| ObjectiveNasopharyngeal carcinoma is the most common head and neck cancer in Southern China. The causes of morbidity may be related to genetic susceptibility, EBV infection, environmental and dietary habits, as well as immune dysfunction. The disease seriously threats the health of people in Southern China. To improve the treatment techniques and methods of nasopharyngeal, in order to ameliorate recovery rate, as well as local control rate, has its practical significance.At present, the standard treatment of nasopharyngeal cancer is recognized as radiation therapy or radiation combined modality therapy. Early nasopharyngeal carcinoma can be able to achieve clinical cure with radiotherapy alone, patients with advanced disease need to select the radiation combined modality therapy.The radiation dose has been basically constant around70Gy in the radiotherapy of nasopharyngeal cance. The goals of improving the treatment techniques and methods are to retain the lesion region dose and to reduce the dose of surrounding normal tissues, the result is the percentage of dry mouth and difficulties in opening mouth are descended. Now we already have the new technique of IMRT and IGRT, targets of precise radiotherapy are gradually being implemented.Since the majority of patients with nasopharyngeal carcinoma are already found locally advanced or late, so most of then need to accept the radiotherapy combined therapy. And malignant disease is the systemic deiease involving the whole body, adjuvant treament as chemotherapy, targeted therapy and biotherapy also play important roles in comprehensive treatment of nasopharyngeal carcinoma. At present, cytotoxic drugs based chemotherapy still occupy an important position in the comprehensive treatment of advanced nasopharyngeal carcinoma. However, cytotoxic drugs often have severe side effects, and patients’tolerance is limited. So the research focuses are increasingly turning to targeted therapy and biological therapy. Because EGFR expression on nasopharyngeal carcinoma cell surface, the study of targeted drugs that block the EGFR pathway is the hot spot. Cetuximab as the example is the drug that blocks the EGFR pathway.PA-MSHA is attenuated preparations of P. aeruginosa. It is the product of the Pseudomonas aeruginosa after recombinant processes. After the process, PA-MSHA retained a high degree of affinity of the mannose and immunogenic, the toxicity was significantly decreased.It is already widely used in clinical application of our country, and also observed significant anti-tumor efficacy. Basic research indicates that PA-MSHA can block the EGFR pathway by combined with the EGFR surface mannose, activates the immune system, both regulates immune function, finally inhibit the growth of target cells. The drug has the functions of inhibiting the EGFR pathway, and immunomodulatory. Because of its lower prices and higher cost-effective, we can expected that it will have broad prospect in the future.In our study, we evaluate the inhibitory effects of pseudomonas eruginosa vaccine (PA-MSHA) on proliferation of human nasopharyngeal cancer cells5-8F and6-10B. Furthermore, the mechanism of PA-MSHA on the inhibiting proliferation of nasopharyngeal nasopharyngeal cancer cells was examined to help obtain more selections in the process of nasopharyngeal carcinoma’s adjuvant therapy.Materials and Methods1. In this experiment, the nasopharyngeal cancer cells5-8F and6-10B were cultured in vitro.Then they were treated with PA-MSHA at different concentrations and different times, and divided into control group and drug goups. Different concentrations of the drug treatment group were selected as0.02×108/ml,0.05×108/ml,0.1×108/ml,0.2×108/ml,0.5×108/ml,1×108/ml,2×108/ml,5×108/ml, processing time are1-7d.2. After the nasopharyngeal cancer cells5-8F and6-10B were treated with PA-MSHA at different concentrations and different times, MTT colorimetric assay was engineered to detect the inhibition ratio of cell proliferation in different goups.Then we calculated the cell survival rate, the half inhibitory concentration of48hours(48h IC5o) and drawn growing curves.Then select the appropriate drug concentration (1×108/ml and2×108/ml) according to the IC50and interaction with the two sets of cell lines1-7d, determinate the absorbing value every day, and drawn growing curves. Analysis time and concentration effect in cell proliferation.3. Cell cycle distribution induced by PA-MSHA was analyzed by flow cytometry in the nasopharyngeal cancer cells5-8F group which were treated with1×108/ml of PAMSHA for48h.Nasopharyngeal cancer cells5-8F were treated with1×108/ml of PAMSHA for48h, then PI staining, flow cytometry detected the cell cycle distribution, and analysised the difference between the percentage of the cell cycle with the control group on the PI fluorescence histogram. 4. The expression level of apoptosis-related and cell cycle-related proteins such as cyclinD1, CDK4, CDK6, Bax and Bcl-2of PA-MSHA treated groups were detected by western blot.5. Statistical analysis was done with SPSS16.0.ANOVA was used in comparison among different groups and t-test was used in cell cycle analysis.P<0.05was accepted as significance.ResultsThe main results and findings are as follows:1. In both cell lines of PA-MSHA treated groups, the proliferation activity was obviously decreased. The cytotoxic effect of PA-MSHA was time-dependent and concentration-dependent. The IC50of the nasopharyngeal cancer cells5-8F treated with PA-MSHA for48h was (1.62±0.31)×108/ml. The IC50of the nasopharyngeal cancer cells6-10B treated with PA-MSHA for48h was (1.95±0.35)×108/ml.Comprehensive analysis found that PA-MSHA significantly inhibited the proliferation of NPC cell lines5-8F, and the effect was time-dependen (F=665.403, P<0.001) and concentration-dependent (F=151.607, P<0.001). Similarly, PA-MSHA significantly inhibited the proliferation of NPC cell lines6-10B, and the effect was time-dependen (F=597.422, P<0.001) and concentration-dependent (F=74.481, P<0.001).2. FCM with PI staining showed that1×108/ml of PA-MSHA can induce the cell cycle arrest of nasopharyngeal cancer cells5-8F.And the percentage of G1phase was increased(t=5.150, P=0.007),the percentage of S phase were decreased(t=-6.820, P=0.002). The differences were statistically significant.3. This result was confirmed by detecting cell cycle-and apoptosis-related proteins.In this section,we treated the both nasopharyngeal cancer cells with1×108/ml and2×108/ml of PA-MSHA for24h and48h. According to the control group, the expression level of apoptosis promoted protein Bax was up-regulated, and the expression level of cyclinD1, CDK4, and CDK6was significantly up-regulated, however the anti-apoptosis protein Bcl-2was down-regulated.And it’s probably time and concentration-dependent.Conclusion1. Pseudomonas eruginosa vaccine (PA-MSHA) can significantly inhibit the cell proliferation of nasopharyngeal cancer cells5-8F and6-10B.2. PA-MSHA can induce the cell cycle arrest and apoptosis of nasopharyngeal cancer cells, and it is the possible mechanism of the inhibition of cell proliferation.3. PA-MSHA can be using as an adjuvant treatment approach in nasopharyngeal carcinoma. It’s worthy to be further clinical studied and researched. |
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