The three major kinds of human colon cancer cell lines, including HCT-116, HCT15, and SW480, were purchased from American Type Culture Collection (https://www.atcc.org/) and cultured in McCoy’s 5A (Gibco, United States), RPMI-1640 (Gibco, United States) and DMEM medium (Gibco, United States), respectively, supplemented with 10% fetal bovine serum (Gibco, United States) and 1% penicillin/streptomycin (Sangon Biotech, China). The colon cancer cell lines were incubated in incubator (Thermo Scientific, United States) with 5% CO₂ at 37 °C. We added 5-MTP (MedChemExpress, United States) in different concentrations during cell culture.

Cells were digested to make single-cell suspensions at logarithmic phase and uniformly seeded in 12-well plates at 1 × 10⁵ cells per well. After 48 h, cells were collected and washed with 1 mL of precooled phosphate buffered saline (PBS). The cell pellet was resuspended with 1 mL of precooled 70% ethanol and fixed overnight at 4 °C. On the second day, 70% ethanol was discarded by centrifugation at 1000 × g for 5 min at 4 °C, washed with 1 mL of precooled PBS. Each sample was added with 500 μL propyl iodide (PI) staining solution, gently mixed, incubated at 37 °C in the dark for 30 min, and the cells were filtered with a 400-mesh cell strainer to detect the cell cycle of each group by flow cytometry.

Colon cancer cells in the logarithmic growth phase were seeded in 6-well plates, and 5-MTP-treated cells were added after the cells attached. Cells in each group were digested with ethylene diamine tetraacetic acid-free trypsin, washed with PBS, and collected at 1000 × g for 5 min. Binding buffer 100 μL was used to resuspend cells, and fluorescein isothiocyanate staining solution 5 μL and PI staining solution 10 μL were added. They were blown and mixed well by the pipette. The cells were allowed to stand at room temperature for 15 min. Before loading the machine, 400 μL of binding buffer solution was added to each tube and mixed well so that the final system was 500 μL. Apoptosis of cells in each group was detected by flow cytometry.

To test the ROS level in each group of cells, the colon cancer cells were inoculated into 6-well plates at logarithmic growth phase. The 2′,7′-dichlorofluorescin diacetate (Sangon Biotech, China) was added, incubated in a cell culture incubator at 37 °C for 20 minutes, and observed under a confocal microscope. Similarly, Hoechst 33342 Viable Cell Staining Solution (Sangon Biotech, China) was added and incubated in a 37 °C cell culture incubator for 10 min and observed under a confocal microscope.

Cells were treated with 5-MTP at various concentrations for 48 h at 12-well plate. Before incubation, 25 μL of 200X JC-10 concentrate (Sangon Biotech, China) was added to a 5 mL assay buffer to dilute JC-10. 500 μL of JC-1 solution was added to each well and incubated at 37 °C for 20 min. After incubated, washed twice with staining buffer, added 500 μL cell culture medium, and observed under an inverted fluorescence microscope.

CRC cell lines were treated with 5-MTP for 48 h at logarithmic growth phase before protein extraction. Then, cells were washed with precooled PBS, 500 μL of RIPA buffer (Beyotime, China) was added to each dish, scraped by cell scraper, and transferred to a new centrifuge tube, lysed on ice for 30 min and centrifuged (12000 × g, 15 min, centrifugation radius 30 cm) to collect the supernatant. After adjusting the protein concentration, 4 × loading buffer was added and placed in a metal bath for heating denaturation (95 °C, 10 min). Sodium-dodecyl sulfate gel electrophoresis electrophoresis was performed to separate proteins (80 V), transferred by wet membranes (300 mA, 90 min), and blocked with 5% skimmed milk overnight (4 °C). Primary antibodies were used to incubate overnight (4 °C), including anti-AKT (1:1000, Cell Signaling Technology, #4685), anti-p-AKT (1:1000, Cell Signaling Technology, #13038), anti-FoxO3a (1:1000, Cell Signaling Technology, #12829), anti-p-FoxO3a (1:1000, Cell Signaling Technology, #9466), anti-β-actin (1:1000, Cell Signaling Technology, #4970), anti-Bax (1:1000, Cell Signaling Technology, #41162), anti-Bcl2 (1:1000, Cell Signaling Technology, #15071), anti-PARP (1:1000, Cell Signaling Technology, #9532), anti-Caspase3 (1:1000, Cell Signaling Technology, #9662), anti-Bim (1:1000, Cell Signaling Technology, #2933), anti-P27 (1:1000, Cell Signaling Technology, #3688) and anti-Cyclin D1 (1:1000, Cell Signaling Technology, #55506). Membranes were washed, incubated with secondary antibodies (1:10000) for 1 h at room temperature, and finally developed with ECL (Jiapeng Biotech, China), cassette luminescence, and band data analysis was performed by ImageJ software.

