X-ray Induces Non-Small Cell Lung Cancer Apoptosis By Upregulating Axin Expression

IntroductionNon-small cell lung cancer(NSCLC) accounts for about 80%of lung cancer,and one of the reasons for unavailable breakthroughs in its treatment is the general hyposensitivity of NSCLC to radiation,which affects the application and therapeutic effects of radiation therapy.However,we presume that relatively sensitive patients may exist in generally hyposensitive patients.Judge the sensitivity of NSCLC to X-ray and enhance the radiosensitity of patients,and thus facilitate us to formulate personalized radiotherapy program for patients that show different sensitivity will undoubtedly play important roles in the improvement in therapeutic effects of radiotherapy on NSCLC.The Wnt pathway has critical roles in the embryonic development stem cell maintenance of animals from hydra to human.The key of this signaling cascade is the regulation of the level of cytoplasmicβ-catenin.Axin is the most important negative regulator in the pathway.In the absence of Wnt signal,β-catenin is assembled to a multiprotein destruction complex typically consisting of Axin,APC,GSK3βand CKIa, and targeted for degradation through the proteasomal machinery,thereby maintaining the cytoplamicβ-catenin at exceedingly low level to preventβ-catenin accumulating in the cytoplasm and translating into nucleus,where it complexes with TCF family of transcription factors and induces cell proliferation.Axin can directly or indirectly bind to and phosphorylate p53,and knockout of Axin can significantly inhibit the cell death. The expression of Axin that has intense apoptosis-inducing functions is significantly down-regulated in non-small cell lung cancer(NSCLC).This study attempts to use X-ray exposure to increase the expression of Axin in NSCLC,and discuss the functions of X-ray in promoting the apoptosis of NSCLC with an aim to judge whether NSCLC is sensitive to X-ray exposure and increase the radiosensitivity of NSCLC to X-ray.Methods 1 PatientsThe paraffin blocks of 95 cases NSCLC tissues and 20 cases of normal lung tissues that were isolated during operations in the First Affiliated Hospital of China Medical University from February 1998 to May 2002 were collected.The calculation for the survival time was counted from the date of operation to the date of death due to recurrence/metastasis or the deadline of follow-up.Of them 72 cases were male and 23 cases were female,and the median age was 58 year(range from 33 to 76 years). According to the TNM classification of International Union Against Cancer(2002),the stage of patients was 40 stageâ… ,18 stageâ…¡,35 stageâ…¢and 2 stageâ…£.Pathological type contained squamous carcinoma in 45,adenocarcinoma in 40 and other types in 10. Differentiation degreed contained well-differentiation in 29,moderate-differentiation in 24 and poor-differentiation in 42.2 Immunohistochemical staining and TUNELImmunohistochemical staining was performed on 4-μm thick tissue sequential sections by Streptavidin-Peroxidase systerm to detect the expression of Axin and p53 in NSCLC and normal lung tissue.Apoptosis in tissues were detected by TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) method using the In Situ Cell Death Detection Kit,with fluorescein,according to manufacturer's(Roche Diagnostics GmbH,Mannheim,Germany) protocol.The immunostained slides were reviewed independently by 2 pathologists.A section was considered negative or positive according to the positive staining rate as described previously:＜40%negative and≥40%positive for Axin;＜5%negative and≥5%positive for p53.3 X-ray exposureFreshly isolated NSCLC tissues during operation were cut into 4mm×4mm×1mm block immediately and maintained in DMEM aseptic medium with 10%fetal calf serum at 37℃.The cancer tissues were exposed to 1 Gy irradiation with 6 MeV X-ray at 37℃using a linear accelerator.The exposed tissues were maintained in medium for 5 h at 37℃in a 5%CO₂ incubator and then harvested for 5 hours.Then half of the tissue was used to prepare for Western blot and RT-PCR to detect the expression of Axin and caspase-3,the other half of the tissue was used to prepare for immunohistochemistry to detect the expression of Axin.The unexposed tissues were used as control.4 Cell culture,transfeetion and X-ray exposureHuman lung adenocarcinoma cells A549 with wild-type p53 and BE1 with p53 mutation were transfected with Axin and AxinΔp53ΔHIPK2.After transfection,the medium was replaced with media containing JNK inhibitor and p53 inhibitor for further culturing followed by exposing to X-ray.After exposure,the cells were assayed by AnnexinV and propidium iodide(PI) staining and detected by flow cytometry then analyzed with Modftit LT V3.0 software.The unexposed cells and transfected with empty plasmid cells were used as control.5 Western blot analysis and RT-PCRWestern blot analysis and RT-PCR were used to detect the change of Axin and the activation of caspase-3 in tissues and A549 and BE1 cells exposed to X-ray.The unexposed tissues and cells were used as control.6 ImmunoflourescenceImmunoflourescence staining was used to detect p-JNK expression in A549 and BE1 cells exposed to X-ray.The unexposed cells were used as control.ResultsWe found that the positive rate of Axin in NSCLC tissues was 45.26%(43/95), which was significantly lower than that of the normal lung tissues.Axin expression was directly correlated with the differentiation of NSCLC(P=0.012),and it was related to TNM staging and lymph node metastasis(P＜0.05) and negatively correlated with the expression of p53(mt)(P=0.000),and it was directly correlated with apoptosis (P=0.002).The prognosis of patients with high expression of Axin was significantly better than those with low expression.X-ray can induce the increased expression of Axin in most of the lung cancer tissues from operational excision(8/15),and caspase-3 expression in the lung cancer tissues with increased Axin expression was significantly higher than those without increased Axin expression(P＜0.05).Both Axin and X-ray showed functions in inducing the apoptosis of A549 cells and BE1 cells,but X-ray can significantly increase the apoptotic rate of cancer cells with high Axin expression,and this function can be significantly blocked by p53 or JNK inhibitors. ConclusionAxin is positively correlated to the apoptosis of NSCLC,and the prognosis in patients with high Axin expression was significantly better than that of patients with low expression.X-ray exposure can induce some of NSCLC to up-regulate the expression of Axin and promote the apoptosis of lung cancer cells with high Axin expression,and the mechanism is mainly realized by p53 and JNK pathways.The increase in the expression of Axin after X-ray exposure is probably an important reference index for the judgment on the sensitivity of NSCLC to X-ray.