Natural compound rhein suppresses colorectal cancer cells growth through mTOR/p70S6 kinase pathway in vitro and in vivo

Background: Rhein is a natural agent isolated from the traditional Chinese medicine rhubarb, which has been used as a medicine in China since ancient times. Although rhein was found to have signicant anticancer effects in different cancer models, the effect and the underlying mechanisms of action of rhein in colorectal cancer (CRC) remain unclear. The mTOR/p70S6 kinase (p70S6K) pathway has been demonstrated as an attractive target for developing novel cancer therapeutics. Methods: The human CRC cell lines HCT116, HCT15, and DLD1 and xenograft mice were used in this study to investigate the effects of rhein. Assessments of cellular morphology, cell proliferation, and anchorage-independent colony formation were performed to examine the effects of rhein on cell growth. Wound healing assay and transwell migration and invasion assay were conducted to detect cell migration and invasion. Cell cycle and apoptosis were investigated by ow cytometry and veried by immunoblotting. Tissue microarray was used to detect mTOR expression in patients with CRC. Gene overexpression and knockdown were implemented to analyze the function of mTOR in CRC. The in vivo effect of rhein was assessed in a xenograft mouse model. Results: Rhein signicantly inhibited CRC cell growth by inducing S phase cell cycle arrest and apoptosis. It also inhibited CRC cell migration and invasion ability through EMT process. mTOR was highly expression in CRC cancer tissues and cells exhibited high mTOR expression. Overexpression of mTOR promoted cell growth, migration, and invasion ability, whereas mTOR knockdown diminished these phenomena of CRC cells in vitro. Moreover, rhein directly targeted mTOR and suppressed the mTOR/p70S6K signaling pathway in CRC cells. Intraperitoneal administration of rhein inhibited CRC cell HCT116 xenograft tumor growth through the mTOR/p70S6K pathway. Conclusions: Rhein exerted anticancer activity in vitro and in vivo through directly targeting mTOR and inhibiting mTOR/p70S6K signaling pathway. These data indicate that rhein is a potent anticancer agent that could be useful for the prevention or c Effect of on the of blotting. d-e with or μM rhein for and ow cytometry. f Effect of rhein on apoptotic marker proteins detected by blotting.

recent years, targeting the mTOR signaling has generated signi cant interest for cancer therapy [6]. In CRC, tandutinib was reported to inhibit colon cancer growth by suppressing Akt/mTOR signaling pathway [7]. Tunicamycin was found to inhibit the growth and aggressiveness of colon cancer cells through down regulation mTOR expression [8]. Inositol-6 phosphate induces autophagy of HT29 colon cancer cells by inhibiting the Akt/mTOR pathway [9]. These results indicated that targeting mTOR is a promising strategy for developing novel cancer therapeutics.
Natural compounds are the major resources of drug development. Rhein (4, 5-dihydroxyanthraquinone-2carboxylic acid) is a natural anthraquinone found in several medicinal plants, including Rheum palmatum, Polygonum multi orum, Aloe barbadensis, and Cassia tora [10]. A previous study suggested that rhein strongly inhibited non-small-cell lung cancer cells growth in vitro and in vivo by suppressing STAT3 pathway [11]. Rhein also induced HepaRG cell death via S phase cell cycle arrest and increased apoptosis [12]. In addition, the anticancer activity of rhein has been reported in breast cancer [13,14], ovarian cancer [15,16], and colon cancer [17], suggesting that it could be a novel agent for the prevention and treatment of CRC.
Although previous studies suggest that rhein exerts potent anticancer ability, the direct target proteins of rhein in CRC have not been identi ed. In the present study, we investigated the anticancer effects of rhein in CRC using in vitro and in vivo experiments and found that rhein suppressed CRC cell growth by directly targeting and inhibiting the mTOR/p70S6K pathway. Our results revealed that rhein might be a potential candidate for CRC treatment.

