Involvement of HDAC2-mediated kcnq2/kcnq3 genes transcription repression activated by EREG/EGFR-ERK-Runx1 signaling in bone cancer pain.

Bone cancer pain (BCP) represents a prevalent symptom among cancer patients with bone metastases, yet its underlying mechanisms remain elusive. This study investigated the transcriptional regulation mechanism of Kv7(KCNQ)/M potassium channels in DRG neurons and its involvement in the development of BCP in rats. We show that HDAC2-mediated transcriptional repression of kcnq2/kcnq3 genes, which encode Kv7(KCNQ)/M potassium channels in dorsal root ganglion (DRG), contributes to the sensitization of DRG neurons and the pathogenesis of BCP in rats. Also, HDAC2 requires the formation of a corepressor complex with MeCP2 and Sin3A to execute transcriptional regulation of kcnq2/kcnq3 genes. Moreover, EREG is identified as an upstream signal molecule for HDAC2-mediated kcnq2/kcnq3 genes transcription repression. Activation of EREG/EGFR-ERK-Runx1 signaling, followed by the induction of HDAC2-mediated transcriptional repression of kcnq2/kcnq3 genes in DRG neurons, leads to neuronal hyperexcitability and pain hypersensitivity in tumor-bearing rats. Consequently, the activation of EREG/EGFR-ERK-Runx1 signaling, along with the subsequent transcriptional repression of kcnq2/kcnq3 genes by HDAC2 in DRG neurons, underlies the sensitization of DRG neurons and the pathogenesis of BCP in rats. These findings uncover a potentially targetable mechanism contributing to bone metastasis-associated pain in cancer patients.

Discovery of a CCR2-targeting pepducin therapy for chronic pain.

Targeting the CCL2/CCR2 chemokine axis has been shown to be effective at relieving pain in rodent models of inflammatory and neuropathic pain, therefore representing a promising avenue for the development of non-opioid analgesics. However, clinical trials targeting this receptor for inflammatory conditions and painful neuropathies have failed to meet expectations and have all been discontinued due to lack of efficacy. To overcome the poor selectivity of CCR2 chemokine receptor antagonists, we generated and characterized the function of intracellular cell-penetrating allosteric modulators targeting CCR2, namely pepducins. In vivo, chronic intrathecal administration of the CCR2-selective pepducin PP101 was effective in alleviating neuropathic and bone cancer pain. In the setting of bone metastases, we found that T cells infiltrate dorsal root ganglia (DRG) and induce long-lasting pain hypersensitivity. By acting on CCR2-expressing DRG neurons, PP101 attenuated the altered phenotype of sensory neurons as well as the neuroinflammatory milieu of DRGs, and reduced bone cancer pain by blocking CD4+ and CD8+ T cell infiltration. Notably, PP101 demonstrated its efficacy in targeting the neuropathic component of bone cancer pain, as evidenced by its anti-nociceptive effects in a model of chronic constriction injury of the sciatic nerve. Importantly, PP101-induced reduction of CCR2 signaling in DRGs did not result in deleterious tumor progression or adverse behavioral effects. Thus, targeting neuroimmune crosstalk through allosteric inhibition of CCR2 could represent an effective and safe avenue for the management of chronic pain.

ALKBH5 modulates bone cancer pain in a rat model by suppressing NR2B expression.

Currently, the clinical treatment of bone cancer pain (BCP) is mainly related to its pathogenesis. The aim of the present study was to elucidate the potential role of N6-methyladenosine (m6A) in BCP in the spinal cord dorsal root ganglia (DRG) of BCP rats and its specific regulatory mechanism in N-methyl-d-aspartate receptor subunit 2B (NR2B). A rat model of BCP was constructed by tibial injection of Walker256 cells, and ALKBH5 and NR2B expression in the spinal cord DRG was detected. ALKBH5 was silenced or overexpressed in PC12 cells to verify the regulatory effect of ALKBH5 on NR2B. The specific mechanism underlying the interaction between ALKBH5 and NR2B was investigated using methylated RNA immunoprecipitation and dual-luciferase reporter gene assays. The results showed increased expression of m6A, decreased expression of ALKBH5, and increased expression of NR2B in the DRG of the BCP rat model. Overexpression of ALKBH5 inhibited NR2B expression, whereas interference with ALKBH5 caused an increase in NR2B expression. In NR2B, interference with ALKBH5 caused an increase in m6A modification, which caused an increase in NR2B. Taken together, ALKBH5 affected the expression of NR2B by influencing the stability of the m6A modification site of central NR2B, revealing that ALKBH5 is a therapeutic target for BCP.

