Bone Cancer Pain Model Research Articles

Targeted therapy: P2X3 receptor silencing in bone cancer pain relief.

Bone cancer pain remains a significant clinical challenge, often refractory to conventional treatments. The upregulation of the P2X3 receptor in the dorsal root ganglia has been implicated in the pathogenesis of bone cancer pain. This study aimed to elucidate the role of the P2X3 receptor in this context and assess the therapeutic potential of receptor silencing. Utilizing a rat model with Walker 256 cells to simulate bone cancer pain, researchers conducted molecular analyses, including semi-quantitative RT-PCR and Western Blot, to investigate P2X3 receptor expression in the dorsal root ganglia. Results demonstrated a marked increase in P2X3 receptor levels in the dorsal root ganglia of the bone cancer pain model. Targeted silencing of the P2X3 receptor using specific shRNA delivered via lentiviral vectors significantly reduced pain sensitivity, underscoring the receptor's potential as a valuable therapeutic target. In addition, a comprehensive gene expression analysis leveraging the GEO data set GSE249443 was performed to explore the underlying biological pathways linked to bone cancer pain. This analysis provided insights into the intricate interplay between bone cancer pain and associated biological processes, offering a deeper understanding of the mechanisms involved in pain modulation and progression. In conclusion, this research identifies the P2X3 receptor as a critical molecular target for mitigating bone cancer pain. The selective silencing of the P2X3 receptor emerges as a promising and innovative therapeutic strategy, presenting novel avenues for managing bone cancer pain and potentially revolutionizing treatment approaches in this challenging domain.

Systemic administration of Resolvin D1 reduces cancer-induced bone pain in mice: Lack of sex dependency in pain development and analgesia.

Bone cancer produces severe pain that is treated with opioids, but serious side effects limit opioid utilization. There is therefore a need to develop effective and safe non-opioid alternatives. The lipid mediator, Resolvin D1 (RvD1), could be a prospective candidate for cancer pain treatment. To assess RvD1 and other potential candidates, appropriate animal models that recapitulate clinical features must be used. Although several preclinical models of cancer pain have been developed, the influence of sex on the development of cancer pain and the effectiveness of RvD1 have not been studied. Using a mouse model of fibrosarcoma growth in and around the calcaneus bone, we demonstrated that the mechanical hyperalgesia in the tumor-bearing hind paw develops independently of sex, except that it developed a little sooner in female mice. A single intravenous injection of RvD1 (0.001-10 μg/kg) decreased hyperalgesia in both sexes with similar potency (ED50 = 0.0015 μg/kg) and efficacy. Repeated daily administration of 10 μg/kg RvD1 prolonged the analgesic effect and completely abolished hyperalgesia. This was also independent of sex. In this preclinical mouse model of bone cancer pain, the development of pain and the analgesic effectiveness of RvD1 are not influenced by sex.

Protein arginine methyltransferase-6 regulates heterogeneous nuclear ribonucleoprotein-F expression and is a potential target for the treatment of neuropathic pain.

JOURNAL/nrgr/04.03/01300535-202509000-00029/figure1/v/2024-11-05T132919Z/r/image-tiff Protein arginine methyltransferase-6 participates in a range of biological functions, particularly RNA processing, transcription, chromatin remodeling, and endosomal trafficking. However, it remains unclear whether protein arginine methyltransferase-6 modifies neuropathic pain and, if so, what the mechanisms of this effect. In this study, protein arginine methyltransferase-6 expression levels and its effect on neuropathic pain were investigated in the spared nerve injury model, chronic constriction injury model and bone cancer pain model, using immunohistochemistry, western blotting, immunoprecipitation, and label-free proteomic analysis. The results showed that protein arginine methyltransferase-6 mostly co-localized with β-tubulin III in the dorsal root ganglion, and that its expression decreased following spared nerve injury, chronic constriction injury and bone cancer pain. In addition, PRMT6 knockout (Prmt6-/-) mice exhibited pain hypersensitivity. Furthermore, the development of spared nerve injury-induced hypersensitivity to mechanical pain was attenuated by blocking the decrease in protein arginine methyltransferase-6 expression. Moreover, when protein arginine methyltransferase-6 expression was downregulated in the dorsal root ganglion in mice without spared nerve injury, increased levels of phosphorylated extracellular signal-regulated kinases were observed in the ipsilateral dorsal horn, and the response to mechanical stimuli was enhanced. Mechanistically, protein arginine methyltransferase-6 appeared to contribute to spared nerve injury-induced neuropathic pain by regulating the expression of heterogeneous nuclear ribonucleoprotein-F. Additionally, protein arginine methyltransferase-6-mediated modulation of heterogeneous nuclear ribonucleoprotein-F expression required amino acids 319 to 388, but not classical H3R2 methylation. These findings indicated that protein arginine methyltransferase-6 is a potential therapeutic target for the treatment of peripheral neuropathic 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.

