Overexpression of Extracellular Superoxide Dismutase 3 Inhibits Cancer Cell Growth and Migration in Colorectal Cancer

Abstract

Background/aims: Incidence of colorectal cancer is rapidly increasing worldwide. Extracellular superoxide dismutase (EcSOD; SOD3) is an antioxidant enzyme. However, SOD3 roles in colorectal cancer progression remain uncertain.

Materials and methods: Superoxide dismutase 3 expression was evaluated, and we analyzed clinical relevance of SOD3 expression in colorectal cancer. Subsequently, SOD3 roles in colorectal cancer progression were detected by gain of function experiments. Changes in subcutaneous tumor and liver nodule size after SOD3 overexpression were examined in nude mice. The expression of proliferation marker Ki67 was assessed by immunohistochemical staining.

Results: Supperoxide dismutase 3 was downregulated in colorectal cancer (P <.01). Downregulation of SOD3 was correlated with unfavorable outcomes (P < .05). Superoxide dismutase 3 upregulation limited the proliferative (P <.05), migrative (P <.01) and invasive actions of colorectal cancer cells (P <.01) by suppressing epithelial-mesenchymal transition. Moreover, SOD3 overexpression reduced Ki67 expression (P <.01) and blocked tumor growth (P <01) and liver metastasis (P <.001) in mouse tumor model.

Conclusion: Superoxide dismutase 3 upregulation attenuates tumor growth and liver metastasis in colorectal cancer, suggesting that SOD3 has potential diagnostic and prognostic values regarding colorectal cancer treatment.

Figure 1.

SOD3 is downregulated in CRC tissues and cells and its downregulation predicts a poor prognosis. (A) RT-qPCR to measure the mRNA level of SOD3 in 80 paired CRC and adjacent normal tissues. (B) Western blotting to measure the protein level of SOD3 in 8 randomly selected paired tissues. (C and D) Overall survival and disease-free survival analysis according to the expression of SOD3. (E-G) RT-qPCR and western blotting to evaluate the mRNA and protein levels of SOD3 in the NCM460 cell line and CRC cell lines (SW620, SW480, DLD-1, and HCT116). Data are shown as mean ± SD. **P < .01, ***P < .001. SOD3, superoxide dismutase 3; RT-qPCR, real-time quantitative polymerase chain reaction.

Figure 2.

Supeoxide dismutase 3 elevation blocks CRC cell proliferation. (A) RT-qPCR was performed to detect the overexpression efficiency of the SOD3 overexpression vector in DLD-1, HCT116, SW480, and SW620 cells. (B) CCK-8 assays were conducted to assess cell viability. (C-F) Colony formation assays were used to evaluate cell proliferation. Data are shown as mean ± SD. *P < .05, **P < .01, ***P < .001.

Figure 3.

Superoxide dismutase 3 overexpression limits CRC cell malignancies by regulating epithelial–mesenchymal transition. (A-D) Transwell assays and wound healing assays to assess cell migration and invasion. (E and F) Western blotting of SOD3, E-cadherin, N-cadherin, and Vimentin protein levels. Data are shown as mean ± SD. **P < .01, ***P < .001. CRC, colorectal cancer; SOD3, superoxide dismutase 3.

Figure 4.

SOD3 overexpression inhibits CRC tumor growth. (A) Macroscopic images of resected tumors. (B) Tumor weight. (C) Tumor volume. (D) Immunohistochemical analysis of Ki67 expression. (E) Quantitative analysis of Ki67 expression. Data are shown as mean ± SD. *P < .05, **P < .01, ***P < .001. SOD3, superoxide dismutase 3.

Figure 5.

SOD3 overexpression suppresses colorectal cancer liver metastasis. (A) Microscopic images of resected livers stained by H&E. (B) Quantification of the number of liver metastatic nodules. Data are shown as mean ± SD. ***P < .001. SOD3, superoxide dismutase 3.