Icariin promoted ferroptosis by activating mitochondrial dysfunction to inhibit colorectal cancer and synergistically enhanced the efficacy of PD-1 inhibitors.
A controlled type of cell death called ferroptosis is linked to increased reactive oxygen species (ROS), lipid peroxidation, and iron buildup. Furthermore, evidence indicates that ferroptosis may act as an immunogenic form of cell death with potential physiological functions in tumors and immunosuppression. Inducing ferroptosis in tumor cells may have the potential to complement cancer immunotherapy strategies. The development of colorectal cancer (CRC) and the poor efficacy of immunotherapy are associated with the crosstalk of cellular ferroptosis. Currently, Icariin (ICA), the main bioactive component extracted from Epimedium, has been shown to inhibit a variety of cancers. However, the specific role and potential mechanism of ICA in regulating ferroptosis in CRC remains unclear.
The aim of this investigation was to clarify the mechanism underlying the anti-CRC cancer properties of ICA and how it induces ferroptosis to enhance immunotherapy.
To evaluate cell viability, the Cell Counting Kit-8 (CCK-8) test was utilized. The transwell test and the wound healing assay were used to assess cell migration. A subcutaneous graft tumor model was constructed with C57BL/6 mice using MC38 colorectal cancer cell lines. The inhibitory effect of ICA on CRC, ferroptosis level and immunomodulatory effects were detected by serum biochemical assay, cytokine assay, hematoxylin-eosin (H&E) staining, immunofluorescence staining, CyTOF mass spectrometry flow screening and Western blotting. Western blotting, proteomics, molecular docking and microscale thermophoresis (MST) were used to forecast and confirm ICA's binding and interaction with HMGA2, STAT3, and HIF-1α. Moreover, the levels of lipid peroxidation and ferroptosis were assessed through the use of the C11-BODIPY fluorescent probe, the FerroOrange fluorescent probe, the iron level, the malondialdehyde (MDA) and reduced glutathione (GSH) assay kit, and Western blotting analysis. To assess alterations in mitochondrial structure and membrane potential, transmission electron microscopy (TEM) and JC-1 immunofluorescence were employed.
It was demonstrated in the current study that ICA treatment inhibits CRC and enhances anti-PD-1 therapy efficacy by inciting ferroptosis. As shown in vitro, ICA inhibits CRC cell proliferation, migration, and apoptosis. As demonstrated in vivo, ICA has a dose-dependent tumor suppressor effect when combined with anti-PD-1, it can significantly inhibit tumor growth, increase the expression of serum TNF-α, IFN-γ, and granzyme B, and promote CD69+CD8+ T, CD69+CD8+Tem, CD69+CD8+Teff, TCRβ+CD8+ T, TCRβ+CD8+ T, TCRβ+CD8+Tem, TCRβ+CD8+Teff. The inhibitory effect of ICA on CRC was associated with the binding of HMGA2, STAT3, and HIF-1α proteins, which inhibited CRC by increasing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), promoting the accumulation of iron (Fe2+), depletion of reduced glutathione (GSH), inhibiting SLC7A11 and GPX4 expressions, thereby inducing ferroptosis in CRC. As a consequence of ICA-induced ferroptosis, mitochondria are dysfunctional, with increased ROS production, membrane potential depolarization (MMP), and ATP production reduced. This process can be efficiently reversed by the mitochondria-targeted antioxidant Mito-Q. It is noteworthy that the ferroptosis inhibitor liproxstatin-1 (lip-1), anti-CD8, and anti-IFN-γ exhibited a significant inhibitory effect on the level of ferroptosis and antitumor capacity of ICA combined with anti-PD-1. This finding suggests that the antitumor immunopotentiating effect of ICA on anti-PD-1 is dependent on the secretion of IFN-γ-induced ferroptosis of CRC cells by the CD8+ T cell.