Cell culture
The human CRC cell lines HCT15, HCT116, DLD1, HT29, SW620 and normal human colon epithelial cells CCD-18Co were purchased from ATCC. HCT116 and HT29 cells were cultured in McCoy's 5A medium. SW620 cells were cultured in L15 medium (Leibovitz). HCT15 and DLD1 were cultured in RPMI1640 medium. CCD-18Co cells were cultured in MEM medium (Leibovitz). All medium was supplemented with 10% FBS (Gibco) and 1% antibiotics. All cells were incubated at 37 °C in a 5% CO 2 humidi ed incubator.
CCK-8 assay CRC cells were plated on 96-well plates (1×10 3 cells /well) to allow attachment and incubated overnight and then treated with various concentrations of rhein or DMSO for 0, 24, 48, 72, and 96 h, followed by incubated with 10 µl CCK-8 solution (Dojindo Japan) per well for an additional 1 h at 37 °C in a 5% CO 2 incubator. The absorbance at 450 nm was assessed using a spectrophotometer (BioTek).
Anchorage-independent cell growth CRC cells (8 × 10 3 cells/well) seeded into complete growth medium containing 0.3% agar with various concentrations of rhein, then overlaid into 6 well plate containing 0.6% agar base and various concentrations of rhein. Plates were incubated at 37 °C in a 5% CO 2 incubator for 2 weeks, then take photographs under microscope (Leica) and count colonies using ImageJ software.
Cell cycle and apoptosis analysis CRC cells were seeded in 60-mm culture dishes (2 × 10 5 cells/dish). After incubated for 12 h, cells were then treated with various concentrations of rhein for 48 h. For cell cycle assay, cells were collected and xed in 70% cold ethanol and stored at -20°C overnight. Cells were dyed using RNase (100 μg/ml) and propidium iodide (PI, 20 μg/ml) staining buffer. For apoptosis assay, cells were collected and then dyed using Annexin V (BioLegnd, USA) and PI. The cell cycle and apoptosis were analyzed by ow cytometry (FACSCalibur; BD Science, California, USA).

Wound-healing assay
The migration ability of the CRC cells was evaluated by wound healing assays. When cells grew to 90%

Western blotting assay
Protein extraction from cells and tumor tissues were performed using the Pro-Prep lysis buffer (Intron Biotechnology, Korea). Protein obtained from lysates were separated by SDS-PAGE and then transferred onto polyvinylidene di uoride membranes. The membranes were incubated overnight with the primary antibodies at 4°C. Subsequently, the membranes were incubated with corresponding secondary antibodies for 1 h at room temperature. Immunoblots were visualized using ECL detection kit (GE Healthcare, Seoul, Korea) by Davinch imaging system (Davinch-K, Seoul, Korea).
Immuno uorescence analysis CRC cells were seeded in 2 well plates, and then treated with various concentrations rhein for 48 h. Then the cells were xed in 4% formaldehyde for 15 min, permeabilized with 0.3% Triton X-100, and incubated with mTOR antibody (1:500; Cat # 2983 CST) overnight at 4 °C. The secondary antibody Alexa Fluor 488conjugated goat anti-rabbit IgG antibody (Invitrogen) was incubated with cells at room temperature for 1 h in the dark. The nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Representative images were captured using a uorescence microscope (Leica).

In vivo xenografts experiments
All animal experiments were performed according to the guidelines and approval of Kyungpook National University. HCT116 cells (1×10 7 cells) suspended in 200 μL PBS were injected subcutaneously in the ank of Balb/c athymic nude mice (male, aged 4-6 weeks). After 6 days of implantation, mice were divided into three groups consisting of eight mice per group. Two groups were treated with rhein at 10 or 50 mg/kg body weight (dissolved in 5% DMSO and 10% Tween-20 in PBS), and the third group was treated with vehicle only. Rhein or vehicle was intraperitoneally injected three times a week for 32 days. Tumor volume and body weights were measured every 4 days. Tumor volume was calculated using the following ellipsoid formula: tumor volume (mm 3 ) (length × width × height × 0.52).