MOTS-c is an effective target for treating cancer-induced bone pain through the induction of AMPK-mediated mitochondrial biogenesis.

Bone cancer pain (BCP), due to cancer bone metastasis and bone destruction, is a common symptom of tumors, including breast, prostate, and lung tumors. Patients often experience severe pain without effective treatment. Here, using a mouse model of bone cancer, we report that MOTS-c, a novel mitochondrial-derived peptide, confers remarkable protection against cancer pain and bone destruction. Briefly, we find that the plasma level of endogenous MOTS-c is significantly lower in the BCP group than in the sham group. Accordingly, intraperitoneal administration of MOTS-c robustly attenuates bone cancer-induced pain. These effects are blocked by compound C, an AMPK inhibitor. Furthermore, MOTS-c treatment significantly enhances AMPKα 1/2 phosphorylation. Interestingly, mechanical studies indicate that at the spinal cord level, MOTS-c relieves pain by restoring mitochondrial biogenesis, suppressing microglial activation, and decreasing the production of inflammatory factors, which directly contribute to neuronal modulation. However, in the periphery, MOTS-c protects against local bone destruction by modulating osteoclast and immune cell function in the tumor microenvironment, providing long-term relief from cancer pain. Additionally, we find that chronic administration of MOTS-c has little effect on liver, renal, lipid or cardiac function in mice. In conclusion, MOTS-c improves BCP through peripheral and central synergistic effects on nociceptors, immune cells, and osteoclasts, providing a pharmacological and biological rationale for the development of mitochondrial peptide-based therapeutic agents for cancer-induced pain.

Netrin-1 Role in Nociceptive Neuron Sprouting through Activation of DCC Signaling in a Rat Model of Bone Cancer Pain.

BACKGROUND: Bone cancer pain (BCP) is a common primary or metastatic bone cancer complication. Netrin-1 plays an essential role in neurite elongation and pain sensitization. This study aimed to determine the role of netrin-1 from the metastatic bone microenvironment in BCP development and identify the associated signaling pathway for the strategy of BCP management. METHODS: The rat BCP model was established by intratibial implantation of Walker 256 cells. Von Frey filaments measured the mechanical pain threshold. Movement-induced pain was assessed using limb use scores. Expressions of associated molecules in the affected tibias or dorsal root ganglia (DRG) were measured by immunofluorescence, immunohistochemistry, real-time quantitative polymerase chain reaction, or western blotting. Transduction of deleted in colorectal cancer (DCC) signaling was inhibited by intrathecal injection of DCC-siRNA. RESULTS: In BCP rats, the presence of calcitonin gene-related peptide (CGRP)-positive nerve fibers increased in the metastatic bone lesions. The metastatic site showed enrichment of well-differentiated osteoclasts and expressions of netrin-1 and its attractive receptor DCC. Upregulation of DCC and increased phosphorylation levels of focal adhesion kinase (FAK) and Rac family small GTPase 1/Cell division cycle 42 (Rac1/Cdc42) were found in the DRG. Intrathecal administration of DCC-siRNA led to a significant reduction in FAK and Rac1/Cdc42 phosphorylation levels in the DRG, decreased nociceptive nerve innervation, and improved pain behaviors. CONCLUSIONS: Netrin-1 may contribute to the activation of the BCP by inducing nociceptive nerve innervation and improving pain behaviors.

JAG-1/Notch signaling axis in the spinal cord contributes to bone cancer pain in rats.

