-Hederin induces paraptosis by targeting GPCRs to activate Ca²⁺/MAPK signaling pathway in colorectal cancer

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 alpha-hederin (alpha-hed) induces caspase-independent paraptotic modes of cell death. Purpose: The present study is aimed to investigate the role of alpha-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 alpha-hed. Molecular docking methods were used to evaluate binding abilities of alpha-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: alpha-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 alpha-hed. Besides, both Swiss prediction and molecular docking showed that the structure of alpha-hed could tightly target to GPCRs. GPCRs were reported to activate the phospholipase C (PLC)-beta 3/ inositol 1,4,5-trisphosphate receptor (IP3R)/ Ca2+/ protein kinase C alpha (PKC alpha) pathway, and we then found all proteins and mRNA expressions of PLC beta 3, IP3R, and PKC alpha were increased by alpha-hed. After blocking the GPCR signaling, alpha-hed could not elevate Ca2+ level and showed less CRC cell cytotoxicity. MAPK cascade is the symbol of paraptosis, and we then demonstrated that alpha-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 alpha-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 alpha-hed as a promising candidate for inducing non-apoptotic cell death, paraptosis. It may overcome the resistance of apoptotic-based chemo-resistance in CRC.