Statistical analysis
All data were presented as mean ± SD from at least three independent experiments. Statistical signi cance was determined using the Student's t-test. A p value < 0.05 was considered to be statistically signi cant.

Rhein exhibits antiproliferative effects in CRC cells
To determine the effect of rhein on CRC cell growth, HCT116, HCT15, and DLD1 cells were used. These CRC cells were treated with various concentrations of rhein (0, 10, 20, 40, and 60 μM) for 24h. At the concentration of 40 μM, there was an obvious cell death accompanied by morphological changes such as round-shaped and transparent in CRC cells (Fig. 1a). These changes in the CRC cells morphology may be due to the inhibition of cell growth and induction of apoptosis. Furthermore, whether rhein was toxic to the normal colon epithelial cells CCD-18Co was examined, and the results demonstrated that the tested concentrations of rhein did not signi cantly inhibition the growth of CCD-18Co cells (Fig. 1b). Next, we evaluated the half-maximal inhibitory concentration (IC50) of rhein in HCT116, HCT15, and DLD1 cells, and the IC50 values were found to be 43.39 μM, 46.08 μM, and 36.33 μM, respectively in these cells at 24 h (Fig. 1c); hence, a concentration of 40μM was chosen for subsequent experiments. The CCK-8 assay results indicated that the proliferation of HCT116, HCT15, and DLD1 cells were signi cantly inhibited in a dose-and time-dependent manners (Fig. 1d). Results of the anchorage-independent colony formation assay revealed a signi cant decrease in the colony number with rhein treatment in CRC cell lines (Fig. 1e, f). Collectively, rhein effectively inhibited CRC cells growth and presented less cytotoxic to the normal colon epithelial cells.

Rhein inhibits the migration and invasion of CRC cells
Cancer metastasis causes the majority of patient deaths from solid tumors [19]. To determine whether rhein could inhibit the migration and invasion of CRC cells, we rstly examined the effect of rhein on the motility of CRC cells using a wound healing assay. The HCT116, HCT15, and DLD1 cells were treated with rhein at 0, 10, 20, and 40 μΜ for 0-24 h. Results demonstrated CRC cells healing over scratch was inhibited by the treatment of rhein in HCT116, HCT15 and DLD1 cells compared to that in control cells at 12 or 24 h (Fig. 2a, b). Next, the effect of rhein on CRC cells migration and invasion was measured by transwell assay. After treatment with rhein (at 0, 10, 20, or 40 μM) for 48 h, the number of migrated or invaded cells were signi cantly decreased in a dose-dependent manner (Fig. 2c-f). Moreover, the migration and invasion of tumor cells were closely related to the epithelial-mesenchymal transition (EMT). To determine the role of rhein in the EMT process, we examined the EMT-related proteins including E-cadherin, N-cadherin, and vimentin using western blot assays. As expected, treatment with rhein upregulated the expression of E-cadherin and downregulated the expression of N-cadherin and vimentin (Fig. 2g). These nding suggested that rhein signi cantly inhibited CRC cells migration and invasion by regulating the expression of EMT-related proteins.

