Treatment Of Bone Cancer Pain Research Articles

Study of the analgesic activities, chronic toxicity and addictive potential of Jia-Yuan-Qing pill in rats.

Jia-Yuan-Qing pill (JYQP) composed of Porcellio laevis Latreille, Corydalis Rhizoma and Radix Cynanchi Paniculati at a ratio of 9:7:7 has been found to be an effective analgesic agent. The present study aimed to evaluate the safety, addictive potential and anti-cancer pain activity of JYQP in a rat model. During the 6-month chronic toxicity test, no significant changes in general behavior, defecation, postural abnormalities, dietary or water intake or blood biochemical parameters were observed in male and female rats. Although a high dose of JYQP (5 g/kg) caused swelling of the liver, spleen and kidney in male and female rats, no pathological changes were observed in all organs examined via hematoxylin and eosin staining. The analgesic effect of JYQP on bone cancer pain was successfully confirmed in a rat model of Walker 256 cell-induced bone cancer. In contrast to morphine, in a physical dependence test, JYQP produced no withdrawal symptoms following chronic administration. The data from this study provide experimental evidence supporting the clinical use of JYQP as an effective, safe and non-addictive agent for the treatment of bone cancer pain.

Intrathecal injection of lentivirus-mediated glial cell line-derived neurotrophic factor RNA interference relieves bone cancer-induced pain in rats.

Bone cancer pain is a common symptom in cancer patients with bone metastases and the underlying mechanisms are largely unknown. The aim of this study is to explore the endogenous analgesic mechanisms to develop new therapeutic strategies for bone-cancer induced pain (BCIP) as a result of metastases. MRMT-1 tumor cells were injected into bilateral tibia of rats and X-rays showed that the area suffered from bone destruction, accompanied by an increase in osteoclast numbers. In addition, rats with bone cancer showed apparent mechanical and thermal hyperalgesia at day 28 after intratibial MRMT-1 inoculation. However, intrathecal injection of morphine or lentivirus-mediated glial cell line-derived neurotrophic factor RNAi (Lvs-siGDNF) significantly attenuated mechanical and thermal hyperalgesia, as shown by increases in paw withdrawal thresholds and tail-flick latencies, respectively. Furthermore, Lvs-siGDNF interference not only substantially downregulated GDNF protein levels, but also reduced substance P immunoreactivity and downregulated the ratio of pERK/ERK, where its activation is crucial for pain signaling, in the spinal dorsal horn of this model of bone-cancer induced pain. In this study, Lvs-siGDNF gene therapy appeared to be a beneficial method for the treatment of bone cancer pain. As the effect of Lvs-siGDNF to relieve pain was similar to morphine, but it is not a narcotic, the use of GDNF RNA interference may be considered as a new therapeutic strategy for the treatment of bone cancer pain in the future.

Sigma-1 Receptor Antagonist BD1047 Reduces Mechanical Allodynia in a Rat Model of Bone Cancer Pain through the Inhibition of Spinal NR1 Phosphorylation and Microglia Activation.

Previous studies have demonstrated that sigma-1 receptor plays important roles in the induction phase of rodent neuropathic pain; however, whether it is involved in bone cancer pain (BCP) and the underlying mechanisms remain elusive. The aim of this study was to examine the potential role of the spinal sigma-1 receptor in the development of bone cancer pain. Walker 256 mammary gland carcinoma cells were implanted into the intramedullary space of the right tibia of Sprague-Dawley rats to induce ongoing bone cancer-related pain behaviors; our findings indicated that, on days 7, 10, 14, and 21 after operation, the expression of sigma-1 receptor in the spinal cord was higher in BCP rats compared to the sham rats. Furthermore, intrathecal injection of 120 nmol of sigma-1 receptor antagonist BD1047 on days 5, 6, and 7 after operation attenuated mechanical allodynia as well as the associated induction of c-Fos and activation of microglial cells, NR1, and the subsequent Ca2+-dependent signals of BCP rats. These results suggest that sigma-1 receptor is involved in the development of bone cancer pain and that targeting sigma-1 receptor may be a new strategy for the treatment of bone cancer pain.

