Effect Of Ionizing Radiation On Invasiveness Of Pulmonary Adenocarcinoma Cells And Its Mechanism

Radiotherapy is one of three therapy approaches for malignant tumor. With the developmentof radiation oncology and the popularity of radiotherapy devices, radiotherapy plays more andmore important role in tumor therapy. According to statistics, 60-70 percentages of tumorpatients were treated with radiotherapy at different stage of tumor for different purpose.Radiotherapy can inhibit the proliferation of tumor cells and induce apoptosis of tumor cellsthrough direct effect or oxygen radicals induced by ray. Consequently, the tumor is killed or thetumor growth is suppressed. Radiation cans inhibit the growth of most type of tumors.However, recently studies have shown that, besides the activation of p53 gene which resultsthe proliferation inhibition and apoptosis of tumor cells, radiation can also activate many otherradiation responsible genes which will subsequently activate the complex network of signaltransduction. This will result in the changes of cytokine secretion and protein expression in cells,and finally alter the microenvironment surrounded the tumor cells. After radiation, thebiological behavior of survived tumor cells, such as cell invasion and cell migration, will changecorrespondingly due to the alternation of gene expression in tumor cells and microenvironmentsurrounded the tumor cells. The changes of tumor invasion and migration will significantlyaffect the prognosis of tumor patients. Many studies have indicated that radiation can enhancethe invasion ability of pancreatic cancer cells, hepatoma carcinoma cells, breast cancer cells,melanoma cells and glioma cells through up regulating the expression of matrixmetalloproteinases (MMPs). The in vivo study in mice confirmed that radiation can promote themetastasis of lewis lung cancer. Moreover, it was reported that invasive local recurrenceincreased after radiation therapy for ductal carcinoma in situ. These studies suggested thatradiotherapy has the potentiality of promoting tumor cell invasion and migration.The incidence of lung cancer is highest among tumors. Radiotherapy is the main therapyapproach for lung cancer. However, the effect of radiation on invasion and migration of lungcancer cells remains unknown. In the current study, we investigated the effect of radiation oninvasion and migration of pulmonary adenocarcinoma cells (A549 cells) and explored themechanism involved in this process.Data are as follows: 1. The effects of radiation on biological behaviour of A549 cellsAfter the A549 cells were irradiated with different dose ofγray, the proliferation ability andviability of A549 cells was detected by WST-8 assay and colony formation assay. The apoptosisof A549 cells was detected using Annexin-Ⅴ/PI double staining after radiation. The adhesion ofcells was detected by cell adhesion assay. The migration of cells was examined by woundinghealing assay. The invasion of cells was detected by transwell invasion assay. The resultsindicated that:(1) The proliferation and viability of A549 cells were inhibited significantly after radiation with2-, 4, or 6-Gy ofγray. 6-Gy ofγray induced A549 cell apoptosis significantly. However, 2- or4-Gy of radiation could not induce apoptosis of A549 cells within 48 hours after treatment.(2) 2- or 4-Gy of radiation promoted the invasiveness and migration of A549 cells, but had noeffect on cell adhesion.2. The mechanism involved in radiation-induced invasion of A549 cells2.1 The effect of radiation on the expression and enzymic activity of MMP-2During the process of cell invasion, tumor cells must degrade the barrier of extracellularmatrix. This will need the participation of many kinds of enzymes, among which MMPs, whichcan degrade basement membrane and most component of ECM, play the most important roles.In the MMPs family, MMP-2 occupies key position during tumor invasion and migration.Moreover, it was reported that radiation can promote the expression of MMP-2 in many kinds oftumor cells.In the current part of study, the MMP-2 mRNA in A549 cells was detected usingsemiquantitative RT-PCT 12 hours after A549 cells were irradiated with 2- of 4-Gy ofγray. TheMMP-2 protein in A549 cells treated with or without irradiation was detected by Western blot.And the enzymic activity of MMP-2 was detected using zymography assay. The resultsindicated that 2- or 4-Gy of radiation promoted the transcription and protein expression ofMMP-2 in a dose dependent manner. At the same time, the enzymic activity of MMP-2 inculture supernatant increased significantly in a dose dependent manner. These results suggestedthat MMP-2 was involved in the radiation-induced invasion of A549 cells.2.2 The signal pathway regulating radiation-induced MMP-2 expressionSignal transducers and activators of transcription 3 (STAT3) belongs to STATs family. Ittransmits the signal of cytokine and growth factor into nucleus. STAT3 is the transcriptionfactor of many genes, including MMP-2,VEGF,Bcl-2. IL-6/JAK-2/STAT3 pathway is thetypical pathway for STAT3 activation. STAT3 can be phosphorylated on tyr705 by JAK-2. Two phosphorylated STAT3 molecules then form a dimmer and translocate into nucleus to regulatethe expression of target genes. Because the effect of radiation on MMP-2 expression occurs intranscriptional level, we subsequently examined the effect of radiation on STAT3.The expression of STAT3 and phosphorylated STAT3 was detected by Western blot. Thedistribution of STAT3 in A549 cells was further investigated using Western blot and indirectimmune fluorescence staining. The results indicated that 2- or 4-Gy of radiation increased thephosphorylation and nucleus localization of STAT3, although the expression of STAT3 remainedunchanged. After AG490, a specific inhibitor of JAK-2, was added in the culture of A549 cells,radiation-induced phosphorylation of STAT3 was suppressed, which implied that thephosphorylation of STAT3 induced bu radiation depended on JAK-2. Further study showed thatthe content of IL-6 in A549 cell cultures increased after 2- or 4-Gy of radiation. Blocking thephosphorylation of STAT3 using AG490 can abolish radiation-induced increase of MMP-2expression in A549 cells. Furthermore, radiation-induced invasion of A549 cells was alsoinhibited after A549 cells were treated with AG490. These results indicated that radiation canactivate IL-6/JAK-2/STAT3 pathway through up regulating the secretion of IL-6, whichpromoted the expression of MMP-2, and this in turn enhanced the invasion of A549 cells.Besides those, the current study also found that radiation-induced migration of A549 cells alsodepended on IL-6/JAK-2/STAT3 pathway.It was also found that radiation-induced activation of STAT3 did not occur in other type oftumor cells, including gastric adenocarcinoma, pancreatic cancer, hepatic carcinoma.3. The effect of radiation on other target gene of JAK-2-STAT3 pathwayBesides MMP-2, STAT3 can regulate the expression of other target genes, such as VEGF,Survivin,Bcl-2. The up regulation of these genes will inhibit the apoptosis of tumor cells andfacilitate angiogenesis, which was disadvantage for radiotherapy. In this section, the expressionsof these genes in A549 cells treated with or without radiation were detected.The results of Western blot and semiquantitative RT-PCR showed that 2- and 4-Gy ofradiation could promote the expression of mRNA and protein of VEGF. The expression ofVEGF induced by radiation was suppressed by AG490. Radiation had no effect on Bcl-2expression, but could up regulate the expression of Survivin and this effect depended on theactivation of STAT3. These results indicated that 2 and 4 Gy of radiation could result in theincrease of VEGF concentration in pulmonary adenocarcinoma, which would facilitate theangiogenesis in tumor. Moreover, the elevation of Survivin would protect the tumor formapoptosis induced by radiation. The innovation of the study included the following: the effects that the invasiveness andmigration of A549 cells could be enhanced by irradiation ofγ-ray were found, and themechanism involved in radiation-induced migration of A549 cells was further found to be thatradiation up regultaed the expression of MMP-2, which finally resulted the increased invasion ofA549 cells, through the activation of IL-6/JAK-2/STAT3 pathway.