ABSTRACT
Metastasis is the main cause of death in colorectal cancer (CRC) patients. However, current treatment options for CRC metastasis are very limited. Lupeol, a triterpene that is widely found in vegetables and fruits, has been reported to possess the cancer-preventive and anti-inflammatory functions. However, the roles of Lupeol in the migration and invasion of colorectal cancer remain unclear. Here, we evaluated the effect of Lupeol treatment on colorectal cancer cell lines, HCT116 and SW620, and delineated its underlying mechanisms. Our results showed that Lupeol induced a dose-dependent inhibition of HCT116 and SW620 cells viability, measured by CCK8 assay. Wound healing and Transwell migration and invasion assays revealed that Lupeol significantly suppressed the migration and invasion of CRC cells. Using laser confocal microscope, we observed that the pseudopods and protrusions of HCT116 and SW620 cells decreased and disrupted after treatment with Lupeol. In addition, the quantitative real-time PCR and Western blotting results showed that Lupeol downregulated the expression of RhoA and RhoC, and their downstream effectors ROCK1, Cofilin, p-MLC, and the associated regulatory protein Cyclin A2. Interestingly, the migration and invasion capacity of CRC cells was reduced after RhoA knockdown. And there were no additional changes in CRC cells with RhoA knockdown to treat with Lupeol. These findings demonstrate that Lupeol can suppress the migration and invasion of colorectal cancer cells by remodeling the actin cytoskeleton via RhoA-ROCK1 pathway inhibition, which may provide an effective anti-metastatic agent for CRC patients.
ABSTRACT
Wnt signaling pathway plays a major role in the progression of colorectal cancer (CRC). Small molecules which can cut off this signal transduction can be promising anti-cancer drugs for CRC therapy. Therefore, we aimed to investigate the mechanisms of FH535, an inhibitor of the Wnt signaling pathway, on inhibiting proliferation and migration of colorectal cancer cells DLD-1 and SW620. We found that FH535 could significantly suppress the growth of DLD-1 and SW620 cells in a concentration-dependent and time-dependent manner. The results of cell cycle tests showed that FH535 could significantly induce G2/M arrest in colorectal cancer cells. Transwell and Wound-healing assays revealed that FH535 notably inhibited cell migration. Moreover, we found that FH535 down-regulated ß-catenin and CyclinA2 expressions while up-regulating Claudin-1 expression at both mRNA and protein levels, which may contribute to the FH535-induced inhibitory effect on proliferation and migration in human colorectal cancer cells. Our study revealed that FH535 inhibited proliferation and migration of colorectal cancer cells by regulating CyclinA2 and Claudin1 gene expression, which enriches regulatory network of FH535 and may contribute to being promising anti-cancer drugs for CRC therapy.
Subject(s)
Claudin-1/metabolism , Colorectal Neoplasms/metabolism , Cyclin A2/metabolism , Sulfonamides/pharmacology , beta Catenin/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Claudin-1/genetics , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Cyclin A2/genetics , Dose-Response Relationship, Drug , Gene Expression Regulation, Neoplastic/drug effects , Humans , Time Factors , Wnt Signaling Pathway/drug effects , beta Catenin/geneticsABSTRACT
BACKGROUND: Lupeol, a triterpene isolated from various herbal plants, possesses an anti-inflammatory function and has been proposed as a candidate for anticancer agents. The purpose of this research was to investigate the effect of lupeol on the viability, apoptosis, cell-cycle distribution, and migration of colorectal cancer cell lines and its molecular mechanism. METHODS: Lupeol was assessed for its anticancer effect using two human colorectal cancer cell lines: SW480 and HCT116. These cells were treated with lupeol, and their viability, apoptosis, migration, and cycle distribution were detected by CCK8, flow cytometry, and the transwell method. Quantitative PCR, Western blot, and immunofluorescence were applied to detect the expressions of CTNNB1, TCF4, cMYC, CCND1, CLDN1, and CCNA2. RESULTS: Lupeol suppressed cell viability and migration and induced cellular apoptosis of both cell lines, with increased p53 and decreased Bcl2 protein levels (P<0.05). Cell cycles of both lupeol-treated cell lines were arrested in the S phase (P<0.05). Quantitative PCR and Western blot analyses showed significantly reduced expressions of CTNNB1, TCF4, and downstream genes of the Wnt-ß-catenin pathway, including the cell-cycle-regulated genes of cMYC and CCND1 of both cell lines upon lupeol treatment (P<0.05). mRNA and protein levels of CLDN1 decreased in HCT116 cells, plus the expression of CCNA2 mRNA and protein decreased in SW480 cells (P<0.05). Immunofluorescence analysis confirmed decreased expression of Wnt-ß-catenin signaling. CONCLUSION: Our findings indicate that lupeol effectively inhibits proliferation and migration and induces apoptosis and cell-cycle arrest of two colorectal cell lines by inactivation of the Wnt-ß-catenin signaling pathway and downregulation of cMYC, CCND1, CCNA2, and CLDN1, thereby making it a promising anticancer candidate.
