Exploring the mechanism of action of sophocarpine in the treatment of prostate cancer based on in vitro cell experiments and network pharmacology
Prostate cancer (PCa) is one of the most common malignant tumors in the urinary system of middle-aged and elderly men worldwide. In 2020, there were 10.06 million new male cancer cases and 5.53 million deaths worldwide, including 1.41 million new PCa cases and 380000 deaths, with a mortality to incidence ratio of 27%. In China, there were 120000 new PCa cases and 50000 deaths, with a PCa mortality to incidence ratio of 41.7%. According to research data from the American Cancer Society in 2023, the incidence of PCa ranks first among male malignant tumors, accounting for approximately 29%. At present, the main treatment options for PCa in clinical practice include androgen deprivation therapy, radical prostatectomy, radiotherapy, chemotherapy, and molecular targeted therapy. However, these treatment methods have many side effects and are prone to drug resistance, resulting in poor prognosis and recurrence or progression in some patients. Therefore, it is of great significance to search for safe and effective new drugs for PCa treatment.
Sophoridine (SRI) is a tetracyclic quinoline alkaloid mainly isolated from Sophora alopecuroides L. Research has shown that SRI has many pharmacological effects, including anti-inflammatory, antiviral, and anti-tumor effects. In 2005, the injection of sophocarpine hydrochloride was approved by the State Food and Drug Administration for the clinical treatment of malignant trophoblastic tumors. At present, SRI has been tried in the treatment of liver metastasis of gastrointestinal tumors. At the same time, a large number of reports have confirmed that SRI has a good inhibitory effect on colorectal cancer, lung cancer, pancreatic cancer and other tumors. However, the impact of SRI on PCa and its possible mechanism of action are still unclear.
Network pharmacology is an emerging discipline based on classic pharmacology, bioinformatics, and computer science theories, widely used in drug development and research on drug mechanisms of action. This article investigates the effects of SRI on the proliferation and apoptosis of DU-145 cells through in vitro cell experiments, and combines network pharmacology and molecular docking techniques to explore its possible mechanism of action, in order to provide theoretical basis and data support for the clinical treatment of PCa with SRI.

PCa is the most common malignant tumor among middle-aged and elderly men, which is prone to drug resistance and metastasis, leading to poor prognosis and high recurrence rate. Therefore, seeking new therapies for the treatment of PCa has become very urgent. The natural chemical active ingredients of traditional Chinese medicine play an important role in the prevention and treatment of malignant tumors. SRI is mainly an active alkaloid isolated from the traditional Chinese medicine Sophora alopecuroides, which has various pharmacological activities, among which anti-tumor activity is particularly prominent. In 2005, sophocarpine hydrochloride injection was approved by the China Food and Drug Administration as an anti-cancer drug. SRI has shown good anti-tumor effects on gastric cancer, liver cancer, lung cancer, colon cancer, pancreatic cancer and other tumors in vivo and in vitro. SRI can inhibit the proliferation and invasion of various tumor cells, promote apoptosis and autophagy of cancer cells. Its function is related to pathways such as PI3K/AKT, Wnt/B-catenin, MAPK/ERK, and cell cycle. As far as current research is concerned, the role and exact mechanism of SRI on PCa are still not fully understood. This study explores the molecular mechanism of SRI therapy for PCa through in vitro cell experiments, network pharmacology, and molecular docking techniques, providing a certain research basis and theoretical basis for its clinical application.
High proliferation rate is an important characteristic of infinite growth of malignant tumor cells, therefore inhibiting cell proliferation is an effective strategy for treating cancer. In this study, it was found that with the increase of SRI concentration, the growth of DU-145 cells was significantly inhibited by detecting cell viability and cell clone formation, indicating that SRI has the ability to inhibit the proliferation of DU-145 cells. The proliferating cell nuclear proteins Ki67 and PCNA are marker proteins for cell proliferation, commonly used as predictive and prognostic markers for cancer diagnosis and treatment. This study detected the expression of Ki67 in DU-145 cells through immunofluorescence and qPCR, and found that SRI could significantly inhibit the expression of Ki67. qPCR and Western blot analysis showed that SRI could significantly reduce the expression of PCNA in DU-145 cells, further proving that SRI has the ability to inhibit the proliferation of DU-145 cells.
