Cytochrome C inhibits tumor growth and predicts favorable prognosis in clear cell renal cell carcinoma

Abstract

Cytochrome C (Cyto C), a multifunctional enzyme, has been demonstrated to be associated with cell apoptosis and respiration. Accumulating evidence has revealed that serum Cyto C is an effective indicator in evaluating the effect of chemotherapy. However, to the best of our knowledge, the clinical significance of Cyto C and its role in cell growth and apoptosis in clear cell renal cell carcinoma (CCRCC) remain unknown. In the present study, Cyto C expression was detected in 150 CCRCC and 30 normal tissues samples via immunohistochemistry. The results demonstrated that Cyto C protein expression levels in CCRCC tissues were downregulated compared with those in corresponding normal tissues. In addition, it was revealed that Cyto C expression was negatively associated with TNM stage. Further analyses revealed that patients with CCRCC and low Cyto C expression levels had a shorter survival time than those with high Cyto C expression. Multivariate analyses indicated that high Cyto C expression levels were an independent prognostic factor for survival. Functionally, overexpression of Cyto C effectively suppressed the growth of CCRCC cells and induced cell apoptosis, and knockdown of Cyto C reversed these effects. Finally, overexpression of Cyto C inhibited the tumor growth of CCRCC cells in vivo. Overall, the data of the present study indicated that Cyto C may be a novel prognostic biomarker and acted as a regulator of tumor growth in CCRCC.

Keywords: apoptosis; clear renal cell carcinoma; cytochrome C; proliferation.

Figure 1.

Cyto C expression is decreased in human CCRCC. (A) Cyto C protein expression levels were examined by western blotting in tumor and adjacent normal tissues from 10 patients with CCRCC. (B) mRNA expression levels of Cyto C in seven matched specimens from the Gene Expression Omnibus database GDS-505. (C) Representative images of IHC analysis for Cyto C in normal renal tissues and CCRCC tissues. Scale bars, 30 µm. (D) IHC scores of tumor and normal tissues from 30 paired CCRCC specimens. ***P<0.001. Cyto C, cytochrome C; CCRCC, clear renal cell carcinoma; IHC, immunohistochemistry; T, tumor; N, normal; H&E, hematoxylin and eosin.

Figure 2.

Low expression levels of Cyto C are associated with advanced TNM stage and predict poor prognosis in CCRCC. (A) Representative images of Cyto C staining in CCRCC specimens at different TNM stages. Scale bar, 20 µm. (B) Immunohistochemistry scores of Cyto C expression in CCRCC tumor tissues of different stages. (C) Kaplan-Meier analysis of overall survival rate in the cohort of 150 patients with CCRCC from the tissue microarray used in the present study. (D) Kaplan-Meier analysis of overall survival rate in a cohort of 877 patients with CCRCC from the TCGA database. *P<0.05. Cyto C, cytochrome C; CCRCC, clear renal cell carcinoma; TCGA, The Cancer Genome Atlas; NS, not significant.

Figure 3.

Cyto C inhibits clear renal cell carcinoma cell proliferation and induces cell apoptosis in vitro. (A) Western blot analysis of Cyto C expression levels in the mock and OECyto C cells, and in the siNC and siCyto C 786-O cells. (B) Cell viability curve of siNC and siCyto C 786-O cells. (C) Cell viability curve of mock and OECyto C 786-O cells. (D) Cell apoptosis was determined via Annexin V-FITC/PI staining and flow cytometry. Data are presented as the mean ± SD of three independent experiments. *P<0.05; **P<0.01. Cyto C, cytochrome C; OE, overexpression; si, small interfering; NC, negative control; PI, propidium iodide.

Figure 4.

Cyto C suppresses CCRCC cell xenograft growth in vivo. (A) Bioluminescent images of tumors in nude mice implanted with OECyto C and mock CCRCC cells at 14 days. (B) Photographs of the harvested xenograft tumors at 28 days post-implantation. (C) Volume and (D) weight of tumors formed by mock and OECyto C 786-O cells in mice. (E) Representative images of Ki67 staining in sections from the xenograft tumors and quantification of the proliferation index. Scale bar, 20 µm. Ki67-positive cells (brown) were counted using microscopy and recognized by total number of nuclei in three high-powered fields. The results were presented as the mean number of Ki67-positive cells ± standard deviation. *P<0.05. Cyto C, cytochrome C; CCRCC, clear renal cell carcinoma; OE, overexpression.