Investigation of low and high dose rate X-ray effects on histopathological changes and prognostic importance of Ki-67 in laryngeal cancer radiotherapy.

The aim of this study is to analyze the effect on histopathological changes and Ki-67 expression levels of Flattening Filter (FF) and Flattening Filter Free (FFF) beams to investigate the radiobiological mechanisms underlying laryngeal cancer (LCa) post-radiotherapy (RT) on mice models. Forty adult NOD SCID gamma (NSG) mice models were randomly divided into four groups; the sham, LCa, FF-RT and FFF-RT groups. The head and neck region of mice in FF-RT and FFF-RT groups (LCa plus RT groups) were irradiated with a single dose of 18 Gy at 400 MU/min and 1400 MU/min. The NSG mice received radiotherapy 30 days after tumor transplantation and sacrificed 2 days after radiotherapy for analysis of histopathology parameters and K-67 expression levels. Comparing the LCa, FF-RT and FFF-RT groups with the sham group, statistically significant differences were observed in histopathological parameters depending on tumor tissue and dose rate (p < 0.05). When the histopathological effects of FF-RT beam on LCa tissue were compared with FFF-RT beam, it was observed that statistically significant differences occurred (p < 0.05). Comparing the LCa group with the sham group, it was observed that the Ki-67 level affected significantly depending on the development of cancer (p < 0.01). It was concluded that FF and FFF beams caused significant changes in the histopathological parameters and Ki-67 expression levels. When the effects of FFF beam on Ki-67 levels, cell nucleus and cytoplasmic findings were compared with FF beam, significant radiobiological differences were observed.

Development and characterization of cancer stem cell-based tumoroids as an osteosarcoma model.

Three-dimensional (3D) cancer tumor models are becoming vital approaches for high-throughput drug screening, drug targeting, development of novel theranostic systems, and personalized medicine. Yet, it is becoming more evident that the tumor progression and metastasis is fueled by a subpopulation of stem-like cells within the tumor that are also called cancer stem cells (CSCs). This study aimed to develop a tumoroid model using CSCs. For this purpose CD133+ cells were isolated from SaOS-2 osteosarcoma cell line with magnetic-activated cell sorting. To evaluate tumoroid formation ability, the cells were incubated in different cell numbers in agar gels produced by 3D Petri Dish® method. Subsequently, CD133+ cells and CD133- cells were co-cultured to investigate CD133+ cell localization in tumoroids. The characterization of tumoroids was performed using Live&Dead staining, immunohistochemistry, and quantitative polymerase chain reaction analysis. The results showed that, CD133+ , CD133- and SaOS-2 cells were all able to form 3D tumoroids regardless of the initial cell number, but, while 72 hr were needed for CD133+ cells to self-assemble, 24 hr were enough for CD133- and SaOS-2 cells. CD133+ cells were located within tumoroids randomly with high cell viability. Finally, when compared to two-dimensional (2D) cultures, there were 5.88, 4.14, 6.95, and 1.68-fold higher messenger RNA expressions for Sox2, OCT3/4, Nanog, and Nestin, respectively, in CD133+ cells that were cultured within 3D tumoroids, showing longer maintenance of stem cell phenotype in 3D, that can allow more relevant screening and targeting efficiency in pharmaceutical testing. It was concluded that CSC-based tumoroids are propitious as 3D tumor models to fill the gap between conventional 2D in vitro culture and in vivo animal experiments for cancer research.

Blocking the Cleavage of Filamin A by Calpain Inhibitor Decreases Tumor Cell Growth.

BACKGROUND/AIM: Filamin A (FLNA) is the most abundant and widely expressed isoform of filamin in human tissues. It is cleaved by calpain at the hinge 1 and 2 domains, producing a 90-kDa carboxyl-terminal fragment (FLNACT). Recently, it has been shown that FLNACT mediates cell signaling and transports transcription factors into the cell nucleus. However, the significance of cleavage of FLNA by calpain has not been studied in cancer cell growth. Calpeptin is a chemical inhibitor of both calpain 1 and 2 that cleaves FLNA. In this study, we questioned if inhibiting calpain using calpeptin would decrease tumor cell proliferation, migration, invasion, and colony formation. MATERIALS AND METHODS: Human melanoma (A7), prostate cancer (PC3), mouse fibrosarcoma (T241) and endothelial (MS1) cells were assayed for proliferation, migration, invasion and colony formation after treatment with calpeptin. Cell lysates were immunoblotted for FLNA and FLNACT Results: Calpeptin treatment of these cells resulted in a decreased production of FLNACT Calpeptin-treated human and mouse tumor cells displayed impaired proliferation, migration, and colony formation. CONCLUSION: These data suggest that the cleavage of FLNA by calpain is an important cellular event in the regulation of tumor cell growth.

Synergistic role of three dimensional niche and hypoxia on conservation of cancer stem cell phenotype.

Hypoxia is a pathalogical condition in which tissues are deprived of adequate oxygen supply. The hypoxia effect on tumors has a critically important role on maintenance of cancer stem cell phenotype. The aim of this study is to investigate the effects of hypoxia on cancer stem cells on three dimensional (3D) in vitro culture models. Osteosarcoma stem cells characterized by CD133 surface protein were isolated from osteosarcoma cell line (SaOS-2) by magnetic-activated cell sorting (MACS) technique. Isolated CD133(+) and CD133(-) cells were cultivated under hypoxic (1% O2) and normoxic conditions (21% O2) for 3 days. For the 3D model, bacterial cellulose scaffold was used as the culture substrate. 3D morphologies of cells were examined by scanning electron microscopy (SEM); RT-PCR and immunocytochemistry staining were used to demonstrate conservation of the cancer stem cell phenotype in 3D environment under hypoxic conditions. Cell viability was shown by MTT assay on 3. and 7. culture days. This study is seen as an introduction to develop a 3D hypoxic cancer stem cell based tumor model to study CSC behavior and tumor genesis in vitro.