research article radiotherapy suppresses bone cancer pain

Research ArticleRadiotherapy Suppresses Bone Cancer Pain throughInhibiting Activation of cAMP Signaling in Rat DorsalRoot Ganglion and Spinal Cord

Guiqin Zhu12 Yanbin Dong1 Xueming He1 Ping Zhao1 Aixing Yang12

Rubing Zhou1 Jianhua Ma1 Zhong Xie1 and Xue-Jun Song134

1Center for Clinical Research and Translational Medicine Lianyungang Oriental Hospital LianyungangJiangsu 222042 China2Department of Anesthesiology Lianyungang Oriental Hospital Lianyungang Jiangsu 222042 China3Division of Anesthesiology amp Pain Medicine Key Laboratory of Carcinogenesis and Translational ResearchMinistry of Education of China Beijing Cancer Hospital Peking University Beijing 100142 China4Department of Anesthesiology Beijing Cancer Hospital Peking University Beijing 100142 China

Correspondence should be addressed to Xue-Jun Song songxuejunbjmueducn

Received 24 July 2015 Revised 10 January 2016 Accepted 18 January 2016

Academic Editor Dianne Cooper

Copyright copy 2016 Guiqin Zhu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Radiotherapy is one of themajor clinical approaches for treatment of bone cancer pain Activation of cAMP-PKA signaling pathwayplays important roles in bone cancer pain Here we examined the effects of radiotherapy on bone cancer pain and accompanyingabnormal activation of cAMP-PKA signaling Female Sprague-Dawley rats were used and received tumor cell implantation (TCI)in rat tibia (TCI cancer pain model) Some of the rats that previously received TCI treatment were treated with X-ray radiation(radiotherapy) Thermal hyperalgesia and mechanical allodynia were measured and used for evaluating level of pain caused byTCI treatment PKA mRNA expression in dorsal root ganglion (DRG) was detected by RT-PCR Concentrations of cAMP IL-1120573and TNF-120572 as well as PKA activity in DRG and the spinal cord were measured by ELISA The results showed that radiotherapysignificantly suppressed TCI-induced thermal hyperalgesia and mechanical allodynia The level of PKA mRNA in DRG cAMPconcentration and PKA activity in DRG and in the spinal cord and concentrations of IL-1120573 and TNF-120572 in the spinal cord weresignificantly reduced by radiotherapy In addition radiotherapy also reduced TCI-induced bone loss These findings suggest thatradiotherapy may suppress bone cancer pain through inhibition of activation of cAMP-PKA signaling pathway in DRG and thespinal cord

1 Introduction

Pain is one of the most prevalent symptoms in patientswith primary bone sarcomas and with the distant metastasesof nonbone primary tumors [1 2] Studies have indicatedthat bone cancer pain has complex and unique mechanismsthat may involve both inflammatory and neuropathic pain[3] Clinically most patients with bone cancer have alreadypassed the optimal time for radical surgery and multidis-ciplinary therapies However with radiotherapy majorityof these patients experience pain relief control of tumor

growth and prolonged survival Radiotherapy is not only aneffective method in the clinical treatment of bone cancerbut also an important approach for treatment of the severepain associated with bone cancer However mechanismsunderlying radiotherapy for bone cancer pain have not beenwell investigated and remain elusive

Cyclic adenosine monophosphate-protein kinase A(cAMP-PKA) signaling pathway plays important roles in anumber of cellular processes including immune function [4]growth [5] differentiation [6] and metabolism [7] and isessential to the plasticity in neural synapses in CNS [8]

Hindawi Publishing CorporationMediators of InflammationVolume 2016 Article ID 5093095 8 pageshttpdxdoiorg10115520165093095

2 Mediators of Inflammation

Activation of cAMP-PKA pathway has been reported toenhance presynaptic neurotransmitter synthesis and vesicu-lar transportation probably through phosphorylation of keytranscriptional factors (ie cAMP response element-bind-ing protein) and synaptic vesicle proteins [9ndash11] In additionrecent studies found that cAMP-PKA signaling pathway isinvolved in both inflammatory pain [12ndash14] and neuropathicpain [15ndash17] The peripheral hyperalgesic actions of inflam-matory mediators are mediated by the cAMP-PKA signalingpathway [18]We have recently demonstrated that the cAMP-PKA pathway is crucial for the maintenance of dorsal rootganglia (DRG) neuronal hyperexcitability and behaviorallyexpressed hyperalgesia in an in vivo neuropathic pain animalmodel of chronic compression of the DRG (CCD model)as well as in an in vitro model of acute DRG dissociation[15 16 18] Recently we have further found that activation ofthe cAMP-PKA signaling pathway plays an important role inboth induction and maintenance of bone cancer pain in rats[19] However it remains unknown whether and then howcAMP-PKA signaling would contribute toward radiotherapytreatment for bone cancer painThis study provides evidencesupporting an idea that radiotherapy may suppress bonecancer pain through inhibition of abnormal activation ofcAMP-PKA signaling pathway in DRG and the spinal cord

2 Materials and Methods

21 Animals and Drugs Female adult Sprague-Dawley rats(160ndash180 g at the start of the experiment) were housed ina controlled lighting environment with free access to foodandwater All experiments were approved by the InstitutionalAnimal Care and Use Committees in Oriental Hospital andconducted in accordancewith theDeclaration of theNationalInstitutes of Health Guide for the Care and Use of LaboratoryAnimals (publication number 85-23 revised 1985) Surgerywas performed under anesthesia with intraperitoneal injec-tion of sodium pentobarbital (50mgkg ip)

