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 Ca release. A Ca chelator significantly downregulated vacuolation in the ER and mitochondria as well as cell death, suggesting that Ca was a key regulator in I3M-induced paraptosis. Our results also revealed that Ca finally transited in mitochondria through mitochondrial Ca 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 Ca release, which was accumulated in the mitochondria via MCU, thus causing paraptosis in MDA-MB-231 breast cancer cells.