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This article has been retracted.

Retraction in: Clin Exp Pharmacol Physiol. 2025 Feb;52(2):e70014 See also: PMC Retraction Policy

. 2020 May 11;47(8):1484–1494. doi: 10.1111/1440-1681.13320

Rotenone restrains the proliferation, motility and epithelial–mesenchymal transition of colon cancer cells and the tumourigenesis in nude mice via PI3K/AKT pathway

Wusong Xue ¹, Siye Men ², Renghai Liu ^1,^✉

PMCID: PMC7384028 PMID: 32282954

Abstract

Rotenone, a toxic rotenoid compound, has anti‐tumour effects on several cancers. This study aims to clarify the effect of rotenone on the proliferation, apoptosis, invasion and migration of colon cancer cells and tumourigenesis in nude mice. The present results show that rotenone significantly inhibited the proliferation, promoted the apoptosis, and suppressed the invasion and migration of colon cancer cells in a dose‐dependent manner. Rotenone inhibited PI3K/AKT pathway in LoVo and SW480 cells in a dose‐dependent manner. In addition, rotenone regulated the proliferation, apoptosis, invasion, migration and EMT of LoVo and SW480 cells through PI3K/AKT pathway. In colon cancer xenograft mice, rotenone inhibited tumour volume and weight in nude mice, inhibited PI3K/AKT pathway and EMT in vivo. Therefore, rotenone inhibited the proliferation, invasion and migration, promoted the apoptosis of colon cancer cells through PI3K/AKT pathway in vitro, and suppressed the tumourigenesis in nude mice in vivo.

Keywords: colon cancer, PI3K/AKT pathway, proliferation, rotenone, tumourigenesis

1. INTRODUCTION

Colon cancer is one of most deadly cancers in the world, causing 50 630 deaths in 2018 in the United States according to Siegel's report. ¹ Although advanced treatments such as surgical resection, targeted drug delivery and adjuvant chemotherapy have been used to treat colon cancer, ² , ³ and to some extent suppress the colon cancer growth and metastasis, ⁴ the five‐year survival rate of colon cancer remains unsatisfactory. ⁵ The occurrence of metastasis is one of most important reasons for the failure of colon cancer treatment, and metastasis to the liver, lymph node and lung are common. ⁶ So, more and more research has been focused on inhibiting the growth and metastasis of colon cancer, however, therapies with good effect for colon cancer are still lacking.

Rotenone, known as an inhibitor of mitochondrial complex I, is a toxic rotenoid compound extracted from leguminosae family plants and can be used as botanical insecticide. ⁷ Studies have shown that rotenone plays an important role in regulating Parkinson's disease through inducing the death and oxidative damage of nerve cells. ⁸ , ⁹ Recent studies have also found that rotenone can suppress the progression of cancers, such as lung cancer and hepatic cancer, through impairing the autophagic flux and inhibiting cancer cell proliferation. ¹⁰ , ¹¹ In addition, deguelin, another rotenoid compound extracted from leguminosae family plants, has been proven to inhibit the proliferation and induce the apoptosis of colon cancer cells, thereby suppressing the growth of colon cancer. ¹² , ¹³ However, studies focusing on the effects of rotenone on the growth and metastasis of colon cancer are still lacking.

The phosphatidylinositol 3‐kinase (PI3K)/Akt pathway can be hyperactivated or altered in a variety of cancers and modulated by multiple molecular mechanisms. ¹⁴ , ¹⁵ Previous studies have reported that the PI3K/Akt pathway regulates the proliferation, autophagy, apoptosis, invasion, migration and cell cycle of cancer cells. ¹⁶ , ¹⁷ These abilities of PI3K/Akt pathway make it critically important in the progression of cancer. Recent studies have also demonstrated that the PI3K/Akt pathway plays an important role in colon cancer. For example, inhibition of phosphorylated (p‐)Akt suppressed the growth and invasion of colon cancer cells. ¹⁸ Activation of the PI3K/Akt pathway promoted metastasis of colon cancer. ¹⁹ The decrease of p‐Akt inhibited the proliferation, migration and invasion of colon cancer cells. ²⁰ These studies indicated that the PI3K/Akt pathway plays an important role in the growth and metastasis of colon cancer.

In this study, we found that rotenone inhibited the proliferation, invasion and migration, promoted the apoptosis of colon cancer cells through the PI3K/AKT pathway in vitro, and suppressed the tumourigenesis in nude mice in vivo, which provides evidence for the use of rotenone in treating colon cancer.

