The Establish Of Radioresistant Subline Of Nasopharyngeal Carcinoma Cell Line CNE-2R Model And The Preliminary Study Of Its Chemotherapy Sensitivity

NPC (nasopharyngeal carcinoma, NPC), also known as "Guang don g cancer" is one of the top ten cancer [1], the World Health Organization ro ugh statistics that there are about 80% of nasopharyngeal carcinoma in China all over the world. NPC is in the top of the head and neck cancer incidence, in south China, especially Guangdong and Guangxi region is the highest incidence (30-80/10 million), give people a tremendous threat for the health .The happen of nasopharyngeal carcinoma is closely related to factors such as genetic, EB virus, environmental pollution .Due to the nasopharynx in the deep base of the skull, the surgery is very difficult, so the combined therapy dominated by radiation therapy (including radiotherapy,concurent chemoradiotherapy (concurrent chemoradiotherapy, CCRT), neoadjuvant chemotherapy (neoadjuvant chemotherapy , NAC)+ radiotherapy and radiotherapy + adjuvant chemotherapy (adjuvant chemotherapy, AC), etc.) is the main treatment for nasopharyngeal carcinoma. In newly diagnosed patients, patients with locally advanced and the late region is about 70%. These patients in clinical T1 + T2 a nd T3 + T4 who received conventional radiotherapy ,the local control rate of that was 64% to 95% and 44% to 68% ^{[2, 3]}, the main reason for treatment failure is local recurrence or distant metastasis, local recurrence rate was about 8% to 20%, regional recurrence rate was about 10% to 21% [4]. In recent years, with the rise of treatment modalities such as IMRT (Intensity-modulated radiation therapy, IMRT) and concurrent chemotherapy to carry out, the 5-year survival rate of the distant metastasis naso pharyngeal carcinoma have achieved a new breakthrough, more than 80% [5-9].In particularly, the meta-analysis have showed chemotherapy had made the 5-year disease-free survival of no metastatic naso pharynxgeal cancer increased by 10%, 5-year survival rate increased 6%, HR value was 0.86 [10]. The position of chemotherapy in the treatment of nasopharyngeal carcinoma has become increasingly prominent. The radiation and chemotherapy is still the main treatment for the recurrence and metastasis after radical cure, however, poor treatment, median survival time after systemic chemotherapy was only 10-20 months [11-13].In summary, the relapse and metastasis after radical radiotherapy and chemotherapy is still the major cause of the treatment failure of locally advanced nasopharyngeal carcinoma, and the tumor cells survive the constantly mutate may be the source of recurrence, then the residual tumor cells induce resistance to radiation [14]. What is the molecular basis of the radiation resistance for these residual cells? How to overcome these remaining cells'resistant to radiation? So many problems have yet to resolve. Although chemotherapy in NPC, especially in the treatment of locally advanced nasopharyngeal carcinoma have becoming more and m ore attention, but the combination of chemotherapy and how effectively,what is the best comprehensive treatment strategy? That has become a hot research field of nasopharyngeal carcinoma, and caused widespread controversy academia. Why the difference between effect of chemotherapy in the patients with Recurrence or metastasis of nasopharyngeal carcinoma after radical radiotherapy and chemotherapy, whether the remaining cells have got the radiation resistance, at the same time produced a multi drug resistance (multidrug resistance, MDR)?Therefore, in order to solve these problems, this project is to apply theγ-ray to the poorly differentiated nasopharyngeal carcinoma cell CNE-2 with the largesplit interval of radiation, to induce the resistance cell line CNE-2R, to take the further study of the relationship between the radiation resistance of nasopharyngeal carcinoma and chemosensitivety. 【objective】To establish a stable radiation-induced cell model of radiationresistance through irradiating the NPC cell line CNE-2 that is relatively sensitive to radiation, so as to provide a good contrast models for the study of the radiation resistance mechanisms and the relationship between radiation resistance and multidrug resistance.【methods】1.To irradiate the nasopharyngeal carcinoma cell line CNE-2 that is relative sensitivity for X-ray ,with intermittent high-doseγ-irradiation (4 Gy/f, 15f, total 60Gy), culture each cell after exposure , take the next exposure to cells that survival still after 3-4 weeks and entered the exponential growth phase, the exposure and training process lasted 12 months, the resulting progeny cells was called CNE-2R (CNE-2 radiation induction ). 