Resveratrol inhibits invasion and metastasis of colorectal cancer cells via MALAT1 mediated Wnt/β-catenin signal pathway

Abstract

Resveratrol, extracted from Chinese herbal medicine Polygonum cuspidatum, is known to inhibit invasion and metastasis of human colorectal cancer (CRC), in which long non-coding Metastasis Associated Lung Adenocarcinoma Transcript 1 (RNA-MALAT1) also plays an important role. Using MALAT1 lentiviral shRNA and over-expression constructs in CRC derived cell lines, LoVo and HCT116, we demonstrated that the anti-tumor effects of resveratrol on CRC are through inhibiting Wnt/β-catenin signaling, thus the expression of its target genes such as c-Myc, MMP-7, as well as the expression of MALAT1. In detail, resveratrol down-regulates MALAT1, resulting in decreased nuclear localization of β-catenin thus attenuated Wnt/β-catenin signaling, which leads to the inhibition of CRC invasion and metastasis. This finding of ours surely provides important pre-clinical evidence supporting future use of resveratrol in prevention and treatment of CRC.

Figure 1. Overexpression of MALAT1 in human colorectal cancer tissues.

In situ hybridization was applied to investigate the MALAT1 expression in 60 paraffin-embedded tumor tissue and adjacent normal tissue samples of CRC patients.

Figure 2. Resveratrol inhibites the proliferation, migration and invasion of CRC LoVo cells.

(A): Twenty-two candidate Chinese medicine monomer anticancer drugs were tested for the effective inhibition doses on the MALAT1 expression in LoVo cells. (B): Correlation of resveratrol drug doses and growth inhibition in LoVo cells. Y axis: percentage of growth inhibition; X axis: resveratrol concentrations (µM). (C): Assessment and quantification of cell migration and invasion of LoVo cells. Values represent the number of migratory/invasive cells per 5 high power fields. *p<0.05 or **p<0.01, compared with control LoVo cells.

Figure 3. Resveratrol decreases the nuclear localization of β-catenin, levels of its downstream proteins, and represses MALAT1 expression in LoVo cells.

(A): Total or cell extracts of different cellular compartments from LoVo cells treated with resveratrol were probed for β-catenin. β-catenin protein levels were quantified. *p<0.05 or **p<0.01, compared with control group. (B): Total cell extracts of LoVo cells treated with resveratrol were probed for c-Myc, MMP-7 and the protein levels were quantified. *p<0.05 or **p<0.01, compared with control LoVo cells. (C): The expression levels of MALAT1 from LoVo cells treated with indicated doses of resveratrol were determined by real time PCR. The relative MALAT1 promoter activities in LoVo cells treated with indicated resveratrol doses were measured. *p<0.05 or **p<0.01, compared with control LoVo cells.

Figure 4. MALAT1 promotes the proliferation, invasion and migration of LoVo cells.

(A): In vitro growth of LoVo cells expressing MALAT1-shRNA, MALAT1-overexpression, empty vector vs. parental cells. (B): Assessment and quantification of cell migration and invasion of indicated LoVo cells in A. Values represent the number of migratory/invasive cells per 5 high power fields. *p<0.05 or **p<0.01, compared with control LoVo group.

Figure 5. MALAT1 increases the nuclear localization of β-catenin and levels of its downstream proteins in LoVo cells.

(A): Whole or cell extracts of different cellular compartments from LoVo cells expressing MALAT1-shRNA, MALAT1-overexpression, empty vector were probed for β-catenin and the protein levels were quantified. *p<0.05 or **p<0.01, compared with vector control. (B): Whole or cell extracts of different cellular compartments from LoVo cells expressing MALAT1-shRNA, MALAT1-overexpressed, empty vector were probed for c-Myc, MMP-7 and the protein levels were quantified. *p<0.05 or **p<0.01, compared with vector control.

Figure 6. Overexpression of MALAT1 attenuates the suppressive effect of resveratrol on migration and invasion in LoVo cells.

(A): LoVo cells expressing MALAT1-overexpressed, empty vector were treated with or without 50 µM resveratrol for 48 hours and cellular migration and invasion ability were measured. (B): Whole or cell extracts of different cellular compartments from LoVo cells expressing MALAT1-overexpressed, empty vector treated with or without 50 µM resveratrol were probed for β-catenin, c-Myc, MMP-7, and the protein levels were quantified. *p<0.05 or **p<0.01, compared with LoVo cells treated with 50 µM resveratrol.

Figure 7. Resveratrol inhibits the Wnt/β-catenin signaling through regulating MALAT1.

(A): Resveratrol negated LiCl induced nuclear translocation of β-catenin. Immunofluorescence staining of β-catenin in LoVo cells treated with LiCl, with or without resveratrol. (B): Nuclear extracts from LoVo cells expressing MALAT1-shRNA, MALAT1-overexpressed, empty vector, treated with LiCl with or without resveratrol, were probed for β-catenin, c-Myc, MMP-7. (C): Quantification of β-catenin, c-Myc, MMP-7 protein levels from the data shown in (A). **p<0.01, compared with blank group or LiCl alone group. (D): The relative LEF/TCF promoter activities in LoVo cells expressing MALAT1-shRNA, MALAT1-overexpressed, empty vector, treated by LiCl, with or without resveratrol. **p<0.01, compared with blank group or LiCl alone group. (E): No interaction between MALAT1 and β-catenin. Insert: RT-PCR result showing amount of RNA pulled-down by RNA-binding protein immunoprecipitation. As negative controls, no cDNA (blank) or IgG alone was used. Lower pane: quantification result of the insert.

Figure 8. Resveratrol inhibits the Wnt/β-catenin signaling through regulating MALAT1.in CRC HCT116 cells.

(A): Resveratrol inhibited the proliferation of HCT116 cells with an IC50 of 100 µM. (B): HCT 116 cells showed lower MALAT1 expression than LoVo cells. **p<0.01, compared with LoVo cells. (C): Overexpression of MALAT1 in HCT116 cells. (D): Overexpression of MALAT1 promoted the in vitro growth of HCT116 cells. (E): Overexpression of MALAT1 attenuated the suppressive effect of resveratrol on migration and invasion in HCT116 cells. HCT116 cells expressing MALAT1-overexpressed, empty vector were treated with or without 50 µM resveratrol for 48 hours and cellular migration and invasion ability were measured. (F): Whole or cell extracts of different cellular compartments from HCT116 cells expressing MALAT1-overexpressed, empty vector treated with or without 50 µM resveratrol for 48 hours were probed for β-catenin, c-Myc, MMP-7 and the protein levels were quantified. *p<0.05 or **p<0.01, compared with HCT116 cells treated with 100 µM resveratrol.