Establishment Of The Tumor Mouse Model Of CNE-1 And CNE-1/taxol And Its Biological Characters

Objectives:1.To establish tumor model in which subcutaneous xenograft were planted in nude mice using human nasopharyngeal carcinoma (NPC) cell lines, CNE-1 and CNE-1/taxol (taxol-resistant cell line), and investigated its biological character.2. To investigate the effects of taxol and DDP on tumor growth in transplanted CNE-1 and CNE-1/taxol tumors in nude mice, and analyze the difference between both CNE-1 and CNE-1/taxol tumors in vivo.3. To explore the role of Bcl-2, VEGF and TSP1 playing in the molecular mechanism of taxol-resistance and resistant reversal in nasopharyngeal carcinoma.Methods:1. Human NPC cell line CNE-1 and its taxol-resistant sub-line CNE-1/taxol were cultured in RPMI 1640 culture medium, supplemented with 14% calf serum,in 37℃,50% CO2 saturated humidity incubate box. Cells were collected after digestion using mixture of 0.25% trypsinase and 0.02% EDTA.2. CNE-1 and CNE-1/taxol cell suspension (1×106 cells,0.2ml) were subcutaneously injected into the armpit of nude mice. Thirty days later, tumor appeared in 50% of mice. Removed tumors out, and cut it into tumor pieces and respectively planted them into the left and right abdomen subcutaneous of 24 nude mice (CNE-1 tumor, left; CNE-1/taxol, right). Fourteen days after tumor engraftment,24 mice were randomly divided into 3 groups, and different treatments were designed as follows: Control group intraperitoneally injected with sterile saline, and taxol group with taxol 10mg/kg, and DDP group with DDP 5mg/kg respectively (0.2ml, once every day, for 5 days).3. Transplant tumor growth was regularly observed, and the volume of tumors was measured at day 0,2,4,6 after different treatments. The rates of growth and growth inhibition were determined by tumor growth curve.4. Two days after treatments, all mice were killed and the tumors were obtained from xenografts. The expression of Bcl-2, VEGF and TSP1 were detected by immunohistochemistry in tumor tissue.5. Data was expressed by the way of "Mean+SD", and statistically analyzed by using SPSS17.0 software. We defined statistical significance as P<0.05.Results:1. In the establishment of CNE-1 and CNE-1/taxol tumor models, there was a low tumor formation rate (50%) with long latency (30 days) using cell suspension inoculation and high tumor formation rate (100%) with shorter latency using tissue explant method.2. In control group, CNE-1 tumor grew faster than CNE-1/taxol in a week, and tumor growth fold was respectively 2.53±0.31 and 1.79±0.27 (P<0.05).3. In taxol group, tumor growth inhibited rate was, respectively, 67.14±13.03% in CNE-1 tumor and 35.84±35.3% in CNE-1/taxol. There was significantly difference between both tumor models (P<0.05).4. In DDP group, tumor growth inhibied rate was respectively 44.71±14.80% in CNE-1 tumor and 50.73±11.38% in CNE-1/taxol (n=8, P>0.05).5. The expression level of Bcl-2, VEGF and TSP1 was as follows: Expression of Bcl-2, VEGF and TSP1 was higher in CNE-1 tumor than that in CNE-1/taxol tumor in control-group; After 7days taxol treatment, their expression of three genes was significantly decreased in CNE-1 tumors, while VEGF expression was significantly increased and TSP1 expression was decreased and Bcl-2 was not changed in CNE-1/taxol tumor. After 7 days DDP treatment, there was no significant change of BCL-2, VEGF and TSP1 expression in CNE-1 tumors, while BCL-2 and TSP1 expression was significantly increased and VEGF expression was not changed in CNE-1/taxol. Conclusion:1. Tissue explant was the preferably way to establish the tumor model of CNE-1 and CNE-1/taxol cells.2. Both taxol and DDP could significantly suppress the growth of CNE-1 tumor, and they are effective drugs for nasopharygeal carcinoma.3. CNE-1/taxol kept the taxol-resistant phenotype after being transplanted in nude mice, which can be reversed by DDP in vivo.4. Higher growth rate in CNE-1 might be associated with higher expression of Bcl-2 and VEGF.5. Taxol resistant phenotype in NPC might relate to decreased TSP1 expression, and DDP could reverse the taxol resistant phenotype through upregulating the TSP1 expresion.