Construction Of Recombinant Caspase3 And Its Analgesic Effect On Bone Cancer Pain In Rats

BackgroundCancer-related pain has always been a difficult clinical issue. As great improvements have been made in the detection and treatment of most types of cancer, cancer pain have played a significant negative role in the quality of lives and the survival rates of cancer patients. Between 75 and 90% of patients with metastatic cancer will experience significant cancer-induced pain. Cancers, including prostate cancer, breast cancer, and lung cancer, have special affinities to metastasize to bone, and produce pathological bone cancer pain. Bone cancer pain is one of the most severe cancer induced pain with high morbidity. The treatment of bone cancer pain now has been largely based on the use of opioids. Opioids are effective in attenuating bone cancer induced pain, but with the increases in frequence and doses, the analgesic effects from opioids are frequently accompanied by side effects such as addiction, tolerance, nausea, vomiting, constipation and respiratory depression. Therefore, it is very necessary to explore a novel mechanism-based treatment which has more effective, long-lasting and less side-effects to relieve the bone cancer pain.The recently published animal models of bone cancer pain have allowed the researchers greater insights into the complicated mechanisms of bone cancer pain and the explore more effective targeted drug managements. The metastatic rat tibia bone cancer pain presents the similar characteristic pain symptoms to human bone cancer pain. In this model, metastative cancer cells were injected into the intramedullary cavity of femur or tibia of rats to produce a serie of behavioral, cellular, and neurochemical changes correlated with the development of cancer growth and bone destruction. Walker 256 carcinoma cells have significant bone metastatic affinities, and are commonly used for the set up of metastatic rat tibia bone cancer pain models. We constructed a model of bone cancer pain induced by the injection of Walker 256 carcinoma cells into the tibia cavity of rats, as the research basis of our study.Some structures in brainstem could elicit desending signals to spinal cord regulate pain signal transmission to central nervous system, and it is one of the superspinal pain medulating structures, which are involved in pain signal integration and modulation. So, to stop the superspinal nociceptive signal up or down transmission could be a novel method for pain management. Rostral ventromedial medulla (RVM) play a critical role in the brainstem descending facilitation system. RVM contains nucleus like 5-HT abundant nucleus raphe magnus (NRM), the adjacent gigantocellularis pars alpha and ventral nucleus reticularis gigantocelluaris. RVM receives the nociceptive information from midbrain periaqueductal gray (PAG), in turn projects to the spinal cord largely along the dorsolateral funiculus (DLF) and terminate in the spinal cord. The PAG, RVM with its spinal projections, constitute the channel of the descending pain control system, which could facilitate or inhibit spinal transmission of pain signal. The ablation of RVM facilitory neurons might attenuate the pain transmission, and thus represent a novel method for pain management.Intra-RVM microinjection of Lidocaine was used to block the function of facilitory neurons in RVM in a rat model of neuropathic pain. The analgesic effects were significant, but as the metabolism of the drug, analgesic effects decreased and finally disappeared. Intra-RVM injection of saporin, which is a neurotoxin, destroyed the RVM neurons and could eventually produce a long-term analgesic effect. However, the necrosis of neurons of RVM could lead to neuroimmune and/or neuroinflammatory responses of adjacent neural tissue. RVM is implicated in multiple important physiological functions, such as respiratory, heart rate, blood pressure, thermoregulation and motion function, the side effects induced from the products of necrosis could be severe and mortal.Caspases3, one of the most downstream caspases, plays a key role in executing programmed cell death, but the activation of wild caspase3 depends on exogenous stimulations and a long procedure of the activation of predomain caspases. A new human recombinant caspase3 was generated by Srinivasula in a reverse order of the big and small subunits by molecular biology methods. Unlike the wild caspase3, this recombinant caspase3 could induce constitutive apoptosis without exogenous stimulations and the activation of predomain caspases. This recombinant caspase3 gene could be used as a novel cell death factor in the genetic engineering. However, as a hetergenetic gene, the human reconstitutive caspase3 gene could induce immunoreactions in experimentary rats. In this study, we will generate rat recombinant caspase3 gene, and inject this self-activated gene with non-viral vector PEI into the RVM in a rat model