IMCA Induces Ferroptosis Mediated by SLC7A11 through the AMPK/mTOR Pathway in Colorectal Cancer.

Ferroptosis, implicated in several diseases, is a new form of programmed and nonapoptotic cell death triggered by iron-dependent lipid peroxidation after inactivation of the cystine/glutamate antiporter system xc–, which is composed of solute carrier family 7 membrane 11 (SLC7A11) and solute carrier family 3 membrane 2 (SLC3A2). Therefore, inducing ferroptosis through inhibiting the cystine/glutamate antiporter system xc– may be an effective way to treat cancer. In previous screening tests, we found that the benzopyran derivative 2-imino-6-methoxy-2H-chromene-3-carbothioamide (IMCA) significantly inhibited the viability of colorectal cancer cells. However, the impact of IMCA on ferroptosis remains unknown. Hence, this study investigated the effect of IMCA on ferroptosis and elucidated the underlying molecular mechanism. Results showed that IMCA significantly inhibited the cell viability of colorectal cancer cells in vitro and inhibited tumor growth with negligible organ toxicity in vivo. Further studies showed that IMCA significantly induced the ferroptosis of colorectal cancer cells. Mechanistically, IMCA downregulated the expression of SLC7A11 and decreased the contents of cysteine and glutathione, which resulted in reactive oxygen species accumulation and ferroptosis. Furthermore, overexpression of SLC7A11 significantly attenuated the ferroptosis caused by IMCA. In addition, IMCA regulated the activity of the AMPK/mTOR/p70S6k signaling pathway, which is related to the activity of SLC7A11 and ferroptosis. Collectively, our research provided experimental evidences on the activity and mechanism of ferroptosis induced by IMCA and revealed that IMCA might be a promising therapeutic drug for colorectal cancer.


Introduction
Colorectal cancer (CRC) is a common malignant tumor and an important health problem worldwide. According to the cancer statistics worldwide for 36 cancers in 185 countries in 2018, both sexes combined, colorectal cancer is the third diagnosed cancer (10.2% of the total cases) globally and the second leading cause (10.2% of total cancer deaths) of cancer-related deaths worldwide [1,2]. Conventional treatment options for cancer include chemotherapy, radiation, and surgery [3]. In addition, new treatment methods, such as biotargeted therapy, immunotherapy, and precise treatment, have been gradually applied for CRC treatment [4,5]. In order to improve the therapeutic effect, chemotherapy, radiation, and surgery are often used in combination. However, conventional treatments are often associated with serious side effects and toxicity, thus significantly affecting patients' quality of life. In addition, cancer cells have also been found to be able to develop resistance toward chemotherapy and radiotherapy over time. Therefore, it is still an important task for researchers to find new drugs with high efficiency and low side effects for CRC.
Identified as a new mode of programmed cell death in 2012, ferroptosis is a unique iron-reliant and reactive oxygen species-(ROS-) dependent form of nonautophagic and nonapoptotic programmed cell death [6,9]. Mitochondrial morphological change is characterized by decreased or vanished mitochondria cristae, a ruptured outer mitochondrial membrane, and a condensed mitochondrial membrane in ferroptotic cells [7,9]. The small molecule erastin induces ferroptosis through inhibiting the import of cystine, which is decomposed into two molecules of cysteine (Cys), resulting in glutathione (GSH) exhaustion and inactivation of the phospholipid peroxidase glutathione peroxidase 4 (GPX4) [8,10]. GPX4 reduces the toxic lipid hydroperoxides (L-OOH) to the nontoxic lipid alcohols (L-OH) by oxidizing GSH to glutathione disulfide (GSSG) [8,11]. Cystine, one of the raw materials for the synthesis of the major antioxidant GSH, is transported to the intracellular space through transporting glutamate to the extracellular space by a heterodimeric cystine/glutamate antiporter system xc -, which is mainly composed of a twelve-pass transmembrane catalytic subunit solute carrier family 7 member 11 (SLC7A11) and a single-pass transmembrane anchoring protein solute carrier family 3 member 2 (SLC3A2) [7,12]. Pharmacological inhibition of the system xc --reliant antioxidant defense system leads to ROS accumulation and ferroptosis, such as erastin, sulfasalazine, and sorafenib [7].
2-Imino-6-methoxy-2H-chromene-3-carbothioamide (IMCA) is a benzopyran derivative, with a wide variety of biological activities for the treatment of cancer, type 2 diabetes, inflammation, skin diseases, Alzheimer's disease (AD), the polycystic kidney disease, and viral and bacterial infections [13]. We report for the first time that IMCA inhibits the viability of medullary thyroid cancer through inducing apoptosis [14]. In the course of studying the anti-CRC effect of IMCA, we found for the first time that IMCA leads to the death of CRC cells.
In the current study, two types of CRC cell lines and xenograft model were utilized to evaluate the anti-CRC effects and the mechanism of IMCA. The effects of IMCA on the biological phenotype of CRC cells were examined, and the underlying molecular mechanisms of IMCAinduced ferroptosis were elucidated. Mechanistically, we found that IMCA downregulated the expression of SLC7A11 and decreased the contents of Cys and glutathione, which resulted in ROS accumulation and ferroptosis. Furthermore, overexpression of SLC7A11 significantly attenuated ferroptosis caused by IMCA through downregulating the expression of SLC7A11. In addition, IMCA regulated the activity of the AMPK/mTOR/p70S6k signaling pathway, which is related to the activity of SLC7A11 and ferroptosis. For the first time, we found a novel small-molecule compound against CRC and demonstrated that IMCA induced ferroptosis mediated by SLC7A11 through the AMPK/mTOR pathway in CRC.

