Induced Bone Cancer Pain Research Articles

Microglia Promote Inhibitory Synapse Phagocytosis in the Spinal Cord Dorsal Horn and Modulate Pain-Like Behaviors in a Murine Cancer-Induced Bone Pain Model.

The microglial activation has been implicated in cancer-induced bone pain. Recent studies have revealed that microglia mediate synaptic pruning in the central nervous system, where the cluster of differentiation 47-signal regulatory protein α (CD47-SIRPα) axis creates a "don't eat me" signal and elicits an antiphagocytic effect to protect synapses against elimination. To date, the synaptic phagocytosis in microglia has never been investigated in the murine cancer-induced bone pain model. The present experiments sought to explore whether microglia phagocytize synapses in mice with bone cancer pain as well as the possible mechanisms. Male C3H/HeN mice were used to induce bone cancer pain. Minocycline and S-ketamine were injected into D14. The number of spontaneous flinches (NSF) and paw withdrawal mechanical thresholds (PWMT) were measured on D0, D4, D7, D10, D14, D21, and D28. Hematoxylin and eosin staining presented bone lesions. Western blotting examined the Gephyrin, CD47, and SIRPα expression. Flow cytometry evaluated the proportion of SIRPα + cells in the spine. Immunofluorescence and 3-dimensional reconstruction showed the Gephyrin puncta inside microglial lysosomes. Mice embedded with tumor cells induced persistent spontaneous pain and mechanical hyperalgesia. Hematoxylin and eosin staining revealed bone destruction and tumor infiltration in marrow cavities. Microglia underwent a responsive and proliferative burst (t = -16.831, P < .001). Western blotting manifested lowered Gephyrin expression in the tumor group (D4, D7, D10, D14, D21, and D28: P < .001). Immunofluorescence and 3-dimensional reconstruction showed larger volumes of Gephyrin puncta inside microglial lysosomes (t = -23.273, P < .001; t = -27.997, P < .001). Treatment with minocycline or S-ketamine exhibited pain relief and antiphagocytic effects (t = -6.191, P < .001, t = -7.083, P < .001; t = -20.767, P < .001, t = -17.080, P < .001; t = 11.789, P < .001, t = 16.777, P < .001; t = 8.868, P < .001, t = 21.319, P < .001). Last but not least, the levels of CD47 and SIRPα proteins were downregulated (D10: P = .004, D14, D21, and D28: P < .001; D10, D14, D21, and D28: P < .001). Flow cytometry and immunofluorescence substantiated reduced microglial SIRPα (t = 11.311, P < .001; t = 12.189, P < .001). Microglia-mediated GABAergic synapse pruning in the spinal cord dorsal horn in bone cancer pain mice, which might be associated with the declined CD47-SIRPα signal. Our research uncovered an innovative mechanism that highlighted microglia-mediated synaptic phagocytosis in a murine cancer-induced bone pain model.

Folic acid relieves bone cancer pain by downregulating P2X2/3 receptors in rats

BackgroundBone cancer pain (BCP) remains a clinical challenge due to the limited and side effects of therapeutic methods. Folic acid has been known as an FDA approved dietary supplement and proved to have an analgesic effect in neuropathic pain. Here we investigate the role and mechanism of folic acid in bone cancer pain of a rat model. MethodsWalker 256 tumor cells were inoculated into the left tibia of rats to induce bone cancer pain model. Pain reflex were assessed by paw withdrawal threshold (PWT) response to Von Frey filaments and paw withdrawal latency (PWL) response to thermal stimulation. Folic acid was injected intraperitoneally to evaluate its analgesic effect in rats with bone cancer pain. Western blotting and qPCR were used to determine P2X2/3 receptor protein and mRNA levels in ipsilateral L4-6 dorsal root ganglion (DRG) and spinal dorsal horn (SDH). ResultsThe PWT and PWL of rats with bone cancer pain were obviously decreased compared to the naïve and sham rats. Interestingly, continuous folic acid treatment significantly increased the PWT and PWL of rats with bone cancer pain. P2X2 and P2X3 receptors were clearly upregulated at both mRNA and protein expression in L4-6 DRG and SDH of rats with bone cancer pain. P2X2 and P2X3 receptors were mainly localized with CGRP (calcitonin gene-related peptide) or IB4 (isolectin B4) positive neurons in L4-6 DRG of rats with bone cancer pain. Notably, continuous folic acid treatment significantly reduced the expression of P2X2 and P2X3 receptors in L4-6 DRG and SDH of rats with bone cancer pain. Finally, intrathecal injection of A317491 (a selective antagonist of P2X2/3 receptors) markedly elevated the PWT and PWL of rats with bone cancer pain. ConclusionThese results suggest that folic acid has an effective antinociceptive effect on bone cancer pain, which is mediated by downregulating P2X2/3 receptors in L4-6 DRG and SDH of rats with bone cancer pain. Folic acid may be a novel therapeutic strategy in cancer patients for pain relief.

