AN INVESTIGATION OF PI3K/AKT/mTOR DUAL INHIBITION AND LOW INTENSITY PULSED ULTRASOUND APPLICATION ON F-ACTIN AND AUTOPHAGY IN GLIOBLASTOMA MULTIFORME CANCER STEM CELLS
Project no. 20897 · Status: Kapanmış · 2019
Summary
Abstract Multiforme (GBM) is a biologically heterogeneous tumor that shows its aggressive effect despite early diagnosis. In this heterogeneous tumor, there are also cancer stem cells which has capability of self-reneweal and differantiation, can express typical stem cell surface markers such as CD133, CD44, stimulate cancer progression and cause cancer cells to gain treatment resistance. PI3K/AKT/mTOR signaling pathway what is responsible for the development of cellular events such as cell growth, survival, migration and carcinogenesis has been reported to be active in GBM and Glioblastoma Multiforme cancer stem cells (GBMKH/CD133 + cancer stem cells) and contribute to tumorigenicity. The cell survival mechanism autophagy and the cellular skeleton element F-actin which causes metastasis of cancer cells by mobilizing during epithelial mesenchymal transition (EMT) are regulated on PI3K/AKT/mTOR pathway. Voxtalisib (XL765, SAR245409), the dual inhibitor of this signaling pathway has been shown to reducing tumor development by increasing tumor cytotoxicity in patients with GBM,consequently, increased the survival rate associated with it. Low-intensity pulsed ultrasound (LIPUS), a non-invasive form of mechanical energy, is an ultrasound type that capable of affecting deep-lying tissues when compared to other mechanical stimulants. Due to low toxicity, deep penetration and high efficacy of LIPUS, there have been current studies that show LIPUS can be used for anti-tumor treatments. It has been reported that LIPUS therapy combined with the drug provides down-regulation of signal proteins that stimulate carcinogenesis and induces apoptosis and autophagy in cancer cells with its mechano-acoustic effect. However, the chemodynamic effect of LIPUS with Voxtalisib has not yet been studied, and there is no adequate study demonstrating F-actin and cell survival mechanisms in GBMKH as a result of Voxtalisib/LIPUS inhibition. In this study, it is aimed to reveal the possible effects of PI3K/AKT/mTOR inhibition with dual inhibitor Voxtalisib and LIPUS combination on F-actin cytoskeleton and cell survival mechanism autophagy. For these purposes, it is realized to isolation of glioblastoma multiforme cancer stem cells using fluorescent activated cell sorting (FACS) method. It has been examined the changes that were occured on F-actin and autophagy in GBM and GBMKH applying Voxtalisib, LIPUS and their dual effects. During the examination of these changes, the appropriate drug dose was determined and cell counts were made. Interpretation of F-actin and signal path inhibition was performed using immunofluorescence methods. Cell migrations were demonstrated with the standard wound healing assay, all parameters that examined in the experimental and control groups were scored and evaluated with the ImageJ programme. As a result, the application of Voxtalisib's increased doses of ligated LIPUS has been shown to inhibit mTOR and decrease viability in both cells. In addition, autophagy has been activated with mTOR inhibition, and in the event of this, it has been observed that LIPUS increases autophagy in GBM cells and GBM cancer stem cells advanced resistance for autophagy at 5 and 10 µM drug doses. Cell invasion has been demonstrated by phalloidin staining and wound healing stratching experiments, eventually it has been resulted that LIPUS negatively affects cell invasion depend on increasing doses. Our studies have shown that co-administration of LIPUS and Voxtalisib coadministration is effective in targeting cancer and cancer stem cells. This study can help development for new methods of treatment and expand the clinical use of LIPUS. Key Words: LIPUS, glioblastoma multiforme cancer stem cell, PI3K, F-aktin, XL765, Autophagy