Colon cancer cell lines SW480 and SW620 as well as the normal human colon cells CRL-1790 were obtained from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences and cultured in DMEM (HyClone; GE Healthcare Life Sciences) supplemented with 10% FBS (Sigma-Aldrich; Merck KGaA) and penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. Rotenone was obtained from Sigma-Aldrich; Merck KGaA, and a stock of 10 mM was diluted in DMSO Hybri-Max (Sigma-Aldrich; Merck KGaA). Colon cells were treated with 0, 0.01, 0.05, 0.1, 1, 5, 10 and 20 µM rotenone for 48 h as previously described (11).

EdU incorporation assay was performed using an EdU Apollo DNA In Vitro kit (Guangzhou RiboBio Co., Ltd.) according to the manufacturer's instructions. Following treatment with rote-none, the cells were incubated with 50 µM EdU as previously described (19,20). Subsequently, the cells were incubated with 1X Hoechst 33342 solution and observed under a confocal microscope (magnification, ×400). The EdU incorporation rate was expressed as the ratio of EdU-positive cells to Hoechst-positive cells.

Cell viability was determined by the Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc.). SW480 and SW620 cells (4×10³) were seeded into a 96-well plate and cultured overnight in an incubator at 37°C. The cells were treated with 10 µM rotenone alone or in presence of10 nMPI3K/AKT signaling activator insulin-like growth factor 1 (IGF-1) in DMEM for 24 h. CCK-8 solution (10 µl) was added into each well and incu-bated at 37°C for 3 h. The OD value of the reaction solution at 450 nm was evaluated using an Anthos 2010 microplate reader (Biochrom, Ltd.).

SW480 and SW620 cells (100 cells/well) were seeded in 6-well plates and incubated overnight at 37°C, followed by treatment with 10 µM rotenone alone or with 10 nMIGF-1 at 37°C. After a 2-week incubation, the cell colonies were stained with 0.5% crystal violet for 10 min at room temperature, the 6-well plate was slowly immersed in tap water to remove the redundant stain and allowed to dry at room temperature for 2 days. The cell colonies (>50 cells) were counted using ImageJ software version 1.48u (National Institutes of Health).

SW480 and SW620 cells were seeded into 6-well plates (~1×10⁶ cells/well) and incubated at 37°C with 5% CO₂ for 24 h until ~100% confluence. Subsequently, the cells were serum starved and treated with 10 µM rote-none for 3 h at room temperature. Wounds were created by scratching the cell monolayers with a 10-µl pipette tip, and the cells were incubated in DMEM containing 1% fetal calf serum (Sigma-Aldrich; Merck KGaA) for 24 h, followed by removal of medium. Images were captured to estimate the cell migration at 0 and 24 h. The scratch width of the cells on both sides of the scratch was measured, and the relative migration rate of cells was calculated as the relative migration distance divided by the scratch width at 0 h.

For cell invasive ability assessment, Transwell chambers (Corning, Inc.) were precoated with Matrigel (BD Biosciences) at 4°C overnight. First, 1×10⁵ SW480 and SW620 cells were treated with 10 µM rotenone and incubated for 24 h in advance on the upper chamber with 8-µm pores with 500 µl serum-free DMEM. The lower chambers were filled with 700 µl DMEM containing 10% FBS. After 24 h, the cells remaining on the upper side of the membrane were removed carefully with a cotton swab, and the cells on the underside of the membrane were incubated with 0.1% crystal violet for 30 min at room temperature and counted under a light microscope in three random high-power fields (magnification, ×200).

Total RNAs from CC cell lines were acquired using the TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and the concentration and purity of the RNA were determined. RNA was reverse-transcribed into cDNA using a RevertAid Fist Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. QPCR was performed using the ABI Q6 detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with 1.0 µl cDNA and a SYBR^® Green Real-Time PCR Master mix (Takara Biotechnology Co., Ltd.). The PCR amplification included initial denaturation at 95°C for 3 min, followed by 40 cycles of 94°C for 30 sec, 60°C for 30 sec and 70°C for 30 sec. Relative quantification and calculations were performed using the comparative quantitation cycle method (2^−ΔΔCq) (21). GAPDH was used as the internal reference. The primer sequences used were as follows: E-cadherin forward, 5′-CCATTCCCAATGAGGCTGGT-3′ and reverse, 5′-GGCTTTTCTGTGACATCCGC-3′; vimentin forward, 5′-ACATGGTGGAAACCGAGGAT-3′ and reverse, 5′-TCCATTTCCCGCATTTGGT-3′; Snail forward, 5′-ACATGGTGGAAACCGAGGAT-3′ and reverse, 5′-GGTGGTGGAAGGAATAACGC-3′; and GAPDH forward, 5′-CGCTCCACCTTCAAGTATGC-3′ and reverse, 5′-GTCCACCACCCTGTTGCTGTAG-3′.

