Effects Of Tyrosine Kinase Etk/BMX On The Radiosensitivity Of Nasopharyngeal Carcinoma

Background:Nasopharyngeal carcinoma(NPC) is one of the most common malignant tumors in south China, there is apparent race, family and district assembling in NPC disease incidence. It is estimated that about 80% of NPC in the world took palce in China, most of which is in Guangdong province and perimeter area. NPC holds the first place in cephalo-trachelian tumor, approximately 78.08% in the malignant tumors. And the age of NPC patient is younger than that of other tumor patient, mainly in 30-50 age range. The incidence of female is not as high as that of male,about 1:3 in proportion. The cure rate of NPC is high to 70% in the earlier period , But about 8%-10% of 5-year-survival rate in advanced stage of NPC. It has become one of the top ten life-threatening malignant tumors in China.The great majority of NPC is poorly differentiated squamous-cell carcinoma, and much of which is temperate sensitive to radiation , while their proximal structure has a higher tolerance to radioactive ray. So the major treatment to NPC nowadays is radiotherapy combined with chemotherapy. But there are different radiosensitivity existing in different patient so as to different therapeutic efficacy. Apoptosis induced by radiation is one of the important factors. Since apoptosis has been found by Kerry in 1972, more apoptosis controlling gene such as bcl-2, p53 et al have been known by humans. In a word, people have been found out the apoptosis mechanism of NPC more and more, especially the apoptosis controlling gene.The endothelial/epithelial tyrosine kinase(Etk/BMX) is one of the non-receptor phosphate tyrosine kinase families of Btk. It was found in prostatic carcinoma cells, and identified in human bone marrow cell, so it is also called bone marrow X kinase(BMX). It is located in chromosomal band Xp22.2 between the DXS197 and DXS207 loci. BMX cDNA consists of a long open reading frame of 675 amino acids, containing a structure resembling the Btk family kinase pleckstrin homology domain (PH), an SH3(Src homology)domain, an SH2(Src homology 2)domain, and a catalytickinase domain.Foreign scientist in prostatic carcinoma and breast cancer research have been found that: Etk/BMX played an important role in carcinoma cells from X ray irradiation. Interestingly, it is found that Etk expresses abnormally in nasopharyngeal carcinoma, so we use the transfeced cell line sune-wt highly expressing Etk to study the relationship between the tyrosine kinases Etk and radiosensitivity in vivo and vitro in nasopharyngeal carcinoma (NPC).Objective:To study the relationship between the tyrosine kinases Etk/BMX and radiosensitiveityin nasopharyngeal carcinoma (NPC).Method :By using Lipofectamine2000 (Invitrogen) following the manufacturer's instruction,recombinant plasmid pcDNA3.0-Etk are transfected into sune-1, and Etk is highly expressed in the transfected cell line sune-wt. After 0, 2, 4, 10, 15Gy X ray radiation, MTT is used to measure survival fraction(SF), and flow cytometer (FCM) assay to examine cell apoptosis rate, cell cycle, proliferation index(PI), and apoptosis-related protein p53, bcl-2, bcl-xl and bak. sune-1, sune-wt and sune-vector are injectd subcutaneously(s.c) into nude mice (BALB/c,nu/nu,4-6 weeks, 18g). Animalsare randomized for therapy in 21 days (n=7 in each group). Tumor volume are determined three times weeks by direct measurement with sliding caliper and calculated by the formula volume V=(4/3)×π×R₁²Ã—R₂, where R₁ is radius 1 and R₂ is radius 2 and R₁2. Radiation was delivered on consecutive 5 days using a linear accelerator as described previously to tumor starting on day 0 when shielding the other body parts (5Gy×5). Dates are presented as mean(±SD or SE). Factorial analysis, Independent samples t test, One-Way ANOVA and ANOVA for repeated measurement are used . The threshold of statistical significance is set at P<0.05. Statistics and fittings of experimental curves are performed with the program SPSS10.0 ( help from the department of Statistics in Southern Medical University, GuangZhou, China).Results:1. we succeeded in getting the new cell line sune-wt highly expressing Etk: FCM was used to measure the expression lever of Etk between pre-transfection and post-transfection and the results were 4.6±1.45%, 75.5±2.34% respectively.2. The SF of sune-wt was higher than that of sune-1 and sune-vector: after treated at dosage of 2Gy, 4Gy, 10 Gy and 15Gy radiation the SF of sune-wt was higher than that of sune-1 and sune-vector. There was no significance in value of SF between sune-1 and sune-vector.3. The rate of apoptosis of sune-wt were lower obviously than sune-1 and sune-vector: After 2Gy, 4Gy, 10 Gy and 15Gy radiation, the rate of apoptosis was increased with the increasing radiation. And at the same dosage of radiation, sune-wt was lower sensitive than sune-1 and sune-vector. But there was not significant between sune-1 and sune-vector.4. After X radiation, the expression of p53 was decreased and bcl-2 increased in sune-wt, but there was no significant difference in bcl-xl and bak.5. After 0Gy, 4Gy, 10Gy X-ray radiation, there were not significance in cell cycle of three cell lines all in Statistics. After 15 Gy irradiation dosage, there was S,G2 phase blockage and highly up-regulated of proliferation index in sune-wt.6. The rate of sune-wt succeeded in forming tumor was 87.5±% in once, but sune-1 was 50%, and sune-vector was 40%. The increased expression of Etk in tumors contributedto be more radiation resistant than sune-1 and sune-vector and obviously increasedthe ability of cells to form tumors.Conclusion:Etk/BMX played an important role in protecting nasopharyngeal carcinoma cells from X-ray irradiation. Its mechanics may ralated to p53 and bcl-2 changing and S, G2 phase blockage. In vivo the study also suggested that the increased expression of Etk in tumors contributed to be more tumorigenic and more radiation resistant than sune-1 and sune-vector.