Application of chonglou saponin II as an immunogenic cell death inducer in treatment of esophageal squamous cell carcinoma
Paris saponin II enhances the immune response in esophageal squamous cell carcinoma by inducing the release of ATP, calreticulin and acetylated HMGB1. When used in combination with PD-1 monoclonal antibodies, it solves the problems of toxicity and limited efficacy of existing ICD inducers in esophageal squamous cell carcinoma, achieving a highly effective combination therapy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GANSU PROVINCIAL PEOPLES HOSPITAL
- Filing Date
- 2026-03-03
- Publication Date
- 2026-06-05
AI Technical Summary
Existing ICD inducers for the treatment of esophageal squamous cell carcinoma suffer from high toxicity, limited efficacy, insufficient mechanistic research, and underutilization of traditional Chinese medicine resources. There is a lack of highly effective, low-toxicity specific inducers and combination therapy regimens.
Polyphyllin II (PPII) was used as an immunogenic cell death inducer to enhance immune activation signals by inducing the release of ATP, calreticulin and acetylated HMGB1. It was also used in combination with the immune checkpoint inhibitor PD-1 monoclonal antibody to remodel the tumor microenvironment.
It significantly enhanced the immune response in esophageal squamous cell carcinoma, achieved efficient tumor suppression, overcame the limitations of monotherapy, and provided a new treatment strategy combining traditional Chinese and Western medicine.
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Figure CN122140739A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to the application of Paris saponin II as an immunogenic cell death inducer in the treatment of esophageal squamous cell carcinoma. Background Technology
[0002] Currently, in the field of cancer treatment, immunogenic cell death (ICD) has gradually transformed from a biological concept into a highly promising therapeutic strategy. Its core value lies in its ability to transform a simple tumor cell killing event into an "in situ vaccine" effect that activates the host's adaptive immune system, thereby generating systemic and durable anti-tumor immune memory and providing new possibilities for overcoming tumor recurrence and metastasis. The hallmark of ICD is the exposure or release of a series of signaling molecules known as damage-associated molecular patterns (DAMPs) during cell death. These mainly include: calreticulin (CRT) translocated to the cell membrane surface (mediating the phagocytosis of dead cells by dendritic cells as an "eat me" signal), large amounts of adenosine triphosphate (ATP) released extracellularly (chemotacticing immune cells as a "find here" signal), and high-mobility group box 1 (HMGB1) released from the cell nucleus or cytoplasm (activating antigen-presenting cells as a "danger" signal). The synergistic effect of these signals is key to initiating an effective anti-tumor T-cell response.
[0003] Based on this theoretical foundation, researchers have screened and identified a few classes of drugs with ICD-inducing capabilities. First are traditional chemotherapy drugs, represented by anthracyclines (such as doxorubicin and epirubicin) and platinum-based drugs (such as oxaliplatin), which are considered the "gold standard" inducers of ICD. These drugs primarily induce DNA damage or endoplasmic reticulum stress, triggering the programmed release of classic DAMPs (CRT, ATP, HMGB1), thus demonstrating that the efficacy of chemotherapy partly stems from its activated immune response. Second are some molecularly targeted drugs, such as vemurafenib, an inhibitor of BRAF V600E mutations. Studies have shown that while inducing apoptosis in specific genotype melanoma cells, it can also upregulate MHC-I molecule expression and promote DAMP release, enhancing tumor immunogenicity and providing a basis for the combination of targeted therapy and immunotherapy. Furthermore, some natural products and active ingredients of traditional Chinese medicine, such as shikonin and tripterygium wilfordii, have also been reported in preclinical studies to trigger ICD-related phenotypes through pathways such as inducing reactive oxygen species bursts and endoplasmic reticulum stress, demonstrating the potential to tap into the natural product library for ICD inducers.
