Bromocriptine and Dopamine Mediate Independent and Synergistic Apoptotic Pathways in Pituitary Cells

Abstract

Dopamine (DA) agonists are the primary treatment choice for prolactinoma, effectively suppressing prolactin expression and reducing tumour size. However, the intracellular pathway(s) through which either DA or its agonists impact on proliferation or lead to tumour shrinkage are incompletely understood. To identify the mediators in the apoptotic cascades after DA or DA agonist challenges we used a well-characterized model system, the rodent somatolactotroph cell line GH3. In these cells, we show that apoptosis induced by the DA agonist bromocriptine (BC), but not DA, is initiated through activation of the JNK pathway. However, both DA and BC activate the terminal effector caspase, caspase-3. Kinetic studies and chemical inhibitor co-incubation experiments support a role for JNK activation preceding caspase-9 activation in BC challenged cells, however, engagement of these mediators was not apparent in DA challenge cells. These studies suggest that apoptosis induced by BC or DA is mediated through distinct and independent pathways that converge with activation of the terminal caspase, caspase-3. These observations were further reinforced by our findings that DA and BC, in co-incubation experiments, synergistically induce apoptosis. These findings raise the possibility that drugs acting through the same pathway as DA may be clinically beneficial when combined with BC.

Reversal of Endogenous Dopamine Receptor Silencing in Pituitary Cells Augments Receptor-Mediated Apoptosis

“…However, the DA-and BC-mediated apoptotic pathways converge to activate the terminal caspase-caspase-3. Coincubation experiments in these cells reinforced and extended these findings where we observed a synergistic increase in apoptotic end points (12).…”

“…Although this transporter represents an attractive alternate mechanism a recent study from our laboratory, using this same model system, namely, the rodent pituitary cell line GH3, reached a different conclusion. In this case, we showed that both DA and BC elicits an apoptotic response in these cells in the absence of a either a D2R or a DAT (12). Moreover, characterization of the intracellular apoptotic pathways engaged by these drugs show them to be distinct.…”

“…As described previously (12,20), low passage GH3 or MMQ cells were seeded into six-well plates (Sarstedt, Leicestershire, UK) until they were between 30 -50% confluence. Cells were then treated with 20 nM of either siD2R or siNT using Lipofectamine 2000 (Invitrogen, Paisley, UK) according to the manufacturer's instructions.…”

“…Cells were then stained using Hoechst 33342 (Molecular Probes, Paisley, UK) or analyzed for DNA fragmentation using a commercial terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) labeling kit (Promega, Southampton UK) as described previously (12).…”

“…Semiconfluent cultures of GH3-D2exp and GH3-D2veh cells, in 96-well plates, were challenged with drugs for 6 h and activated caspases detected using a commercial kit that specifically detects the terminal caspases (Caspase 3 and 7) as previously described (12,20). Assays were performed as suggested by the suppliers (Promega, Southampton UK) in triplicate determinations and experiments repeated at least three times.…”