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. 2016 Apr 28;12(6):746-56.
doi: 10.7150/ijbs.13988. eCollection 2016.

Chemoprevention of Low-Molecular-Weight Citrus Pectin (LCP) in Gastrointestinal Cancer Cells

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Free PMC article

Chemoprevention of Low-Molecular-Weight Citrus Pectin (LCP) in Gastrointestinal Cancer Cells

Shi Wang et al. Int J Biol Sci. .
Free PMC article

Abstract

Background & aims: Low-molecular-weight citrus pectin (LCP) is a complex polysaccharide that displays abundant galactosyl (i.e., sugar carbohydrate) residues. In this study, we evaluated the anti-tumor properties of LCP that lead to Bcl-xL -mediated dampening of apoptosis in gastrointestinal cancer cells.

Methods: We used AGS gastric cancer and SW-480 colorectal cancer cells to elucidate the effects of LCP on cell viability, cell cycle and apoptosis in cultured cells and tumor xenografts.

Results: Significantly decreased cell viabilities were observed in LCP treated AGS and SW-480 cells (P<0.05). Cell cycle-related protein expression, such as Cyclin B1, was also decreased in LCP treated groups as compared to the untreated group. The AGS or SW-480 cell-line tumor xenografts were significantly smaller in the LCP treated group as compared the untreated group (P<0.05). LCP treatment decreased Galectin-3 (GAL-3) expression levels, which is an important gene in cancer metastasis that results in reversion of the epithelial-mesenchymal transition (EMT), and increased suppression of Bcl-xL and Survivin to promote apoptosis. Moreover, results demonstrated synergistic tumor suppressor activity of LCP and 5-FU against gastrointestinal cancer cells both in vivo and in vitro.

Conclusions: LCP effectively inhibits the growth and metastasis of gastrointestinal cancer cells, and does so in part by down-regulating Bcl-xL and Cyclin B to promote apoptosis, and suppress EMT. Thus, LCP alone or in combination with other treatments has a high potential as a novel therapeutic strategy to improve the clinical therapy of gastrointestinal cancer.

Keywords: Low-molecular-weight citrus pectin (LCP); apoptosis; caspases; epithelial- mesenchymal transition (EMT).; gastrointestinal cancer cells.

Conflict of interest statement

Conflicts of interest: This study is not related to any potentially competing financial or other interests.