Naringenin alleviates bone cancer pain via NF-κB/uPA/PAR2 pathway in mice.

This investigation aims to explore the protective role of Naringenin (Nar) in bone cancer pain (BCP) via TNF-α-mediated NF-κB/uPA/PAR2 pathway. BCP model was manipulated by the injection of LL2 cells into femur of mice. The levels of TNF-α and uPA in bone tissue and serum were studied by ELISA. The expressions of PAR2, PKC-γ, PKA and TRPV1 were determined by qPCR and western blot. Levels of p-IKKβ, IKKβ, p-p65, p65 were determined by western blot. Levels of p-p65 and uPA in bone tissue were studied by immunohistochemistry. Behavior tests in this investigation included paw withdrawal latency (PWL) and the paw withdrawal threshold (PWT). Radiological analysis and micro-CT were used to study bone structure. The lesions of bone tissue were determined by HE staining. The Dorsal root ganglia (DRG) isolated from mice were used to determine the level of PAR2 pathway. Naringenin improved the BCP-induced bone damage based on the increases of BV/TV, Conn. D, BMD and BMC and the decrease of bone destruction score. Naringenin repressed the reductions of PWT and PWL in BCP mice. Naringenin decreased the levels of PAR2, PKC-γ, PKA and TRPV1 of DRG and reduced the levels of p-IKKβ, p-p65, and uPA in serum and bone tissue in BCP. Importantly, naringenin suppressed the enhancement of TNF-α in serum and bone tissue in BCP mice. Naringenin alleviated pain sensitization and bone damage of mice with BCP via TNF-α-mediated NF-κB/uPA/PAR2 pathway. We demonstrated a novel pathway for anti-BCP treatment with naringenin.

Analysis of Ionomic Profiles of Spinal Cords in a Rat Model with Bone Cancer Pain.

Ionomics is used to study levels of ionome in different states of organisms and their correlations. Bone cancer pain (BCP) severely reduces quality of life of patients or their lifespan. However, the relationship between BCP and ionome remains unclear. The BCP rat model was constructed through inoculation of Walker 256 cells into the left tibia. Von Frey test, whole-cell patch-clamp recording and inductively coupled plasma mass spectrometry (ICP-MS) technologies were conducted for measuring tactile hypersensitivity, the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) of neurons of spinal slices, and ionome of spinal cord samples, respectively. Principal component analysis (PCA) was used to explore ionomic patterns of the spinal cord. The BCP rat model was successfully constructed through implantation of Walker 256 cells into the left tibia. The frequency and amplitude of mEPSCs of neurons in the spinal cord slices from the BCP model rats were notably greater than those in the sham control. In terms of ionomics, the spinal cord levels of two macroelements (Ca and S), four microelements (Fe, Mn, Li and Sr) and the toxic element Ti in the BCP group of rats were significantly increased by inoculation of Walker 256 cancer cells, compared to the sham control. In addition, the correlation patterns between the elements were greatly changed between the sham control and BCP groups. PCA showed that inoculation of Walker 256 cells into the tibia altered the overall ionomic profiles of the spinal cord. There was a significant separation trend between the two groups. Taken together, inoculation of Walker 256 cells into the left tibia contributes to BCP, which could be closely correlated by some elements. The findings provided novel information on the relationship between the ionome and BCP.

