Epithelial-mesenchymal transition and metastasis of colon cancer cells induced by the FAK pathway in cancer-associated fibroblasts.

OBJECTIVE: The role and mechanism of tetrathiomolybdate (TM) in cancer-associated fibroblasts (CAFs) in colon cancer using three-dimensional (3D) culture were investigated, and the associations between the focal adhesion kinase (FAK) pathway and epithelial-mesenchymal transition (EMT) in CAFs were explored. METHODS: A 3D co-culture model of colon cancer LOVO cells with CAFs and normal fibroblasts (NFs) was established using Matrigel as a scaffold material. The differential expression of LOXL2 (lysyl oxidase-like 2) in the supernatant of CAFs and NFs was determined using ELISA, and expression levels of EMT-related proteins and FAK signaling pathway-related proteins were determined using western blot. RESULTS: LOXL2 levels secreted by CAFs were higher compared with that secreted by NFs. In the CAF + LOVO group, compared with the LOVO group, E-cadherin expression decreased significantly, while N-cadherin and F-PAK expression increased significantly. TM results were opposite compared with the above results. CONCLUSIONS: CAFs stimulate EMT in human colon cancer LOVO cells by secreting LOXL2 to activate the FAK signaling pathway, thereby promoting tumor metastasis. TM inhibited the occurrence of EMT in the CAF-induced colon cancer LOVO cell line, thereby reducing the invasion and metastasis of colon cancer cells.

RRS1 silencing suppresses colorectal cancer cell proliferation and tumorigenesis by inhibiting G2/M progression and angiogenesis.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Ribosome biogenesis regulatory protein homolog (RRS1) is an essential factor involved in ribosome biogenesis, while its role in CRC remains largely unclear. Here, we found that RRS1 expression was significantly higher in CRC tissues compared with adjacent normal tissues. RRS1 High expression also predicted poor overall survival of CRC patients. Knockdown of RRS1 induced the G2/M cell cycle arrest, apoptosis and suppressed the proliferation of RKO and HCT-116 CRC cells. Furthermore, angiogenesis was also reduced in CRC cells after RRS1 knockdown. In addition, suppression of RRS1 blunted the tumor formation of CRC cells in nude mice. At the molecular level, silencing of RRS1 decreased the expression of M-phase inducer phosphatase 3 (CDC25C), Cyclin-dependent kinase 1 (CDK1), antigen KI-67 (KI67) and increased the protein level of cyclin-dependent kinase inhibitor 1 (CDKN1A) and tumor suppressor p53 (p53). Taken together, our findings provide evidence that RRS1 may promote the development of colon cancer. Therefore, targeting RRS1 may be a promising therapeutic strategy for CRC patients.

Osteopontin knockdown suppresses the growth and angiogenesis of colon cancer cells.