Protein samples were preprocessed with RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.). The protein (30 µg/lane) was separated by 10% SDS-PAGE and transferred to PVDF membranes (EMD Millipore). The membranes were blocked with 10% non-fat milk in 1X TBS + 0.1% Tween-20 (TBST) for 2 h at room temperature. Subsequently, the membranes were incubated with primary antibodies against cleaved caspase-3 (1:1,000; ab2302), Bax (1:1,000; ab32503), Bcl-2 (1:1,000; ab32124), E-cadherin (1:50; ab1416), vimentin (1:1,000; ab92547), Snail (1:1,000; ab229701), AKT (1:10,000; ab179463), phosphor (p)-AKT (T308; 1:1,000; ab38449), mTOR (1:2,000; ab2732), p-mTOR (1:1,000; ab109268) and anti-GAPDH (1:10,000; ab181602) at 4°C overnight. All primary antibodies were obtained from Abcam. Subsequently, the membranes were washed three times with TBST and incubated with a horse-radish peroxidase-conjugated goat-anti-mouse IgG (1:1,000; A7007; Beyotime Institute of Biotechnology) for 1 h at room temperature. Specific protein bands were developed using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.) and visualized using a ChemiDoc MP system (Bio-Rad Laboratories, Inc.).

SW480 and SW620 cells (1×10⁵ cells/dish) were fixed in 4% paraformaldehyde at 25°C for 15 min and treated with 0.1% Triton X-100 for 15 min. After blocking with 5% FBS for 1 h at room temperature, the cells were incubated with an anti-vimentin antibody (1:1,000; ab92547) for 2 h at room temperature. After washing three times with PBS, the cells were fixed with an anti-quenching sealer Fluoromount-G (Southern Biotech) and observed under fluorescence microscopyin three random high-power fields (magnification, ×400).

Male BALB/c athymic nude mice (6-week-old) were obtained from the Chongqing Medical University Animal Center and maintained in specific pathogen-free grade filter-top cages with a 12-h light/dark cycle in a controlled temperature (24±1°C) and 55% humidity, and received sterile rodent chow and water ad libitum. Rotenone (3 mg/kg; treated group) or vehicle (untreated group) was administered by intraperitoneal injection once a day for three days prior to inoculation of the cells into mice (n=6 per treatment group) as previously described (20). In addition, the nude mice were subcutaneously injected with 1×10⁷ SW480 cells in 0.1 ml DMEM in the right axillary region. Animals were monitored three times a week to observe tumor growth. Tumor sizes were measured using a caliper, and the tumor volumes were calculated as follows: Tumor volume (mm³)=1/2 × length × width². When tumors reached sufficient size (length ~18 mm, width ~13 mm and volume ~1,500 mm³), animals were euthanized by an overdose of pentobarbital (intraperitoneal, 120 mg/kg) on day 28, and tumor tissues were obtained. The protocol was approved by the Laboratory Animal Management Committee of Chongqing Medical University.

Data were analyzed using SPSS 19.0 (IBM Corp.) and are presented as the mean ± SD of three independent experiments. Student's t-test was used for comparisons between two groups. Multiple comparisons were performed by two-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To identify the cytotoxicity of rotenone on CC cells, human CC cell lines SW480 and SW620 and normal human colon CRL-1790 cells were treated with rotenone for 2 days and analyzed by the CCK-8 assay. The viability of CC cells decreased as the concentration of rotenone increased; by contrast, rotenone exhibited no significant effects on CRL-1790 cell viability (Fig. 1A). In subsequent experiments, cells were treated with 10 µM rote-none. In the colony formation assay, the numbers of colonies in the rotenone-treated wells were significantly lower compared with those in the untreated wells (Fig. 1B). In addition, EdU incorporation assays were used to investigate the effects of rotenone on CC cell proliferation. The results showed that rotenone notably inhibited CC cell proliferation compared with the untreated cells (Fig. 1C). The rotenone-mediated reduction of CC cell proliferation was also confirmed by western blot assay, as the protein level of Bcl-2 was decreased, whereas the levels of Bax and cleaved caspase-3 were increased following rotenone treatment compared with the untreated cells (Fig. 1D).

