The role of thymosin beta 4 in actin cytoskeleton and mitochondrial regulation in human colon cancer cells

• Main Authors: Mei-Chuan Tang, 唐美娟
• Other Authors: Yeu Su
• Format: Others
• Language: en_US
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/87369260410444228640

博士 === 國立陽明大學 === 生物藥學研究所 === 99 === Thymosin β4 (Tβ4), the most abundant member of β-thymosin family, is regarded as the main G-actin sequestering peptide in mammalian cells. This 43-amino acid peptide forms a 1:1 complex with G-actin and thereby regulates F-actin polymerization by modulating the free actin pool. In addition, Tβ4, expressed ubiquitously and overexpressed in various tumors, also plays a key role in cell migration, tumor metastasis, angiogenesis, blood coagulation, anti-inflammation, apoptosis and wound healing. Colorectal carcinoma (CRC) is a highly fatal malignancy due mainly to its metastasis despite reduced tumor relapse might be achieved by surgery and combined chemotherapy. Moreover, currently available treatments do not greatly improve the prognosis of metastatic CRC (mCRC) patients which could be attributed at least in part to the development of drug resistance. In this regard, upregulation of Tβ4 has been demonstrated not only in the original tumors of majority of CRC patients but also their liver metastases, suggesting a critical role of this G-actin binding protein in promoting the malignant progression of CRC. Interestingly, downregulation of Tβ4 has been reported to reduce the tumorgenicity and even sensitize tumor cells to drug-induced apoptosis. Hence, Tβ4 might be a good therapeutic target for CRC, especially mCRC. CRC patients with higher Tβ4 expression have significantly shorter progression -free and overall survivals as well as the elevated incidence of subsequent distant metastasis. Hence, understanding the mechanism of cancer cell migration induced by Tβ4 may provide means to inhibit their metastasis. We demonstrated herein that Tβ4, ILK (integrin linked kinase) and IQGAP1 (IQ motif containing GTPase activating protein 1) form a complex to stimulate the migration of SW480 human colon cancer cells via a rapid actin cytoskeleton re-organization by maintaining higher Rac1 activities. To assess the influences of Tβ4 downregulation on the malignancy of colon cancer cells, recombinant adenoviruses carrying DNA fragments encoding two different Tβ4 shRNAs were generated and used to infect SW480 human colon cancer cells. Real-time RT-PCR analysis and immunoflourescent staining demonstrated effective knockdown of RNA and protein levels of Tβ4, respectively, after virus infection. Tβ4 knockdown triggered a detachment of SW480 cells whose microfilaments were severely disrupted and a reduction in migration ability of Tβ4-overexpressing SW480 cells whose ILK and IQGAP1 protein levels were simultaneously reduced. Protein levels of phosphor-cofilin (inactive form) and gelsolin, two main F-actin severing proteins, were reduced and increased, respectively, after Tβ4 downregulation. Rather than reducing the expression of cofilin or gelsolin or both in Tβ4-downregulating SW480 cells by their respective shRNAs, the changes of focal adhesion complex might result in dramatic decrease of floated cells which was associated with a restoration of F-actin levels. These findings suggest that Tβ4 increases the migration of colon cancer cells via activating Rac1 by elevating IQGAP1/ILK complexes. On the other hand, Tβ4 downregulation reduces IQGAP1/ILK protein levels as well as F-actin levels in similar cells. Tβ4 not only can be oxidized to sulfoxide by H2O2 directly but also increases the expression of anti-oxidative enzymes and the latter protects cells from oxidative damage. To assess the anti-oxidative role of endogenous Tβ4 in tumor cells, two recombinant adenoviruses expressing different Tβ4 shRNAs were used to infect SW480 cells. This treatment induced significant increases of ROS (reactive oxygen species) that lasted for 72 hrs in SW480 cells. Interestingly, some cristae-loss and several electron-dense mitochondria appeared in cells with Tβ4 knockdown which was accompanied by a marked decline of the membrane potential of these organelles. Strikingly, while the ATP and lactate levels in SW480 cells were notably elevated by Tβ4 downregulation, such treatment significantly diminished the mitochondrial DNA copy number and the protein levels of several subunits of the electron transport complexes. Finally, immnuofluorescent staining results suggested the presence of Tβ4 in mitochondria. To our best knowledge, this is the first report to demonstrate that Tβ4 stimulates the migration of human CRC cells by complexing with ILK and IQGAP1 which maintains the active Rac1. In the meantime, knockdown of this G-actin sequestering peptide disrupts the morphology and some crucial functions of mitochondria in similar tumor cells. However, more work is required to dissect the precise role of Tβ4 in mitochondria.