Spinal sirtuin 2 attenuates bone cancer pain by deacetylating FoxO3a

Bone cancer pain (BCP) is refractory to currently used analgesics. Recently, sirtuin 2 (SIRT2) was reported to play a vital role in neuropathic pain but its role in BCP remains unknown. It was hypothesized that spinal SIRT2 attenuates BCP by deacetylating FoxO3a and suppressing oxidative stress. The mouse model of BCP established by injecting tumor cells into the intramedullary space of the femur demonstrated that spinal SIRT2 and FoxO3a were downregulated in BCP development. Intrathecal administration of LV-SIRT2 reduced pain hypersensitivity (mechanical and thermal nociception) in BCP mice. Spinal SIRT2 overexpression upregulated FoxO3a and antioxidant genes (SOD2 and catalase) and inhibited FoxO3a acetylation, phosphorylation, and ubiquitination. Moreover, intrathecal administration of SIRT2 shRNA induced pain hypersensitivity in normal mice. Spinal SIRT2 knockdown downregulated FoxO3a and antioxidant genes and increased FoxO3a acetylation, phosphorylation, and ubiquitination. In summary, spinal SIRT2 increases FoxO3a expression in BCP mice and inhibits oxidative stress by deacetylating FoxO3a and further reducing FoxO3a phosphorylation, ubiquitination, and degradation, leading to BCP relief.

Activation of the BMP2-SMAD1-CGRP pathway in dorsal root ganglia contributes to bone cancer pain in a rat model

Peripheral nerve remodeling and sensitization are involved in cancer-related bone pain. As a member of the transforming growth factor-β class, bone morphogenetic protein 2 (BMP2) is recognized to have a role in the development of the neurological and skeletal systems. Our previous work showed that BMP2 is critical for bone cancer pain (BCP) sensitization. However, the mechanism remains unknown. In the current study, we demonstrated a substantial increase in BMP2 expression in the dorsal root ganglia (DRG) in a rat model of BCP. Knockdown of BMP2 expression ameliorated BCP in rats. Furthermore, the DRG neurons of rats with BCP expressed higher levels of calcitonin gene-related peptide (CGRP), and BCP was successfully suppressed by intrathecal injection of a CGRP receptor blocker (CGRP8-37). Downregulation of BMP2 expression reduced the expression of CGRP in the DRG of rats with BCP and relieved pain behavior. Moreover, we revealed that upregulation of CGRP expression in the DRG may be induced by activation of the BMPR/Smad1 signaling pathway. These findings suggest that BMP2 contributes to BCP by upregulating CGRP in DRG neurons via activating BMPR/Smad1 signaling pathway and that therapeutic targeting of the BMP2-Smad1-CGRP pathway may ameliorate BCP in the context of advanced cancer.

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

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. 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. 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. Netrin-1 may contribute to the activation of the BCP by inducing nociceptive nerve innervation and improving pain behaviors.

LncRNA 51325 Alleviates Bone Cancer Induced Hyperalgesia Through Inhibition of Pum2.

