Cancer Pain Model Research Articles

PAR2 on oral cancer cells and nociceptors contributes to oral cancer pain that can be relieved by nanoparticle-encapsulated AZ3451

Oral cancer is notoriously painful. Activation of protease-activated receptor 2 (PAR2, encoded by F2RL1) by proteases in the cancer microenvironment is implicated in oral cancer pain. PAR2 is a G protein-coupled receptor (GPCR) expressed on neurons and cells in the cancer microenvironment. Sustained signaling of PAR2 from endosomes of neurons mediates sensitization and nociception. We focused on the differential contribution of PAR2 on oral cancer cells and neurons to oral cancer pain and whether encapsulation of a PAR2 inhibitor, AZ3451 in nanoparticles (NP) more effectively reverses PAR2 activation. We report that F2RL1 was overexpressed in human oral cancers and cancer cell lines. Deletion of F2RL1 on cancer cells reduced cancer-associated mechanical allodynia. A third-generation polyamidoamine dendrimer, functionalized with cholesterol was self-assembled into NPs encapsulating AZ3451. NP encapsulated AZ3451 (PAMAM-Chol-AZ NPs) more effectively reversed activation of PAR2 at the plasma membrane and early endosomes than free drug. The PAMAM-Chol-AZ NPs showed greater efficacy in reversing nociception than free drug, with respect to both level and duration, in three preclinical mouse models of oral cancer pain. The antinociceptive efficacy was confirmed with an operant orofacial assay. Genetic deletion of F2RL1 on cancer cells or F2rl1 on neurons each partially reversed mechanical cancer allodynia. The remaining nociception could be effectively reversed by PAMAM-Chol-AZ NPs. These findings suggest that PAR2 on oral cancer cells and neurons contribute to oral cancer nociception and NPs loaded with a PAR2 antagonist provide increased antinociception and improved oral function compared to free drug.

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.

Molecular mechanism of basolateral amygdala involved in electroacupuncture-induced amelioration of cancer pain and concomitant depression based on transcriptomics techniques.

To observe the effect of electroacupuncture (EA) of "Zusanli" (ST36) and "Sanyinjiao" (SP6) on cancer pain and concomitant negative emotion in cancer pain model mice, and to explore its molecular mechanisms in the basolateral amygdala (BLA) by using transcriptomics techniques. C57BL/6 mice were randomized into sham operation, model and EA groups, with 10 mice in each group. The cancer pain model was established by injecting PBS suspension containing Lewis lung cancer cells into the femur. The mice in the EA group received EA stimulation(1 mA, 2 Hz) on ST36 and SP6 from the 10th day after modeling, 20 min per day for 12 successive days. The bone damage of the distal femur was observed with X-ray and H.E. staining, respectively. The mechanical pain threshold (MPT) was detected by using von Frey. The depression-like behavior was detected by using sucrose-preference test (sucrose preference index in 12 h), and the immobility (feeling of despair) duration of forced swimming within 4 min. The BLA tissue was extracted for RNA sequencing (RNA library construction, and screening differential gene profiling by transcriptomic sequencing) and bioinformatics analysis. The real-time PCR was used to validate the mRNA expression of differentially expressed genes：tumor necrosis factor superfamily 8 (Tnfsf8), bone marrow stromal cell antigen 1 (Bst1), prodynorphin (Pdyn) and voltage-gated sodium channelβ4 (Scn4b). H.E. staining and X-ray showed significant bone damage in the distal femur in cancer pain mice. In contrast to the sham operation group, the MPT on the 1st , 4th, 7th , 10th, 14th and 21st day after modeling and sucrose preference index were significantly decreased (P<0.001, P<0.000 1), and the immobility time of the forced swimming was considerably increased in the model group (P<0.001). In contrast to the model group, the MPT values on the 14th and 21st day and sucrose preference index were obviously increased (P<0.000 1, P<0.05), and the immobility time was strikingly decreased in the EA group (P<0.01). RNA sequencing showed that a total of 404 differentially expressed genes (205 up-regulated, 199 down-regulated) were screened in the model group compared with the sham operation group, and a total of 329 differentially expressed genes (206 up-regulated and 123 down-regulated) were screened in the EA group compared with the model group. Venn diagram analysis of the differentially expressed genes showed that 45 up-regulated and 28 down-regulated genes in the model group were completely reversed by EA. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the screened differentially expressed genes revealed that the above differential genes were mainly enriched in the ligand receptor activity, cytokine receptor binding, and cytokine activity related to neuro-inflammation, as well as in neuropeptide signaling pathways related to neuronal excitability, and calcium ion mediated signal transduction. The analysis of KEGG pathway showed that the differentially expressed genes were mainly enriched in the inflammation-related pathways, such as interleukin-17 pathway. Validation analysis of the differentially expressed genes showed that the expression levels of Tnfsf8 and Bst1 were significantly up-regulated in the model group compared with the sham operation group (P<0.01, P<0.05), and down-regulated by EA (P<0.01, P<0.05), while the expression levels of Pdyn and Scn4b were down-regulated in the model group in comparison with the sham operation group (P<0.01), and up-regulated by EA (P<0.05, P<0.01), which was consistent with the changing trend of the gene sequencing results. Acupuncture of ST36 and SP6 can significantly relieve cancer pain and concomitant negative emotion in cancer pain mice, which may be related to its functions in alleviating neuro-inflammation and relieving the abnormal activities of specific neurons in the BLA.