Cell Lines and Cell
Culture. Human CRC cell lines DLD-1 and HCT-116 were purchased from a typical cell culture collection committee of the Chinese Academy of Sciences Library (Shanghai, China). These cells were grown in Roswell Park Memorial Institute 1640 (Gibco, 11875119) medium containing 10% heat-inactivated fetal bovine serum (Gibco, 16000-044), 100 U/mL penicillin and 100 μg/mL streptomycin (Solarbio, P1400), and 5% CO 2 at 37°C. The solvent dimethyl sulfoxide (DMSO) used in the experiments was less than 0.1%. 2.3. Cell Viability Assay. IMCA cytotoxicity was detected with the MTT cell proliferation and cytotoxicity detection kit in accordance with the manufacturer's instructions. In brief, DLD-1 and HCT-116 cells were seeded into 96-well cell culture plates with 5 × 10 3 cells per well and cultured continuously for 12 h. Different intervention reagents were added to the cultural plates and cultured continuously for 48 h. A 10 μL MTT solution (10 mg/mL in PBS) was added to the cultural plates and cultured continuously for 4 h. Then, 100 μL of DMSO was added to the cultural plates and the absorbance of the samples was measured using a multifunctional enzyme marker (Varioskan Flash, Thermo Scientific) at the wave length of 570 nm.

Chemicals and
2.4. ROS Analysis. ROS was detected with the ROS detection kit in accordance with the manufacturer's instructions. Briefly, DLD-1 and HCT-116 cells were seeded into 6-well cell culture plates with 2 × 10 5 cells per well and cultured continuously for 12 h. Different intervention reagents were added to the cultural plates and cultured continuously for 48 h.

Detection with Multifunctional Enzyme
Marker. Cells were harvested and washed once with PBS. The harvested cells were suspended in DCFH-DA, diluted 1000 times in serum-free medium, and incubated for 20 min. Subsequently, the cells were washed three times with serum-free medium and were then detected with a multifunctional enzyme marker (Varioskan Flash, Thermo Scientific) at the excitation wavelength of 488 nm and emission wavelength of 525 nm.
2.4.2. Detection by Confocal Microscopy. As previously described [15], the cells were washed once with PBS, incubated in DCFH-DA, and diluted 1000 times in serum-free medium for 20 min. Then, the cells were washed three times with serum-free medium and detected via confocal microscopy (A1R+Storm, Nikon).

