Investigation of sonic hedgehog signaling pathway with cylopamin inhibition and relationship between related mechanisms with molecular methods on glioblastoma multiforme and glioblastoma multiforme cancer stem cells and astrocyte co-cultures

Cancer is a disease caused by uncontrolled cell division, in which cells have the ability to invade and metastase. During embryogenesis, controlled cell division, differentiation, migration, and programmed cell death occur, as well. Embryo development and tumor formation comprimise similar behaviours of cells like cell migration, invasion, differentiation, and also escaping from immune system. Moreover, these two cell types are similar in gene expression, protein profiling and signaling pathways. Among the members of the Hedgehog gene family, Sonic Hedgehog (Shh) has the most prominent effects on the embryonic development at the brain and extremities. The Sonic Hedgehog signaling pathway is one of the important signaling pathways that active in both embryogenesis and carcinogenesis. Many cancer types, such as medulloblastoma, rhabdomyosarcoma, colorectal carcinoma, have increased activity of this pathway. Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adult humans and is a rapidly growing, infiltrating tumor, even if it does not metastase to the distant organs. Tumors are composed of a heterogeneous group of cells. Tumor stem cells, like stem cells, are characterized by self-renewal and differentiation and resistant to treatment regimens such as radiotherapy and chemotherapy besides, they can spread distant organs and are thought to be affected by cancer recurrence also found in tumor mass. Cancer stem cells have also been identified in tumor masses of patients with Glioblastoma multiforme, and these cells may cause worsening prognosis. In recent years, Hedgehog signaling pathway has become an important target for Glioblastoma multiforme treatment. Cyclopamine is an important agent used in the inhibition of the Shh signaling pathway by binding to the Smoothened (Smo) receptor and has shown to inhibit tumor growth in gliomas, pancreatic and colon carcinomas in animal studies. Various co-culture and microenvironmental researches related to GBM are not yet sufficient. Based on the literature, it is thought that the Sonic Hedgehog signaling pathway plays an important role in the embryonic period, is also active in cancer and cancer stem cells, and may also be effective in treatment resistance. In the context of literature information, we prospectively investigated the effect of Shh signaling pathway and its inhibition on GBM and GBM cancer stem cells cocultured with astrocytes. Cell-cell interactions in the co-culture conditions were evaluated to determine whether the Shh signaling pathway active in GBM and GBM cancer stem cells, will undergo a change in the inhibition effect of cyclopamine in the presence of astrocytes. At the same time, the ability of the cells to secrete Shh protein as paracrine signalling, and cell-cell interactions were expected to answer the question whether Shh signaling pathway has a positive or negative effect on cyclopamine inhibition. As a result, when the Shh signaling pathway was inhibited in GBM and GBM cancer stem cells, the differential changes in intracellular mechanisms such as apoptosis and cell cycle were examined by PCR, immunofluorescence, ELISA, TUNEL and cell cycle analysis. Although cyclopamine is an effective agent to suppress the Shh pathway, it does not cause sufficient cell death alone. Even so the presence of Shh pathway activation in GBM and GBM KaKH revealed the need to suppress Shh pathway in combination cancer therapies. Results of GBM and GBM KaKH control groups and cyclopamine effect on them are different at RNA and protein levels. Different results were often obtained when co-culture with these cells was obtained. As a result of this study, especially when the differences in single cell culture and co-culture results are considered, the importance of micro-environment effect in diseases and treatments has been revealed.