22 Animal Model of Bone Cancer Pain The protocols ofthe bone cancer pain model were similar to that describedpreviously [19ndash21] In brief following induction of generalanesthesiawith intraperitoneal injection of sodiumpentobar-bital rats were placed abdominal side up After disinfectingwith 75 vv ethanol a one-centimeter rostrocaudal incisionwas made in the skin directly above the top half of the tibiaTumor cells (1 times 105 cells120583L 5 120583L) extracted from the asceticfluid of female rats that receivedWalker 256 mammary glandcarcinoma cells were injected (tumor cell implantation TCI)into the intramedullary space of the right tibia to inducebone cancer Injection site was closed with bone wax whilethe syringe was removed The incision was then dusted withpenicillin powder and closed Rats in the sham group wereinjected with the same number of boiled tumor cells

23 Radiotherapy A single dose of radiationwas given 9 daysafter TCI Rats were immobilized in an acryl jig and a doseof 6Gy was delivered to the right tibia area using a collimatorsystem with 6 MV X-rays [22] Sham radiotherapy applied in

TCI rats was using the same protocol except that they werenot given the real X-ray radiation

24 Radiographic Observation On the 17th day after TCItreatment radiographic images were taken (exposure setting12ms 31 KVp) using a Philips Digital Radiographer System(Digital DiagnostVM PhilipsMedical SystemsDMCGmbHHamburg Germany) Bone destruction was evaluated on ascale of 0ndash5 [20 23] 0 = normal bone structure withoutany sign of deterioration 1 = small radiolucent lesions in theproximal epiphysis (lt3) 2 = increased number of radiolucentlesions (gt3) indicating loss of medullary bone 3 = lossof medullary bone plus erosion of the cortical bone 4 =full-thickness unicortical bone loss and 5 = full-thicknessbicortical bone loss and displaced fractureThe experimenterwas blinded to the treatment of the samples

25 Behavioral Test Thermal hyperalgesia was indicated by asignificantly shortened latency of foot withdrawal in responseto heat stimulation To determine thermal hyperalgesia aradiant heat source was focused and delivered on a portion ofthe hind paw the thermal stimuli shut off automatically whenhind paw moved (or after 20 s to prevent tissue damage)Thermal stimuli were delivered 3 times to each hind paw at5ndash8-minute intervals Mechanical allodynia was indicated bya significant decrease in the threshold of paw withdrawal tomechanical indentation of the plantar surface of each hindpaw with a sharp cylindrical probe The probe was appliedto 6 designated loci distributed over the plantar surface ofthe foot The minimal force that induced paw withdrawalwas read off the display [19 24] The experimenters who per-formed these behavioral tests were blinded to the treatmentcondition of the animals

26 mRNA Isolation and RT-PCR The total RNA isolatedwith TRIzol Reagent (Invitrogen USA) was reverse tran-scribed using M-MLV reverse transcriptase (Takara) Primersets were synthesized by Integrated DNA Technologies (San-gon Biotech) their sequences are shown in Table 1 Theamplification conditions were set as follows 94∘C for 2minand 30 cycles of 94∘C for 30 s and 58∘C for 40 s PCRproducts were analyzed on agarose gel electrophoresis andwere verified by DNA sequencing The gels were imagedwith Tanon 2500 Imaging Systems and analyzed by analysissoftware ImageJ 148u

27 Measurement of cAMP IL-1120573 and TNF-120572 Levels and PKAActivity The DRG and the spinal cord at segments of L

4-L5

ipsilateral to TCI were collected on postoperative days 10 and14 for further neurochemical analysis Commercial enzyme-linked immunosorbent assay kits were used to determine theconcentrations of cAMP IL-1120573 and TNF-120572 and activity ofPKA according to the manufacturersrsquo instructions ELISAkit for cAMP was purchased from Cayman Chemical (AnnArbor Michigan USA) ELISA kits for IL-1120573 TNF-120572 andactivated PKA were purchased from RampD Systems (Min-neapolis Minnesota USA)

Mediators of Inflammation 3

Table 1 Primer sequences of the genes studies for RT-PCR

Primer name Forward ReversePKA-RII 5-ACCTCAGACGGCTCCCTTTG-3 5-CGTCTCCAACCGCATAAGCAG-3PKA-C 5-ACCTTGGGAACGGGTTCCTTCG-3 5-TACACCCAATGCCCACCAGTCC-3120573-actin 5-TCTACAATGAGCTGCGTGTG-3 5-AATGTCACGCACGATTTCCC-3

Ther

mal

with

draw

al(ip

silat

eral

thre

shol

d (s

))

Sham

468

1012141618

3 5 7 9 11 13 15 171Postoperative day

TCI + radiotherapyTCI + sham

lowastlowastlowast

lowastlowastlowastlowast lowastlowast lowastlowast lowastlowast lowastlowast

(a)Th

erm

al w

ithdr

awal

(con

tral

ater

al th

resh

old

(s))

Sham


10

12

14

16

18


(b)

1 3 5 7 9 11 13 15 17

Mec

hani

cal w

ithdr

awal

(ipsil

ater

al th

resh

old

(mN

))

Postoperative daySham


lowastlowast

lowastlowast lowastlowast lowastlowast lowastlowast lowastlowast lowastlowast

120150180210240270300330

lowast

(c)

1 3 5 7 9 11 13 15 17

Mec

hani

cal w

ithdr

awal

(con

tral

ater

al th

resh

old

(mN

))



210

240

270

300

330

(d)

Figure 1 Radiotherapy relieved thermal hyperalgesia and mechanical allodynia in TCI ratsThermal hyperalgesia of the hind paw ipsilateral(a) and contralateral (b) to TCI Mechanical allodynia of the hind paw ipsilateral (c) and contralateral (d) to TCI Arrows indicateadministration of X-radiation (6Gy) on the 9th day after surgery Eight rats were included in each group lowast119875 lt 005 lowastlowast119875 lt 001 versussham 119875 lt 005 119875 lt 001 versus TCI + sham radiotherapy