Rhein induces cell cycle S phase arrest and apoptosis of CRC cells
To further explore the mechanism of rhein on the proliferation of CRC cells, we investigated the cell cycle distribution after 48 h treatment with rhein by ow cytometry assays. Results indicated that rhein treatment markedly increased cell number of CRC cells in S phases (Fig. 3a, b). To con rm this change, we examined the levels of S phase regulatory proteins by western blotting. Results showed that rhein treatment signi cantly decreased the expressions of Cyclin A1, Cyclin E1, and CDK2 in HCT15 and HCT116 cells (Fig. 3c). The mTOR pathway has been reported to regulate the translation of Cyclin D1 [20,21], and hence we examined whether rhein treatment can in uence the expression of Cyclin D1 and found that the expression of Cyclin D1 was downregulated (Fig. 3c). These results suggested that rhein effectively induced S phase cell cycle arrest by inhibiting the expressions of Cyclin A1, Cyclin E1, CDK2 and Cyclin D1. We next examined whether rhein induces apoptosis of CRC cells using Annexin V/PI staining and ow cytometry analysis. The HCT116, HCT15, and DLD1 CRC cells were treated with 0, 10, 20 and 40 μM of rhein for 48 h and then the apoptosis was analyzed. Results demonstrated that rhein induced apoptosis in HCT116, HCT15, and DLD1 cells in a dose-dependent manner (Fig. 3d, e). In addition, the apoptotic marker proteins p53, p-p53, cleaved caspase 3 and Bax were strongly upregulated in CRC cells treated with rhein (Fig. 3f).

mTOR is highly expressed in CRC tissues and cells
Studies have demonstrated that a high expression of mTOR, as a biomarker in various cancers, was associated with poor prognosis [22][23][24]. An immunohistochemical study performed in 154 patients, showed that p-mTOR (Ser2448) and p-p70S6K (Thr389) were overexpressed in CRC tumor tissues compared to that in normal colon tissues [25]. In the present study, we evaluated the role of mTOR in CRC by examining the expression of mTOR in a CRC tumor microarray that included 70 pairs of cancer tissues and adjacent cancer tissues (Fig. 4a). We observed that total mTOR was signi cantly overexpressed in cancer compared to that in adjacent cancer tissues (Fig. 4a). We also evaluated the mTOR expression in CRC cell lines and found that mTOR was highly expressed in CRC cell lines, especially in HCT116 and HCT15 cells, compared to that in the normal colon epithelial cell line CCD-18Co (Fig. 4b). These results demonstrate that mTOR is a potential therapeutic molecular target for CRC treatment.

Rhein directly targets mTOR and suppresses mTOR signaling pathway in CRC cells
It has been reported rhein induces apoptosis through regulation of the PI3K/AKT/mTOR signaling pathway in human lung cancer A549 cells [26] and inhibits autophagy by regulating AMPK/mTOR signaling in rat renal tubular cells [27]. To determine whether rhein can directly targeting mTOR protein, we carried out in vitro pull-down assays using rhein-conjugated Sepharose 4B beads (or Sepharose 4B beads only as a negative control) and HCT116 or HCT15 cell lysate (Fig. 4c). Our results demonstrated that rhein directly binds to mTOR protein (Fig. 4c). We then investigated the effect of rhein on mTOR/p70S6K pathways in CRC cells. Results showed that treatment with rhein inhibited p-mTOR and p-p70S6K in both HCT15 and HCT116 cells (Fig. 4d). Studies have demonstrated that HSF1 activation in multiple cancers is strongly associated with tumor metastasis and death [28] and that mTOR is essential for HSF1 activation and HSP90 synthesis [29]. Therefore, we detected the HSF1 and HSP90 expression after treatment with rhein in HCT15 and HCT116 cells. As expected, the protein levels of HSF1 and HSP90 were also downregulated by rhein treatment in CRC cells (Fig. 4d). In addition, we conducted quantitative real-time PCR to evaluate the mRNA levels of HSF1, HSP90, and LDHA. The mRNA levels of HSP90 and LDHA were decreased in the rhein-treated HCT116 cells (Additional le 1: Fig. S1). Results of immuno uorescence analysis showed that mTOR expression was suppressed after rhein treatment compared to that in control (Fig. 4e). These data indicate that rhein directly targets mTOR and suppresses mTOR/p70S6K signaling pathway in CRC cells.