Apoptosis is the main cause of tumor cell death, therefore, avoiding apoptosis can lead to resistance in tumor treatment. Bcl-2, as an inhibitor of apoptosis, plays a decisive role in the regulation of cell apoptosis. Bax is a homologous conjugate of Bcl-2 and a pro apoptotic protein. The ratio of Bcl-2 to Bax is an important indicator for measuring the effect of cell apoptosis. Caspase-3 and Caspase-7 are the executors of cell apoptosis. When apoptosis occurs, Caspase-3 and Caspase-7 are sequentially cleaved and activated by upstream Caspase proteins. Then, activated Caspase-3 and Caspase-7 will cleave multiple downstream substrates, ultimately triggering an apoptotic cascade reaction. This study detected the apoptosis of DU-145 cells after 48 hours of SRI treatment through flow cytometry experiments, and the results showed that SRI could significantly promote the apoptosis of DU-145 cells. Therefore, we further examined the expression of apoptosis related factors. The qPCR results showed that SRI could upregulate the mRNA expression of Caspase-3 and Caspase-7, downregulate the mRNA expression of Bcl-2, and downregulate the mRNA expression level of Bax, but the Bcl-2/Bax ratio was significantly reduced (P<0.05). In addition, Western blot results showed that SRI could upregulate the expression of cleaned-Caspase-3 protein and downregulate the expression of Bcl-2 protein. Although there was no significant difference in the upregulation of Bax protein expression, the protein expression ratio of Bcl-2/Bax was significantly reduced (P<0.05). These results suggest that SRI can regulate the expression of apoptosis related factors, activate apoptosis signaling pathways, and induce apoptosis in DU-145 cells.
In addition, in order to investigate the mechanism of SRI affecting the proliferation and apoptosis of DU-145 cells, this study analyzed it through network pharmacology research methods. We collected a total of 127 SRI targets through HERB, TCMSP, Pharm Mapper, and Swiss Target Prediction databases, and obtained 60 common targets of SRI acting on PCa through Venny 2.1.0 online tool. Through PPI network analysis, we identified 16 core targets (top 10 degree values) including ALB, TNF, IL6, CASP3, MAPK1, PPARG, MDM2, PGR, MAPK8, NR3C1, ABL1, MAPK14, PARP1, HDAC2, CDK2, GSK3B, etc. MAPK8, CASP3, MAPK1, MAPK14, and TNF core targets are enriched in the MAPK signaling pathway. GO enrichment analysis yielded 158 entries (P<0.01), indicating that SRI may mainly exert therapeutic effects on PCa by regulating gene expression, apoptosis process, protein phosphorylation, and protein serine/threonine/tyrosine kinase activity. The KEGG pathway enrichment analysis results showed 84 signaling pathways, including cancer signaling pathway, proteoglycans in cancer, MAPK signaling pathway, chemocarcinogenesis reactive oxygen species, PI3K-AKT signaling pathway, apoptosis, etc. (P<0.01). The most concerning aspect is the role of the MAPK signaling pathway in the anti PCa process of SRI. Mitogen activated protein kinase (MAPK) can transmit extracellular information to the nucleus and plays an important role in physiological and pathological processes. There are three types of MAPK family members in mammalian cells: extracellular signal regulated kinase (ERK), c-JunN-terminal protein kinase/stress activated protein kinase (JNK/SAPK), and p38 mitogen activated protein kinase (p38MAPK, also known as MAPK14).
P38MAPK is an activated protein kinase that participates in numerous biological processes such as cell proliferation, differentiation, and apoptosis. Its activation can inhibit cell proliferation and promote apoptosis. Research has found that activation of the p38 pathway can reduce the proliferation activity of BGC-823 gastric cancer cells and block them in the G2/M phase. Activation of p38MAPK can inhibit the proliferation of SMMC-7721 cells and initiate the intracellular Caspase signaling cascade, ultimately inducing apoptosis. In addition, studies have shown that traditional Chinese medicine extracts can activate p38MAPK, trigger Caspase mediated apoptotic cascade reactions, and induce apoptosis in Lewis lung cancer cells as well as HSC-3 and SCC-9 oral cancer cells. P38MAPK kinase also plays an important role in the occurrence and development of PCa. Research has found that activation of p38MAPK can resist cell proliferation, promote apoptosis, and induce tumor dormancy in malignant transformation of prostate epithelium or bone metastatic prostate cancer. Meanwhile, other studies have shown that ingredients such as docetaxel and ursolic acid can increase the phosphorylation level of p38MAPK, induce aging of PCa cells, inhibit the proliferation of various PCa cells such as PC-3 and 22Rv1, and promote their apoptosis. Therefore, this study focuses on the role of p38MAPK protein kinase in SRI anti PCa. Using molecular docking technology to verify the binding of SRI to p38MAPK protein, the results showed that SRI can stably bind to p38MAPK protein, with a binding energy of -6.83 kcal/mol. The detection results of p-p38MAPK and p38MAPK protein expression levels indicate that SRI can activate p38MAPK protein, and the phosphorylation level of p38MAPK protein is upregulated, suggesting that SRI can exert anti PCa effects by activating p38MAPK protein and inducing apoptosis pathway.
In summary, SRI can inhibit the proliferation of DU-145 cells and promote their apoptosis, which may be related to its activation of p38MAPK. However, this study only explored the effect of SRI on DU-145 cells in vitro. The next step will be to conduct in vivo animal experiments to further investigate the mechanism of SRI’s anti PCa effect.