Our study represents the inaugural demonstration of the mechanism whereby ICA exerts anti-CRC effects and synergistically enhances the efficacy of anti-PD-1, inducing mitochondrial damage and leading to ferroptosis. ICA promotes ferroptosis of CRC cells by inducing mitochondrial dysfunction, and ICA combined with anti-PD-1 significantly promotes CD69, TCRβ signalling, activates effector CD8+ T cells to secrete IFN-γ, and achieves immunopotentiation by promoting ferroptosis of CRC cells, thus inhibiting CRC development. This study is built upon existing research into the pharmacodynamic mechanisms of ICA in the context of CRC, and offers a novel therapeutic approach in addressing the issue of CRC immunotherapy potentiation.
Haoyue W
，Kexiang S
，Shan TW
，Jiamin G
，Luyun Y
，Junkai W
，Wanli D
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Euphorbia Pekinensis Rupr. sensitizes colorectal cancer to PD-1 blockade by remodeling the tumor microenvironment and enhancing peripheral immunity.
Immune checkpoint blockade, such as monoclonal antibodies targeting programmed cell death protein 1 (PD-1), has been a major breakthrough in the treatment of several cancers, but has limited effect in colorectal cancer (CRC), which is a highly prevalent cancer worldwide. Current chemotherapy-based strategies to boost PD-1 response have many limitations. And the role of peripheral immunity in boosting PD-1 response continues to attract attention. Therefore, candidate combinations of PD-1 blockade need to be drugs with multi-targets and multi-modulatory functions. However, it is still unknown whether traditional Chinese medicines with such property can enhance the applicability and efficacy of PD-1 blockade in colorectal cancer.
Euphorbia Pekinensis extract (EP) was prepared and the constituents were analyzed by HPLC. CRC cells were used for in vitro experiments, including cell viability assay, colony formation assay, flow cytometry for 7-AAD staining, western blotting for caspase 3 and caspase 7, HMGB1 and ATP detection. An orthotopic CT26 mouse model was subsequently used to investigate the combination of EP and PD-1 blockade therapy. Tumor volume and tumor weight were assessed, tumor tissues were subjected to histopathological HE staining and TUNEL staining, and tumor-infiltrating immune cells were evaluated by immunofluorescence staining. RNA-sequencing, target prediction and pathway analysis were further employed to explore the mechanism. Molecular docking and cellular thermal shift assay (CETSA) were utilized to verify the direct target of the core component of EP. And, loss-of-function analysis was carried to confirm the upstream-downstream relationship. Flow cytometry was employed to analyze CD8+ T cells in the peripheral blood and spleen.
The main constituents of EP are diterpenoids and flavonoids. EP dramatically suppresses CRC cell growth and exerts its cytotoxic effect by triggering immunogenic cell death in vitro. Moreover, EP synergizes with PD-1 blockade to inhibit tumorigenesis in tumor-bearing mice. Disruption of ISX nuclear localization by helioscopinolide E is a central mechanism of EP-induced apoptosis in CRC cell. Meanwhile, EP activates immune response by upregulating Phox2b to reshape the immune microenvironment. In addition, EP regulates peripheral immunity by regulating the T cell activation and proliferation, and the ratio of CD8+ T cells in peripheral blood is drastically increased, thereby enhancing the therapeutic efficacy of anti-PD1 immunotherapy.
EP triggers intra-tumor immunogenic cell death and modulates the immunoregulatory signaling to elicit the tumor immunogenicity. Moreover, EP participates in transcriptional activation of immune response-related pathways. Consequently, multiple stimulating functions of EP on macro- and micro-immune potentiates the anti-tumor effect of PD-1 blockade in CRC.
Chen YY
，Zeng XT
，Gong ZC
，Zhang MM
，Wang KQ
，Tang YP
，Huang ZH
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Comparison of efficacy and safety of PD-1/PD-L1 combination therapy in first-line treatment of advanced NSCLC: an updated systematic review and network meta-analysis.
