Arctigenin inhibits the progression of colorectal cancer through epithelial-mesenchymal transition via PI3K/Akt/mTOR signaling pathway

Abstract

Background

Colorectal cancer (CRC) is a significant disease worldwide, with high mortality rates. Conventional treatment methods often lead to metastasis and drug resistance, highlighting the need to explore new drugs and their potential molecular mechanisms. In this study, we investigated the effects of arctigenin on CRC cell proliferation, migration, invasion, apoptosis, and related protein expression, as well as its potential molecular mechanisms.

Methods

The CCK-8 assay, transwell migration and invasion assays, flow cytometry, immunoblotting and immunofluorescence staining, western blot and an allograft tumor transplantation model was used.

Results

Our study revealed that arctigenin effectively inhibited CRC cell proliferation, migration, and invasion in a dose-dependent manner, while also inducing apoptosis. At the molecular level, arctigenin significantly downregulated the expressions of PCNA, Bcl2, MMP-2, and MMP-9 and upregulated the expressions of Bax and cleaved caspase-3. Additionally, arctigenin demonstrated the ability to inhibit the epithelial-mesenchymal transition (EMT) process by upregulating E-cadherin and downregulating mesenchymal markers, such as N-cadherin, Vimentin, Snail, and Slug. Furthermore, arctigenin could inhibit the activation of the PI3K-AKT-mTOR signaling pathway, which has been implicated in cancer progression. In vivo experiments also showed that arctigenin significantly reduced tumor volume and size compared to the control group, with no significant adverse effects on the liver.

Conclusions

This is the first study to elucidate the mechanism by which arctigenin inhibits colorectal cancer metastasis through the PI3K-AKT-mTOR signaling pathway by suppressing the EMT process at the molecular level.

1. Introduction

Colorectal cancer (CRC) is one of the most common digestive system malignancies worldwide and the third most prevalent malignant tumor globally [1,2]. CRC-related deaths account for 8%-9% of all cancer deaths [3]. With the adjustment of lifestyle and dietary habits, the incidence of colorectal cancer in China has been increasing year by year and tends to affect younger individuals [4]. Early diagnosis of CRC is difficult, and most patients present with clinical symptoms in the middle to late stage of the disease. Treatment for CRC is still primarily surgery, with chemotherapy and radiation therapy as auxiliary methods. In terms of treatment effectiveness, there are significant individual differences among CRC patients, and the aforementioned therapies have significant drawbacks and a poor prognosis, with the potential for cell toxicity and resistance [5,6]. Some patients are unable to tolerate the side effects of radiation and chemotherapy. Therefore, it is necessary to find effective drugs that can inhibit CRC with fewer side effects and explore their molecular mechanisms.

Metastasis is the primary cause of cancer incidence and mortality. The process of epithelial-mesenchymal transition (EMT) is believed to play a crucial role in tumor invasion and metastasis [7,8]. EMT alters the type of tumor cells, enhances their ability to degrade the extracellular matrix, weakens their intercellular adhesion, and thus acquires migratory and invasive characteristics, thereby participating in the process of cancer metastasis. During the EMT process, the expression of mesenchymal cell markers, such as N-cadherin and Vimentin, typically increases, while the expression of epithelial cell markers, such as E-cadherin, decreases [9,10]. Current research suggests that activating the PI3K/AKT/mTOR pathway can promote the EMT process, thereby increasing the metastatic ability of tumor cells [11,12].

Arctigenin, a dibenzyl butyrolactone lignin derived from the Compositae plant Arctium lappa, has various pharmacological effects, such as anti-inflammatory, antiviral, immunomodulatory, and anti-tumor activities, as demonstrated by numerous studies [13]. These effects have been confirmed in animal and cell models. Arctigenin can exert its anticancer effects by targeting various intracellular pathways involved in cancer treatment. Studies have shown that arctigenin can inhibit cell cycle progression by regulating the expression of cell cycle proteins and cyclin-dependent kinases [14]. Additionally, arctigenin can accelerate the senescence of gallbladder cancer cells by inhibiting the RAF-MEK-ERK signaling pathway [15].

Literature reports suggest that arctigenin induces apoptosis in CRC cells via the ROS/p38MAPK pathway [16], but its mechanism of action in CRC cell proliferation, invasion, and migration remains unclear. Therefore, this study aimed to elucidate how arctigenin inhibited CRC through the PI3K-AKT-mTOR signaling pathway using in vivo experiments (establishing tumor xenograft and liver-lung metastasis models) and in vitro experiments (cell biology experiments). The results of this study would provide a foundation of evidence for the clinical application of arctigenin in the treatment of CRC.

2 Materials and methods

All methods were performed in accordance with the relevant guidelines and regulations.

2.1 Reagents and antibodies

arctigenin (CAS:7770-78-7) and 740 Y-P were purchased from MedChemExpres, Anti-E-cadherin (ab231303), anti-N-cadherin (ab98952), anti-PCNA (ab29), anti-Bcl2 (ab32124), anti-Bax (ab32503), anti-cleaved caspase 3 (ab32042), anti-caspase 3 (ab32351), anti-MMP2 (ab92536), anti-MMP9 (ab76003) and anti-GAPDH (ab9485) were purchased from Abcam (Cambridge, Britain). Anti-Snail (A5243), anti-Vimentin (A11952) were purchased from Abclonal (Wuhan, China). Anti-slug (PA5-73015), anti-p-PI3K (PA5-17387), anti-PI3K (MA1-74183), anti-p-AKT (PA5-95669), anti-AKT (MA5-14916), anti-p-mTOR (44-1125G), anti-mTOR (PA5-34663) were purchased from Thermo Fisher Scientific. Goat anti-mouse and goat anti-rabbit secondary antibodies were purchased from LI-COR (Lincoln, USA).