2. RESULTS

2.1. Rotenone inhibited the proliferation and promoted the apoptosis of colon cancer cells

To observe the effect of rotenone on the proliferation and apoptosis of colon cancer cells, different concentrations of rotenone were used to treat colon cancer cells LoVo and SW480. The molecular structural formula of rotenone was shown in Figure 1A. As shown in Figure 1B,C, rotenone significantly inhibited the proliferation of LoVo and SW480 cells at the concentration of 100 μg/mL, and reached the maximum inhibitory effect at the level of 1000 μg/mL. The effect of rotenone on normal colon epithelial cell line FHC was also detected by CCK8, which showed that there was no significant difference on normal colon epithelial cells treated with or without rotenone (Figure 1D). Brdu staining also showed the proliferation of LoVo cells and SW480 was significantly inhibited at the concentration of 100 and 1000 μg/mL (Figure 1E). In addition, the apoptosis of LoVo and SW480 cells was significantly promoted under the treatment of rotenone (100 and 1000 μg/mL; Figure 1F). In LoVo and SW480 cells, the protein level of cleaved caspase 3 was significantly up‐regulated under the treatment of rotenone (100 and 1000 μg/mL; Figure 1G,H).

Rotenone affected the proliferation and apoptosis of colon cancer cells. Colon cancer cells LoVo and SW480 were divided into control group, solvent control group (veh), and rotenone groups (Rot‐10 μg/mL, Rot‐100 μg/mL and Rot‐1000 μg/mL). A, Molecular structural formula of rotenone. B and C, The proliferation of LoVo and SW480 cells was detected using CCK8 assay. D, The proliferation of normal colon epithelial cell line FHC was detected using CCK8 assay. E, The proliferation of LoVo and SW480 cells was detected using Brdu staining. F, The apoptosis of LoVo and SW480 cells was detected using flow cytometry. G and H, Cleaved caspase 3 protein from LoVo and SW480 cells was detected using Western blot, **P < .01, ***P < .001 vs veh group. B and C, , Rot‐1000 μg/mL; , Rot‐100 μg/mL; , Rot‐10 μg/mL; , veh; , control

Inline graphic — Rotenone affected the proliferation and apoptosis of colon cancer cells. Colon cancer cells LoVo and SW480 were divided into control group, solvent control group (veh), and rotenone groups (Rot‐10 μg/mL, Rot‐100 μg/mL and Rot‐1000 μg/mL). A, Molecular structural formula of rotenone. B and C, The proliferation of LoVo and SW480 cells was detected using CCK8 assay. D, The proliferation of normal colon epithelial cell line FHC was detected using CCK8 assay. E, The proliferation of LoVo and SW480 cells was detected using Brdu staining. F, The apoptosis of LoVo and SW480 cells was detected using flow cytometry. G and H, Cleaved caspase 3 protein from LoVo and SW480 cells was detected using Western blot, **P < .01, ***P < .001 vs veh group. B and C, , Rot‐1000 μg/mL; , Rot‐100 μg/mL; , Rot‐10 μg/mL; , veh; , control

2.2. Rotenone suppressed the invasion and migration of LoVo and SW480 cells

As shown in Figure 2A, rotenone significantly suppressed the invasion of LoVo and SW480 cells at the concentration of 100 and 1000 μg/mL. Wound healing assay showed the scratch width was wider in Rot‐100 μg/mL group and Rot‐1000 μg/mL group than the solvent control group (veh) group, indicating the motility of LoVo and SW480 cells was suppressed by rotenone (100 and 1000 μg/mL; Figure 2B,C). We also found that protein level of epithelial marker E‐cadherin was significantly up‐regulated after the treatment of rotenone (100 and 1000 μg/mL), protein levels of mesenchymal markers Vimentin and N‐cadherin were significantly down‐regulated after the treatment of rotenone (100 and 1000 μg/mL), indicating rotenone suppressed EMT of colon cancer cells (Figure 2D,E).

Rotenone suppressed the invasion and migration of LoVo and SW480 cells. LoVo and SW480 cells were divided into control, veh, and rotenone groups (Rot‐10 μg/mL, Rot‐100 μg/mL and Rot‐1000 μg/mL). A, The invasion of LoVo and SW480 cells was detected using Transwell assay. B and C, The motility of LoVo and SW480 cells was detected using wound healing assay. D and E, Protein levels of epithelial marker E‐cadherin, mesenchymal markers Vimentin and N‐cadherin were detected by western blot. **P < .01, ***P < .001 vs veh group. D and E, , control; , veh; , Rot‐10 μg/mL; , Rot‐100 μg/mL; , Rot‐1000 μg/mL

