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. 2024 Jul 2;9(28):30698–30707. doi: 10.1021/acsomega.4c03101

Synthesis and Biological Activity of 2-Chloro-8-methoxy-5-methyl-5H-indolo [2,3-b] Quinoline for the Treatment of Colorectal Cancer by Modulating PI3K/AKT/mTOR Pathways

Yunhao Ma †, Hongmei Zhu †, Xinrong Jiang †, Zhongkun Zhou †, Yong Zhou †, Yanan Tian ‡, Lixue Tu †, Juan Lu †, Yuqing Niu †, Liqian Du †, Zhenzhen Si †, Hong Fang †, Huanxiang Liu ‡, Yingqian Liu †,*, Peng Chen †,*
PMCID: PMC11256334  PMID: 39035959

Abstract

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Developing novel drugs from natural products has proven to be a very effective strategy. Neocryptolepine was isolated from Cryptolepis sanguinolenta, a traditional endemic African herb, which exerts a wide range of biological activities such as antimalaria, antibacterial, and antitumor. 2-Chloro-8-methoxy-5-methyl-5H-indolo [2,3-b] quinoline (compound 49) was synthesized, and its cytotoxicity was assessed on pancreatic cancer PANC-1 cells, colorectal cancer HCT116 cells, liver cancer SMMC-7721 cells, and gastric cancer AGS cells in vitro. The results of the in vitro assay showed that compound 49 exerted remarkable cytotoxicity on colorectal cancer HCT116 and Caco-2 cells. The cytotoxicity of compound 49 to colorectal cancer HCT116 cells was 17 times higher than that of neocryptolepine and to human normal intestinal epithelial HIEC cells was significantly reduced. Compound 49 exhibited significant cytotoxicity against the colorectal cancer HCT116 and Caco-2 cells, with IC50 of 0.35 and 0.54 μM, respectively. The mechanism of cytotoxicity of compound 49 to colorectal cancer HCT116 and Caco-2 cells was further investigated. The results showed that compound 49 could inhibit colony formation and cell migration. Moreover, compound 49 could arrest the cell cycle at the G2/M phase, promote the production of reactive oxygen species, reduce mitochondrial membrane potential, and induce apoptosis. The results of Western blot indicated that compound 49 showed cytotoxicity on HCT116 and Caco-2 cells by modulating the PI3K/AKT/mTOR signaling pathway. In conclusion, these results suggested that compound 49 may be a potentially promising lead compound for the treatment of colorectal cancer.

1. Introduction

Colorectal cancer (CRC) is a highly aggressive and common malignancy in the world.1−3 Global cancer statistics for 2020 showed that there are approximately 1.93 million new cases of colorectal cancer and approximately 0.93 million colorectal cancer deaths worldwide, ranking third in incidence and second in mortality of all malignancies.4 Currently, the main treatments for CRC include surgery, chemotherapy, radiotherapy, immunotherapy, and targeted therapies.5−7 Chemotherapy remains the most commonly used treatment method in clinical.8 At present, the commonly used chemotherapy drugs mainly include 5-fluorouracil (5-Fu), oxaliplatin, irinotecan, capecitabine, and so on. Combination therapy strategies are widely used in clinical treatment.9 Although chemotherapy has improved the survival rate of patients with colorectal cancer, its disadvantages, such as high toxicity and side effects, easy to produce drug resistance, and so on, cannot be ignored,10,11 Therefore, it is necessary to develop novel high-efficiency and low-toxic chemotherapy drugs against colorectal cancer.12

Natural products are considered a promising source for novel anticancer drugs.13,14 The analysis of anticancer drugs showed that most of the approved drugs are unmodified natural products or semisynthetic derivatives, or molecules synthesized based on the pharmacophores of natural product compounds.15−17 It has been reported that the indole quinoline alkaloids have good antibacterial and antitumor activities.18,19 Neocryptolepine is a promising alkaloid isolated from the traditional endemic African herb Cryptolepis sanguinolenta, whose broad bioactivity including cytotoxicity, antibacterial, antifungal, and antiplasmodial has been confirmed by several studies.20,21 Also, structural modification of compounds is an effective method to enhance pharmacological activity.22,23 Therefore, it is valuable to design and evaluate the cytotoxic activity of a neocryptolepine derivative against colorectal cancer cells.