Notch signal plays an important role in regulating cell-cell interactions with the adjacent cells. However, it remains unknown whether Jagged1 (JAG-1) mediated Notch signaling regulates bone cancer pain (BCP) via the spinal cell interactions mechanism. Here, we showed that intramedullary injection of Walker 256 breast cancer cells increased the expression of JAG-1 in spinal astrocytes and knockdown of JAG-1 reduced BCP. The supplementation of exogenous JAG-1 to the spinal cord induced BCP-like behavior and promoted expression of c-Fos and hairy and enhancer of split homolog-1 (Hes-1) in the spinal cord of the naïve rats. These effects were reversed when the rats were administered intrathecal injections of N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). The intrathecal injection of DAPT reduced BCP and inhibited Hes-1 and c-Fos expression in the spinal cord. Furthermore, our results showed that JAG-1 up-regulated Hes-1 expression by inducing the recruitment of Notch intracellular domain (NICD) to the RBP-J/CSL-binding site located within the Hes-1 promoter sequence. Finally, the intrathecal injection of c-Fos-antisense oligonucleotides (c-Fos-ASO) and administration of sh-Hes-1 to the spinal dorsal horn also alleviated BCP. The study indicates that inhibition of the JAG-1/Notch signaling axis may be a potential strategy for the treatment of BCP.

Co-Administration of nalbuphine to improve morphine tolerance in mice with bone cancer pain.

BACKGROUND: Kappa-opioid receptor (KOR) agonists are known for having opposite and/or different effects compared with Mu-opioid receptor (MOR) agonists. This study is aimed at clarifying the analgesic effect and tolerance of nalbuphine combined with morphine, and quantifying the mRNA and protein expression of spinal MOR and KOR in a mouse bone cancer pain (BCP) model treated with nalbuphine and morphine. METHOD: BCP model was prepared in C3H/HeNCrlVr Mice by implanting the sarcoma cells into the intramedullary space of the femur. The paw withdrawal thermal latency (PWL) measured by thermal radiometer was used to assess thermal hyperalgesia. PWL testing was performed after implantation and drug administration according to the protocol. Hematoxylin-eosin staining in the spinal cord and x-ray in the femoral intramedullary canal was detected. Real-time PCR and western blot analysis played a role in detecting spinal MOR and KOR expression changes. RESULTS: In tumor-implanted mice, the spinal MOR and KOR protein and mRNA expression was down-regulated when compared to that in sham-implanted mice (p < 0.05). Morphine therapy can lead to a decrease in spinal µ receptor expression. Similarly, the nalbuphine therapy can lead to a decrease in the expression of κ receptor protein and mRNA at the spinal cord level (p < 0.05). Morphine, nalbuphine, or nalbuphine co-administration with morphine all can extend the paw withdrawal thermal latency (PWL) to radiant thermal stimulation in tumor-implanted mice (p < 0.05). Compared with the morphine treatment group, nalbuphine co-administration with morphine delayed the reduction of PWL value again (p < 0.05). DISCUSSION: BCP itself may induce down-regulation of the spinal MOR and KOR expression. A low dose of nalbuphine co-administration with morphine led to the delayed emergence of morphine tolerance. The part of the mechanism may be due to the regulation of spinal opioid receptors expression.

Rac1/PAK1 signaling contributes to bone cancer pain by Regulation dendritic spine remodeling in rats.

Bone cancer pain (BCP) is severe chronic pain caused by tumor metastasis to the bones, often resulting in significant skeletal remodeling and fractures. Currently, there is no curative treatment. Therefore, insight into the underlying mechanisms could guide the development of mechanism-based therapeutic strategies for BCP. We speculated that Rac1/PAK1 signaling plays a critical role in the development of BCP. Tumor cells implantation (TCI) into the tibial cavity resulted in bone cancer-associated mechanical allodynia. Golgi staining revealed changes in the excitatory synaptic structure of WDR (Wide-dynamic range) neurons in the spinal cord, including increased postsynaptic density (PSD) length and thickness, and width of the cleft. Behavioral and western blotting test revealed that the development and persistence of pain correlated with Rac1/PAK1 signaling activation in primary sensory neurons. Intrathecal injection of NSC23766, a Rac1 inhibitor, reduced the persistence of BCP as well as reversed the remodeling of dendrites. Therefore, we concluded that activation of the Rac1/PAK1 signaling pathway in the spinal cord plays an important role in the development of BCP through remodeling of dendritic spines. Modulation of the Rac1/PAK1 pathway may be a potential strategy for BCP treatment.

miR-199a-3p mediates bone cancer pain through upregulation of dnmt3a expression in spinal dorsal horn neurons.

