Peripheral Mechanism Underlying Bone Cancer Pain In Rats: Modulation Of Trpv1 Current By Lpa And The Signal Pathway Analysis

Cancer, also known as malignant neoplasm, is a disease caused by cell proliferation aberration. Besides proliferation aberration, cancer cells invade and destroy the ambient normal tissues. In the advanced cancer patients, cancer cells metastasize to other organs, in which bone is one of the most common sites of metastasis. Bone cancer pain is difficult to treat and severely affects the patients'life quality.Lysophosphatidic acid (LPA) is a simple and ubiquitous phospholipid that exists in all eukaryotic tissues. The effects of LPA are widespread and diverse, regulating many biological processes, includes cell proliferation, migration, wound healing, cadiovascular function, adhesion and regulation of cytokine release etc. There are five LPA receptor subtypes, LPAj1-5, all of which are G protein-coupled receptors (GPCR). Dorsal root ganglion (DRG) neurons predominantly express LPA1 receptor. Recent studies showed that LPA involves in cancer cell proliferation and migration, as well as the development of neuropathic pain. However, the role of LPA in cancer pain remains unknown.The Vanilloid Receptor (transient receptor potential vanilloid receptor 1, TRPV1), an important molecule conducting peripheral pain signal, is a non-selective cation channel expressed in nociceptors. It can be activated by capsaicin, nociceptive thermal stimuli, acidic PH and many endogenous ligands. It can be regulated by many inflammatory mediators such as bradykinin, chemotactic factors and substance P (SP). Recent studies suggested that TRPV1 plays an important role in bone cancer pain.In this study, bone cancer model was established by injection of WALKER 256 mammary gland carcinoma cells into the rat tibia. By means of X-ray, transmission electron microscope scanning, immunohistochemistry, western blot, whole-cell patch recording and behavioral test, the behavioral and peripheral morphologic changes and the role of LPA in bone cancer pain were examined. Furthermore, the regulation of TRPV1 receptor by LPA and the possible downstream signal pathways were analyzed. The main results are as follows:1. Bone cancer pain model:the behavioral and peripheral morphologic changes1.1. Establishment of rat's bone cancer pain modelAfter inoculation of WALKER 256 mammary gland carcinoma cells into the tibia, rats behaved similar pathological features as clinical patients, such as allodynia, bone destruction and weight loss. Fifteen days after inoculation, X-ray results showed that ipsilateral tibias with cancer were destroyed severely, rats were very thin and weak. High death rate happened at this stage.Bone cancer-induced mechanical allodynia was examined by von Frey test. Compared to sham-operated rats, paw withdrawl threshold (PWT) of bone cancer rats reduced significantly since 7 days after cancer cell injection, and the maximal decrease was on the 16th day post-operation, suggesting that mechanical allodynia occurred in bone cancer rats.1.2. Injury of peripheral nerve induced by bone cancerTransmission electron microscope scanning and immunohistochemistry were used to detect the peripheral nerve system changes. The posterior articular nerve (PAN), a trunk of sciatic nerve, projects to the tibial marrow and innervates the periosteum and substantia ossea. The PANs of bone cancer rats and sham-operated rats were detected on the 14th day post-operation. PANs of bone cancer rats were demyelinated, indicating significant injury happened in the bone cancer rats.Activating transcription factor 3 (ATF3) is a member of activating transcription factor/cAMP-responsive element binding protein (ATF/CREB) family, often used as a marker of nerve injury. On the 14th day after inoculation, ATF3 expression in the ipsilateral and contralateral L4-6 DRGs of bone cancer rats and sham-operated rats were detected. The immunohistochemistry results showed that ATF3 expression were up-regulated in the ipsilateral L4-6 DRGs of bone cancer rats, suggesting bone cancer induced peripheral nerve injury. ATF3 were co-stained with neurofilament 200 (NF200, large cell marker), calcitonin gene-related peptide (CGRP, peptidergic small cell marker) but not with griffonia simplicifolia isolectin B4 (IB4, non-peptidergic small cell marker). High percentage of the large cells and peptidergic small cells were found to be injuried in the ipsilateral L4-5 DRGs of bone cancer rats, while few injuried non-peptidergic small cells were