Pathophysiological mechanisms contributing to prostate cancer-induced bone pain

Muralidharan, Arjun (2013). Pathophysiological mechanisms contributing to prostate cancer-induced bone pain PhD Thesis, School of Pharmacy, The University of Queensland.

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Author Muralidharan, Arjun
Thesis Title Pathophysiological mechanisms contributing to prostate cancer-induced bone pain
Formatted title
School, Centre or Institute School of Pharmacy
Institution The University of Queensland
Publication date 2013
Thesis type PhD Thesis
Supervisor Maree T. Smith
Bruce Wyse
Total pages 197
Total colour pages 25
Total black and white pages 172
Language eng
Subjects 1109 Neurosciences
1115 Pharmacology and Pharmaceutical Sciences
Formatted abstract
My PhD research project was designed to optimise a rat model of prostate cancer-induced bone pain (PCIBP) and to characterise this model using pharmacological, radiological, histological, molecular biological and immunohistochemical methods. I then used this optimised rat model to gain novel insight into the pathophysiology of PCIBP.

In the first part of my project, I systematically varied the number of AT3B prostate cancer cells (APCCs) administered by unilateral intratibial injection in groups of rats, and assessed the comparative effects on temporal development of pain behaviour and general health in these animals. The main finding was that unilateral intratibial injection (ITI) of 4×104 APCCs
produced bilateral hindpaw hypersensitivity that was fully developed between days 14 and 21, secondary to confined development of osteosclerotic bone tumours, whilst maintaining good general animal health for at least 90 days post- ITI. Additionally, there was apparent spontaneous resolution of bilateral hindpaw hypersensitivity from day 21 that was maintained until at least day 90 post-ITI. Importantly, as single bolus dose of a prototypic opioid receptor antagonist, naloxone, administered during the intervals, 28-37 days and 85-90 days post-ITI rescued the hindpaw hypersensitivity, my findings implicate a role for upregulated endogenous opioidergic signaling, in masking pain associated with advanced prostate cancerinduced bony metastases.

Next, I pharmacologically characterised this optimised rat model of PCIBP using commonly available analgesic and adjuvant drugs. The estimated mean ED50 doses for morphine, gabapentin, meloxicam and amitriptyline for relief of mechanical allodynia in the ipsilateral hindpaws were 1.9, 78.0, 2.6 and 14.9 mg/kg respectively. The estimated mean ED50 doses for gabapentin and meloxicam for relief of thermal hyperalgesia in the ipsilateral hindpaws were 37.4 and 2.7 mg/kg respectively.

The optimized rat model of PCIBP was further characterized using immunohistochemical methods. Specifically, I examined the expression levels of markers of neuronal and nonneuronal cell activation in the lumbar dorsal root ganglia (DRGs) of these animals using specific immunofluorescently-labelled antibodies. Markers investigated included angiotensin II, angiotensin II type 2 receptor (AT2R), substance P (SubP), nerve growth factor (NGF) and its high affinity receptor, tyrosine kinase A (TrkA), phosphorylated-p38 mitogen-activated protein kinases (MAPK) (pp38 MAPK) and phosphorylated-p44/p42 MAPK (pp44/pp42 MAPK), all of which are implicated in DRG sensory neuron hyper-excitability. The mean levels of immunofluorescence (IF) for angiotensin II, TrkA, NGF, pp38 MAPK and pp44/pp42 MAPK were significantly increased in the bilateral lumbar DRGs of PCIBP-rats c.f. the ipsilateral lumbar DRGs of sham-control rats. My findings showing augmented levels of angiotensin II and activation of p38 MAPK and p44/p42 MAPK in the lumbar DRGs of rats with PCIBP, are highly reminiscent of previous findings from our laboratory in the unilateral chronic constriction injury (CCI) of the sciatic nerve rat model of neuropathic pain.  Specifically, our laboratory’s previous work showed that angiotensin II signaling via the AT2R induced p38 MAPK and p44/p42 MAPK activation in the lumbar DRGs and that this was blocked at the time of peak effect of an analgesic dose of EMA300, a small molecule AT2R antagonist.

To assess a possible role for angiotensin II signalling via the AT2R in the pathobiology of PCIBP, I next examined the efficacy of single bolus intravenous (i.v.) doses of PD123319, a small molecule AT2R antagonist with >1000-fold selectivity over the angiotensin type 1 receptor, in rats with fully developed bilateral hindpaw hypersensitivity. My results show for the first time that PD123319 produced dose-dependent relief of mechanical allodynia and thermal hyperalgesia in the ipsilateral hindpaws with estimated mean ED50 values of 0.8 and 3.9 mg/kg, respectively.

Finally, the effects of PD123319 on the expression levels of putative chronic pain markers in the ipsilateral lumbar DRGs of rats with prostate cancer-induced bone pain, were examined. Briefly, at the time of peak effect of a single bolus dose of PD123319 (3mg/kg i.v.), the mean IF levels for angiotensin II, NGF, pp38 MAPK and pp44/pp42 MAPK, but not TrkA, were significantly (p<0.05) decreased when assessed using immunohistochemical and/or western blotting methods. Additionally, there were no significant changes (p>0.05) in the IF levels for the AT2R or Sub P in the ipsilateral lumbar DRGs of PCIBP-rats administered PD123319 c.f. drug-naïve rats.

In summary, during the course of my PhD, I have successfully established and characterised an optimised rat model of prostate cancer-induced bone pain. Additionally, I have shown that a selective small molecule AT2R antagonist produced dose-dependent relief of hindpaw hypersensitivity in this rat model by a mechanism involving inhibition of augmented angiotensin II signalling via the AT2R to attenuate NGF/TrkA signaling, with the net result being inhibition of p38 MAPK and p44/p42 MAPK activation, in the lumbar DRGs. Thus my findings, for the first time, implicate a role for augmented angiotensin II/AT2R signaling in the pathophysiology of prostate cancer-induced bone pain. Hence, small molecule AT2R
antagonists are worthy of further investigation as novel analgesics for the treatment of metastatic bone pain associated with prostate cancer.
Keyword Prostate cancer induced bone pain (PCIBP)
Mechanical allodynia
Thermal hyperalgesia
Bone histology
Angiotensin II
Angiotensin II type 2 receptor (AT2R)
Phospho-p38 mitogen activated protein kinase (MAPK)
Phospho-p44/p42 MAPK

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Created: Fri, 28 Jun 2013, 22:53:49 EST by Mr Arjun Muralidharan on behalf of Scholarly Communication and Digitisation Service