The use of immune checkpoint inhibitors has led to an increase in randomized controlled trials exploring various first-line combination treatment regimens. With the introduction of new PD-1/PD-L1 inhibitors, there are now more clinical options available. For the first time, the AK105 monoclonal antibody Penpulimab, developed in China, was included. The AK105-302 Phase III trial studied the efficacy and safety of Penpulimab combined with chemotherapy in patients with advanced or metastatic squamous NSCLC. To determine the optimal treatment options, we conducted an updated network meta-analysis to compare the effectiveness and safety of these regimens.
The system retrieves data from Chinese and English electronic databases, Clinical Trials, and the gov Clinical Trial Registration website up to September 6, 2023. The study indirectly compared the efficacy and safety of PD-1/PD-L1 combination regimens, including overall survival (OS), progression-free survival (PFS), objective response rate (ORR), all-grade adverse events, and above-grade III adverse events. Subgroup analyses were conducted based on programmed death ligand 1 (PD-L1) level, histological type, ECOG score, sex, and smoking history.
Nineteen RCTS were included, with a total of ten thousand eight hundred patients. Penpulimab plus chemotherapy (Pen + CT) provided the best OS (HR = 0.55, 95% CI 0.38-0.81) for PD-L1 patients with non-selective advanced NSCLC. Except Nivolumab plus Ipilimumab (Niv + Ipi), other PD-1/PD-L1 combination therapies significantly extended PFS compared with CT, and Nivolumab plus Bevacizumab combined with chemotherapy (Niv + Bev + CT) (HR = 0.43, 95% CI 0.26-0.74) provided the best PFS benefit and was comparable to Pen + CT (HR = 1.0) for PFS prolongation. For ORR, except Niv + Ipi, all the other regimens significantly improved ORR compared with CT. In terms of safety, except Tor + CT, the incidence of any-grade AEs or grade ≥ 3 adverse events may be higher than those of chemotherapy. The subgroup analysis revealed that for patients with PD-L1 levels below 1%, treatment with Tor + CT resulted in the best progression-free survival (HR = 0.47, 95% CI 0.25-0.86). For patients with PD-L1 levels of 1% or higher, Sintilimab plus chemotherapy (Sin + CT) (HR = 0.56, 95% CI 0.31-0.99) and Camrelizumab plus chemotherapy (Cam + CT) (HR = 0.43, 95% CI 0.28-0.64) were associated with the best overall survival and progression-free survival, respectively. For patients with SqNSCLC, combined immunotherapy may provide greater survival benefits. For patients with Non-sqNSCLC, Niv + Bev + CT and Tor + CT were associated with optimal PFS and OS, respectively. Cam + CT provided the best PFS in male patients with a history of smoking and an ECOG score of 0. In both female and non-smoking patient subgroups, Pem + CT was associated with the best PFS and OS benefits.
For patients with advanced non-selective PD-L1 NSCLC, two effective regimens are Pen + CT and Niv + Bev + CT, which rank first in OS and PFS among all patients. Cam + CT and Tor + CT have advantages for OS in patients with SqNSCLC and Non-sqNSCLC, respectively. Niv + Ipi + CT provided the best OS benefit for patients with an ECOG score of 0, while Pem + CT may be the most effective treatment for patients with an ECOG score of 1. Pem + CT has a better effect on female patients and non-smokers. Sin + CT was found to be the most effective treatment for male patients and the smoking subgroup, while Cam + CT was found to be the most effective for PFS. In addition, Tor + CT was associated with the best PFS for patients with negative PD-L1 expression. Pem + CT was found to significantly improve both PFS and OS compared to CT alone. For patients with positive PD-L1 expression, Sin + CT and Cam + CT were found to be optimal for OS and PFS, respectively. It is important to note that, with the exception of Tor + CT, the toxicity of the other combinations was higher than that of CT alone.
Yang Y
，Chen W
，Dong L
，Duan L
，Gao P
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ResearchGate
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