Inline graphic — Rotenone suppressed the invasion and migration of LoVo and SW480 cells. LoVo and SW480 cells were divided into control, veh, and rotenone groups (Rot‐10 μg/mL, Rot‐100 μg/mL and Rot‐1000 μg/mL). A, The invasion of LoVo and SW480 cells was detected using Transwell assay. B and C, The motility of LoVo and SW480 cells was detected using wound healing assay. D and E, Protein levels of epithelial marker E‐cadherin, mesenchymal markers Vimentin and N‐cadherin were detected by western blot. **P < .01, ***P < .001 vs veh group. D and E, , control; , veh; , Rot‐10 μg/mL; , Rot‐100 μg/mL; , Rot‐1000 μg/mL

2.3. Rotenone inhibited PI3K/AKT pathway in LoVo and SW480 cells

It has been reported that overexpression of the PI3K/AKT pathway plays an important role in the promotion of colon cancer progression. ²¹ So, we determined the effect of rotenone on the PI3K/AKT pathway in LoVo and SW480 cells. We found that the protein levels of p‐PI3K and p‐AKT were down‐regulated after the treatment of rotenone (100 and 1000 μg/mL; Figure 3A,B). Rotenone (100 and 1000 μg/mL) also suppressed the ratio of p‐PI3K to PI3K and the ratio of p‐AKT to AKT (Figure 3A,B).The mRNA expressions of AKT downstream signal molecules IKK, TSC1 and GSK3 were detected by RT‐PCR. Results showed that rotenone (100 and 1000 μg/mL) significantly decreased IKK mRNA expression (Figure 3C,D), while rotenone significantly promoted TSC1 and GSK3 mRNA expressions (Figure 3C,D).

Rotenone inhibited PI3K/AKT pathway in LoVo and SW480 cells. LoVo and SW480 cells were divided into control, veh, and rotenone groups (Rot‐10 μg/mL, Rot‐100 μg/mL and Rot‐1000 μg/mL). A and B, The protein levels of p‐PI3K, PI3K, p‐AKT, and AKT were detected by western blot. C and D, AKT downstream signal molecules IKK, TSC1 and GSK3 mRNA expressions were detected by RT‐PCR P > .05, P < .05, **P < .01, ***P < .001 vs veh group. A‐D, , control; , veh; , Rot‐10 μg/mL; , Rot‐100 μg/mL; , Rot‐1000 μg/mL

Inline graphic — Rotenone inhibited PI3K/AKT pathway in LoVo and SW480 cells. LoVo and SW480 cells were divided into control, veh, and rotenone groups (Rot‐10 μg/mL, Rot‐100 μg/mL and Rot‐1000 μg/mL). A and B, The protein levels of p‐PI3K, PI3K, p‐AKT, and AKT were detected by western blot. C and D, AKT downstream signal molecules IKK, TSC1 and GSK3 mRNA expressions were detected by RT‐PCR P > .05, P < .05, **P < .01, ***P < .001 vs veh group. A‐D, , control; , veh; , Rot‐10 μg/mL; , Rot‐100 μg/mL; , Rot‐1000 μg/mL

2.4. Rotenone regulated the proliferation, apoptosis, invasion and migration of LoVo cells through PI3K/AKT pathway

Next, we investigated whether rotenone regulated the proliferation, apoptosis, invasion and migration of LoVo cells through the PI3K/AKT pathway. PI3K overexpressing vector or silencing PI3K (si‐PI3K) was transfected into LoVo cells to overexpress or inhibit PI3K expression. As shown in Figure 4A, the proliferation of LoVo cells was promoted in the PI3K group compared to the control group, whereas the proliferation of LoVo cells was inhibited in the si‐PI3K group compared to the control group (Figure 5A). Rotenone significantly increased the apoptosis of LoVo cells, the apoptosis of LoVo cells was significantly inhibited in the PI3K group (Figure 4B), whereas the apoptosis of LoVo cells was significantly promoted in the si‐PI3K group compared to the control group (Figure 5B). Rotenone significantly inhibited the invasion of LoVo cells, the invasion of LoVo cells was facilitated in the PI3K group (Figure 4C), whereas the invasion of LoVo cells was inhibited in the si‐PI3K group (Figure 5C). Rotenone significantly enlarged the scratch width; the scratch width was narrower in the PI3K group (Figure 4D), whereas the scratch width was wider in the si‐PI3K group, indicating the motility of LoVo cells was promoted by si‐PI3K (Figure 5D). The protein level of epithelial marker E‐cadherin was down‐regulated in the PI3K group compared with the control group, and protein levels of mesenchymal markers Vimentin and N‐cadherin were up‐regulated in the PI3K group (Figure 4F).While the protein level of E‐cadherin was up‐regulated in the si‐PI3K group compared with the control group, and protein levels of Vimentin and N‐cadherin were down‐regulated in the si‐PI3K group compared with the control group (Figure 5E). We also found the protein levels of p‐PI3K and p‐AKT were up‐regulated in the PI3K group compared with the control group, and the ratio of p‐PI3K to PI3K and the ratio of p‐AKT to AKT were increased in the PI3K group (Figure 4E). While the protein levels of p‐PI3K and p‐AKT were down‐regulated in the si‐PI3K group than control group, and the ratio of p‐PI3K to PI3K and the ratio of p‐AKT to AKT were decreased in the si‐PI3K group compared with the control group (Figure 5F).