Bone cancer pain (BCP) represents one of the most challenging comorbidities associated with cancer metastasis. Long non-coding RNAs (lncRNAs) have garnered attention as potential therapeutic agents in managing neuropathic pain. However, their role in the regulation of nociceptive information processing remains poorly understood. In this study, we observed a significant down-regulation of the spinal lncRNA ENSRNOG00000051325 (lncRNA51325) in a rat model of bone cancer pain. Our study sought to elucidate the potential involvement of lncRNA51325 in the development of BCP by modulating the expression of molecules associated with pain modulation. We established the BCP model by injecting Walker 256 cells into the tibial plateau of rats. We conducted tests on the pain behaviors and anxiety-like responses of rats through von-Frey test, Gait analysis, and Open Field Test. Spinal lumbar expansion was harvested for molecular biology experiments to explore the relationship between lncRNA51325 and Pumilio RNA binding family member 2 (Pum2). Notably, the overexpression of lncRNA51325 effectively attenuated mechanical allodynia in rats afflicted with BCP, whereas the knockdown of lncRNA51325 induced pain behaviors and anxiety-like responses in naïve rats. Additionally, we observed a time-dependent increase in the expression of Pum2 in BCP-afflicted rats, and intrathecal injection of Pum2-siRNA alleviated hyperalgesia. Furthermore, our investigations revealed that lncRNA51325 exerts a negative modulatory effect on Pum2 expression. The overexpression of lncRNA51325 significantly suppressed Pum2 expression in BCP rats, while the knockdown of lncRNA51325 led to elevated Pum2 protein levels in the spinal cord of naïve rats. Subsequent treatment with Pum2-siRNA mitigated the downregulation of lncRNA51325-induced mechanical allodynia in naïve rats. Our findings indicate that lncRNA51325 plays a role in regulating bone cancer pain by inhibiting Pum2 expression, offering a promising avenue for novel treatments targeting nociceptive hypersensitivity induced by bone metastatic cancer.

Characterization of commercially available murine fibrosarcoma NCTC-2472 cells both in vitro and as a model of bone cancer pain in vivo.

For many cancer patients tumor burden negatively impacts quality of life due to associated pain onset. Neuropathic pain is commonly associated with late cancer stages, and is resultant of tumor metastasis to bone, herein referred to as cancer-induced bone pain. Given the severe impact on quality of life and clinical treatment strategies focusing on symptom management, novel therapeutics are needed to alleviate cancer-induced bone pain and/or reduce cancer burden. In the current study we characterized a commercially available murine fibrosarcoma cell line, NCTC-2472 in vitro, which can be used to assess the capacity of novel compounds to impact cellular viability. We found that dimethyl sulfoxide, a known cytotoxic agent and common drug preparation compound, significantly decreased cell viability in a dose-related manner. We then characterized the in vivo tumor development and associated pain behavior characteristics following implantation of NCTC-2472 fibrosarcoma into male and female C3H/HeJ mice. The C3H/HeJ strain was utilized as these mice are syngeneic with NCTC-2472 fibrosarcoma and their use reduces potential implantation failure. We found that tumor development in mice resulted in the development of mechanical allodynia but not thermal hyperalgesia. Gabapentin, a clinically relevant analgesic, produced dose-related mechanical allodynia reversal. These studies provide further characterization of a cancer-induced bone pain model that can be used to examine novel compounds as anti-cancer and analgesic therapeutics.

Blockade of spinal dopamine D1/D2 receptor heteromers by levo-Corydalmine suppressed calcium signaling cascade in spinal neurons to alleviate bone cancer pain in rats.

Background: Dopamine receptors have been reported to be involved in pain, while the exact effects and mechanism in bone cancer pain have not been fully explored. Methods: Bone cancer pain model was created by implanting walker 256 mammary gland carcinoma into right tibia bone cavity. Primary cultured spinal neurons were used for in vitro evaluation. FLIPR, western-blot, immunofluorescence, and Co-IP were used to detect cell signaling pathway. Results: Our results indicated that spinal dopamine D1 receptor (D1DR) and spinal dopamine D2 receptor (D2DR) could form heteromers in TCI rats, and antagonizing spinal D1DR and D2DR reduced heteromers formation and alleviated TCI-induced bone cancer pain. Further results indicated that D1DR or D2DR antagonist induced antinociception in TCI rats could be reversed by D1DR, D2DR, and D1/D2DR heteromer agonists. And Gq, IP3, and PLC inhibitors also attenuated TCI-induced bone cancer pain. In vitro results indicated that D1DR or D2DR antagonist decreased the Ca2+ oscillations upregulated by D1DR, D2DR, and D1/D2DR heteromer agonists in activated primary cultured spinal neurons. Moreover, inhibition of D1/D2DR heteromers induced antinociception in TCI rats was partially mediated by the CaMKII and MAPKs pathway. In addition, a natural compound levo-Corydalmine (l-CDL), could inhibit D1/D2DR heteromers and attenuate bone cancer pain. Results: Inhibition of spinal D1/D2DR heteromers via l-CDL decreases excitability in spinal neurons, which might present new therapeutic strategy for bone cancer pain.