Levo-Tetrahydropalmatine Attenuates Bone Cancer Pain by Inhibiting Microglial Cells Activation.

Objective. The present study is to investigate the analgesic roles of L-THP in rats with bone cancer pain caused by tumor cell implantation (TCI). Methods. Thermal hyperalgesia and mechanical allodynia were measured at different time points before and after operation. L-THP (20, 40, and 60 mg/kg) were administrated intragastrically at early phase of postoperation (before pain appearance) and later phase of postoperation (after pain appearance), respectively. The concentrations of TNF-α, IL-1β, and IL-18 in spinal cord were measured by enzyme-linked immunosorbent assay. Western blot was used to test the activation of astrocytes and microglial cells in spinal cord after TCI treatment. Results. TCI treatment induced significant thermal hyperalgesia and mechanical allodynia. Administration of L-THP at high doses significantly prevented and/or reversed bone cancer-related pain behaviors. Besides, TCI-induced activation of microglial cells and the increased levels of TNF-α and IL-18 were inhibited by L-THP administration. However, L-THP failed to affect TCI-induced astrocytes activation and IL-1β increase. Conclusion. This study suggests the possible clinical utility of L-THP in the treatment of bone cancer pain. The analgesic effects of L-THP on bone cancer pain maybe underlying the inhibition of microglial cells activation and proinflammatory cytokines increase.

Protease-activated receptor 2 antagonist potentiates analgesic effects of systemic morphine in a rat model of bone cancer pain.

Bone cancer pain affects the quality of life of cancer patients. This study was aimed at investigating the analgesic effects of combined therapies with an antagonist of proteinase-activated receptor (PAR) 2 and morphine on pain-related behaviors in a rat model of bone cancer pain. Female Wistar rats were inoculated intramedullarily with Walker 256 cells into their tibias. The analgesic effects of intraperitoneal treatment with morphine and/or intrathecal with the PAR2 antagonist, FSLLRY-NH2, on bone cancer pain-related behaviors in rats were examined. Treatment with morphine at 3 or 10 mg/kg significantly improved limb-use and weight-bearing scores and reduced the number of spontaneous flinches in rats. Treatment with FSLLRY-NH2 at 10 mmol/L also significantly improved limb use and weight bearing scores, and reduced the number of spontaneous flinches in rats. Combination a sub-analgesic dose of FSLLRY-NH2 (0.1 mmol/L) and morphine further elevated limb-use and weight-bearing scores and reduced the number of flinches compared with the effects of morphine alone in rats. Our data indicate that the combination of morphine and FSLLRY-NH2 has potent analgesic effects on bone cancer pain and our findings may aid in design of new strategies for the treatment of bone cancer pain.

Inducible Lentivirus‐Mediated siRNA against TLR4 Reduces Nociception in a Rat Model of Bone Cancer Pain

Although bone cancer pain is still not fully understood by scientists and clinicians alike, studies suggest that toll like receptor 4 (TLR4) plays an important role in the initiation and/or maintenance of pathological pain state in bone cancer pain. A promising treatment for bone cancer pain is the downregulation of TLR4 by RNA interference; however, naked siRNA (small interference RNA) is not effective in long-term treatments. In order to concoct a viable prolonged treatment for bone cancer pain, an inducible lentivirus LvOn-siTLR4 (tetracycline inducible lentivirus carrying siRNA targeting TLR4) was prepared and the antinociception effects were observed in bone cancer pain rats induced by Walker 256 cells injection in left leg. Results showed that LvOn-siTLR4 intrathecal injection with doxycycline (Dox) oral administration effectively reduced the nociception induced by Walker 256 cells while inhibiting the mRNA and protein expression of TLR4. Proinflammatory cytokines as TNF-α and IL-1β in spinal cord were also decreased. These findings suggest that TLR4 could be a target for bone cancer pain treatment and tetracycline inducible lentivirus LvOn-siTLR4 represents a new potential option for long-term treatment of bone cancer pain.

