Study On The Mechanism Of Camptothecin On Neurotoxicity Of Mouse Cortical Neurons

As we all know, camptothecin (CPT) was an effective anti-neoplastic agent. Pharmacological studies confirmed that the main antitumor mechanism of CPT was inhibiting topoisomerase Ⅰ (topoisomerase Ⅰ, Topo-Ⅰ) activity in tumor cells during DNA replication, that was in S phase of cell cycle. Neurons were highly differentiated cells and cells were non-division. The cell cycle only stayed in G1 phase and there was no DNA replication process. So, in theory CPT was no cytotoxic effect to the neurons. However, both clinical and experimental reported that CPT had certain neurological side effects. Malignant tumor patients often appeared nervous system symptoms during the period of chemotherapy, including dysarthria,body ataxia, etc, especially long-term chemotherapy patients. Nowadays, the mechanism of CPT neurotoxicity was unclear. Neurons were highly differentiated cells, and the brain was often at a high oxygen environment.Therefore, the DNA transcription in neurons was more active than other cells. Therefore, whether theneuron-virulent mechanism of CPT which related to DNA transcription in neurons. CPT inhibited the Topo-Ⅰ activity during the DNA transcription in neurons, and eventually caused single-break DNA. So the accumulation of a large number of single-break DNA influenced the stability of the whole DNA, eventually led to the double-stranded DNA break, as well as caused the neuronal apoptosis. To this end, this experiment observed the DNA damage and neuronal apoptosis of original generation of culturing mouse neurons after been intervened by CPT and CPT-DRB respectively. In order to further reveal the internal central neurotoxicity mechanism of CPT.Objective:This experiment was mainly done by the primary cultured mouse cerebral cortex neurons as the breakthrough point which was intervened by CPT and the CPT-DRB respectively,and observed the neuronal activity and apoptosis. To interpret scientifically the mechanism of CPT’s neurotoxin from the molecular level, as well as provided experimental basis in clinical therapy.Methods:1.To cultivate the original generation of mouse nerve cells in the cerebral cortex and purification.2.Select the concentration gradient of CPT affect the activity of neurons, followings:CCK-8 tested the neuronal activity, Cells were divided into 7 groups, and intervened by different concentrations of CPT, which final concentration were 0、2.5、5、10、20、40、80UM respectively, and set up six complex hole in each concentration. After culturing 12h、24h、48h respectively. Observed the neurotoxicity of CPT, and screening out the suitable drug concentration (IC50) which was been the following experimental drug concentration. 3.To observe the neuronal activity and apoptosis which were intervened by CPT:after then neuron matured and set up three groups respectively:normal group, CPT group, CPT joint DRB (transcription inhibitors) group. The Normal group of neurons did not do any processing, the CPT group were intervened with 41.8 UM CPT (IC50 concentration of preliminary experiment), the CPT joint DRB group were intervened with 41.8 UM CPT and 10UM DRB. After 24h, CCK-8 tested the change of neurons activity, and then TUNEL staining, γ-H2AX immunofluorescence labeling and flow cytometry method tested the detection of DNA damage and apoptosis of neurons.Results:1. It was concluded that IC50 and the suitable time:41.8UM and 24h.2. The marked time-dependency of induction of neurons by CPT:change of each neurons activity was observed when they had been intervened by different concentration of CPT:12 h time points, compared with the normal control group, OD value of the treatment group decreased, decreased of neurons activity in 2.5 UM group was not obvious, cell activity decreased significantly in 5 UM group (P<0.01). Withthe increase of the concentration of CPT, cell activity decreased more significantly.24h,48h time point, compared with normal control group, the OD value of each drug group declined significantly (P<0.01, P<0.001), and with the increase of concentration, the more significantly the neurons activity decreased. On the other hand, under the same concentration of CPT, with the extension of time, the more significantly the neurons activity decreased.3. The effects of neurons activation after intervened by CPT:compared with the normal control group, the CPT group OD value decreased significantly (P<0.01), prompted that CPT could significantly inhibit the activity of neurons. Compared with the CPT group, the OD value of the CPT joint DRB group increased significantly(P<0.001), Prompt that the inhibition activity of neurons of CPT decreased obviously after joining DRB transcription inhibitor.4. The expression of the three groups neuronal γ-H2AX after intervened by CPT:Neurons in normal group were hardly seen green fluorescent, illustrated that the normal neurons γ-H2AX negative expression. The γ-H2AX expression of CPT group neurons significantly increased, and the tag γ-H2AX green fluorescent particles mainly distributed in the nuclei of neurons, prompted that the neurons have appeared DNA damage. The γ-H2AX expression of neurons in the CPT-DRB group were less than CPT groups, illustrated that add DRB could reduce the DNA damage in neurons.5. The TUNEL staining results of the three groups neurons:the nucleus of the normal group neurons were round and neat, and were not seen the fluorescent particles. The nucleus shape of the CPT group became irregular, nucleus area marked green fluorescent particles with intensity, and the number of positive staining neurons notably increased. The nucleus shape of add DRB group neurons were irregular, but the green fluorescent particles in add CPT-DRB group were less obviously than in CPT group.6. The results of flow cytometry instrument test:compared with the control group, the normal cells in CPT group was significantly lower (2.9%). Thepercentage of early apoptosis cells(40.1%), late apoptosis cells(56.8%) increased obviously. Compared with the CPT group, the percentage of late apoptosis cells(15.3%)in CPT-DRB group decreased obviously, the percentage of normal cells (12.0%) increased obviously.Conclusions:1. CPT has inhibited obviously the cortex neuron activity, and has the apparent concentration-time dependence.2. CPT could obviously inhibit neuron activity, as well as caused DNA damage and apoptosis. The results confirmed that CPT has neurotoxicity.3. The DNA transcription inhibitor DRB could effectively reduce the neuronal injury and apoptosis which were induced by CPT, suggested that neurotoxicity of CPT could be associated with DNA transcription process.