Abstract
1-(2-Chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195) is a proven enhancer of apoptotic cell death in a variety of cellular models. This effect is independent of its established cellular target, the mitochondrial benzodiazepine receptor (mBzR), since it is able to promote cell death also in mBzR knockout cells. Thus recently it was suggested that PK11195 might exert its effect by modulating the expression and function of the oncogene Bcl-2. We have previously demonstrated that Bcl-2 modulates cellular Ca2+ homeostasis as its overexpression reduces the Ca2+ concentration in the endoplasmic reticulum (ER) ([Ca2+]er), impairing mitochondrial and cytosolic Ca2+ overload during cellular stress and therefore inhibiting the induction of the apoptotic cascade. Here, using ER, mitochondria and cytosolic targeted aequorin probes, we show that cellular treatment with PK11195 induces opposite changes in cellular Ca2+ homeostasis, increasing the [Ca2+]er and amplifying IP3 induced Ca2+ transients in mitochondria ([Ca2+]m) and cytosol ([Ca2+]c). This work provides evidence for a novel pharmacological effect of PK11195 on Ca2+ signalling which may be linked to its effect on Bcl-2 and account for its role in apoptotic cell death.
Highlights
1-(2-Chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195) is a proven enhancer of apoptotic cell death in a variety of cellular models
To avoid consumption of the aequorin, the luminal [Ca2+] of these organelles must be reduced prior to the reconstitution of the probe with prostethic group coelenterazine. This was obtained by incubation of the cells in Krebs Ringer Buffer (KRB) supplemented with the low affinity coelenterazinen and 2.5 μMionomycin, a Ca2+ ionophore, in the absence of extracellular Ca2+
Using genetically encoded Ca2+ indicators, we have demonstrated that pharmacological treatment with PK11195 modulates intracellular Ca2+ signaling in HeLa cells independently of mitochondrial benzodiazepine receptor (mBzR) binding and consistently with a direct inhibition of Bcl-2 effect on cellular Ca2+ homeostasis
Summary
1-(2-Chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195) is a proven enhancer of apoptotic cell death in a variety of cellular models. Members of the Bcl-2 gene family are known to exert their pro- or anti-apoptotic effect determining the state of mitochondrial permeability, by promoting or inhibiting respectively the release of proapoptotic factors like cytochrome c (cyt-c) [21] and apoptosis inducing factor (AIF) from the intermembrane space [22,23,24]. To this aim, a tuned regulation of Ca2+ fluxes is essential and a role for this family of proteins in regulating ion fluxes has been long proposed [25]. We propose that the PK11195 induced alterations in Ca2+ signaling might be consequence of a direct blockade of Bcl-2 interaction with other Bcl-2 family members, relevant to cell death through mitochondrial membrane permeabilization
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