2
Oxidative Medicine and Cellular Longevity 2.5. Cys Analysis. Cys was detected with the Cys detection kit according to the manufacturer's instructions. Briefly, DLD-1 and HCT-116 cells were seeded into 6-well cell culture plates with 2 × 10 5 cells per well and cultured continuously for 12 h. Different intervention reagents were added to the cultural plates and cultured continuously for 48 h. The cells were crushed by ultrasound and centrifuged at 8000 g for 10 min. Then, 100 μL of the supernatant, 500 μL of reagent I, and 500 μL of reagent II were mixed and detected with a multifunctional enzyme marker (Varioskan Flash, Thermo Scientific) at the wavelength of 600 nm.
2.6. GSH Analysis. GSH was detected with the GSH detection kit in accordance with the manufacturer's instructions. Briefly, DLD-1 and HCT-116 cells were seeded into 6-well cell culture plates with 2 × 10 5 cells per well and cultured continuously for 12 h. Different intervention reagents were added to the cultural plates and cultured continuously for 48 h. Cells were harvested and washed twice with PBS. The cells were resuspended in reagent I and frozen and thawed three times with liquid nitrogen. A 20 μL supernatant from the cell suspension centrifuged at 8000 rpm was mixed with 140 μL of reagent II and 40 μL of reagent III and detected with a multifunctional enzyme marker (Varioskan Flash, Thermo Scientific) at the wavelength of 412 nm.
2.7. qRT-PCR Assay. As previously described [16], total RNA was extracted using Trizol reagent in accordance with the manufacturer's instructions. mRNA was reversed transcribed into cDNA with the PrimeScript RT reagent kit (Takara, DRR047A) in a 20 μL reaction system. qPCR analysis was conducted with on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The PCR primers are listed in Table 1.
2.8. Western Blot Analysis. As previously described [17], protein expression was determined by Western blot in accordance with standard protocols. Briefly, cells were harvested and lysed in RIPA buffer with a protease inhibitor. Cell lysates were quantitated with a Bradford reagent, separated with a denatured sodium dodecyl sulfate 4%-20% polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a polyvinylidene fluoride (PVDF) membrane through wet electroblotting. The PVDF membrane was blocked with dried skimmed milk and incubated with primary antibodies specific for GPX4, GSS, and SLC7A11. A HRP-conjugated secondary antibody was incubated at room temperature for 2 h, and the blot analysis was visualized with a chemiluminescence analyzer (Amersham Biosciences, Boston, MA, USA).

2.9.
Overexpression of SLC7A11. The recombinant overexpression plasmid of SLC7A11 was constructed by Hanbio Technical Co., Ltd. (Shanghai, China). The overexpression plasmid of SLC7A11 was transfected into DLD-1 and HCT-116 cells by Lipofectamine 3000 and P3000 to produce SLC7A11-overexpressing transient cell lines. After 48 h of transfection, the cells were collected and transfection efficiency was determined using Western blot.
2.10. In Vivo Experiments. As previously described [18], the animal experiments involved in this project have been approved by the Medical and Scientific Research Ethics Committee of Henan University School of Basic Medical Sciences. Five-week-old female BALB/c nude mice were purchased from Beijing Weitong Lihua Experimental Animal Technical Co., Ltd. (Beijing, China). 10 6 DLD-1 cells suspended in normal saline were injected subcutaneously into nude mice. The volume of tumors was calculated using the following formula: L ðthe long diameterÞ × W ðthe short diameterÞ × W × 1/2. The mice were randomly assigned to the treatment and control groups until the tumor size reached approximately 100 mm 3 . The mice in the treatment group were injected with 0.174 mg/mL IMCA (100 μL), and those in the control group were injected with an equal volume of normal saline. The nude mice were euthanized, and samples were obtained from their tumor, heart, hepar, kidney, and blood after 33 days of IMCA treatment. The serum was separated, and alanine aminotransferase and urea nitrogen were determined using the animal specific automatic biochemical analyzer (Catalyst Dx, IDEXX, Maine, USA) to evaluate the effects of IMCA on liver and kidney functions. Blood was obtained, and the number of blood cells was determined by an animal hematology analyzer (BC-5000 vet, Mindray, Shenzhen, China) to evaluate the effect of IMCA on blood routine. Organ index was calculated by dividing organ mass by body weight and multiplying by 100% to evaluate the effect of IMCA on organs.
2.11. Transmission Electron Microscopy. DLD-1 and HCT-116 cells were seeded into 6-well cell culture plates with 2 × 10 5 cells per well and cultured continuously for 12 h. Different intervention reagents were added to the cultural plates and cultured continuously for 48 h. Cells were harvested and fixed in 2% glutaraldehyde. Samples were treated and detected in the electron microscopy room of Xi'an Jiaotong University.
3 Oxidative Medicine and Cellular Longevity 2.12. Statistical Analysis. All statistical analyses were completed by SPSS16.0. The statistical difference between the treatment and control groups of IMCA was analyzed by Student's t-test.