28 Statistical Analysis All statistical analyses were carriedout using Statistical Product and Service Solutions ver 150(SPSS Inc Chicago Illinois USA) Alterations in cAMPmRNA and concentration PKA activity and the levels of IL-1120573 and TNF-120572were tested using one-way analysis of variance(ANOVA) followed by Bonferroni post hoc tests Two-wayrepeated-measures ANOVA (days times groups) was used to testthe behavioral responses to thermal and mechanical stimulifollowed by Bonferroni post hoc testsThe nonparametric sta-tistical method was used for analyzing the bone radiographsAll data were presented as means plusmn SEM Statistical resultswere deemed significant if 119875 value was less than 005

3 Results

31 Radiotherapy Attenuated TCI-Induced Thermal Hyperal-gesia and Mechanical Allodynia TCI-treated rats exhibited

significant thermal hyperalgesia and mechanical allodyniaduring postoperative days 5ndash17The thresholds of thermal andmechanical withdrawal ipsilateral but not contralateral tothe TCI treatment decreased approximately by 50 duringpostoperative days 9ndash17 compared to sham control To deter-mine whether radiotherapy treatment affects bone cancerpain a single dose of X-radiation (6Gy) was applied on the9th day after operation Such radiotherapy produced long-lasting inhibition of TCI-induced thermal hyperalgesia andmechanical allodynia The inhibition lasted for a week with1 d delay following the X-radiation application The peakvalues of inhibition of the thermal hyperalgesia and mechan-ical allodynia were approximately 25ndash50 The thermal andmechanical withdrawal of the hind paw contralateral to TCItreatment was not altered following the radiotherapy Dataare summarized in Figure 1


(a) (b) (c)

Sham0

1

2

3

4

5

Bone

des

truc

tion

scor

e


lowast

lowastlowast

(d)

Figure 2 Radiotherapy alleviated TCI-induced tibia bone destruction Radiographic images were taken on the 17th day after TCI (a b c)Representative bone destruction and tumor growth in groups of sham (a) TCI + sham radiotherapy (b) and TCI + radiotherapy (c) Dataare summarized in (d) Eight rats were included in each group Arrows in (andashc) point to the areas of the bone destruction lowastlowast119875 lt 001 versussham 119875 lt 005 versus TCI + sham lowast119875 lt 005 versus sham

32 Radiotherapy Alleviated TCI-Induced Bone DestructionBone structure integrity was evaluated by radiographic obser-vation on the 17th day after TCI No radiographic change(score = 0 no bone destruction) was found in the groupof sham without radiotherapy Bone destruction was seenclearly in groups of TCI with and without radiotherapyrespectively Score of the TCI group with sham therapy was375 plusmn 043 (range 3ndash5) Radiotherapy greatly reduced TCI-induced tibia bone destruction The score in the group ofTCI with radiotherapy dropped to 238 plusmn 052 (range 1ndash3)which was significantly less than the score in TCI group withsham therapy Radiological changes of the tibia are shown inFigure 2

33 Radiotherapy Inhibited TCI-Induced PKA mRNA Expres-sion in DRG Our previous studies have shown that expres-sion of PKA-RII and PKA-CmRNAs was increased after TCItreatment in a time-dependent manner [19] To test whetherradiotherapy has an effect on this TCI-induced PKA mRNAexpression we measured the levels of PKA-RII and PKA-C mRNAs on day 1 and day 5 after radiotherapy treatment(postoperative days 10 and 14 resp) using RT-PCRWe foundthat radiotherapy treatment greatly inhibited TCI-inducedincrease of expression of PKA-RII and PKA-C mRNA Theexpression of PKA-RII and PKA-C mRNA in the TCI +

radiotherapy group was significantly reduced compared withthe TCI + sham group (Figure 3)

34 Radiotherapy Reduced TCI-Induced Increase of cAMPLevel and PKA Activity in DRG and the Spinal Cord We havepreviously shown that cAMP concentration and PKG activityin DRG and the spinal cord were significantly increased ina time-dependent manner after TCI treatment [19] To testthe hypothesis that cAMP-PKA signaling pathway might bealtered by radiotherapy we measured cAMP level and PKAactivity in DRG and the spinal cord following radiotherapytreatment The results showed that radiotherapy treatmentsignificantly reducedTCI-induced increase of cAMP concen-tration as well as PKA activity in DRG and the spinal cord(Figure 4)

35 Radiotherapy Reduced TCI-Induced Activity of IL-1120573 andTNF-120572 in the Spinal Cord We have recently shown that IL-1120573 and TNF-120572 levels in the spinal cord were significantlyincreased in a time-dependent manner after TCI treatment[25] To test whether radiotherapy could affect TCI-inducedincrease of IL-1120573 and TNF-120572 wemeasured levels of IL-1120573 andTNF-120572 in the spinal cord after radiotherapy on postoperativedays 10 and 14 (1 and 5 days after radiotherapy) Our resultsshowed that radiotherapy treatment significantly reduced


PKA-C

PKA-RII

Sham

Postoperative day 14 Postoperative day 10

TCI + radiotherapyTCI + sham TCI + radiotherapyTCI + sham

120573-actin

(a)

10 14

PKA-

RII r

elativ

e mRN

A le

vel

(fold

of s

ham

)

Postoperative day

012345

lowastlowast


Sham

(b)

Sham

Postoperative day

10 14

PKC

relat

ive m

RNA

leve

l(fo

ld o

f sha

m)

lowast

lowast

0

1

2

3

4

5


(c)

Figure 3 Levels of PKA-RII and PKA-C mRNA in DRG (a) Representative bands showing levels of PKA-RII and PKA-C mRNA analyzedby RT-PCR (b and c) Data quantification Four samples were used for each group with two ganglia in each sample lowast119875 lt 005 versus sham119875 lt 005 versus TCI + sham

TCI-induced increase of IL-1120573 and TNF-120572 in the spinal cord(Figure 5)