2.2 Cell culture

The condition of cell culture was accordance with my study published previously [17]. In brifiely, human colon cells (NCM460) were obtained from Shanghai Yaji Biotechnology Co., Ltd., while human CRC cell lines HCT116, SW620, and DLD-1 were procured from the National Collection of Authenticated Cell Cultures. NCM460 and HCT116 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. SW620 cells were cultured in L-15 medium supplemented with 10% FBS at 37°C. DLD-1 cells were cultured in RPMI-1640 medium supplemented with 10% FBS at 37°C in a 5% CO₂ incubator.

2.3 CCK-8 assay

A CCK-8 assay was utilized to investigate the impact of arctigenin on the proliferation of CRC cell lines (HCT116, SW620, and DLD-1). The cells were initially seeded in 96-well plates at a density of 5 x 10⁴ cells per well and allowed to incubate overnight. Subsequently, the cells were exposed to various concentrations of arctigenin (1, 5, 10, 20 and 40 μM) for 48 h. Following treatment, 10 μL of CCK-8 reagent was added to each well and incubated for 4 h in a CO₂ incubator. The absorbance of each well was determined at a wavelength of 450 nm via a microplate reader. Cell viability was calculated using the formula as follows: (OD_drug-OD_blank)/ (OD_control-OD_blank) x 100%.

2.4 Transwell assay

The impact of arctigenin on CRC cell migration was evaluated using the Transwell migration assay (without matrix gel). In contrast, the Transwell invasion assay (with matrix gel) was used to assess its effect on CRC cell invasion. Briefly, CRC cells were exposed to arctigenin at concentrations of 1, 5, and 10 μM for 24 h and then seeded onto the upper chamber. The lower chamber contained DMEM (500 μL) supplemented with 10% FBS. After 48 h of incubation, the upper chamber was removed, and the cells were fixed with 4% paraformaldehyde (PFA) for 5 min and stained with crystal violet for 5 min. Finally, five random fields were selected under a microscope to count cells and calculate the average value.

2.5 Flow cytometry

The Annexin V-FITC/PI apoptosis detection kit (ThermoFisher, E-CK-A211) was used to examine the effect of arctigenin on the apoptosis of CRC cells (HCT116, SW620, and DLD-1). Cells were seeded in six-well plates and exposed to arctigenin at different concentrations (0, 1, 5, and 10 μM) for 48 h. The cells were then collected after trypsinization, washed with PBS, and resuspended in Annexin V binding buffer. A total of 5 x 10⁵ cells were collected and centrifuged at 300 g for 5 min, and the supernatant was discarded. Next, the cells were washed with pre-cooled PBS, centrifuged, and resuspended in 100 μL of diluted Annexin V binding buffer. Then, 2.5 μL of Annexin V-FITC and 2.5 μL of PI staining solution were added to the cell suspension. The mixture was gently vortexed and incubated at room temperature in the dark for 15–20 min. Finally, the cell suspension was mixed with 400 μL of diluted 1 × Annexin V binding buffer and immediately analyzed by flow cytometry.

2.6 Western blotting analysis

The cells were exposed to various concentrations of arctigenin for 24 h in a culture incubator. After incubation, the cells were collected and washed with cold PBS. They were then lysed with RIPA buffer, and the cell lysates were transferred to a 1.5-mL EP tube and sonicated three times at 10-min intervals. Subsequently, the cells were centrifuged at 15,000 rpm at 4°C for 15 min, the supernatant was collected, and the protein concentration was determined using the BCA method. Next, equal amounts of proteins were subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a nitrocellulose (NC) membrane. The NC membrane was blocked with 5% (w/v) skim milk for 2 h and then incubated with the primary antibodies overnight at 4°C. The blots were washed three times with PBST and incubated with the corresponding secondary antibody at room temperature in the dark for 1 h. Subsequently, the membrane was analyzed using the Odyssey Infrared Fluorescence Imaging System (LI-COR, Lincoln, NE), and the intensity of the protein bands was quantified using Scion image software.

2.7 In vivo tumor model

An ectopic tumor transplantation model was used to evaluate the effect of arctigenin on in vivo tumor growth. BALB/c nude mice used in the experiment were purchased from the Nantong University Laboratory Animal Center. This animal experiment was approved by the Nantong University Animal Experiment Ethics Committee (S20220221-015). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. The ectopic tumor transplantation model was established as follows: briefly, 1×10⁶ HCT116 cells were resuspended in 1 mL of PBS and subcutaneously injected (0.2 mL) into the left or right axilla of 6-week-old male BALB/c mice. Tumor volume was measured every 3 days after the tumors became macroscopic. When the tumor volume reached 100 mm³, the mice were divided into different groups (n = 5, respectively). The experimental groups were administered arctigenin at doses of 20 and 40 mg/kg. The control group was given physiological saline. It was then decided to sacrifice the mice, which received an intraperitoneal injection of ketamine (100 mg/kg) prepared with a physiological saline solution by cervical dislocation, and harvest the tumors after 14 days of using the arctigenin. Tumor volume and weight were measured, and immunohistochemical analysis was performed. During the entire duration of this study, there were no instances of mice being found to be in poor health or experiencing mortality. Studies involving animals were performed in accordance with the recommendations in the ARRIVE guidelines.

2.8 Hematoxylin and eosin (H&E) staining

Following the completion of the experiment, the mice were euthanized, and their liver and tumor tissues were removed intact. The tissues were fixed in 4% PFA for 24 h and then dehydrated using different concentrations of ethanol solution. After dehydration, the tissues were embedded in paraffin and cut into 5-μm-thick sections using a microtome for routine staining. Pathological changes in the tissue sections were subsequently observed under a microscope.