Nerve Growth Factor/Tyrosine Kinase A Receptor Pathway Enhances Analgesia in an Experimental Mouse Model of Bone Cancer Pain by Increasing Membrane Levels of δ-Opioid Receptors.

The role of nerve growth factor (NGF)/tyrosine kinase A receptor (TrKA) signaling, which is activated in a variety of pain states, in regulating membrane-associated δ-opioid receptor (mDOR) expression is poorly understood. The hypothesis was that elevated NGF in bone cancer tumors could upregulate mDOR expression in spinal cord neurons and that mDOR agonism might alleviate bone cancer pain. Bone cancer pain (BCP) was induced by inoculating Lewis lung carcinoma cells into the femoral marrow cavity of adult C57BL/6J mice of both sexes. Nociceptive behaviors were evaluated by the von Frey and Hargreaves tests. Protein expression in the spinal dorsal horn of animals was measured by biochemical analyses, and excitatory synaptic transmission was recorded in miniature excitatory synaptic currents. The authors found that mDOR expression was increased in BCP mice (BCP vs. sham, mean ± SD: 0.18 ± 0.01 g vs. mean ± SD: 0.13 ± 0.01 g, n = 4, P < 0.001) and that administration of the DOR agonist deltorphin 2 (Del2) increased nociceptive thresholds (Del2 vs. vehicle, median [25th, 75th percentiles]: 1.00 [0.60, 1.40] g vs. median [25th, 75th percentiles]: 0.40 [0.16, 0.45] g, n = 10, P = 0.001) and reduced miniature excitatory synaptic current frequency in lamina II outer neurons (Del2 vs. baseline, mean ± SD: 2.21 ± 0.81 Hz vs. mean ± SD: 2.43 ± 0.90 Hz, n = 12, P < 0.001). Additionally, NGF expression was increased in BCP mice (BCP vs. sham, mean ± SD: 0.36 ± 0.03 vs. mean ± SD: 0.16 ± 0.02, n = 4, P < 0.001), and elevated NGF was associated with enhanced mDOR expression via TrKA signaling. Activation of mDOR produces analgesia that is dependent on the upregulation of the NGF/TrKA pathway by increasing mDOR levels under conditions of BCP in mice.

MiR-199a-3p suppresses neuroinflammation by directly targeting MyD88 in a mouse model of bone cancer pain

AimsPain is a profoundly debilitating symptom in cancer patients, leading to disability, immobility, and a marked decline in their quality of life. This study aimed to investigate the potential roles of miR-199a-3p in a murine model of bone cancer pain induced by tumor cell implantation in the medullary cavity of the femur. Materials and methodsWe assessed pain-related behaviors, including the paw withdrawal mechanical threshold (PWMT) and the number of spontaneous flinches (NSF). To investigate miRNA expression and its targets in astrocytes, we employed a combination of RNA-seq analysis, qRT-PCR, Western blotting, EdU, TUNEL, ChIP, ELISA, and luciferase reporter assays in mice (C3H/HeJ) with bone cancer pain and control groups. Key findingsOn days 10, 14, 21, and 28 post-surgery, we observed significant differences in PWTL, PWMT, and NSF when compared to the sham group (P < 0.001). qRT-PCR assays and miRNA sequencing results confirmed reduced miR-199a-3p expression in astrocytes of mice with bone cancer pain. Gain- and loss-of-function experiments demonstrated that miR-199a-3p suppressed astrocyte activation and the expression of inflammatory cytokines. In vitro investigations revealed that miR-199a-3p mimics reduced the levels of inflammatory factors in astrocytes and MyD88/NF-κB proteins. Furthermore, treatment with a miR-199a-3p agonist resulted in reduced expression of MyD88, TAK1, p-p65, and inflammatory mediators, along with decreased astrocyte activation in the spinal cord. SignificanceCollectively, these findings demonstrate that upregulation of miR-199a-3p may offer a therapeutic avenue for mitigating bone cancer pain in mice by suppressing neuroinflammation and inhibiting the MyD88/NF-κB signaling pathway.