TRPV1 in dorsal root ganglion contributed to bone cancer pain.

Tumor growth in situ or bone metastases in cancer patients all can induce bone cancer pain. It is frequently occurred in patients with breast, prostate, and lung cancer. Because of the lack of effective treatment, bone cancer pain causes depression, anxiety, fatigue, and sleep disturbances in cancer patients, disrupts the daily quality of life, and results in huge economic and psychological burden. Over the past years, transient receptor potential channels (TRPs), especially TRP vanilloid 1 (TRPV1) in dorsal root ganglion (DRG), have been considered to be involved in bone cancer pain. The characteristic of TRPV1 had been well studied. The mechanisms under TRPV1 regulation in DRG with bone cancer pain are complex, including inflammatory mediators, endogenous formaldehyde, and other mechanisms. In the present review, we summarize the role and potential mechanism of TRPV1 in DRG in bone cancer pain. As the primary sensory neurons, targeting the TRPV1 channel in DRG, might have fewer side effects than in central. We hope systematically understand of TRPV1 modulation in DRG will bring more effective strategy.

Activation of the STING pathway induces peripheral sensitization via neuroinflammation in a rat model of bone cancer pain

BackgroundNeuroinflammation in the peripheral nervous system has been linked to cancer metastasis-induced bone pain. The stimulator of interferon genes (STING), an innate immune sensor for cytosolic DNA, plays an important role in inflammation and cancer metastasis and is reported to be a critical regulator of nociception. Here, we examined the role of STING in primary nociceptive neurons and chronic pain to determine if it could be a new target for treating bone cancer pain (BCP).MethodsWalker 256 cancer cells were injected intratibially to induce bone cancer pain in rats. STING and its downstream inflammatory factors in dorsal root ganglia (DRG) were detected using western blotting and immunofluorescent staining. Transmission electron microscopy and the BCL2-associated X (Bax) expression were used to detect the mitochondrial stress in DRG neurons. C-176, a specific inhibitor of STING, was used to block STING activation and to test the pain behavior.ResultsMechanical hyperalgesia and spontaneous pain were observed in BCP rats, accompanied by the upregulation of the STING expression in the ipsilateral L4-5 DRG neurons which showed significant mitochondrion stress. The STING/TANK-binding kinase 1 (TBK1)/nuclear factor-kappa B (NF-κB) pathway activation was observed in the DRGs of BCP rats as well as increased IL-1β, IL-6, and TNF-α expression. C-176 alleviated bone cancer pain and reduced the STING and its downstream inflammatory pathway.ConclusionWe provide evidence that STING pathway activation leads to neuroinflammation and peripheral sensitization. Pharmacological blockade of STING may be a promising novel strategy for preventing BCP.

Evidence of the Involvement of Spinal EZH2 in the Development of Bone Cancer Pain in Rats.

IntroductionBone cancer pain (BCP) seriously affects the quality of life of patients with advanced cancer, but effective treatment methods are lacking. This study mainly investigates the role of EZH2 in a well-established BCP model induced by Walker 256 breast cancer cells in rats.MethodsFemale Sprague–Dawley rats of the same age weighing approximately 160 g were selected for the experiment. The BCP model was established by injecting inactivated Walker 256 breast cancer cells into the tibia. von Frey filaments were used to measure the paw withdrawal threshold, and bone destruction in the rat was observed using x-ray. The spinal EZH2 and H3K27Tm levels were measured using Western blotting and RT–qPCR analysis. Intrathecal injection of an EZH2 inhibitor was performed to examine the role of EZH2 in trigeminal BCP.ResultsExperimental results showed that injecting Walker 256 breast cancer cells into the tibia induced bone cancer pain. Spinal EZH2 and H3K27Tm levels were significantly increased over time in BCP rats. An intrathecal injection of 3-deazaneplanocin A (DZNep), a selective EZH2 inhibitor, downregulated the expression of EZH2 and attenuate the BCP-induced mechanical allodynia state.ConclusionIntrathecal injection of DZNep relieve bone cancer pain in rats. EZH2 expressed in spinal cord tissue may be involved in the process of bone cancer pain in rats.