Cells were seeded in six-well plates at 800 cells/well in cell suspension, and 5-MTP was added when the cells were completely attached and cultured in an incubator for about 10 d, and > 50 cells/colony were considered as one clone, stained with crystal violet, photographed and counted.

After adjusting the cell density of each group to 1 × 10⁴ cells/mL, 200 μL of cell suspension was added to a 96-well plate and cultured at 37 °C for 0, 24, 48, and 72 h. Following this, 10 μL of Cell Counting Kit 8 (CCK-8) solution was added to each well. After incubation at 37 °C for 2 h, absorbance values were measured using a microplate reader (Flash Biotech, China) at 450 nm.

Invasion assays were performed at 37 °C using transwell chambers coated with matrigel. The cell density was adjusted to 4 × 10⁵ cells/mL, 100 μL was added to the upper transwell chamber, and then 600 μL of medium containing 10% foetal bovine serum was added to the lower chamber. After incubation at 37 °C for 24 h, the cells on the lower surface of the membrane were fixed with 4% paraformaldehyde (Sangon Biotech, China) for 20 min and stained with 500 μL, 1% crystal violet (Sangon Biotech, China) for 30 min. Five fields were randomly selected to observe the number of cells in invasion using an inverted light microscope and photographed.

GraphPad Prism (v9.0.0) software was used for data processing. Measurement data were expressed as mean ± SD. An independent sample t-test was performed between two groups. A one-way analysis of variance was used to compare the means between multiple groups. P < 0.05 was considered with statistically significant.

ATL was selected for further analysis, and its structure is shown in Figure 1A. To validate the role of 5-MTP in CRC, we selected HCT116, HCT15, and SW480 for subsequent experiments. In HCT116, HCT15, and SW480 cells, the results of CCK8 and colony formation assay showed that 5-MTP inhibited CRC cell proliferation gradually with increasing drug concentration (Figures 1B-J). These data suggested that 5-MTP inhibited human CRC cell proliferation.

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Figure 1 5-methoxytryptophan inhibited human colorectal cancer cell proliferation. A: The structure of 5-methoxytryptophan (5-MTP) is shown; B-D: The Cell Counting Kit 8 assays were used to measure the viability of the colorectal cancer cells treated with 5-MTP at the dose of 5, 25 and 100 μM; E-J: Colony formation assay were used to measure the viability of the HCT-116 (E and F), HCT-15 (G and H), SW480 (I and J) cells treated with 5-MTP at the dose of 5, 25 and 100 μM. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. 5-MTP: 5-Methoxytryptophan.

Next, we examined the effects of 5-MTP on CRC cell apoptosis by measuring levels of JC-1 and ROS, among others. In HCT-116 cells, Hoechst staining showed that 5-MTP inhibited CRC cell activity progressively (Figures 2A and B). Flow cytometry results showed that 5-MTP significantly inhibited JC-1 levels and promoted the apoptosis rate and ROS levels (Figures 2C-H).