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.

Electroacupuncture attenuates nociceptive behaviors in a mouse model of cancer pain.

Pain is a major symptom in cancer patients, and cancer-induced bone pain (CIBP) is the most common type of moderate and severe cancer-related pain. The current available analgesic treatments for CIBP have adverse effects as well as limited therapeutic effects. Acupuncture is proved effective in pain management as a safe alternative therapy. We evaluated the analgesic effect of acupuncture in treatment of cancer pain and try to explore the underlying analgesic mechanisms. Nude mice were inoculated with cancer cells into the left distal femur to establish cancer pain model. Electroacupuncture (EA) treatment was applied for the xenograft animals. Pain behaviors of mice were evaluated, followed by the detections of neuropeptide-related and inflammation-related indicators in peripheral and central levels. EA treatment alleviated cancer-induced pain behaviors covering mechanical allodynia, thermal hyperalgesia and spontaneous pain, and also down-regulated immunofluorescence expressions of neuropeptide CGRP and p75 in the skin of affected plantar area in xenograft mice, and inhibited expressions of overexpressed neuropeptide-related and inflammation-related protein in the lumbar spinal cord of xenograft mice. Overall, our findings suggest that EA treatment ameliorated cancer-induced pain behaviors in the mouse xenograft model of cancer pain, possibly through inhibiting the expressions of neuropeptide-related and inflammation-related protein in central level following tumor cell xenografts.

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.

Activation of Transient Receptor Potential Vanilloid 1 Is Involved in Both Pain and Tumor Growth in a Mouse Model of Cancer Pain

Activation of calcitonin gene-related peptide (CGRP)-positive sensory neurons in the tumor microenvironment has been shown to be involved in tumor growth. However, how CGRP-positive sensory neurons are activated requires elucidation. In this study, we focused on transient receptor potential vanilloid 1 (TRPV1) and examined the contribution of TRPV1 to tumor growth and cancer pain in a mouse cancer model in which Lewis lung carcinoma was subcutaneously inoculated in the left plantar region. Tumor inoculation gradually increased the volumes of the hind paws of wild type (WT) mice over time, but those of both αCGRP knockout mice and TRPV1 knockout mice were significantly smaller than those of WT mice after tumor inoculation. Both TRPV1 and CGRP are therefore suggested to be involved in tumor growth. In an immunohistochemical study, the percentage of phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB)-positive profiles in CGRP-positive dorsal root ganglion (DRG) neurons in WT mice was significantly increased after tumor inoculation. The percentage of p-CREB-positive profiles in CGRP-positive DRG neurons in TRPV1 knockout mice was also increased after tumor inoculation, but was significantly lower than that in WT mice, indicating the contribution of TRPV1 to activation of CGRP-positive DRG neurons. Cancer pain in TRPV1 knockout mice was significantly lower than that in WT mice. In conclusion, TRPV1 is involved in both tumor growth and cancer pain, potentially leading to a novel strategy for the treatment of cancer pain and cancer development. Cancer pain is also suggested to facilitate tumor growth.

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.

GFAP palmitoylcation mediated by ZDHHC23 in spinal astrocytes contributes to the development of neuropathic pain

BackgroundCancer pain has a significant impact on patient’s quality of life. Astrocytes play an important role in cancer pain signaling. The direct targeting of astrocytes can effectively suppress cancer pain,...

