The three cell lines used in this study (MU41, U87MG
and LN229) form functional invadopodia that degrade the
FITC-labelled gelatin which can be seen to co-localize with
rhodamine phalloidin-stained actin puncta (Fig.1a). The
Radiotherapy is a very effective treatment against many types of cancer, which involves the use of high-energy radiation to target and eradicate cancer cells (25). This process disrupts the ability to divide and proliferate by inducing breaks in the DNA strands. While some cells may naturally be resistant to radiation, this could be because of DNA repair mechanisms, protective microenvironments, or genetic changes (4, 25, 26, 27, 28). Despite its effectiveness, radiotherapy may have unanticipated negative effects because it paradoxically promotes the progression, migration, and invasion of cancer cells (5, 29). Exposure to ionizing radiation can trigger survival mechanisms that result in enhanced aggressiveness of cancer cells (5, 29). Numerous studies have been investigating the intricate relationship between the ionizing radiation and cytoskeletal proteins within various types of cancers, including CRC (30, 31).
FliI is a cytoskeletal protein that influences tumorigenesis and cancer progression. In breast cancer cells, elevated FliI levels have been associated with increased tumor invasiveness and metastasis (18, 32). Choi et al. (2020) found that FliI acts as an inhibitor of ER-stress induced cell apoptosis in CRC cells (33). By modulating cellular Ca2 + balance, FliI reduces the apoptotic responses triggered by ER stress. Furthermore, previous studies investigated the role of FliI in skin cancer, which regulate the cell motility and tissue invasion (19, 21). The present study aims to investigate the relationship between FliI expression and the resistance of HCT116 CRC cells to ionizing radiation. A significant increase in FliI expression was observed in irradiated HCT116 colorectal cancer cells. Then, the expression of FliI was significantly reduced through the transfection of siRNA targeting FliI. Suppression of FliI expression was shown to enhance the radiosensitivity of HCT116 cancer cells, with the highest SER at SF6 (2.06), with the value (WT and siControl) being close to the expected value from a previous study by Chen et al. (2010) (34). This result indicates that FliI plays a role in enhancing the radio-sensitization of HCT116 cells.
Previously, FliIs have been demonstrated to be involved in cell motility and tissue invasion (19, 21). The transwell migration and invasion assay indicates that ionizing radiation alone reduced the number of migrated and invaded cells, most probably due to the reduced cell viability caused by radiation itself. However, cancer cells that could resist the effects of ionizing radiation can become more invasive. As evident from the gelatin degradation assay analysis, the data obtained in this study indicate that irradiated HCT116 cells significantly increased invadopodia formation. These results are consistent with previous studies demonstrating a significant increase in invadopodia formation when glioma and glioblastoma cells were irradiated (35, 36, 37). Inhibition of FliI protein significantly inhibits the migration and invasion abilities of irradiated HCT116 cells. We found that reduced expression of FliI suppressed invasion by reducing the formation of invadopodia and reducing the ability of HCT116 cells to degrade gelatin.
Studies have shown that ionizing irradiation can enhance the invasiveness and metastatic potential of cancer cells through various mechanisms. For instance, irradiation-induced DNA damage and cellular stress responses can trigger signaling pathways involved in epithelial-mesenchymal transition (EMT), a biological process wherein epithelial cells undergo changes leading to a mesenchymal phenotype. This transition is characterized by the loss of cell adhesion and polarity, which allows cells to acquire enhanced migratory and invasive properties (38, 39). As cells lose their epithelial characteristics, epithelial markers like E-cadherin and cytokeratins are typically downregulated, while mesenchymal markers like N-cadherin, vimentin, and fibronectin are upregulated, reflecting the acquisition of a mesenchymal phenotype (40, 41). Numerous studies have provided evidence supporting the idea that ionizing radiation can induce changes promoting EMT in diverse cancer types. This includes instances observed in glioblastoma, lung cancer and CRC that showed a low level of epithelial markers and a high level of mesenchymal markers upon radiation exposure (42, 43, 44, 45). Additionally, ionizing irradiation promotes the expression of matrix metalloproteinases (MMPs), which enhance the invasive capability of cancer cells. Studies have shown increased MMP expression in irradiated CRC and breast cancer cells (13, 15). In this study, irradiated HCT116 cells were found to increase the invasiveness of the cells, as the percentage of cells forming invadopodia showed a significant increase. This is in line with the upregulation of FliI protein expression in irradiated HCT116 cells as a result of ionizing radiation, which enhances the invasive characteristics of HCT116 cells.
When invadopodia formation was reduced following FliI silencing, this situation led to an investigation of β-catenin, cortactin and MMP2 expression levels. β-Catenin signaling has induced metastasis in cancers such as breast cancer, hepatocellular carcinoma (HCC), and melanoma by facilitating metastatic spread through increased of cell migration and invasion (46, 47, 48). Furthermore, cortactin and MMP2 plays a crucial role in matrix degradation during cancer cell invasion, exhibit increased localization at invadopodia. (49, 50). The results indicated that a significant reduction in FliI protein levels has not significantly impacted β-catenin expression, although there was a slight reduction. Previous studies have shown that when FliI is silenced, β-catenin expression reduce significantly (21). This is because of the way FliI and β-catenin are controlled. The observed discrepancy could be caused by a variety of factors, such as differences in experimental conditions, the specific cell lines used, or the degree of FliI silencing achieved. These findings also shows that cortactin and MMP2 expression remains unchanged upon FliI silencing. This suggests that the regulation of cortactin and MMP2 may be FliI-independent, thus, cortactin and MMP2 are transported and targeted via different molecular mechanisms. In addition, this independent regulation might affect other types of MMPs, such as MMP9 and MMP14, which are also involved in invadopodia formation, particularly in HCT116 cells (49, 51, 52). Further research is necessary to elucidate another actin cytoskeleton related to EMT, such as E-cadherin and vimentin, to determine the specific conditions under which FliI influences these pathways. Furthermore, studies have shown that inhibiting Flii suppresses CRC proliferation, migration, and invasion in vitro. These findings suggest that FliI may be important in the development of new treatments that will make radiotherapy more effective and lessen the impact of aggressive metastatic CRC.