[0004] However, currently known inducing agents still have some drawbacks in their application for treatment, such as: First, the toxicity spectrum of known highly effective ICD inducers is generally quite severe. Taking doxorubicin as the classic example, the dose required to induce ICD is often accompanied by significant dose-limiting toxicities, especially cumulative cardiotoxicity, which seriously limits its long-term or widespread clinical safety window as an "immunoadjuvant." Similarly, platinum-based drugs have the risks of neurotoxicity and nephrotoxicity. This makes the development of novel, highly effective but low-toxicity ICD inducers an urgent need. Second, the effects of existing ICD inducers exhibit significant tumor type heterogeneity and are not universally applicable. For example, oxaliplatin has a clear ICD effect in colorectal cancer, but not all chemotherapy drugs can effectively induce ICD in all cancer types. Especially in esophageal squamous cell carcinoma (ESCC), which has a high incidence and poor prognosis, highly effective and specific ICD inducers remain scarce. The immunogenic potential of existing chemotherapy regimens (such as paclitaxel combined with platinum-based drugs) has not been fully elucidated and utilized, resulting in a lack of effective immune initiation methods in this field. Third, current research on the mechanisms of ICD remains at a relatively superficial level. The vast majority of work focuses on confirming the "presence" or "total" changes of DAMPs such as CRT, ATP, and HMGB1, while paying serious insufficient attention to key post-translational modification events that determine the functional activity of these signaling molecules. For example, the immune adjuvant activity of HMGB1 is highly dependent on its acetylation, phosphorylation, and other modification states; different modification forms may mediate completely opposite immune effects. Current techniques have almost no in-depth analysis of the fine regulation of the modification states of molecules such as HMGB1 during ICD and their biological significance, resulting in a shortcoming in mechanistic research: "knowing what happens, but not why." Fourth, from the perspective of original innovation, the existing technological system has failed to fully utilize my country's unique treasure trove of medicinal resources. Although some natural products have been reported sporadically, research on systematically discovering single compounds (TCM monomers) from TCM with clear ICD-inducing activity, well-defined chemical structures, and unique mechanisms of action remains lacking. This results in insufficient diversity of chemical structures and novelty of mechanisms of action in the existing ICD inducer library, and also fails to reflect the potential value of TCM theory in modern tumor immunotherapy. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a novel anti-esophageal squamous cell carcinoma treatment strategy that is derived from traditional Chinese medicine, has a clear mechanism of action, and has significant efficacy.
[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides the application of Paris saponin II in the preparation of immunogenic cell death inducers.
[0007] For esophageal squamous cell carcinoma with poor response to existing immunotherapies, this invention provides a novel, highly effective, and relatively safe ICD inducer. This invention discovers a novel immunomodulatory active monomer, Polyphyllin II (hereinafter referred to as PPII), extracted from the traditional Chinese medicine *Paris polyphylla*. *Paris polyphylla* is commonly used in traditional Chinese medicine for clearing heat and detoxifying, reducing swelling and relieving pain. This invention is the first to discover and confirm PPII as a highly effective ICD inducer from a single Chinese medicinal herb, providing a novel candidate drug molecule for tumor immunotherapy.
[0008] PPII-induced ICD is a complete process involving multiple levels of biological events, programmatically releasing a series of powerful immune-activating signals, namely damage-associated molecular patterns (DAMPs). This signaling cascade manifests itself as follows: (1) ATP release: PPII treatment can lead to the active and large-scale release of intracellular ATP from the extracellular environment. Extracellular ATP, as a key "find here" signal, exerts a strong chemotactic effect by binding to P2Y2 and P2X7 receptors on the surface of antigen-presenting cells (such as dendritic cells), and promotes the maturation of dendritic cells and the release of inflammatory factors.
[0009] (2) Extravasated calreticulin: PPII can induce endoplasmic reticulum stress, leading to unfolded protein responses and causing calreticulin (CRT), a molecular chaperone protein originally located in the endoplasmic reticulum lumen, to translocate to the cell membrane surface. CRT exposed on the surface of apoptotic cells acts as a "eat me" signal, significantly enhancing the phagocytic capacity of dendritic cells for tumor antigens by binding to receptors such as CD91, a low-density lipoprotein receptor-associated protein on dendritic cells. This is a key first step in initiating a specific immune response.
