Carbenoxolone upregulates TRAIL\TRAILR2 expression and enhances the anti-neoplastic effect of doxorubicin in experimentally induced hepatocellular carcinoma in rats.
This study investigates the in vivo anticancer activity of carbenoxolone (CBX) and its role in fighting hepatocellular carcinoma (HCC) progression and alleviating resistance against doxorubicin (DOX). Moreover, the molecular mechanism of action of CBX is explored.
HCC was induced in Sprague Dawley rats via biweekly administration of thioacetamide (TAA) (200 mg/kg) intraperitoneally (i.p.) for 16 weeks after administering a single dose of diethylnitrosamine (DEN) (200 mg/kg, i.p.). A prophylactic model was established by treating rats with i.p. CBX (20 mg/kg/day) for 4 weeks starting on week 13 post-TAA injection. A therapeutic model was established by treating rats with CBX, DOX, or their combination for 7 weeks following 16 weeks of TAA administration. Serum Alpha-fetoprotein (AFP) and biochemical markers of hepatic functions were assessed. Histopathological examinations of hepatic tissues were performed. Immunohistochemical and qRT-PCR analyses were applied to assess the differential expressions of TRAIL/TRAILR2, Bcl-2, TGF-β1, and caspases 3, 8, and 9.
CBX markedly improved hepatic functions, reduced serum AFP levels, and alleviated TAA-induced hepatic histopathological alterations. CBX triggered apoptosis as evident by upregulating apoptotic markers: TRAIL/TRAILR2, caspases 3, 8, and 9, and downregulating the antiapoptotic protein Bcl-2. CBX downregulated TGF-β1. Interestingly, CBX/DOX combination mitigated hepatic damage and induced apoptosis in a way that surpassed DOX-only treatment.
The current study proposes that CBX is a promising anti-tumor compound, which can work effectively under prophylactic and therapeutic modes. Interestingly, CBX enhanced the anti-tumor effect of DOX. CBX exerted these effects via, in part, stimulating TRAIL-induced apoptosis along with attenuating fibrosis.
El-Zehery IM
，El-Mesery M
，El-Sherbiny M
，El Gayar AM
，Eisa NH
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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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Albiflorin Alleviates Severe Acute Pancreatitis-Associated Liver Injury by Inactivating P38MAPK/NF-κB Signaling Pathway.
Albiflorin (Alb) is a monoterpenoid component that is commonly found in Paeonia lactiflora Pall. or Paeonia veitchii Lynch. It is known for its impressive anti-oxidant and anti-inflammatory properties. However, the effect of Alb on severe acute pancreatitis (SAP)-associated liver injury has not been fully understood. To investigate this, we conducted a study using a rat model of SAP induced by administering two intraperitoneal injections of 20% L-arginine (3.3 g/kg) over a period of 2 h. Subsequently, the SAP-induced rats were randomly assigned into different groups with the treatment of gradient doses of Alb (5, 10, and 20 mg/kg), with the normal saline as the sham group. The pathological changes in rat livers were evaluated through hematoxylin-eosin staining. Furthermore, the levels of amylase (AMY), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were determined using specific enzyme-linked immunosorbent assay kits. Moreover, the serum levels of inflammatory factors, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β, were quantified. Finally, immunohistochemical and Western blot analyses were conducted to determine phosphorylation levels of nuclear factor kappa B (NF-κB) p65 and mitogen-associated protein kianse (MAPK) p38 in the liver tissues. TNF-α stimulated liver cells were used as a cell model to further confirm the involvement of NF-κB and p38 in the effect of Alb. Our study revealed that Alb effectively mitigated the hepatic pathological damage in a dose-dependent manner and reduced the levels of indicators associated with hepatic malfunction (AMY, AST, and ALT) in rats with SAP-induced liver injury. Additionally, Alb demonstrated its ability to suppress inflammation and oxidative stress markers in the liver tissues. Alb exerted dose-dependent inhibitory effects by modulating the P38MAPK/NF-κB signaling pathway. Overall, our findings strongly support the hepatoprotective effect of Alb in rats with SAP-induced liver injury, suggesting that Alb protects against SAP-induced liver injury through the suppression of inflammation and oxidative stress via the P38MAPK/NF-κB signaling pathway.
Li H
，Zeng X
，Sun D
，Qi X
，Li D
，Wang W
，Lin Y
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