Results
3.1. IMCA Reduced the Viability of CRC Cell Lines. The cell viability of human CRC cell lines DLD-1 and HCT116 treated with different concentrations of IMCA for 48 h was determined using the MTT method to confirm the inhibition efficiency of IMCA on the proliferation of CRC cell lines. The viability of the two CRC cell lines significantly reduced in a dose-dependent manner compared with that of the control cells (Figures 1(a) and 1(b)). The IC50 values of IMCA for DLD-1 and HCT116 cells were 50.2 μM and 44.5 μM, respectively. The cell morphology was photographed with an inverted microscope (Figure 1(c)). Compared with the control group cells, the CRC cells treated with IMCA for 48 h were characterized by shattered, metamorphous, and multidirectional cell morphology. The above results showed that IMCA significantly reduced the viability of CRC cell in vitro.
3.2. IMCA Inhibited the Growth of Xenograft In Vivo. Since we observed a significantly inhibitory effect of IMCA on CRC cell viability, we next dissected the antitumor effects of IMCA using the BALB/c nude mouse xenografts bearing DLD-1 cells in vivo. As shown in Figures 2(a) and 2(b), the tumor volume and weight in the IMCA-treated group mice dramatically reduced and the inhibition rate reached 76.4% compared with those in the saline negative control group. We monitored the body weight of nude mice every three days during the treatment period to determine the impact of IMCA on the health of the mice. The nude mice did not significantly lose weight during the entire treatment period compared with the control group (Figure 2(c)). For health measurements, we also measured liver and kidney function index, organ index, and blood routine, including alanine aminotransferase (ALT), blood urea nitrogen (BUN), heart index, liver index, kidney index, red blood cells (RBC), white blood cells (WBC), lymphocytes (Lym), and monocytes (Mon) (Figures 2(d)-2(g)). The results showed that no significant health figure changes were observed. To further assess the toxicity of IMCA on healthy animals, nontumorbearing nude mice were injected with equal doses of IMCA and normal saline through the tail vein, and the body weight of nude mice was monitored every three days during the treatment period. Results showed that IMCA did not significantly affect the weight gain of nude mice, compared with the normal saline groups (Figures 2(h) and 2(i)). There were no significant changes in liver, kidney, heart, and spleen indices, compared with the normal saline groups (Figure 2(j)).  Oxidative Medicine and Cellular Longevity Collectively, the above data indicate that IMCA significantly inhibits tumor growth with negligible organ toxicity in vivo.

IMCA Induced ROS-Mediated Ferroptosis of CRC Cell
Lines. Ferroptosis is a unique iron-reliant and nonapoptotic form of programmed cell death, which is characterized by ROS accumulation induced by lipid peroxidation and ineffective GPX4 [19,20]. In order to determine the cause that Collectively, these data suggest that IMCA induces CRC cell ferroptosis in vitro.

IMCA Inhibited the Expression of SLC7A11 In Vitro.
Ferroptosis is characterized by the accumulation of ROS, which is scavenged by GPX4 through conversion of reduced GSH into the oxidized form GSSG [24][25][26]. Therefore, the expression of GPX4 and the GSH level were explored and we found that IMCA significantly reduced GSH levels with negligible impact on the expression of GPX4 in DLD-1 and HCT116 cells (Figures 4(a) and 4(b); Fig. S1). GSH is synthesized from glutamate, Cys, and glycine by the ATP-dependent catalysis of glutathione synthetase (GSS) [27]. The rate of GSH synthesis is primarily limited by the Cys content [28]. The expression of GSS and the Cys level were determined to elucidate the mechanism of GSH reduction triggered by IMCA. Results showed that IMCA significantly reduced Cys levels with negligible impact on the expression of GSS in DLD-1 and HCT116 cells with negligible changes in the expression of GSS (Figures 4(c)-4(h)). The heterodimeric cystine/glutamate antiporter system xctransports Cys into the intracellular space to synthesize GSH, which inhibited ferroptosis. SLC7A11 is the catalytic subunit of system xc - [29]. The expression of SLC7A11was determined to dissect the mecha-nism by which IMCA triggers Cys reduction. Results showed that IMCA significantly reduced the expression of SLC7A11 in DLD-1 and HCT116 cells (Figures 4(i)-4(m)). Collectively, these data suggest that IMCA induces CRC cell ROS accumulation and ferroptosis by downregulating SLC7A11 expression, inhibiting Cys transport and reducing GSH synthesis in vitro.

