2.1. Reagents and cell lines
The Leiws cell line was provided by Cancer Center and State Key Laboratory of Biotherapy, West China Hospital of Sichuan, phloretin (purity, 97%, BR) was purchased from Dalian Meilun Company. Dimethyl sulfoxide (DMSO), methyl thiazole blue (MTT), crystal violet all were purchased from Sigma Company. DMEM medium and Australian fetal bovine serum were purchased from Waltham Company. Annexin V-FITC Apoptosis Detection kit was purchased from BD Company. Polyclonal antibodies against Ki-67 purchased from Bioworld Technology Co. Ltd. (Nanjing, China).
Lewis lung cancer (LLC) cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator.
C57BL/6J mice (female, 5 weeks old) were purchased from Chengdu Dashuo Biotechnology Co. Ltd. All animal experiments were implemented in accordance with the Institutional Animal Care and Use Guidelines, and approved by the Institutional Animal Southwest Medical Care and Use Committee (Chendu, China).
2.2. Cytotoxicity test
They are treated with concentration gradients of and each group has 6 replicates. The phloretin powder was dissolved in cell culture medium. Incubate to the set time, then add 20 μL of MTT to each well, terminate the incubation after 4 hours, add 200 μL of DMSO to each well, and set it at 480 μL in a microplate reader.
2.3. Colony forming assay
Ph were added to the LLC cells and the medium was replaced after 2h to remove the drug. One hour after adding the Ph, the cells were irradiated up to 10 Gy using a linear accelerator (varian) at the dose rate of 60 cGy/min. To assess colony formation, the variously treated Lewis cells were seeded into 6-well plates and cultured for 10-14 days. Plating efficiency (PE) ,surviving fraction (SF) and he sensitivity enhancement ratio (SER) were calculated by the multi-target model fitting of the data using nonlinear regression.
2.4. Establishment of lung cancer model and treatment protocol
A lung cancer model was established by subcutaneously injecting each mouse with a 100µl suspension of LLC cells in the dorsal aspect of the right foot. After the tumors volumes were ~100-200 mm3, the tumor-bearing mice were randomized into the following four treatment groups (n = 12 each): control (0.9% normal saline), Phloretin, RT, RT + Phloretin. The phloretin solution was injected intraperitoneally at a dose of 20mg/kg based on the previous report[10] , once every 2 days, for a total of 6 treatments, and radiotherapy was given after the third intraperitoneal injection. The truncal region of the mice harboring the tumor xenograft were irradiated before the third injection at the dose rate of 60 cGy/min and source–subject distance of 70 cm, to a total dose of 10 Gy. After treatment, half of the animals in each group were randomly euthanized, and the tumors were harvested for various analyses. A tumor growth curve was plotted based on tumor size against days after treatment. The survival time of each mouse was recorded. Tumor growth curve (TGC) was calculated with (Ti5-T5) as the time taken for the treated tumors (Ti5) and the control tumors (T5) to increase to 5 times their initial tumor volume.
2.5. Micro 18F-FDG PET/CT imaging
The metabolic status of the tumors in response to different treatments were evaluated in terms of 18F-FDG uptake by using Inveon micro PET/CT (Siemens, Munich, Germany). The mice were fasted for 12 hours, anesthetized with 1% pentobarbital at the dose of 5 ml/kg, and then injected with 100-200 mCi FDG into their tail veins. Tracer uptake values of the tumors were measured in attenuation-corrected lateral chromatographic sections by calculating standard uptake values (SUVs) measured by ROI.
2.6. Apoptosis analysis
The soy bean-sized tumors were digested into individual tumor cells. For apoptosis analysis, the cells were stained with PI and annexin V-FITC, and the apoptotic cells were detected by flow cytometry (BD FACSVerse, Piscatway, NJ).
2.7. Immunohistochemistry (IHC)
The tissue sections were immuno-stained using antibodies against Ki-67 according to the manufacturer’s instructions, and observed under an optical microscope (Leica TE2000-S microscope, Tokyo, Japan). The Ki-67 positive and total numbers of cells were counted in 5 randomly selected regions in each tumor section under 400x magnification, and the percentage of positive cells was calculated.
2.8. Statistical analysis
All data are expressed as the mean±standard deviation (SD). One-way analysis of variance (ANOVA) was used to compare different groups, and survival curves were plotted according to the Kaplan–Meier method. P values less than 0.05 and 0.01 were considered statistically significant. Data analysis was performed using SPSS statistics 17.0 software (SPSS Inc., Chicago, IL).