AIM: To investigate the effects of osteopontin (OPN) gene expression knockdown on colon cancer Lovo cells in vitro. METHODS: Four candidate small interfering RNA (siRNA) constructs targeting the OPN gene and a scrambled control sequence (NC-siRNA) were synthesized and inserted into a pGPU6/GFP/Neo expression vector. After confirmation by restriction enzyme digestion and DNA sequencing, the recombinant plasmids were subsequently transfected into a human colon cancer cell line (Lovo) using a liposome transfection method. Stably transfected cells were maintained with G418 selection and referred to as Lovo-OPN-1, -2, -3, -4, and Lovo-NC cells. Knockdown efficiency of each of the four siRNA constructs was determined by real-time reverse transcription polymerase chain reaction assays and western blotting, and the construct with the most effective silencing was used for subsequent experiments. Cell proliferation, adhesion, and Matrigel invasion assays were performed to analyze the effects of OPN knockdown in stably transfected Lovo cells. The levels of four angiogenic factors, namely vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, MMP-9 and urokinase plasminogen activator were detected by enzyme-linked immunosorbent assays (ELISA). RESULTS: Recombinant vectors containing OPN-specific and scrambled siRNA sequences were successfully constructed and stably transfected into Lovo cells. Compared with the control Lovo and Lovo-NC cells, the levels of OPN mRNA and protein expression in Lovo-OPN-1, -2, -3, and -4 were significantly reduced (all P < 0.05), with the most efficient reduction observed in Lovo-OPN-4 cells (P < 0.05). Relative to untransfected Lovo cells, OPN mRNA expression levels in Lovo-NC and Lovo-OPN-4 cells were 1.008 ± 0.067 and 0.160 ± 0.023, respectively. The relative OPN protein expression levels in Lovo, Lovo-NC, and Lovo-OPN-4 cells were 3.024 ± 0.211, 2.974 ± 0.630, and 0.121 ± 0.008, respectively. Moreover, transfection with the scrambled sequence had no effect on the expression of OPN. After 24, 48, 72, and 96 h of cultivation, absorption values at 450 nm to assess proliferation of Lovo-OPN-4 cells were 0.210 ± 0.017, 0.247 ± 0.024, 0.314 ± 0.037, and 0.359 ± 0.043, respectively, which were significantly lower than those of Lovo (0.244 ± 0.031, 0.313 ± 0.024, 0.513 ± 0.048 and 0.783 ± 0.051) and Lovo-NC cells (0.241 ± 0.029, 0.309 ± 0.022, 0.563 ± 0.023, and 0.735 ± 0.067) (all P < 0.05). The absorption values at 595 nm, which were measured in a cell adhesion assay, showed that adhesion of Lovo-OPN-4 cells (0.215 ± 0.036) was significantly decreased compared to Lovo (0.490 ± 0.037) and Lovo-NC cells (0.462 ± 0.043) (P < 0.05). The number of invasive Lovo-OPN-4 cells (16.1 ± 1.9) was also significantly decreased compared to Lovo (49.9 ± 5.4) and Lovo-NC cells (48.8 ± 4.5) (P < 0.05). ELISA assays showed significant reductions in Lovo-OPN-4 cells compared to Lovo and Lovo-NC cells with regard to the expression of VEGF (1687.85 ± 167.84 ng/L vs 2348.54 ± 143.80 ng/L and 2284.39 ± 138.62 ng/L, respectively), MMP-2 (2966.07 ± 177.36 µg/L vs 4084.74 ± 349.54 µg/L and 4011.41 ± 424.48 µg/L, respectively), MMP-9 (3782.89 ± 300.64 µg/L vs 5062.90 ± 303.02 µg/L and 4986.38 ± 300.75 µg/L, respectively) and uPA (1152.69 ± 120.79 µg/L vs 1380.90 ± 147.25 µg/L and 1449.80 ± 189.92 µg/L, respectively) (all P < 0.05). CONCLUSION: Knockdown of OPN gene expression suppresses colon cancer cell growth, adherence, invasion, and expression of angiogenic factors.

[Effect of human colon carcinoma-associated fibroblasts on biological behavior of colon carcinoma Lovo cells].

OBJECTIVE: To investigate the effects of human colon carcinoma-associated fibroblasts (CAFs) on proliferation, adhesion, migration and invasion of colon carcinoma Lovo cells. METHODS: The co-culture models among colon CAFs, NFs and Lovo cell were established by conditioned medium (CM) of human colon CAFs and colon normal fibroblasts (NFs). Lovo cells in the blank control group was treated with serum-free culture medium. The effects of human colon CAFs on proliferation, adhesion, migration and invasion of colon carcinoma Lovo cells were detected by cell proliferation assay, adhesion assay, migration assay and Transwell invasion assay. RESULTS: After co-culture with colon CAFs, the absorbance (A) value of Lovo cells was (0.667±0.059) in 48 h and (0.709±0.030) in 72 h. The A value of Lovo cells adhesion to fibronectin was (0.588±0.067). The cell mobility rates were (35.2±8.7)% in 12 h and (64.6±7.1)% in 24 h. The number of invasive cell was (56.2±4.8). All the above parameters were increased compared with those in the blank control group and NFs group (all P<0.01). CONCLUSION: Human colon CAFs can promote the proliferation, adhesion, migration and invasion of colon carcinoma Lovo cells.