| Background:Nasopharyngeal carcinoma (NPC) is the most common cancer originating in the nasopharynx. The etiology of NPC seems to follow a multi-step process, in which Epstein-Barr virus, ethnic background, salt-cured food and environmental carcinogens all seem to play an important role. Due to the anatomical position of NPC and its tendency to present with cervical lymph node metastases, it is not amenable to surgery. Radiotherapy is the most important method for NPC. This treatment failure is mainly due to a portion of radioresistant phenotype of NPC, and the dose of radiation is also limited by the damage caused to surrounding normal tissues. So, it is a hotspot for NPC treatment research to look for safe and effective antitumor drugs having radiosensitizing effect.Icaritin is a derivative of icariin, which is extraxted from the traditional Chinese herbal medicine Empimedium. Previous studies showed that icaritin possesses multiple kinds of biological activities, such as antioxidation, cardiovascular function improvement, osteoporosis prevention and protection of neurodegenerative injury. Recent researches have showed that icaritin also has antitumor activities in several cancers, including hepatocellular carcinoma, endometrial cancer, breast cancer, prostate cancer and hematologic malignancy cells. In this study, we used CNE-2cell line as a model system to observe the effect of icaritin on cell proliferation, apoptosis and radiosensitivization of human nasopharyngeal carcinoma cells and reveal its possible radiosensitizing mechanisms. The data may provide experimental basis for further clinical research of using icaritin as a novel agent for nasopharyngeal carcinoma treatment.Part I Proliferation-inhibiting and apoptosis-inducing effect of icaritin on CNE-2cellsObjective:To investigate the effect of proliferation and apoptosis of icaritin on CNE-2cells.Methods:Different concentrations of icaritin were given to CNE-2cells to observe cell morphology by inverted phase contrast microscope. The inhibition of proliferation was analyzed by tetrazolium blue (MTT) assay. The apoptosis rate of CNE-2cells was analyzed by flow cytometry.Results:1. CNE-2cells underwent dramatic changes in morphology after treated with different concentrations of icaritin, the cells turned round with lower refraction and the volume became smaller.2. MTT assay indicated that the inhibition effect on CNE-2cells was in a dose and time-dependent manner, the difference showed significant (P<0.05). The IC50at24h,48h.72h was (93.63±1.60)μmol/L,(52.90±0.32)μmol/L,(32.62±1.17)μmol/L.3. Flow cytometry assay indicated that the apoptosis rate was significantly increased after treated by icaritin for24h and48h. With the increasing of concentration, the apoptosis rate was also increased, ranging from (4.59±1.49)%to (30.73±4.06)%at24h,(6.15±0.97)%to (52.65±2.07)%at48h.Conclusions:Icaritin could inhibit the proliferation and induce the apoptosis of CNE-2cells in a concentration and time-dependent manner.Part Ⅱ Radiosensitizing effect of icaritin on CNE-2cells and its possible mechanismsObjective:To investigate the effect of radiosensitization of icaritin on CNE-2cells and explore its mechanisms.Methods:Under the different radiation doses were given to CNE-2cells treated with low cytotoxic icaritin, MTT assay was used to detect the cell proliferation inhibitory effects. The radiosensitizing effect of icaritin on CNE-2cells was observed by clonogenic assay. The radiation dose-cell survival curve was evaluated by the mathematic model of one-hit multi-target. Flow cytometric analysis was used to detect the change of cell cycle of CNE-2cells induced by low cytotoxic icaritin for24h.Results:1. MTT assay indicated that in the same group, the inhibition effect was increased when the radiation dose became higher. At the same radiation dose, the group of5.0μmol/L icaritin significantly had higher suppression rate than the control group (icaritin,0.0μmol/L). 2. Clonogenic assay indicated that icaritin could enhance radiation on CNE-2cells. According to the cell survival curve, D0,SF2,Dq and N values of the5.0μmol/L icaritin combined radiation group were all lower than the radiation group, and the SER was (1.71±0.05).3. Flow cytometry assay indicated that the distribution of cell cycle could be affected by icaritin. After CNE-2cells were incubated with icaritin of5.0μmol/L for24h, G2/M phase fraction which was sensitive to radiation was higher significantly, while the G0/G1phase was decreased.Conclusions:At a low concentration, icaritin had radiosensitizing effect on CNE-2cells, and its mechanisms might be related to that the repair ability to sublethal injury was decreased and the cell cycle was blocked in G2/M phase.
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