The occurrence and development of colorectal cancer are related to a variety of signal transduction pathways.24 The PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B)/mTOR (mammalian target of rapamycin) signaling pathway is one of the most commonly abnormally activated signaling pathways in colorectal cancer.25 This signaling pathway can regulate the vital activities of cancer cells such as autophagy, proliferation, apoptosis, drug resistance, and metastasis.26 Studies have been done to develop drugs that target the significant protein of the PI3K/AKT/mTOR signaling pathway; a number of drugs have been evaluated in clinical trials, and it is becoming increasingly clear that inhibitors of PI3K/AKT/mTOR can effectively inhibit tumor progression.27,28 However, few inhibitor drugs have been approved for clinical use, and those that have been approved for clinical use are mainly PI3K and mTOR inhibitors. The modulation of the PI3K/AKT/mTOR signaling pathway may be a good strategy for the treatment of colorectal cancer.

In the present study, attracted by rational drug design, 2-chloro-8-methoxy-5-methyl-5H-indolo [2,3-b] quinoline (compound 49) was designed and synthesized based on the core structure of neocryptolepine. We performed cytotoxicity evaluation on several cell lines and found that compound 49 showed better cytotoxicity on colorectal cancer HCT116 and Caco-2 cells. Subsequently, the potential cytotoxic mechanism of compound 49 was studied. This work may lay the foundation for further structural modification of compound 49 and its development into a promising clinical anticancer drug.

2. Materials and Methods

2.1. Synthesis

All of the reagents and solvents used in the synthesis of compound 49 were commercially purchased from suppliers and used directly without further purification. The synthetic route of compound 49 is outlined in Scheme 1. The synthesized compound 49 was characterized by 13C NMR, 1H NMR, mass spectrometry, and purity analysis (Supporting Information). 1H and 13C NMR spectra were recorded at 400 and 100 MHz on a Bruker AM-400 and referenced to the solvent signals. Mass spectrometry was recorded using a Bruker Daltonics APEXII49 spectrometer.

Scheme 1. Synthetic Route of Compound 49.

Scheme 1

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Reagents and conditions: (a) IPA, CH3I, 3 h; (b) KOH, H2O2, 48 h; (c) 6-methoxyindole, p-TSA, EtOH, 24 h.

2.2. Cell Culture

The human colorectal cancer HCT116 cells, pancreatic cancer PANC-1 cells, and gastric cancer AGS cells were purchased from the American Type Culture Collection (HCT116, LOT:70019042, PANC-1, LOT:70018880, AGS, LOT:70012225). Human liver cancer SMMC-7721 cells, human normal intestinal epithelial HIEC cells, and human colorectal cancer SW480 and Caco-2 cells were stored in our own laboratory. HCT116, SMMC-7721, Caco-2, and AGS cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution. SW480, HIEC, and PANC-1 cells were incubated in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution. All cell lines were maintained in a humidified atmosphere at 37 °C with 5% CO2.

2.3. MTT Assay

When cells were in the logarithmic phase, 1 × 104 cells/well (100 μL) were incubated in 96-well plates. The plate was incubated overnight at 37 °C; after exposure of compound 49 (0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 μM) (for 0, 24, 48, and 72 h), 10 μL of MTT solution (5 mg/mL MTT in PBS, pH 7.2) was added in plates for 48 h at 37 °C. Then, the reagent was removed, and 100 μL of DMSO was added to each well. The absorbance of the wells was measured using a microplate reader at 490 nm. The viability of cells was calculated by the formula = 100% × (sample absorbance)/ (control absorbance).

