Purpose: Indirubin-3'-monoxime (I3M) induces cell death in many cancer cells; however, whether I3M regulates paraptosis is unclear. The present study aimed to investigate I3M-induced paraptosis.

Methods: We treated various cancer cells with I3M, and measured vacuole formation (a paraptosis marker) and the regulating signaling pathway such as endoplasmic reticulum (ER) stress, reactive oxygen species, and proteasomal dysfunction.

Results: We found that I3M induced small vacuole formation in MDA-MB-231 breast cancer cells and transient knockdown of eIF2α and CHOP significantly downregulated vacuolation in the ER and mitochondria, as well as cell death in response to I3M, indicating that I3M-meditaed paraptosis was upregulated by ER stress. Moreover, I3M accumulated ubiquitinylated proteins via proteasome dysfunction, which stimulated ER stress-mediated Ca²⁺ release. A Ca²⁺ chelator significantly downregulated vacuolation in the ER and mitochondria as well as cell death, suggesting that Ca²⁺ was a key regulator in I3M-induced paraptosis. Our results also revealed that Ca²⁺ finally transited in mitochondria through mitochondrial Ca²⁺ uniporter (MCU), causing I3M-mediated paraptosis; however, the paraptosis was completely inhibited by, ruthenium red, an MCU inhibitor.

Conclusion: I3M induced proteasomal dysfunction-mediated ER stress and subsequently promoted Ca²⁺ release, which was accumulated in the mitochondria via MCU, thus causing paraptosis in MDA-MB-231 breast cancer cells.