Rotenone regulated the proliferation, apoptosis, invasion and migration of LoVo cells through PI3K/AKT pathway. LoVo cells were transfected with PI3K overexpressing vector, then LoVo cells were treated with 1000 μg/mL rotenone. A, The proliferation of LoVo cells was detected using CCK8 assay. B, The apoptosis of LoVo cells was detected using flow cytometry. C, The invasion of LoVo cells was detected using Transwell assay. D, The motility of LoVo cells was detected using wound healing assay. E, The protein levels of p‐PI3K, PI3K, p‐AKT, and AKT were detected by western blot. F, The protein levels of epithelial marker E‐cadherin, mesenchymal markers Vimentin and N‐cadherin were detected by western blot. *P < .05, **P < .01, ***P < .001 vs control. NS P > .05 vs PI3K group. A, , PI3K + Rot‐1000 μg/mL; , PI3K; , Rot‐1000 μg/mL; , control; E and F, , control; , Rot‐1000 μg/mL; , PI3K; , PI3K + Rot‐1000 μg/mL

Inline graphic — Rotenone regulated the proliferation, apoptosis, invasion and migration of LoVo cells through PI3K/AKT pathway. LoVo cells were transfected with PI3K overexpressing vector, then LoVo cells were treated with 1000 μg/mL rotenone. A, The proliferation of LoVo cells was detected using CCK8 assay. B, The apoptosis of LoVo cells was detected using flow cytometry. C, The invasion of LoVo cells was detected using Transwell assay. D, The motility of LoVo cells was detected using wound healing assay. E, The protein levels of p‐PI3K, PI3K, p‐AKT, and AKT were detected by western blot. F, The protein levels of epithelial marker E‐cadherin, mesenchymal markers Vimentin and N‐cadherin were detected by western blot. *P < .05, **P < .01, ***P < .001 vs control. NS P > .05 vs PI3K group. A, , PI3K + Rot‐1000 μg/mL; , PI3K; , Rot‐1000 μg/mL; , control; E and F, , control; , Rot‐1000 μg/mL; , PI3K; , PI3K + Rot‐1000 μg/mL

Rotenone regulated the proliferation, apoptosis, invasion and migration of LoVo cells through PI3K/AKT pathway. LoVo cells were transfected with silencing PI3K (si‐PI3K), then LoVo cells were treated with 1000 μg/ml rotenone. A. The proliferation of LoVo cells was detected using CCK8 assay. B. The apoptosis of LoVo cells was detected using flow cytometry. C. The invasion of LoVo cells was detected using Transwell assay. D. The motility of LoVo cells was detected using wound healing assay. E. The protein levels of p‐PI3K, PI3K, p‐AKT, and AKT were detected by western blot. F. The protein levels of epithelial marker E‐cadherin, mesenchymal markers Vimentin and N‐cadherin were detected by western blot. *P < .05, **P < .01, ***P < .001 vs control. NS P > .05 vs si‐PI3K group. (A, E, F) , si‐PI3K+Rot‐1000 μg/mL; , si‐PI3K; , Rot‐1000 μg/mL; , control

Inline graphic — Rotenone regulated the proliferation, apoptosis, invasion and migration of LoVo cells through PI3K/AKT pathway. LoVo cells were transfected with silencing PI3K (si‐PI3K), then LoVo cells were treated with 1000 μg/ml rotenone. A. The proliferation of LoVo cells was detected using CCK8 assay. B. The apoptosis of LoVo cells was detected using flow cytometry. C. The invasion of LoVo cells was detected using Transwell assay. D. The motility of LoVo cells was detected using wound healing assay. E. The protein levels of p‐PI3K, PI3K, p‐AKT, and AKT were detected by western blot. F. The protein levels of epithelial marker E‐cadherin, mesenchymal markers Vimentin and N‐cadherin were detected by western blot. *P < .05, **P < .01, ***P < .001 vs control. NS P > .05 vs si‐PI3K group. (A, E, F) , si‐PI3K+Rot‐1000 μg/mL; , si‐PI3K; , Rot‐1000 μg/mL; , control