detected.2. The peripheral mechanism of bone cancer pain: involvement of LPA2.1. Involvement of LPA in the development of bone cancer painLPA1 expression was detected in the ipsilateral L4-6 DRGs of bone cancer rats and sham-operated rats on the 14th day after inoculation. LPA1 was found up-expressed in the ipsilateral L4-6 DRGs of bone cancer rats. LPA1 receptor antagonist VPC32183 was used to examine the possible role of LPA in bone cancer pain. Bone cancer rats were intrathecally injected with VPC32183 or saline (control) by lumber puncture on the day before inoculation, and the 2nd,4th,7th and 9th day post-operation respectively. VPC32183 was found to attenuate the mechanical allodynia and thermal hyperalgesia, suggesting that activation of LPA1 involves in bone cancer pain.2.2. Potentiation of TRPV1 channel activity by LPAThe changes of TRPV1 activity were examined to study its possible role in bone cancer pain. Expression of TRPV1 was detected in the ipsilateral L4-6 DRGs of bone cancer rats and sham-operated rats during the 14th-16th days after operation. Western blot results showed that TRPV1 expression were up-regulated in the ipsilateral L4-6 DRGs of bone cancer rats 14-16 days after cancer cell injection.Capsaicin induced currents were analyzed by whole-cell patch-clamp recording on acutely dissociated DRG neurons. A low concentration (0.5μM) of capsaicin was used to minimize the desensitization of TRPV1. The amplitude of TRPV1 currents induced by 0.5μM of capsaicin was compared between bone cancer rats and sham-operated rats during the 14th-16th days after operation. Owing to a large diversity of amplitude of TRPV1 currents among neurons tested, DRG neurons responsive to capsaicin were divided into the low current amplitude group and the high current amplitude group according to the amplitude of TRPV1 currents. In both groups, the average amplitudes of TRPV1 currents in cancer rats were higher than that in sham-operated rats, suggesting that the neurons in bone cancer rats were more sensitive to capsaicin.2.3. Downstream signal pathway analysisTo investigate the possible crosstalk between TRPV1 and LPA1, LPA1 and TRPV1 were double-stained in L4-6 DRG neurons. LPA1 were highly co-expressed with TRPV1 in L4-6 DRG neurons, which provides a morphological substrate for their crosstalk.Potentiation of TRPV1 currents by LPA were detected by whole-cell patch-clamp recording on acutely dissociated DRG neurons. LPA was found to potentiate the TRPV1 currents in a dose-dependent manner. Following 1μM,0.1μM or 0.01μM LPA perfusion, TRPV1 currents were increased by 221%,109%and 58% respectively. The currents were at maximal 3 min after LPA perfusion and reduced slowly to the control level 9 minutes after LPA perfusion under all of the three concentrations. No obvious potentiation of the currents was observed by 0.001μM LPA.Furthermore, potentiation of TRPV1 currents by LPA was enhanced in the bone cancer state. A very low concentration of capsaicin (0.5 nM) failed to evoke TRPV1 currents in all of the neurons tested in sham-operated rats, whereas 40%of the neurons tested in bone cancer rats were responsive to capsaicin. The effect of LPA on 12 neurons which did not respond to 0.5 nM capsaicin (sub-threshold concentration) in bone cancer rats was examined.0.01μM LPA could potentiate capsaicin to evoke TRPV1 currents in 8 of 12 neurons, indicating LPA-induced potentiation of TRPV1 currents under bone cancer state. However, this phenomenon was not observed in sham-operated rats.LPA1 antagonist VPC32183, protein kinase C (PKC) antagonist BIM, PKCεinhibitorεV1-2, protein kinase A (PKA) antagonist H89 and Rho (a small G protein) inhibitor BoTXC3 were used to examine the downstream signal pathway of potentiation of TRPV1 currents by LPA. VPC32183, BIM andεV1-2, but not H89 or BoTXC3, could block the potentiation of TRPV1 currents by LPA, indicating LPA potentiates TRPV1 receptor via PKC pathway, particularly by PKCε, depending on LPA1 activation, while PKA or Rho-Rock pathway doesn't involve in this effect.In conclusion, (1) Bone cancer induces peripheral nerve injury and mechanical allodynia; (2) Bone cancer induces up-regulation of LPA1 receptor in DRG neurons and LPA1 receptor antagonist alleviates bone cancer-induced mechanical allodynia and thermal hyperalgesia; (3) Activation of LPA1 potentiates TRPV1 currents in DRG neurons, amplifies pain signals from nociceptors to spinal cord in bone cancer rats; (4) LPA potentiates TRPV1 currents via PKC, particularly PKCε, but not PKA or Rho-Rock pathway.