Background: Non‐apoptotic cell death is presently emerging as a potential direction to overcome the apoptosis resistance of cancer cells. In the current study, a natural plant agent α‐hederin (α‐hed) induces caspase‐independent paraptotic modes of cell death.Purpose: The present study is aimed to investigate the role of α‐hed induces paraptosis and the associated mechanism of it.Methods: The cell proliferation was detected by CCK‐8. The cytoplasm organelles were observed under electron microscope. Calcium (Ca²⁺) level was detected by flow cytometry. Swiss Target Prediction tool analyzed the potential molecule targets of α‐hed. Molecular docking methods were used to evaluate binding abilities of α‐hed with targets. The expressions of genes and proteins were analyzed by RT‐qPCR, western blotting, immunofluorescence, and immunohistochemistry. Xenograft models in nude mice were established to evaluate the anticancer effects in vivo.Results: α‐hed exerted significant cytotoxicity against a panel of CRC cell lines by inhibiting proliferation. Besides, it induced cytoplasmic vacuolation in all CRC cells. Electron microscopy images showed the aberrant dilation of endoplasmic reticulum and mitochondria. Both mRNA and protein expressions of Alg‐2 interacting proteinX (Alix), the marker of paraptosis, were inhibited by α‐hed. Besides, both Swiss prediction and molecular docking showed that the structure of α‐hed could tightly target to GPCRs. GPCRs were reported to activate the phospholipase C (PLC)‐β3/ inositol 1,4,5‐trisphosphate receptor (IP3R)/ Ca²⁺/ protein kinase C alpha (PKCα) pathway, and we then found all proteins and mRNA expressions of PLCβ3, IP3R, and PKCα were increased by α‐hed. After blocking the GPCR signaling, α‐hed could not elevate Ca²⁺ level and showed less CRC cell cytotoxicity. MAPK cascade is the symbol of paraptosis, and we then demonstrated that α‐hed activated MAPK cascade by elevating Ca²⁺ flux. Since non‐apoptotic cell death is presently emerging as a potential direction to overcome chemo‐drug resistance, we then found α‐hed also induced paraptosis in 5‐fluorouracil‐resistant (5‐FU‐R) CRC cells, and it reduced the growth of 5‐FU‐R CRC xenografts.Conclusions: Collectively, our findings proved α‐hed as a promising candidate for inducing non‐apoptotic cell death, paraptosis. It may overcome the resistance of apoptotic‐based chemo‐resistance in CRC.

Background: Helicobacter pylori (H. pylori) accounts for the majority of gastric cancer (GC) cases globally. The present study found that H. pylori promoted GC stem cell (CSC)‐like properties, therefore, the regulatory mechanism of how H. pylori promotes GC stemness was explored.Methods: Spheroid‐formation experiments were performed to explore the self‐renewal capacity of GC cells. The expression of R‐spondin 3 (RSPO3), Nanog homeobox, organic cation/carnitine transporter‐4 (OCT‐4), SRY‐box transcription factor 2 (SOX‐2), CD44, Akt, glycogen synthase kinase‐3β (GSK‐3β), p‐Akt, p‐GSK‐3β, β‐catenin, and G protein subunit gamma 7 (GNG7) were detected by RT‐qPCR, western blotting, immunohistochemistry (IHC), and immunofluorescence. Co‐immunoprecipitation (CoIP) and liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) were performed to identify proteins interacting with RSPO3. Lentivirus‐based RNA interference constructed short hairpin (sh)‐RSPO3 GC cells. Small interfering RNA transfection was performed to inhibit GNG7. The in vivo mechanism was verified using a tumor peritoneal seeding model in nude mice.Results: H. pylori extracts promoted a CSC‐like phenotype in GC cells and elevated the expression of RSPO3. RSPO3 knockdown significantly reduced the CSC‐like properties induced by H. pylori. Previous studies have demonstrated that RSPO3 potentiates the Wnt/β‐catenin signaling pathway, but the inhibitor of Wnt cannot diminish the RSPO3‐induced activation of β‐catenin. CoIP and LC–MS/MS revealed that GNG7 is one of the transmembrane proteins interacting with RSPO3, and it was confirmed that RSPO3 directly interacted with GNG7. Recombinant RSPO3 protein increased the phosphorylation level of Akt and GSK‐3β, and the expression of β‐catenin in GC cells, but this regulatory effect of RSPO3 could be blocked by GNG7 knockdown. Of note, GNG7 suppression could diminish the promoting effect of RSPO3 to CSC‐like properties. In addition, RSPO3 suppression inhibited MKN45 tumor peritoneal seeding in vivo. IHC staining also showed that RSPO3, CD44, OCT‐4, and SOX‐2 were elevated in H. pylori GC tissues.Conclusion: RSPO3 enhanced the stemness of H. pylori extracts‐infected GC cells through the GNG7/β‐catenin signaling pathway.

The histone methyltransferase DOT1L, the sole enzyme catalyzing H3K79 methylation, is increasingly implicated in cancer progression, yet its role in shaping the tumor immune microenvironment (TME) remains unclear. Here, we demonstrate that DOT1L orchestrates immune evasion in lung adenocarcinoma (LUAD) through epigenetic activation of multiple immune checkpoints. Integrative analysis of TCGA and single-cell RNA-seq data revealed that high DOT1L expression correlates with poor prognosis, diminished cytotoxic immune-cell infiltration, and upregulation of inhibitory checkpoints (PD-L1, PD-1, LAG3, CD276, etc.). Mechanistically, ChIP-seq identified DOT1L-mediated H3K79me2 enrichment at promoters of JAK1/STAT3 genes, and some immune checkpoints, including LAG3, CD276, etc. Pharmacological DOT1L inhibition (SGC0946) suppressed the JAK1/STAT3/PD-L1 axis, reduced PD-1+ T cells in a vitro immune microenvironment. In vivo , SGC0946 attenuated lung metastasis, improved survival, and remodeled the TME by downregulating PD-L1, LAG3, and CD276 expression, reduced PD-1+ T cells subsets, and alongside with enhanced TNF-α, IFN-γ production. Clinical LUAD specimens further validated the correlation between DOT1L expression, STAT3 activation, and checkpoint upregulation, particularly in metastatic disease. Our findings identify DOT1L as an epigenetic linchpin of immune suppression.