A Traditional Chinese Medicine Xiao-Ai-Tong Suppresses Pain through Modulation of Cytokines and Prevents Adverse Reactions of Morphine Treatment in Bone Cancer Pain Patients.

Treating cancer pain continues to possess a major challenge. Here, we report that a traditional Chinese medicine Xiao-Ai-Tong (XAT) can effectively suppress pain and adverse reactions following morphine treatment in patients with bone cancer pain. Visual Analogue Scale (VAS) and Quality of Life Questionnaire (EORTC QLQ-C30) were used for patient's self-evaluation of pain intensity and evaluating changes of adverse reactions including constipation, nausea, fatigue, and anorexia, respectively, before and after treatment prescriptions. The clinical trials showed that repetitive oral administration of XAT (200 mL, bid, for 7 consecutive days) alone greatly reduced cancer pain. Repetitive treatment with a combination of XAT and morphine (20 mg and 30 mg, resp.) produced significant synergistic analgesic effects. Meanwhile, XAT greatly reduced the adverse reactions associated with cancer and/or morphine treatment. In addition, XAT treatment significantly reduced the proinflammatory cytokines interleukin-1β and tumor necrosis factor-α and increased the endogenous anti-inflammatory cytokine interleukin-10 in blood. These findings demonstrate that XAT can effectively reduce bone cancer pain probably mediated by the cytokine mechanisms, facilitate analgesic effect of morphine, and prevent or reduce the associated adverse reactions, supporting a use of XAT, alone or with morphine, in treating bone cancer pain in clinic.

Relief of cancer pain by glycine transporter inhibitors.

Recent studies have revealed the antinociceptive effects of glycine transporter (GlyT) inhibitors in neuropathic pain models such as sciatic nerve-injured and diabetic animals. Bone cancer can cause the most severe pain according to complex mechanisms in which a neuropathic element is included. Bone cancer modifies the analgesic action of opioids and limits their effectiveness, and thus novel medicament for bone cancer pain is desired. For the femur bone cancer model, NCTC 2472 tumor cells were injected into the medullary cavity of the distal femur of C3H/HeN mice. Effects of GlyT2 inhibitors, ORG 25543 and ALX 1393, and GlyT1 inhibitors, ORG 25935, and knockdown of the expression of spinal GlyTs protein by GlyTs siRNA on pain-like behaviors, such as allodynia, withdrawal threshold, guarding behavior, and limb-use abnormality, were examined in the femur bone cancer model mice. Effects of morphine in combination with GlyT inhibitor were examined. GlyT2 inhibitors, ORG 25543 and ALX 1393, and GlyT1 inhibitor ORG 25935 by IV or oral administration or knockdown of the expression of spinal GlyTs protein improved pain-like behaviors at 11 days after tumor transplantation. The pain-relief activity was potent and long lasting. Morphine at a dose with no analgesic activity combined with ORG 25543 further promoted the ORG 25543-induced pain-relief activity. Injection of ORG 25543 on the second day after tumor implantation caused 3 phases of pain responses; pain-like behaviors were initially accelerated (at 2-4 days) and subsequently almost disappeared (5-7 days) and then reappeared. Intrathecal injection of strychnine 1 day after injection of ORG 25543 transiently antagonized the pain-relief activity of ORG 25543. In control mice, strychnine improved pain-like behaviors 4 days after tumor implantation and aggravated the behaviors between 4 and 5 days. The evidence suggests that the different mechanisms are phase-dependently involved. GlyT inhibitors with or without morphine may be a new strategy for the treatment of bone cancer pain and lead to further investigations of the mechanisms underlying the development of bone cancer pain.

CGMP and cGMP-dependent protein kinase I pathway in dorsal root ganglia contributes to bone cancer pain in rats.

