Proline-rich acidic protein 1 upregulates mitotic arrest deficient 1 to promote cisplatin-resistance of colorectal carcinoma by restraining mitotic checkpoint complex assembly

Abstract

Background: The mechanism underlying cisplatin resistance in colorectal carcinoma (CRC) has not yet been elucidated. This study is aimed to illustrate the indispensable role of proline-rich acidic protein 1 (PRAP1) in cisplatin-resistant CRC. Methods: Cell viability and apoptosis were monitored using cell counting kit-8 and flow cytometry. Immunofluorescence and morphological analysis were used to determine mitotic arrest in cells. In vivo drug resistance was evaluated using a tumor xenograft assay. Results: PRAP1 was highly expressed in cisplatin-resistant CRC. PRAP1-upregulation in HCT-116 cells increased chemoresistance to cisplatin, whereas RNAi-mediated knockdown of PRAP1 sensitized cisplatin-resistant HCT-116 cells (HCT-116/DDP) to cisplatin. PRAP1-upregulation in HCT-116 cells hindered mitotic arrest and the formation of mitotic checkpoint complexes (MCC), followed by an increase in multidrug-resistant proteins such as p-glycoprotein 1 and multidrug resistance-associated protein 1, while PRAP1-knockdown in HCT-116/DDP cells partly restored colcemid-induced mitotic arrest and MCC assembly, resulting in decreased multidrug-resistant protein levels. PRAP1 downregulation-mediated sensitization to cisplatin in HCT-116/DDP cells was abolished by the inhibition of mitotic kinase activity by limiting MCC assembly. Additionally, PRAP1-upregulation increased cisplatin-resistance in CRC in vivo. Mechanistically, PRAP1 increased the expression of mitotic arrest deficient 1 (MAD1), that competitively binds to mitotic arrest deficient 2 (MAD2) in cisplatin-resistant CRC cells, leading to failed assembly of MCC and subsequent chemotherapy resistance. Conclusion: PRAP1-overexpression caused cisplatin resistance in CRC. Possibly, PRAP1 induced an increase in MAD1, which competitively interacted with MAD2 and subsequently restrained the formation of MCC, resulting in CRC cells escape from the supervision of MCC and chemotherapy resistance.

Keywords: Proline-rich acidic protein 1; cisplatin resistance; colorectal carcinoma; mitotic arrest deficient 1; spindle assembly checkpoint.

Figure 1

Expression pattern of proline-rich acidic protein 1 (PRAP1) in human colorectal cancer. (A) PRAP1 expression in clinical colorectal carcinoma (CRC) tissues (n=275) and control group (n=349) was detected by using Gene Expression Profiling Interactive Analysis (GEPIA 2). (B) The mRNA expression of PARP1 in several CRC cell lines including HCT-116, HT-29, Lovo and SW480 and normal colon cell line CRL-1790 were determined by qRT-PCR. (C) Based on the Oncomine database, PRAP1 expression was detected in the cisplatin-treated CRC clinic cell line at 0, 6, 12, and 24 h (n=9, 12, 12 and 12). (D) The PARP1 protein level was evaluated by western blotting in cisplatin-resistant HCT-116 cells (HCT-116/DDP) and HT-29 cells (HT-29/DDP), 5-Fluorouracil (5-FU)-resistant HCT116 cells (HCT-116/5-Fu) and the corresponding control groups, quantitative analysis is presented on the right. *P<0.05; **P<0.01.

Figure 2

Effects of PRAP1 on cell viability and apoptosis in HCT-116 and HCT-116/DDP cells. (A)-(B) EGFP-PRAP1 plasmid and control vector were transfected in HCT-116 cells. Cells were induced with different concentrations of cisplatin (5 and 10 μM) for 24, 48, 72, and 96 h. Cell viability was determined by Cell Counting Kit-8 (CCK8) Assay kit. (C-D) siPRAP1 and negative control siRNA (siNC) were transfected in HCT-116/DDP cells. Cells were induced with 10, and 20 μM cisplatin for 24, 48, 72, and 96 h. Cell viability was monitored using CCK8 Assay kit. (E) EGFP-PRAP1 plasmid and control vector were transfected in HCT-116 cells, and then cells were treated with 5 μM cisplatin for 72 h. Cell apoptosis was conducted by flow cytometry using the Annexin V-FITC cell apoptosis detection kit. (F) siPRAP1 and siNC were transfected in HCT-116/DDP cells and cells were incubated with 10 μM cisplatin for 72 h. Cell apoptosis was detected by flow cytometry using the Annexin V-FITC cell apoptosis detection kit. *P<0.05; **P<0.01.