2.5. Rotenone inhibited the tumourigenesis in nude mice

Finally, we explored the effect of rotenone on the inhibition of tumourigenesis through the PI3K/AKT pathway. LoVo cells transfected with PI3K overexpressing vector or si‐PI3K were subcutaneously inoculated into nude mice, then rotenone (1 mg/kg) was intraperitoneally injected into the mice every other day. As shown in Figure 6A‐C, rotenone significantly reduced tumour volumes and tumour weight. Tumour volumes and tumour weight were significantly facilitated in the PI3K group compared to the control group, whereas tumour volumes and tumour weight were significantly inhibited in the si‐PI3K group as compared to the control group (Figure 7A‐C). Rotenone significantly promoted cell apoptosis in tumour tissue, and cell apoptosis in tumour tissue was significantly inhibited in PI3K group than control group (Figure 6D). Rotenone up‐regulated cleaved caspase 3 protein level in tumour tissues, and cleaved caspase 3 protein level was also up‐regulated in si‐PI3K group (Figure 7D). In addition, rotenone decreased vimentin expression, and the expression of vimentin was increased in PI3K group than control group (Figure 6E). Rotenone increased E‐cadherin expression, and the expression of E‐cadherin was down‐regulated in the PI3K group (Figure 6G), whereas the expression of E‐cadherin was up‐regulated in the si‐PI3K group compared to the control group (Figure 7E). In addition, rotenone decreased vimentin and N‐cadherin expressions, the expressions of vimentin and N‐cadherin were up‐regulated in PI3K group (Figure 6G), whereas the expressions of vimentin and N‐cadherin were decreased in the si‐PI3K group compared to the control group (Figure 7E), indicating rotenone inhibited EMT in vivo. The protein levels of p‐PI3K, p‐AKT in tumour tissues demonstrated that rotenone inhibited PI3K/AKT pathway in vivo (Figure 6F and 7F).

Rotenone inhibited the tumourigenesis in nude mice. LoVo cells were transfected with PI3K overexpressing vector, then LoVo cells were subcutaneously inoculated into nude mice. To observe the effect of rotenone on tumourigenesis in nude mice, rotenone (1 mg/kg) was intraperitoneally injected into the mice every other day. So, nude mice were divided into control group, Rot group, PI3K group and PI3K + Rot group, with five mice in each group. Six weeks later, mice were sacrificed and tumour tissues were collected. A, Tumours collected from mice in the four groups. B, Tumour volumes at day 14, 21, 28, 35, 42 in the four groups. C, Tumour weight in the four groups. D, The apoptosis was detected in tumour tissue using TUNEL assay. E, The expression of vimentin was detected using immunohistochemistry. F and G, The protein levels of p‐PI3K, PI3K, p‐AKT, AKT, epithelial marker E‐cadherin, mesenchymal markers Vimentin and N‐cadherin were detected by western blot. **P < .01, ***P < .001 vs control. NS P > .05 vs PI3K group. B, , PI3K + Rot; , PI3K; , Rot; , control; F and G, , control; , Rot; , PI3K; , PI3K + Rot

Inline graphic — Rotenone inhibited the tumourigenesis in nude mice. LoVo cells were transfected with PI3K overexpressing vector, then LoVo cells were subcutaneously inoculated into nude mice. To observe the effect of rotenone on tumourigenesis in nude mice, rotenone (1 mg/kg) was intraperitoneally injected into the mice every other day. So, nude mice were divided into control group, Rot group, PI3K group and PI3K + Rot group, with five mice in each group. Six weeks later, mice were sacrificed and tumour tissues were collected. A, Tumours collected from mice in the four groups. B, Tumour volumes at day 14, 21, 28, 35, 42 in the four groups. C, Tumour weight in the four groups. D, The apoptosis was detected in tumour tissue using TUNEL assay. E, The expression of vimentin was detected using immunohistochemistry. F and G, The protein levels of p‐PI3K, PI3K, p‐AKT, AKT, epithelial marker E‐cadherin, mesenchymal markers Vimentin and N‐cadherin were detected by western blot. **P < .01, ***P < .001 vs control. NS P > .05 vs PI3K group. B, , PI3K + Rot; , PI3K; , Rot; , control; F and G, , control; , Rot; , PI3K; , PI3K + Rot