Regulatory role of PI3K/Akt/WNK1 signal pathway in mouse model of bone cancer pain

In the advanced stage of cancer, the pain caused by bone metastasis is unbearable, but the mechanism of bone cancer pain (BCP) is very complicated and remains unclear. In this study, we used 4T1 mouse breast cancer cells to establish a bone cancer pain model to study the mechanism of BCP. Then the paw withdrawal mechanical threshold (PWMT) and the hematoxylin-eosin staining were used to reflect the erosion of cancer cells on tibia tissue. We also determined the role of proinflammatory factors (TNF-α, IL-17, etc.) in BCP by the enzyme-linked immunosorbent assay in mouse serum. When GSK690693, a new Akt inhibitor, was given and the absence of intermediate signal dominated by Akt is found, pain may be relieved by blocking the transmission of pain signal and raising the PWMT. In addition, we also found that GSK690693 inhibited the phosphorylation of Akt protein, resulting in a significant decrease in with-nolysinekinases 1 (WNK1) expression in the spinal cord tissue. In the BCP model, we confirmed that GSK690693 has a relieving effect on BCP, which may play an analgesic effect through PI3K-WNK1 signal pathway. At the same time, there is a close relationship between inflammatory factors and PI3K-WNK1 signal pathway. The PI3K/Akt pathway in the dorsal horn of the mouse spinal cord activates the downstream WNK1 protein, which promotes the release of inflammatory cytokines, which leads to the formation of BCP in mice. Inhibition of Akt can reduce the levels of IL-17 and TNF-α, cut off the downstream WNK1 protein signal receiving pathway, increase the PWMT and relieve BCP in mice. To clarify the analgesic target of BCP, to provide reference and theoretical support for the clinical effective treatment of BCP and the development of new high-efficiency analgesics.

Downregulated CAV-1 in mouse spinal cord may alleviate bone cancer pain by inhibiting the ERK/CREB pathway

BackgroundThis study aimed to assess the potential function of Caveolin-1 (CAV-1) in mice with bone cancer pain. Method: Using a mice bone cancer pain model we explored the contribution of CAV-1 expression to bone cancer pain on the 14th day after surgery, mice in the tumor group were randomized and treated with increasing doses of the CAV-1 inhibitor, methyl-beta-cyclodextrin. Pain was assessed by monitoring the number of spontaneous flinches (NSF) and paw withdrawal mechanical threshold (PMWT)mechanical withdrawal threshold (MWT). The localization and expression of CAV-1 in mouse neurons was also determined. Additionally, the protein levels of CAV-1, extracellular signal regulated kinase (ERK) 1/2, cAMP response element-binding protein (CREB) were monitored in mouse spinal cord tissues by western blotting. Results: CAV-1 was remarkably upregulated in the spinal cord of the tumor group on the 4th day after surgery, then downregulated on day 10, and upregulated again at day 14. Such CAV-1 levels were maintained until day 28. In the tumor group, the expression of p-ERK1/2 and p-CERB were upregulated at day 14 after surgery. Intrathecal injection of methyl-beta-cyclodextrin (MCD) downregulated p-ERK1/2 and p-CERB expression which correlated with alleviation of pain. Conclusion: Inhibition of CAV-1 in the spinal cord alleviates bone cancer pain in mice which correlates with inhibition of the ERK/CREB pathway.

Lactobacillus rhamnosus GG and butyrate supplementation in rats with bone cancer reduces mechanical allodynia and increases expression of μ-opioid receptor in the spinal cord.