2.4. Migration Assay

HCT116 cells (1 × 105) were seeded in a serum-free medium into the upper chamber, and the nether chamber was filled with 500 μL of complete cell culture medium with 20% fetal bovine serum. After the incubation of 48 h at the atmosphere of 37 °C and 5% CO2, the HCT116 cells were fixed with 4% (w/v) paraformaldehyde for 40 min. Then, the cells were stained with 1% crystal violet for another 15 min. After being washed with PBS, the numbers of HCT116 cells were observed and counted with ImageJ.

2.5. Analysis of Cell Cycle Arrest

The HCT116 and Caco-2 cells were treated with compound 49 in the different concentrations (0.2 and 0.4 μM) for 24 h. Cells (1 × 106) were fixed 75% ethanol overnight. Then, the HCT116 and Caco-2 cells were slowly washed with PBS and incubated with 100 μL of RNase A about 30 min at 37 °C. PI (400 μL) was used for staining at 4 °C for 30 min. The cellular distribution was measured by using a flow cytometer (BD Biosciences, USA).

2.6. Hoechst 33258 Staining

Sterile round cover slides (12 mm) were placed in 24-well plate, and 1.5 × 105 HCT116 cells were seeded on the slide and incubated for 24 h. Then, the HCT116 cells were exposed to different concentrations of compound 49 (0, 0.2, 0.4, and 0.8 μM) for 48 h. The HCT116 cells were stained with Hoechst 33258 dye (10 μg/mL) (C0021, Solarbio) for about 20 min. Finally, the circular cover slide with HCT116 cells was placed on the slide, and three fields of view were randomly selected for observation and photography (20×) with a Nikon-ECLIPSE 80i fluorescence microscope.

2.7. Analysis of Cell Apoptosis

HCT116 and Caco-2 cells of 8 × 105 cells per well were plated in a 6-well plate before different concentrations of compound 49 (0, 0.2, 0.4, and 0.8 μM) were added for 48 h. Then, the HCT116 and Caco-2 cells were harvested with trypsin without EDTA. Annexin V/Alexa Fluor 488 staining solution (5 μL) were added to the cells and incubated for 5 min at room temperature in the dark. Then, 10 μL of PI staining solution and 400 μL of PBS were added for analysis by flow cytometry (BD Biosciences, USA) and FlowJo V10.

2.8. Plate Clone Formation Assay

For plate clone formation assays, HCT116 and Caco-2 cells (500 cells/well) were seeded in 24-well plates. After attachment, cells were treated with compound 49 of various concentrations (0, 0.05, 0.1, and 0.2 μM) for 7 days at the cultured condition. Then, cells were fixed by 4% (w/v) paraformaldehyde for 2 h and stained with 0.1% crystal violet dye solution for 40 min at room temperature, and the cells were washed twice with PBS and observed.

2.9. JC-10 Staining Analysis

When cells were in the logarithmic phase, 8 × 105 HCT116 and Caco-2 cells/well were inoculated in 6-well plates for 24 h, exposed to indicated concentrations of compound 49 (0, 0.2, 0.4, and 0.8 μM). After incubation for 48 h, cells were harvested and washed with PBS twice. Next, JC-10 staining dye solution was added and the HCT116 and Caco-2 cells were rinsed with JC-10 binding buffer. Measurements were performed by flow cytometry (BD Biosciences, USA). FACS data were processed using the FlowJo V10.

2.10. ROS Assay

HCT116 and Caco-2 cells of 8 × 105 cells/well were seeded in a 6-well plate for 24 h. After attachment, cells were treated with compound 49 (0, 0.2, 0.4, and 0.8 μM) for 48 h. Then, the cells were harvested by trypsin without EDTA and washed twice with PBS. After cells were stained with DCFH-DA staining dye solution for 30 min, the fluorescence of DCF was detected by a flow cytometer (BD Biosciences, USA). FACS data were processed using the FlowJo V10.