AbobotulinumtoxinA (Dysport®) shows efficacy in a model of MRMT-1–induced bone cancer pain in the rat

AbobotulinumtoxinA (Dysport®) shows efficacy in a model of MRMT-1–induced bone cancer pain in the rat

Simultaneous inhibition of NMDA and mGlu1/5 receptors by levo‐corydalmine in rat spinal cord attenuates bone cancer pain

Bone cancer pain is a challenge for its not completely clarified mechanism and broad clinical morbidity. Therefore, novel and more effective drugs are urgent needed for improvement of patients' quality of life. Glutamate receptors have been associated with the development of the central sensitization of chronic pain. Inhibition of N-methyl-d-aspartate (NMDA) and metabotropic glutamate (mGlu) receptors can effectively attenuate bone cancer pain, respectively. Herein, our results indicated that levo-Corydalmine (l-CDL), a compound from Corydalis yanhusuo W.T. Wang, which has been used in traditional Chinese medicine for pain relief could effectively attenuate bone cancer pain induced by tibia bone cavity tumor cell implantation (TCI) through simultaneously inhibiting the NMDA and mGlu1/5 receptors in rat spinal cord without notable side effects. Both intragastric and intrathecal administration of l-CDL significantly alleviated the mechanical hypersensitivity induced by TCI in rats, and the analgesic effect of l-CDL could be reversed by intrathecal administration of NMDA receptor agonist NMDA and mGlu1/5 receptor agonist DHPG but not AMPA receptor agonist AMPA. l-CDL could also selectively suppress NMDA and DHPG induced rapid rise in Ca2+ oscillations in primary cultures neurons of spinal cord in vitro. The antinociception of l-CDL were partially mediated by the reduced phosphorylation of PKC γ and ERK1/2 in spinal cord of TCI rats in a NMDA and mGlu1/5 dependent manner. In conclusion, these results suggested that l-CDL attenuates TCI induced bone cancer pain through simultaneously inhibiting the NMDA and mGlu1/5 receptors and the downstream PKC γ, ERK1/2 signaling pathways in the spinal cord.

Effect of intrathecal injection of magnesium sulfate on pain behavior in bone cancer pain mouse

Objective To investigate the effect of intrathecal injection of magnesium sulfate(MgSO4) on pain behavior in mouse with bone cancer pain. Methods 56 male 8-10 week old C3H/HeJ mice weighing 18-22 g were divided randomly into 7 groups(n=8): sham group(S group), control group(C group) and MgSO4 plus morphine treat groups(T1-T5 group). Group C and T mice were induced bone cancer pain models by intra-right-femur inoculation of osteolytic NCTC2472 cells while group S were injected of only α-MEM. On the 14d after inoculation, group S and C received intrathecal injection of artificial cerebrospinal fluid 5μl, while group T1-T5 received intrathecal injection of MgSO4 14.4μg, 43.2μg, 86.4μg, morphine 0.36μg, MgSO4 14.4μg-morphine 0.36μg, which were dissolved in 5μl artificial cerebrospinal fluid. Mice received pain behavior tests including quantification of spontaneous flinches, paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) at 0.5h before and 0.5h, 2h, 4h, 8h after administration. Results Treatment with MgSO4(14.4μg), morphine (0.36μg) have no effect on bone cancer pain, while treatment with MgSO4 (43.2μg, 86.4μg)can dose-dependently reverse quantification of spontaneous flinches, mechanical allodynia and thermal hyperalgesia which were induced by inoculation as well as MgSO4 14.4μg-morphine 0.36μg. At 0.5h after administration, the quantification of spontaneous flinches of the three groups((10.08±1.66), (7.35±1.36), (10.54±1.32)) were decreased when compared with control group (13.05±2.06), PWMT ((0.81±0.22) g; (1.33±0.19)g; (0.93±0.26)g), PWTL((10.57±1.53)s; (13.12±1.71)s; (11.46±1.83)s) were increased when compared with control group((0.42±0.23)g, (8.87±1.27)s)(P<0.05). The effect reached maximum level at 2h, lasted for at least 4h and disappeared at 8h. Conclusion Intrathecal injection MgSO4 can effectively attenuate bone cancer pain dose-dependently.At the same time MgSO4 can amplify the analgesic effect of subliminal morphine. Key words: Bone cancer pain; Magnesium sulfate; Morphine; Behavior; Mouse