Rotenone inhibited the tumourigenesis in nude mice. LoVo cells were transfected with si‐PI3K, then LoVo cells were subcutaneously inoculated into nude mice. To observe the effect of rotenone on tumourigenesis in nude mice, rotenone (1 mg/kg) was intraperitoneally injected into the mice every other day. So, nude mice were divided into control group, Rot group, si‐PI3K group and si‐PI3K + Rot group, with five mice in each group. Six weeks later, mice were sacrificed and tumour tissues were collected. A, Tumours collected from mice in the four groups. B, Tumour volumes at day 14, 21, 28, 35, 42 in the four groups. C, Tumour weight in the four groups. D, Cleaved caspase 3 protein level in tumour tissue was detected by Western blot. E and F, The protein levels of p‐PI3K, PI3K, p‐AKT, AKT, epithelial marker E‐cadherin, mesenchymal markers Vimentin and N‐cadherin were detected by western blot. **P < .01, ***P < .001 vs control. NS P > .05 vs si‐PI3K group. (B, E, F) , si‐PI3K+Rot; , si‐PI3K; , Rot; , control

Inline graphic — Rotenone inhibited the tumourigenesis in nude mice. LoVo cells were transfected with si‐PI3K, then LoVo cells were subcutaneously inoculated into nude mice. To observe the effect of rotenone on tumourigenesis in nude mice, rotenone (1 mg/kg) was intraperitoneally injected into the mice every other day. So, nude mice were divided into control group, Rot group, si‐PI3K group and si‐PI3K + Rot group, with five mice in each group. Six weeks later, mice were sacrificed and tumour tissues were collected. A, Tumours collected from mice in the four groups. B, Tumour volumes at day 14, 21, 28, 35, 42 in the four groups. C, Tumour weight in the four groups. D, Cleaved caspase 3 protein level in tumour tissue was detected by Western blot. E and F, The protein levels of p‐PI3K, PI3K, p‐AKT, AKT, epithelial marker E‐cadherin, mesenchymal markers Vimentin and N‐cadherin were detected by western blot. **P < .01, ***P < .001 vs control. NS P > .05 vs si‐PI3K group. (B, E, F) , si‐PI3K+Rot; , si‐PI3K; , Rot; , control

3. DISCUSSION

Rotenone, a natural rotenoid compound, has an anti‐tumour effect through inhibiting the growth of cancers. For example, rotenone could alter the radiation responses of murine sarcoma tumour and fibrosarcoma, and inhibited tumour growth. ²² Rotenone suppressed the activation of STAT3 in lung cancer cells, which inhibited lung cancer cell survival and reduced chemoresistance. ²³ However, the effect of rotenone on the growth of colon cancer is still not known. In this study, we found that rotenone remarkably inhibited the proliferation and increased the apoptosis of colon cancer cells in a dose‐dependent manner in vitro. Therefore, we demonstrated for the first time that rotenone played an anti‐tumour role in colon cancer through inhibiting the growth of colon cancer cells.

Rotenone exerts its promotion or inhibition effect on the invasion and migration of cancers. For example, rotenone inhibited the invasion and migration of hepatoma cells, thereby suppressing hepatoma development. ²¹ In human lung adenocarcinoma, rotenone remarkably promoted the migration and invasion of lung adenocarcinoma cells to facilitate tumour metastasis. ²⁴ However, the effect of rotenone on the invasion and migration of colon cancer is largely unknown. In this study, we found that rotenone remarkably inhibited the invasion and migration of colon cancer cells in a dose‐dependent manner in vitro. Therefore, we clarified for the first time that rotenone exerted its anti‐tumour role in colon cancer through inhibiting the invasion and migration of colon cancer cells.

Previous studies have also shown that rotenone could inhibit or promote EMT of cancer cells. For example, rotenone obtained a similar effect like HSP60 knockdown, for it increased reactive oxygen species production and further activated EMT of clear cell renal cell carcinoma and glioblastoma cells. ²⁵ , ²⁶ On the contrary, rotenone inhibited EMT of metastatic canine mammary gland tumour cells, indicating rotenone might be an anti‐tumour agent against metastatic canine mammary gland tumour. ²⁷ In this study, rotenone significantly up‐regulated epithelial marker E‐cadherin expression and down‐regulated mesenchymal markers Vimentin and N‐cadherin expressions in LoVo cells, indicating rotenone suppressed EMT of colon cancer cells. Therefore, we illustrated for the first time that rotenone exerted its anti‐tumour role in colon cancer through suppressing EMT of colon cancer cells.

Mechanistically, researchers have found that rotenone induced the apoptosis of cancer cells through inhibiting the PI3K/Akt pathway in lung cancer. ¹⁰ However, the role of rotenone in the PI3K/Akt pathway in colon cancer cells is not known. In this study, we found that rotenone inhibited the PI3K/Akt pathway in colon cancer cells in a dose‐dependent manner. In addition, rotenone inhibited the proliferation, invasion, migration and EMT, and promoted the apoptosis of colon cancer cells through PI3K/AKT pathway in vitro. Therefore, we interpreted for the first time that rotenone played an anti‐tumour role in colon cancer through inhibiting the PI3K/Akt pathway.