2.11. Molecular Docking

To find the target of compound 49 exerting cytotoxicity to colorectal cancer cells, molecular docking was performed by Schrödinger 10.1. The protein crystal structure of PI3KCA (PDB:6JVF) was obtained from the Protein Data Bank. First, the complex structure was treated, removing crystal water, side chains, and hydrogen atoms from the protein structure and minimizing the energy of the entire structure. Then, compound 49 and protein PI3KCA were docked and the center of binding between small molecules and proteins was used as the docking site of compound 49 in the process, thus completing the whole docking process.

2.12. Western Blot

HCT116 cells (8 × 105) and Caco-2 cells/well were inoculated in 6-well plates for 24 h. After attachment, cells were treated with compound 49 of 0.4 μM. Then, HCT116 and Caco-2 cells were harvested and lysed in RIPA lysis buffer mixed with protease inhibitor buffer on ice for 30 min. Total protein (25 μg) was resolved by SDS–PAGE and transferred on the polyvinylidene difluoride (PVDF) membrane before blocking by 5% dry nonfat milk in tris-buffered saline plus 0.1% tween for 2 h at room temperature and probing by primary antibody overnight at 4 °C. Then, the membrane was washed with Tris-Buffered Saline Tween (TBST) three times and incubated with the secondary HRP-conjugated antibody for another 1 h at room temperature. Then, the membrane was washed with TBST and scanned using the BCL luminescence system for the protein and Tanon imaging system, and the protein levels were analyzed with ImageJ. All antibodies were purchased from Bioss at a dilute concentration of 1:2000 for primary antibody and 1:10,000 for secondary antibody. All experiments were performed three times. p <0.05 was considered significant.

2.13. Statistical Analysis

All the results and statistical analyses were showed by mean ± standard deviation, and the data were analyzed by IBM SPSS statistical software. The differences of analysis components were analyzed by Student’s t test (t test), and p <0.05 indicated statistically significant differences between two groups.

3. Results and Discussion

3.1. Synthesis and Structure Identification of Compound 49

The compound 49 was obtained under the mild conditions by the work of Vecchione et al.29 The 6-chlor-1-methylquinolinium iodide was obtained by methylation with 6-chloroquinoline, IPA, and CH3I for 4 h. Then, 2-(methylamino)-5-chlor-benzaldehyde was synthesized by oxidation with the 6-chlor-1-methylquinolinium iodide and H2O2 in KOH for 48 h. Finally, 2-(methylamino)-5-chlor-benzaldehyde was cyclized with 6-methoxyindole and p-TSA for 24 h to obtain compound 49. The structure of compound 49 was confirmed by 1H NMR, 13C NMR, and mass spectrometry (Supporting Information, Figures S1–S3). The purity of compound 49 was above 99% by high-performance liquid chromatography (HPLC), and the results are shown in Figure S4, with methanol: acetonitrile = 80:20 as a mobile phase for 30 min. Physical properties of compound 49: yield, 62%; red solid, mp 180.11–181.23 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 1H), 8.08 (s, 1H), 7.91 (d, J = 6.0 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.72 (d, J = 9.1 Hz, 1H), 7.07 (s, 1H), 6.71 (d, J = 8.4 Hz, 1H), 4.20 (s, 3H), 3.81 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 161.88, 158.05, 156.16, 134.95, 129.92, 128.47, 126.36, 125.64, 122.85, 122.15, 117.45, 116.39, 108.01, 102.83, 55.82, 32.87. MS-ESI m/z: calcd for C17H13ClN2O [M + H]+ 297.0789; found: 297.0791.

3.2. Cytotoxic and Structure–Activity Relationship Analysis of Compound 49

The cytotoxicity of compound 49 was evaluated against cancer cell lines and human normal intestinal epithelial HIEC cell. The IC50 values of compound 49 are shown in Table 1 on different cell lines for 48 h. Compound 49 exhibited good and selective cytotoxicity on CRC cell lines, especially HCT116 and Caco-2 cells.

