Activation of spinal TDAG8 and its downstream PKA signaling pathway contribute to bone cancer pain in rats.

Bone cancer pain is difficult to treat and has a strong impact on the quality of life of patients. Few therapies have emerged because the molecular mechanisms underlying bone cancer pain are poorly understood. Recently, T-cell death-associated gene 8 (TDAG8) has been shown to participate in complete Freund's adjuvant-induced chronic inflammatory pain. In this study, we aimed to examine whether TDAG8 and its downstream protein kinase A (PKA) pathway are involved in bone cancer pain. A bone cancer pain model was made by inoculation of Walker 256 cells into the intramedullary space of rat tibia. Spinal TDAG8 expression was increased after inoculation with tumor cells. Intrathecal TDAG8 siRNA attenuated bone cancer pain behaviors during the initiation and maintenance phases; there were also concomitant decreases in TDAG8 mRNA and protein levels in spinal cord. Moreover, we found spinal PKA and phosphorylated cAMP response element-binding (pCREB) protein levels were up-regulated in the rat model of bone cancer pain. Knockdown of TDAG8 resulted in reduced bone cancer pain-induced spinal PKA and pCREB protein expression in two procedures. Furthermore, intrathecal H-89 (a PKA inhibitor) significantly attenuated bone cancer pain behaviors in rats. Our results suggest a causal relationship between TDAG8 expression and the initiation and maintenance of bone cancer pain. Activation of spinal TDAG8 contributes to bone cancer pain through the PKA signaling pathway in rats. These findings may lead to novel strategies for the treatment of bone cancer pain.

Brain-derived neurotrophic factor modulates N-methyl-D-aspartate receptor activation in a rat model of cancer-induced bone pain.

Brain-derived neurotrophic factor (BDNF) released within the spinal cord induces phosphorylation of N-methyl-D-aspartate (NMDA) receptors on the spinal cord neurons. This process is necessary for maintaining pain hypersensitivity after nerve injury. However, little is known about the role of BDNF and NMDA receptors in cancer-induced bone pain (CIBP), whose features are unique. This study demonstrates a critical role of the BDNF-modulated NMDA subunit 1 (NR1) in the induction and maintenance of behavioral hypersensitivity in a rat model of CIBP, both in the spinal cord and in the dorsal root ganglia (DRG). We selectively suppressed BDNF expression by RNA interference (RNAi) using intrathecal administration of BDNF small interfering RNA (siRNA). Then, we assessed mechanical threshold and spontaneous pain in CIBP rats. Real-time PCR, Western blotting, and fluorescent immunohistochemical staining were used to detect BDNF or NR1 both in vivo and in vitro. BDNF and phospho-NR1 were expressed under CIBP experimental conditions, with expression levels peaking at day 6 (BDNF) or 9 (NR1). Intrathecal BDNF siRNA prevented CIBP at an early stage of tumor growth (days 4-6). However, at later stages (days 10-12), intrathecal BDNF siRNA only attenuated, but did not completely block, the established CIBP. BDNF-induced NMDA receptor activation in the spinal cord or DRG leads to central sensitization and behavioral hypersensitivity. Thus, BDNF might provide a targeting opportunity for alleviating CIBP.

Minocycline-induced reduction of brain-derived neurotrophic factor expression in relation to cancer-induced bone pain in rats.

Previous studies have suggested that the release of brain-derived neurotrophic factor (BDNF) from microglia in spinal cord is necessary for maintaining pain hypersensitivity after nerve injury. However, little is known about its role in cancer-induced bone pain (CIBP), which is in some ways unique. This study demonstrates a critical role of minocycline (a potent inhibitor of microglial activation)-modulated BDNF in the induction and maintenance of behavioral hypersensitivity in a rat model of CIBP. We assessed mechanical threshold and spontaneous pain of CIBP rats. Moreover, minocycline was administered intrathecally from day 4 to day 6 (early stage) or from day 10 to day 12 (later stage), after carcinoma cell inoculation. Real-time PCR, Western blots, and double immunofluorescence were used to detect the expression of OX-42 (marker of activated microglia), phosphorylated p38-MAPK (p-p38), and BDNF. We found that intrathecal minocycline could prevent CIBP at an early stage of tumor growth (from day 4 to day 6). However, at the late stage (from day 10 to day 12), intrathecal minocycline had no effect. Moreover, the expression of OX-42 and BDNF under CIBP, peaking on day 6, were all reduced after minocycline injection from day 4 to day 6. The ability of minocycline-induced reduction of BDNF in the induction of behavioral hypersensitivity could provide an opportunity for alleviating CIBP.

Antiallodynic effects of propentofylline Elicited by interrupting spinal glial function in a rat model of bone cancer pain.