Due to its complex pathological mechanisms, bone cancer pain (BCP) has become an increasingly challenging clinical issue, there is an urgent need to identify the underlying mechanisms of BCP. In our present study, we found that decreased expression of miR-199a-3p in spinal dorsal horn (SDH) neurons contributed to BCP hypersensitivity. Intrathecal administration of miR-199a-3p agomir alleviated the initiation of tumor inoculation induced pain hypersensitivity and suppressed the expression of DNMT3A. Subsequently, luciferase assays confirmed direct binding between miR-199a-3p and Dnmt3a mRNA. AAV-DNMT3A-shRNA microinjection relieved mechanical hyperalgesia and upregulated the expression of Nrf2 levels in BCP. In naïve rats, Overexpression of DNMT3A yielded the opposite effects. Finally, increase of DNMT3A by lentiviral vector abolished miR-199a-3p-mediated alleviation hypersensitivity effects on BCP progression. Taken these together, our findings highlighted a novel contribution of miR-199a-3p to BCP and provided a fresh outlook on potential mechanism research for BCP.

Schwann cell insulin-like growth factor receptor type-1 mediates metastatic bone cancer pain in mice.

Insulin growth factor-1 (IGF-1), an osteoclast-dependent osteolysis biomarker, contributes to metastatic bone cancer pain (MBCP), but the underlying mechanism is poorly understood. In mice, the femur metastasis caused by intramammary inoculation of breast cancer cells resulted in IGF-1 increase in femur and sciatic nerve, and IGF-1-dependent stimulus/non-stimulus-evoked pain-like behaviors. Adeno-associated virus-based shRNA selective silencing of IGF-1 receptor (IGF-1R) in Schwann cells, but not in dorsal root ganglion (DRG) neurons, attenuated pain-like behaviors. Intraplantar IGF-1 evoked acute nociception and mechanical/cold allodynia, which were reduced by selective IGF-1R silencing in DRG neurons and Schwann cells, respectively. Schwann cell IGF-1R signaling promoted an endothelial nitric oxide synthase-mediated transient receptor potential ankyrin 1 (TRPA1) activation and release of reactive oxygen species that, via macrophage-colony stimulating factor-dependent endoneurial macrophage expansion, sustained pain-like behaviors. Osteoclast derived IGF-1 initiates a Schwann cell-dependent neuroinflammatory response that sustains a proalgesic pathway that provides new options for MBCP treatment.

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

BACKGROUND: Neuroinflammation 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). METHODS: Walker 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. RESULTS: Mechanical 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-1ß, IL-6, and TNF-α expression. C-176 alleviated bone cancer pain and reduced the STING and its downstream inflammatory pathway. CONCLUSION: We 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.

miR-155-5p in the spinal cord regulates hypersensitivity in a rat model of bone cancer pain.

BACKGROUND: Noncoding microRNAs have emerged as critical players of gene expression in the nervous system, where they contribute to regulating nervous disease. As stated in previous research, the miR-155-5p upregulation happens in the spinal cord at the nociceptive state. It was unclear if miR-155-5p is linked to bone cancer pain (BCP). Herein, we aimed at investigating the miR-155-5p functional regulatory function in BCP process and delineating the underlying mechanism. METHODS: The miRNA-155-5p levels and cellular distribution were determined by RNA sequencing, fluorescent in situ hybridization (FISH), and quantitative real-time PCR (qPCR). Immunoblotting, qPCR, dual-luciferase reporter gene assays, immunofluorescence, recombinant overexpression adeno-associated virus, small interfering RNA, intraspinal administration, and behavioral tests were utilized for exploring the downstream signaling pathway. RESULTS: The miR-155-5p high expression in spinal neurons contributes to BCP maintenance. The miR-155-5p blockage via the intrathecal injection of miR-155-5p antagomir alleviated the pain behavior; in contrast, upregulating miR-155-5p by agomir induced pain hypersensitivity. The miR-155-5p bounds directly to TCF4 mRNA's 3' UTR. BCP significantly reduced protein expression of TCF4 versus the Sham group. The miR-155-5p inhibition relieved the spinal TCF4 protein's down-expression level, while miR-155-5p upregulation by miR-155-5p agomir intrathecal injection decreased TCF4 protein expression in naïve rats. Additionally, TCF4 overexpression in BCP rats could increase Kv1.1. Moreover, TCF4 knockdown inhibited Kv1.1 expression in BCP rats. Indeed, TCF4 and Kv1.1 were co-expressed in BCP spinal cord neurons. CONCLUSION: The study findings stated the miR-155-5p pivotal role in regulating BCP by directly targeting TCF4 in spinal neurons and suggested that miR-155-5p could be a promising target in treating BCP.