2.9 Immunofluorescence staining

The methods of immunofluorescence staining was accordance with our work published perviously [17]. To detect the main proteins related to EMT, including E-cadherin, N-cadherin, and p-PI3K proteins, cells were exposed to different concentrations of arctigenin for 24 h. Cells were then collected from each group and fixed with 4% PFA at room temperature for 20 min. After washing with PBS three times, the cells were permeabilized with 0.1% Triton X-100 at room temperature for 10 min, followed by washing with PBS three times (5 min each). The cells were then blocked with 3% bovine serum albumin (BSA) for 60 min and incubated with primary antibodies against E-cadherin, N-cadherin, and p-PI3K overnight at 4°C. Next, the cells were incubated with secondary antibodies at room temperature for 1 h and stained with DAPI (1 μg/mL) at room temperature for 3 min in the dark. Finally, the protein fluorescence was observed under a fluorescence microscope, and images were acquired. The protein fluorescence intensity of each group of cells was measured using Image-Pro Plus 6.0 software.

2.10 Statistical analysis

In the in vivo experiments, tumor volume (V) was calculated using the formula V = 0.5×L×W2, where L represents the length of the tumor and W represents the width. Data analysis was performed using GraphPad Prism 7, and the results were expressed as mean ± standard deviation. one-way ANOVA was used to determine differences between groups, and a P-value less than 0.05 was considered statistically significant.

3 Results

3.1 Effects of arctigenin on the proliferation of CRC cell lines

The CCK-8 assay was used to evaluate the effect of arctigenin on the viability of CRC cells. As shown in Fig 1A–1D, compared with the control group, arctigenin had no significant effect on the viability of normal human colonic epithelial cells at concentrations of 1, 5, 10, 20 and 40 μM. However, it significantly inhibited the viability of CRC cells. The inhibition was dose-dependent, with IC50 values of 15.54μM for HCT116, 17.43μM for SW620 and 20.41μM for DLD-1, with the most significant inhibition observed in HCT116 cells. Therefore, HCT116 cells were selected for further experiments. Western blotting analysis (Fig 1E and 1F) showed that arctigenin significantly reduced the expression of proliferating cell nuclear antigen (PCNA) in CRC cells compared to the control group in a dose-dependent manner. These results indicated that arctigenin inhibited the proliferation of CRC cells in a concentration-dependent manner.

[Figure omitted. See PDF.]

A,B,C,D CCK-8 detection of the effffect of arctigenin on the survival rate of colorectal cancer cells (HCT116, SW620, DLD-1). E Western blot detection of the effffect of arctigenin on PCNA protein expression in HCT116 cells. “*” indicates the difffference compared with the control group (P < 0.05), n = 3.

3.2 Arctigenin induces apoptosis in CRC cell lines

The results of the flow cytometry presented in Fig 2A–2C demonstrated that arctigenin had a significant dose-dependent effect on promoting apoptosis in CRC cell lines (HCT116, SW620 and DLD-1) compared to the control group. Additionally, arctigenin was observed to modulate the expression of apoptotic proteins (HCT116 cell lines), resulting in a dose-dependent inhibition of Bcl-2 expression and an increase in the expression of Bax and cleaved caspase-3 proteins (Fig 2D–2F). However, the expression of caspase-3 did not show a significant change. These findings provided strong evidence that arctigenin could induce apoptosis in CRC cell lines.

[Figure omitted. See PDF.]

A,B,C HCT116, SW620 and DLD-1 cells apoptosis was detected by flow cytometry and quantified. D,E,F The expression level of Bcl-2, Bax, Cleaved caspase-3 and caspase-3 proteins were assessed by western blot and quantifified. “*” indicates the difffference compared with the control group (P < 0.05), n = 3.

3.3 Arctigenin inhibits migration of CRC cell lines

The migration ability of CRC cells was evaluated using the Transwell migration assay in the presence of arctigenin. As depicted in Fig 3A–3C, arctigenin treatment at doses of 1, 5, and 10 μM for 48 h significantly inhibited the migration ability of CRC cells (HCT116, SW620, and DLD-1) in a dose-dependent manner, as compared to the control group.

[Figure omitted. See PDF.]

A,B,C Transwell assay was used to evaluate the effect of arctigenin on migration on HCT116, SW620 and DLD-1. “*” indicates the difffference compared with the control group (P < 0.05), n = 3.

3.4 Arctigenin inhibits invasion of CRC cell lines

As demonstrated in Fig 4A–4C, the Transwell invasion assay revealed that exposure to arctigenin at doses of 1, 5, and 10 μM for 48 h significantly inhibited the invasive potential of CRC cells (HCT116, SW620, and DLD-1) in a dose-dependent manner compared to the control group. This outcome was consistent with the migration results, supporting the suppressive impact of arctigenin on the movement capacity of CRC cells. Matrix metalloproteinases (MMPs) can disintegrate the extracellular matrix and promote cancer cell EMT. In the MMP family, MMP-2 and MMP-9 are known to be associated with tumor metastasis and overexpressed. Therefore, in this research, we investigated the expressions of MMP-2 and MMP-9 to explore the potential anti-invasive activity of arctigenin. The Western blotting analysis (HCT116 cell lines) (Fig 4D) results indicated that exposure to arctigenin for 48 h significantly reduced the expressions of MMP-2 and MMP-9 in CRC cells, and this inhibition of MMP-2 and MMP-9 proteins was dose-dependent.

[Figure omitted. See PDF.]

A,B,C Transwell assay was used to evaluate the effect of arctigenin on invasion on HCT116, SW620 and DLD-1. D Western blot detection of the effffect of arctigenin on MMP-2 and MMP-9 protein expression in HCT116 cells. “*” indicates the difffference compared with the control group (P < 0.05), n = 3.

3.5 Arctigenin inhibits tumor growth in vivo in mice

We employed a BALB/c mouse xenograft tumor model to investigate the in vivo anticancer efficacy of arctigenin. Our results indicated that arctigenin treatment significantly reduced the volume and weight of tumors compared to the control group (Fig 5A–5C). Additionally, a decreased Ki-67 level was observed in immunohistochemistry (IHC) staining in the arctigenin group (20 and 40 mg/kg) (Fig 5D and 5E). As shown in Fig 5F, in the model group, nuclear division is easy to see, but necrosis is not obvious. As the dose increases (20mg/kg, 40mg/kg), nuclear division becomes increasingly rare and the necrotic lesion becomes larger. In summary, the model group had strong proliferative ability, while the drug treatment group suppressed the increase. Furthermore, H&E staining results did not reveal any significant adverse reactions of arctigenin on the liver and intestines (Fig 5G and 5H).