Table 1. Antiproliferative Properties for Compound 49 on Different Cell Lines for 48 h.

  IC50 (μM)
cell lines compound 49 neocryptolepine
HCT116 0.35 ± 0.04 6.26 ± 1.22
Caco-2 0.54 ± 0.04 13.54 ± 0.83
AGS 26.9 ± 0.00 5.81 ± 1.91
PANC-1 >50 >50
SMMC-7721 22.92 ± 2.16 19.31 ± 2.71
HIEC >50 31.37 ± 0.44
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According to Figure 1A, compound 49 exerted cytotoxicity on HCT116 cells in a time-dependent and concentration-dependent manner. The IC50 of compound 49 against Caco-2 cells was 0.54 μM, and compound 49 showed better cytotoxic activity than 5-Fu on Caco-2 cells (Figure 1B). For comparison, the IC50 values of neocryptolepine on HCT116 cells and human normal intestinal epithelial HIEC cells were 6.26 and 31.37 μM (Figure 1C). The IC50 of compound 49 against HCT116 cells was 0.35 μM, while 5-Fu, a CRC chemotherapy drug commonly used in the clinic, had little cytotoxicity on HCT116 cells at the same concentration (Figure 1D). Moreover, The IC50 of compound 49 in human normal intestinal epithelial HIEC cells was greater than 50 μM (Figure 1F). Therefore, compared with neocryptolepine, the introduction of 2-site Cl atom and 8-site methoxyl substituents made the cytotoxicity of compound 49 better in colorectal cancer cells. The 2-site Cl and 8-site methoxyl could be important substituents for compound 49 to exert good cytotoxic effects. Taken together, compound 49 displayed excellent cytotoxic activity to CRC cells and low toxicity to normal intestinal epithelial cells.

Figure 1.

Figure 1

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Effects of compound 49 on HCT116 and Caco-2 cells. (A) Concentration and time-dependent effects of compound 49 on HCT116 cells. (B–D) Compound 49 had a better anticancer activity than 5-Fu on HCT116 and Caco-2 cells. (E) The OD490 nm values of cells treated with compound 49 in HCT116 cell lines. Values were shown as the mean ± SD, n = 3. *p <0.05, **p <0.01, ***p <0.001.

3.3. Compound 49 Inhibited the Plate Clone Formation of Colorectal Cells and Blocked the Colorectal Cell Cycle at the G2/M Stage

To further verify the cytotoxicity of compound 49in vitro, plate clone formation assay was performed. According to Figure 2A,B, compound 49 inhibited the formation of HCT116 and Caco-2 cell clones. These results suggested that compound 49 could inhibit the proliferation of HCT116 and Caco-2 cells. Anticancer drugs of small molecules often interfere with the cancer cell cycle.30 It has been reported that neocryptolepine intercalated into DNA and interfered with the catalytic activity of human topoisomerase II, provoked a massive accumulation of P388 murine leukemia cells at the G2/M phase.31 To determine the mechanisms by which compound 49 exerted cytotoxicity, HCT116 and Caco-2 cell cycle distribution was examined by flow cytometry. According to Figure 2C,D, the HCT116 and Caco-2 cell cycle progression was significantly arrested at the G2/M phase in a concentration-dependent manner. The percentage of the G2/M phase HCT116 and Caco-2 cells increased from 15.21 to 82.53% and from 23.22 to 86.48% after the treatment of compound 49 at different concentrations (0, 0.2, and 0.4 μM) for 24 h (Table 2). These results suggested that compound 49 could induce cell cycle arrest at the G2/M phase.

Figure 2.