Positive feedback regulation between microRNA-132 and CREB in spinal cord contributes to bone cancer pain in mice.

cAMP response element-binding protein (CREB)-dependent gene expression plays an important role in central sensitization. CREB-regulated transcription coactivator 1 (CRTC1) dramatically increase CREB-mediated transcriptional activity. microRNA-132 (miR-132), which is highly CREB-responsive, functions downstream from CREB/CRTC1 to mediate activity-dependent synaptic plasticity and in turn loops back to amplify CREB/CRTC1 signalling. This study aimed to investigate the positive feedback regulation between miR-132 and CREB in spinal cord in the maintenance of bone cancer pain. Osteosarcoma cells were implanted into the intramedullary space of the right femurs of C3H/HeNCrlVr mice to induce bone cancer pain. We further investigated effects of repeated intrathecal administration with Adenoviruses expressing CREB-siRNA or miR-132 antisense locked nucleic acid (LNA), respectively, on nociceptive behaviours and on the activity of CREB/CRTC1 signalling. Intramedullary inoculation of osteosarcoma cells resulted in up-regulation of spinal p-CREB, CRTC1 and CREB-target genes (NR2B and miR-132). Repeated intrathecal administration with Adenoviruses expressing CREB-siRNA or miR-132 LNA-AS, respectively, attenuated bone cancer-evoked pain behaviours, reduced the activity of CREB/CRTC1 signalling and down-regulated CREB-target gene NR2B expression in spinal cord. These findings suggest that activation of spinal CREB/CRTC1 signalling may play an important role in bone cancer pain. Interruption to the positive feedback regulation between CREB/CRTC1 and its target gene miR-132 can effectively relieved the bone cancer-induced mechanical allodynia and spontaneous pain. WHAT DOES THIS STUDY ADD?: The positive feedback regulation between CREB/CRTC1 and its target gene miR-132 in spinal cord plays an important role in bone cancer pain.

New Mechanism of Bone Cancer Pain: Tumor Tissue-Derived Endogenous Formaldehyde Induced Bone Cancer Pain via TRPV1 Activation.

In recent years, our serial investigations focused on the role of cancer cells-derived endogenous formaldehyde in bone cancer pain. We found that cancer cells produced formaldehyde through demethylation process by serine hydroxymethyltransferase (SHMT1 and SHMT2) and lysine-specific histone demethylase 1 (LSD1). When the cancer cells metastasized into bone marrow, the elevated endogenous formaldehyde induced bone cancer pain through activation on the transient receptor potential vanilloid subfamily member 1 (TRPV1) in the peripheral nerve fibers. More interestingly, TRPV1 expressions in the peripheral fibers were upregulated by the local insulin-like growth factor I (IGF-I) produced by the activated osteoblasts. In conclusion, tumor tissue-derived endogenous formaldehyde induced bone cancer pain via TRPV1 activation.

CREB-regulated transcription coactivator 1 enhances CREB-dependent gene expression in spinal cord to maintain the bone cancer pain in mice.