of metastatic tibia bone cancer pain. Rat recombinant caspase3 could induce neuron apotosis in the RVM of bone cancer pain rats without immuo or inflammatory responses, but produce a long-term analgesic effect against bone cancer pain.Methods and Results1,Construction of rat constitutively active recombinant caspase3 gene and investigation of its pro-apoptotic effect in vitroMethods:Total RNA of rat astroglioma C6 cells was extracted and the reverse transcription PCR was performed to gain cDNA. According to Genbank, the primers of large and small subunits of wild caspase3 were designed and reversed rat caspase3 gene was gained by settling the small subunit prior to the large one through recombinant PCR, and cloned into the expression vector pcDNA3.1+-Remcasp3 and C1-Remcasp3 to transfect human 293T cells and rat immortalized neural progenitor cells (INPC). The expression and pro-apoptotic effect of recombinant caspase-3 was observed by the changes of morphology of the transfected cells through immunofluorescence and analyzed by Annexin V-FITC staining, Flowcytometry and MTT assay. Transmission electron microscope was used to detect the micro morphology changes of apoptosis of the transfected cell.Results:The rat recombinant caspase3 with reversed large and small subunits was successfully contructured. The sequencing result was the same as designed. After transfected with C1-Remcasp3, INPC and 293T cells presented obvious apoptosis. Transmission electron microscope detected cytoplasm concentration, chromatin condensation and the formation of apoptotic bodies. MTT assay showed that the proliferation of recombinant caspase-3 transfected cells was significantly inhibited(INPC: 44.61±0.15%; 293T:48.35±0.16%) (P<0.05).Annexin V-FITC staining revealed that the percentage of apoptotic cells in the transfectants of recombinant caspase3 gene was(INPC: 16.00±0.99%; 293T:30.67±1.52%),which was much higher than that of control cells (P<0.05)2,Contruction and evaluation of the rat model of bone cancer painMethods:Cultured rat Walker 256 mammary gland carcinoma cells was injected into the rat abdominal cavity.6-7 days after injection, the rat peritoneal fluid was extracted and prepared for tibia injection. Thirty-two female SD rats were divided into 4 groups at random,8 rats in each group:ⅠNaive group;ⅡD-Hank's group:10μl sterilized D-Hank's solution was injected into the tibia cavity of the right leg of rats; III Heat-killed cells group: 10μl heat-killed Walker 256 carcinoma cell solution was injected into the tibia cavity of the right leg of rats, and IV Bone cancer pain group:10μl live Walker 256 carcinoma cell solution was injected into the tibia cavity of the right leg of rats. Mechanical allodynia, mechanical hyperalgesia and ambulatory pain were measured one day before sugery, the day of surgery, and each day to the 15th day after sugery. X ray was used on the 15th day after sugery. After radiological detection, the rats were sacrificed and the GFAP and OX42 immunofluorescence were used and high performance liquid chromatography (HPLC) method was taken to measure the glutamate concentrations in RVM and spinal cord.Results:Significant behavioral changes were observed in the rats of BCP group:the mechanical withdrawal threshold and latency were significantly decreased, as compared with other groups.15 days after injection with live carcinoma cells, tibia bone destruction was monitored using radiological methods and showed the signs of radiolucent and deterioration with medullary bone loss, close to the injection site, While No radiological changes were observed on the normal tibia bone. In the immunofluoresence test, GFAP and OX42 were used to label the astrocytes and microglia in spinal cord. In bone cancer pain group, the number of immunoreactive astrocytes and microglia were significantly increased, and fluorescence intensity were much higher than the other control groups. The results of high performance liquid chromatography showed that the concentration of Glutamate in RVM and spinal cord were significantly higher than the other control groups (P<0.05).3. Analgesic effects of targeted intra-RVM injection of In-vivo jetPEIin a rat model of bone cancer painMethods:After being identified, the plasmid pcDNA3.1+-Remcasp3 was prepared in large scale. The concentration and purity were measured. The preparation of the in-vivo jetPEI/DNA complexes were performed under sterile conditions:the DNA and non-viral vector were mixed under certain proportion and added into sterilized 10% glucose solution. Sterile water was added into the mixture to obtain a final concentration of 5% glucose. 