[Figure omitted. See PDF.]

A Representative images of tumors in mice treated with arctigenin (20 mg/kg and 40mg/ kg). B,C Tumor volume and tumor weight was reduced in arctigenin-treated mice. D,E Ki-67 IHC staining of xenograft tumors. Original magnifcation 400×. Scale bar = 50 μm. H-scores of the Ki67 staining. F,G,H Representative images of tumor, liver and intestines with H&E staining, Scale bars = 50 μm. n = 5. *^,#p<0.05 vs. the control group.

3.6 Arctigenin inhibits the EMT process in CRC cells

EMT is known to play a critical role in the metastasis of CRC. During this process, the expression of the E-cadherin protein decreases, while the expression of the N-cadherin protein increases in tumor cells. To investigate whether arctigenin had an impact on the EMT process of HCT116 cells, we conducted further experiments. The western blot results revealed that arctigenin increased the expression of E-cadherin protein while decreasing the expression of N-cadherin protein in HCT116 cells compared to the control group (Fig 6A and 6B). The immunofluorescence results further confirmed this finding, showing that arctigenin increased the expression of E-cadherin protein while decreasing the expression of N-cadherin protein (Fig 6C–6F). Additionally, we investigated the expressions of Vimentin, Snail, and Slug proteins, which are key regulatory factors in the EMT process. We found that arctigenin reduced the expressions of these proteins, further demonstrating its inhibitory effect on the EMT process of HCT116 cells (Fig 6G–6I).

[Figure omitted. See PDF.]

A,B Western blot analysis showed that the expression of E-cadherin in HCT116 cells treated with arctigenin was signifcantly higher than that in the control group, while the expression of N-cadherin was lower than that of the control group. C,D,E,F Immunofuorescence staining showed that E-cadherin was upregulated while N-cadherin was downregulated. G,H,I Western blot analysis showed that the expression of Vimentin, Snail and slug were lower than that of the control group. n = 3. *p<0.05 vs. the control group.

3.7 Arctigenin inhibits activation of PI3K/Akt/mTOR signaling pathway

It has been reported that the PI3K/Akt/mTOR signaling pathway plays a crucial role in the EMT process. Therefore, we studied whether arctigenin inhibited the EMT process through the PI3K/Akt/mTOR signaling pathway using western blot. The results showed that arctigenin inhibited the expressions of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR in a dose-dependent manner (Fig 7A, 7D and 7E). Immunofluorescence results also showed that arctigenin inhibited the expression of p-PI3K (Fig 7B and 7C). These findings demonstrated that arctigenin inhibited the activation of the PI3K/Akt/mTOR signaling pathway.

[Figure omitted. See PDF.]

A, D and E Western blot detection of the effffect of arctigenin on p-PI3K, PI3K, p-Akt, Akt, p-mTOR and mTOR protein expression in HCT116 cells. B,C Immunofuorescence staining showed that p-PI3K was downregulated. “*” indicates a difffference compared to the control group (P < 0.05), n = 3.

3.8 Arctigenin exerts anti-tumor effects in CRC by inhibiting the PI3K/Akt/mTOR signaling pathway

In the present study, we found that arctigenin exhibited anti-tumor properties in CRC by inhibiting the PI3K/Akt/mTOR pathway. To verify this finding, we used 740 Y-P (10 μM) as a PI3K activator to increase its expression. Our results indicated that arctigenin at 5 μM reduced the levels of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR. However, after treating the cells with 740 Y-P, the levels of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR were significantly increased (Fig 8A–8C).

[Figure omitted. See PDF.]

A,B,C Western blot analysis showed that after treating the cells with 740 Y-P, the levels of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR were significantly increased. D,E,F,G 740 Y-P could reverse the effect of arctigenin on the proliferation, apoptosis, migration and invasion of HCT116 cells. “*” indicates a difffference compared to the control group (P < 0.05), “#” indicates a difffference compared to the arctigenin group, n = 3.

As depicted in Fig 8D, the CCK-8 assay demonstrated that arctigenin at 5 μM significantly inhibited the proliferation of HCT116 CRC cells. Nevertheless, when given the PI3K activator 740 Y-P, the inhibitory effect of arctigenin on cell proliferation was reversed. In Fig 8E and 8F, the Transwell migration and invasion assays revealed that arctigenin at 5 μM significantly suppressed the migration and invasion of HCT116 cells. Nonetheless, when the cells were treated with the PI3K activator 740 Y-P, the inhibitory effect of arctigenin on cell migration and invasion was significantly reduced. As illustrated in Fig 8G, the flow cytometry results showed that arctigenin at 5 μM could induce apoptosis in HCT116 cells. However, the pro-apoptotic effect of arctigenin was hindered when the cells were treated with the PI3K activator 740 Y-P. Taken together, these results confirmed that arctigenin exerted an anti-tumor effect in CRC by inhibiting the PI3K/Akt/mTOR signaling pathway.

4 Discussion

Natural products have been used to treat many malignant diseases, and a growing body of research has demonstrated the significant role of natural medicines in treating these diseases. Pharmacological studies have confirmed that traditional Chinese medicine has a significant preventive effect on colon cancer, especially on the recurrence of colon cancer in late stage [18,19]. Most patients are diagnosed with advanced-stage CRC, which limits treatment options and has a high rate of postoperative recurrence and metastasis [20–22]. Therefore, there is an urgent need to discover new therapeutic drugs that can play a new role in the treatment of CRC. Arctigenin is an active ingredient derived from burdock, which is widely distributed in China, Korea, and Japan [23]. As an extract of burdock seeds, arctigenin has substantial anticancer potential and has been reported to have a therapeutic effect in treating cancer. Previous studies have confirmed that arctigenin can promote the apoptosis of colorectal cancer cells, but its effect on the proliferation and metastasis of colorectal cancer and the related molecular mechanisms are still unclear.