Overexpression of mTOR promotes the proliferation, anchorage-independent colony formation, migration and invasion of CRC cells
For assessing the functional role of mTOR in CRC progression, we established three stable mTORoverexpressing CRC cell lines. Results of western blotting con rmed a signi cant increase of mTOR expression in HCT116, HCT15, and DLD1 cells compared with control cells (Fig. 5a). Results of the CCK-8 assays and anchorage-independent colony formation assay results demonstrated that overexpressing mTOR promoted the proliferation and anchorage-independent colony formation abilities of CRC cells (Fig.  5b-d). Moreover, overexpression of mTOR remarkably enhanced the migration and invasion abilities of CRC cells as measured by transwell assays (Fig. 5e-h).
Knockdown of mTOR suppressed the proliferation, anchorage-independent colony formation, migration, and invasion of CRC cells To elucidate the effect of knockdown of mTOR in CRC cells, endogenous mTOR expression in HCT116 and HCT15 cells was silenced using a lentiviral vector carrying shRNA speci cally targeting mTOR (Fig.   6a). As shown in Fig. 6b-d, cell proliferation and anchorage-independent colony formation abilities were signi cantly inhibited after the downregulation of mTOR expression. However, treatment with rhein failed to further reduces the colony numbers in mTOR-knockdown cells (Additional le 2: Fig. S2), indicating that mTOR is the primary target of rhein in CRC cells proliferation. Furthermore, the migration and invasion abilities were suppressed in the mTOR-knockdown cells compared to that in control cells (Fig.   6e-h).

Rhein suppresses HCT116 CRC tumor growth in a xenograft mouse model
To con rm the antitumor activity of rhein in vivo, we established HCT116 xenografts model by injecting human HCT116 cells subcutaneously into the anks of nude mice to initiate tumor formation. Tumorbearing mice were divided into three groups and intraperitoneally injected with 2 dosages of rhein (10 and 50 mg/kg) and vehicle 3 times per week for 34 days. We observed that treatment with rhein (10 or 50mg/kg body weight) signi cantly inhibited tumor growth compared to vehicle-treated mice (Fig. 7a, c). Meanwhile, treatment with rhein had no effect on body weight as no signi cant differences between the vehicle-and rhein-treated mice (Fig. 9b). These data demonstrated that rhein could effectively inhibit the tumor growth in vivo without exhibiting an obvious toxicity. In addition, we detected the expression of p-mTOR, p-p70S6K, p70S6K, Cyclin D1 proteins in the tumor tissues. The expression of p-mTOR, p-p70S6K, p70S6K, Cyclin D1 were signi cantly inhibited after treatment with rhein in xenograft tumors at doses of 10 mg/kg and 50 mg/kg body weight compared to those in vehicle-treated tumors (Fig. 7d). H&E staining results showed no histological abnormalities in the liver and lung in the treatment groups (Fig. 7e). In addition, IHC analysis was conducted to evaluate the protein levels of Ki67, HSF1, Cyclin D1, or Cyclin A1 in HCT116 xenograft tumors, and the results showed that rhein decreased the expression of these protein markers (Fig. 7f, g). Overall, these results demonstrated that rhein effectively inhibited CRC tumor growth through mTOR/p70S6K pathway in vivo and has the potential to be used as a chemotherapeutic agent for CRC.

