Evaluation Of Breast Cancer Resistance And Albendazole Therapy With ¹⁸F-FDG Uptake

Part 1Evaluation of the mechanisms of breast cancer resistance with ¹⁸F-FDG uptakeObjective:To establish the acquired tamoxifen?TAM?-resistant human breast cancer cell T47D-TamR,compare the ¹⁸F-FDG cell uptake rate between T47D-TamR and its parental cell T47D,and study the preliminary mechanism.Methods:Long-term step rise drug stimulation was used for cell line T47D-TamR establishment and then the cell proliferation and resistance index?RI?was determined by MTT assay.The uptake rate of 18F-FDG between T47D-TamR cell and T47D cell were measured in the setting of different cell count,reaction time,18F-FDG dosage and glucose concentration.The LDH activity,cellular ATP level and lactic acid concentration in cell supernatant of T47D-TamR cell and T47D cell were detected.Western blot was used to examine the expression of ER?,GLUT1,phosphorylated AMPK?p-AMPK?and phosphorylated mTOR?p-mTOR?between T47D-TamR cell and T47D cell.Student t-test and two-way were used to analyze the data.Results:T47D-TamR cell line was successfully established and its drug RI was1.97�0.08,which showed a significantly decreased cell proliferation efficacy?P<0.05?.Significantly differences were found between T47D-TamR cell and T47D cell when changing the cell count,reaction time,and 18F-FDG dosage?P<0.05?.The LDH activities of T47D-TamR cell and T47D cell were?0.42�0.04?and?0.32�0.02?U/mg,cellular ATP level were?19.99�0.32?and?14.01�0.70?nmol/mg,lactic acid concentration in cell supernatant were?2.95�0.05?and?2.02�0.07?mmol/L,respectively,which showed a significant difference?P<0.05?.The relative expression of ER?,p-AMPK,p-mTOR,GLUT1 were 0.26�0.03,0.36�0.06,0.75�0.11,0.35�0.07 in T47D cell,and 0.17�0.02,0.61�0.09,0.52�0.08,0.21�0.04 in T47D-TamR cell,respectively,which showed a significant difference?P<0.05?.Conclusions:T47D-TamR cell shows lower cell uptake rate of ¹⁸F-FDG,LDH activity,cellular ATP level and lactic acid secretion,increased expression of p-AMPK and decreased expression of ER?,p-mTOR,GLUT1 than parental cell,indicating the decreased glycolysis ability in TAM-resistant breast cancer cell.Part 2Evaluation on the mechanisms of the therapeutic effect of ABZ in breast cancer with ¹⁸F-FDG uptakeObjective:To investigate the killing effect of albendazole?ABZ?on breast cancer cells MCF-7 and MDA-MB-231,and to further analyze the value and mechanisms of¹⁸F-FDG uptake in evaluating the therapeutic effect of ABZ on breast cancer.Methods:The experiments were carried out under the condition of ABZ concentration gradient.The changes of ¹⁸F-FDG cell uptake rate of breast cancer cells with ABZ treatment were measured by gamma counter.The cellular ATP levels and lactate concentrations in cell supernatant of ABZ-treated breast cancer cells were detected.MTT and colony formation assays were used to determine the inhibitory effect of ABZ on the proliferation of normal breast cells and breast cancer cell lines.Flow cytometry?FCM?was used to detect the changes of cell cycle distribution of breast cancer cells after ABZ treatment,and apoptosis was detected by FCM and DAPI staining.The effect of ABZ on the migration of breast cancer cells was detected by wound-healing assay.Western blot was used to detect the protein expression of GLUT1,phosphorylated AMPK?p-AMPK?and phosphorylated mTOR?p-mTOR?in breast cancer cells after ABZ treatment.Student t-test was used to analyze the data.Results:The uptake rate of ¹⁸F-FDG,intracellular ATP level and lactate concentration in supernatant were decreased in ABZ in dose dependently.The results of MTT assay showed that ABZ could inhibit the proliferation of breast cancer cell line MCF-7 and MDA-MB-231 cells,but had no significant effect on normal breast cells MCF-10A.Colony formation assay also showed that ABZ inhibited the growth of breast cancer cells in a concentration dependent manner.PI single staining showed that ABZ induces cell cycle arrest in the G2/M phase,FCM analysis and DAPI staining showed that ABZ can lead to cell apoptosis.The results of wound-healing assay showed that ABZ could inhibit the migration ability of breast cancer cell lines.The protein expression of GLUT1 and p-mTOR in breast cancer cells was decreased after ABZ treatment,while the protein expression of p-AMPK was increased.Conclusions:ABZ can effectively inhibit the proliferation,migration,and glycolysis of breast cancer cells.The mechanisms may be related to the decrease of glucose uptake mediated by GLUT1,and sequential activation of AMPK/mTOR signaling pathway,leading to cell cycle arrest and apoptosis.The study on mechanisms of reduction of¹⁸F-FDG cell uptake treated with ABZ laid the experimental foundation for evaluating the therapeutic effect of ¹⁸F-FDG PET imaging of ABZ on breast cancer.