From Plant to Patient: Thapsigargin, a Tool for Understanding …
83
9.2 Thapsigargin Induces Cell death, ER Stress and Growth
Arrest, and Inhibits Autophagy
Inhibition of SERCA causes efflux of Ca
2+ from the ER into the cytosol [129]. The
ER Ca
2+ depletion provokes SOCE and an inflow of extracellular Ca
2+ to give a high
nanomolar cytosolic concentration. Studies at the single-cell level have indicated
that after the initial Ca
2+ rise, the cytosolic Ca
2+ levels decrease to baseline, but after
12 to 36 h a second rise to micromolar cytosolic concentration occurs [130–132].
This second rise in Ca
2+ concentration occurs asynchronously within the cell population and is associated with rapidly ensuing morphological and biochemical changes
related to apoptosis [130–133]. These studies, together with general indications of
the cytotoxic actions of increased cytosolic Ca
2+ levels [134], suggested a role for
cytosolic Ca
2+ in thapsigargin-induced cell death, which may involve calmodulin
activation [132]. However, this mechanism may be restricted to certain cell types
since studies in S49 T-lymphoma cells, LNCaP and PC3 prostate cancer cells, and
MCF7 breast cancer cells indicate that a rise in cytosolic Ca
2+ levels is not required
for thapsigargin to induce cell death [135–138]. Instead, more general critical initiating factors of thapsigargin-induced cell death appear to be ER Ca
2+ depletion and
the resulting unfolded protein response (UPR) [129, 138, 139]. The absence of Ca
2+
in the ER prevents the proteins from folding correctly. Initially, the cell stops protein
expression, degrades misfolded proteins and mobilizes chaperones involved in the
protein-folding process [140].
Prolonged ER stress, however, initiates the apoptotic switch. Thapsigargininduced apoptosis requires prolonged ER Ca
2+ depletion and a sustained UPR [129,
138] and involves distinct contributions from UPR components (Fig. 9) [139]. The
factors ATF4 and CHOP upregulate the expression of death receptor 5 [139], which
is strictly required for thapsigargin-mediated activation of caspase-8, caspase-3, and
cell death in LNCaP cells and HCT116 colorectal cancer cells [139, 141, 142]. The
UPR transcription factors ATF4 and CHOP also upregulate MAP1LC3B (LC3B),
which through a non-autophagic mechanism, contributes to caspase-8 activation
[139]. For thapsigargin-induced cell death, PERK is required in both LNCaP and
HCT116 cells but acts independently of ATF4, CHOP, DR5, and LC3B [139]. Also,
IRE1 appears to play a cell-type-dependent role since it is required for thapsigargininduced cell death in LNCaP cells [139] and mouse embryonic fibroblasts [143],
but not in HCT116 cells [139] or MCF10A breast epithelial cells [144]. Part of the
explanation for this is suggested by the recent discovery of an IRE1-XBP1-dependent
pathway that leads to sustained activation of JNK, which acts in a pro-apoptotic
manner in LNCaP cells but not in HCT116 cells [139, 145].
Treatment of cells with thapsigargin blocks the intracellular lysosomal degradation pathway autophagy via perturbation of intracellular Ca
2+ , and independently
of apoptosis and the UPR [146]. Thapsigargin also inhibits cell proliferation, and
it can do this even at sub-cytotoxic concentrations [128, 138]. Detailed analyses
of thapsigargin-mediated effects in LNCaP and PC3 cells indicate that partial ER
Ca
2+ depletion (obtained with low concentrations of thapsigargin) is sufficient to
83
9.2 Thapsigargin Induces Cell death, ER Stress and Growth
Arrest, and Inhibits Autophagy
Inhibition of SERCA causes efflux of Ca
2+ from the ER into the cytosol [129]. The
ER Ca
2+ depletion provokes SOCE and an inflow of extracellular Ca
2+ to give a high
nanomolar cytosolic concentration. Studies at the single-cell level have indicated
that after the initial Ca
2+ rise, the cytosolic Ca
2+ levels decrease to baseline, but after
12 to 36 h a second rise to micromolar cytosolic concentration occurs [130–132].
This second rise in Ca
2+ concentration occurs asynchronously within the cell population and is associated with rapidly ensuing morphological and biochemical changes
related to apoptosis [130–133]. These studies, together with general indications of
the cytotoxic actions of increased cytosolic Ca
2+ levels [134], suggested a role for
cytosolic Ca
2+ in thapsigargin-induced cell death, which may involve calmodulin
activation [132]. However, this mechanism may be restricted to certain cell types
since studies in S49 T-lymphoma cells, LNCaP and PC3 prostate cancer cells, and
MCF7 breast cancer cells indicate that a rise in cytosolic Ca
2+ levels is not required
for thapsigargin to induce cell death [135–138]. Instead, more general critical initiating factors of thapsigargin-induced cell death appear to be ER Ca
2+ depletion and
the resulting unfolded protein response (UPR) [129, 138, 139]. The absence of Ca
2+
in the ER prevents the proteins from folding correctly. Initially, the cell stops protein
expression, degrades misfolded proteins and mobilizes chaperones involved in the
protein-folding process [140].
Prolonged ER stress, however, initiates the apoptotic switch. Thapsigargininduced apoptosis requires prolonged ER Ca
2+ depletion and a sustained UPR [129,
138] and involves distinct contributions from UPR components (Fig. 9) [139]. The
factors ATF4 and CHOP upregulate the expression of death receptor 5 [139], which
is strictly required for thapsigargin-mediated activation of caspase-8, caspase-3, and
cell death in LNCaP cells and HCT116 colorectal cancer cells [139, 141, 142]. The
UPR transcription factors ATF4 and CHOP also upregulate MAP1LC3B (LC3B),
which through a non-autophagic mechanism, contributes to caspase-8 activation
[139]. For thapsigargin-induced cell death, PERK is required in both LNCaP and
HCT116 cells but acts independently of ATF4, CHOP, DR5, and LC3B [139]. Also,
IRE1 appears to play a cell-type-dependent role since it is required for thapsigargininduced cell death in LNCaP cells [139] and mouse embryonic fibroblasts [143],
but not in HCT116 cells [139] or MCF10A breast epithelial cells [144]. Part of the
explanation for this is suggested by the recent discovery of an IRE1-XBP1-dependent
pathway that leads to sustained activation of JNK, which acts in a pro-apoptotic
manner in LNCaP cells but not in HCT116 cells [139, 145].
Treatment of cells with thapsigargin blocks the intracellular lysosomal degradation pathway autophagy via perturbation of intracellular Ca
2+ , and independently
of apoptosis and the UPR [146]. Thapsigargin also inhibits cell proliferation, and
it can do this even at sub-cytotoxic concentrations [128, 138]. Detailed analyses
of thapsigargin-mediated effects in LNCaP and PC3 cells indicate that partial ER
Ca
2+ depletion (obtained with low concentrations of thapsigargin) is sufficient to