Discussion
Rhein is a natural anthraquinone found in several medicinal plants [10], and it has been reported to possess a wide range of biological activities. Recent studies have demonstrated that rhein exhibited potent e cacy in inhibiting tumor growth [11,12,17,30]. However, the mechanisms underlying its anticancer effects of rhein remain poorly elucidated. In the present study, we found that mTOR was highly expressed in the CRC patients tumor tissues (Fig. 4a) and CRC cells (Fig. 4b). We further demonstrated that rhein exerted an anticancer activity by suppressing mTOR/p70S6K signal pathway in vitro and in vivo.
Dysregulation of the Akt/mTOR pathway has been found in various cancer types, including CRC. The Akt/mTOR signaling is essential for the proliferation, metabolism and angiogenesis [31]. Studies have reported that inhibition of mTOR effectively suppressed tumor growth in several cancers, including bladder [32], breast [33], pancreatic [34,35], and colon [6,9] cancers. mTORC1 and mTORC2 are the two different complexes of mTOR. mTORC1 phosphorylates p70S6K and eukaryotic initiation factor 4Ebinding protein1 (4E-BP1), which leads to increased cell proliferation [36,37]. mTORC2 and its associated protein rictor phosphorylates Akt at Ser 473, thereby leding to activation of the Akt pathway [38]. In our study, we found that rhein treatment effectively downregulated the p-mTOR (Ser2448) and p-p70S6K (Thr389) in CRC cells (Fig. 4d) and xenograft tumor tissues (Fig. 7d), thereby suggesting that rhein inhibits the functions of mTOR and p70S6K.
HSF1 is a master regulator of the heat shock response, which can promote malignant transformation, cancer cell proliferation, and survival [28]. Recent studies showed that HSF1 can act as an oncogene and be strongly related to advanced tumor progression and poor prognosis in gastric cancer [39], and breast cancer [40,41]. A previous study demonstrated that mTOR is essential for the HSF1 activation and heat shock protein synthesis [29]. In the present study, we found HSF1 is highly expression in CRC cells, and rhein treatment downregulated the expression of HSF1 and its downstream HSP90 protein level through mTOR inhibition.
Cell cycle progression is monitored strictly by cyclin-dependent kinases (CDKs) and their partner cyclins [42]. The Cyclin A/CDK2 complex is required for progression through the S phase [43]. S phase cell cycle arrest was observed in cells with hypoxia [44], DNA damage [45], and chemotherapy [46,47]. Moreover, the mTOR/p70S6K pathway has been reported to be involved in DNA damage [48]. Previous research has demonstrated that rhein suppresses HepaRG cell growth through S phase cell cycle arrest [12], which is consistent with the results of the present study. Our results showed that rhein induced S phase cell cycle arrest in CRC cells through downregulation of Cyclin A1, Cyclin E1, and CDK2.
Cancer metastasis is known to be a major cause of treatment failure. The epithelial-mesenchymal transition (EMT) is a key mechanism involved in cancer metastasis [49]. During EMT process, epithelial cells transform into migrating and in ltrating cells, and cancer cells appear to lose epithelial markers such as E-cadherin and acquire mesenchymal markers such as N-cadherin [50]. In the present study, we found that rhein decreased the levels of N-cadherin and vimentin and increased the levels of E-cadherin in CRC cells, showing that rhein can suppress the EMT process of CRC cells (Fig. 2). In addition, our results indicated that mTOR plays an important role in the EMT process. Overexpression of mTOR could promote CRC cells migration and invasion (Fig. 5), whereas knockdown of mTOR could inhibit CRC cells migration and invasion (Fig. 6). To our knowledge, our study is the rst to report that mTOR promotes the migration and invasion of CRC cells.
Cell line-derived tumor xenograft models are the most commonly used for assessing cancer therapeutic e cacy. To further explore the anticancer effect of rhein in vivo, we established the HCT116 xenografts model. Our results showed that intraperitoneal injection of rhein dramatically suppressed CRC tumor growth in xenograft models (Fig. 7a) without causing toxicity based on our observations of no signi cant loss of body weight loss, and histological lesions of liver or lung tissues compared to those in vehicle group mice (Fig. 7b, e). p-mTOR and p-p70S6K were found to be downregulated in tumor tissues lysis, which further con rmed that rhein suppressed CRC cell growth through mTOR/p70S6K pathway.

Conclusions
In summary, our study reveals that mTOR/p70S6K signaling plays a vital role in the progression of CRC.
We demonstrated that rhein inhibited CRC cells growth in vitro and in vivo by direct targeting mTOR and suppressing mTOR/p70S6K signaling pathway. Our study indicates that rhein might be a potential antitumor agent for CRC prevention and treatment.
Abbreviations CRC: Colorectal cancer PI3K: Phosphoinositide 3-kinase experimental; HBZ analysed the data and wrote the manuscript. MOK and ZYR supervised the experiments and revised the manuscript. All authors read and approved the nal manuscript.

