| Part 1 Study on the antitumor efficacy of albendazole in triple negative breast cancerObjective:This study further explored the antitumor effect of albendazole(ABZ)on triple negative breast cancer(TNBC)in vivo and in vitro,to lay the important evidence for subsequent research.Methods:In vitro experiments,the MTT method and clone formation experiments were used to determine the inhibitory effect of ABZ on the proliferation of breast cancer cell lines(MCF-7 and MDA-MB-231).The apoptosis effect of ABZ on breast cancer cell lines was measured by apoptosis experiments.Wound-healing assay and Transwell test were used to detect the effect of ABZ on the migration ability of breast cancer cell lines.In vivo experiments,MDA-MB-231 cells were subcutaneously injected into the right axillary region of 6～8-week-old female BALB/c athymic nude mice.When the tumor volume reached about 100 mm3,tumor-bearing mice were randomly divided into two groups(control group and the ABZ treatment group).Administration was performed daily,and tumor size and mouse weight were monitored every two days.Survival analysis was performed.At the end of the experiment,blood was collected through the abdominal aorta for blood routine and biochemical analysis.Student t-test and two-way were used to analyze the data.Results:The results of MTT and clone formation experiments showed that ABZ inhibited the proliferation of breast cancer cell lines(MCF-7 and MDA-MB-231 cells)in a time-and concentration-dependent manner.In Annexin V-FITC/PI flow cytometry experiment,two Breast cancer cells(MCF-7 and MDA-MB-231)were treated with 0 and 1.0 μM ABZ for 24 hours,and the apoptosis rates of flow cytometry were(9.92±0.40)%,(21.47±0.38)%,and(12.73 ± 0.55)%and(31.17±1.36)%,suggesting that ABZ could induce apoptosis in two cell lines.Breast cancer cells were treated with 0 and 1.0 μM ABZ,the number of migrated cells in the cell scratch test was 93.33 ± 0.38,28.00 ± 3.00,and 124.00±32.05,25.67±5.69.The ratio of the number of cells passing through the chamber in the Transwell experiment were:(100.00±10.16)%,(62.47±2.64)%,and(100.00 ±15.06)%,(58.94±1.96)%,the results showed that ABZ inhibited the migration ability of two cell lines.At the same time,all in vitro experiments showed that ABZ had a more significant inhibitory effect on MDA-MB-231 cells than MCF-7 cells,and the difference is statistically significant(P<0.05).In vivo experiments found that the tumor volume of the control group and the experimental group on the 9th day of the experiment were:1278.70 ±3798.80 mm3 and 724.18 ±255.62 mm3.Compared with the control group,the tumor volume of the tumor-bearing mice became slower and smaller after ABZ treatment.The weight of the mice was stable and the survival time of the mice was prolonged.The blood routine and biochemical results showed that there were no significant differences in RBC,PLT,CRE,and UREA.WBC,ALT,and AST were significantly lower in the ABZ treatment group than in the control group.The differences between the groups were statistically significant(P<0.05).Conclusions:In vitro,ABZ can inhibit the proliferation and migration of triple-negative breast cancer cells and induce their apoptosis.In vivo,it can inhibit the growth of triple-negative breast cancer,prolong the survival of mice,has no obvious toxic side effects,and may inhibit tumor metastasis.Part 2 18F-FDG PET imaging for monitoring the early anti-tumor effect of albendazole on triple-negative breast cancer and the study for its mechanismObjective:To explore the feasibility of using 18F-FDG PET to evaluate the early antitumor effect of ABZ on triple-negative breast cancer and a preliminary study of its related mechanism.Methods:Female nude mice carrying MDA-MB-231 xenografts were randomly divided into a control group and an ABZ treatment group.18F-FDG PET imaging was performed on day 0(before administration)and day 3(after administration)of the drug treatment.The results of the corresponding tumor site SUVmean and SUVmax values were obtained after image processing to detect the early tumor suppression effect of ABZ on MDA-MB-231 tumors.After the imaging was completed,the tumor tissue was removed for sectioning,and the early inhibition of MDA-MB-231 tumors proliferation and the promotion of apoptosis by ABZ were detected by fluorescent immunoassay.Then,immunofluorescence and Western blot were used to determine the expression of CLUT1 in vivo and in vitro when ABZ acted in the early stage of MDA-MB-231 cells.Western blot was used to detect the proteins in some pathways of ABZ on MDA-MB-231 tumors(PI3K,p-AKT,p-p65,p-AMPK,p-mTOR,p-P70S6K,mutP53,Cleaved-caspase3,PARP and internal reference GAPDH)levels.Results:18F-FDG PET imaging was performed after ABZ treatment of tumor-bearing nude mice carrying MDA-MB-231 xenografts.The results correspond to those in the control group and the ABZ treatment group.On day 0,SUVmean values were 5.17±0.85 and 4.03 ±0.42,while the SUVmax values were 7.83 ± 1.35 and 6.33 ± 0.83,respectively,which had no significant difference between the two groups(P>0.05).On the third day,the SUVmean values were 8.60±1.37 and 5.53 ± 0.58,and the SUVmax values were 11.67 ± 1.80 and 7.60 ± 0.42,respectively,which showed a significant difference between the two groups(P<0.05).Compared with the control group,the SUVmean and SUVmax values of the ABZ treatment group were significantly reduced on the third day.The results of Immunofluorescence revealed that ABZ inhibited the proliferation and promoted apoptosis of MDA-MB-231 cells in the early stage.Furthermore,in vivo and in vitro experiments,immunofluorescence and Western blot analysis showed that ABZ inhibited the expression of GLUT 1 in MDA-MB-231 cells.Simultaneously,further mechanistic studies found that the expression of PI3K,AKT,P56,and P53 was decreased and p-A_MPK was increased in MDA-MB-231 cells after ABZ treatment.The differences between the groups were statistically significant(P<0.05).Conclusions:Our study suggested that ABZ has an early antitumor effect on MDA-MB-231 tumors.The mechanism may be related to reducing the expression of GLUT1 by inhibiting the PI3K/AKT signaling pathway after ABZ action,thereby reducing glucose uptake,inhibiting the glycolysis process,activating the AMPK/P53 signaling pathway,and causing cell proliferation inhibition and increased apoptosis.At the same time,18F-FDG PET imaging can be used to evaluate the early tumor suppressive effect of breast cancer ABZ on MDA-MB-231 tumors. |
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