The activation of microglia and astrocytes in the spinal cord is involved in the progress of cancer pain. Propentofylline (PPF), a glial modulating agent, alleviates pain hypersensitivity in neuropathic pain models. The present study investigated the potential roles of PPF in a preclinical rat model of bone caner pain established by inoculating Walker 256 cells into the left tibia. At day 9 postinoculation, single administration of PPF (10 µg/10 µl, i.t.) significantly but transiently suppressed mechanical allodynia induced by bone cancer. Repeated application of PPF (10 µg/10 µl, i.t., once daily from days 9 to 12) persistently relieved mechanical allodynia on the side ipsilateral to surgery. Immunohistochemistry and ELISA showed that microglia and astrocytes in the spinal cord were activated, and the production of glia-derived proinflammatory cytokines interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α) markedly increased at day 12 postinoculation in the cancer group. Intrathecal injection of PPF (10 µg/10 µl) significantly inhibited the activation of spinal glial cells and the expression of proinflammatory cytokines. These results suggest that the glial modulating agent PPF has antiallodynic effects on bone cancer pain and has potential utility for clinical treatment of cancer pain.

Cancer-induced bone pain sequentially activates the ERK/MAPK pathway in different cell types in the rat spinal cord.

BACKGROUND: Previous studies have demonstrates that, after nerve injury, extracellular signal-regulated protein kinase (ERK) activation in the spinal cord-initially in neurons, then microglia, and finally astrocytes. In addition, phosphorylation of ERK (p-ERK) contributes to nociceptive responses following inflammation and/or nerve injury. However, the role of spinal cells and the ERK/MAPK pathway in cancer-induced bone pain (CIBP) remains poorly understood. The present study analyzed activation of spinal cells and the ERK/MAPK pathway in a rat model of bone cancer pain. RESULTS: A Sprague Dawley rat model of bone cancer pain was established and the model was evaluated by a series of tests. Moreover, fluorocitrate (reversible glial metabolic inhibitor) and U0126 (a MEK inhibitor) was administered intrathecally. Western blots and double immunofluorescence were used to detect the expression and location of phosphorylation of ERK (p-ERK). Our studies on pain behavior show that the time between day 6 and day 18 is a reasonable period ("time window" as the remaining stages) to investigate bone cancer pain mechanisms and to research analgesic drugs. Double-labeling immunofluorescence revealed that p-ERK was sequentially expressed in neurons, microglia, and astrocytes in the L4-5 superficial spinal cord following inoculation of Walker 256 cells. Phosphorylation of ERK (p-ERK) and the transcription factor cAMP response element-binding protein (p-CREB) increased in the spinal cord of CIBP rats, which was attenuated by intrathecal injection of fluorocitrate or U0126. CONCLUSIONS: The ERK inhibitors could have a useful role in CIBP management, because the same target is expressed in various cells at different times.

[The role of brain-derived neurotrophic factor in pain facilitation and spinal mechanism in rat model of bone cancer pain].

OBJECTIVE: To investigate the role of brain-derived neurotrophic factor (BDNF) in pain facilitation and spinal mechanisms in the rat model of bone cancer pain. METHODS: The bone cancer pain model was developed by inoculated Walker 256 mammary gland carcinoma cells into the tibia medullary cavity. Sixty SD female rats were divided into 5 groups (n = 12 each) randomly; group I: control group (sham operation); group II: model group; group III: control group + anti-BDNF intrathecal (i.t.); group IV: model group + control IgG i.t.; group V: model group + anti-BDNF i.t.. Anti-BDNF or control IgG was injected i.t. during 7 to 9th day. Von-Frey threshold was measured one day before operation and every 2 days after operation. On the 9th day after threshold tested, rats were sacrificed after i.t. injection of either anti-BDNF or control IgG, the lumbar 4-6 spinal cord was removed. The expression of the spinal BDNF and the phosphorylation of extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) were detected by immunohistochemistry assay and Western-Blot. Co-expression pattern of BDNF and p-ERK1/2 were determined by double-labeling immunofluorescence. RESULTS: We demonstrated the coexistence of BDNF and p-ERK1/2 in the spinal cord of rats. From the 7 to 9th day after operation, von-Frey threshold in groups II and IV was significantly lower than that in group I and group V (P < 0.01), group V was remarkly higher than that in group IV (P < 0.01). The spinal BDNF and p-ERK1/2 expression in group II or IV were significantly increased compared with that in group I or V (P < 0.01), intrathecal anti-BDNF was significantly suppressed BDNF and p-ERK1/2 expression (P < 0.01). CONCLUSION: BDNF and p-ERK1/2 was coexistence in the spinal cord of rats, and it maybe involved in the bone cancer pain.