Figure 2

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Effect of compound 49 on HCT116 cell colony formation and cell cycle. (A) Compound 49 inhibited colony formation of HCT116 cells. (B) Compound 49 inhibited colony formation of Caco-2 cells. (C) HCT116 cells were treated with compound 49 (0, 0.2, and 0.4 μM) for 24 h and analyzed by flowed cytometry. (D) Caco-2 cells were treated with compound 49 (0, 0.2, and 0.4 μM) for 24 h and analyzed by flowed cytometry. Values were shown as the means ± SD, n = 3, *p <0.05, **p <0.01, ***p <0.001.

Table 2. Effects of Compound 49 on the Cell Cycle Distribution of HCT116 and Caco-2 Cells Treated for 24 h.

  the distribution of HCT116 cells in each phase (%)
 the distribution of Caco-2 cells in each phase (%)
compound 49 G0/G1 S G2/M G0/G1 S G2/M
control 42.56 ± 3.31 42.23 ± 2.37 15.21 ± 0.94 32.04 ± 0.97 44.75 ± 0.47 23.22 ± 1.44
0.2 μM 34.12 ± 2.20 44.88 ± 2.00 21.01 ± 0.20 36.68 ± 1.57 33.52 ± 0.45 29.81 ± 1.12
0.4 μM 3.92 ± 0.04 13.55 ± 3.61 82.53 ± 3.56 4.54 ± 0.67 8.99 ± 0.89 86.48 ± 1.56
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3.4. Compound 49 Inhibited the Migration of Colorectal Cells

Cell migration is critical in cancer invasion and progression,32 and metastases have been the major cause of cancer-associated deaths in general.33 The transwell assay was performed to investigate the effect of compound 49 on HCT116 cell migration. According to Figure 4B, after treatment of compound 49, the number of migratory cells of HCT116 was significantly reduced in a dose-dependent manner. The results suggested that compound 49 inhibited the migration of HCT116 cells.

Figure 4.

Figure 4

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Compound 49 induced early apoptosis and inhibited cell migration of HCT116 and increased the ROS level of HCT116 and Caco-2 cells. (A) Compound 49 induced early apoptosis of HCT116. (B) Compound 49 inhibited cell migration of HCT116 cells. (C, D) Analysis of ROS induced by compound 49 (0, 0.2, 0.4, and 0.8 μM) for 48 h using the DCFH-DA assay.

3.5. Compound 49 Produced ROS, Disrupted MMP, and Induced Cell Apoptosis

To determine whether compound 49 affects HCT116 apoptosis, we analyzed HCT116 cells treated by compound 49 using the qualitative approach of the Hoechst 33258 staining assay. The result showed that the number of brightly fluorescent cells increased in a concentration-dependent manner (Figure 4A). That means compound 49 could induce the apoptosis of HCT116 cells. To further quantitatively test the effect of compound 49 on apoptosis of HCT116 cells, an Annexin V-Alexa Fluor 488/PI staining assay was performed. As shown in Figure 3A,B, compound 49 mainly caused late apoptosis; the percentage of late apoptotic HCT116 cells increased from 3.7 to 45.3%, and the percentage of late apoptotic Caco-2 cells increased from 11.6 to 43.0%. The percentage of early and late stages apoptotic cells increased from 4.89 to 54.00% in HCT116 cells and from 7.86 to 71.50% in Caco-2 cells after the treatment of compound 49 at different concentrations (0, 0.2, 0.4, and 0.8 μM) for 48 h, which suggested that compound 49 significantly increased the cellular apoptosis in a concentration-dependent manner (Table 3).

Figure 3.

Figure 3

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Effect of compound 49 on colorectal cell apoptosis. (A) Analysis of HCT116 cell apoptosis induced by compound 49 (0, 0.2, 0.4, and 0.8 μM) using the Annexin V/PI assay. (B) Analysis of Caco-2 cell apoptosis induced by compound 49 (0, 0.2, 0.4, and 0.8 μM) using the Annexin V/PI assay. (C, D) The depolarization of MMP in HCT116 and Caco-2 cells after treatment with compound 49 (0, 0.2, 0.4, and 0.8 μM) for 48 h by the JC-10 assay. Values were shown as the means ± SD, n = 3, *p <0.05, **p <0.01, ***p <0.001.

