P2X7 and Cancer
Presented by:
Extracellular ATP as a messenger molecule
P2 receptors
P2X7 receptor
P2X7 receptor and Apoptosis
P2X7 splice variants
P2X7 as a novel biomarker
Outline
Simultaneously or one after the other, three significant independent functions of adenine compounds have been developed in living cells early in the course of evolution.
Containing high-energy phosphate groups,
As part of adenine nucleotides,
They have served as interacellular signaling molecules.
Adenine compounds
The concept of a purinergic signaling system, using purine nucleotides and nucleosides as extracellular messengers, was first proposed over 30 years ago.
purines and pyrimidines mediate:
short-term (acute) signaling functions :
Neurotransmission, secretion
long-term (chronic) signaling functions:
in cell proliferation, differentiation, and death involved in development and regeneration.
Purinergic signaling
ATP is not only the universal currency of energy metabolism but also a ubiquitous extracellular messenger.
Platelet aggregation
Pain sensation
Cytokine secretion
Cell proliferation
Cell differentiation
Cell death
…
ATP (Adenosine nucleotide triphosphate)
P receptors = purinergic receptor = purinoreceptor
P receptors
P1 receptors
P2 receptors
A1
A2A
A2B
A3
P2X1-7
P2Y1,2,4,6,11,12,13,14
P2X
P2X receptors are ATP-gated ion channels that mediate sodium influx, potassium efflux and, to varying extents, calcium influx, leading to depolarization of the cell membrane.
P2Y
P2Y receptors are G-protein-coupled receptors (GPCRs)
that are activated by purine and/or pyrimidine nucleotides.
P2 receptors
N-terminal
C-terminal
TM1
TM2
P2X7 is unique among the family in that
Has a much larger carboxy terminus than other members of the P2X family.
Continued stimulation results in the formation of a larger pore.
Further activation of P2X7 in several cell types results in the induction of apoptosis.
The P2X7 gene comprises 13 exons and is located on chromosome 12q24. The P2X7 gene product is a 595 amino acid protein.
P2X7 receptor
12
13
C-terminal
Activation of P2X7 receptor lead to:
Formation of a large pore in the cell membrane permeable to molecules of up to 900 Da in molecular weight.
Release of numerous molecules such as caspases (caspase-1, caspase-3, caspase-9), IL-1β, kinases and phosphatases.
Re-organization of cytoskeletal structures which are directly connected to the long intracellular C-terminal ‘tail’ of the P2X7 receptor.
P2X7 receptor function
APOPTOSIS
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Stimulation of P2X7R triggers all of the known morphological changes of apoptosis:
Membrane blebbing
Shrinkage
Nuclear condensation
Fragmentation
Formation of apoptotic bodies
P2X7 receptor and Apoptosis
These morphological changes are paralleled by the biochemical hallmarks:
DNA fragmentation,
Disruption of nuclear lamins,
Exposure of phosphatidylserine
P2X7R is a membrane channel/pore that would be an ideal system for the investigation of the role of intracellular ions in apoptosis.
P2X7 receptor and Apoptosis
Activation of P2X7 receptor cause Apoptosis through:
Perturbing intracellular ions concentration
Ca2+
K+
Direct activation of Caspases by P2X7
Death Domain
P2X7 receptor and Apoptosis
P2X7 receptor and Apoptosis
Cellular ionic homeostasis, fundamentally K+ homeostasis, has been implicated as a critical regulator of apoptosis.
K+, maintained at ~150mM, is a major ion in healthy cells.
High concentrations of extracellular K+ or K+ channel blockers suppress apoptosis.
K+ efflux and Apoptosis
K+ physiological concentration:
Inhibited the formation of the apoptosome
Inhibited the processing of procaspase-3
Inhibited the nucleosomal DNA fragmentation
Inhibited the nuclear fragmentation
P2X7 receptor and Apoptosis
K+ efflux and Apoptosis
P2X7 receptor and Apoptosis
K+ efflux and Apoptosis
P2X7 receptor and Apoptosis
Ca2+ influx and Apoptosis
Calcineurin
Inactive
Ca 2+
Ca 2+
Ca 2+
Calcineurin
Active
BAD
P
Bcl-2
Bcl-xL
BAD
Cyto C
Cyto C
Cyto C
Apoptosome
Apoptosis
P2X7 receptor and Apoptosis
Death Domain
caspase3
caspase8
Apoptosis
MAPk
Promote
Apoptosis
?
P2X7 receptor splice variants
P2X7-j splice variant
Was identified in
cancer cervical cells.
lacks:
the entire intracellular carboxy terminus,
the second transmembrane domain,
the distal third of the extracellular loop of the full-length
P2X7 receptor.
it showed:
Diminished ligand-binding,
Failed to form pores,
Failed to mediate apoptosis.
The P2X7-j interacted with the full-length P2X7 in a manner suggesting hetero-oligomerization and blocked the P2X7-mediated actions such as apoptosis.
Because in cancer cervical cells the P2X7-j is co-expressed with the wild-type P2X7, it is hypothesized that co-expression of the P2X7-j could lead to defective apoptosis and enhance the growth of the cancer cervical cells.
P2X7-j splice variant
= P2x7
= P2x7-j
+
+
+
Apoptosis
Apoptosis
P2X7-mediated apoptosis are lower in cancer cells than in normal cells.
The differences were not the result of ligand availability since steady-state levels of ATP in conditioned media of cancer epithelial cells were similar to those of normal epithelial cells.
Similarly, there were no significant differences in P2X7 receptor activation, oligomerization or cycling between normal and cancer cells.
P2x7 and cervix cancer
In contrast, the differences were associated with lower P2X7 mRNA and protein levels in the cancer cells than in the corresponding normal epithelial cells.
These findings are biologically and clinically important because defective apoptosis may lead to cancer.
P2x7 and cancer
The data suggest that the reduced expression of P2X7 in cancer epithelial cells is the result of high steady state levels of miR-186 and miR-150 in cancer cells which activate the instability domains and decrease P2X7 mRNA levels possibly by inducing degradation of the transcript.
These data suggest that miR-186 and miR-150 stimulate a decrease in P2X7 mRNA steady state levels by targeting instability.
P2x7 and miRNA
The P2X7 Receptor: A Novel Biomarker of
Uterine Epithelial Cancers
Endometrial epithelial cancers
Cervical cancer
P2X7 as a novel biomarker
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