Availability of data and materials
The datasets used and analyzed in the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate All animal experiments were performed in accordance with the ethical standards of the Animal Care and Use Committee of Kyungpook National University.

Consent for publication
The authors declare that they agree to submit the article for publication.
(magni cation, 50×). b Anchorage-independent colony number was decreased with or without rhein treatment after mTOR knockdown in HCT116 and HCT15 cells. *p < 0.05, **p < 0.01, ***p < 0.001. Figure 1 Rhein inhibits the growth of CRC cells. a Morphological changes after treatment with rhein (magni cation, 50×). b Cytotoxic effect of rhein on CCD-18Co cells. CCK-8 assay was used after various concentrations of rhein treatment for 72 h. c Cytotoxic effects of rhein on HCT15, HCT116, and DLD1 cells. CCK-8 assay was used after various concentrations of rhein treatment for 24 h. d Cell proliferation was determined by CCK-8 assay after rhein treatment for 0, 24, 48, 72, and 96 h. e-f, Effect of rhein on anchorage-independent growth of CRC cells. *P < 0.05, **P < 0.01, ***P < 0.001. ow cytometry. c Effect of rhein on the expression of S phase-related proteins were detected by western blotting. d-e Cells were treated with 0, 10, 20, or 40 μM rhein for 48 h, and apoptosis was was detected by ow cytometry. f Effect of rhein on apoptotic marker proteins was detected by western blotting. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 4 mTOR is highly expressed in CRC tissues and cells and rhein directly targeting mTOR and suppresses mTOR signaling pathway in CRC cells. a The expression of mTOR was evaluated by IHC analysis on a CRC tumor microarray. b mTOR, p-mTOR, and HSF1 expression levels in CRC cell lines and normal colon normal cell line CCD-18Co were measured by western blotting. c The binding of rhein to mTOR in HCT116 or HCT15 cell lysates was determined by western bloting. d The effects of rhein on mTOR signaling pathway in CRC cells were assessed by western blot analysis. e Immuno uorescence results showed mTOR level is decreased after rhein treatment compared to control (magni cation, 200×). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5
Overexpression of mTOR promotes CRC cells growth. a Overexpression of mTOR in HCT15, HCT116, and DLD1 cell lines was con rmed using western blot assay. b Effects of mTOR overexpression on the proliferation of HCT15, HCT116, and DLD1 cells were assessed by CCK-8 assay. c-d, Anchorageindependent colony formation assays in mTOR-overexpressing cells and control cells of HCT15, HCT116, Figure 6 Knockdown of mTOR suppresses CRC cells growth. a mTOR expression was knocked down in HCT116 and HCT15 cell lines. b Knockdown of mTOR inhibited the proliferation in HCT116 and HCT15 cells, as assessed by CCK-8 assay. c-d, Anchorage-independent colony formation assays in mTOR-knockdown and control cells of HCT15 and HCT116. e,g Representative images of the transwell assay results of cell migration and invasion in HCT15 and HCT116 cells (sh-mTOR compared with Mock). f, h Quanti cation of the migrated or invaded cells. Magni cation, 50×. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7
Rhein suppresses HCT116 CRC tumor growth in a xenograft mouse model. a Tumor volumes were plotted over 38 days after inoculation of cells. b Body weights. c The images presented tumors from mice treated with vehicle or rhein (10 or 50 mg/kg). d The expressions of p-mTOR, mTOR, p-p70 S6K, p70S6K, Cyclin D1, CDK2 and β-Actin was detected by western blotting. e Histopathology conducted by H&E staining of liver and lung samples. f-g, Immunohistochemistry staining analysis of Ki-67, HSF1, Cyclin D1 and Cyclin