BackgroundcAMP response element binding protein (CREB)-dependent gene expression plays an important role in central sensitization. CREB-regulated transcription coactivator 1 (CRTC1) dramatically increases CREB-mediated transcriptional activity. Brain-derived neurotrophic factor, N-methyl-d-aspartate receptor subunit 2B, and miRNA-212/132, which are highly CREB responsive, function downstream from CREB/CRTC1 to mediate activity-dependent synaptic plasticity and in turn loops back to amplify CREB/CRTC1 signaling. This study aimed to investigate the role of spinal CRTC1 in the maintenance of bone cancer pain using an RNA interference method.ResultsOsteosarcoma cells were implanted into the intramedullary space of the right femurs of C3H/HeNCrlVr mice to induce bone cancer pain. Western blotting was applied to examine the expression of spinal phospho-Ser133 CREB and CRTC1. We further investigated effects of repeated intrathecal administration with Adenoviruses expressing CRTC1-small interfering RNA (siRNA) on nociceptive behaviors and on the upregulation of CREB/CRTC1-target genes associated with bone cancer pain. Inoculation of osteosarcoma cells induced progressive mechanical allodynia and spontaneous pain, and resulted in upregulation of spinal p-CREB and CRTC1. Repeated intrathecal administration with Adenoviruses expressing CRTC1-siRNA attenuated bone cancer–evoked pain behaviors, and reduced CREB/CRTC1-target genes expression in spinal cord, including BDNF, NR2B, and miR-212/132.ConclusionsUpregulation of CRTC1 enhancing CREB-dependent gene transcription in spinal cord may play an important role in bone cancer pain. Inhibition of spinal CRTC1 expression reduced bone cancer pain. Interruption to the positive feedback circuit between CREB/CRTC1 and its targets may contribute to the analgesic effects. These findings may provide further insight into the mechanisms and treatment of bone cancer pain.

EphrinB–EphB signaling regulates spinal pain processing via PKCγ

Spinal ephrinB–EphB signaling is involved in the modulation of pain processing. The aim of the present study was to investigate whether protein kinase C-γ (PKCγ) acts as a downstream effector in regulating spinal pain processing associated with ephrinB–EphB signaling in mice. The intrathecal injection of ephrinB2-Fc, an EphB receptor activator, caused thermal hyperalgesia and mechanical allodynia, as well as increased activation of spinal PKCγ. Knockdown of spinal PKCγ prevented the pain behaviors induced by ephrinB2-Fc. Furthermore, the intrathecal injection of EphB2-Fc, an EphB receptor blocker, suppressed formalin-induced inflammatory, chronic constriction injury (CCI)-induced neuropathic, and tibia bone cavity tumor cell implantation (TCI)-induced bone cancer pain behaviors, in addition to reducing the activation of spinal PKCγ. Finally, the intrathecal injection of MK801, an N-methyl-d-aspartate (NMDA) receptor blocker, prevented the pain behaviors and spinal PKCγ activation induced by ephrinB2-Fc. Overall, the results confirm the important role of PKCγ in the regulation of spinal pain processing associated with ephrinB–EphB signaling.

Kinesin superfamily protein 17 contributes to the development of bone cancer pain by participating in NR2B transport in the spinal cord of mice

Τreatment of bone cancer pain remains a challenge, while the mechanisms causing the pain remain elusive. We demonstrated that the expression of the N‑methyl‑D‑aspartate (NMDA) receptor NR2B subunit was upregulated in mice with bone cancer pain. Kinesin superfamily protein 17 (KIF17), a recently characterized member of the kinesin superfamily proteins, has been demonstrated to transport and deliver the NR2B subunit to dendrites in mammalian neurons. In the present study, we induced bone cancer pain via femur bone cavity osteosarcoma NCTC 2472 tumor cell implantation (TCI) in mice. The results showed that TCI in mice increased the number of spontaneous flinches, mechanical allodynia events, expression of spinal KIF17 and NR2B subunits. Intrathecal administration of KIF17 antisense oligodeoxynucleotide (ODN) attenuated the behavioral signs of bone cancer pain and suppressed the increased expression of NR2B induced by TCI. In addition, KIF17 binds to a protein complex that contains mLin‑10 to transport NR2B, and we determined that the increase of mLin‑10 was suppressed following admini-stration. Thus, these findings suggested that KIF17 contributed to the development of bone cancer pain in the spinal cord through NR2B transport and that mLin‑10 may also play a role in pain development.

PKA is required for the modulation of spinal nociceptive information related to ephrinB–EphB signaling in mice

EphB receptors and their ephrinB ligands are implicated in modulating of spinal nociceptive information processing. Here, we investigated whether protein kinase A (PKA), acts as a downstream effector, participates in the modulation spinal nociceptive information related to ephrinB–EphB signaling. Intrathecal injection of ephrinB2-Fc caused thermal hyperalgesia and mechanical allodynia, which were accompanied by increased expression of spinal PKA catalytic subunit (PKAca) and phosphorylated cAMP-response element-binding protein (p-CREB). Pre-treatment with H89, a PKA inhibitor, prevented the activation of CREB by ephrinB2-Fc. Inhibition of spinal PKA signaling prevented and reversed pain behaviors induced by the intrathecal injection of ephrinB2-Fc. Furthermore, blockade of the EphB receptors by intrathecal injection of EphB2-Fc reduced formalin-induced inflammatory, chronic constrictive injury (CCI)-induced neuropathic, and tibia bone cavity tumor cell implantation (TCI)-induced bone cancer pain behaviors, which were accompanied by decreased expression of spinal PKAca and p-CREB. Overall, these results confirmed the important involvement of PKA in the modulation of spinal nociceptive information related to ephrinBs–EphBs signaling. This finding may have important implications for exploring the roles and mechanisms of ephrinB–EphB signaling in physiologic and pathologic pain.

