Spinal TDAG8and Its Downstream Signaling Pathways Contribute To Bone Cancer Pain In Rats

Bone cancer pain (BCP) significantly affects the quality of life in many patients withbone metastasis, as current therapies leave some patients with insufficient pain relief. Thelimited success of current therapies for BCP may be related, at least in part, to theinadequate understanding of the potential mechanisms of BCP. T cell death associated gene8(TDAG8) is a novel acid sensing G-protein-coupled receptor. Recently, expression ofTDAG8has been shown in spinal cord and dorsal root ganglion (DRG), and possibleinvolvement of TDAG8in chronic inflammatory pain has been investigated. BCP is notsimilar to inflammatory pain state, and tissue acidosis is an important feature of cancer. Ithas been suggested that a relatively high proton concentration, known as acidosis, is directlink between disease and pain. Whether TDAG8is involved in bone cancer pain remainsunclear. In the present study, Walker256cells were inoculated into the intramedullaryspace of rat tibia to establish a model of BCP. Spinal TDAG8and its downstream proteinexpression were examined after inoculation with tumor cells. Then, the pain behaviors,TDAG8, and its downstream protein expression were examined by intrathecal TDAG8siRNA. Furthermore, a method of neuropharmacology was used to investigate whether itsdownstream signaling pathways were involved in the bone cancer pain. The presentresearch may lead to novel strategies for the treatment of bone cancer pain.Part one: Establish a rat model of bone cancer painObjective To establish a rat model of bone cancer pain.Methods Female SD rats weighing150-180g were randomly divided into3groups:normal group, sham group, and BCP group. Walker256cells were inoculated into theintramedullary space of rat let tibia in BCP group. The boiled cells were injected into the bone cavity in sham group. The ambulatory score (AS) and the paw withdrawal threshold(PWT) to von Frey filaments were measured at1day before and1，3，6，9,12,15,18dayafter inoculation (n=8). Radiology and histochemical staining were performed in normal,sham, BCP6d,12d, and18d groups to confirm bone destruction and cancer cell infiltration(n=3).Results There were no significant differences in AS and PWT between NS andsham groups. In contrast,6days after Walker256inoculation, obvious increases in the ASto spontaneous pain were observed in the BCP group compared with NS and sham groups.At the same time, the PWT to mechanical stimulation markedly decreased. Subsequently,the AS progressively increased and the PWT progressively decreased in the tumor-bearinggroup. Radiographs of the left tibia of BCP rats showed that signs of radiolucent lesion inthe proximal epiphysis on day six and twelve after inoculation. On day eighteen, furtherdeterioration was found with medullary bone loss and/or bicortical bone loss. Histologyshowed that tumor growth and various degrees of bone destruction were observed on daysix, twelve and eighteen after inoculation. Eighteen days after inoculation, tumor cellswere densely packed in the marrow cavity.Conclusion We successfully set up a rat model of bone cancer pain by tibiainoculation of Walker256mammary gland carcinoma cells. Part two: Expression of spinal TDAG8and its downstream proteins in arat model of bone cancer painObjective To observe the expression of spinal TDAG8and its downstream PKA,CREB, PKC and ERK1/2proteins in a rat model of bone cancer pain.Methods The expression of spinal TDAG8was detected by immunofluorescence,real-time PCR and Western blot in normal, sham, BCP6d, BCP12d and BCP18d rats. Theexpression of spinal PKA, CREB, PKC and ERK1/2proteins were detected by Westernblot in the same goups.Results On days6,12and18after inoculation, the number of TDAG8-positive cells in superficial spinal dorsal horn increased in BCP rats compared to sham or normal rats.The relative levels of spinal TDAG8mRNA and protein significantly andtime-dependently increased in bone cancer pain rats compared to sham or normal rats. Astatistically significant increase of PKAca, p-CREB, p-PKCγ and p-ERK1/2(P <0.05or0.01) protein levels were observed in bone cancer pain rats compared to normal or shamrats.Conclusion Spinal TDAG8and its downstream PKA, CREB, PKC and ERK1/2proteins were up-regulated in a rat model of bone cancer pain. Part three: SiRNA inhibits the expression of TDAG8on ratneurons-dorsal spinal cord cellsObjective To investigate the ability of chemically synthesized siRNA to knockdownTDAG8on rat neurons-dorsal spinal cord (RNdsc) cells.Methods Four siRNAs were chemically synthesized: three of them were used toinhibit TDAG8expression in RNdsc cells, the rest was fluorescence-labeled mismatchsiRNA as a nagative control. They were all transfected into RNdsc cells with jetPEI,respectively. RNdsc cells were randomly divided into5groups: normal,vehicle, mismatch,and