The Role Of Ephrinb2-ephb1 Receptor Signaling In Bone Cancer Pain

Background and ObjectiveIt is estimated by the World Health Organization (WHO) that each year over 10 million new cases of cancer will be reported, 20%-50% of which complained that pain is the first symptom of them. Pain is the one of the most frequent complications for patients with cancer. Over 50% cancer patients suffered moderate-to-severe pain. Despite the diffusion of several guidelines for cancer pain management, including the well-known"analgesic ladder"recommended by the WHO, undertreatment is well documented and can involve up to 40%-80%. Cancer-induced pain is most disruptive to cancer patient's quality of life. Among the different types of cancer pains, bone cancer pain is the most common, severe and refractory. Therefore, further investigating the mechanisms responsible for the generation and development of bone cancer pain and developing new effective therapy is the urgent problem to be solved. In the present study, rat model of tibia bone cancer pain was used to investigate the role of ephrinB2-EphB1 receptor signaling in regulation of bone cancer pain, which would provide a new impetus for the development of effective approaches for bone cancer pain managements.Materials and Methods(1) According to the previous description by Medhurst, Walker 256 rat mammary gland carcinoma cells were used to establish rat model of tibia bone cancer pain. Tumor cell implantation (TCI)-induced behavioral changes were tested. The expressions of ephirinB2 and EphB1 in spinal cord were detected by western blot. Immunofluorescence was used to identify the distribution and location of ephrinB2 and EphB1.(2) To investigate the role of ephrinB2-EphB1 signaling in bone cancer pain, exogenous blocking reagent EphB2-Fc was used (5μg/20μl, i.t.) to test the effect of EphB1 receptor blockade on pain-related behaviors induced by TCI. And the expression of c-fos, GFAP, and IBA were identified by Immunofluorescence. Meanwhile, western blot was used to detected the activation (phosphorylation) of NR1, NR2A, NR2B, Src, and the subsequent Ca2+-dependent signal molecules, CREB, CaMKII and ERK. Additionally, the effect of EphB2-Fc on tumor growth and bone tissue destruction were test by hematoxylin-eosin (HE) staining and X-ray.(3) To investigate whether the forward signaling or the reverse signaling contribute to the regulation of pain process, firstly, we detected the effects of EphB2-Fc (5μg/20μl , i.t.) on the activation of EphB1 and ephrinB2 by western blot. Further, we examined the effects of ephrinB2 (2μg/20μl, i.t.), an exogenous EphB receptor activator, on expression and activation of EphB1, ephrinB2 and NR1, NR2B receptors, and on behavioral changes. Lastly, to examine whether EphB1 receptor activation is required for ephrinB2-Fc's effects, ephrinB2-Fc was given after EphB2-Fc administration and tested the activation of EphB1, ephrinB2 NR1, NR2B receptors by western blot and behavioral changes by thermal stimuli. To investigate the mechanism of EphB2-Fc-induced EphB1 receptor down-regulation, MMP-2 inhibitors (MMP2i, 10μg/20μl) and MMP-9 inhibitor ( FN439, 100μg/20μl ) were used, and the expression of EphB1 receptor was detected by western blot.(4) Opioid is widely used for cancer pain in clinic. To investigate the role EphB1 signaling on opioid tolerance, firstly, on rat bone cancer pain model, morphine (10 mg/kg, s.c., twice a day,) were used combined with low dose of EphB2-Fc (2μg/20μl, i.t., once a day), and tested the analgesic effects of morphine. Further, the role of EphB1 receptor in morphine tolerance was tested by using the standard protocols of the acute and chronic morphine tolerance in na?ve and EphB1 gene knockout (EphB1-/-) mice. Additionally, the expression and activation of EphB1 receptor were tested by western blot.Results(1) Rats that received TCI exhibited obvious thermal hyperalgesia and mechanical allodynia on ipsilateral hindpaw from day 7 after operation, and the extent were more and more severe with the progress of the disease. The contralateral hindpaw exhibited significant hyperalgesia and allodynia from day 14 after operation. Expression of EphB1 receptor and its ligand ephrinB2 in spinal cord, which was at low level in sham rats, was increased significantly in a time-dependent manner after TCI. Time courses of the up-regulation of EphB1 and ephrinB2 were well correlated with TCI-induced pain behaviors. The increased EphB1 and ephrinB2 were distributed predominatly in the superficial dorsal horn (DH) ipsilateral to TCI. Both of them were colocalized primarily with neurons (NeuN) and astrocytes (GFAP). In primary afferent terminals within the DH and the nociceptors within the DRG, ephrinB2 was colocalized with CGRP-, but not IB4-fibers and cells.(2) Production and persistence of TCI-induced pain behaviors were greatly delayed and suppressed by intrathecal administration of an EphB receptor blocking reagent EphB2-Fc (5μg/20μl). Meanwhile, TCI caused significant induction of c-Fos and activation of astrocyte (GFAP) and microglia (IBA), and these alterations were inhibited by the treatment of EphB2-Fc. In addition, repetitive treatment with EphB2-Fc produced a complete and long-lasting inhibition of phosphorylation of NMDA receptor subunits NR1 and NR2B, Src, ERK, CaMKII and CREB. Phosphorylation of NR2A was altered by neither TCI nor EphB2-Fc administration. EphrinB2-EphB1 signaling inhibition showed no effect on tumor growth or bone destruction.(3) EphB2-Fc administration obviously inhibited up-regulation and activation of EphB1 receptor induced by TCI, but showed no effect on ephrinB2. EphrinB2-Fc administration increased the activation of EphB1 receptor. EphrinB2-Fc, in EphB1-dependent manner, induced phosphorylation of NR1 and NR2B and thermal hyperalgesia. EphB2-Fc-induced down-regulation of EphB1 was prevented by spinal administration of an MMP2 inhibitor MMP2i (10μg), or an MMP9 inhibitor FN439 (100μg).(4) Administration of EphB2-Fc at lower dose (2μg/20μl) successfully prevented morphine tolerance, and rescued the analgesic effect of morphine in treating bone cancer hyperalgesia. In na?ve mice, both acute and chronic morphine tolerance were reduced by administration of low dose EphB2-Fc (2μg/10μl, i.t.). These pharmacological effects were further confirmed in EphB1-/-and its WT (EphB1+/+) mice. Expression and phosphorylation of EphB1 receptor were significantly up-regulated following chronic and acute morphine treatment, respectively. Such up-regulation was inhibited by EphB2-Fc treatment. Conclusion(1) TCI can increase the expression and activation of ephrinB2 and EphB1 in spinal cord, and both of them were colocalized primarily with neurons and astrocytes in the superficial of DH.(2) EphrinB2-EphB1 receptor signaling plays a key role in the development and maintenance of bone cancer pain. EphrinB2-EphB1 forward signaling contributes to regulation of bone cancer pain by activating NMDA receptor subunits NR1 and NR2B and the subsequent Ca2+-dependent signaling through Src kinase phosphorylation, which could be one of the mechanisms of bone cancer pain.(3) Matrix metalloproteinase MMP-2 and MMP-9 mediated the cleavage of EphB1 receptor induced by EphB2-Fc.(4) EphB1 receptor inhibition prevents morphine tolerance and rescues the analgesia of morphine.