Sophoridine Inhibits Human Colorectal Cancer Progression via Targeting MAPKAPK2

Colorectal cancer is one of the most frequent causes of cancer-related morbidity and mortality globally (1–3). Despite the benefits of high-quality early screening and detection, surgery, and new chemotherapeutic agents for improving the treatment of advanced and metastatic colorectal cancers, the 5-year survival rate for advanced colorectal cancers is less than 10% (4). There is still a lack of optimal treatment strategies for colorectal cancers. Currently, anti-EGFR agents (bevacizumab, ramucirumab, regorafenib, ziv-aflibercept) are used in combination with chemotherapy as a standard of care for the first-line therapy of metastatic colorectal cancer (5–7). Although these agents are designed to restrain tumor-selective proliferation, they can cause serious toxic effects, affect normal tissues, and sometimes severely interfere with therapeutic success and the quality of life of patients (8). Therefore, the development of novel, effective treatment approaches is urgently needed to improve clinical outcomes of colorectal cancer patients.

Traditional Chinese medicine (TCM) has a long history of application and a significant contribution to modern medicine (9, 10). As important sources of active natural products, TCM shows unique advantages (11–13). Radix Sophorae Flavescentis (the dried roots of Sophora Flavescens Ait) is widely used in China, Japan, and some European countries for its various physiologic functions (14, 15). It contains several major effective components such as Matrine, Sophoridine, Oxymatrine, etc. Sophoridine, an active quinolizidine alkaloid compound, displays various biological properties such as anticancer activity, antiviral activity, antifibrotic activity, antimicrobial activity, antiinflammatory activity, etc. (16–19). In particular, recent studies have revealed that it exhibits potent anticancer effects in different tumor cell lines and animal models (20); however, the exact underlying mechanism of the anticancer effect of Sophoridine is remaining unclear.

In this study, we screened the pharmacokinetic parameters of the main chemical constituents of Radian Sophorae flavescentis by Traditional Chinese Medicine Systems Pharmacology (TCMSP) Database and Analysis Platform database and found that Sophoridine is one of the best antitumor active ingredients. We have identified that Sophoridine is an effective inhibitor of MAPKAPK2 by directly interacting with the ATP site of MAPKAPK2, leading to the repression of multiple oncogenic processes in colorectal cancers. Clinical studies have shown that a higher level of MAPKAPK2 is associated with a poorer percent survival rate (prognosis). Furthermore, a higher level of MAPKAPK2 was positively associated with the enrichment of downregulation of apoptosis and autophagy by gene set enrichment analysis (GSEA), as well as upregulation of proliferation and cell-cycle arrest.

Taken all together, these results show that Sophoridine may be recognized as an attractive drug candidate by targeting MAPKAPK2 for colorectal cancers therapy.

Human colorectal cancer cell lines HCT116, RKO, and SW480 were obtained from Cell Resource Center of the Chinese Academy of Sciences in 2017 (CAMS, PUMC, Beijing, China). The cells being used were used within 1 month after resuscitation (passage number between 9 and 30). The cell lines were identified using a short tandem repeat analysis and tested for mycoplasma using MycoAlert (Lonza) in these cell lines. All cell lines were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified atmosphere under 5% CO₂ at 37°C. Sophoridine (purity ≥98%) was purchased from Shanghai Yuanye Bio-Technology and dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mmol/L stock solution for storage at −20°C.

Cell viability was measured using CCK-8 assay (Dojindo). Human colorectal cancer cells were seeded into flat-bottom 96-well plates (5 × 10³ cells/well) and treated with Sophoridine at indicated concentrations for another 48 hours after plating for 24 hours. Subsequently, medium was discarded and added solution of cell counting to each well, followed by 1 hour of incubation. The absorbance was detected at 450 nm using a microplate reader.

Human colorectal cancer cells (1 × 10³) were seeded into 6-well plates. After 24 hours, cells were treated with Sophoridine at the indicated concentrations for 48 hours. Cells were then cultured in fresh medium for another week. Colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Photographs were acquired in indicated time periods, and the cell numbers were counted.

Cells were seeded in 24-well plates and treated with Sophoridine at indicated concentrations for 48 hours. The cells were washed in cold PBS and then fixed with 4% paraformaldehyde followed by 5 minutes of permeabilization with 0.1% Triton X-100. Then we blocked with 1% BSA containing 1% goat serum for 30 minutes. After incubation with anti-LC3 overnight at 4°C, cells were exposed to corresponding secondary antibodies for 1 hour at room temperature, and then stained with DAPI (4′,6-diamidino-2-phenylindole). Cells were observed by confocal laser scanning microscopy and quantified manually the acquired images with Image J software.

