| Section 1:Effect of lidocaine on the proliferation,migration,invasion,and colony formation ability of MCF-7 breast cancer(BC)cellsObjective: To compare the effects of lidocaine on the growth of MCF-10A(a normal breast cell line)and MCF-7(a BC cell line),to observe the effects of lidocaine on the apoptosis,migration,invasion,and colony formation ability of MCF-7 BC cells,and to explore the possible underlying mechanisms.Methods: In the in-vitro experiment,the effects of lidocaine treatment at different concentrations(0.1,0.5,1,2,4 and 8)and at different time points(12,24,36,48,60,72 h)on the proliferation of MCF-10 A and MCF-7 cells were measured by CCK-8 assay.Thereafter,changes in the apoptosis,migration,invasion and colony formation ability of MCF-7 cells treated with lidocaine at different concentrations were detected by flow cytometry and TUNEL assay,Transwell invasion assay,cell scratch assay and colony formation assay,respectively.In addition,changes in the expression of apoptotic proteins Bax,Bcl-2,Cleaved caspase3 were detected by Western blotting assay.Results: Lidocaine at >4 m M exerted a great inhibitory effect on the proliferation of MCF-10 A cells in a dose-dependent manner,and the difference was statistically significant(P < 0.05).Lidocaine at >0.5 m M significantly inhibited the proliferation ability of MCF-7 cells,with statistically significant differences(P < 0.05).Compared with control group,lidocaine treatment significantly increased the apoptosis,diminished the migration and invasion,and decreased the number of colonies formed by MCF-7 cells,and there were statistically significant differences(P < 0.05).Additionally,the expression of apoptotic proteins Bax and Cleaved caspase3 was up-regulated,while that of Bcl-2 was down-regulated,and the differences were of statistical significance(P< 0.05).Conclusion: Lidocaine not only has an inhibitory effect on the proliferation,migration,invasion,and colony formation of MCF-7 cells,but also has a pro-apoptotic effect,which promotes the expression of apoptosis-related proteins Bax and Cleaved caspase3,but decreases that of Bcl-2.Section 2: Mechanism of lidocaine in affecting the biological behavior of MCF-7 breast cancer(BC)cells via the PTEN/PI3K/AKT signaling pathwayObjective: Phosphatase and tensin homolog deleted on chromosome ten(PTEN)is involved in the development of multiple tumors,which also represents an upstream regulator gene of apoptotic proteins such as Bax and Bcl-2.This sections aimed to explore the mechanism of lidocaine in mediating the biological behavior of MCF-7 cells via the PTEN/PI3K/AKT signaling pathway.Methods: We used SF1670 to inhibit the expression of PTEN in cells.The expression of PTEN and the downstream pathway molecules in MCF-7 breast cancer(BC)cells in control and lidocaine treatment groups;the expression of downstream pathway molecules in MCF-7 BC cells with PTEN inhibition and in MCF-7 BC cells with PTEN inhibition+lidocaine treatment;and changes in the lidocaine-induced inhibition of MCF-7 growth before and after interference with PTEN expression were observed by CCK-8 assay,flow cytometry(FACS),TUNEL assay,Transwell invasion assay,wound healing assay,and colony formation assay.Results: Compared with control group,the expression of PTEN,Bax,and Cleaved caspase3 was significantly higher,whereas that of p-AKT and Bcl-2was lower in lidocaine treatment group,and there were statistically significant differences(P < 0.05).Relative to control group,the expression of PTEN,Bax and Cleaved caspase3 significantly decreased,while that of p-AKT and Bcl-2increased in PTEN inhibitor group,with the difference being statistically significant(P < 0.05).Compared with lidocaine group,the expression of PTEN,Bax,and Cleaved caspase3 was dramatically down-regulated,whereas that of p-AKT and Bcl-2 was up-regulated in lidocaine + PTEN inhibitor group,and the difference was of statistical significance(P < 0.05).In comparison with lidocaine group,cell proliferation was promoted in lidocaine + PTEN inhibitor group,with the difference being statistically significant(P < 0.05).In addition,apoptosis significantly decreased in lidocaine + PTEN inhibitor group,with the difference being of statistical significance(P < 0.05).Moreover,the invasion,migration and colony formation ability of cancer cells were significantly enhanced in lidocaine + PTEN inhibitor group,and the