A prospective, randomized experimental research. To demonstrate the role of cGMP (cyclic guanosine monophosphate)-cGKI (cGMP-dependent protein kinase I) pathway in dorsal root ganglia (DRG) in bone cancer pain. Treating bone cancer pain continues to possess a major clinical challenge because the specific cellular and molecular mechanisms underlying bone cancer pain remain elusive. cGMP and cGMP-dependent protein kinases pathway in DRG plays important role in nerve injury-induced hyperexcitability of DRG neurons, as well as neuropathic pain, however, whether this pathway participates in bone cancer pain is unknown. The rat model of bone cancer pain was produced by intramedullary injection of rat breast cancer cells (Walker 256) into right tibia. Thermal hyperalgesia and mechanical allodynia were measured before and after administration of inhibitor of cGMP-cGKs pathway (Rp-8-pCPT-cGMPS). Immunofluorescence and reverse transcription-polymerase chain reaction were used to reflect expression of cGKI in DRG neurons, whereas the concentration of cGMP in DRG was tested using enzyme-linked immunosorbent assay method. Whole-cell patch clamp was used to record the hyperexcitability of small neurons in DRG with or without cGKs inhibitor after tumor cell implantation (TCI). TCI treatment significantly increased the concentration of cGMP in DRG and activity of cGKs in DRG and the spinal cord. TCI treatment also induced upregulation of cGKI messenger ribonucleic acid and protein in DRG, as well as enhanced hyperexcitability in DRG neurons. Spinal administration of Rp-8-pCPT-cGMPS, cGMP-cGKs inhibitor, significantly suppressed TCI-induced activation of cGMP-cGKI signaling, and hyperexcitability of DRG neurons. Meanwhile, in vivo intrathecal delivery of the Rp-8-pCPT-cGMPS significantly prevented and suppressed TCI-induced hyperalgesia and allodynia. From these results, we confirm that TCI treatment activates cGMP-cGKI signaling pathway and continuing activation of this pathway in DRG is required for hyperalgesia and/or hyperalgesia and allodynia after TCI treatment. N/A.

Intrathecal injection of the peptide myr-NR2B9c attenuates bone cancer pain via perturbing N-methyl-D-aspartate receptor-PSD-95 protein interactions in mice.

N-methyl-D-aspartate receptor (NMDARs)-dependent central sensitization plays an important role in cancer pain. Binding of NMDAR subunit 2B (NR2B) by postsynaptic density protein-95 (PSD-95) can couple NMDAR activity to intracellular enzymes, such as neuronal nitric oxide synthase (nNOS), facilitate downstream signaling pathways, and modulate NMDAR stability, contributing to synaptic plasticity. In this study, we investigated whether perturbing the specific interaction between spinal NR2B-containing NMDAR and PSD-95, using a peptide-mimetic strategy, could attenuate bone cancer-related pain behaviors. Osteosarcoma cells were implanted into the intramedullary space of the right femurs of C3H/HeJ mice to induce progressive bone cancer-related pain behaviors. Western blotting was applied to examine the expression of spinal phospho-Tyr1472 NR2B, nNOS, and PSD-95. We further investigated the effects of intrathecal injection of the mimetic peptide Myr-NR2B9c, which competitively disrupts the interaction between PSD-95 and NR2B, on nociceptive behaviors and on the upregulation of phospho-Tyr1472 NR2B, nNOS, and PSD-95 associated with bone cancer pain in the spinal cord. Inoculation of osteosarcoma cells induced progressive bone cancer pain and resulted in a significant upregulation of phospho-Tyr1472 NR2B, nNOS, and PSD-95. Intrathecal administration of Myr-NR2B9c attenuated bone cancer-evoked mechanical allodynia, thermal hyperalgesia, and reduced spinal phospho-Tyr1472 NR2B, nNOS, and PSD-95 expression. Intrathecal administration of Myr-NR2B9c reduced bone cancer pain. Internalization of spinal NR2B and dissociation NR2B-containing NMDARs activation from downstream nNOS signaling may contribute to the analgesic effects of Myr-NR2B9c. This approach may circumvent the negative consequences associated with blocking NMDARs, and may be a novel strategy for the treatment of bone cancer pain.

