Loss of AKR1C1 is a good prognostic factor in advanced NPC cases and increases chemosensitivity to cisplatin in NPC cells

Abstract

Cisplatin resistance is one of the main obstacles in the treatment of advanced nasopharyngeal carcinoma (NPC). AKR1C1 is a member of the Aldo-keto reductase superfamily (AKRs), which converts aldehydes and ketones to their corresponding alcohols and has been reported to be involved in chemotherapeutic resistance of multiple drugs. The expression and function of AKR1C1 in NPC have not been reported until now. The aim of this research was to investigate the expression of AKR1C1 and it is role in cisplatin resistance in NPC. AKR1C1 protein expression was detected by immunohistochemistry in human NPC tissues and by Western blot assays in NPC and immortalized nasopharyngeal epithelial cells. The effects of AKR1C1 knock-down by siRNA on proliferation, migration and invasion in NPC cells were evaluated by CCK8, wound healing and transwell assays. To evaluate the effects of AKR1C1 silencing on cisplatin sensitivity in NPC cells, CCK8 assays were used to detect cell proliferation, flow cytometry was used to detect cell cycle distribution, and flow cytometry and DAPI staining were used to detect cell apoptosis. AKR1C1 down-regulation was associated with advanced clinicopathological characters such as larger tumor size, more lymphatic nodes involvement, with metastasis and later clinical stages, while AKR1C1 down-regulation was a good prognostic factor for overall survival (OS) in NPC patients. In vitro study showed that AKR1C1 was not directly involved in the malignant biological behaviours such as proliferation, cell cycle progression and migration of NPC cells, whereas AKR1C1 knock-down could enhance cisplatin sensitivity of NPC cells. These results suggest that AKR1C1 is a potential marker for predicting cisplatin response and could serve as a molecular target to increase cisplatin sensitivity in NPC.

Keywords: AKR1C1; chemosensitivity; cisplatin; nasopharyngeal carcinoma.

Figure 1

AKR1C1 expression decreased in NPC tissues and frequently lost in NPC cells. (A) AKR1C1 expression in non‐cancerous nasopharyngeal biopsies and NPC tissues based on IHC. a: High expression of AKR1C1 in non‐cancerous nasopharyngeal epithelia; b: high expression of AKR1C1 in NPC biopsies; c: low expression of AKR1C1 in NPC biopsies. The brown staining indicates AKR1C1 immunoreactivity. (B) AKR1C1 expression was significantly lower in the NPC biopsies than in the non‐cancerous nasopharyngeal biopsies. (C) Expression levels of AKR1C1 in the indicated immortalized nasopharyngeal epithelial cells and NPC cells were examined by Western blotting. Abbreviations: NT, non‐cancerous nasopharyngeal epithelia; NPC, nasopharyngeal carcinoma; AKR1C1, Human 20‐keto reductase family 1 member C1; IHC, immunohistochemical staining

Figure 2

Loss of AKR1C1 was associated with advanced TNM stage while with good prognosis in NPC patients. (A) Representative images of AKR1C1 expression in NPC biopsies with different TNM stages. High expression of AKR1C1 was observed in the T1, N0, M0, and I stage and differentiated histological subtype (DNKC) of NPC biopsies, while low expression of AKR1C1 was detected in the T4, N2, M1, and IV stage and differentiated histological subtype (UDC) of tumours. (B) Numbers and percentages of cases with high or low expression of AKR1C1 according to different clinicopathological features. (C) Low AKR1C1 expression was associated with a good prognosis in NPC patients. *P < .05, **P < .01, ^# P < .001. Abbreviations: NPC, nasopharyngeal carcinoma; AKR1C1, human 20‐keto reductase family 1 member C1; DNKC, differentiated non‐keratinized squamous carcinoma; UDC, undifferentiated non‐keratinized carcinoma

Figure 3

Knock‐down of AKR1C1 by siRNA was not involved in cell proliferation, migration and invasion in NPC cells. (A) Western blot assay showed that AKR1C1 was successfully knocked down by siRNA in CNE1 and CNE2 cells. (B‐C) The effects of AKR1C1 silencing on cell proliferation were measured by CCK8 assay. (D‐E) Migrative and invasive activities of the si‐AKR1C1 and si‐ctrl groups of NPC cells were based on transwell assays. The average number of cells per field was determined from three repeated independent experiments. NS, not significant, P > .05. Abbreviations: NPC, nasopharyngeal carcinoma; AKR1C1, human 20‐keto reductase family 1 member C1

Figure 4

Knock‐down of AKR1C1 by siRNA enhanced cisplatin‐induced inhibition of cell proliferation and arrest of cell cycle. (A) Down‐regulation of AKR1C1 sensitized NPC cells to cisplatin's toxicity on cell proliferation according to CCK8 assays. (B and C) Cisplatin blocked G2/M and G1/S transition in CNE1 and CNE2 cells, respectively. Meanwhile, AKR1C1 silencing sensitized NPC cells to cisplatin's inhibiting effects on cell cycle according to flow cytometry. The average number of cells per field was determined from three repeated independent experiments. *P < .05, **P < .01, ^# P < .001, NS, not significant, P > .05. Abbreviations: AKR1C1, human 20‐keto reductase family 1 member C1; DDP, cisplatin

Figure 5

Knock‐down of AKR1C1 by siRNA reversed cisplatin resistance and induced apoptosis in NPC cells. (A and B) After treated with cisplatin for 48 hours, si‐ctrl and si‐AKR1C1 transfected cells were assayed by flow cytometry for calculating apoptotic cells. Results are presented as the mean ± standard error of the mean. The average number of cells per field was determined from three repeated independent experiments.*P < .05, **P < .01; NS, not significant, P > .05. Abbreviations: AKR1C1, human 20‐keto reductase family 1 member C1; DDP, cisplatin