Abstract
Calcium (Ca2+) is a fundamental regulator of cell signaling and function. Thapsigargin (Tg) blocks the sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA), disrupts Ca2+ homeostasis, and causes cell death. However, the exact mechanisms whereby SERCA inhibition induces cell death are incompletely understood. Here, we report that low (0.1 μm) concentrations of Tg and Tg analogs with various long-chain substitutions at the O-8 position extensively inhibit SERCA1a-mediated Ca2+ transport. We also found that, in both prostate and breast cancer cells, exposure to Tg or Tg analogs for 1 day caused extensive drainage of the ER Ca2+ stores. This Ca2+ depletion was followed by markedly reduced cell proliferation rates and morphological changes that developed over 2-4 days and culminated in cell death. Interestingly, these changes were not accompanied by bulk increases in cytosolic Ca2+ levels. Moreover, knockdown of two key store-operated Ca2+ entry (SOCE) components, Orai1 and STIM1, did not reduce Tg cytotoxicity, indicating that SOCE and Ca2+ entry are not critical for Tg-induced cell death. However, we observed a correlation between the abilities of Tg and Tg analogs to deplete ER Ca2+ stores and their detrimental effects on cell viability. Furthermore, caspase activation and cell death were associated with a sustained unfolded protein response. We conclude that ER Ca2+ drainage and sustained unfolded protein response activation are key for initiation of apoptosis at low concentrations of Tg and Tg analogs, whereas high cytosolic Ca2+ levels and SOCE are not required.
Highlights
Calcium (Ca2؉) is a fundamental regulator of cell signaling and function
The first part of the Tg binding curve is linear (Fig. 2A) because it represents virtually complete binding of Tg to sarco/endoplasmic reticulum (ER) Ca2؉-ATPase (SERCA) such that the activity decreases linearly with the added concentration of Tg and can be used to estimate the fraction of SERCA in the sample that is complexed by Tg
When matching LD50(Tg-max) values for Tg and individual Tg analogs across the three cell lines, we found that PC3 cells had slightly lower or similar LD50(Tg-max) values compared with MCF7 cells (0.8 –1.9-fold difference compared with PC3 cells where among the Tg analogs only epoxy derivative of thapsigargin (EpoTg) showed a lower LD50(Tg-max) in MCF7 cells), whereas LNCaP cells had much higher LD50(Tg-max) values (6 –20-fold higher LD50(Tg-max) values compared with those observed in PC3 cells)
Summary
Inhibition of ATPase activity—The effect of the Tg analogs and Tg on Ca2ϩ-ATPase activity of purified SERCA1a was studied by a protocol that allows the measurement of both high affinity and kinetic aspects of the binding process after preincubation of the protein with gradually increasing inhibitor concentrations (Fig. 2). Inspection and analysis of the semilogarithmic data for Leu8ADT and Asp-8ADT show that their mechanism of action is consistent with a sequential reaction scheme where first one Ca2ϩ is removed from Ca2E1, accompanied by binding of the inhibitor, to form a Ca2ϩ plus inhibitor complex (CaE–Inh), which in a following reaction involves the dissociation of the second bound Ca2ϩ, leading to the formation of an E2–Inh complex as shown by Reactions 1 and 2 below. This can be compared with the less than 1 min required for 50% conversion of Ca2E1 to CaE–Inh with 12 M Leu-8ADT or
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