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Figure 2 5-methoxytryptophan induced apoptosis and reactive oxygen species in HCT-116 cells. A and B: The Hoechst staining was used to measure the viability of HCT-116 cells treated with 5-methoxytryptophan (5-MTP) at the dose of 5, 25 and 100 μM; C and D: Apoptosis was detected by flow cytometry by double staining with PI and Annexin V of HCT-116 cells treated with 5-MTP at the dose of 5, 25 and 100 μM; E and F: Flow cytometry were used to analyses the JC-1 level of HCT-116 cells treated with 5-MTP at the dose of 5, 25 and 100 μM; G and H: 2’,7’-dichlorofluorescin diacetate assays were used to test the reactive oxygen species production of HCT-116 cells treated with 5-MTP at the dose of 5, 25 and 100 μM. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. 5-MTP: 5-Methoxytryptophan; ROS: Reactive oxygen species; PI: Propyl iodide.

Flow cytometry results showed that the peak value became higher in the G2/M phase, that is, 5-MTP-induced HCT116 cell cycle arrest (Figures 3A and B). These results demonstrated that 5-MTP induced cell cycle arrest in HCT-116 cells.

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Figure 3 5-methoxytryptophan induced cell cycle arrest in HCT-116 cells. A and B: The HCT-116 cells were treated with 5-methoxytryptophan at the dose of 5, 25, and 100 μM. The cycle arrest was detected by flow cytometry.

To further investigate the effect of 5-MTP on CRC cells, invasion and migration assays showed that 5-MTP inhibited CRC cell invasion and migration (Figures 4A-C). Ly294002 (PI3K) inhibitors have been shown to play a role in various tumors, including proliferation, metastasis, and apoptosis[13-15]. However, the effects of 5-MTP combined with ly294002 on invasion and migration in CRC cells are further enhanced is unknown. It was found that 5-MTP combined with ly294002 resulted in significantly less cell invasion and migration than 5-MTP (Figures 4D and F).

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Figure 4 5-methoxytryptophan inhibited migration and invasion in HCT-116 cells. A-C: Detection of migration and invasion in HCT-116 cells were treated with 5-methoxytryptophan (5-MTP) at the dose of 5, 25 and 100 μM by transwell test; D-F: The HCT-116 cells were treated with 5-MTP and/or LY294002. Detection of migration and invasion in HCT-116 cells by transwell test. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. 5-MTP: 5-Methoxytryptophan.

The PI3K/Akt/FoxO3a signaling pathwa plays an important role in various pathological processes in various tumors, including proliferation, metastasis, and apoptosis[16-18]. However, Figure 4 confirmed that 5-MTP combined with ly294002 inhibited CRC cell invasion and migration. The next experimental results showed that 5-MTP inhibited the relative protein levels of p-Akt/t-AKT and p-FoxO3a/t-FoxO3a with increasing drug concentration in HCT116 cells (Figures 5A-D). It was further found that the relative protein levels of p-Akt/t-AKT and p-FoxO3a/t-FoxO3a were also inhibited over time when the 5-MTP concentration was fixed, confirming the previous conclusions (Figures 5E-H). Meanwhile, western blot results showed that Caspase3, PARP, and BAX protein levels were increased, while Bcl2 protein levels were significantly decreased (Figures 6A-D). These results are the same as those in the Figure 1. Overall, our data indicated that 5-MTP induced dose-response and time course inhibition of PI3K/Akt/FoxO3a signaling pathway in HCT-116 cells.

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Figure 5 5-methoxytryptophan induced dose-response and time course inhibition of PI3K/Akt/FoxO3a signaling pathway in HCT-116 cells. A-D: The HCT-116 cells were treated with 5-methoxytryptophan (5-MTP) at the dose of 0, 1, 5, 25, 50 and 100 μM. The expression of PI3K/Akt/FoxO3a signaling pathway protein was detected by western blotting; E-H: The HCT-116 cells were treated with the dose of 25 μM of 5-MTP at the time of 0, 3, 6, 12, 24 and 72 h. The expression of p-Akt, t-AKT, p-FoxO3a and t-FoxO3a was detected by western blotting. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. 5-MTP: 5-Methoxytryptophan.

