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. 2018 Dec;39(12):1885-1893.
doi: 10.1038/s41401-018-0004-z. Epub 2018 May 16.

Modified Citrus Pectin Inhibited Bladder Tumor Growth Through Downregulation of galectin-3

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Free PMC article

Modified Citrus Pectin Inhibited Bladder Tumor Growth Through Downregulation of galectin-3

Tian Fang et al. Acta Pharmacol Sin. .
Free PMC article

Abstract

Modified citrus pectin (MCP) is a carbohydrate enriched complex, which has been implicated in cancer treatment and prevention. However, the effects of MCP on urinary bladder cancer (UBC) are unknown. In this study, MCP was first tested in T24 and J82 human UBC cells and showed the inhibition of cell viability by the sulforhodamine B (SRB) assay. The MCP-treated UBC cells exhibited G2/M phase arrest with the decrease of Cyclin B1 and phosphorylated Cdc2. Caspase-3 was also activated, leading to the cleavage of Caspase-3 and PARP. We further explored the possible molecular mechanisms upon MCP treatment in UBC cells. Reduction of galectin-3 was observed and followed with the inactivation of Akt signaling pathway. Of note, galectin-3 knockdown by RNA interference recapitulated the MCP-mediated anti-proliferation, cell cycle arrest and apoptosis. Moreover, oral administration of MCP to the T24 xenograft-bearing nude mice inhibited the tumor growth significantly (P < 0.05). Quantification analysis of immunohistochemistry staining for Ki67 and cleaved Caspase-3 confirmed the decrease of proliferation index (P < 0.05) and the increase of apoptosis index (P < 0.01) in 700 mg/kg MCP-fed UBC xenografts. Using the information from TCGA database, we revealed that the overexpression of galectin-3 was associated with high tumor grade with lymph node metastasis, poor overall survival in UBC patients. Considering the remarkable inhibitory effects of MCP on UBC cell proliferation and survival in vitro and in vivo mainly through galectin-3, which is upregulated in UBCs, MCP may become an attractive agent, as a natural dietary fiber, for prevention and therapy of UBCs.

Keywords: cell survival; galectin-3; modified citrus pectin; urinary bladder cancer; xenograft.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
MCP impaired the viability of UBC cells. a Morphological changes of T24 and J82 UBC cells upon MCP treatment for 48 h. b Effects of MCP on cell viability by the SRB assay. T24 and J82 UBC were treated with MCP at the indicated concentrations for 72 h. All experiments were independently repeated three times. Bars represented mean ± SD vs. vehicle control. **P < 0.01; ***P < 0.001
Fig. 2
Fig. 2
MCP induced G2/M cell cycle arrest in UBC cells. Effects of MCP on cell cycle distribution of T24 cells (a, b) and J82 cells (c, d). The cell cycle distribution was analyzed by FACS after propidium iodide staining and a representative experiment was shown (a, c). Quantification results from three independent experiments were presented (b, d). Bars represented mean ± SD. *P < 0.05; ***P < 0.001. (e) Effects of MCP on G2/M transition regulators by Western blotting assay
Fig. 3
Fig. 3
MCP induced apoptosis in UBC cells. a Caspase-3 activities in T24 and J82 cells treated with MCP at indicated concentrations for 48 h. Bars represented mean ± SD vs. vehicle control. *P < 0.05; **P < 0.01; ***P < 0.001. b Effects of MCP on apoptotic proteins by Western blotting assay. c Representative images showed morphological changes of MCP-treated T24 cells using Hoechst 33342 staining. Scale bars, 25 μm
Fig. 4
Fig. 4
MCP suppressed Galectin 3 expression and Akt signaling pathway. T24 and J82 cells were treated with MCP with the indicated concentrations for 48 h, followed by Western blotting assay
Fig. 5
Fig. 5
Galectin-3 knockdown induced cell cycle arrest and apoptosis in T24 cells. a Comparison of normalized galectin-3 mRNA expression levels in patients without lymph node metastasis (N0, n = 73) and with lymph node metastasis ( ≥ N1, n = 43) in the TCGA Nature 2014 cohort. b Kaplan-Meier plots of cumulative overall survival (OS) were calculated for patients with low and high levels of galectin-3 expression in TCGA Nature 2014 cohort (n = 129, left panel) and TCGA provisional cohort (n = 405, right panel). Lower quartile was determined as the cut-point. c Knockdown of galectin-3 by two different siRNAs (siGal3-1 and siGal3-2), using nontarget siRNA as a negative control (siNC). d Galectin-3 knockdown impaired the viability of UBC cells. eg Galectin-3 knockdown induced G2/M cell cycle arrest in UBC cells. Quantification of cell cycle distribution (e), a representative cell cycle distribution pattern (f) and expression levels of G2/M phase transition regulators (g) were shown for the T24 cells with galectin-3 siRNA treatment. h Galectin-3 knockdown triggered Caspase-3 activities in UBC cells. i Effects of galectin-3 knockdown on apoptotic proteins and Akt signalings by Western blotting assay. j Galectin-3 knockdown partially rescued the inhibitory effect of MCP on cell viability in T24 cells. 48 h after siGal3-1 or siNC transfection, 1% MCP was applied to T24 cells for additional 48 h. Cell viability was assessed using the SRB assay. Bars represented mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001
Fig. 6
Fig. 6
MCP inhibited the growth of UBC xenografts in vivo. Tumor volume (a) and body weight (b) of T24 xenograft-bearing mice with MCP treatment for 6 weeks. Three experimental groups, including 700 mg/kg MCP, 350 mg/kg MCP and Vehicle control, were measured. Photograph (c) and the average weights (d) of T24 xenografts harvested at the endpoint. e Representative images of IHC staining for Ki67, cleaved Caspase-3 (cv Caspase-3) and Galectin-3 on tissue sections from MCP and vehicle-treated T24 xenografts. f Proliferation index and apoptosis index were quantified by the positive IHC staining for Ki67 and cleaved Caspase-3, respectively. Bars represented the mean ± SD. n.s., P ≥ 0.05; *P < 0.05; **P < 0.01

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