Overexpression of SLC7A11 Rescues IMCA-Induced
Ferroptosis of CRC Cells In Vitro. SLC7A11 plays an important role in regulating ROS-mediated ferroptosis. Knocking down the expression of SLC7A11 results in elevated levels of endogenous ROS levels. Overexpression of SLC7A11 results in a cancer stem cell phenotype that contributes to severe chemoresistance [30,31]. SLC7A11-overexpressing DLD-1 and HCT116 CRC cells were generated. Cys, GSH, ROS, and cell viability were detected, and results showed that the overexpression of SLC7A11 significantly rescued the IMCA-induced reduction of Cys, GSH, and cell viability and increased the ROS levels in SLC7A11-overexpressing
3.6. IMCA Inhibits mTOR/P70S6K Activity through Phosphorylating AMPK. AMPK phosphorylation at Thr172 blocks the activity of SLC7A11, which inhibits the activity of system xcto transport cystine into cells and eventually leads to ferroptosis [12,32]. As expected, the present studies showed that IMCA promoted the phosphorylation of AMPK in DLD-1 and HCT116 cells (Figures 6(a) and 6(b)). AMPK activation inhibits mTOR activity, which counteracts the elevated expression of SLC7A11 induced by APR246 and the protective cellular responses, and eventually results in cell death [33][34][35]. Furthermore, our results showed that the phosphorylation of mTOR and the downstream target protein P70S6K had been significantly decreased by 50 μM IMCA treatment in DLD-1 and HCT116 cells.