2.Use colony formation assay and the linear quadratic model to fit the dose-survival curve of CNE-2R and CNE-2 cells and calculate the radiation biology parameters, to detect its radiation resistant. Culture cells for 8 days in a row and draw the cell growth curve of CNE-2 and CNE-2R. To detect the change of the cell cycle of CNE-2R and its parent cell CNE-2 after irradiation by flow cytometry.3.While the two cells been frozen for three months and passaged 10 times,carry colony formation experiments again and refit dose survival curve, to test the stability of the radiation resistance.【results】1.Theα/βvalues of the CNE-2R generated from intermittent high dose irradiation and its parent cell CNE-2 were 3.947±0.314 and 22.3 33±4.786 (P <0.05), SF2 values were 0.609±0.018 and 0.260±0.012 ( P <0.05).2.The doubling time of CNE-2 cell is 24.8h, CNE-2R is 40.67h. The cycle distribution of CNE-2 cell was that G0/G1 phase was 59.4%±1.00, S phase was 29.1%±1.23, G2/M phase was 12.7%±0.80; the cell cycle distribution of CNE-2R was that G0/G1 phase was 69.6%±1.96, S phase was 23.6%±1.91, G2 of 6.7%±0.78. The exponential doubling time of CNE-2 was 40.6%, CNE-2R was 30.4%. After 12h of 4Gy irradiation , S phase of CNE-2 have increased significantly (P<0.05), after 24h the G2/M phase cells increased significantly (P<0.05), after 48h returned to normal state. CNE-2R cell cycle is not change significantly over time.3.After the two cells cryopreserved 3 months and continued passage 10 times,α/βvalues of CNE-2R and CNE-2 cells were 4.049±1.122 and 21.637±1.203 (P<0.05), SF2 values were 0.605±0.055 and 0.226±0.008 (P<0.05).【conclusions】1.Compared to parental cells CNE-2,α/βvalue of the CNE-2R induced with intermittent high-dose radiation was significantly smaller, SF2 was significantly increased, showing a significant resistance to radiation.2.Cell cycle analysis results indicate that the proportion of CNE -2R strain on proliferation was less than the CNE-2, the doubling time was longer than the CNE-2 cell lines. After irradiation by a single 4Gy, CNE-2 cells showed a redistibution phenomenon,the irradiation has little effect on cell cycle distribution of CNE-2R cells.3.After frozen 3 months and continued passageing 10 times,the CNE-2R line successfully maintained its resistance to radiation. 【objective】The study was to research the relationship between radiation resistance and chemosensitivity of nasopharyngeal carcinoma CNE-2R cell lines ,to explore changes in sensitivity to chemotherapy of the CNE-2 cells before and after hypofractionated radiationinduced, whether produce multidrug resistance.【methods】Take NPC radiation resistance cell lines CNE-2R induced in the early experiments as the experimental group,the parental cell line CNE-2 as the control group, use MTT assay to detect killing rate of the two cells from cisplatin, 5-fluorouracil, carboplatin, paclitaxel , gemcitabine, doxorubicin and other drugs,and find the half inhibitory concentration, then calculate the relative resistance index of the resistance cell line CNE-2R; take the same concentration of cisplatin and 5-fluorouracil to act on the two sets of cells, 48 hours treatment ,use flow cytometry to detect the apoptosis. 【results】1.MTT assay to detect cytotoxicity resultsThe resistance index of the NPC radioresistance cell line CNE-2R induced by intermittent high-dose radiation to cisplatin, 5-fluorouracil, carboplatin, paclitaxel,gemcitabine,doxorubicin were 3.26±0.31,3.65±0.09,10.04±1.34, 1.51±0.03,8.95±0.20,3.16±0.39,compared with CNE-2 cells,half inhibitory concentr ation IC50 were significantly incresed the differences were statistically significant (P <0.05).2.The apoptosis induced by cisplatin or 5-fluorouracilThe apoptosis of CNE-2 and CNE-2R cultured in normal after 48h were 5.827%±0.033 and 6.297%±0.045, the difference was not statistically significant.The apoptosis of CNE-2 and CNE-2R cultured in 10μg/ml 5-Fu after 48h were 31.735%±2.529 and 16.18%±1.281, the difference was significant (P<0.05). The apoptosis of CNE-2 and CNE -2R cultured in 1μg/ml DDP after 48h were 56.98%±3.738 and 36. 897%±3.290, the difference was significant (P <0.05).【conclusions】In this study, NPC radiation resistance cell lines CNE -2R induced in the early experiments, have produced radiation resistant, also have got multidrug resistance.