In our study, we first studied the effects of arctigenin on human colon epithelial cells and three types of colon cancer cells (HCT116, SW620 and DLD-1), and found that arctigenin can inhibit the proliferation of colon cancer cells, with IC50 values of 15.54μM, 17.43μM, and 20.41μM, respectively. In addition, arctigenin did not induce cytotoxicity on human colon epithelial cells. PCNA is a marker protein for tumor cell proliferation [24], and arctigenin inhibited the expression of the PCNA protein. In addition, we verified the effect of arctigenin on apoptosis, and the results were consistent with previous studies that arctigenin promotes apoptosis of CRC cells. At the molecular level, arctigenin increased the expressions of the pro-apoptotic protein Bax and cleaved caspase-3 protein while reducing the expression of the anti-apoptotic protein Bcl2. In addition, we also studied the effect of arctigenin on tumor-bearing nude mice. Compared with the control group, arctigenin effectively inhibited tumor growth, and the tumor volume was significantly decreased than that of the control group. However, we found that the effects of 20mg/kg and 40mg/kg arctigenin on the volume and weight of the tumor were similar, indicating that 20mg/kg arctigenin arctigenin can achieve the maximum inhibitory effect, and beyond 20mg/kg, there is no concentration-dependent effect. H&E staining results showed that arctigenin inhibited tumor growth while having no significant toxicity to the liver. These results were consistent with cell experiments, confirming that arctigenin could inhibit tumor growth while maintaining good safety.

Tumor metastasis is a significant challenge in cancer treatment, and inhibiting the migration and invasion of tumor cells is crucial in cancer therapy [25]. Our research demonstrated that arctigenin could effectively inhibit the migration and invasion of CRC cells. MMPs can break down various extracellular matrix and basement membrane components, promoting tumor metastasis and angiogenesis [26]. Gelatinase activity, particularly MMP-2 and MMP-9, is strongly correlated with the invasive ability of malignant tumors. Our results revealed that arctigenin significantly decreased the expressions of MMP-2 and MMP-9.

The EMT in cancer is linked to increased cell motility and invasiveness, playing a pivotal role in tumor cell metastasis [27]. High expression of N-cadherin among EMT markers is positively correlated with CRC tissue metastasis and lower patient survival rates [28]. The decreased expression of E-cadherin is associated with the invasion of CRC cells and can enhance the resistance of tumor cells to standardized chemotherapy drugs [29]. In addition, Vimentin, Snail, and Slug proteins are mesenchymal markers positively correlated with tumor proliferation, metastasis, and decreased patient survival rates [30]. E-cadherin is an epithelial marker, while N-cadherin, Vimentin, Snail, and Slug proteins are mesenchymal markers. Our results demonstrated that arctigenin inhibited EMT.

The PI3K/AKT/mTOR pathway is a critical player in cell biology and has been identified as a promising target for cancer treatment [31]. In addition to the classical Wnt/β-catenin pathway, the PI3K/AKT signaling pathway has been reported to directly or cooperatively affect EMT and induce tumor invasiveness [32]. As such, the PI3K/AKT signaling pathway is expected to become a potential target for preventing and treating metastatic tumors by mediating the EMT process. Some natural and synthetic small molecules have demonstrated significant potential in suppressing colon cancer by reducing the activity of the PI3K/Akt/mTOR signaling pathway [33,34]. We observed the suppression of PI3K/Akt/mTOR pathway activation upon arctigenin by assessing the protein expression levels associated with this signaling cascade.

5. Conclusion

In summary, our research demonstrated that arctigenin had the ability to inhibit the proliferation, migration, and invasion of CRC cells while promoting apoptosis. This effect was achieved by suppressing the PI3K-AKT-mTOR signaling pathway, as well as inhibiting the EMT process. These findings provided a strong theoretical basis for the potential clinical application of arctigenin as a treatment for CRC.

Supporting information

S1 Data.

https://doi.org/10.1371/journal.pone.0308947.s001

(XLSX)

S1 Raw images.

https://doi.org/10.1371/journal.pone.0308947.s002

References

1. 1. Li J, Huang L, Zhao H, Yan Y, Lu J. The Role of Interleukins in Colorectal Cancer. Int J Biol Sci. 2020;16:2323–2339. pmid:32760201

* View Article

* PubMed/NCBI

* Google Scholar

2. 2. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin. 2020;70:7–30. pmid:31912902

* View Article

* PubMed/NCBI

* Google Scholar

3. 3. Novoa Díaz MB, Martín MJ, Gentili C. Tumor microenvironment involvement in colorectal cancer progression via Wnt/β-catenin pathway: Providing understanding of the complex mechanisms of chemoresistance. World J Gastroenterol. 2022; 28(26):3027–3046.

* View Article

* Google Scholar

4. 4. Zhang Y, Shi J, Huang H, et al. Burden of colorectal cancer in China. Zhonghua Liu Xing Bing Xue Za Zhi. 2015;36(7):709–714.

* View Article

* Google Scholar

5. 5. Li C, Gao S, Li XP et al. Procaine inhibits the proliferation and migration of colon cancer cells through inactivation of the ERK/MAPK/FAK pathways by regulation of RhoA. Oncol Res. 2018;26(2):209–217. pmid:28492141

* View Article

* PubMed/NCBI

* Google Scholar

6. 6. Li G, Zhang C, Liang W et al. Berberine regulates the Notch1/ PTEN/PI3K/AKT/mTOR pathway and acts synergistically with 17-AAG and SAHA in SW480 colon cancer cells. Phram Biol. 2021;59(1):21–30. pmid:33417512

* View Article

* PubMed/NCBI

* Google Scholar

7. 7. Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol. 2014; 15(3):178–196. pmid:24556840

* View Article

* PubMed/NCBI

* Google Scholar

8. 8. Yue B, Song C, Yang L, et al. METTL3-mediated N6-methyladenosine modification is critical for epithelialmesenchymal transition and metastasis of gastric cancer. Mol Cancer. 2019;18(1):142. pmid:31607270

* View Article

* PubMed/NCBI

* Google Scholar

9. 9. Li T, Huang H, Shi G, et al. TGF-β1-SOX9 axis-inducible COL10A1 promotes invasion and metastasis in gastric cancer via epithelial-to-mesenchymal transition. Cell Death Dis. 2018;9(9):849.