Standard Western blot analysis was performed as previously described (21, 22). Antibodies against SQSTM1/p62 (D5E2), Bax (D2E11), Bcl-2, and Cyclin D1 were purchased from Cell Signaling Technology. Antibodies against p27, LC-3, and MAPKAPK2 were purchased from Proteintech. β-Actin and Phospho-MAPKAPK2 (Thr222) antibodies were purchased from ABClonal. Full scans of Western blot assays are shown in Supplementary Figs. S4 to S7.

Expression vector of human MAPKAPK2 was designed and purchased from Genechem. We transfected with 0.8 mg of DNA construct using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

Colorectal cancer cells were treated with sop for 48 hours. The cells were collected with EDTA-free trypsin and washed with ice-cold PBS for 2 times. For cell apoptosis analysis, all cells were resuspended with binding buffer and incubated with Annexin V–FITC and propidium iodide (BD Biosciences) according to the manufacturer's instructions. In a limited time, the percentages of apoptotic cells were analyzed using flow cytometry (Attune NxT; Invitrogen). For cell-cycle analysis, harvested cells were fixed in 75% ethanol overnight at −20°C. On the following day, the cells were recovered by centrifugation and washed in cold PBS. Thereafter, they were incubated in 0.5 mL PI/RNase staining Solution (Invitrogen) in the dark at room temperature for 30 minutes. Cell cycle was determined and analyzed by flow cytometry.

Total RNA was extracted as previously described (23). RNA quantity and purity were determined by using a NanoDrop 2000 (Thermo Scientific). Total RNA was reverse transcribed with HiFiScript cDNA Synthesis Kit (Cowin Biotech). Then, real-time PCR was performed in triplicate with UltraSYBR mixture (Cowin Biotech) using 7500 RT-PCR System (Applied Biosystems, Life Technologies). The expression of genes was normalized to the Actin gene. The primers used are listed in the Supplementary Table S1.

Cellular thermal shift assays (CETSA) were performed to determine the direct binding between Sophoridine and MAPKAPK2 in cellular. Colorectal cancer cells were pretreated with DMSO or Sophoridine for 48 hours, chilled on ice, washed with PBS plus protease inhibitor cocktail, and then collected and heated for 3 minutes at appropriate temperature. Subsequently, cells were lysed and proteins were separated, and the corresponding index was determined by Western blot assays.

The drug affinity responsive targets stability (DARTS) assay was conducted as described above (24, 25). To prepare DARTS samples, 1 × 10⁷ colorectal cancer cells were lysed in 2.4 mL M-PER buffer with protease inhibitors, centrifuged, collected proteins, and then added 10 × TNC buffer. Lysates were equally divided into two parts for 1 hour at room temperature with DMSO or Sophoridine, and incubated with 1 mg/mL pronase at room temperature for 5 minutes. The reaction was stopped by adding protease inhibitors, and samples were stored at −20°C standby.

Docking simulations were operated using the DiscoveryStudio 2017 R2 molecular modeling software. The three-dimensional (3D) structures of the Sophoridine molecule were generated with ChemDraw and were energy minimized with CHARMm force field. The initial 3D geometric coordinates of MAPKAP kinase 2 (PDBcode: 2JBO) were obtained from the Protein Databank (PDB). Then, the protein structure was prepared by removing water molecules and adding hydrogen. CDOCKER protocols were employed as docking approaches and calculated the predicted binding energy (kcal mol⁻¹). The complex structure with the most favorable binding-free energies was selected as the optimal docked conformation for later experimental verification.

Clinical data can be obtained via the publically available The Cancer Genome Atlas and Gene Expression Omnibus (GEO) datasets. The expression level of MAPKAPK2 in colorectal cancer patients was analyzed by Kaplan–Meier estimate. GSEA was used to identify the association of MAPKAPK2 expression with biological processes of colorectal cancer cells by GSEA 3.0 software (http://www.broadinstitute.org/gsea/).

The data were represented as mean ± SD. Two-tailed unpaired Student t test was used for comparing two groups of data. One-ANOVA was used to compare multiple groups of data. Survival analysis was determined using the Kaplan–Meier estimates and the log-rank test. The variation (P < 0.05) was considered statistically significant.