Figure 3

Effect of PRAP1 on the assembly of mitotic checkpoint complex (MCC). (A) Representative photographs of colcemid-challenged HCT-116 cells with or without EGFP-PRAP1 transfection which were examined by Livecyte Cell Analysis System. The rounded-up cell morphology was accepted to be under mitotic arrest (top). Expression and distribution of pH3-positived and apoptotic cells as monitored by IF staining. Red: pH3, Blue: Hoechst, Scale bar: 20 μm. (B) Representative photographs of colcemid-induced siPRAP1-treated HCT-116/DDP cells (the top picture). pH3-positive and apoptotic cells were detected by IF staining (the last two pictures). Red: pH3, Blue: Hoechst, Scale bar: 20 μm. (C-D) HCT-116 cells were transfected with PRAP1 recombinant overexpressed plasmid and HCT-116/DDP cells were transfected with siPRAP1. The interaction between budding uninhibited by benzimidazole-related 1 (BUBR1) with other MCC-related protein including cell-division cycle protein 20 (Cdc20), mitotic arrest deficient 2 (MAD2) and budding uninhibited by benzimidazoles 3 (Bub3) as determined by Co-IP in these cell groups.

Figure 4

Role of MCC in PRAP1-mediated cisplatin resistance. (A) In PRAP1 overexpressed HCT-116 cells and siPRAP1 plasmid-transfected HCT-116/DDP cells, western blotting assay was used to detect expressions of multidrug resistant protein of p-glycoprotein 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1). (B) Quantitative analysis of panel A as shown on the right. (C-D) PRAP1-depleted HCT-116/DDP cells were treated with 20 μM cisplatin and Mps1-IN-1. Cell apoptosis was evaluated by Annexin V-FITC cell apoptosis detection kit using flow cytometry (C) and quantitative analysis as presented in panel D (D). (E) Cell viability was measured by CCK8 Assay kit. (F) siPRAP1-transfected HCT-116/DDP cells were treated with 20 μM cisplatin and Mps1-IN-1, multidrug resistant protein as estimated by western blotting. (G) Quantitative analysis is shown on the right. *P<0.05; **P<0.01.

Figure 5

Role of mitotic arrest deficient 1 (MAD1) in PRAP1-mediated MCC assembly inhibition and cisplatin-resistant CRC cells. (A) HCT-116 and HCT-116/DDP cells were transfected with EGFP-PRAP1 and siPRAP1 plasmid, respectively, MAD1 expression was measured by western blotting assay. (B) Quantitative analysis of panel A is shown on the right. (C) Colcemid-challenged HCT-116/DDP cells were transfected with siPRAP1 or MAD1 overexpression plasmid, the interaction of MAD2 with MAD1, and with BUBR1 as explored by Co-IP. (D) Representative photographs of colcemid-challenged HCT-116/DDP cells with siPRAP1 or MAD1 overexpressing plasmid transfection which were examined using Livecyte Cell Analysis System. The rounded-up cell morphology was accepted to be under mitotic arrest (the top picture). (E) Expression and distribution of pH3-positive and apoptotic cells as monitored by IF staining. Red: pH3, Blue: Hoechst, Scale bar: 20 μm. (F) Multidrug resistant protein as estimated via western blotting and quantitative analysis as displayed below. *P<0.05; **P<0.01.

Figure 6

Effects of PRAP1 on cisplatin-resistance of colorectal carcinoma in vivo. (A) All mice were sacrificed by cervical dislocation and the tumors of mice from the four groups were harvested and images captured (n=6 per group). (B) The tumor growth curve of xenograft mice after initial cell injection and drug-treatment. Cisplatin was taken orally on day 15 after initial cell injection. (C) The differences in tumor weight among the four groups. (D) IHC staining of tumors with Ki67 antibody in the four groups. Relative quantitative analysis of Ki67-positive cell number is shown in the left panel. Scale bar, 50 μm. (E) Drug resistance related protein levels including MDR1 and MAD1, and protein level of PRAP1 in tumors as determined by western blotting. Relative quantitative analysis of protein levels is shown on the left panel. (F) The interaction of MAD2 with MAD1, and with BUBR1 in tumors as explored by Co-IP. ns, no significant. * P<0.05, ** P<0.01.

Figure 7

Schematic diagram of PRAP1-mediated cisplatin-resistance. PRAP1 overexpression caused the cisplatin resistance in CRC. Mechanically, PRAP1 could positively regulate MAD1 expression, and the binding of MAD1 to MAD2 destroyed MCC assembly by weakening the interaction between MAD2 and BUBR1, which in turn allowed tumor cells to escape from the control of MCC, resulting in the drug-resistant phenotype.