* View Article

* Google Scholar

10. 10. Dong J, Wang R, Ren G, et al. HMGA2-FOXL2 axis regulates metastases and epithelial-to-mesenchymal transition of chemoresistant gastric cancer. Clin Cancer Res. 2017;23(13):3461–3473. pmid:28119367

* View Article

* PubMed/NCBI

* Google Scholar

11. 11. Liao H, Zhang L, Lu S, et al. KIFC3 Promotes Proliferation, Migration, and Invasion in Colorectal Cancer viaPI3K/AKT/mTOR Signaling Pathway. Front Genet. 2022;13:848926. pmid:35812733

* View Article

* PubMed/NCBI

* Google Scholar

12. 12. Shen J, Zhai J, You Q, et al. Cancer-associated fibroblasts-derived VCAM1 induced by H. pylori infection facilitates tumor invasion in gastric cancer. Oncogene. 2020;39(14):2961–2974. pmid:32034307

* View Article

* PubMed/NCBI

* Google Scholar

13. 13. He Y, Fan Q, Cai T, et al. Molecular mechanisms of the action of Arctigenin in cancer. Biomed Pharmacother. 2018;108:403–407. pmid:30236849

* View Article

* PubMed/NCBI

* Google Scholar

14. 14. Zheng JLi Q, Wang Wet al. Apoptosis-related protein-1 acts as a tumor suppressor in cholangiocarcinoma cells by inducing cell cycle arrest viadownregulation of cyclin-dependent kinase subunits. Oncol. Rep. 2016;35(2):809–816. pmid:26572808

* View Article

* PubMed/NCBI

* Google Scholar

15. 15. Zhang M, Cai S, Zuo B, Gong W, Tang Z, Zhou D, et al. Arctigenin induced gallbladder cancer senescence through modulating epidermal growth factor receptor pathway. Tumour Biol. 2017; 39(5): 1010428317698359. pmid:28459363

* View Article

* PubMed/NCBI

* Google Scholar

16. 16. Li QC, Liang Y, Tian Y, et al. Arctigenin induces apoptosis in colon cancer cells through ROS/p38MAPK pathway. J BUON. 2016;21(1):87–94. pmid:27061535

* View Article

* PubMed/NCBI

* Google Scholar

17. 17. Feng P, Zhu L, Jie J, et al. Cannabidiol inhibits invasion and metastasis in colorectal cancer cells by reversing epithelial-mesenchymal transition through the Wnt/β-catenin signaling pathway. J Cancer Res Clin Oncol. 2023;149(7):3587–3598.

* View Article

* Google Scholar

18. 18. Banik K, Ranaware AM, Deshpande V, et al. Honokiol for cancer therapeutics: A traditional medicine that can modulate multiple oncogenic targets. Pharmacol Res. 2019;144:192–209. pmid:31002949

* View Article

* PubMed/NCBI

* Google Scholar

19. 19. Sun X, Ng TTH, Sham KWY, et al. Bufalin,a Traditional Chinese Medicine Compound, Prevents Tumor Formation in Two Murine Models of Colorectal Cancer. Cancer Prev Res (Phila). 2019;12(10):653–666. pmid:31431500

* View Article

* PubMed/NCBI

* Google Scholar

20. 20. Zhou G, Yang J, Song P. Correlation of ERK/MAPK Signaling Pathway With Proliferation and Apoptosis of Colon Cancer Cells. Oncol Lett. 2019;17 (2):2266–2270. pmid:30675292

* View Article

* PubMed/NCBI

* Google Scholar

21. 21. Hao T, Xu J, Fang S, et al. Overexpression of ZNF460 Predicts Worse Survival and Promotes Metastasis through JAK2/STAT3 Signaling Pathway in Patient with Colon Cancer. J. Cancer. 2021;12 (11). pmid:33976729

* View Article

* PubMed/NCBI

* Google Scholar

22. 22. Li X, Wen D, Li X, et al. Identification of an Immune Signature Predicting Prognosis Risk and Lymphocyte Infiltration in Colon Cancer. Front Immunol. 2020;11, 1678. pmid:33013820

* View Article

* PubMed/NCBI

* Google Scholar

23. 23. Fang R, Cui Q, Sun J, et al. PDK1/Akt/PDE4D axis identifified as a target for asthma remedy synergistic with beta2 AR agonists by a natural agent arctigenin. Allergy. 2015; 70:1622–1632.

* View Article

* Google Scholar

24. 24. Thomas FM, Sudi S, Muhamad Salih FA, et al. Modulation of POPDC1 Expression by Phenothiazine and Trifluoperazine Suppress Colon Cancer Growth and Migration. Asian Pac J Cancer Prev. 2022;23(8):2863–2871. pmid:36037145

* View Article

* PubMed/NCBI

* Google Scholar

25. 25. Jiang YA, Liu JY, Hong WW, et al. Arctigenin Inhibits Glioblastoma Proliferation through the AKT/mTOR Pathway and Induces Autophagy. Biomed Res Int. 2020; 2020: 3542613. pmid:33015162

* View Article

* PubMed/NCBI

* Google Scholar

26. 26. AF Chambers LM Matrisian. Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst. 1997;89: 1260–1270.