Role of cannabinoid 2 receptor in the development of bone cancer pain

To explore the effects of cannabinoid 2 receptor (CB2) in the development of bone cancer pain in mice. A total of 84 mice (C3H/HeJ) were randomly divided into 4 groups:tumor group (Group T, n = 30), medication administration group (Group J, n = 12), vehicle group (Group D, n = 12) and sham group (Group S, n = 30). And 2 × 10(5) osteolytic NCTC2472 cells in α-MEM were injected into medullary cavity of right distal femur to induce bone cancer pain in a murine model while sham mice received an injection of only α-MEM. All mice were tested for pain-related behaviors at pre-inoculation and at Days 5, 7, 10 and 14 post-inoculation. The tests included paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL). Group J and Group D were injected intrathecally with 2 µg JWH015 dissolved in 4% DMSO and only 4% DMSO respectively in a 5 µl. volume. Pain behavior tests were performed before and at 1, 6, 24, 48 and 72 h after an intrathecal injection. Lumbar intumescentia of mice in each group were harvested to examine the expression level of CB2 by Western blot after pain behavior tests at Days 5, 7, 10 and 14 post-inoculation and 12 h after an intrathecal injection. (1) Pain behavior tests:Mechanical allodynia appeared at Day 7 post-inoculation. The value of PWMT was (1.27 ± 0.28) g (P < 0.05) and it declined gradually to (0.53 ± 0.20) g at Day 14. The threshold of mechanical hyperalgesia increased to (1.00 ± 0.20) g at 6 h after an intrathecal injection of JWH015, peaked at (1.40 ± 0.39) g at 12 h, became alleviated after 48 h and recovered to the pre-dosing levels at 72 h. Thermal hyperalgesia appeared at Day 10 post-inoculation. The value of PWTL was (16.9 ± 0.4) s (P < 0.05) at Day 10 and declined to (11.5 ± 0.7) s at Day 14 post-inoculation. The threshold of thermal hyperalgesia increased to (15.7 ± 1.9) g at 6 h after an intrathecal injection of JWH015, peaked at (18.6 ± 2.3) g at 12 h, became alleviated after 48 h and recovered to the pre-dosing levels at 72 h. (2) Western blot: From Day 5 post-inoculation, the ratio of CB2/β-actin increased gradually. Compared with the ratio of 0.190 ± 0.010 at Day 5 post-inoculation, the ratio of CB2/β-actin increased to 0.660 ± 0.010 at Day 14 post-inoculation (P < 0.05); compared with the ratio of 0.903 ± 0.006 in group D at 12 h after an intrathecal injection of JWH015, the ratio of CB2/β-actin 0.510 ± 0.010 significantly decreased (P < 0.05). The cannabinoid 2 receptor plays an important role in the formation of bone cancer pain.

Intrathecal Injection of Metabotropic Glutamate Receptor Subtype 3 and 5 Agonist/Antagonist Attenuates Bone Cancer Pain by Inhibition of Spinal Astrocyte Activation in a Mouse Model

