Arenobufagin enhances T-cell anti-tumor immunity in colorectal cancer by modulating HSP90ß accessibility.

BACKGROUND: Colorectal cancer (CRC) is a significant public health issue, ranking as one of the predominant cancer types globally in terms of incidence. Intriguingly, Arenobufagin (Are), a compound extracted from toad venom, has demonstrated the potential to inhibit tumor growth effectively. PURPOSE: This study aimed to explore Are's molecular targets and unravel its antitumor mechanism in CRC. Specifically, we were interested in its impact on immune checkpoint modulation and correlations with HSP90ß-STAT3-PD-L1 axis activity. METHODS: We investigated the in vivo antitumor effects of Are by constructing a colorectalcancer subcutaneous xenograft mouse model. Subsequently, we employed single-cell multi-omics technology to study the potential mechanism by which Are inhibits CRC. Utilizing target-responsive accessibility profiling (TRAP) technology, we identified heatshock protein 90ß (HSP90ß) as the direct target of Are, and confirmed this through a microscale thermophoresis experiment (MST). Further downstream mechanisms were explored through techniques such as co-immunoprecipitation, Western blotting, qPCR, and immunofluorescence. Concurrently, we arrived at the same research conclusion at the organoid level by co-cultivating with immune cells. RESULTS: We observed that Are inhibits PD-Ll expression in CRC tumor xenografts at low concentrations. Moreover, TRAP revealed that HSP90ß's accessibility significantly decreased upon Are binding. We demonstrated a decrease in the activity of the HSP90ß-STAT3-PD-Ll axis following low-concentration Are treatment in vivo. The PDO analysis showed improved enrichment of lymphocytes, particularly T cells, on the PDOs following Are treatment. CONCLUSION: Contrary to previous research focusing on the direct cytotoxicity of Are towards tumor cells, our findings indicate that it can also inhibit tumor growth at lower concentrations through the modulation of immune checkpoints. This study unveils a novel anti-tumor mechanism of Are and stimulates contemplation on the dose-response relationship of natural products, which is beneficial for the clinical translational application of Are.

α-hederin regulates glucose metabolism in intestinal epithelial cells by increasing SNX10 expression.

BACKGROUND: Sorting nexin 10 (SNX10) has recently been identified as a critical regulator of colorectal carcinogenesis, whose deletion promoted cell proliferation and survival in human CRC cells, and promoted colorectal tumor growth and upregulated amino-acid metabolism in mice. However, what happens when silencing SNX10 in normal human intestinal epithelial cells (IECs) remains unknown, and no drugs targeting SNX10 have been reported. Here, we first investigated the biological function and underlying mechanisms of SNX10 in normal human IECs, and found that α-hederin, a pentacyclic triterpenoid saponin, has a regulatory effect on SNX10 expression. PURPOSE: This study aimed to explore the function of SNX10 in IECs to provide a new target for the prevention and treatment of malignant transformation and the intervention mechanism of α-hederin for further development of potential novel agents targeting SNX10. METHODS: The transfection approach was used to construct SNX10 stable knockdown cells. Cell proliferation was detected by CCK8, clone formation, EdU, flow cytometry, and wound healing assays. Enzyme activity assays for glucose metabolism, qRT-PCR, western blotting, and immunofluorescence staining were performed to investigate the protein expression of signaling pathways. RESULTS: Silencing SNX10 promoted cell proliferation and cycle transition in IECs and increased the activity of key enzymes involved in glucose metabolism. Moreover, DEPDC5 expression was significantly decreased following SNX10 knockdown, followed by activation of the mTORC1 pathway. α-hederin reversed the accelerated cell proliferation, cycle progression, and glucose metabolic activity, as well as the activated mTORC1 pathway caused by SNX10 knockdown, by notably increasing SNX10 expression in a dose-dependent manner. CONCLUSION: We first reported that knockdown of SNX10 in normal human IECs promoted cell proliferation and activated glucose metabolism by activating the mTORC1 pathway. Meanwhile, we first found that α-hederin down-regulated glucose metabolism activity and slowed cell proliferation by increasing SNX10 expression in IECs.

Anti-inflammatory actions of Caesalpinin M2 in experimental colitis as a selective glucocoricoid receptor modulator.

Although repression of inflammatory gene expression makes glucocorticoids (GCs) powerful anti-inflammatory agents, side effects limit usage and drive the search for improved glucocorticoid receptor (GR) ligands. It has been postulated that the anti-inflammatory effects of GCs are primarily mediated by GR's activity in transrepressing major inflammation pathways such as NF-κB pathway, whereas their side effects are mostly mediated by GR's transactivation. In this study, we found that Caesalpinin M2 (C-M2), a cassane furanoditerpene isolated from a Chinese medical plant, exerts an anti-inflammatory potential both in vitro and in vivo. C-M2 inhibited the expression of proinflammatory cytokine IL-1ß and IL-6 in LPS-activated bone marrow-derived macrophages. Meanwhile, C-M2 treatment attenuated DSS-induced experimental acute colitis in mice and did not cause side effects, such as spleen involution, like dexamethasone treatment. Molecular docking and cellular thermal shift assay demonstrated that C-M2 could bind to GR in the ligand binding site. We showed that C-M2 mediates gene-inhibitory effects by activating GR. More importantly, C-M2 failed to induce GR binding to glucocorticoid response element-dependent genes and in turn activate their transcription. But it did repress NF-κB-dependent transcription by facilitating the interaction between GR and p65. Taken together, this non-steroidal compound of plant origin may exert anti-inflammatory actions as a selective GR modulator and might hold great potential for therapeutic use in inflammatory diseases.

Small-molecule RL71-triggered excessive autophagic cell death as a potential therapeutic strategy in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) has an aggressive phenotype and a poor prognosis owing to the high propensity for metastatic progression and the absence of specific targeted treatment. Here, we revealed that small-molecule RL71 targeting sarco/endoplasmic reticulum calcium-ATPase 2 (SERCA2) exhibited potent anti-cancer activity on all TNBC cells tested. Apart from apoptosis induction, RL71 triggered excessive autophagic cell death, the main contributor to RL71-induced TNBC cell death. RL71 augmented the release of Ca2+ from the endoplasmic reticulum (ER) into the cytosol by inhibiting SERCA2 activity. The disruption of calcium homeostasis induced ER stress, leading to apoptosis. More importantly, the elevated intracellular calcium signals induced autophagy through the activation of the CaMKK-AMPK-mTOR pathway and mitochondrial damage. In two TNBC xenograft mouse models, RL71 also displayed strong efficacy including the inhibition of tumor growth, the reduction of metastasis, as well as the prolongation of survival time. These findings suggest SERCA2 as a previous unknown target candidate for TNBC treatment and support the idea that autophagy inducers could be useful as new therapeutics in TNBC treatment.