4 Discussion

This study demonstrates that radiotherapy is an effectivetreatment approach for treating bone cancer painThe cAMP-PKA signaling pathway may be a mechanism that underliesthe analgesic effect of radiotherapy in bone cancer painRadiotherapy suppresses TCI-induced painful behaviorsthermal hyperalgesia and mechanical allodynia and allevi-ates TCI-induced massive bone destruction Radiotherapyreduces TCI-induced increased expression of PKAmRNAs inDRGaswell as the increased level of cAMPconcentration andPKA activity in both DRG and the spinal cord In additionradiotherapy results in a significant decrease of IL-1120573 andTNF-120572 activity in the spinal cordThese findings suggest thatradiotherapy treatment may suppress TCI-induced hyperal-gesia and allodynia by inhibiting the cAMP-PKA signalingpathway in DRG and the spinal cord

Pain is one of the most prominent symptoms in clini-cally advanced cancer patients Approximately 50 of thesepatients experience moderate to severe pain [1] Mechanisms

of cancer pain are thought to be complex and may involve acombination of inflammation nerve injury and other uniquefactors [26] Considerable evidence of cancer pain treatmentshows that radiotherapy effectively relieves pain in up to 95of patients and can maintain the level of analgesia in morethan 70 of the patients for up to three months [27ndash29] Pre-vious studies have shown that activation of the cAMP-PKAsignaling pathway contributes to noxious stimulus-inducedperipheral and central sensitization [30ndash32] We have foundthat in vivo chronic compression of DRG or in vitro acuteDRG dissociation resulted in activation of the cAMP-PKAsignaling pathway Continued activation of the cAMP-PKAsignaling pathway is required to maintain hyperexcitabilityof the DRG neurons and behaviorally expressed hyperalgesiain these two different injury-related stress conditions [15 17]Recently we have confirmed that activation of the cAMP-PKA pathway plays an essential role in the induction andmaintenance of bone cancer pain [19 25] Here we providedirect evidence that the elevated activity of cAMP-PKAsignaling pathway is significantly decreased by radiotherapyin a bone cancer pain model These results support an ideathat radiotherapy may suppress bone cancer pain throughinhibition of abnormal activation of cAMP-PKA signaling


Sham 10 14

Postoperative day

DRG

lowast

lowast

0102030405060

cAM

P co

ncen

trat

ion

(pm

olm

g pr

otei

n)


(a)

Sham 10 14

cAM

P co

ncen

trat

ion

(pm

olm

g pr

otei

n)

Postoperative day

SC

lowast

lowast

05

101520253035404550


(b)

Sham 10 14Postoperative day

DRG

lowastlowast

0

02

04

06

08


PKA

activ

ity(p

mol

120583g

prot

ein

min

)

(c)


SC

lowastlowast

0

02

04

06

08


PKA

activ

ity(p

mol

120583g

prot

ein

min

)

(d)

Figure 4 Alterations of cAMP concentration and PKA activity in DRG and the spinal cord after TCI with or without radiotherapy (a and b)cAMP concentration (c and d) PKA activityThe tissues were collected on postoperative days 10 and 14 that is 1 and 5 days after radiotherapyrespectively Each group included four samples with two ganglia in each sample lowast119875 lt 005 versus sham 119875 lt 005 versus TCI + sham


lowastlowast

0

100

200

300

400

500

(pg

mg

prot

ein)

IL-1120573

conc

entr

atio

n


(a)


lowast

lowast

0

100

200

300

400

500

600

(pg

mg

prot

ein)

TNF-120572

conc

entr

atio

n


(b)

Figure 5 Radiotherapy reduced TCI-induced increase of IL-1120573 and TNF-120572 in the spinal cord IL-1120573 and TNF-120572 concentrations were analyzedby ELISA The tissues were collected on postoperative days 10 and 14 that is 1 and 5 days after radiotherapy respectively Four samples wereincluded in each group and each sample included a segment of the spinal cord at L

4-L5 lowast119875 lt 005 versus sham 119875 lt 005 versus TCI + sham


pathway suggesting a new mechanism for the radiotherapyof bone cancer pain

The proinflammatory cytokines are activators of thecAMP-PKA pathway in primary afferent neurons [25 33ndash35] During tumor growth and development of bone cancerpain certain proinflammatory cytokines such as TNF-120572 andIL-1120573 are activated and released from the astrocytes andmicroglial cells and contribute to bone cancer pain [2536 37] Radiotherapy can reduce these proinflammatorycytokines and inhibit activation of the cAMP-PKA signalingpathway and thus result in relief of bone cancer pain Thesefindings suggest that the cytokines are also targets thatmay beresponsible for radiotherapy-induced analgesia In additionour results showed that radiotherapy resulted in less boneloss in TCI rats This is consistent with the previous findingthat radiotherapy with high dose may kill part of the tumorcells or reduce their activation and thus delay the destructionof the bone structure [38 39] These findings indicate thatradiotherapymay reduce both pain and loss of bone structurefollowing TCI treatment

5 Conclusions

Our results demonstrate that radiotherapy can effectivelysuppress bone cancer pain probably through inhibition ofactivation of cAMP-PKA signaling pathway in the primarysensory neurons and the spinal cordThis studymay suggest anew mechanism underlying radiotherapy-induced analgesiaof bone cancer pain and support the clinical use of radiother-apy in treatment of certain cancer pain conditions

Disclosure

This study should be attributed equally to the institutions

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Guiqin Zhu and Yanbin Dong contributed equally to thisstudy

Acknowledgments

This work was partly supported by grants from JiangsuProvince Distinguished Medical Expert (JPDME2014-123)Lianyungang City Project 555-2 (LYG-555-2) and the Munic-ipal Science and Technology Bureau Foundation (SH1338SH1420) and the National Nature Science Foundation ofChina (NSFC-81271241 and 81320108012)

References

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attitudesrdquo Annals of Oncology vol 20 no 8 pp 1420ndash14332009

[2] M H J van den Beuken-van Everdingen J M de Rijke A GKessels H C Schouten M van Kleef and J Patijn ldquoPrevalenceof pain in patients with cancer a systematic review of the past40 yearsrdquo Annals of Oncology vol 18 no 9 pp 1437ndash1449 2007

