H Liu1, Y-J Wang, L Bian, Z-H Fang, Q-Y Zhang, J-X Cheng. 1. Department of Gynecologic Oncology, the Fourth Hospital of Hebei Medical University, Shijiazhuang, China. chengcjianxinjx@sina.com.
Abstract
OBJECTIVE: The aim of the study is to investigate the radiosensitivity of CD44+/CD24+ cervical cancer cells and to explore its mechanism of radiotherapy resistance. Moreover, we further to test whether the CD44+/CD24+ cervical cancer cells had the characteristics of stem cells. MATERIALS AND METHODS: The human squamous cell carcinoma SiHa cells were cultured in vitro, and CD44+/CD24+ SiHa cells were sorted by FACS analysis. CD44+/CD24+ SiHa cells and the parental SiHa cells were given several fractionated irradiation at a cumulative dose of 8 Gy, 16 Gy, 30 Gy, respectively. Survival curves were obtained and fitted using clonogenic assays, and the radiosensitivity of tumor cells was compared according to the radiobiological parameters, including Do, Dq, N and SF 2. Morphological changes of cell apoptosis were determined using Hoechst 33258 fluorescence staining. The ultrastructural changes in cells with apoptosis were observed by transmission electron microscopy. Cell apoptosis rate was determined by FCAS analysis. DNA "ladder" in apoptotic cells was detected by gel electrophoresis. The mRNA levels of cell apoptosis-related genes were detected by RT-PCR assay. Balling capacities of CD44+/CD24+ SiHa cells and parental SiHa cells were detected by suspension culture without FBS. The in vivo tumorigenicity was detected by inoculating CD44+/CD24+ SiHa and parental SiHa cells into nude mice. RESULTS: The FACS analysis results demonstrated that there was a concomitant increase in the percentage of CD44+/CD24+ cells as the increasing irradiation doses. Colony formation assay results showed that the colony formation rate of CD44+/CD24+ SiHa cells was significantly higher than that of parental SiHa cells (p < 0.05). Moreover, the data from Hoechst 33258 staining, DNA fragment gel electrophoresis, transmission electron microscopy and FACS analysis showed that CD44+/CD24+ SiHa cells had no cell apoptosis after irradiation treatment. RT-PCR results showed that the mRNA levels of bcl-2, surviving and OCT4 were significantly higher in CD44+/CD24+ SiHa cells than that of parental SiHa cells (p < 0.01). CD44+/CD24+ SiHa cells could form more compact cell spheres with a larger volume than that of parental SiHa cells (p < 0.05). CD44+/CD24+ cervical cancer cells had more potent tumorigenicity than that of parental cervical cancer cells. CONCLUSIONS: CD44+/CD24+ cervical cancer resist cell apoptosis induced by irradiation therapy and possessed the characteristics of stem cells.
OBJECTIVE: The aim of the study is to investigate the radiosensitivity of CD44+/CD24+ cervical cancer cells and to explore its mechanism of radiotherapy resistance. Moreover, we further to test whether the CD44+/CD24+ cervical cancer cells had the characteristics of stem cells. MATERIALS AND METHODS: The humansquamous cell carcinoma SiHa cells were cultured in vitro, and CD44+/CD24+ SiHa cells were sorted by FACS analysis. CD44+/CD24+ SiHa cells and the parental SiHa cells were given several fractionated irradiation at a cumulative dose of 8 Gy, 16 Gy, 30 Gy, respectively. Survival curves were obtained and fitted using clonogenic assays, and the radiosensitivity of tumor cells was compared according to the radiobiological parameters, including Do, Dq, N and SF 2. Morphological changes of cell apoptosis were determined using Hoechst 33258 fluorescence staining. The ultrastructural changes in cells with apoptosis were observed by transmission electron microscopy. Cell apoptosis rate was determined by FCAS analysis. DNA "ladder" in apoptotic cells was detected by gel electrophoresis. The mRNA levels of cell apoptosis-related genes were detected by RT-PCR assay. Balling capacities of CD44+/CD24+ SiHa cells and parental SiHa cells were detected by suspension culture without FBS. The in vivo tumorigenicity was detected by inoculating CD44+/CD24+ SiHa and parental SiHa cells into nude mice. RESULTS: The FACS analysis results demonstrated that there was a concomitant increase in the percentage of CD44+/CD24+ cells as the increasing irradiation doses. Colony formation assay results showed that the colony formation rate of CD44+/CD24+ SiHa cells was significantly higher than that of parental SiHa cells (p < 0.05). Moreover, the data from Hoechst 33258 staining, DNA fragment gel electrophoresis, transmission electron microscopy and FACS analysis showed that CD44+/CD24+ SiHa cells had no cell apoptosis after irradiation treatment. RT-PCR results showed that the mRNA levels of bcl-2, surviving and OCT4 were significantly higher in CD44+/CD24+ SiHa cells than that of parental SiHa cells (p < 0.01). CD44+/CD24+ SiHa cells could form more compact cell spheres with a larger volume than that of parental SiHa cells (p < 0.05). CD44+/CD24+ cervical cancer cells had more potent tumorigenicity than that of parental cervical cancer cells. CONCLUSIONS:CD44+/CD24+ cervical cancer resist cell apoptosis induced by irradiation therapy and possessed the characteristics of stem cells.
Authors: Margaretha A Skowron; Teresa K Becker; Lukas Kurz; Sina Jostes; Felix Bremmer; Florian Fronhoffs; Kai Funke; Gamal A Wakileh; Melanie R Müller; Aaron Burmeister; Thomas Lenz; Anja Stefanski; Kai Stühler; Patrick Petzsch; Karl Köhrer; Peter Altevogt; Peter Albers; Glen Kristiansen; Hubert Schorle; Daniel Nettersheim Journal: Mol Oncol Date: 2021-08-02 Impact factor: 6.603