CAC1 knockdown reverses drug resistance through the downregulation of P-gp and MRP-1 expression in colorectal cancer

Abstract

CDK2-associated cullin domain 1 (CAC1) is as a novel cell cycle regulator widely expressed in colorectal cancer (CRC). However, its expression and function in drug resistant CRC cells remains elusive. Therefore, the present study aimed to assess the biochemical function and relevance of CAC1 in drug resistant CRC cells, and detect the potential mechanism. For this purpose, a total of 83 CRC cases were collected for the immunohistochemical analysis of CAC1 expression. Functional studies (stable transfection, flow cytometry, colony formation, and invasion and migration assays) were performed in SW480, LoVo and their corresponding 5-FU resistant cells. In addition, a nude mice xenograft model was established for further observation in vivo. In the present study, CAC1 protein expression was higher in CRC tissues than that in normal tissues (P<0.05). Furthermore, CAC1 protein expression was higher in SW480/5-FU cells than in SW480 cells. CAC1 knockdown arrested 5-FU resistant cells at the G1/S phase and increased the sensitivity of 5-FU resistant cells to 5-FU by inducing apoptosis. In addition, CAC1 reduced the invasive and migration ability of SW480/5-FU and LoVo/5-FU cells in vitro, and reduced their tumorigenicity and metastatic ability in vivo. Finally, CAC1 knockdown resulted in decreased P-glycoprotein and MRP-1 protein expression. Based on these results, it can be concluded that CAC1 plays an important role in the occurrence and promotion of drug resistance in CRC. Therefore, the knockdown of CAC1 may be considered as a new strategy for the development of CRC drug resistance treatments in the future.

Fig 1. CAC1 protein expression in CRC tissues and cell lines.

(A) The immunohistochemistry analysis of CRC tissues and its corresponding normal tissue samples are shown, and the tissue microarray sections were classified into four groups, according to staining intensity (×200). (B) The western blot analysis of CAC1 expression in CRC cell lines and drug resistance cell line SW480/5-FU is shown.

Fig 2. The overall survival curve of colorectal cancer patients with different CAC1 expression.

(A) The overall survival curve of the 83 enrolled patients. (B) The overall survival curves of patients with CAC1 positive expression and CAC1 negative expression.

Fig 3. CAC1 knockdown induced the arrest of SW480/5-FU cells in the G1/S phase.

(A) The stable shCAC1 transfection of SW480 and SW480/5-FU cells for 24–48 hours. (B) The quantitative RT-PCR analysis of CAC1 mRNA expression in SW480 and SW480/5-FU cells following CAC1 knockdown. (C) The western blot analysis of CAC1 protein expression in SW480 and SW480/5-FU cells following CAC1 knockdown. CAC1 knockdown induced cell cycle arrest at the G1 phase in SW480, SW480/5-FU cells (D) and LoVo, LoVo/5-FU cells (E). Cells were stably transfected with shCAC1 for one week, and the cell cycle profile was detected using FACS analysis following propidium iodide staining. The representative results of three independent experiments are shown. *P<0.05, **P<0.01.

Fig 4. CAC1 knockdown increased sensitivity of SW480/5-FU cells to 5-FU by influencing P-glycoprotein and MRP-1 expression.

(A) SW480 and SW480/5-FU cells were transfected with shCAC1 and shCON for one week, and subjected to FACS analysis with Annexin V-FITC and propidium iodide staining. (B) The expression of drug resistance-associated proteins, P-glycoprotein and MRP-1, in SW480 and SW480/5-FU cells after transfection with shCAC1 and shCON was examined by western blot. *P<0.05, **P<0.01.

Fig 5. CAC1 knockdown increased sensitivity of LoVo/5-FU cells to 5-FU by influencing P-glycoprotein and MRP-1 expression.

(A) LoVo and LoVo/5-FU cells transfected with shCAC1 and shCON were subjected to FACS analysis with Annexin V-FITC and propidium iodide staining. (B) The expression of drug resistance-associated proteins, P-glycoprotein and MRP-1, in LoVo and LoVo/5-FU cells after transfection with shCAC1 and shCON was examined by western blot. *P<0.05, **P<0.01.

Fig 6. CAC1 knockdown reduced colony formation and invasive and migration ability of SW480 and SW480/5-FU cells.

(A) Colony formation assay, (B) cell migration assay and (C) cell invasion assay were detected in SW480 and SW480/5-FU cells were stably transfected with shCAC1 and shCON for one week. *P<0.05, **P<0.01.

Fig 7. CAC1 knockdown reduced the invasive and migration ability of LoVo and LoVo/5-FU cells.

(A) Cell invasion assay and (B) cell migration assay were detected in LoVo and LoVo/5-FU cells transfected with shCAC1 and shCON, respectively. *P<0.05, **P<0.01.

Fig 8. CAC1 knockdown reduced the growth of SW480/5-FU cell xenografts and liver metastatic lesions in vivo.

(A) Tumor growth curve and (B) tumor weight in each group. (C) Representative photographs of tumors and (D) liver metastatic lesions isolated from nude mice in each group at six weeks after inoculation with SW480-shCON, SW480-shCAC1, SW480/5FU-shCON and SW480/5FU -shCAC1. (E) The levels of CAC1, P-gp and MRP-1 in transplanted tumors were detected by IHC. The expression of CAC1, P-gp and MRP-1 was lower in SW480/5FU-shCAC1 transplanted tumors, when compared to the SW480/5FU-shCON group. *P<0.05, **P<0.01.