[3] M J Goblirsch P Zwolak and D R Clohisy ldquoAdvances inunderstanding bone cancer painrdquo Journal of Cellular Biochem-istry vol 96 no 4 pp 682ndash688 2005

[4] C H Serezani M N Ballinger D M Aronoff and M Peters-Golden ldquoCyclic AMP master regulator of innate immune cellfunctionrdquo American Journal of Respiratory Cell and MolecularBiology vol 39 no 2 pp 127ndash132 2008

[5] P J S Stork and J M Schmitt ldquoCrosstalk between cAMP andMAP kinase signaling in the regulation of cell proliferationrdquoTrends in Cell Biology vol 12 no 6 pp 258ndash266 2002

[6] K Yamamizu and J K Yamashita ldquoRoles of cyclic adenosinemonophosphate signaling in endothelial cell differentiationand arterial-venous specification during vascular developmentrdquoCirculation Journal vol 75 no 2 pp 253ndash260 2011

[7] G G Holz E Heart and C A Leech ldquoSynchronizing Ca2+and cAMP oscillations in pancreatic 120573-cells a role for glucosemetabolism and GLP-1 receptors Focus on lsquoRegulation ofcAMP dynamics by Ca2+ and G protein-coupled receptors inthe pancreatic 120573-cell a computational approachrsquordquoTheAmericanJournal of PhysiologymdashCell Physiology vol 294 no 1 pp C4ndashC6 2008

[8] R Waltereit and M Weller ldquoSignaling from cAMPPKA toMAPK and synaptic plasticityrdquoMolecular Neurobiology vol 27no 1 pp 99ndash106 2003

[9] W Koppert ldquoOpioid-induced hyperalgesia Pathophysiologyand clinical relevancerdquo Anaesthesist vol 53 no 5 pp 455ndash4662004

[10] T King M H Ossipov T W Vanderah F Porreca and J LaildquoIs paradoxical pain induced by sustained opioid exposure anunderlying mechanism of opioid antinociceptive tolerancerdquoNeurosignals vol 14 no 4 pp 194ndash205 2004

[11] S Tumati W R Roeske T M Largent-Milnes T W Vanderahand E V Varga ldquoIntrathecal PKA-selective siRNA treatmentblocks sustained morphine-mediated pain sensitization andantinociceptive tolerance in ratsrdquo Journal of NeuroscienceMeth-ods vol 199 no 1 pp 62ndash68 2011

[12] C M Hingtgen K J Waite and M R Vasko ldquoProstaglandinsfacilitate peptide release from rat sensory neurons by activatingthe adenosine 31015840 51015840-cyclic monophosphate transduction cas-caderdquo The Journal of Neuroscience vol 15 no 7 pp 5411ndash54191995

[13] A B Malmberg E P Brandon R L Idzerda H Liu G SMcKnight and A I Basbaum ldquoDiminished inflammation andnociceptive pain with preservation of neuropathic pain in micewith a targeted mutation of the type I regulatory subunit ofcAMP-dependent protein kinaserdquo The Journal of Neurosciencevol 17 no 19 pp 7462ndash7470 1997

[14] M R Lewin and E T Walters ldquoCyclic GMP pathway is criticalfor inducing long-term sensitization of nociceptive sensoryneuronsrdquo Nature Neuroscience vol 2 no 1 pp 18ndash23 1999

[15] X-J Song Z-B Wang Q Gan and E T Walters ldquocAMPand cGMP contribute to sensory neuron hyperexcitability andhyperalgesia in rats with dorsal root ganglia compressionrdquoJournal of Neurophysiology vol 95 no 1 pp 479ndash492 2006

[16] Z-JHuangH-C Li AACowan S Liu Y-K Zhang andX-JSong ldquoChronic compression or acute dissociation of dorsal root


ganglion induces cAMP-dependent neuronal hyperexcitabilitythrough activation of PAR2rdquo Pain vol 153 no 7 pp 1426ndash14372012

[17] J-H Zheng E T Walters and X-J Song ldquoDissociation ofdorsal root ganglion neurons induces hyperexcitability that ismaintained by increased responsiveness to cAMP and cGMPrdquoJournal of Neurophysiology vol 97 no 1 pp 15ndash25 2007

[18] K O Aley and J D Levine ldquoRole of protein kinase A inthe maintenance of inflammatory painrdquo The Journal of Neuro-science vol 19 no 6 pp 2181ndash2186 1999

[19] G-Q Zhu S Liu D-DHe Y-P Liu andX-J Song ldquoActivationof the cAMP-PKA signaling pathway in rat dorsal root ganglionand spinal cord contributes toward induction and maintenanceof bone cancer painrdquo Behavioural Pharmacology vol 25 no 4pp 267ndash276 2014

[20] S J Medhurst K Walker M Bowes et al ldquoA rat model of bonecancer painrdquo Pain vol 96 no 1-2 pp 129ndash140 2002

[21] Q-L Mao-Ying J Zhao Z-Q Dong et al ldquoA rat model of bonecancer pain induced by intra-tibia inoculation of Walker 256mammary gland carcinoma cellsrdquo Biochemical and BiophysicalResearch Communications vol 345 no 4 pp 1292ndash1298 2006

[22] H C Park J Seong J H An J Kim U J Kim and B WLee ldquoAlteration of cancer pain-related signals by radiationproteomic analysis in an animal model with cancer boneinvasionrdquo International Journal of Radiation Oncology BiologyPhysics vol 61 no 5 pp 1523ndash1534 2005

[23] NM Luger D BMachM A Sevcik and PWMantyh ldquoBonecancer pain from model to mechanism to therapyrdquo Journal ofPain and Symptom Management vol 29 no 5 pp S32ndashS462005

[24] X-J Song J-H Zheng J-L Cao W-T Liu X-S Song andZ-J Huang ldquoEphrinB-EphB receptor signaling contributes toneuropathic pain by regulating neural excitability and spinalsynaptic plasticity in ratsrdquoPain vol 139 no 1 pp 168ndash180 2008