Bone cancer pain: from mechanism to therapy.

To review how common cancers such as breast, lung, and prostate cancers drive significant and frequently life-altering pain when the cells metastasize to bones. Similar to cancer, the factors that drive bone cancer pain evolve and change with disease progression. Bone cancer pain has both a nociceptive and neuropathic component. The nociceptive component is driven by the release of algogenic substances by tumor and their associated stromal cells, acidosis caused by bone-destroying osteoclasts, and mechanical destabilization and fracture of the bone. The neuropathic component is induced by tumor cell growth which injures and destroys the distal ends of nerve fibers that normally innervate the bone, as well as by inducing a highly pathological sprouting of both sensory and sympathetic nerve fibers. There is both a nociceptive and neuropathic component of bone cancer pain. In bone cancer pain, there is frequently a continual afferent drive of sensory nerve fibers that induces a peripheral and central sensitization. These mechanistic insights have begun to lead to advances in not only how we understand bone cancer pain but to the development of new therapies to treat bone cancer pain.

Evidence for involvement of spinal RANTES in the antinociceptive effects of triptolide, a diterpene triepoxide, in a rat model of bone cancer pain.

It has been shown that triptolide has beneficial effects in the treatment of neuropathic pain, but its effects on bone cancer pain (BCP) remain unclear. In this study, we aimed to explore the potential role of spinal regulated activation of normal T cell expressed and secreted (RANTES) in the antinociceptive effects of triptolide on BCP. A BCP model was induced by injecting Walker 256 mammary gland carcinoma cells into the intramedullary space of rat tibia. Intrathecal administration of triptolide (0.5, 1, 2μg) could dose-dependently alleviate mechanical hyperalgesia and spontaneous pain. In addition, there were also concomitant decreases in RANTES mRNA and protein expression levels in spinal dorsal horn. These results suggest that the antinociceptive effects of triptolide are related with inhibition of spinal RANTES expression in BCP rats. The findings of this study may provide a promising drug for the treatment of BCP.

CXCL12 in astrocytes contributes to bone cancer pain through CXCR4-mediated neuronal sensitization and glial activation in rat spinal cord.

BackgroundPrevious studies have demonstrated that chemokine CXCL12 and its receptor CXCR4 are critical for pain sensitization, but the mechanisms involved are not clear. In this study, we investigated the specific cellular mechanisms of CXCL12/CXCR4 chemokine signaling in the development and maintenance of bone cancer pain after tumor cell implantation (TCI).MethodsTCI in the tibial cavity of rats was used to establish a bone cancer pain model. Mechanical allodynia and thermal hyperalgesia were determined by measuring the paw withdrawal threshold and latency, respectively. The protein expression and cellular localization of CXCL12 and CXCR4 were detected by western blot and immunofluorescence staining. The sensitization of neurons, activation of astrocytes and microglia were examined by observing the immunofluorescence intensity of c-Fos, GFAP and IBA1.ResultsOur results demonstrated that CXCL12 was upregulated in a time-related manner, both in the dorsal root ganglia and spinal cord after TCI. Spinal CXCL12 was predominately expressed in astrocytes, and an intrathecal injection of astrocyte metabolic inhibitor fluorocitrate or selective JNK inhibitor SP600125 abolished TCI-induced CXCL12 production. A single intrathecal injection of a CXCL12 neutralizing antibody (10 μg/10 μl) at day 10 after TCI transiently reversed bone cancer pain in a dose-dependent manner. Whereas repetitive intrathecal administration of a CXCL12 neutralizing antibody (10 μg/10 μl, once a day from day 3 to 5 after TCI) significantly delayed the onset of TCI-induced pain behaviors for nearly five days. Spinal CXCR4 was also upregulated after TCI and colocalized with neurons, astrocytes and microglia. Blocking CXCR4 suppressed TCI-induced activation of neurons, astrocytes and microglia in the spinal cord at day 14. Repeated intrathecal administration of AMD3100 (5 μg/10 μl, once a day for three days) significantly delayed and suppressed the initiation and persistence of bone cancer pain in the early phase (at day 5, 6 and 7 after TCI) and in the late phase (at day 12, 13 and 14 after TCI) of bone cancer, respectively.ConclusionsTaken together, these results demonstrate that CXCL12/CXCR4 signaling contributed to the development and maintenance of bone cancer pain via sensitizing neurons and activating astrocytes and microglia. Additionally, this chemokine signaling may be a potential target for treating bone cancer pain.