* View Article

* Google Scholar

27. 27. Brabletz T, Kalluri R, Nieto MA, et al. EMT in cancer. Nat Rev Cancer. 2018;18:128–134. pmid:29326430

* View Article

* PubMed/NCBI

* Google Scholar

28. 28. Sebastian A, Pandey V, Mohan CD, et al. Novel adamantanyl-based thiadiazolyl pyrazoles targeting EGFR in triple-negative breast cancer. ACS Omega. 2016;1(6):1412–1424. pmid:30023509

* View Article

* PubMed/NCBI

* Google Scholar

29. 29. Lee J, Mohan CD, Deivasigamani A, et al. Brusatol suppresses STAT3-driven metastasis by downregulating epithelial- mesenchymal transition in hepatocellular carcinoma. Journal of Advanced Research. 2020;26:83–94. pmid:33133685

* View Article

* PubMed/NCBI

* Google Scholar

30. 30. Satelli A, Li S. Vimentin in cancer and its potential as a molecular target for cancer therapy. Cellular and Molecular Life Sciences: CMLS. 2011;68(18):3033–3046.

* View Article

* Google Scholar

31. 31. Mannella V, Boehm K, Celik S, et al. Growth and Viability of Cutaneous Squamous Cell Carcinoma Cell Lines Display Different Sensitivities to Isoform-specific Phosphoinositide 3-Kinase Inhibitors. Int J Mol Sci. 2021;22 (7):3567. pmid:33808215

* View Article

* PubMed/NCBI

* Google Scholar

32. 32. Xu W, Yang Z, Lu N. A new role for the PI3K/Akt signaling pathway in the epithelial-mesenchymal transition. Cell adhesion migration. 2015;9(4):317–24. pmid:26241004

* View Article

* PubMed/NCBI

* Google Scholar

33. 33. Narayanankutty A. PI3K/ Akt/ mTOR Pathway as a therapeutic target for colorectal cancer: a review of preclinical and clinical evidence. Curr Drug Targets. 2019;20(12):1217–1226. pmid:31215384

* View Article

* PubMed/NCBI

* Google Scholar

34. 34. Zhu ML, Zhang PM, Jiang M, et al. Myricetin induces apoptosis and autophagy by inhibiting PI3K/Akt/mTOR signalling in human colon cancer cells. 2020;20(1):209. pmid:32631392

* View Article

* PubMed/NCBI

* Google Scholar

Citation: Chen X-F, Liu P-G, Sheng N, Li X-S, Hu R-K, Zhu L-X, et al. (2024) Arctigenin inhibits the progression of colorectal cancer through epithelial-mesenchymal transition via PI3K/Akt/mTOR signaling pathway. PLoS ONE 19(9): e0308947. https://doi.org/10.1371/journal.pone.0308947

About the Authors:

Xiang-Fan Chen

Contributed equally to this work with: Xiang-Fan Chen, Pei-Gen Liu

Roles: Data curation

Affiliation: Biological Sample Bank, Afliated Hospital 2 of Nantong University, Nantong, China

Pei-Gen Liu

Contributed equally to this work with: Xiang-Fan Chen, Pei-Gen Liu

Roles: Data curation

Affiliation: Department of General Surgery, Central Hospital of Panzhihua City, Panzhihua, Sichuan Province, China

Nan Sheng

Roles: Data curation

Affiliation: Department of Clinical Laboratory, Afliated Hospital 2 of Nantong University, Nantong, China

Xin-Shuai Li

Roles: Data curation

Affiliation: Department of Pharmacy, Afliated Hospital 2 of Nantong University, Nantong, China

Rui-Kun Hu

Roles: Data curation

Affiliation: Personnel Department, Affiliated Maternity and Child Health Care Hospital of Nantong University, Nantong, China

Long-Xun Zhu

Roles: Data curation

E-mail: [email protected] (PF); [email protected] (L-XZ)

Affiliation: Department of Pharmacy, Afliated Hospital 2 of Nantong University, Nantong, China

Panfeng Feng

Roles: Conceptualization, Data curation

E-mail: [email protected] (PF); [email protected] (L-XZ)

Affiliation: Department of Pharmacy, Afliated Hospital 2 of Nantong University, Nantong, China

ORICD: https://orcid.org/0000-0001-7817-7971

References

1. Li J, Huang L, Zhao H, Yan Y, Lu J. The Role of Interleukins in Colorectal Cancer. Int J Biol Sci. 2020;16:2323–2339. pmid:32760201

2. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin. 2020;70:7–30. pmid:31912902

3. Novoa Díaz MB, Martín MJ, Gentili C. Tumor microenvironment involvement in colorectal cancer progression via Wnt/β-catenin pathway: Providing understanding of the complex mechanisms of chemoresistance. World J Gastroenterol. 2022; 28(26):3027–3046.

4. Zhang Y, Shi J, Huang H, et al. Burden of colorectal cancer in China. Zhonghua Liu Xing Bing Xue Za Zhi. 2015;36(7):709–714.

5. Li C, Gao S, Li XP et al. Procaine inhibits the proliferation and migration of colon cancer cells through inactivation of the ERK/MAPK/FAK pathways by regulation of RhoA. Oncol Res. 2018;26(2):209–217. pmid:28492141

6. Li G, Zhang C, Liang W et al. Berberine regulates the Notch1/ PTEN/PI3K/AKT/mTOR pathway and acts synergistically with 17-AAG and SAHA in SW480 colon cancer cells. Phram Biol. 2021;59(1):21–30. pmid:33417512

7. Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol. 2014; 15(3):178–196. pmid:24556840

8. Yue B, Song C, Yang L, et al. METTL3-mediated N6-methyladenosine modification is critical for epithelialmesenchymal transition and metastasis of gastric cancer. Mol Cancer. 2019;18(1):142. pmid:31607270

9. Li T, Huang H, Shi G, et al. TGF-β1-SOX9 axis-inducible COL10A1 promotes invasion and metastasis in gastric cancer via epithelial-to-mesenchymal transition. Cell Death Dis. 2018;9(9):849.

