 195
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Fig. 5. Akt and MAPK activation in CT26 cells. CT26 cells were treated with HAP, HT-HAP, HT-HAP-P or HT-HAP-S for the indicated times. Whole-cell extracts were prepared and subjected to western blotting using specific antibodies. (A) representative immunoblots. (B) densitometric analysis of the ratio of p-Akt/Akt, p-ERK/ ERK, p-JNK/JNK and p-p38/p38. Each value represents the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 compared to each 0 h group (oneway ANOVA followed by LSD's test).
activity) produced a robust decrease in Akt phosphorylation in CT26 tumors, whereas HT-HAP-P (no anti-tumor activity) exhibited no obvious changes in Akt phosphorylation (Fig. 7A–B). Collectively, both in vitro and in vivo data indicate that inhibition of Akt activation is a mechanism for the inhibition effect of HT-HAP on tumors. The spleen and thymus of tumor-bearing mice were lysed and subjected to western blot analysis. In the HT-HAP group, Akt activation was increased in both spleen and thymus (Fig. 7C–F), in contrast to the decrease of Akt activation in tumors. Furthermore, HT-HAP-P and HTHAP-S, both of which showed immune-enhancing activity, also promoted Akt activation in the spleen and thymus, although to different extents (Fig. 7C–F). Thus, the increase of Akt activation is a mechanism
inactivation and MAPK activation are related to the anti-tumor effect. Consistent with these in vitro data, Akt activation in CT26 tumors in mice was inhibited by HT-HAP (with anti-tumor activity), but not by HAP (without anti-tumor activity) (Fig. 6). However, the involvement of MAPKs was excluded by this in vivo study (Fig. 6) for two reasons: (1) phosphorylation of ERK in tumors was up-regulated by both HAP (without anti-tumor activity) and HT-HAP (with anti-tumor activity), albeit to different levels, and (2) p38 phosphorylation was largely unchanged, while JNK phosphorylation was decreased in tumors in contrast to results from our in vitro cell studies. Thus, the inhibition of Akt activation could be a mechanism for HT-HAP’s anti-tumor action. Supporting this proposal, we found that HT-HAP-S (with anti-tumor 196
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Fig. 6. Akt and MAPK activation in CT26 tumors. Tumors of mice treated with HAP or HT-HAP were dissected, lysed and subjected to western blotting. (A) representative immunoblots. (B) densitometric analysis of the ratio of p-Akt/Akt, p-ERK/ERK, p-JNK/JNK and p-p38/p38. Each value represents the mean ± SD of four tumors in each group. *P < 0.05, **P < 0.01 compared to each control group (one-way ANOVA followed by LSD's test).
pectin also showed no correlation between immune-enhancement and anti-tumor activity in a mouse sarcoma 180 tumor model (Hao et al., 2013). However, these studies did not exclude the possibility that, in a long term, enhanced immunity may help the body combat cancer.
of action for the HT-HAP-mediated enhancement of immunity in mice. 4. Discussion Chemotherapeutic drugs are widely used in the treatment of various cancers; however their therapeutic effectiveness is usually accompanied by adverse side effects (Chabner & Roberts, 2005; Dezern et al., 2013; Verstappen, Heimans, Hoekman, & Postma, 2003). Chemotherapeutic drugs, particularly when used in high-doses and for long periods of time, can therefore lead to a decline in body weight, splenocyte proliferation, organ index, macrophage phagocytosis and natural killer (NK) cell activity (Dezern et al., 2013; Hayashi, Nakano, Hashimoto, Kanekiyo, & Hayashi, 2008; Zhang, Nie, Huang, Li, & Xie, 2013). Thus, it is important for patients to avoid the damage that occurs with the use of immunosuppressive drugs. Nutritional supplements and dietary changes are deemed crucial to regulating immune responses (Keusch, 2003; Maslowski & Mackay, 2011). Here we investigated the effects of HT-HAP on both tumor growth and immunity using a xenograft CT26 tumor model in normal Balb/c mice that are widely used in tumor and immunological studies (Anderson, & Grey, 1974; Besedovsky, & Sorkin, 1974; Hori, Nomura, & Sakaguchi, 2003; Song, Baik, Hong, & Sung, 2016; Yuan, Song, Li, Li, & Dai, 2006; Zhang et al., 2012). We report that HT-HAP inhibits tumor growth, yet promotes immunity, in tumor-bearing mice. Furthermore, we identified the effective components and a signaling molecule related to these activities. Even though the anti-tumor activity of HT-HAP may result from immune-enhancement, we believe that this is unlikely, primarily because HT-HAP-P can enhance immunity and not anti-tumor activity, whereas HT-HAP-S exhibited less immune stimulating activity and better anti-tumor activity than HT-HAP-P. As further confirmation, we investigated cytokine expression in tumors. ELISA results show that both HAP and HT-HAP have little effect on IL-12 and IFN-γ expression (Supplementary Fig. 1). Our previous study with heat-modified citrus
5. Conclusion Our results demonstrate that HT-HAP plays an inhibitory role on CT26 tumor growth and an enhanced effect on immunity. The inhibitory effect on tumor was exerted by small molecules in HT-HAP while the enhancement effect on immunity was primarily exerted by pectin fragments in HT-HAP. The effects of HT-HAP on tumor and immunity are correlated with the Akt activation, which might be the underlying mechanism of action. The protective effect of HT-HAP on immunity in tumor-bearing mice prompts us to propose that ingestion of heat-modified pectin in the daily diet may be beneficial to cancer patients being treated with immunosuppressive drugs. Our study also suggests that the unfractionated sample may have an advantage over individual components, because the mixture exerts multiple actions. 6. Conflict of interest The authors declare no conflict of interest. 7. Ethics statements The research did not include any human subjects. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals. Acknowledgment This work was supported by the University S&T Innovation Platform 197
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Fig. 7. Akt activation in tumors, spleen and thymus. The tumor, spleen and thymus from mice treated with HT-HAP, HT-HAP-P or HT-HAP-S were lysed and subjected to western blotting. (A, C and E) representative immunoblots. (B, D and F) densitometric analysis of the ratio of p-Akt/Akt. Each value represents the mean ± SD of four animals in each group. *P < 0.05, **P < 0.01 compared to each control group (one-way ANOVA followed by LSD's test).
of Jilin Province for Economic Fungi (#2014B-1), the Fundamental Research Funds for the Central Universities (No.: 2412016KJ044), the National Natural Science Foundation of China (Nos. 31370805 and 31470798). We are grateful to Prof. Kevin Mayo for critical reading and editing of this manuscript.
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