P-Rex2 mediation of synaptic plasticity contributes to bone cancer pain.

Bone cancer pain (BCP) seriously affects the quality of life; however, due to its complex mechanism, the clinical treatment was unsatisfactory. Recent studies have showed several Rac-specific guanine nucleotide exchange factors (GEFs) that affect development and structure of neuronal processes play a vital role in the regulation of chronic pain. P-Rex2 is one of GEFs that regulate spine density, and the present study was performed to examine the effect of P-Rex2 on the development of BCP. Tumor cells implantation induced the mechanical hyperalgesia, which was accompanied by an increase in spinal protein P-Rex2, phosphorylated Rac1 (p-Rac1) and phosphorylated GluR1 (p-GluR1), and number of spines. Intrathecal injection a P-Rex2-targeting RNAi lentivirus relieved BCP and reduced the expression of P-Rex2, p-Rac1, p-GluR1, and number of spines in the BCP mice. Meanwhile, P-Rex2 knockdown reversed BCP-enhanced AMPA receptor (AMPAR)-induced current in dorsal horn neurons. In summary, this study suggested that P-Rex2 regulated GluR1-containing AMPAR trafficking and spine morphology via Rac1/pGluR1 pathway is a fundamental pathogenesis of BCP. Our findings provide a better understanding of the function of P-Rex2 as a possible therapeutic target for relieving BCP.

CXCR1 participates in bone cancer pain induced by Walker 256 breast cancer cells in female rats.

Bone cancer pain (BCP) is a clinically intractable mixed pain, involving inflammation and neuropathic pain, and its mechanisms remain unclear. CXC chemokine receptor 1 (CXCR1, IL-8RA) and 2 (CXCR2, IL-8RB) are high-affinity receptors for interleukin 8 (IL8). According to previous studies, CXCR2 plays a crucial role in BCP between astrocytes and neurons, while the role of CXCR1 remains unclear. The objective of this study was to investigate the role of CXCR1 in BCP. We found that CXCR1 expression increased in the spinal dorsal horn. Intrathecal injection of CXCR1 siRNA effectively attenuated mechanical allodynia and pain-related behaviors in rats. It was found that CXCR1 was predominantly co-localized with neurons. Intrathecal injection of CXCR1-siRNA reduced phosphorylated JAK2/STAT3 protein levels and the NLRP3 inflammasome (NLRP3, caspase1, and IL-1ß) levels. Furthermore, in vitro cytological experiments confirmed this conclusion. The study results suggest that the spinal chemokine receptor CXCR1 activation mediates BCP through JAK2/STAT3 signaling pathway and NLRP3 inflammasome (NLRP3, caspase1, and IL-1ß).

LncRNA NONRATT009773.2 promotes bone cancer pain progression through the miR-708-5p/CXCL13 axis.

Bone cancer pain (BCP) is the most frequently observed chronic cancer pain, and its development remains largely unexplored. Dysregulation of non-coding RNAs greatly contributes to the pathogenesis of BCP. In the present study, we found a new long noncoding RNA (lncRNA), NONRATT009773.2, and investigated its role in the spinal cord of BCP rats. Our results showed that NONRATT009773.2 was significantly up-regulated in BCP model rats, whereas depletion of NONRATT009773.2 attenuated BCP. In contrast, overexpression of NONRATT009773.2 triggered pain-like symptoms in normal animals. Moreover, NONRATT009773.2 functioned as a microRNA (miRNA) sponge to absorb miR-708-5p and up-regulated miRNA downstream target CXCL13, which plays fundamental roles in the initiation and maintenance of neuroinflammation and hyperalgesia. Collectively, our current findings indicated that NONRATT009773.2 could be employed as a new therapeutic target for BCP.

Spinal Nrf2 translocation may inhibit neuronal NF-κB activation and alleviate allodynia in a rat model of bone cancer pain.