Figures

Figure 1
Figure 1
Cytotoxicity of LCP on both AGS gastric cancer and SW-480 colorectal cancer cell growth in vitro. 1A, AGS and SW-480 cells were treated with different concentration of LCP for 24 hrs and the cell viability was measured by MTT method. The percentage of inhibition on cell viability with LCP treatment demonstrated a concentration dependent manner and having significant difference as compared with parallel untreated cells, respectively (*: P<0.05). 1B, AGS and SW-480 cells were treated with different concentration of 5-FU for 24 hrs and the cell viability was measured by MTT method. The percentage of inhibition on cell viability with 5-FU treatment also demonstrated a concentration dependent manner and having significant difference as compared with parallel untreated cells, respectively (*: P<0.05). Moreover, both cell-lines were relatively more sensitive to 5-FU treatment compared to that treated by LCP. 1C, Compared with the control group (Negative), there were significant effects of single LCP (5.0 mg/ml) or single 5-FU (200 µM) or their combination (5.0 mg/ml LCP + 200 µM 5-FU) treatment on both AGS and SW-480 cells, respectively (* P<0.05). The inhibitory ability of both AGS and SW-480 cells by their combination (5.0 mg/ml LCP + 200 µM 5-FU) was higher than that by single 5-FU (200 µM) treatment, but there was no significant difference between them (P>0.05).The comparison of the percentage of inhibition on cell viability among 5.0 mg/ml LCP, 200 μM 5-FU and their combination (5.0 mg/ml LCP + 200 μM 5-FU) treatment. The inhibitory ability of both AGS and SW-480 cells by their combination LCP (5.0 mg/ml) and 5-FU (200 μM) or single 5-FU (200 μM) was significantly increased than that mediated by single LCP (5 mg/ml) (**: P<0.05). 1D, The effect of LCP on cell proliferation in AGS and SW-480 cells was tested by colony assay. 10 days after a different range of LCP treatment, each colony which contained more than 50 cells was considered to represent a viable clonogenic cell. The inhibitory ability of colony formation showed a concentration dependent manner. All assays represented the mean ± SD of two independent experiments with triplicate dishes.
Figure 2
Figure 2
Flow cytometry analysis of cell cycle in AGS and SW-480 cells after LCP treatment. The percentage of phase population of cell cycle in AGS cells with different concentration of LCP treatment for 24 hrs (2A-1, 0 mg/ml; 2A-2, 5.0 mg/ml; 2A-3, 10.0 mg/ml LCP). Flow cytometry analysis of the phase population of AGS cells (2B-1) and SW-480 cells (2B-2) after LCP treatment for 24 hrs, showing the effect of different concentrations of LCP on the cell cycle. 2B-3, Effect of different concentration of 5-FU or the combination of 5-FU and LCP on the cell cycle in AGS cells. All assays represented the mean ± SD of triplicate independent experiments.
Figure 3
Figure 3
Western blot analysis of the expression of cell cycle-related enzymes (Cyclin A and Cyclin B1) in AGS cells (3A) and SW-480 cells (3B) according to different concentration of LCP treatment. 5-FU treatment was used as a comparison. The amount of protein was normalized by comparing the intensity of the α-tubulin band.
Figure 4
Figure 4
Effect of LCP on tumor xenografts growth. 4A, shows the tumor mass (g) that was measured on the final experiment day immediately after the tumor tissue was removed from the mouse by surgical excision. The average tumor mass is indicated as a bold bar in each group. P value was compared with untreated group (Negative). The average tumor weight of LCP treated group was significantly smaller than that of untreated control group, being similar to 5-FU treated group. 4B, during this period, each mouse was manually examined for body weight every week and there were not significant differences between untreated group mice and treated group mice. All experiments represented the mean ± SD of triplicate independent experiments.
Figure 5
Figure 5
Effect of LCP treatment on Galcetin-3 and EMT-related gene expression. Alteration of Galcetin-3 and EMT markers expression after different concentration of LCP treatment for 24 hrs in AGS cells (5A) and SW-480 cells (5B). The amount of protein was normalized by comparing the intensity of the α-tubulin band. 5C, In AGS and SW-480 xenograft nude mice experiment, when the tumor was measurable, mice were treated daily with 5-FU at 25 mg/kg by i.p. injection, or different dose of LCP by oral gavage, or by their combination, respectively. Results showed that 5.0% (wt/vol) LCP treatment significantly alters the expression of Galcetin-3, E-cadherin and Twist at mRNA level as compared with controls (all P<0.05). GAPDH was used as reference. All experiments represented the mean ± SD of triplicate independent experiments.
Figure 6
Figure 6
Effect of LCP on apoptosis in gastrointestinal cancer cells. The expression of apoptotic-related protein levels which including two anti-apoptotic proteins (Bcl-xL and Survivin) and two pro-apoptotic proteins (Caspase-3 and Caspase-8) were determined by Western blot in AGS cells (6A) and SW-480 cells (6B) according to LCP (10.0 mg/ml) or 5-FU (200 μM) treatment. 6C, Immunohistochemical staining of Bcl-xL and TUNEL analysis of apoptosis in AGS xenograft tissues. Control group (without LCP treatment): ① Bcl-xL protein by IHC; ② Apoptosis by TUNEL. LCP treated group (10.0 mg/ml LCP): ③ Bcl-xL protein by IHC; ④ apoptosis by TUNEL.

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