Rhein Suppresses Colorectal Cancer Cell Growth by Inhibiting the mTOR Pathway In Vitro and In Vivo

Abstract

Colorectal cancer (CRC) is one of the leading causes of mortality and morbidity in the world. Rhein has demonstrated therapeutic effects in various cancer models. However, its effects and underlying mechanisms of action in CRC remain poorly understood. We investigated the potential anticancer activity and underlying mechanisms of rhein in CRC in vitro and in vivo. Cell viability and anchorage-independent colony formation assays were performed to examine the antigrowth effects of rhein on CRC cells. Wound-healing and Transwell assays were conducted to assess cell migration and invasion capacity. Cell cycle and apoptosis were investigated by flow cytometry and verified by immunoblotting. A tissue microarray was used to detect mTOR expression in CRC patient tissues. Gene overexpression and knockdown were done to analyze the function of mTOR in CRC. The anticancer effect of rhein in vivo was assessed in a CRC xenograft mouse model. The results show that rhein significantly inhibited CRC cell growth by inducing S-phase cell cycle arrest and apoptosis. Rhein inhibited CRC cell migration and invasion through the epithelial-mesenchymal transition (EMT) process. mTOR was highly expressed in CRC cancer tissues and cells. Overexpression of mTOR promoted cell growth, migration, and invasion, whereas mTOR knockdown diminished these phenomena in CRC cells in vitro. In addition, rhein directly targeted mTOR and inhibited the mTOR signaling pathway in CRC cells. Rhein promoted mTOR degradation through the ubiquitin-proteasome pathway. Intraperitoneal administration of rhein inhibited HCT116 xenograft tumor growth through the mTOR pathway. In conclusion, rhein exerts anticancer activity in vitro and in vivo by targeting mTOR and inhibiting the mTOR signaling pathway in CRC. Our results indicate that rhein is a potent anticancer agent that may be useful for the prevention and treatment of CRC.

Keywords: Rhein; colorectal cancer; mTOR; xenograft.

Figure 1

Rhein inhibits the growth of CRC cells. (A) Chemical structure of rhein. (B) Morphological changes of HCT15, HCT116, and DLD1 cells following treatment with 0, 10, 20, 40, and 60 μM rhein for 24 h (Scale bar = 128 μm). (C) HCT15, HCT116, and DLD1 cells were treated with rhein (0, 10, 20, and 40 μM) for 0, 24, 48, and 72 h. Cell viability was determined using the CCK-8 assay. (D,E) Effect of rhein on anchorage-independent growth of CRC cells (Scale bar = 250 μm). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Rhein inhibits the CRC cells migration and invasion. (A) A wound-healing assay was performed to evaluate cell migration in HCT15, HCT116, and DLD1 cells. (B) Quantitative analysis of the migration of HCT15, HCT116, and DLD1 cells. (C) Representative images of migrating cells in HCT15, HCT116, and DLD1 cells. (D) Cell migration presented as a percentage of the control. (E) Representative images of invading cells representing HCT15, HCT116, and DLD1 cells. (F) Cell invasion presented as a percentage of the control. (G) Western blot analysis of EMT-related proteins in HCT15, HCT116, and DLD1 cells treated with rhein compared with the control. Scale bar = 128 μm. * p < 0.05, ** p < 0.01, *** p < 0.001. Uncropped western blots figures are shown in Figure S2.

Figure 3

Rhein induces S-phase cell cycle arrest and apoptosis in CRC cells. (A,B) Flow cytometry was used to determine the cell cycle distribution of rhein-treated CRC cells. Cells were treated with 0, 10, 20, and 40 μM rhein for 48 h and cell cycle was analyzed by flow cytometry. (C) The expression of cyclin A1, cyclin E1, cyclin D1, and CDK2 in CRC cells were measured by Western blot analysis. (D,E) Cells were treated with 0, 10, 20, or 40 μM rhein for 48 h and apoptosis was assessed by flow cytometry. (F) Expression of p-p53, p53, Bax, and cleaved caspase-3 proteins were measured by Western blot analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. Uncropped western blots figures are shown in Figure S2.