The photo of Radian Sophorae flavescentis is shown in Fig. 1A. The pharmacokinetic parameters of the main chemical constituents of Radian Sophorae flavescentis were screened by TCMSP database in Supplementary Table S2, and Sophoridine is one of the best antitumor active ingredients. As shown in Supplementary Fig. S1A and Supplementary Table S3, the pharmacokinetics properties were elucidated of Sophoridine in rat by high performance liquid chromatography. The result could provide meaningful reference for further clinical medication of Sophoridine. The chemical structure of Sophoridine is shown in Fig. 1B. As shown in Fig. 1C, clonogenicity of colorectal cancer cell lines (RKO, SW480, and HCT116) was dramatically reduced by Sophoridine for 48 hours. The CCK8 assay was used to detect the cytotoxic effects of Sophoridine against colorectal cancer cell lines and two nontumorigenic cell lines (HKC and LX-2). Consistently, as evidenced by decreased cell viability, Sophoridine strongly inhibited cell proliferation in colorectal cancer cells (Fig. 1D–F). However, Sophoridine did not affect the cell viability of HKC and LX-2 cells (Supplementary Fig. S1B and S1C). In vivo, the indexes had no statistical difference of routine blood test and serum biochemical measurements, compared with the control group, in Supplementary Tables S4 and S5, and no obvious abnormalities in histopathology after the Sophoridine administration in mice (Supplementary Fig. S1D). These results suggest that Sophoridine showed no obvious drug toxicity under conditions of potent antitumor efficacy. To investigate cell apoptosis regulation effect of Sophoridine on colorectal cancer cells, we performed Western blot assays. As shown in Fig. 1G, after exposure to Sophoridine, colorectal cancer cells showed a downregulation of Bcl-2 and an upregulation of Bax, which result in a dose-dependent increase of the ratio of Bax/Bcl-2. The apoptotic effects were further confirmed by employing Annexin V staining by treating colorectal cancer cells with Sophoridine (Fig. 1H). Taken together, these results suggest that Sophoridine suppresses growth and induces apoptosis of colorectal cancer cells.

To investigate cell-cycle arrest promotion effect of Sophoridine on colorectal cancer cells, we performed Western blot assays. As indicated in Fig. 2A and B, Sophoridine significantly reduced the expression of Cyclin D1, while markedly increased the expression of p27 in a dose-dependent manner. The cell-cycle arrest effects were further confirmed by employing flow cytometry detection. As shown in Fig. 2C, Sophoridine significantly raised cell number at G₀–G₁ phase after 48-hour exposure, accompanied by decreased cell number at G₂–M phase. Moreover, we also explored the protein levels of classical autophagy markers by Western blot assays and immunofluorescence staining. As shown in Fig. 2D–F, Sophoridine markedly reduced the expression of p62, whereas dramatically increased the expression of LC-3B puncta dose-dependently. Collectively, these results indicate that Sophoridine induces cell-cycle arrest and promotes autophagy of colorectal cancer cells.

To investigate potential targets of Sophoridine in colorectal cancer cells, 116 pharmacophore candidates were predicted via pharmMapper (http://www.lilab-ecust.cn/pharmmapper/). The ranked list of hit target pharmacophore models is sorted by normalized fit score in descending order (Supplementary Table S6), and the top ten were displayed in Table 1. To improve the specificity, 3,298 colorectal cancer–associated genes were retrieved from the disGeNET (http://www.disgenet.org) database (Supplementary Table S7). A total of 67 potential targets of Sophoridine identified in colorectal cancer–associated genes were selected for constructing the drug-target (D-T) network (Fig. 3A). Thus, we examined changes in the transcriptional levels of these genes in colon cancer cells after treatment with Sophoridine. As shown in Fig. 3B, Sophoridine can significantly inhibit the expression of MAPK14, BRAF, FGFR1, and other genes. To make a deep exploring of the action mechanism of Sophoridine in colorectal cancer cells, we used String (https://string-db.org/) to obtain protein interactions and then constructed protein–protein interaction (PPI) network of genes associated with drug mediated by Cytoscape 3.2.1 (Fig. 3C), and the key topological parameter degree was analyzed. The biological functions of these potential targets were performed by Cytoscape plugin, ClueGO. These results suggest that these genes are involved in the development of various cancers and are closely related to MAPK VENTS, signaling through FGFR, VEGF pathways, etc. (Fig. 3D). Based on the above analysis, we predict that Sophoridine can inhibit the development of colorectal cancer cells by targeting MAPKAPK2.