A previous report showed that rotenone combined with 2‐Deoxy‐D‐glucose effectively suppressed tumour growth of colon cancer cell (HT29) xenografts. ²⁸ However, the underlying mechanism of rotenone in inhibiting tumour growth in xenograft mice was not clearly illustrated. In this in vivo experiment, rotenone reduced tumour volume and weight in nude mice, promoted cell apoptosis in tumour tissue, and decreased mesenchymal marker vimentin expression through the PI3K/Akt pathway, indicating rotenone suppressed the growth and metastasis of colon cancer through the PI3K/Akt pathway in vivo.

In conclusion, our study demonstrated that rotenone inhibited the proliferation, invasion, migration and EMT of colon cancer cells through PI3K/AKT pathway in vitro, and suppressed the tumourigenesis in colon cancer xenograft mice in vivo. Rotenone might be a therapeutic drug for the treatment of colon cancer.

4. MATERIALS AND METHODS

4.1. Cell lines, treatment and transfection

Colon cancer cell lines LoVo and SW480 were obtained from the Cell Bank of Chinese Academy of Sciences. LoVo cells were cultured in Dulbecco's Modified Eagle Medium medium (DMEM; Gibco) supplemented with 2.5 g/L NaHCO₃ and 10% fetal bovine serum (Gibco) under an atmosphere of 95% air and 5% CO₂ at 37℃. SW480 cells were cultured in Leibovitz's L‐15 Medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) under an atmosphere of 100% air at 37℃. Normal colon epithelial cell line FHC was obtained from the American Type Culture Collection (ATCC), and cultured in DMEM:F12 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 25 mmol/L HEPES, 10 ng/mL cholera toxin, 0.005 mg/mL insulin, 0.005 mg/mL transferrin, 100 ng/mL hydrocortisone, and 20 ng/mL human recombinant EGF (ThermoFisher Scientific) under an atmosphere of 95% air and 5% CO₂ at 37℃.

Rotenone was obtained from Sigma with the purity >95%. LoVo and SW480 cells were treated with different concentrations of rotenone (10, 100, 1000 μg/mL). For solvent control group (veh), dimethylsulfoxide (DMSO; Sigma) was used to treat LoVo and SW480 cells.

LoVo cells were seeded into a culture plate and grown to 80% confluence for cell transfection. PI3K overexpressing vector was synthesised by Genechem and was transfected into LoVo cells using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions.

4.2. Cell Counting Kit‐8 (CCK8) assay

The CCK8 kit (Beyotime Biotechnology) was used to detect the proliferation of LoVo and SW480 cells. Cells (2 × 10³/well) were seeded into 96‐well plates and cultured for indicated time (6, 24, 48 and 72 hours). Then, 10 μL of CCK8 solution was added into each well and the absorbance at 450 nm was measured by a microplate reader (Thermo Scientific).

4.3. Bromodeoxyuridine (BRDU) staining

The BrdU staining kit (Sigma) was used to detect the proliferation of LoVo cells. BrdU was added to cells at the concentration of 10 μmol/L. After two hours’ incubation, treated cells were fixed with 4% paraformaldehyde for 25 minutes at 25℃. Then, immunoreactive cells were observed using a fluorescent microscope (Nikon, Japan), and cells were counted in five visual fields.

4.4. Flow cytometry

Annexin V‐FITC Apoptosis Detection Kit (Beyotime Biotechnology) was used to determine the apoptotic cells. Cells were centrifuged at 670 g for 5 minutes and washed twice with pre‐cooling PBS. Binding buffer (195 μL) was added to the cells. Then, the cells were treated with 5 μL Annexin V‐FITC and 10 μL PI at 25℃ for 20 minutes in the dark. Cell apoptosis was determined by a flow cytometry (FACSCanto II; BD).

4.5. Transwell assay

Transwell chamber (pore size 8 μm; Corning) was used to determine the invasion of LoVo cells according to previous report. ²⁹ LoVo cells (1 × 10⁴) were placed in serum‐free DMEM (Gibco) in the upper chamber coated with matrigel. And the lower chamber was added with DMEM medium supplemented with 10% fetal bovine serum. Twenty‐four hours later, cells in the lower chamber were collected and stained with crystal violet. The number of invasion cells was counted using a microscope (Nikon).