Discussion
Chemotherapy is increasingly used in CRC as a complementary treatment strategy for CRC after surgery [36,37]. In consideration of the high morbidity and mortality of CRC [2], new therapeutic drugs with high efficiency and low side effects for CRC must be developed. The present study showed that IMCA significantly inhibited the viability of human CRC cell lines DLD-1 and HCT116 (Figure 1). Further in vivo experiments showed that IMCA significantly inhibited the growth of xenograft and did not significantly affect the main organ index and blood biochemical parameters, such as aspartate transaminase (AST) and urea nitrogen (BUN). In vitro and in vivo results revealed that IMCA may be an effective drug candidate for CRC. IMCA is a benzopyran derivative, provided with a wide variety of biological activities, including regulating cell death by ferroptosis execution [38]. For example, benzopyran derivative vitamin E hydroquinone is an endogenous regulator of ferroptosis [38]. Further transcript profile   The results also showed that apoptosis inhibitor z-Vad-FMK (3 μM) did not rescue the cell viability inhibited by IMCA in DLD-1 (e) and HCT116 (f) cell lines. (g) Confocal laser scanning microscope images of ROS generation were obtained and compared between the IMCA treatment and control groups in DLD-1 and HCT116 cells. Relative ROS accumulation was determined with a multifunctional enzyme marker in DLD-1 (h) and HCT116 (i) cells. Relative mRNA expression of ferroptosis markers CHAC1 and PTGS2 was determined and compared between the IMCA treatment and control groups in DLD-1 (j) and HCT116 (k) cells.
Transmission electron microscopy images of mitochondrial morphology were obtained and compared between the IMCA treatment groups (m) and control groups (l) in DLD-1 cells. The black tip points to mitochondria. * p < 0:05; * * p < 0:01; * * * p < 0:001. 9 Oxidative Medicine and Cellular Longevity our results showed that IMCA at 50 μM significantly promoted the mRNA expression of CHAC1 and PTGS2 in DLD-1 and HCT116 cells. Overall, our results provided evidence that IMCA causes cell death through ferroptosis.
GSH is a momentous intracellular antioxidant that acts as a reducing substrate of GPX4 to mitigate the accumula-tion of ROS and protect cells from oxidative damage [7,41]. Results showed that IMCA at 50 μM significantly reduced the content of GSH in DLD-1 and HCT116 cells. One of the committed substrates used to synthesize GSH catalyzed by GSS is Cys, which has limited intracellular content owing to neurotoxicity [42][43][44]. Cys should be  Figure 4: IMCA inhibited the expression of SLC7A11. The relative GSH contents were determined and compared between the IMCA treatment and control groups in DLD-1 (a) and HCT116 (b) cells. Relative mRNA expression of GSS was determined and compared between the IMCA treatment and control groups in DLD-1 (c) and HCT116 (d) cells. Relative protein expression of GSS was determined and compared between the IMCA treatment and control groups in DLD-1 (e) and HCT116 (f) cells. The relative Cys contents were determined and compared between the IMCA treatment and control groups in DLD-1 (g) and HCT116 (h) cells. Relative mRNA expression of SLC7A11 was determined and compared between the IMCA treatment and control groups in DLD-1 (i) and HCT116 (j) cells. Relative protein expression of SLC7A11 was determined and compared between the IMCA treatment and control groups in DLD-1 (k) and HCT116 (l) cells. * p < 0:05; * * p < 0:01; * * * p < 0:001.
supplemented extracellularly to ensure that sufficient GSH is synthesized to protect cells from oxidative damage. Cys is derived from the decomposition of cystine, which is imported into the cells through the cystine/glutamate antiporter system xc - [45,46]. Inhibition of system xcsignificantly depletes the intracellular Cys, retards GSH synthesis, and eventually results in ferroptosis [47]. Erastin [6], sulfasalazine [48], sorafenib [6], Artesunate [49], Lanperisone [50], and Piperazine erastin [10] are the most investigated inhibitors targeting system xcand inducing ferroptosis. In the present study, IMCA did not significantly downregulate GSS expression but depleted the intracellular Cys. These results revealed that IMCA depleted the intracellular Cys and GSH and induced ferroptosis. We overexpressed SLC7A11 in DLD-1 and HCT116 cell lines to determine the role of SLC7A11 in ferroptosis induced by IMCA. We next determined the effect of SLC7A11 overexpression on cell Cys, GSH, and ROS amounts and cell viability regulated by IMCA in DLD-1 and HCT116 cells. As a result, overexpression of SLC7A11 recovered the Cys and GSH depleted by IMCA and inhibited the ROS levels enhanced by IMCA. Ultimately, overexpression of SLC7A11 restored the viability of DLD-1 and HCT116 cells inhibited by IMCA at 50 μM. Taken together, our results provide a novel mechanism that IMCA induces cell death by ferroptosis through downregulating the expression of SLC7A11.
As a central energy metabolic switch, AMP-activated protein kinase (AMPK) exerts a paramount effect in cellular physiology and the pathological development of chronic diseases including cancer [51]. The activity of SLC7A11 is inhibited by AMPK phosphorylation through phosphorylating BECN1, which plays distinct roles in regulating cell ferroptosis [12,32]. In addition, the activity of SLC7A11 is inhibited by the mTORC pharmacological inhibitor rapamycin, which counteracts the elevated expression of SLC7A11 induced by APR246 and the protective cellular responses, leading to cell death [34]. Consistent with these two mechanisms, DHA induces the lethal ROS accumulation and ferroptosis of leukemia cells through the AMPK/mTOR pathway [25]. The present study showed that IMCA at 50 μM induced AMPK phosphorylation activation, mTOR dephosphorylation inhibition, and ultimately lethal ROS accumulation and ferroptosis in DLD-1 and HCT116 cells. Therefore, the SLC7A11 downregulation and ferroptosis induced by IMCA are related to the AMPK/mTOR pathway.
This study discovered a novel small-molecule compound (IMCA) for CRC treatment in vitro and in vivo, and elucidated that IMCA induces ferroptosis by downregulating SLC7A11 expression through the AMPK/m-TOR pathway. These results provided a new therapeutic potential compound for CRC and new insights to induce ferroptosis.  Figure 6: IMCA inhibited the mTOR/P70S6K activity through phosphorylating AMPK. DLD-1 (a) and HCT116 (b) cells were treated with 50 μM IMCA for 48 h, and the protein expression was assessed by Western blot. The intensities of the p-AMPK, p-mTOR, and p-P70S6K bands were quantified by densitometry analyses and normalized by the amount of AMPK, mTOR, and P70S6K in DLD-1 (c) and HCT116 (d) cells, respectively (n = 3). * p < 0:05, * * p < 0:01, and * * * p < 0:001 compared with the control group.