A Side-Effect-Free Interventional Therapy for Precisely Eliminating Unresectable Cancer Pain.

Of patients bearing unresectable tumors at advanced stages, most undergo serious pain. For unresectable tumors adjacent to vital organs or nerves, eliminating local cancer pain without adverse effects remains a formidable challenge. Interventional ablative therapies (IATs), such as radio frequency ablation (RFA), microwave ablation, and irreversible electroporation, have been clinically adopted to treat various carcinomas. In this study, we established another palliative interventional therapy to eliminate local cancer pain, instead of relieving nociception temporarily. Here, we developed another interventional ablative therapy (termed nanoparticle-mediated microknife ablation) to locoregionally eliminate cancer pain and tumors. The IAT system was composed of self-assembled nanodrugs, infusion catheters, puncture needles, injection pump, and an empirical tumor ablation formula. Notably, the ablation formula established in the IAT system enables us to predict the essential nanoparticle (NP) numbers used for completely destroying tumors. In a mouse model of cancer pain, tumor-targeted nanodrugs made of Paclitaxel and Hematoporphyrin, which have an extremely high drug-loading efficiency (more than 60%), were infused into tumors through injection pumps under imaging guidance. In conclusion, when compared to classic chemotherapeutic agents, IAT showed significantly higher effectiveness in cancer pain removal. It also presented no damage to the nervous, sensory, and motor capabilities of the treated mice. All of these merits resulted from NPs' long-lasting retention, targeted ablation, and confined diffusion in tumor stroma. Therefore, this safe treatment modality has great potential to eradicate local cancer pain in the clinic.

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.

Calcitonin Related Polypeptide Alpha Mediates Oral Cancer Pain.

Oral cancer patients suffer pain at the site of the cancer. Calcitonin gene related polypeptide (CGRP), a neuropeptide expressed by a subset of primary afferent neurons, promotes oral cancer growth. CGRP also mediates trigeminal pain (migraine) and neurogenic inflammation. The contribution of CGRP to oral cancer pain is investigated in the present study. The findings demonstrate that CGRP-immunoreactive (-ir) neurons and neurites innervate orthotopic oral cancer xenograft tumors in mice. Cancer increases anterograde transport of CGRP in axons innervating the tumor, supporting neurogenic secretion as the source of CGRP in the oral cancer microenvironment. CGRP antagonism reverses oral cancer nociception in preclinical oral cancer pain models. Single-cell RNA-sequencing is used to identify cell types in the cancer microenvironment expressing the CGRP receptor components, receptor activity modifying protein 1 Ramp1 and calcitonin receptor like receptor (CLR, encoded by Calcrl). Ramp1 and Calcrl transcripts are detected in cells expressing marker genes for Schwann cells, endothelial cells, fibroblasts and immune cells. Ramp1 and Calcrl transcripts are more frequently detected in cells expressing fibroblast and immune cell markers. This work identifies CGRP as mediator of oral cancer pain and suggests the antagonism of CGRP to alleviate oral cancer pain.

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.

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.

Antinociceptive Effect of a p-Cymene/β-Cyclodextrin Inclusion Complex in a Murine Cancer Pain Model: Characterization Aided through a Docking Study.

Pain is one of the most prevalent and difficult to manage symptoms in cancer patients, and conventional drugs present a range of adverse reactions. The development of β-cyclodextrins (β-CD) complexes has been used to avoid physicochemical and pharmacological limitations due to the lipophilicity of compounds such as p-Cymene (PC), a monoterpene with antinociceptive effects. Our aim was to obtain, characterize, and measure the effect of the complex of p-cymene and β-cyclodextrin (PC/β-CD) in a cancer pain model. Initially, molecular docking was performed to predict the viability of complex formation. Afterward, PC/β-CD was obtained by slurry complexation, characterized by HPLC and NMR. Finally, PC/β-CD was tested in a Sarcoma 180 (S180)-induced pain model. Molecular docking indicated that the occurrence of interaction between PC and β-CD is favorable. PC/β-CD showed complexation efficiency of 82.61%, and NMR demonstrated PC complexation in the β-CD cavity. In the S180 cancer pain model, PC/β-CD significantly reduced the mechanical hyperalgesia, spontaneous nociception, and nociception induced by non-noxious palpation at the doses tested (p < 0.05) when compared to vehicle differently from free PC (p > 0.05). Therefore, the complexation of PC in β-CD was shown to improve the pharmacological effect of the drug as well as reducing the required dose.

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.