10～12 days after intra tibia injection of carcinoma cells, rats were anesthetized with propofol (50μg/100g i.p.). Then they were positioned in a stereotaxic apparatus and a single microinjection into RVM was performed according to its anatomical position (anteroposterior,-11.0 mm from bregma; lateral,±0.6mm from midline; dorsoventral,-8.5 mm from the cranium). Drug administration into the RVM was performed by slowly and carefully expelling 1μl of the solution into RVM for both sides. The needle was left in position for a minute to allow sufficient diffusion of drugs before the needle was withdrawn. Behavioral tests were taken each day on one day before intra-RVM injection, the day after injection, to the 7th day after intracranial injection.48h after intra-RVM injection of DNA/PEI mixure, rats were sacrificed and paraffin sections of RVM were prepared for TUNEL detection. Spinal cord GFAP and OX42 immunofluorescence tests were taken and high performance liquid chromatography (HPLC) method was taken to measure the glutamate concentrations in RVM and spinal cord.Results:Before intraRVM injection, there were no significant differences in withdrawal thresholds, withdrawal duration and score of ambulatory pain among different groups of rats.24h after intraRVM injection of recombinant caspase3 and PEI mixture, the withdrawal thresholds were significantly increased, withdrawal durations and the score of ambulatory pain were significantly down regulated (P<0.05). A lot of apoptotic cells were found in RVM. The concentration of glutamate in RVM and spinal cord of BCP rats were significantly down regulated after intraRVM injection of Remcasp3/PEI mixture, as compared with BCP rats without intraRVM injection of recombinant caspase3 (P<0.05). In the immunofluoresence test, the number of immunoreactive astrocytes and microglia were significantly increased in bone cancer pain group of rats. After treated with recombinant caspase3 and PEI mixture in RVM, the number of activated glial cells decreased, and the level of both astrocytes and microglia activation were significantly down-regulated.4. Safty evaluation of targeted intraRVM injection of Recombinant caspase3/in-vivo jetPEI mixtureMethods:From one day before intraRVM injection, the day of the injection, to 7th day after surgery, the basic physiological function parameters were measured in Naive group, PEI group,5%Glucose group, PEI+5%Glucose group and Remcasp3 group of rats. The weight of body, respiratory rate, rectal temperature, and heart rate were measured. Results:There were no significant differences on physiological parameters before intra RVM injection. After RVM injection, the body weight, rectal temperature, respiratory rates and heart rates were lack of significant differences among 5 groups of rats in the whole observation time(P>0.05).5. Statistical analysisAll of the analyses were performed by SigmaStat 3.0 software package. All data were expressed as the mean±standard deviation (SD). Group comparisons and the other results using one-way analysis of variance. P-value of< 0.05 was considered statistically significant.Summary1. Major results(1) The rat recombinant caspase3 can be expressed in eukaryocytes, and induce apoptosis without exogenous stimulation.(2) IntraRVM injection of In-vivo jetPEI/recombinant caspase3 mixture could induce apoptosis to the neurons in RVM and produce significant, complete and long lasting analgesic effects in bone cancer pain rats.(3) The concentration of excitatory amino acids-Glutamate in the center nervous system and the number of activated astrocytes and microgial cells in spinal cord of bone cancer pain rats are significantly higher than normal rats. After inducing apoptosis into the neurons in RVM, the concentrations of glutamate and the activation of glial cells were significantly down regulated.(4) A single intraRVM injection and inducing apoptosis to neurons of RVM could not produce significant changes in the motor and basic physiological functions of experiment rats. 2. Conclusions(1) The rat recombinant caspase3 was successfully constructed in-vitro, and could be expressed in eukaryocytes. It can induce constitutely self apoptosis to neural and non-neural cells without sitmulaitons or predomain caspases. It could be used safely in rats with normal immunosystem and provide a new "cell death" gene for the research and management of gene therapy for central nervous system diseases.(2) Similar to neuropathic pain and inflammatory pain, RVM is critical in the initaion and mantance of bone cancer pain. Targeted ablation of the neurons in RVM by means of apoptosis could produce effective and safe analgesic effects.(3) Non-viral catonic PEI could be used as a high effective and safe vector for central nervous system transfection.(4) The level of excitatory amino acids glutamate and the activation of spinal glial cells are closed involved in the intiation and maintance of bone cancer pain.(5) Stereotactic microinjection of DNA/PEI mixture into RVM did not induce significant changes to the physiological functions of experiment animals. This suggested that targeted ablation of RVM could produce effective, long lasting and safe analgesic effects and subsequently provide us a new approach for superspinal pain management targets.