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. 2020 Aug;46(2):700-708.
doi: 10.3892/ijmm.2020.4637. Epub 2020 Jun 10.

Rotenone restrains colon cancer cell viability, motility and epithelial‑mesenchymal transition and tumorigenesis in nude mice via the PI3K/AKT pathway

Wenbo Xiao  1 , Yongwei Liu  2 , Maolin Dai  3 , Yu Li  4 , Renqun Peng  4 , Shuangjiang Yu  5 , Hao Liu  4
Affiliations

Rotenone restrains colon cancer cell viability, motility and epithelial‑mesenchymal transition and tumorigenesis in nude mice via the PI3K/AKT pathway

Wenbo Xiao et al. Int J Mol Med. 2020 Aug.

Retraction in

Abstract

Rotenone, a natural hydrophobic pesticide, has been reported to display anticancer activity in a variety of cancer cells. However, the mechanism of rotenone on colon cancer (CC) cell migration, invasion and metastasis is still unknown. In the present study, the cytotoxicity of rotenone on CC cells were detected by the Cell Counting Kit‑8 assay and confirmed by clone formation assay. The effects of rotenone on CC cell invasion and migration activity were determined in vitro by Transwell invasion and wound healing assays, respectively. In addition, to reveal whether rotenone affected the epithelial‑mesenchymal‑transition (EMT) process, reverse transcription‑quantitative PCR, western blotting and immunofluorescence assays were used to detect the expression of EMT markers. The expression levels of the key markers of the PI3K/AKT pathway after rotenone treatment alone or in combination with a PI3K/AKT signaling activator in CC were also detected by western blotting. Finally, the in vivo antitumor effects of rotenone were evaluated in a subcutaneous xenotransplant tumor model treated with an intraperitoneal injection of rotenone. The results of the present study demonstrated that rotenone treatment induced CC cell cytotoxicity and greater effects were observed with increasing concentrations and inhibited cell proliferation compared with untreated cells. In vitro cell function assays revealed that rotenone inhibited CC cell migration, invasion and EMT compared with untreated cells. Mechanically, the phosphorylation levels of AKT and mTOR were downregulated in rotenone‑treated CC cells compared with untreated cells. Additionally, AKT and mTOR phosphorylation levels were increased by the PI3K/AKT signaling activator insulin‑like growth factor 1 (IGF‑1), which was reversed by rotenone treatment. The cell function assays confirmed that the IGF‑1‑activated cell proliferation, migration and invasion were decreased by rotenone treatment. These results indicated that rotenone affected CC cell proliferation and metastatic capabilities by inhibiting the PI3K/AKT/mTOR signaling pathway. In addition, rotenone inhibited tumor growth and metastatic capability of CC, which was confirmed in a xenograft mouse model. In conclusion, the present study revealed that rotenone inhibited CC cell viability, motility, EMT and metastasis in vitro and in vivo by inhibiting the PI3K/AKT/mTOR signaling pathway.

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Figures

Figure 1
Figure 1
Effects of rotenone on cell viability and clone formation in human CC cell lines. (A) SW480, SW620 and CRL-1790 cells were treated with increasing concentrations of rotenone, and the cytotoxicity was detected by the Cell Counting Kit-8 assay at 24 h. (B) SW480 and SW620 cells were treated with 10 µM rotenone, and the numbers of cell colonies were determined by colony formation assay after 20 days (n=3, mean ± SD). (C) Cells treated with 10 µM rotenone were subjected to EdU incorporation assay and analyzed by confocal microscopy. Scale bar, 50 µm. (D) The expression levels of cell proliferation markers caspase-3, cleaved-caspase-3, Bax and Bcl-2 were determined by western blot assay in CC cells treated with rotenone. *P<0.05 vs. untreated. CC, colon cancer; EdU, ethynyl-2-deoxyuridine; U, untreated; T, treated.
Figure 2
Figure 2
Migratory and invasive abilities of colon cancer cell lines treated with rotenone. (A) SW480 and SW620 cells were incubated with rotenone, and cell migration was determined by wound healing assay. (B) The invasion of SW480 and SW620 was determined by the Transwell invasion assay after treatment with rotenone. All experiments were repeated three times. *P<0.05 vs. untreated.
Figure 3
Figure 3
Rotenone regulates epithelial-mesenchymal transition in CC cells. (A and B) Rotenone affected the expression of (A) mRNA and (B) protein of EMT hallmarks, E-cadherin, vimentin and Snail, in SW480 and SW620 cells was determined by Real-time qPCR and western blotting. U, untreated; T, treated. (C) The positive expression of vimentin in SW480 and SW620 cells treated with rotenone was analyzed by immunofluorescence. Blue, DAPI; green, vimentin. *P<0.05 vs. untreated. CC, colon cancer; U, untreated; T, treated.
Figure 4
Figure 4
Rotenone inhibits CC progression via the PI3K/AKT/mTOR signaling pathway. (A and B) The expression of p-AKT, total AKT, p-mTOR and total mTOR was detected by western blotting analysis in cells treated with (A) rotenone alone or (B) rotenone and IGF-1. (C) CC cell viability was determined by Cell Counting Kit-8 assay following treatment with the PI3K/AKT signaling activator IGF-1 alone or co-treatment with rotenone. (D) Migration and (E) invasion of CC cells were detected by wound healing and Transwell invasion assays following treatment with IGF-1 alone or co-treatment with rotenone. (F) The expression of epithelial-to-mesenchymal transition markers E-cadherin, vimentin and Snail in CC cells treated with IGF-1 alone or co-treatment with rotenone was determined by western blotting.*P<0.05 vs. untreated;#P<0.05 vs. IGF-1. CC, colon cancer; IGF-1, insulin-like growth factor 1; p, phosphorylated; U, untreated; T, treated; OD, optical density.
Figure 5
Figure 5
Rotenone inhibits tumor growth and metastasis of colon cancer in vivo. (A) Representative tumors and tumor volume rom mice injected with rotenone or vehicle. (B) The positive expression of caspase-3, Bcl-2, E-cadherin and Vimentin was analyzed by immunohistochemical staining in harvested tumor samples. Magnification, ×400. (C) The expression of cell proliferation markers caspase-3, cleaved-caspase-3, Bax and Bcl-2, epithelial-to-mesenchymal transition markers E-cadherin and vimentin, as well as p-AKT and AKT, was verified by western blotting in tumor samples. p, phosphorylated.
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