Palliation of Bone Cancer Pain by Antagonists of Platelet-Activating Factor Receptors

Bone cancer pain is the most severe among cancer pain and is often resistant to current analgesics. Thus, the development of novel analgesics effective at treating bone cancer pain are desired. Platelet-activating factor (PAF) receptor antagonists were recently demonstrated to have effective pain relieving effects on neuropathic pain in several animal models. The present study examined the pain relieving effect of PAF receptor antagonists on bone cancer pain using the femur bone cancer (FBC) model in mice. Animals were injected with osteolytic NCTC2472 cells into the tibia, and subsequently the effects of PAF receptor antagonists on pain behaviors were evaluated. Chemical structurally different type of antagonists, TCV-309, BN 50739 and WEB 2086 ameliorated the allodynia and improved pain behaviors such as guarding behavior and limb-use abnormalities in FBC model mice. The pain relieving effects of these antagonists were achieved with low doses and were long lasting. Blockade of spinal PAF receptors by intrathecal injection of TCV-309 and WEB 2086 or knockdown of the expression of spinal PAF receptor protein by intrathecal transfer of PAF receptor siRNA also produced a pain relieving effect. The amount of an inducible PAF synthesis enzyme, lysophosphatidylcholine acyltransferase 2 (LPCAT2) protein significantly increased in the spinal cord after transplantation of NCTC 2472 tumor cells into mouse tibia. The combination of morphine with PAF receptor antagonists develops marked enhancement of the analgesic effect against bone cancer pain without affecting morphine-induced constipation. Repeated administration of TCV-309 suppressed the appearance of pain behaviors and prolonged survival of FBC mice. The present results suggest that PAF receptor antagonists in combination with, or without, opioids may represent a new strategy for the treatment of persistent bone cancer pain and improve the quality of life of patients.

EphrinB-EphB receptor signaling contributes to bone cancer pain via Toll-like receptor and proinflammatory cytokines in rat spinal cord

Treating bone cancer pain poses a major clinical challenge, and the mechanisms underlying bone cancer pain remain elusive. EphrinB-EphB receptor signaling may contribute to bone cancer pain through N-methyl-d-aspartate receptor neuronal mechanisms. Here, we report that ephrinB-EphB signaling may also act through a Toll-like receptor 4 (TLR4)-glial cell mechanism in the spinal cord. Bone cancer pain was induced by tibia bone cavity tumor cell implantation (TCI) in rats. TCI increased the expression of TLR4 and the EphB1 receptor, the activation of astrocytes and microglial cells, and increased levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). The increased expression of TLR4 and EphB1 were colocalized with each other in astrocytes and microglial cells. Spinal knockdown of TLR4 suppressed TCI-induced behavioral signs of bone cancer pain. The TCI-induced activation of astrocytes and microglial cells, as well as the increased levels of IL-1β and TNF-α, were inhibited by intrathecal administration of TLR4-targeting siRNA2 and the EphB receptor antagonist EphB2-Fc, respectively. The administration of EphB2-Fc suppressed the TCI-induced increase of TLR4 expression but siRNA2 failed to affect TCI-induced EphB1 expression. Intrathecal administration of an exogenous EphB1 receptor activator, ephrinB2-Fc, increased the expression of TLR4 and the levels of IL-1β and TNF-α, activated astrocytes and microglial cells, and induced thermal hypersensitivity. These ephrinB2-Fc-induced alterations were suppressed by spinal knockdown of TLR4. This study suggests that TLR4 may be a potential target for preventing or reversing bone cancer pain and other similar painful processes mediated by ephrinB-EphB receptor signaling.