To detect whether MAPKAPK2 is a direct target of Sophoridine, we employed the CETSAs. As shown in Fig. 4A–C, Sophoridine treatment significantly shifted the MAPKAPK2 melting curve compared with control. Moreover, our DARTS data suggested that Sophoridine binds to MAPKAPK2, protecting it from proteolytic cleavage (Fig. 4D). To search the promising target for binding mode of Sophoridine in MAPKAPK2, molecular docking simulation experiments were performed between Sophoridine and MAPKAPK2 by employing Discovery Studio 2017 R2 software. The CDOCKER docking result revealed that Sophoridine can bound into the ATP site of MAPKAP kinase 2 (PDBcode: 2JBO), and extend into the sub pockets for the adenine moiety and the α-phosphate, surrounded by key residues (LEU193, LEU70, ALA91, VAL78, LYS93), thus blocking the ATP-binding site fully (Fig. 4E–G). Furthermore, to further evaluate the MAPKAPK2 inhibitory effect, we detected the constitutive activation of MAPKAPK2 in colorectal cancer cells by the specific antibodies against phospho-MAPKAPK2 Thr222. As shown in Fig. 4H and Supplementary Fig. S2A, Sophoridine significantly reduced the phosphorylation level of MAPKAPK2 (Thr222) and MAPKAPK2 activity, but there was no big difference in its total expression in colorectal cancer cells. Interestingly, Sophoridine did not affect the p38 (upstream activators of MAPKAPK2) activity and significantly reduced the docking interaction of p38-MAPKAPK2 in Supplementary Fig. S2B and S2C.

Next, we found that MAPKAPK2 overexpression in colorectal cancer cells strongly attenuated the inhibitory effect of Sophoridine on colony formation and cell viability (Fig. 5A–C). In addition, ectopic MAPKAPK2 expression dramatically recovered MAPKAPK2-regulated cell-cycle arrest and MAPKAPK2-induced apoptosis and autophagy (Fig. 5D–I). Moreover, we found that knockdown of endogenous MAPKAPK2 by siRNA has a similar effect with Sophoridine-regulated cell-cycle arrest, apoptosis, and autophagy. Furthermore, knockdown of endogenous MAPKAPK2 further enhanced the anticolorectal cancer effect of Sophoridine (Supplementary Fig. S3A–S3D). Taken together, these results indicate that Sophoridine promotes apoptosis and autophagy and induces cell-cycle arrest through targeting MAPKAPK2.

To further investigate the clinical outcome of MAPKAPK2 in colorectal cancer patient, we subjected them to Kaplan–Meier analysis in GEO data set. Data revealed that higher MAPKAPK2 expression was associated with poorer percent disease-specific survival (DSS) (GSE17536, P = 0.020, Fig. 6A), disease-free survival (DFS) (GSE17536, P = 0.039, Fig. 6B), and overall survival (OS) (GSE17536, P = 0.025, Fig. 6C). Moreover, GEO database revealed that MAPKAPK2 level is high in colorectal cancer tissue compared with that in normal colon tissue (GSE110225, P = 0.043, Fig. 6D). Furthermore, according to the level of MAPKAPK2 from GSE17536, we estimated that higher level of MAPKAPK2 was positively correlated with enrichment of downregulation of apoptosis and autophagy by GSEA, as well as upregulation of proliferation and cell-cycle arrest (Fig. 6E–H). Taken together, these results indicate that MAPKAPK2 may be a prognosis marker in colorectal cancer patients.

Collectively, our results show that the MAPKAPK2 plays an important role in Sophoridine-regulated apoptosis, autophagy, and cell-cycle arrest in colorectal cancer.

Sophoridine, an active quinolizidine alkaloid compound, has been proven to possess extensive physiologic activities (16, 17). Previous studies have revealed that Sophoridine displays prominent anticancer biological effects (20). However, the underlying molecular mechanisms are still elucidated. In this study, we have illustrated that Sophoridine promotes apoptosis and autophagy and induces cell-cycle arrest via targeting MAPKAPK2, leading to the blocking of the growth and development of colorectal cancers.

Network pharmacology is one of the strategies for discovering new drugs (26–28). In recent years, it has played an increasingly important role in the research and development of new drugs, such as target identification, mechanism of action, discovery and optimization of drug lead, and preclinical efficacy and safety evaluation (29–31). Network pharmacology is often studied by integrating multidisciplinary molecular networks, such as chemical informatics, bioinformatics, and systems biology (32, 33). Radix Sophorae Flavescentis is very commonly used in China, Japan, and some European countries for its various physiologic functions (17). Sophoridine is one of major bioactive components from Radix Sophorae Flavescentis. Previous studies have identified Sophoridine contains several biological properties (16, 20). In this study, we predicted 116 pharmacophore candidates via PharmMapper (http://lilab.ecust.edu.cn/pharmmapper) and found 67 potentially targets of Sophoridine from 3,298 colorectal cancer–associated genes, and then we constructed the D-T network through these targets. Moreover, we have found that Sophoridine can significantly inhibit the expression of MAPK14, BRAF, FGFR1, and other genes by real-time PCR. Furthermore, we used String (https://string-db.org/) to obtain protein interactions and then constructed PPI network of genes associated with drug mediated by Cytoscape 3.2.1. And we analyzed the key topological parameter degree and performed the biological functions of these potential targets by Cytoscape plugin, ClueGO. Taken together, the results suggest that these genes are involved in the development of various cancers and are closely related to MAPK EVENTS, signaling through FGFR, VEGF pathways, etc., and we predicted that MAPKAPK2 may target colorectal cancer cells involved in the treatment with Sophoridine.