[Feasibility of establishment of rat model of bone cancer pain by using Walker 256 cells cultured in vitro or in vivo].

OBJECTIVE: To investigate the possibility of establishing rat model of bone cancer pain using cancer cells cultured in vitro or by ascites passaging and verify the reliability of this method. METHODS: Syngeneic SD rat carcinoma cells of the line Walker 256 were cultured in vitro and inoculated into the peritoneal cavity of SD rats respectively. Two kinds of Walker 256 cell suspension were made. Thirty-two SD rats were randomly divided into 4 equal groups: Group N (undergoing injection of Hank's solution into cavitas medullaris of left tibia), Group K (undergoing injection of heat-killed Walker 256 cells), Group V (injected with Walker 256 cells cultured in vitro), and Group A (injected with Walker 256 cells passaged in ascites). After 6, 12, and 18 days, the rats underwent roentgenography. Radio nuclide emission computed tomography (ECT) was conducted 12 days later. And MRI was conducted 15 days later. Thermal withdrawal latency (TWL) and pressure withdrawal threshold (PWT) were measured. Von Frey threshold and weight bearing of left hind limb were examined. On the 18th day after the establishment of model the rats were killed with their left tibia taken out to undergo microscopy. RESULTS: The rats of Groups A and V began to display decrease of left hint limb activity and roentgenography showed minute defect of bone trabecula in the proximal epiphysis by day 6. By day 12 roentgenography showed multiple defect of bone trabecula, ECT showed reactive bone formation. The rats of Groups A and V displayed signs of weight loss by day 14, and by day 18 roentgenography showed full thickness bicortical bone loss and formation of soft tissue tumor. Histological examination 18 days later revealed that the bones inoculated with live cells showed infiltration of bone marrow spaces by malignant tumor. The PWT values gradually decreased since day 6 to day 18, and the PWT values in this period of Groups A and V were all significantly lower than those of Groups K and N (all P < 0.01). The von Frey values gradually decreased since day 6 to day 18, and the von Frey values in this period of Groups A and V were all significantly lower than those of Groups K and N (all P < 0.01). The weight bearing value of left hind limb gradually decreased and the weight bearing difference between the 2 hind limbs gradually increased since day 6 to day 18, and the values of weight bearing difference between the 2 hind limbs of Groups A and V were significantly higher than those of Groups K and N (all P < 0.01). Sixteen rats underwent subcutaneous injection of morphine, and the PWT values increased 20, 30, and 40 min later in a dose-dependent manner (P < 0.01). Naloxone injected 1 h later antagonized the analgesic effect of morphine. CONCLUSION: A SD rat model of bone cancer pain has been successfully established by using syngeneic rat bone carcinoma cells cultured in vitro or in vivo, and the latter being more convenient.

[Celastrol in the inhibition of neovascularization].

OBJECTIVE: To study the inhibition effect of celastrol on neovascularization. METHODS: The effect of celastrol on the in vitro proliferation of endothelial cell of vessel (ECV) was examined by MTT assay. The effect of celastrol on endothelial cell migration, tube formation on Matrigel and Chick chorioallantoic membrane angiogenesis was also examined. Matrigel plug assay was used to evaluate the effect of celastrol on angiogenesis in vivo. RESULTS: The proliferation of ECV was inhibited significantly by celastrol with IC(50) being 1.33 microg/ml. Celastrol inhibited endothelial cell migration and tube formation in a dose-dependent manner. Celastrol also inhibited angiogenesis both in Matrigel plug of mouse model and in chick chorioallantoic membranes. CONCLUSION: Celastrol, which can inhibit angiogenesis, could be developed as an antiangiogenic drug.

[Several monomes from Tripterygium wilfordii inhibit proliferation of glioma cells in vitro].

BACKGROUND AND OBJECTIVE: Researches indicated that Tripterygium wilfordii possess antitumor activity. The current study was designed to investigate inhibitive effect of several monomes of Tripterygium wilfordii on the proliferation of glioma cells. METHODS: The effect of three monomes from Tripterygium wilfordii on the proliferation of glioma cell lines SHG44, C6, and U251 in vitro was examined by using MTT assay. Immunohistochemistry was used to examine the expression of Bax, Bcl-2 after treatment of triptolide and celastrol. RESULT: The proliferation of glioma cells was remarkably inhibited by triptolide and celastrol. They both increased expression of Bax and decreased expression of Bcl-2 in the SHG44 cells. CONCLUSION: Triptolide and celastrol inhibit the proliferation of glioma cells in vitro, which was associated with promoting the expression of Bax and inhibiting the expression of Bcl-2 and accelerating cell apotosis.