Chronic cancer pain is one of the most unbearable symptoms for the patients with advanced cancer. The treatment of cancer pain continues to possess a major challenge. Here, we report that adjusting gut microbiota via probiotics can reduce bone cancer pain (BCP) in rats. The model of BCP was produced by tumor cell implantation (TCI) to the tibia in rats. Continuous feeding of Lactobacillus rhamnosus GG (LGG) was used to modulate the gut microbiota. Mechanical allodynia, bone destruction, fecal microbiota, and neurochemical changes in the primary dorsal root ganglion (DRG) and the spinal dorsal horn (DH) were assessed. LGG supplementation (109 CFU/rat/day) delayed the production of BCP for 3-4 days and significantly alleviated mechanical allodynia within the first 2 weeks after TCI. TCI-induced proinflammatory cytokines TNF-α and IL-β in the DH, and TCI-induced bone destruction in the tibia were both significantly reduced following LGG supplementation examined on day 8 after TCI. Meanwhile, we found that LGG supplementation, in addition to inhibiting TCI-induced pain, resulted in a significantly increased expression of the μ-opioid receptor (MOR) in the DH, but not in the DRG. LGG supplementation significantly potentiated the analgesic effect of morphine. Furthermore, LGG supplementation led to an increase in butyrate levels in the feces and serum and a decrease in histone deacetylase 2 (HDAC2) expression in the DH. Feeding TCI-rats with sodium butyrate solution alone, at a dose of 100 mg/kg, resulted in decreased pain, as well as decreased HDAC2 expression and increased MOR expression in the DH. The increased expression of MOR and decreased HDAC2 were also observed in neuro-2a cells when we treated the cells with serum from TCI rats with supplementation of LGG or sodium butyrate. This study provides evidence that reshaping the gut microbiota with probiotics LGG can delay the onset of cancer pain. The butyrate-HDAC2-MOR pathway may be the underlying mechanism for the analgesic effect of LGG. These findings shed light on an effective, safe, and non-invasive approach for cancer pain control and support the clinical implication of probiotics supplementation for patients with BCP.

Exosome-associated lysophosphatidic acid signaling contributes to cancer pain.

Pain associated with bone cancer remains poorly managed, and chemotherapeutic drugs used to treat cancer usually increase pain. The discovery of dual-acting drugs that reduce cancer and produce analgesia is an optimal approach. The mechanisms underlying bone cancer pain involve interactions between cancer cells and nociceptive neurons. We demonstrated that fibrosarcoma cells express high levels of autotaxin (ATX), the enzyme synthetizing lysophosphatidic acid (LPA). Lysophosphatidic acid increased proliferation of fibrosarcoma cells in vitro. Lysophosphatidic acid is also a pain-signaling molecule, which activates LPA receptors (LPARs) located on nociceptive neurons and satellite cells in dorsal root ganglia. We therefore investigated the contribution of the ATX-LPA-LPAR signaling to pain in a mouse model of bone cancer pain in which fibrosarcoma cells are implanted into and around the calcaneus bone, resulting in tumor growth and hypersensitivity. LPA was elevated in serum of tumor-bearing mice, and blockade of ATX or LPAR reduced tumor-evoked hypersensitivity. Because cancer cell-secreted exosomes contribute to hypersensitivity and ATX is bound to exosomes, we determined the role of exosome-associated ATX-LPA-LPAR signaling in hypersensitivity produced by cancer exosomes. Intraplantar injection of cancer exosomes into naive mice produced hypersensitivity by sensitizing C-fiber nociceptors. Inhibition of ATX or blockade of LPAR attenuated cancer exosome-evoked hypersensitivity in an ATX-LPA-LPAR-dependent manner. Parallel in vitro studies revealed the involvement of ATX-LPA-LPAR signaling in direct sensitization of dorsal root ganglion neurons by cancer exosomes. Thus, our study identified a cancer exosome-mediated pathway, which may represent a therapeutic target for treating tumor growth and pain in patients with bone cancer.

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

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. 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. 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). 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.

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.