MAPKAPK2, also called MK2, is known to be acted as a downstream signaling protein of p38MAPK and regulates a cascade of critical biological processes including inflammatory responses, nuclear export, stress, and DNA damage (34). Depending on these processes, MAPKAPK2 regulates transcript stability, expression of diverse proteins, and the phosphorylation involved in numerous important cellular phenomenon, such as cell cycle, senescence, cell migration, cell proliferation, and apoptosis (35–37). Systemic side effects are a major obstacle to the conversion of developed p38MAPK inhibitors into successful new drugs. This is the foremost cause of failure in the clinical trials. To solve this problem and effectively inhibit p38MAPK, researchers turned their focus to many of its downstream targets, such as MAPKAPK2. Previous studies have shown that targeting MAPKAPK2 to interdict its downstream events is as good as direct upstream inhibition of the p38MAPK pathway without obvious side effects of p38MAPK inhibitors (38–40). In the present study, we have demonstrated a direct binding between MAPKAPK2 and Sophoridine by DARTS assay and CETSA. Furthermore, we have found that Sophoridine selectively inactivates phospho-MAPKAPK2 (Thr222) and directly binds into the ATP site of MAPKAPK2 by molecular docking. The blockage effects are further determined by Western blot: Sophoridine markedly reduces phospho-MAPKAPK2 (Thr222) in a dose-dependent manner, but there is no obvious deference in its total expression in colorectal cancer cells.

Past reports have demonstrated the expression of MAPKAPK2 in a multitude of cell types like cancers, smooth muscle cells, and endothelial cells (41–44). A recent study has elucidated that MAPKAPK2 plays a key role in colon cancer processes via axis inhibition of Hsp27, which finally results in promoting cell angiogenesis, migration, survival, and proliferation (35). Literature reports have shown that deletion of MAPKAPK2 conduces to DNA damage and apoptosis through impaired phosphorylation of MDM2 and subsequently enhances the primary regulator of p53 stability in skin cancer (45). Past studies have reported that MAPKAPK2 promotes the invasion and metastasis via regulation of MMP-2/9 mRNA half-life in bladder cancer (46). In the present study, we have found that ectopic MAPKAPK2 expression significantly blocks Sophoridine-regulated apoptosis, autophagy, and cell-cycle arrest of colorectal cancer. Clinical studies have shown that MAPKAPK2 expression is associated with poor prognosis. Furthermore, we estimated that a higher level of MAPKAPK2 was positively associated with the enrichment of downregulation of apoptosis and autophagy by GSEA, as well as upregulation of proliferation and cell-cycle arrest. Collectively, our results demonstrate that the MAPKAPK2 plays a major role in Sophoridine-regulated tumorigenesis in colorectal cancers.

In summary, we have screened the pharmacokinetic parameters of the main chemical constituents of Radian Sophorae flavescentis by TCMSP database and have found that Sophoridine is one of the best antitumor active ingredients. Our study elucidates that Sophoridine induces apoptosis, autophagy, and cell-cycle arrest through targeting MAPKAPK2, which leads to inhibiting the tumor development and progression of colorectal cancers. These studies show that Sophoridine may be a promising therapeutic strategy that blocks tumorigenesis in colorectal cancers.

No potential conflicts of interest were disclosed.

Conception and design: H. Yu

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R. Wang, H. Liu, Y. Shao, K. Wang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Yin, Y. Qiu, H. Wu, E. Liu, T. Wang, X. Gao, H. Yu

Writing, review, and/or revision of the manuscript: H. Yu

Study supervision: H. Yu

This work was supported by grants from National Natural Science Foundation of China (81603253, 21711540293, and 81873089 to H. Yu, and 81602614 and 81973570 to Y. Qiu), Important Drug Development Fund, Ministry of Science and Technology of China (2018ZX09735-002 to T. Wang), and Natural Science Foundation of Tianjin City (No. 15PTCYSY00030 to Z. Li).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.