[0010] (3) Release and modification of HMGB1: PPII treatment induces the release of high-mobility group box 1 (HMB1) from the nucleus to the extracellular space. The most crucial finding of this invention is that the HMGB1 released after PPII induction undergoes significant histone acetylation modification. This post-translational modification is not accidental; it is a key switch determining the immune activity of HMGB1. Acetylation modification enhances the binding ability of HMGB1 to TLR4 (Toll-like receptor 4) and promotes its transport from the nucleus to the cytoplasm, thereby greatly enhancing its efficacy as a "danger" signal to stimulate dendritic cell maturation, secretion of pro-inflammatory cytokines (such as TNF-α and IL-6), and cross-presentation of antigens. The discovery and verification of "acetylated HMGB1" is a fundamental characteristic that distinguishes PPII from classic inducers such as doxorubicin and oxaliplatin, which only cause passive release of HMGB1. It elucidates the unique molecular mechanism by which PPII induces a more potent and higher-quality immune response from the perspective of epigenetic regulation.
[0011] Preferably, the immunogenic cell death inducer is used to induce immunogenic cell death in esophageal squamous cell carcinoma cells.
[0012] Preferably, the immunogenic cell death includes at least one of the following features: (1) inducing ATP release; (2) inducing calreticulin to evert to the cell membrane surface; and (3) inducing the release of high-mobility group box 1.
[0013] Preferably, the high-mobility group B1 protein has undergone acetylation modification.
[0014] This invention is the first to discover and verify that PPII induces ICD in esophageal squamous cell carcinoma cells and specifically releases acetylated HMGB1.
[0015] Secondly, this invention provides the application of Paris saponin II in the preparation of a drug for treating esophageal squamous cell carcinoma.
[0016] Thirdly, this invention provides the application of Paris saponin II in combination with immune checkpoint inhibitors in the preparation of drugs for treating esophageal squamous cell carcinoma.
[0017] Based on the role of PPII in reshaping the tumor immune microenvironment by inducing ICD (transforming "immunely cold tumors" into "immunely hot tumors"), this invention prospectively proposes and protects a combination therapy regimen with significant clinical value. This regimen uses the core of this invention—a pharmaceutical composition containing PPII—as the first therapeutic component, combined with at least one immune checkpoint inhibitor as the second therapeutic component. The biological basis for its synergistic effect lies in the fact that the large amount of tumor antigens and newly activated T cells generated by the PPII-induced ICD effect precisely require immune checkpoint inhibitors to relieve immunosuppression in the tumor microenvironment (such as the PD-1 / PD-L1 signaling pathway), thereby freeing the "brake-down" T cells and enabling them to effectively attack tumor cells. In vivo pharmacodynamic experiments have clearly demonstrated that this combination regimen has a significantly better growth-inhibiting effect on esophageal squamous cell carcinoma xenografts than any single-agent therapy, achieving a synergistic anti-tumor effect of "1+1>2".
[0018] Preferably, the immune checkpoint inhibitor includes PD-1.
[0019] Fourthly, the present invention provides a pharmaceutical composition for treating esophageal squamous cell carcinoma, comprising Paris saponin II and a pharmaceutically acceptable carrier.
[0020] Preferably, it also includes immune checkpoint inhibitors.
[0021] Preferably, the immune checkpoint inhibitor includes PD-1.
[0022] The beneficial effects of this invention are as follows: This invention reveals for the first time a novel mechanism by which PPII enhances immunogenicity by inducing the release of acetylated HMGB1, which has significant scientific value. The active ingredient of this traditional Chinese medicine directly targets clinically refractory esophageal squamous cell carcinoma, with conclusive in vitro and in vivo experimental evidence, making it of great significance in translational medicine.
[0023] In vivo experiments have demonstrated that the combination of PPII and PD-1 monoclonal antibody produces a synergistic anti-tumor effect, providing a novel and effective strategy to address the current clinical challenge of limited response rates of immune checkpoint inhibitors in esophageal squamous cell carcinoma. The proposed treatment regimen of "Paris polyphylla saponin II (PPII) combined with PD-1 monoclonal antibody" is not a simple drug additive approach, but rather establishes a profound synergistic mechanism between the traditional Chinese medicine philosophy of "strengthening the body's resistance and eliminating pathogenic factors" and the principles of modern tumor immunology. This "integration of Chinese and Western medicine" strategy demonstrates significant advantages over single-therapy approaches in both theoretical logic and practical efficacy. Attached Figure Description
[0024] Figure 1 This is the result of apoptosis detection.
[0025] Figure 2 The results are from the ATP release assay.
[0026] Figure 3 This is the result of CRT eversion detection.
[0027] Figure 4 The results are for the release of acetylated HMGB1.