10. Dong J, Wang R, Ren G, et al. HMGA2-FOXL2 axis regulates metastases and epithelial-to-mesenchymal transition of chemoresistant gastric cancer. Clin Cancer Res. 2017;23(13):3461–3473. pmid:28119367

11. Liao H, Zhang L, Lu S, et al. KIFC3 Promotes Proliferation, Migration, and Invasion in Colorectal Cancer viaPI3K/AKT/mTOR Signaling Pathway. Front Genet. 2022;13:848926. pmid:35812733

12. Shen J, Zhai J, You Q, et al. Cancer-associated fibroblasts-derived VCAM1 induced by H. pylori infection facilitates tumor invasion in gastric cancer. Oncogene. 2020;39(14):2961–2974. pmid:32034307

13. He Y, Fan Q, Cai T, et al. Molecular mechanisms of the action of Arctigenin in cancer. Biomed Pharmacother. 2018;108:403–407. pmid:30236849

14. Zheng JLi Q, Wang Wet al. Apoptosis-related protein-1 acts as a tumor suppressor in cholangiocarcinoma cells by inducing cell cycle arrest viadownregulation of cyclin-dependent kinase subunits. Oncol. Rep. 2016;35(2):809–816. pmid:26572808

15. Zhang M, Cai S, Zuo B, Gong W, Tang Z, Zhou D, et al. Arctigenin induced gallbladder cancer senescence through modulating epidermal growth factor receptor pathway. Tumour Biol. 2017; 39(5): 1010428317698359. pmid:28459363

16. Li QC, Liang Y, Tian Y, et al. Arctigenin induces apoptosis in colon cancer cells through ROS/p38MAPK pathway. J BUON. 2016;21(1):87–94. pmid:27061535

17. Feng P, Zhu L, Jie J, et al. Cannabidiol inhibits invasion and metastasis in colorectal cancer cells by reversing epithelial-mesenchymal transition through the Wnt/β-catenin signaling pathway. J Cancer Res Clin Oncol. 2023;149(7):3587–3598.

18. Banik K, Ranaware AM, Deshpande V, et al. Honokiol for cancer therapeutics: A traditional medicine that can modulate multiple oncogenic targets. Pharmacol Res. 2019;144:192–209. pmid:31002949

19. Sun X, Ng TTH, Sham KWY, et al. Bufalin,a Traditional Chinese Medicine Compound, Prevents Tumor Formation in Two Murine Models of Colorectal Cancer. Cancer Prev Res (Phila). 2019;12(10):653–666. pmid:31431500

20. Zhou G, Yang J, Song P. Correlation of ERK/MAPK Signaling Pathway With Proliferation and Apoptosis of Colon Cancer Cells. Oncol Lett. 2019;17 (2):2266–2270. pmid:30675292

21. Hao T, Xu J, Fang S, et al. Overexpression of ZNF460 Predicts Worse Survival and Promotes Metastasis through JAK2/STAT3 Signaling Pathway in Patient with Colon Cancer. J. Cancer. 2021;12 (11). pmid:33976729

22. Li X, Wen D, Li X, et al. Identification of an Immune Signature Predicting Prognosis Risk and Lymphocyte Infiltration in Colon Cancer. Front Immunol. 2020;11, 1678. pmid:33013820

23. Fang R, Cui Q, Sun J, et al. PDK1/Akt/PDE4D axis identifified as a target for asthma remedy synergistic with beta2 AR agonists by a natural agent arctigenin. Allergy. 2015; 70:1622–1632.

24. Thomas FM, Sudi S, Muhamad Salih FA, et al. Modulation of POPDC1 Expression by Phenothiazine and Trifluoperazine Suppress Colon Cancer Growth and Migration. Asian Pac J Cancer Prev. 2022;23(8):2863–2871. pmid:36037145

25. Jiang YA, Liu JY, Hong WW, et al. Arctigenin Inhibits Glioblastoma Proliferation through the AKT/mTOR Pathway and Induces Autophagy. Biomed Res Int. 2020; 2020: 3542613. pmid:33015162

26. AF Chambers LM Matrisian. Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst. 1997;89: 1260–1270.

27. Brabletz T, Kalluri R, Nieto MA, et al. EMT in cancer. Nat Rev Cancer. 2018;18:128–134. pmid:29326430

28. Sebastian A, Pandey V, Mohan CD, et al. Novel adamantanyl-based thiadiazolyl pyrazoles targeting EGFR in triple-negative breast cancer. ACS Omega. 2016;1(6):1412–1424. pmid:30023509

29. Lee J, Mohan CD, Deivasigamani A, et al. Brusatol suppresses STAT3-driven metastasis by downregulating epithelial- mesenchymal transition in hepatocellular carcinoma. Journal of Advanced Research. 2020;26:83–94. pmid:33133685

30. Satelli A, Li S. Vimentin in cancer and its potential as a molecular target for cancer therapy. Cellular and Molecular Life Sciences: CMLS. 2011;68(18):3033–3046.

31. Mannella V, Boehm K, Celik S, et al. Growth and Viability of Cutaneous Squamous Cell Carcinoma Cell Lines Display Different Sensitivities to Isoform-specific Phosphoinositide 3-Kinase Inhibitors. Int J Mol Sci. 2021;22 (7):3567. pmid:33808215

32. Xu W, Yang Z, Lu N. A new role for the PI3K/Akt signaling pathway in the epithelial-mesenchymal transition. Cell adhesion migration. 2015;9(4):317–24. pmid:26241004

33. Narayanankutty A. PI3K/ Akt/ mTOR Pathway as a therapeutic target for colorectal cancer: a review of preclinical and clinical evidence. Curr Drug Targets. 2019;20(12):1217–1226. pmid:31215384

34. Zhu ML, Zhang PM, Jiang M, et al. Myricetin induces apoptosis and autophagy by inhibiting PI3K/Akt/mTOR signalling in human colon cancer cells. 2020;20(1):209. pmid:32631392