Astrocytes and metabotropic glutamate receptors play important roles in nociceptive processing. However, their roles in bone cancer pain were not well understood. This study sought to investigate whether selective mGluR3 and mGluR5 agonist or antagonist develop antinociceptive effects on bone cancer pain by inhibition of spinal astrocyte activation. C3H/HeNCrlVr mice were inoculated into the intramedullary space of the femur with sarcoma NCTC 2472 cells to induce bone cancer pain. Quantitative real-time reverse transcription-polymerase chain reaction and Western blot experiments examined messenger RNA and protein expression of spinal glial fibrillary acidic protein, mGluR3, and mGluR5. The authors further investigated effects of intrathecal treatment with the mGluR3 agonist (APDC), the mGluR3 antagonist (LY341495), the mGluR5 agonist (CHPG), or the mGluR5 antagonist (MTEP) on nociceptive behaviors and spinal astrocyte activation associated with bone cancer pain. Inoculation of sarcoma cells, but not α-MEM solution, induced progressive bone cancer pain and resulted in up-regulation of glial fibrillary acidic protein, mGluR3, and mGluR5 expression on days 10, 14, and 21 postinoculation. Intrathecal administration of APDC and MTEP attenuated bone cancer-evoked spontaneous pain, mechanical allodynia, thermal hyperalgesia, and reduced spinal glial fibrillary acidic protein expression. However, treatment with LY341495 and CHPG induced thermal hyperalgesia and spinal glial fibrillary acidic protein expression in control mice. Spinal mGluR3 activation or mGluR5 inhibition reduced bone cancer pain. Inhibition of spinal astrocyte activation may contribute to the analgesic effects. These findings may lead to novel strategies for the treatment of bone cancer pain.

Tumor Tissue-Derived Formaldehyde and Acidic Microenvironment Synergistically Induce Bone Cancer Pain

BackgroundThere is current interest in understanding the molecular mechanisms of tumor-induced bone pain. Accumulated evidence shows that endogenous formaldehyde concentrations are elevated in the blood or urine of patients with breast, prostate or bladder cancer. These cancers are frequently associated with cancer pain especially after bone metastasis. It is well known that transient receptor potential vanilloid receptor 1 (TRPV1) participates in cancer pain. The present study aims to demonstrate that the tumor tissue-derived endogenous formaldehyde induces bone cancer pain via TRPV1 activation under tumor acidic environment.Methodology/Principal FindingsEndogenous formaldehyde concentration increased significantly in the cultured breast cancer cell lines in vitro, in the bone marrow of breast MRMT-1 bone cancer pain model in rats and in tissues from breast cancer and lung cancer patients in vivo. Low concentrations (1∼5 mM) of formaldehyde induced pain responses in rat via TRPV1 and this pain response could be significantly enhanced by pH 6.0 (mimicking the acidic tumor microenvironment). Formaldehyde at low concentrations (1 mM to 100 mM) induced a concentration-dependent increase of [Ca2+]i in the freshly isolated rat dorsal root ganglion neurons and TRPV1-transfected CHO cells. Furthermore, electrophysiological experiments showed that low concentration formaldehyde-elicited TRPV1 currents could be significantly potentiated by low pH (6.0). TRPV1 antagonists and formaldehyde scavengers attenuated bone cancer pain responses.Conclusions/SignificanceOur data suggest that cancer tissues directly secrete endogenous formaldehyde, and this formaldehyde at low concentration induces metastatic bone cancer pain through TRPV1 activation especially under tumor acidic environment.

A Blocking Antibody to Nerve Growth Factor Attenuates Skeletal Pain Induced by Prostate Tumor Cells Growing in Bone

Prostate cancer is unique in that bone is often the only clinically detectable site of metastasis. Prostate tumors that have metastasized to bone frequently induce bone pain which can be difficult to fully control as it seems to be driven simultaneously by inflammatory, neuropathic, and tumorigenic mechanisms. As nerve growth factor (NGF) has been shown to modulate inflammatory and some neuropathic pain states in animal models, an NGF-sequestering antibody was administered in a prostate model of bone cancer where significant bone formation and bone destruction occur simultaneously in the mouse femur. Administration of a blocking antibody to NGF produced a significant reduction in both early and late stage bone cancer pain-related behaviors that was greater than or equivalent to that achieved with acute administration of 10 or 30 mg/kg of morphine sulfate. In contrast, this therapy did not influence tumor-induced bone remodeling, osteoblast proliferation, osteoclastogenesis, tumor growth, or markers of sensory or sympathetic innervation in the skin or bone. One rather unique aspect of the sensory innervation of bone, that may partially explain the analgesic efficacy of anti-NGF therapy in relieving prostate cancer-induced bone pain, is that nearly all nerve fibers that innervate the bone express trkA and p75, and these are the receptors through which NGF sensitizes and/or activates nociceptors. The present results suggest that anti-NGF therapy may be effective in reducing pain and enhancing the quality of life in patients with prostate tumor-induced bone cancer pain.