[25] S Liu Y-P Liu W B Song and X-J Song ldquoEphrinB-EphBreceptor signaling contributes to bone cancer pain via Toll-like receptor and proinflammatory cytokines in rat spinal cordrdquoPain vol 154 no 12 pp 2823ndash2835 2013

[26] J R Ghilardi H Rohrich T H Lindsay et al ldquoSelectiveblockade of the capsaicin receptor TRPV1 attenuates bonecancer painrdquo The Journal of Neuroscience vol 25 no 12 pp3126ndash3131 2005

[27] A Lin and M E Ray ldquoTargeted and systemic radiotherapyin the treatment of bone metastasisrdquo Cancer and MetastasisReviews vol 25 no 4 pp 669ndash675 2006

[28] D Rades S E Schild and J L Abrahm ldquoTreatment of painfulbone metastasesrdquo Nature Reviews Clinical Oncology vol 7 no4 pp 220ndash229 2010

[29] E Chow K Harris G Fan M Tsao and W M Sze ldquoPalliativeradiotherapy trials for bone metastases a systematic reviewrdquoJournal of Clinical Oncology vol 25 no 11 pp 1423ndash1436 2007

[30] Q-P Ma and C J Woolf ldquoBasal and touch-evoked fos-likeimmunoreactivity during experimental inflammation in theratrdquo Pain vol 67 no 2-3 pp 307ndash316 1996

[31] C-N Liu P D Wall E Ben-Dor M Michaelis R Amir andMDevor ldquoTactile allodynia in the absence ofC-fiber activationaltered firing properties of DRG neurons following spinal nerveinjuryrdquo Pain vol 85 no 3 pp 503ndash521 2000

[32] S-J Hu X-J Song K W Greenquist J-M Zhang and R HLaMotte ldquoProtein kinase A modulates spontaneous activity inchronically compressed dorsal root ganglion neurons in the ratrdquoPain vol 94 no 1 pp 39ndash46 2001

[33] M Cui and G D Nicol ldquoCyclic AMP mediates the prosta-glandin E2-induced potentiation of bradykinin excitation inrat sensory neuronsrdquo Neuroscience vol 66 no 2 pp 459ndash4661995

[34] S Neumann T P Doubell T Leslie and C J Woolf ldquoInflam-matory pain hypersensitivity mediated by phenotypic switchin myelinated primary sensory neuronsrdquo Nature vol 384 no6607 pp 360ndash364 1996

[35] J A M Smith C L Davis and G M Burgess ldquoProstaglandinE2-induced sensitization of bradykinin-evoked responses in ratdorsal root ganglion neurons is mediated by cAMP-dependentprotein kinase Ardquo European Journal of Neuroscience vol 12 no9 pp 3250ndash3258 2000

[36] M Yao X-Y Chang Y-X Chu et al ldquoAntiallodynic effectsof propentofylline elicited by interrupting spinal glial functionin a rat model of bone cancer painrdquo Journal of NeuroscienceResearch vol 89 no 11 pp 1877ndash1886 2011

[37] R-X Zhang B Liu L Wang et al ldquoSpinal glial activation in anew rat model of bone cancer pain produced by prostate cancercell inoculation of the tibiardquo Pain vol 118 no 1-2 pp 125ndash1362005

[38] M Goblirsch C Lynch W Mathews J C Manivel P W Man-tyh and D R Clohisy ldquoRadiation treatment decreases bonecancer pain through direct effect on tumor cellsrdquo RadiationResearch vol 164 no 4 pp 400ndash408 2005

[39] G D Roodman ldquoMechanisms of bone metastasirdquo The NewEngland Journal of Medicine vol 350 no 16 pp 1655ndash16982004

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

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Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Activation of cAMP-PKA pathway has been reported toenhance presynaptic neurotransmitter synthesis and vesicu-lar transportation probably through phosphorylation of keytranscriptional factors (ie cAMP response element-bind-ing protein) and synaptic vesicle proteins [9ndash11] In additionrecent studies found that cAMP-PKA signaling pathway isinvolved in both inflammatory pain [12ndash14] and neuropathicpain [15ndash17] The peripheral hyperalgesic actions of inflam-matory mediators are mediated by the cAMP-PKA signalingpathway [18]We have recently demonstrated that the cAMP-PKA pathway is crucial for the maintenance of dorsal rootganglia (DRG) neuronal hyperexcitability and behaviorallyexpressed hyperalgesia in an in vivo neuropathic pain animalmodel of chronic compression of the DRG (CCD model)as well as in an in vitro model of acute DRG dissociation[15 16 18] Recently we have further found that activation ofthe cAMP-PKA signaling pathway plays an important role inboth induction and maintenance of bone cancer pain in rats[19] However it remains unknown whether and then howcAMP-PKA signaling would contribute toward radiotherapytreatment for bone cancer painThis study provides evidencesupporting an idea that radiotherapy may suppress bonecancer pain through inhibition of abnormal activation ofcAMP-PKA signaling pathway in DRG and the spinal cord

2 Materials and Methods

21 Animals and Drugs Female adult Sprague-Dawley rats(160ndash180 g at the start of the experiment) were housed ina controlled lighting environment with free access to foodandwater All experiments were approved by the InstitutionalAnimal Care and Use Committees in Oriental Hospital andconducted in accordancewith theDeclaration of theNationalInstitutes of Health Guide for the Care and Use of LaboratoryAnimals (publication number 85-23 revised 1985) Surgerywas performed under anesthesia with intraperitoneal injec-tion of sodium pentobarbital (50mgkg ip)

22 Animal Model of Bone Cancer Pain The protocols ofthe bone cancer pain model were similar to that describedpreviously [19ndash21] In brief following induction of generalanesthesiawith intraperitoneal injection of sodiumpentobar-bital rats were placed abdominal side up After disinfectingwith 75 vv ethanol a one-centimeter rostrocaudal incisionwas made in the skin directly above the top half of the tibiaTumor cells (1 times 105 cells120583L 5 120583L) extracted from the asceticfluid of female rats that receivedWalker 256 mammary glandcarcinoma cells were injected (tumor cell implantation TCI)into the intramedullary space of the right tibia to inducebone cancer Injection site was closed with bone wax whilethe syringe was removed The incision was then dusted withpenicillin powder and closed Rats in the sham group wereinjected with the same number of boiled tumor cells