Figure 4

Rhein directly targets mTOR and suppresses the mTOR signaling pathway in CRC cells. (A) The expression of mTOR was evaluated by IHC analysis using a CRC tumor microarray. (B) mTOR, p-mTOR, and HSF1 expression levels in CRC cell lines and the CCD-18Co normal colon cell line were measured by Western blot analysis. (C) The binding of rhein to mTOR in HCT15 and HCT116 cell lysates was determined by Western blot analysis. (D) The effects of rhein (48 h) on the mTOR signaling pathway in CRC cells were determined by Western blot analysis. (E,F) Immunofluorescence results showed that mTOR levels are decreased after rhein treatment for 48 h compared with the control (Scale bars = 50 μm). * p < 0.05, ** p < 0.01, *** p < 0.001. Uncropped western blots figures are shown in Figure S2.

Figure 5

Rhein promotes mTOR protein degradation by the ubiquitin–proteasome pathway. (A) HCT116 cells were treated with cycloheximide (CHX; 10 μM) and MG 132 (15 μM) for 1, 2, and 3 h and mTOR protein expression was measured by Western blot analysis. (B) HCT116 cells were treated with MG132 in the absence or presence of 40 μM rhein for 3 or 6 h and mTOR protein expression was measured by Western blot analysis. (C,D) HCT116 cells were transfected with Flag-tagged ubiquitin plasmid for 24 h followed by treatment with MG132 alone or in combination with 40 μM rhein. Protein ubiquitination was analyzed by Western blot analysis. Uncropped western blots figures are shown in Figure S2.

Figure 6

Overexpression of mTOR promotes the proliferation, anchorage-independent colony formation, migration, and invasion of CRC cells. (A) Overexpression of mTOR in HCT15, HCT116, and DLD1 cell lines was confirmed using Western blot analysis. (B) Cell viability was assessed by CCK-8 assay in mTOR overexpressing CRC cells. (C,D) Anchorage-independent colony formation assays in mTOR-overexpressing and control HCT15, HCT116, and DLD1 cells (Scale bars = 250 μm). (E,G) Representative images of migrating and invading cells are shown in the vector or mTOR overexpressing HCT15, HCT116, and DLD1 cells (Scale bars = 128 μm). (F,H) Quantification of the migrating or invading cells. (I,J) Anchorage-independent colony formation assays in mTOR-overexpressing and control HCT116 cells after treated with rhein (Scale bars = 200 μm). * p < 0.05, ** p < 0.01, *** p < 0.001. Uncropped western blots figures are shown in Figure S2.

Figure 7

mTOR knockdown suppresses the proliferation, anchorage-independent colony formation, migration, and invasion of CRC cells. (A) Western blot analysis showing knockdown of mTOR following shRNA transfection in HCT15 and HCT116 cell lines. (B) Cell viability of HCT15 and HCT116 was assessed using the CCK-8 assay. (C,D) Anchorage-independent colony formation assays in mTOR knockdown and control HCT15 and HCT116 cells (Scale bars = 250 μm). (E,G) Representative images of migrating and invading cells are shown in HCT15 and HCT116 cells (Scale bars = 128 μm). (F,H) Quantification of the migrating and invading cells. (I,J) Anchorage-independent colony formation assays in mTOR knockdown and control HCT116 cells after treated with rhein (Scale bars = 250 μm). * p < 0.05, ** p < 0.01, *** p < 0.001. Uncropped western blots figures are shown in Figure S2.

Figure 8

Rhein suppresses HCT116 CRC tumor growth in a xenograft mouse model. (A) Nude mice were injected subcutaneously with HCT116 cells. Tumor volumes were plotted over 38 days after inoculation. (B) Tumor weights. (C) The images show tumors from mice treated with vehicle or rhein (10 or 50 mg/kg). (D) Body weights. (E) Histopathology conducted by H&E staining of liver and lung samples. (F) The expression of p-mTOR, mTOR, p-p70S6K, p70S6K, HSF1, HSP90, cyclin D1, CDK2 and β-Actin was detected by Western blot analysis. (G,H) Immunohistochemical staining of Ki-67, cyclin D1, HSF1, and cyclin A1 in HCT116 xenograft tumors (Scale bars = 64 μm). (I) Schematic diagram of the mechanism of action of rhein based on this study. * p < 0.05, ** p < 0.01, *** p < 0.001. Uncropped western blots figures are shown in Figure S2.