[0028] Figure 5 The changes in tumor volume in each treatment group are shown. Detailed Implementation
[0029] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.
[0030] Unless otherwise specified, the experimental methods used in this invention are conventional methods, and the materials and reagents used are commercially available products that can be obtained through commercial channels.
[0031] The PPII of this invention is a commercially available product. PPII with a purity of not less than 98% confirmed by modern analytical techniques such as high performance liquid chromatography (HPLC) meets the requirements of the embodiments of this invention.
[0032] Example 1: In vitro validation of PPII-induced immunogenic cell death in esophageal squamous cell carcinoma cells Human esophageal squamous cell carcinoma cells KYSE-150 were seeded at a density of 60% in six-well plates. After 24 hours, the cells were cultured in medium containing PPII (final concentration 1.5 μM) for 48 hours.
[0033] Apoptosis was detected using Annexin V-FITC / PI flow cytometry, and the results are as follows: Figure 1 As shown, after 48 hours of PPII treatment, the apoptosis rate increased significantly (P<0.001).
[0034] The release of ATP from cell supernatant was detected using spectrophotometry, and the results are as follows: Figure 2 As shown, after 48 hours of PPII treatment, the ATP concentration in the supernatant was significantly higher than that in the control group (P<0.001).
[0035] Flow cytometry staining of cell surface was used to detect CRT eversion, and the results were as follows: Figure 3 As shown, the PPII-treated group showed stronger CRT fluorescence intensity on the cell membrane surface (P<0.001).
[0036] Western blotting was used to detect the release of acetylated HMGB1 from the cell supernatant. The results are as follows: Figure 4 As shown, HMGB1 release was confirmed. Further detection with acetylated lysine antibody confirmed that the HMGB1 released induced by PPII underwent acetylation modification (Ace-HMGB1).
[0037] Example 2: Antitumor efficacy of PPII combined with PD-1 monoclonal antibody in a mouse model of esophageal squamous cell carcinoma Model establishment: esophageal squamous cell carcinoma cells from mEC25 mice were subcutaneously inoculated into C57BL / 6 mice to form xenografts.
[0038] Grouping and drug administration: When the tumor volume is approximately 150-100 mm 3 At that time, they were randomly divided into 4 groups (n=5): (1) Solvent control group (physiological saline); (2) PPII monotherapy group (5 μg / animal intratumoral injection, once every 3 days); (3) PD-1 monoclonal antibody group (200 μg / animal intraperitoneal injection, once every 3 days); (4) PPII + PD-1 monoclonal antibody combination group (5 μg / animal intratumoral injection of PPII, 200 μg / animal intraperitoneal injection of PD-1 monoclonal antibody).
[0039] Mice were treated four times (12-day cycles). After treatment, mice were sacrificed when the longest diameter of the tumor reached 1.5 cm, and the tumors were harvested for analysis. Results are as follows: Figure 5 As shown, the tumor volume in the combination therapy group was significantly smaller than that in any single-drug group (P<0.001), indicating that the combination therapy could significantly inhibit tumor growth.
[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.
Claims
1. Application of Paris saponin II in the preparation of immunogenic cell death inducers.
2. The application as described in claim 1, characterized in that, The immunogenic cell death inducer is used to induce immunogenic cell death in esophageal squamous cell carcinoma cells.
3. The application as described in claim 2, characterized in that, The immunogenic cell death includes at least one of the following features: (1) induction of ATP release; (2) induction of calreticulin eversion to the cell membrane surface; (3) induction of release of high-mobility group box 1.
4. The application as described in claim 3, characterized in that, The high-mobility group box protein B1 was acetylated.
5. Application of Paris saponin II in the preparation of drugs for treating esophageal squamous cell carcinoma.
6. Application of Paris saponin II in combination with immune checkpoint inhibitors in the preparation of drugs for the treatment of esophageal squamous cell carcinoma.
7. The application as described in claim 6, characterized in that, The immune checkpoint inhibitors include PD-1.
8. A pharmaceutical composition for treating esophageal squamous cell carcinoma, characterized in that, This includes Paris saponin II and a pharmaceutically acceptable carrier.
9. The pharmaceutical composition according to claim 8, characterized in that, It also includes immune checkpoint inhibitors.
10. The pharmaceutical composition according to claim 9, characterized in that, The immune checkpoint inhibitors include PD-1.