Table 3. Apoptosis Rate of HCT116 and Caco-2 Cells Treated with Compound 49 for 48 h.

compound 49 the apoptosis rate of HCT116 cells (%) the apoptosis rate of Caco-2 cells (%)
control 4.89 ± 0.06 7.86 ± 0.44
0.2 μM 24.35 ± 0.21 35.05 ± 4.03
0.4 μM 42.20 ± 4.10 50.40 ± 1.84
0.8 μM 54.00 ± 1.56 71.50 ± 2.83
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Early apoptosis is often accompanied by the reduction of the mitochondrial membrane potential, so we performed JC-10 staining experiments to detect the effect of compound 49 on the mitochondrial membrane potential of HCT116 and Caco-2 cells. According to Figure 3C,D, HCT116 cells with reduced mitochondrial membrane potential increased from 7.2 to 76.1%, and Caco-2 cells with reduced mitochondrial membrane potential increased from 19.2 to 54.0%. These results indicated that compound 49 could significantly cause a reduction in mitochondrial membrane potential in HCT116 and Caco-2 cells.

The production of ROS can affect the integrity of mitochondrial membranes, which is the basis of maintaining a normal level of mitochondrial membrane potential. To verify whether ROS was involved in the decrease in mitochondrial membrane potential, changes in ROS production were detected in HCT116 and Caco-2 cells treated with different concentrations (0, 0.2, 0.4, and 0.8 μM) of compound 49 for 48 h. As shown in Figure 4C,D, the relative fluorescence intensity of DCF of HCT116 cells increased from 1.00 to 5.02 and the relative fluorescence intensity of DCF of Caco-2 cells increased from 1.00 to 1.46 after treatment with compound 49 (Table 4).

Table 4. Relative Mean Fluorescent Intensity of HCT116 and Caco-2 Cells Treated with Compound 49 for 48 h.

compound 49 the relative mean fluorescent intensity of HCT116 cells the relative mean fluorescent intensity of Caco-2 cells
control 1.00 ± 0.00 1.00 ± 0.00
0.2 μM 1.87 ± 0.03 1.33 ± 0.03
0.4 μM 3.87 ± 0.38 1.23 ± 0.01
0.8 μM 5.02 ± 0.49 1.46 ± 0.05
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These results indicated a significant and concentration-dependent increase in cellular ROS production after the treatment of compound 49. Subsequently, the mitochondrial apoptosis pathway was verified by examining the expression of related proteins. According to Figure 5B, the expression level of cleaved-caspase 9 protein was significantly upregulated after treatment of compound 49 for 48 h. In conclusion, compound 49 might induce the apoptosis of HCT116 and Caco-2 cells by activating the mitochondrial pathway.

Figure 5.

Figure 5

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Result of molecular docking between compound 49 and PI3KCA receptor and compound 49 regulated the PI3K/Akt signaling pathway and cell cycle-related protein expression. (A) The result of molecular docking between compound 49 and the PI3KCA receptor. (B, C) The expression levels of related proteins in HCT116 and Caco-2 cells after treatment with compound 49 for 48 h. Values are shown as the means ± SD, n = 3, *p <0.05, **p <0.01, ***p <0.001.