Protease‐activated receptor 2 in dorsal root ganglion contributes to peripheral sensitization of bone cancer pain

Treating bone cancer pain continues to be a major clinical challenge, and the underlying mechanisms of bone cancer pain remain elusive. Protease-activated receptor 2 (PAR2) has been reported to be involved in neurogenic inflammation, nociceptive pain and hyperalgesia. Here, we investigated the role of PAR2 in bone cancer pain development. Expression of PAR2, mechanical allodynia, thermal hyperalgesia and neurochemical alterations induced by bone cancer pain were analysed in male, adult C3H/HeJ mice with tumour cell implantation (TCI). To investigate the contribution of PAR2 to bone cancer pain, PAR2 antagonist peptide and PAR2 knockout mice were used. TCI produced bone cancer-related pain behaviours. Production and persistence of these pain behaviours were well correlated with TCI-induced up-regulation of PAR2 in sciatic nerve and dorsal root ganglia (DRG). PAR2 knockout and spinal administration of PAR2 antagonist peptide prevented and/or reversed bone cancer-related pain behaviours and associated neurochemical changes in DRG and dorsal horn (DH). TCI also induced proteases release in tumour-bearing tibia, sciatic nerve and DRG. Plantar injection of supernatant from sarcoma cells induced PAR2 up-regulation and intracellular calcium [Ca(2+) ]i increase in DRG, and calcitonin gene-related peptide accumulation in DH, as well as significant thermal and mechanical hyperalgesia, which were all in PAR2-dependent manners. These findings suggest that PAR2 may be a key mediator for peripheral sensitization of bone cancer pain. Inhibiting PAR2 activation, especially during the early phase, may be a new therapy for preventing/suppressing development of bone cancer pain.

Upregulation of P2X3 receptors by neuronal calcium sensor protein VILIP-1 in dorsal root ganglions contributes to the bone cancer pain in rats

Primary and metastatic cancers that affect bone are frequently associated with severe and intractable pain. The mechanisms underlying the development of bone cancer pain are largely unknown. In this study, we first demonstrated that a functional upregulation of P2X3 receptors in dorsal root ganglion (DRG) neurons is closely associated with the neuronal hyperexcitability and the cancer-induced bone pain in MRMT-1 tumor cell–inoculated rats. Second, we revealed that visinin-like protein 1 (VILIP-1), a member of visinin-like proteins that belong to the family of neuronal calcium sensor proteins is responsible for the observed upregulation of P2X3 receptors in DRG neurons. The interaction between the amino terminus of VLIP-1 and the carboxyl terminus of the P2X3 receptor is critical for the surface expression and functional enhancement of the receptor. Finally, overexpression of VILIP-1 increases the expression of functional P2X3 receptors and enhances the neuronal excitability in naive rat DRG neurons. In contrast, knockdown of VILIP-1 inhibits the development of bone cancer pain via downregulation of P2X3 receptors and repression of DRG excitability in MRMT-1 rats. Taken together, these results suggest that functional upregulation of P2X3 receptors by VILIP-1 in DRG neurons contributes to the development of cancer-induced bone pain in MRMT-1 rats. Hence, P2X3 receptors and VILIP-1 could serve as potential targets for therapeutic interventions in cancer patients for pain management. Pharmacological blockade of P2X3 receptors or knockdown of VILIP-1 in DRGs would be used as innovative strategies for the treatment of bone cancer pain.