Bone cancer pain (BCP) is a clinical pathology that urgently needs to be solved, but research on the mechanism of BCP has so far achieved limited success. Nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) has been shown to be involved in pain, but its involvement in BCP and the specific mechanism have yet to be examined. This study aimed to test the hypothesis that BCP induces the transfer of Nrf2 from the cytoplasm to the nucleus and further promotes nuclear transcription to activate heme oxygenase-1 (HO-1) and inhibit the activation of nuclear factor-kappa B (NF-κB) signalling, ultimately regulating the neuroinflammatory response. Von-Frey was used for behavioural analysis in rats with BCP, whereas western blotting, real-time quantitative PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect molecular expression changes, and immunofluorescence was used to detect cellular localization. We demonstrated that BCP induced increased Nrf2 nuclear protein expression with decreased cytoplasmic protein expression in the spinal cord. Further increases in Nrf2 nuclear protein expression can alleviate hyperalgesia and activate HO-1 to inhibit the expression of NF-κB nuclear protein and inflammatory factors. Strikingly, intrathecal administration of the corresponding siRNA reversed the above effects. In addition, the results of double immune labelling revealed that Nrf2 and NF-κB were coexpressed in spinal cord neurons of rats with BCP. In summary, these findings suggest that the entry of Nrf2 into the nucleus promotes the expression of HO-1, inhibiting activation of the NF-κB signalling pathway, reducing neuroinflammation and ultimately exerting an anti-nociceptive effect.

Contribution of TRESK two-pore domain potassium channel to bone cancer-induced spontaneous pain and evoked cutaneous pain in rats.

Cancer-associated pain is debilitating. However, the mechanism underlying cancer-induced spontaneous pain and evoked pain remains unclear. Here, using behavioral tests with immunofluorescent staining, overexpression, and knockdown of TRESK methods, we found an extensive distribution of TRESK potassium channel on both CGRP+ and IB4+ nerve fibers in the hindpaw skin, on CGRP+ nerve fibers in the tibial periosteum which lacks IB4+ fibers innervation, and on CGRP+ and IB4+ dorsal root ganglion (DRG) neurons in rats. Moreover, we found a decreased expression of TRESK in the corresponding nerve fibers within the hindpaw skin, the tibial periosteum and the DRG neurons in bone cancer rats. Overexpression of TRESK in DRG neurons attenuated both cancer-induced spontaneous pain (partly reflect skeletal pain) and evoked pain (reflect cutaneous pain) in tumor-bearing rats, in which the relief of evoked pain is time delayed than spontaneous pain. In contrast, knockdown of TRESK in DRG neurons produced both spontaneous pain and evoked pain in naïve rats. These results suggested that the differential distribution and decreased expression of TRESK in the periosteum and skin, which is attributed to the lack of IB4+ fibers innervation within the periosteum of the tibia, probably contribute to the behavioral divergence of cancer-induced spontaneous pain and evoked pain in bone cancer rats. Thus, the assessment of spontaneous pain and evoked pain should be accomplished simultaneously when evaluating the effect of some novel analgesics in animal models. Also, this study provides solid evidence for the role of peripheral TRESK in both cancer-induced spontaneous pain and evoked cutaneous pain.

Effects of dezocine on morphine tolerance and opioid receptor expression in a rat model of bone cancer pain.

BACKGROUND: Clinically, the coadministration of opioids to enhance antinociception and decrease tolerance has attracted increasing research attention. We investigated the effects of dezocine, a mu- and kappa-opioid receptor agonist/antagonist, on morphine tolerance and explored the involvement of opioid receptor expression in a rat model of bone cancer pain. METHODS: Thermal nociceptive thresholds were measured after the subcutaneous injection of morphine (10 mg/kg) alone or combined with dezocine (10 or 1 mg/kg) for 7 consecutive days. Real-time PCR and western blot analysis were used to examine opioid receptor expression in the periaqueductal gray (PAG) and spinal cord. RESULTS: The analgesic effect was significantly decreased after 4 days of morphine administration. We observed that low-dose dezocine significantly attenuated morphine tolerance without reducing the analgesic effect of morphine. Low-dose dezocine coadministration significantly reversed the downregulated expression of mu (MOR) and delta (DOR) opioid receptors in the PAG and the upregulated expression of kappa (KOR) and DOR in the spinal cord induced by morphine. Moreover, low-dose dezocine coadministered with morphine significantly inhibited KOR expression in both the PAG and spinal cord. CONCLUSIONS: The combination of low-dose dezocine with morphine may prevent or delay the development of morphine tolerance in a rat model of bone cancer pain. The regulation of opioid receptor expression in the PAG and spinal cord may be part of the mechanism.