23 Radiotherapy A single dose of radiationwas given 9 daysafter TCI Rats were immobilized in an acryl jig and a doseof 6Gy was delivered to the right tibia area using a collimatorsystem with 6 MV X-rays [22] Sham radiotherapy applied in

TCI rats was using the same protocol except that they werenot given the real X-ray radiation

24 Radiographic Observation On the 17th day after TCItreatment radiographic images were taken (exposure setting12ms 31 KVp) using a Philips Digital Radiographer System(Digital DiagnostVM PhilipsMedical SystemsDMCGmbHHamburg Germany) Bone destruction was evaluated on ascale of 0ndash5 [20 23] 0 = normal bone structure withoutany sign of deterioration 1 = small radiolucent lesions in theproximal epiphysis (lt3) 2 = increased number of radiolucentlesions (gt3) indicating loss of medullary bone 3 = lossof medullary bone plus erosion of the cortical bone 4 =full-thickness unicortical bone loss and 5 = full-thicknessbicortical bone loss and displaced fractureThe experimenterwas blinded to the treatment of the samples

25 Behavioral Test Thermal hyperalgesia was indicated by asignificantly shortened latency of foot withdrawal in responseto heat stimulation To determine thermal hyperalgesia aradiant heat source was focused and delivered on a portion ofthe hind paw the thermal stimuli shut off automatically whenhind paw moved (or after 20 s to prevent tissue damage)Thermal stimuli were delivered 3 times to each hind paw at5ndash8-minute intervals Mechanical allodynia was indicated bya significant decrease in the threshold of paw withdrawal tomechanical indentation of the plantar surface of each hindpaw with a sharp cylindrical probe The probe was appliedto 6 designated loci distributed over the plantar surface ofthe foot The minimal force that induced paw withdrawalwas read off the display [19 24] The experimenters who per-formed these behavioral tests were blinded to the treatmentcondition of the animals

26 mRNA Isolation and RT-PCR The total RNA isolatedwith TRIzol Reagent (Invitrogen USA) was reverse tran-scribed using M-MLV reverse transcriptase (Takara) Primersets were synthesized by Integrated DNA Technologies (San-gon Biotech) their sequences are shown in Table 1 Theamplification conditions were set as follows 94∘C for 2minand 30 cycles of 94∘C for 30 s and 58∘C for 40 s PCRproducts were analyzed on agarose gel electrophoresis andwere verified by DNA sequencing The gels were imagedwith Tanon 2500 Imaging Systems and analyzed by analysissoftware ImageJ 148u

27 Measurement of cAMP IL-1120573 and TNF-120572 Levels and PKAActivity The DRG and the spinal cord at segments of L

4-L5

ipsilateral to TCI were collected on postoperative days 10 and14 for further neurochemical analysis Commercial enzyme-linked immunosorbent assay kits were used to determine theconcentrations of cAMP IL-1120573 and TNF-120572 and activity ofPKA according to the manufacturersrsquo instructions ELISAkit for cAMP was purchased from Cayman Chemical (AnnArbor Michigan USA) ELISA kits for IL-1120573 TNF-120572 andactivated PKA were purchased from RampD Systems (Min-neapolis Minnesota USA)




Ther

mal

with

draw

al(ip

silat

eral

thre

shol

d (s

))

Sham

468

1012141618



lowastlowastlowast


(a)Th

erm

al w

ithdr

awal

(con

tral

ater

al th

resh

old

(s))

Sham


10

12

14

16

18


(b)

1 3 5 7 9 11 13 15 17

Mec

hani

cal w

ithdr

awal

(ipsil

ater

al th

resh

old

(mN

))



lowastlowast


120150180210240270300330

lowast

(c)

1 3 5 7 9 11 13 15 17

Mec

hani

cal w

ithdr

awal

(con

tral

ater

al th

resh

old

(mN

))



210

240

270

300

330

(d)



3 Results




(a) (b) (c)

Sham0

1

2

3

4

5

Bone

des

truc

tion

scor

e


lowast

lowastlowast

(d)








PKA-C

PKA-RII

Sham



120573-actin

(a)

10 14

PKA-

RII r

elativ

e mRN

A le

vel

(fold

of s

ham

)

Postoperative day

012345

lowastlowast


Sham

(b)

Sham

Postoperative day

10 14

PKC

relat

ive m

RNA

leve

l(fo

ld o

f sha

m)

lowast

lowast

0

1

2

3

4

5


(c)



4 Discussion





Sham 10 14

Postoperative day

DRG

lowast

lowast

0102030405060

cAM

P co

ncen

trat

ion

(pm

olm

g pr

otei

n)


(a)

Sham 10 14

cAM

P co

ncen

trat

ion

(pm

olm

g pr

otei

n)

Postoperative day

SC

lowast

lowast

05

101520253035404550


(b)


DRG

lowastlowast

0

02

04

06

08


PKA

activ

ity(p

mol

120583g

prot

ein

min

)

(c)


SC

lowastlowast

0

02

04

06

08


PKA

activ

ity(p

mol

120583g

prot

ein

min

)

(d)



lowastlowast

0

100

200

300

400

500

(pg

mg

prot

ein)

IL-1120573

conc

entr

atio

n


(a)


lowast

lowast

0

100

200

300

400

500

600

(pg

mg

prot

ein)

TNF-120572

conc

entr

atio

n


(b)






5 Conclusions


Disclosure






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Ther

mal

with

draw

al(ip

silat

eral

thre

shol

d (s

))

Sham

468

1012141618



lowastlowastlowast


(a)Th

erm

al w

ithdr

awal

(con

tral

ater

al th

resh

old

(s))