Spinal RhoA/Rho Kinase Signalling Pathway may Participate in the Development of Bone Cancer Pain

It has been shown that activation of spinal RhoA/Rho kinase (ROCK) signalling pathway facilitates nociception in neuropathic and inflammatory pain, but its effects on bone cancer pain (BCP) have not previously been studied. This study was designed to examine the potential role of the spinal RhoA/ROCK signalling pathway in the development of BCP. A model for bone cancer was induced by injecting Walker 256 cells into the tibia of rats. On days 6, 9 and 15 after inoculation, the expression of spinal RhoA and ROCK2 protein levels was higher in the Walker 256 cells injected rats compared to the sham rats. On day 9, intrathecal injection of C3 exoenzyme (a RhoA inhibitor, 10pg) significantly attenuated BCP behaviour as well as up-regulation of spinal RhoA and ROCK2 protein levels. These effects were completely abolished by intrathecal pretreatment with U-46619 (a RhoA agonist, 1.5pg). These results suggest that the spinal RhoA/ROCK signalling pathway may be involved in the development of BCP. The findings of this study may lead to novel therapeutic strategies for prevention and/or treatment of BCP.

Changes in protein expression and distribution of spinal CCR2 in a rat model of bone cancer pain

Accumulating evidence suggests that chemokine CC motif receptor 2 (CCR2) plays an important role in neuropathic pain. It has been shown that spinal CCR2 is upregulated in several neuropathic pain models and expressed by neuronal and glial cells in the spinal cord. In this study, we investigated the expression changes and cellular localization of spinal CCR2 in a rat model of bone cancer induced by Walker 256 cell inoculation. The present results indicated that mechanical allodynia progressively increased in bone cancer pain (BCP) rats. Western blot and immunohistochemical analysis demonstrated that the expression of CCR2 in the spinal cord was significantly increased on day 6, 12, and 18 in BCP rats, with a peak on day 6. Furthermore, double immunofluorescence labeling indicated that CCR2 was expressed by both microglia and neurons in the spinal cord. These results suggest that CCR2 may be involved in the development of BCP, and that targeting CCR2 may be a new strategy for the treatment of BCP.

Enhanced SCN7A/Nax expression contributes to bone cancer pain by increasing excitability of neurons in dorsal root ganglion

Bone pain is one of the most common complications in cancer patients with bone metastases, and has the most significant impact on quality of life for patients. Patients with bone cancer pain may be difficult to treat due to the poor understanding of the mechanisms; therefore, the mechanisms of bone cancer pain required elucidation for developing new therapeutics. Recent studies show that SCN7A/Nax channel serves as a sodium-level sensor of the body fluid that controls the Na-intake behavior by changing the excitability of neurons. In the current study, the expression of SCN7A/Nax and the excitability of primary sensory neurons in bone cancer pain rats were examined. The analgesic effects of knockdown SCN7A/Nax channel using RNAi lentivirus intrathecal treatment were evaluated with a behavioral test. The results showed that implantation of sarcoma induced ongoing and movement-evoked pain behaviors, whereas SCN7A/Nax knockdown prevented the onset of these hyperalgesia. Immunohistochemistry showed that SCN7A/Nax was located in the medium- to large-sized neurons in dorsal root ganglions (DRGs). The proportion of SCN7A/Nax-positive cells was significantly increased in DRGs ipsilateral to sarcoma implantation. Immunostaining results were further confirmed by Western blot and real time-polymerase chain reaction (RT-PCR) analyses. Recording from primary sensory neurons in excised rat dorsal root ganglias, we found that most of SCN7A/Nax-positive neurons exhibited subthreshold oscillations, depolarized resting membrane potential and more negative threshold of action potential. These electrophysiological changes of neurons increased ectopic spike discharge which was thought to be an important generator of chronic pain, however, the hyperexcitability was completely reversed by SCN7A/Nax knockdown. These results demonstrate that enhanced expression of SCN7A/Nax channel within distinct subpopulation of DRG neurons contributes to bone cancer pain by increasing the excitability of these neurons. These findings may lead to novel strategies for the treatment of bone cancer pain.