The circular RNA circSlc7a11 promotes bone cancer pain pathogenesis in rats by modulating LLC-WRC 256 cell proliferation and apoptosis.

Treatment of bone cancer pain (BCP) caused by bone metastasis in advanced cancers remains a challenge in clinical oncology, and the underlying mechanisms of BCP are poorly understood. This study aimed to investigate the pathogenic roles of circular RNAs (circRNAs) in regulating cancer cell proliferation and BCP development. Eight differentially expressed circRNAs in the rat spinal cord were validated by agarose gel electrophoresis and Sanger sequencing. Expression of circRNAs and mRNAs was detected by quantitative RT-PCR. MTS assay and flow cytometry were performed to analyze cell proliferation and apoptosis, respectively. Differentially expressed mRNA profiles were characterized by deep RNA sequencing, hierarchical clustering, and functional categorization. The interactions among circRNAs, microRNAs (miRNAs), and mRNAs were predicted using TargetScan. Additionally, western blot was performed to determine the protein levels of Pax8, Isg15, and Cxcl10. Multiple circRNAs were differentially expressed in the spinal cords of BCP model rats; of these, circSlc7a11 showed the greatest increase in expression. The overexpression of circSlc7a11 significantly promoted cell proliferation and repressed apoptosis of LLC-WRC 256 and UMR-106 cells, whereas circSlc7a11 silencing produced the opposite effects. Altered expression of circSlc7a11 also induced substantial changes in the mRNA expression profiles of LLC-WRC 256 cells; these changes were linked to multiple apoptotic processes and signaling pathways, such as the chemokine signaling pathway, and formed a complex circRNA/miRNA/mRNA network. Additionally, Pax8, Isg15, and Cxc110 protein level in LLC-WRC 256 cells was consistent with the mRNA results. The circRNA circSlc7a11 regulates rat BCP development by modulating LLC-WRC 256 cell proliferation and apoptosis through multiple-signaling mechanisms.

Transcriptional Regulation of Voltage-Gated Sodium Channels Contributes to GM-CSF-Induced Pain.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the production of granulocyte and macrophage populations from the hematopoietic progenitor cells; it is one of the most common growth factors in the blood. GM-CSF is also involved in bone cancer pain development by regulating tumor-nerve interactions, remodeling of peripheral nerves, and sensitization of damage-sensing (nociceptive) nerves. However, the precise mechanism for GM-CSF-dependent pain is unclear. In this study, we found that GM-CSF is highly expressed in human malignant osteosarcoma. Female Sprague Dawley rats implanted with bone cancer cells develop mechanical and thermal hyperalgesia, but antagonizing GM-CSF in these animals significantly reduced such hypersensitivity. The voltage-gated Na+ channels Nav1.7, Nav1.8, and Nav1.9 were found to be selectively upregulated in rat DRG neurons treated with GM-CSF, which resulted in enhanced excitability. GM-CSF activated the Janus kinase 2 (Jak2)-signal transducer and activator of transcription protein 3 (Stat3) signaling pathway, which promoted the transcription of Nav1.7-1.9 in DRG neurons. Accordingly, targeted knocking down of either Nav1.7-1.9 or Jak2/Stat3 in DRG neurons in vivo alleviated the hyperalgesia in male Sprague Dawley rats. Our findings describe a novel bone cancer pain mechanism and provide a new insight into the physiological and pathological functions of GM-CSF.SIGNIFICANCE STATEMENT It has been reported that granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a key role in bone cancer pain, yet the underlying mechanisms involved in the GM-CSF-mediated signaling pathway in nociceptors is not fully understood. Here, we showed that GM-CSF promotes bone cancer-associated pain by enhancing the excitability of DRG neurons via the Janus kinase 2 (Jak2)-signal transducer and activator of transcription protein 3 (Stat3)-mediated upregulation of expression of nociceptor-specific voltage-gated sodium channels. Our study provides a detailed understanding of the roles that sodium channels and the Jak2/Stat3 pathway play in the GM-CSF-mediated bone cancer pain; our data also highlight the therapeutic potential of targeting GM-CSF.