Sham


10

12

14

16

18


(b)

1 3 5 7 9 11 13 15 17

Mec

hani

cal w

ithdr

awal

(ipsil

ater

al th

resh

old

(mN

))



lowastlowast


120150180210240270300330

lowast

(c)

1 3 5 7 9 11 13 15 17

Mec

hani

cal w

ithdr

awal

(con

tral

ater

al th

resh

old

(mN

))



210

240

270

300

330

(d)



3 Results




(a) (b) (c)

Sham0

1

2

3

4

5

Bone

des

truc

tion

scor

e


lowast

lowastlowast

(d)








PKA-C

PKA-RII

Sham



120573-actin

(a)

10 14

PKA-

RII r

elativ

e mRN

A le

vel

(fold

of s

ham

)

Postoperative day

012345

lowastlowast


Sham

(b)

Sham

Postoperative day

10 14

PKC

relat

ive m

RNA

leve

l(fo

ld o

f sha

m)

lowast

lowast

0

1

2

3

4

5


(c)



4 Discussion





Sham 10 14

Postoperative day

DRG

lowast

lowast

0102030405060

cAM

P co

ncen

trat

ion

(pm

olm

g pr

otei

n)


(a)

Sham 10 14

cAM

P co

ncen

trat

ion

(pm

olm

g pr

otei

n)

Postoperative day

SC

lowast

lowast

05

101520253035404550


(b)


DRG

lowastlowast

0

02

04

06

08


PKA

activ

ity(p

mol

120583g

prot

ein

min

)

(c)


SC

lowastlowast

0

02

04

06

08


PKA

activ

ity(p

mol

120583g

prot

ein

min

)

(d)



lowastlowast

0

100

200

300

400

500

(pg

mg

prot

ein)

IL-1120573

conc

entr

atio

n


(a)


lowast

lowast

0

100

200

300

400

500

600

(pg

mg

prot

ein)

TNF-120572

conc

entr

atio

n


(b)






5 Conclusions


Disclosure






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b) (c)

Sham0

1

2

3

4

5

Bone

des

truc

tion

scor

e


lowast

lowastlowast

(d)








PKA-C

PKA-RII

Sham



120573-actin

(a)

10 14

PKA-

RII r

elativ

e mRN

A le

vel

(fold

of s

ham

)

Postoperative day

012345

lowastlowast


Sham

(b)

Sham

Postoperative day

10 14

PKC

relat

ive m

RNA

leve

l(fo

ld o

f sha

m)

lowast

lowast

0

1

2

3

4

5


(c)



4 Discussion





Sham 10 14

Postoperative day

DRG

lowast

lowast

0102030405060

cAM

P co

ncen

trat

ion

(pm

olm

g pr

otei

n)


(a)

Sham 10 14

cAM

P co

ncen

trat

ion

(pm

olm

g pr

otei

n)

Postoperative day

SC

lowast

lowast

05

101520253035404550


(b)


DRG

lowastlowast

0

02

04

06

08


PKA

activ

ity(p

mol

120583g

prot

ein

min

)

(c)


SC

lowastlowast

0

02

04

06

08


PKA

activ

ity(p

mol

120583g

prot

ein

min

)

(d)



lowastlowast

0

100

200

300

400

500

(pg

mg

prot

ein)

IL-1120573

conc

entr

atio

n


(a)


lowast

lowast

0

100

200

300

400

500

600

(pg

mg

prot

ein)

TNF-120572

conc

entr

atio

n


(b)






5 Conclusions


Disclosure






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















PKA-C

PKA-RII

Sham



120573-actin

(a)

10 14

PKA-

RII r

elativ

e mRN

A le

vel

(fold

of s

ham

)

Postoperative day

012345

lowastlowast


Sham

(b)

Sham

Postoperative day

10 14

PKC

relat

ive m

RNA

leve

l(fo

ld o

f sha

m)

lowast

lowast

0

1

2

3

4

5


(c)



4 Discussion





Sham 10 14

Postoperative day

DRG

lowast

lowast

0102030405060

cAM

P co

ncen

trat

ion

(pm

olm

g pr

otei

n)


(a)

Sham 10 14

cAM

P co

ncen

trat

ion

(pm

olm

g pr

otei

n)

Postoperative day

SC

lowast

lowast

05

101520253035404550


(b)


DRG

lowastlowast

0

02

04

06

08


PKA

activ

ity(p

mol

120583g

prot

ein

min

)

(c)


SC

lowastlowast

0

02

04

06

08


PKA

activ

ity(p

mol

120583g

prot

ein

min

)

(d)



lowastlowast

0

100

200

300

400

500

(pg

mg

prot

ein)

IL-1120573

conc

entr

atio

n


(a)


lowast

lowast

0

100

200

300

400

500

600

(pg

mg

prot

ein)

TNF-120572

conc

entr

atio

n


(b)






5 Conclusions


Disclosure






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Sham 10 14

Postoperative day

DRG

lowast

lowast

0102030405060

cAM

P co

ncen

trat

ion

(pm

olm

g pr

otei

n)


(a)

Sham 10 14

cAM

P co

ncen

trat

ion

(pm

olm

g pr

otei

n)

Postoperative day

SC

lowast

lowast

05

101520253035404550


(b)


DRG

lowastlowast

0

02

04

06

08


PKA

activ

ity(p

mol

120583g

prot

ein

min

)

(c)


SC

lowastlowast

0

02

04

06

08


PKA

activ

ity(p

mol

120583g

prot

ein

min

)

(d)



lowastlowast

0

100

200

300

400

500

(pg

mg

prot

ein)

IL-1120573

conc

entr

atio

n


(a)


lowast

lowast

0

100

200

300

400

500

600

(pg

mg

prot

ein)

TNF-120572

conc

entr

atio

n


(b)






5 Conclusions


Disclosure






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















5 Conclusions


Disclosure






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article radiotherapy suppresses bone cancer pain

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