BCP is a kind of chronic pain with unique and complicated mechanism, which is the most common symptom of pain for the patient with cancer. For a long time, it has seriously affect the life quality of patients with cancer and brought oppressive pain to patients and their families. Spinal cord is the primary central of pain information transmission and integration, and its sensitization is the main mechanism of chronic pain. Rat spinal cord contains thousands of proteins, which may be closely related to the development of BCP. Therefore, it may be a promising way to analyze the profile of spinal cord protein alterations in order to try finding potential biomarker to elucidate the pathogenesis of BCP [10]. Nowadays, proteomic analysis is widely considered as a valuable strategy to conduct mechanistic studies [11]. In order to find potential molecules and elucidate the regulatory mechanisms of BCP, Proteomic approaches were applied.
In this study, we first implanted carcinoma cells into the tibial plateau of rats to establish a model of BCP. The PWT of rats ipsilateral paw was measured on the day 0, 7, 14 and 21 after inoculation. With the tumor growth, the PWT decreased obviously in BCP rats, it suggested that the bone cancer pain animal model was successfully established.
Proteomic method of 2-DE gels electrophoresis was used to analyze the rat lumbar spinal cord. For the results, a total of 60 DEPs were identified, including 32 downregulated proteins and 28 upregulated proteins. The 60 protein spots with significant differential expression were selected for MALDI-TOF-MS analysis. Among 60 protein spots, 34 DEPs were successfully identified by PMF.
We analyzed the differentially expressed proteins, and taking into consideration their function annotation, identified several proteins that may be related to the molecular mechanism of the BCP. For example, cofilin1, tropomodulin-2 (Tmod2) and tropomyosin beta chain (Tpms), which are associated with actin filament organization function. Cofilin1 is an actin-binding protein, the primary function of which is dynamic reorganization of actin cytoskeleton. It plays an important role in regulating actin dynamic, affecting synaptic transmission and plasticity. A previous study reported that the expression of cofilin1 is down-regulated in DRG and spinal cord of BCP, suggesting that cofilin1 is involved in the formation and development of BCP [12]. Our results also showed cofilin downregulation on the day 7 after inoculation in the BCP model. Actin filaments have a wide range of functions in membrane morphology, cell movement, vesicle transport, neurite development, and synaptic communication, and are considered to play an important role in synaptic plasticity [13]. The state of actin (as monomers or polymeric strands) and the length of the filaments are controlled by numerous actin-regulatory proteins. Tmod2 is a protein that binds to the slow-growing (pointed) ends of actin filaments (F-actin), preventing both elongation and depolymerization. Tpm bind along the length of actin filaments and prevents filament depolymerization. In fact, the pointed-end capping activity of Tmod2 is greatly enhanced by association with Tpm, which suggests that the two proteins function as a complex to stabilize the filament and regulate its length. In our results, Tmod2 was downregulated and Tpm was upregulated in the BCP model, suggesting that actin filaments were activated and lose staility, which increased the growth rate of actin filaments, and finally contributed to enhanced of information synaptic transmission of dendritc spines in neurons, inducing the occurrence of BCP. It has been reported knockout of Tmod2 increased long –term potentiation (LTP) in brain slices [14]. This fully suggests that Tmod2 is associated with synaptic information transmission and may be involved in the occurrence and development of BCP.
Glia maturation factor-β (GMFB) is considered to be a growth and differentiation factor for both glia and neurons, Which is expressed predominantly in the central nervous system (CNS) [15]. The major cells that express GMFB in the nervous system are glial cells, including astrocytes, Bergmann glia in the cerebellum, Schwann cells in the peripheral nervous system (PNS). It is commonly accepted that reactive oxygen species (ROS) are important by-products of metabolism and participate in many pathophysiological processes. Recent studies have shown that ROS are involved in sensory transmission and regulation and are an important factor leading to carcinogenic pain. As part of the cellular antioxidant system, Copper-zinc superoxide dismutase (Cu-Zn SOD) can scavence ROS, reduce oxidative stress at the spinal cord level and relieve bone cancer pain through its own antioxidant action and protective antioxidant action of prdx4 [16]. A previous study reported that overexpression of GMFB using an adenovirus vector carrying GMFB cDNA in C6 rat glioma cells led to the activation of NF-κB as well as an increase in Cu-Zn SOD expression [17]. In our study, GMFB is downregulated on the day 7 after surgery in the BCP model. It suggest that the production of reactive oxygen species increases in the BCP, while the stimulation of Cu-Zn SOD is weakened by the down-regulation of GMFB, and the expression of Cu-Zn SOD is reduced, which forms oxidative stress and stimulates the production of various harmful substances by the cells, causing the abnormal release of neurotransmitters as well as the abnormal activation of corresponding receptors, resulting in bone cancer pain. Therefore, GMFB is a valuable molecule for further functional research in BCP.
Calpain is a family of calcium-dependent intracellular cysteine protease, the activity of which is dependent on intracellular calcium levels. They participate in a variety of cellular processes such as signal transduction, cell proliferation, cell cycle progression, differentiation, apoptosis, membrance fusion, necrosis and platelet activation, etc [18]. Among them, Calpain is known to exert restrictive proteolysis on its specific substrates, particularly IκBα, c‑Jun, and c‑Fos, all of which are essential molecular players in osteoclastogenesis [19]. Studies have suggested that inhibition of the activity of the protease calpain reduce the development of acute and chronic inflammation [20]. On the other hand, a previous study also reported that calpain inhibitor reduces BCP possibly through inhibition of osteoclastogenesis in rat BCP model [21]. However, Calpain was downregulated in our study. Therefore, the downregulation of Calpain may suggest activation of a otective mechanism in BCP model, and could reduce the extent of pain.
The N-myc downstream–regulated gene (NDRG) family consists of four members (NDRG1~4) that are differentially expressed in various organs and important cellular processes, such as cell proliferation, apoptosis, differentiation, development, and stress reponse [22]. As a member of the NDRG family, NDRG family member 2 (NDRG2) is predominantly expressed in astrocytes of the central nervous system (CNS). Its function in the CNS is mainly related to its role in astrocyte activity. The expression of NDRG2 is ubiquitous in astrocytes, and mostly colocalizes with the astrocyte marker glial fibrillary acidic protein (GFAP). NDRG2, which is involved in not only the regulation of apoptosis, blood–brain barrier integrity, glutamate clearance and other cellular metabolic processes, but also stress responses, is commonly regarded as a tumor suppressor [23, 24]. NDRG2 has been found to be expressed in neurons, playing an important in neural differentiation, synapse formation, and axon survival [25]. A recent study revealed the correlations between Glutamatergic and GABAergic nerve terminals and NDRG2 immunopositive astrocytic processes [26]. The expression of the glutamate transporters glutamate aspartate transporter (GLAST) and glutamate transporter 1 (GLT-1) were increased by deletion of NDRG2 in glialtransmission. A previous study demonstrated that NDRG2 is implicated in many neurological diseases including glioma, stroke, neurodegeneration and psychiatric disorders. However, the expression changes of NDRG2 in BCP have been rarely reported. Our study found that the expression of NDRG2 in BCP model was up-regulated, Considering the role of NDRG2 in astrocytes, NDRG2 may induce the activation of spinal astrocytes to participate in the occurrence and maintenance of bone cancer pain.
GO enrichment analysis revealed that the biological functions of the differentially expressed proteins relate mainly to actin filament organization, hippocampus development, oxidation-reduction process and give an insight into the underlying molecular mechanisms of the BCP. The functions of these proteins in biological processes should be further validated.