To determine the effects of rotenone on CC cell migration and invasion in vitro, wound healing and Transwell assays were respectively used with SW480 and SW620 cells treated with 10 µM rotenone. As demonstrated in Fig. 2A, 24-h rotenone incubation inhibited the migration of CC cells compared with that of the untreated cells. In addition, following incubation with rotenone for 24 h, the invasive ability of CC cells was decreased when compared with the untreated control group (Fig. 2C).

To determine whether rotenone affected the EMT process in CC cells, RT-qPCR and western blotting assays were performed to detect the mRNA and protein levels of the EMT markers E-cadherin, vimentin and Snail. SW480 and SW620 cells treated with 10 µM rotenone for 24 h exhibited upregulated E-cadherin and downregulated Snail and vimentin mRNA and protein levels compared with the untreated cells (Fig. 3A and B). In addition, the detection of vimentin expression by fluorescent immunocytostaining was also consistent with the above results as rotenone-treated cells exhibited lower fluorescence levels compared with the untreated controls (Fig. 3C). Therefore, these results indicated that rotenone contributed to EMT in CC cells.

To further investigate the molecular mechanism and pathway by which rotenone suppressed CC development and metastasis, the involvement of the PI3K/AKT signaling pathway in the anticancer effect of rotenone was determined. As presented in Fig. 4A, p-AKT (Ser473) and p-mTOR protein expression were downregulated in rotenone-treated CC cells. These results suggested that rotenone may exert its antitumor effect by inhibiting the activity of the PI3K/AKT/mTOR signaling pathway. To address this hypothesis, rotenone-treated or untreated CC cells were incubated with a PI3K/AKT signaling activatorIGF-1. The results demonstrated that the protein levels of p-AKT and p-mTOR were upregulated byIGF-1 compared with the untreated control cells, whereas rotenone reversed this effect (Fig. 4B). In addition, the proliferative, migratory and invasive abilities of rotenone and IGF-1 co-treated CC cells were determined. Of note, the SW480 and SW620 cell proliferation ofIGF-1-treatedcells was reversed by rotenone treatment, which was confirmed by the CCK-8 assay (Fig. 4C). Similarly, the wound healing and Transwell assays indicated that the IGF-1-induced CC cell migratory and invasive abilities were decreased following rotenone treatment compared with those of cells treated with IGF-1 alone (Fig. 4D and E). In addition, co-treatment with IGF-1 and rotenone resulted in the upregulation of E-cadherin and downregulation of vimentin and Snail protein expression in CC cells compared with that in cells treated with IGF-1 alone (Fig. 4F). In summary, these results suggested that rotenone decreased CC proliferation, migration, invasion and EMT in vitro by inhibiting the PI3K/AKT/mTOR pathway.

To confirm the in vitro anti-tumor effect of rotenone, the in vivo effects of rotenone were evaluated using the subcutaneous xenotransplant tumor model. Mice were injected intraperitoneally once a day with 3 mg/kg/day rotenone as previously described (22) for three days, and SW480 tumor cells were inoculated into nude mice. Rotenone-treated mice exhibited significantly inhibited tumor growth compared with the untreated control group (Fig. 5A). Immunohistochemical staining of the xenograft tissue sections revealed decreased expression of vimentin and increased expression of caspase-3, Bcl-2, E-cadherin after rotenone treatment (Fig. 5B). Subsequently, rotenone-mediated inhibition of mouse tumor tissue proliferative and metastatic abilities in vivo was confirmed by western blot assay, as the protein level of Bcl-2 was downregulated, whereas those of Bax and cleaved caspase-3 were upregulated following rotenone treatment compared with the tissues from untreated mice. The expression levels of vimentin were decreased, whereas the levels of E-cadherin were increased following rotenone treatment compared with those detected in the untreated mice. Of note, p-AKT expression was downregulated by rotenone compared with that in the untreated mouse tissues (Fig. 5C). These findings revealed that rotenone inhibited CC cell proliferation and metastasis in vivo.