3.6. Compound 49 Regulated the PI3K/AKT/mTOR Signaling Pathway in Colorectal Cancer HCT116 and Caco-2 Cells

As shown in Figure 5A, molecular docking results supported the possibility that PI3KCA was the target of the action of compound 49. The positively charged amino group on lysine 802 formed a stable hydrogen bond interaction with the oxygen atom on the methoxy of the acceptor, and the phenyl ring on compound 49 formed a π–π stacking interaction with tryptophan 780. Moreover, the ligand small molecule compound 49 was in a hydrophobic cavity consisting of 772MET, 850VAL, 922MET, 930PHE, 851VAL, 836TYR, 800ILE, 848ILE, 932ILE, 807LEU, and 934PHE, forming a strong hydrophobic interaction. The CDK1 plays an important role at the G2/M transition in the cell cycle, and the activity of CDK1 depends on the binding of CDK to cyclin B1. Western blot experiments were used to evaluated the protein expression levels of related signaling pathways after the treatment of compound 49. As shown in Figure 5B,C, the results revealed that the expression level of PI3KCA and p-AKT proteins was downregulated in HCT116 and Caco-2 cells after treatment of compound 49. In addition, the expression level of the CDK1 protein was upregulated, while the expression level of the cyclin B1 protein was downregulated. These results suggested that compound 49 might induce cell apoptosis of HCT116 and Caco-2 cells by regulating the PI3K/AKT/mTOR cell signaling pathway, thereby exhibiting cytotoxic effects.

Significant changes in cellular signaling pathways occur during cancer development.24 Numerous studies have shown that targeting signaling pathways to treat cancer is a promising strategy.34,35 The PI3K/AKT/mTOR signaling pathway is one of the most frequently activated signaling pathways in human cancers. As multiple genes of the PI3K/AKT/mTOR signaling pathway are frequently altered in human cancers, the genes related with this pathway are important molecular therapeutic targets in cancer treatment.25 Therefore, the regulatory role of compound 49 in this pathway was examined. In this study, MTT results showed that compound 49 could exert better cytotoxic effects on HCT116 and Caco-2 cells than the positive drug 5-Fu and have less toxicity to normal cells. Cell cloning and cell migration assays showed that compound 49 could inhibit colorectal cell proliferation and migration. Flow cytometry results indicated that compound 49 could block the cell cycle in the G2/M phase and induce cell apoptosis by the mitochondrial pathway. Molecular docking and Western blot results suggested that compound 49 might exert cytotoxicity by affecting the protein expression related PI3K/AKT/mTOR signaling pathway and cell cycle. Considering these results, compound 49 may be a promising lead compound for the treatment of colorectal cancer and provide support for subsequent structural modifications of the compounds.

4. Conclusions

In this study, MTT results showed that compound 49 could exert better cytotoxic effects than the positive drug 5-Fu and less toxicity to normal cells. Plate clone formation and cell migration assays showed that compound 49 could inhibit colorectal cell proliferation and migration. Flow cytometry results indicated that compound 49 could arrest the cell cycle in the G2/M phase and induce cell apoptosis by the mitochondrial pathway in HCT116 and Caco-2 cells. Molecular docking and Western blot results suggested that compound 49 might exert cytotoxicity by regulating the key protein expression related to the PI3K/AKT/mTOR signaling pathway and cell cycle. Considering these results, compound 49 might be a promising lead compound for the treatment of colorectal cancer and provide support for subsequent structural modifications of the compounds.

Acknowledgments

This work was supported by the Technological Innovation Guidance Program of Gansu Province (Grant No. 21CX6QA127), Scientific Research Project of Gansu Medical Products Administration (Grant No. 2022GSMPA008), Gansu Intellectual Property Project (Grant No. 22ZSCQ065), Technology Program of Lanzhou City (Grant No. 2022-2-52), and the College Students’ Innovation and Entrepreneurship Program of Lanzhou University, China (Grant Nos. 20240260001 and 20240260017).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c03101.

  • 1H and 13C NMR spectra of compound 49 and its high-resolution mass spectrogram, and the purity test of compound 49 by HPLC (PDF)

Author Contributions

§ Y.M. and H.Z. contributed equally to this work.

The authors declare no competing financial interest.

Supplementary Material

ao4c03101_si_001.pdf (337.7KB, pdf)

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