4.6. Wound healing assay

Wound healing assay was conducted according to previous report. ²² LoVo cells were cultured in DMEM medium supplemented with 10% fetal bovine serum in 60‐mm culture dishes until 100% confluence to form a monolayer. Then, the monolayer was scratched by a pipette tip. At 0 and 48 hours, the scratch width was observed by photographs.

4.7. Western blot

Proteins of LoVo cells were isolated using RIPA lysis buffer (Beyotime Biotechnology), and the concentration of proteins was measured using BCA Protein Assay Kit (Beyotime Biotechnology). Proteins were separated on a 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes, and the membranes were blocked with 5% skim milk for 1.5 hours. Then, the membranes were incubated with primary antibodies against E‐cadherin (Cell Signaling Technology), N‐cadherin (Cell Signaling Technology), Vimentin (Cell Signaling Technology), p‐PI3K (Cell Signaling Technology), PI3K (Cell Signaling Technology), p‐AKT (Cell Signaling Technology), AKT (Cell Signaling Technology), and cleaved caspase 3 (Cell Signaling Technology). After washing three times, the membranes were incubated with horseradish peroxidase‐conjugated secondary antibody (Abcam). The bands were visualized and quantified using iBright Imaging System (Invitrogen). β‐actin was used as an internal control.

4.8. Real time PCR

Total RNA was isolated from LoVo cells using Trizol (Invitrogen). SuperScript VILO cDNA Synthesis Kit (Invitrogen) was used to reverse RNA into first‐strand cDNA. RT‐PCR was conducted to measure IKK, TSC1 and GSK3 mRNA expressions using SuperScript IV One‐Step RT‐PCR System (Invitrogen). GAPDH was used as internal control. The 2^−ΔΔCt method was used to evaluate the relative mRNA expressions.

4.9. Terminal dUTP nick‐end labeling (TUNEL) assay

The TUNEL Apoptosis Assay Kit (Beyotime Biotechnology) was used to detect cell apoptosis in tumour tissues. ²³ Frozen sections were fixed with 4% paraformaldehyde for 30 minutes at 25℃, and washed with PBS for two times. Subsequently, the sections were labeled with TdT labeling reaction mix for 1 hour at 37℃. Then, the sections were observed using a fluorescent microscope (Nikon).

4.10. Immunohistochemistry

Paraffin sections of tumour tissues were sliced into 4 μm‐thick sections. Dewaxing sections were microwaved for antigen retrieval with Tri‐EDTA. Then, the sections were blocked with endogenous peroxidase, and incubated with primary antibody against vimentin (Cell Signaling Technology) at 4℃ overnight. The sections were then incubated with secondary antibody for 30 minutes. Finally, the sections were developed using 3, 3′‐diaminobenzidine (DAB) chromogen.

4.11. In vivo tumour formation assay

Female BALB/c nude mice (6 weeks old) were obtained from Shanghai experimental animal research centre, and maintained in a specific pathogen‐free facility with free access to water and food. LoVo cells transfected with PI3K overexpressing vector (1 × 10⁶) were subcutaneously inoculated into the nude mice. To observe the effect of rotenone on tumourigenesis in nude mice, rotenone (1 mg/kg) was intraperitoneally injected into the nude mice every other day. Nude mice were divided into control group, Rot group, PI3K group and PI3K + Rot group, with five mice in each group. The length and width of tumours were measured using a caliper every seven days. Six weeks later, mice were sacrificed and tumour tissues were collected and weighed. Tumour volumes (mm³) = length × width²/2. All animal experiments were approved by the Institutional Animal Care and Use Committee of Beijing University of Chinese Medicine.

4.12. Statistical analysis

SPSS 18.0 (Chicago, IL, USA) was used to analyze the data, and all data were presented as mean ± standard deviation (SD). Student t test was used to compare the differences between two groups, and one‐way analysis of variance (ANOVA) test was used to compare the differences among groups. P value less than .05 was considered statistically significant.

CONFLICT OF INTEREST

None.

ACKNOWLEDGEMENTS

We received supports from Department of Anoretal and Department of General Surgery at Dongfang Hospital, Beijing University of Chinese Medicine.

Xue W, Men S, Liu R. Rotenone restrains the proliferation, motility and epithelial–mesenchymal transition of colon cancer cells and the tumourigenesis in nude mice via PI3K/AKT pathway. Clin Exp Pharmacol Physiol. 2020;47:1484–1494. 10.1111/1440-1681.13320

First author: Wusong Xue

The peer review history for this article is available at https://publons.com/publon/10.1111/1440-1681.13320

[Correction added on 17 June 2020, after first online publication: URL for peer review history has been corrected.]

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