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Figure 6 5-methoxytryptophan induced apoptosis in HCT-116 cells. A-D: The expression of apoptosis-associated protein was detected by western blotting. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. 5-MTP: 5-Methoxytryptophan; Bcl2: B-cell lymphoma-2.

To further verify that 5-MTP combined with PI3K/Akt/p-FoxO3a signaling pathway inhibitors played a better therapeutic effect, ly294002 (PI3K) inhibitor and AKT small interfering RNA (siRNA) were transfected into HCT116 cells. The results showed that the relative protein levels of p-Akt/AKT and p-FoxO3a/FoxO3a were significantly decreased in the 5-MTP combined with the ly294002 group compared with the 5-MTP group. At the same time, we also found that Cyclin D1 and P27 protein levels were significantly decreased while Bim was significantly increased in the 5-MTP combined ly294002 group compared with the 5-MTP group, suggesting that 5-MTP-induced cell cycle-related proteins are associated with the PI3K/Akt/p-FoxO3a signaling pathway (Figures 7A-D). It was further found that the 5-MTP combined with the AKT siRNA group came to the same conclusion as the 5-MTP group (Figures 7E-H).

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Figure 7 Apparent correlations between 5-methoxytryptophan-induced cell cycle and apoptosis-associated proteins or inhibition of PI3K/Akt/FoxO3a signaling pathway in HCT-116 cells. A-D: The HCT-116 cells were treated with 5-methoxytryptophan (5-MTP) and/or LY294002. The expression of PI3K/Akt/FoxO3a signaling pathway and cycle arrest protein was detected by Western blotting; E-H: The HCT-116 cells were treated with the dose of 25 μM of 5-MTP and/or AKT siRNA. The expression of p-Akt, t-AKT, p-FoxO3a, t-FoxO3a, Bim, Cyclin D1 and P27 was detected by western blotting. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. 5-MTP: 5-Methoxytryptophan; NC: Normal control; siRNA: Small interfering RNA.

Colorectal cancer (CRC) is a highly prevalent malignant tumor. Research is needed to find and develop effective drugs for the treatment of CRC. 5-methoxytryptophan (5-MTP) is a tryptophan metabolite found in animals and humans. The effect of 5-MTP on the proliferation and apoptosis of CRC cells is still unclear.

Our work explored the effects of 5-MTP on the proliferation, migration, invasion and apoptosis of CRC cells. We tried to explore the potential of 5-MTP as a drug for the treatment of CRC.

Here, we studied that 5-MTP combined with PI3K/Akt/FOXO3a signaling pathway inhibitor can significantly promote apoptosis and cell cycle arrest of CRC cells, and inhibit cell proliferation. 5-MTP can be used as an effective drug in the treatment of CRC.

In this study, a series of experiments were carried out. Colony forming assay and Cell Counting Kit were used to detect the effect of 5-MTP on the proliferation of CRC cell lines. The effect of 5-MTP on cell growth was detected by cell cycle analysis. In addition, the effects of 5-MTP on apoptosis and reactive oxygen species of HCT-116 cells were studied. In order to further explore the effect of 5-MTP on CRC, we studied the effect of 5-MTP on PI3K/Akt signaling pathway in CRC cells.

5-MTP can promote apoptosis and cell cycle arrest of CRC cells, and inhibit cell proliferation. However, compared with 5-MTP alone, 5-MTP combined with PI3K/Akt/FOXO3a signaling pathway inhibitors significantly promoted apoptosis and cell cycle arrest of CRC cells, and inhibited cell proliferation. It provides new insights into the mechanism of drug action.

5-MTP combined with PI3K/Akt/FOXO3a signaling pathway inhibitor significantly promoted apoptosis and cell cycle arrest of CRC cells, and inhibited cell proliferation.

This study confirmed that 5-MTP combined with PI3K/Akt/FOXO3a signaling pathway inhibitor could significantly promote the apoptosis and cell cycle arrest of CRC cells, and inhibit cell proliferation. These results provide a new direction for the drug treatment of CRC. However, the study of mechanism in this study is relatively limited. Therefore, further analysis, nude mouse experiments and more cell experiments are needed to explore its mechanism.