Ionizing Radiation Promotes Transforming Growth Factor-beta-mediated Epithelial-mesenchymal Transition Of Caner Cells

Radiotherapy is an important means of cancer treatment currently, but the emergence of metastases seriously influences the curative effect of radiotherapy. Whether radiotherapy can directly induce the metastasis of cancer cells is still unsettled, so a key element in the process of tumor metastasis is target at investigating the relationship between ionizing radiation (IR) and tumor metastasis, which will provide some new ideas for understanding the mechanism of tumor metastasis after radiotherapy and optimize the clinical radiation strategy.Some researches indicate that epithelial-mesenchymal transition (EMT) is one of the important mechanisms of tumor metastasis. Cancer cells can acquire the capabilities of migration and invasion by EMT stimulated by some extracellular signals, such as cytokines and growth factors, and then form distant metastases. Transforming growth factor-beta (TGF-β) is one of the important factors that could induce EMT in cancer cells. Notably, some clinical and experimental studies showed that IR could activate TGF-βin cancer cells and surrounding tissues, and induce inflammation, fibrosis, even tumor metastasis and recurrence. Therefore, investigating the effect of ionizing radiation on EMT and the role of TGF-βin this process will provide some important evidence to clarify the relationship between ionizing radiation and tumor metastasis. Here, six kinds of cancer cell lines with different metastatic potential and originated from different human organs were exposed to IR, and the effects of IR on EMT were evaluated by morphology, EMT markers and functional indexes. Based on the expression of TGF-βin cancer cells after irradiation, we investigated the role of TGF-βand its downstream pathways on IR induced-EMT in cancer cells by using specific pathway inhibitor.Methods:1. The human colorectal adenocarcinoma cell lines SW480 and SW620, human breast adenocarcinoma cell lines MCF-7 and MDA-MB-231, human lung adenocarcinoma cell line A549 and PC14 were irradiated by ⁶⁰Coγray at the total dose of 2 Gy, then the changes associated with EMT, including morphology, EMT markers, migration and invasion were observed by microscope, western blotting, immunofluorescence, scratch assay and transwell chamber assay respectively.2. ELISA was used to detect the expression of TGF-βin six cancer cell lines after the irradiation byγray at the total dose of 2 Gy.3. Western blotting was used to test the effect of IR on the expression of p-Smad2/3 in A549. Then the role of TGF-β/Smad signaling in IR-induced EMT was investigated through using the specific inhibitor of ALK-5, SB 431542.4. The radioresistance of IR-induced cancer cells was evaluated by clone formation assay.5. The expression of p-Akt in A549 was test after irradiation ofγray at the total dose of 2 Gy. Then the specific inhibitor of PI3K/Akt, LY294002 was used to investigate the role of Akt pathway in IR-induced EMT, and study the relationship between TGF-βand Akt combined with SB431542.Results:1. After the irradiation byγray at the total dose of 2 Gy, six cancer cell lines presented the mesenchymal phenotype, and compared with sham irradiation group, the expressions of epithelial markers E-Cadherin and Cytokeratin were descended and mesenchymal markers N-Cadherin and Vimentin were enhanced, the areas of scratch wound healing were enlarged and the numbers of the cells transferred to the lower surface in transwell chamber were increased. The results suggested that IR could promote EMT of cancer cells.2. The levels of TGF-βin all of the six cancer lines were enhanced following the irradiation byγray at the total dose of 2 Gy.3. Following the irradiation byγray at the total dose of 2 Gy, the expression of p-Smad 2/3 in A549 was increased. After the treatment of SB431542, a specific inhibitor of ALK-5, the p-Smad 2/3 level felled back, epithelial marker E-Cadherin was raised, mesenchymal marker N-Cadherin was decreased, the healing areas and the numbers of the cells transferred to the transwell chamber lower surface were also reduced. The results suggested that TGF-β/Smad put some effects on the process of IR-induced EMT of cancer cells.4. The survival rates of IR-induced A549 cell were enhanced following irradiation compared with control cells, which indicated the effects induceded by IR could strengthen radioresistance of cancer cells. 5. Following the irradiation byγray at the total dose of 2 Gy, the expression of p-Akt in A549 was increased. After the treatment of LY 294002, a specific inhibitor of PI3K/Akt pathway, compared with the IR group, the p-Akt level was felled back, epithelial marker E-Cadherin was raised, mesenchymal marker N-Cadherin was decreased, the healing areas and the numbers of the cells transferred to the transwell chamber lower surface were also reduced. The results suggested that Akt put some effects on the process of IR-induced EMT of cancer cells. The futher studies show that, the expression of p-Smad 2/3 and p-Akt in irradiated A549 cells were both descended through the treatment of SB431542, while p-Akt was decreased and p-Smad 2/3 was increased by the action of LY 294002. The results indicated that the effects of TGF-βin IR-induced EMT of cancer cells could be transited by the activation of Akt pathway.Conclusion:Ionizing radiation can promote EMT of cancer cells, the Smad signaling and Akt pathway stimulated by TGF-βplayed important roles in this process.