Ca2+ influx and ROS generation trigger CDDO-Me-induced dilation of the ER and apoptosis in breast cancer cells

OBJECTIVE The synthetic triterpenoid 2-cyano-3,12-dioxoolean-1,9(11)-dien-C28-methyl ester(CDDO-Me)is considered a promising anti-tumorigenic compound.In this study,we investigated the anti-cancer effect of CDDO-Me on breast cancer cells and its underlying mechanisms.METHODS To investigate the effect of CDDO-Me on various breast cancer cells,cell viability assay using calcein-AM and EthD-1 as well as MTT assay was performed.To clarify the origin of CDDO-Me-induced vacuoles,electron microscopy as well as fluorescence microscopy using YFP-ER or YFP-Mito construct was performed.To measure the changes in intracellular Ca2+and ROS levels,flow cytometry using Fluo-3 and H2DCF-DA was performed.RESULTS CDDO-Me treatment induces progressive ER-derived vacuolation and subsequent apoptosis in various breast cancer cells.CDDO-Me-induced increases in intracellular Ca2+ levels,reflecting influx from the extracellular milieu,make a critical contribution to ER-derived vacuolation and subsequent cell death.In parallel with increasing 2+ Calevels,CDDO-Me markedly increases the generation of reactive oxygen species(ROS).Interestingly,we found that there exists a reciprocal positive-regulatory loop between Ca2+ influx and ROS generation that triggers ER stress and ER dilation in response to CDDO-Me.CONCLUSION ER-derived vacuolation via Ca2+ influx and ROS generation is responsible for the potent anticancer effects of CDDOMe on breast cancer cells.

Combination of kaempferol and chloroquine induces glioma cell death via expansion and subsequent rupture of lysosomes

OBJECTIVE Chloroquine is considered as a potential chemotherapy and radiotherapy sensitizer,but the anticancer effect of chloroquine alone is limited.Since we found that the flavonoid kaempferol effectively sensitizes glioma cells to chloroquine-mediated cell death,we investigated the underlying mechanisms of glioma cell death induced by the combination of kaempferol and chloroquine.METHODS To examine the effect of kaempferol and/or chloroquine on various glioma cells,cell viability assay using calcein-AM and EthD-1was performed.The changes in the lysosomal structures following treatment with kaempferol and/or chloroquine were observed by electron microscopy and fluorescence microscopy using acridine orange or Lyso-tracker Red.The changes in cathepsin D proteins were analyzed by Western blotting,immunocytochemistry,and fluorescence microscopy using BODIPY FL-pepstatin.RESULTS Treatment with subtoxic doses of chloroquine,when combined with kaempferol,effectively induced cell death in various glioma cells,but not in normal astrocytes.While kaempferol treatment increased the numbers of lysosome,chloroquine treatment increased lysosomal masses.Combined treatment with kaempferol and chloroquine induced the expansion and subsequent rupture of lysosomes,leading to the spillage of the lysosomal contents into the cytosol.We found that while kaemfperol treatment increased the active mature forms of cathepsin D,chloroquine treatment completely blocked the processing of cathepsin D.The processing of cathepsin D was also blocked by the combined treatment,but the activity of cathepsin D,which was released from the lysosomes,was restored.The cell death induced by kaempferol and chloroquine in U251 MG cells was accompanied by mitochondrial dysfunction,ER stress,and DNA damage.CONCLUSION Disruption of lysosomal membrane integrity and a resultant release of lysosomal proteases may critically contribute to the irreparable damage of various organelles and glioma cell death by chloroquine plus kaempferol.

副凋亡:肿瘤细胞克服凋亡抵抗的途径(英文)

Since inherent or acquired cellular resistance to various pro-apoptotic treatments often leads to therapeutic failure,a better understanding of alternative non-apoptotic pathways may facilitate the design of novel therapeutics against malignant cancer cells.Paraptosis is a cell death mode characterized by extensive vacuolization that arises via dilation of the endoplasmic reticulum(ER)and mitochondria,without any apoptotic characteristics.We found that curcumin,dimethoxycurcumin(a more stable analog of curcumin),and celastol kill malignant cancer cells via induction of paraptosis as a main cell death mode.Study on the underlying mechanisms of paraptosis revealed that simultaneous proteasomal inhibition and mitochondrial Ca2+ overload can effectively induce paraptosis in cancer cells.Mitochondrial Ca2+ overload can be achieved not only by inhibition of mitochondrial Na+/Ca2+ exchanger but also by IP3 receptor-or ryanodine receptor-mediated release of Ca2+ from the ER and its subsequent mitochondrial Ca2+ uniporter-mediated Ca2+ influx into mitochondria.Since malignant cancer cells are more vulnerable to oxidative stress and ER stress than normal cells,clarification of the molecular basis of paraptosis that targets mitochondria and the ER at the same time may provide a rational therapeutic strategy for effectively killing resistant cancer cells.