siRNA50,100,200pmol group. After transfection with siRNA for24h, the rate oftransfection was calculated under fluorescence microscope and the cytotoxicity of complexwas detected using MTT. The expression of TDAG8was detected by real-time PCR andWestern blot analysis.Results The transfection rate was high enough to reach92.9%and the cytotoxicityof complex was very low. Meanwhile, TDAG8-siRNA50,100,200pmol candose-dependently inhibit the expression of TDAG8mRNA and protein levels. The TDAG8siRNA200pmol could reduce the expression of TDAG8mRNA and protein up to88%and73%, individually.Conclusion Chemically synthesized siRNA can effectively inhibit the expression ofTDAG8on RNdsc cells. Part four: Intrathecal TDAG8siRNA could attenuate bone cancer painin the rat modelObjective To investigate the effects of intrathecal TDAG8siRNA on bone cancerpain at different time points in the rat model.Methods The rats were randomly divided into pre-treatment and treatment groups,and each group was further divided into five sub-groups: sham, BCP+NS, BCP+mismatch, BCP+vehicle and BCP+siRNA group. Pre-treatment and treatment groupswhich intrathecal injection was starting on day3or6for three consecutive days aftertumor inoculation. Pain was measured by using ambulatory score and paw withdrawalthreshold（n=8）. The tissue was harvested at24h after the last injection. The expressionof spinal TDAG8was detected by real-time PCR and Western blot analysis, and theexpression of PKA, CREB, PKC and ERK1/2protein levels were detected by Western blot（n=4）.Results In pre-treatment group, intrathecal TDAG8siRNA could prevent the initiationof bone cancer pain. However, in treatment group, intrathecal injection of TDAG8siRNAcould alleviate, but not reverse, the maintenance of bone cancer pain. In both groups,intrathecal injection of TDAG8siRNA could significantly decrease the expression ofTDAG8mRNA and protein levels. Meanwhile, the expression of PKA, CREB, PKC andERK1/2protein levels also significantly decreased.Conclusion Intrathecal injection of TDAG8could effectively inhibit the expressionof TDAG8and it downstream PKA, CREB, PKC and ERK1/2protein levels. It couldsignificantly attenuate bone cancer pain in the rat model. Part five: Involvement of the spinal TDAG8-PKA-CREB signalingpathway in bone cancer pain in ratsObjective To investigate the role of TDAG8-PKA-CREB signaling pathway on bone cancer pain in rats.Methods The experiments divided into tow sections, one was intrathecal H-89andthe other was intrathecal TDAG8-siRNA. In intrathecal H-89section, the bone cancer ratswere randomly divided into three groups: NS group, DMSO group and H-89group (n=8). H-89group intrathecal injected H-898nmol. The volume of intrathecal injection was10μl for each group. To observe the changes of AS and PWT before intrathecal injectionand15,30,45min after intrathecal injection. The Western blot analysis was used bydetected the expression of spinal PKAca and p-CREB protein levels, one hour afterintrathecal injection. In intrathecal TDAG8-siRNA section, the methods were the same aspart two and four.Results Spinal PKAca and p-CREB protein levels were up-regulated in the ratmodel of bone cancer pain. Intrathecal H-89could significantly attenuate pain behaviorsand the expression of spinal PKAca and p-CREB protein levels. IntrathecalTDAG8-siRNA could attenuate bone cancer pain behaviors, together with concomitantdecrease of spinal PKAca and p-CREB protein levels.Conclusion TDAG8-PKA-CREB signaling pathway may participate in bone cancerpain in rats. Part six: Involvement of the spinal TDAG8-PKC-ERK signalingpathway in bone cancer pain in ratsObjective To investigate the role of TDAG8-PKC-ERK signaling pathway on bonecancer pain in rats.Methods The experiments divided into tow sections, one was intrathecalGF109203X and the other was intrathecal TDAG8-siRNA. In intrathecal GF109203Xsection, the bone cancer rats were randomly divided into three groups: NS group, DMSOgroup and GF109203X group (n=8). GF109203X group intrathecal injected GF109203X0.4nmol. The volume of intrathecal injection was10μl for each group. To observe thechanges of AS and PWT before intrathecal injection and60,120,180min after intrathecal injection. The Western blot analysis was used by detected the expression of spinal p-PKCγand p-ERK1/2protein levels, three hours after intrathecal injection. In intrathecal TDAG8-siRNA section, the methods were the same as part two and four.Results Spinal p-PKCγ and p-ERK1/2protein levels were up-regulated in the ratmodel of bone cancer pain. Intrathecal GF109203X could significantly attenuate painbehaviors and the expression of spinal p-PKCγ and p-ERK1/2protein levels. IntrathecalTDAG8-siRNA could attenuate bone cancer pain behaviors, together with concomitantdecrease of spinal p-PKCγ and p-ERK1/2protein levels.Conclusion TDAG8-PKC-ERK signaling pathway may participate in bone cancerpain in rats.