Study on the Mechanism of Cell Cycle Checkpoint Kinase 2 (CHEK2) Gene Dysfunction in Chemotherapeutic Drug Resistance of Triple Negative Breast Cancer Cells

Abstract

BACKGROUND This study aimed to investigate the mechanism of CHEK2 gene dysfunction in drug resistance of triple negative breast cancer (TNBC) cells. MATERIAL AND METHODS To perform our study, a stable CHEK2 wild type (CHEK2 WT) or CHEK2 Y390C mutation (CHEK2 Y390C) expressed MDA-MB-231 cell line was established. MTT assay, cell apoptosis assay and cell cycle assay were carried out to analyze the cell viability, apoptosis, and cell cycle respectively. Western blotting and qRT-PCR were applied for related protein and gene expression detection. RESULTS We found that the IC50 value of DDP (Cisplatin) to CHEK2 Y390C expressed MDA-MB-231 cells was significantly higher than that of the CHEK2 WT expressed cells and the control cells. After treatment with DDP for 48 h, cells expressing CHEK2 WT showed lower cell viability than that of the CHEK2 Y390C expressed cells and the control cells; compared with the CHEK2 Y390C expressed cells and the control cells, cells expressing CHEK2 WT showed significant G1/S arrest. Meanwhile, we found that compared with the CHEK2 Y390C expressed cells and the control cells, cell apoptosis was significantly increased in CHEK2 WT expressed cells. Moreover, our results suggested that cells expressing CHEK2 WT showed higher level of p-CDC25A, p-p53, p21, Bax, PUMA, and Noxa than that of the CHEK2 Y390C expressed cells and the control cells. CONCLUSIONS Our findings indicated that CHEK2 Y390C mutation induced the drug resistance of TNBC cells to chemotherapeutic drugs through administrating cell apoptosis and cell cycle arrest via regulating p53 activation and CHEK2-p53 apoptosis pathway.

Figure 1

Suppression of CHEK2 in MDA-MB-231 cells. CHEK2 gene was silenced in MDA-MB-231 cells by transfection with CHEK2-shRNA. Forty-eight hours after transfection, the transfection efficiency was detected using western blotting (A) and qRT-PCR (B), respectively. And IC₅₀ value of DDP to MDA-MB-231 cells was detected (C). Data were presented as mean ±SD; ** p<0.01 versus control.

Figure 2

Re-expression of CHEK2 WT and CHEK2 Y394C in CHEK2-knockdown MDA-MB-231 cells. CHEK2 WT (wild-type CHEK2) or CHEK2 Y390C (CHEK2 Y390C mutation) was re-expressed in CHEK2 knockdown MDA-MB-231 cells. CHEK2 knockdown MDA-MB-231 cells transfected with vector control were used as the control group. CHEK2 expression was detected using western blotting (A) and qRT-PCR (B), respectively. The IC₅₀ value of DDP to MDA-MB-231 cells was detected (C). Data were presented as mean ±SD; ** p<0.01 versus control.

Figure 3

Effect of CHEK2 Y390C mutation on MDA-MB-231 cell viability. CHEK2 WT (wild-type CHEK2) or CHEK2 Y390C (CHEK2 Y390C mutation) was re-expressed in CHEK2 knockdown MDA-MB-231 cells. CHEK2 knockdown MDA-MB-231 cells transfected with vector control were used as the control group. After treatment with 3 μM DDP for 48 hours, the cell viability was detected using MTT assay. Data are presented as mean ±SD; ** p<0.01 versus control.

Figure 4

Effect of CHEK2 Y390C mutation on MDA-MB-231 cell apoptosis. CHEK2 WT (wild-type CHEK2) or CHEK2 Y390C (CHEK2 Y390C mutation) was re-expressed in CHEK2 knockdown MDA-MB-231 cells. CHEK2 knockdown MDA-MB-231 cells transfected with vector control were used as the control group. After treatment with 3 μM DDP for 48 hours, the cell apoptosis was analyzed using FCM. Data are presented as mean ±SD; ** p<0.01 versus control.

Figure 5

Effect of CHEK2 Y390C mutation on CHEK2-p53 apoptosis pathway in MDA-MB-231 cell apoptosis. CHEK2 WT (wild-type CHEK2) or CHEK2 Y390C (CHEK2 Y390C mutation) was re-expressed in CHEK2 knockdown MDA-MB-231 cells. CHEK2 knockdown MDA-MB-231 cells transfected with vector control were used as the control group. After treatment with 3 μM DDP for 48 hours, the protein (A) and mRNA (B–E) level of p21, Bax, PUMA, and Noxa were determined using western blot and qRT-PCR, respectively. Data are presented as mean ±SD; ** p<0.01 versus control.

Figure 6

Effect of CHEK2 Y390C mutation on MDA-MB-231 cell cycle. CHEK2 WT (wild-type CHEK2) or CHEK2 Y390C (CHEK2 Y390C mutation) was re-expressed in CHEK2 knockdown MDA-MB-231 cells. CHEK2 knockdown MDA-MB-231 cells transfected with vector control were used as the control group. After treatment with 3 μM DDP for 48 hours, cell cycle was analyzed by FCM. Meanwhile, the protein level of p-CDC25 was determined using western blot. Data are presented as mean ±SD; ** p<0.01 versus control.

Figure 7

Effect of CHEK2 Y390C mutation on p53 activation in MDA-MB-231 cells. CHEK2 WT (wild-type CHEK2) or CHEK2 Y390C (CHEK2 Y390C mutation) was re-expressed in the CHEK2 knockdown MDA-MB-231 cells. CHEK2 knockdown MDA-MB-231 cells transfected with vector control were used as the control group. After treatment with 3 μM DDP for 48 hours, the protein level of p-p53 was determined using western blot. Data are presented as mean ±SD; ** p<0.01 versus control.