differences were statistically significant(P < 0.05).Conclusion: Lidocaine inhibits the biological behavior of MCF-7 BC cells via the PTEN/PI3K/AKT signaling pathway.Section 3: Mechanism of lidocaine in inhibiting the biological behavior of MCF-7 breast cancer(BC)cells through the mi R-130b-3p-targeted binding to PTENObjective: To investigate the mechanism of lidocaine in inhibiting the growth of MCF-7 BC cells through the mi R-130b-3p-targeted binding to PTEN.Methods: The current section adopted bioinformatic approaches to explore the targeting relationship between mi R-130b-3p and PTEN and to detect the differential expression of mi R-130b-3p between BC tissues and normal tissues.The expression of mi R-130b-3p in lidocaine-treated MCF-7 BC cells was then observed by q RT-PCR.Additionally,the targeting relationship between mi R-130b-3p and PTEN was verified by dual-luciferase assay.Meanwhile,cells were transfected with mi R-130b-3p mimic and inhibitor to up-regulate or down-regulate mi R-130b-3p expression.The expression of PTEN and the downstream pathway molecules in control MCF-7 BC cells was examined by Western blotting analysis.In the meantime,the expression of PTEN and the downstream pathway molecules in MCF-BC cells after regulation of mi R-130b-3p expression,that after regulation of mi R-130b-3p expression+lidocaine treatment,and that after lidocaine treatment were also detected.The Changes in the biological behavior of MCF-7 BC cells before and after lidocaine-induced regulation of mi R-130b-3p expression were analyzed by CCK-8,FACS and TUNEL,Transwell invasion,cell scratch and colony formation assays.Results: Biochemical analysis preliminarily revealed that mi R-130b-3p was differentially expressed between BC and normal tissues,and it targeted PTEN gene.In addition,mi R-130b-3p was experimentally demonstrated to be lowly expressed in MCF-7 BC cells of lidocaine-treated group,and there were statistically significant differences(P < 0.05).As shown by results of dual-luciferase assay,mi R-130b-3p targeted PTEN gene.mi R-130b-3p mimic down-regulated PTEN expression in MCF-7 BC cells compared with control group,whereas the inhibitor up-regulated PTEN expression,and the difference was statistically significant(P < 0.05).Compared with lidocaine group,mi R-130b-3p mimic combined with lidocaine treatment group showed decreased expression of PTEN and Cleaved caspase3 but increased expression of p-AKT,and the difference was statistically significant(P < 0.05).The proliferation of MCF-7 BC cells in combination group significantly increased,while cell apoptosis decreased,and the number of cells passing through the vesicles also increased.In the meantime,the proportion of cell scratch healing increased and the number of cell colonies also increased.By contrast,the expression of PTEN and Cleaved caspase3 was significantly up-regulated,while that of p-AKT was down-regulated in mi R-130b-3p inhibitor + lidocaine treatment group compared with lidocaine group,and the difference was statistically significant(P < 0.05).In the mi R-130b-3p inhibitor + lidocaine treatment group,MCF-7 BC cells showed significantly decreased proliferation,increased apoptosis,a decreased number of cells passing through the vesicles,a decreased proportion of cell scratch healing,and a decreased number of cell colonies.These results suggested that mi R-130b-3p was involved in the mechanism by which lidocaine inhibited the biological behavior of MCF-7 BC cells by targeting the binding to PTEN gene.Conclusion: Lidocaine can inhibit MCF-7 BC cell biological behavior through mi R-130b-3p-targeted binding to PTEN.Section 4: Inhibitory effects of lidocaine on the growth of breast cancer(BC)xenograft in nude mice and the underlying mechanismsObjective: To investigate the effect of lidocaine on the growth of BC and the underlying mechanism by animal experiments.Methods: A human BC xenograft nude mouse model was established,and the changes in tumor weight and tumor volume were observed under the intervention with locally injected lidocaine.Hematoxylin eosin(HE)staining was conducted to observe the effect of lidocaine on the morphology of BC cells,while immunohistochemical(IHC)staining was performed to measure the expression of PTEN and Cleaved caspase3 in the transplanted tumors.Results: Compared with control group,local injection of lidocaine significantly inhibited tumor growth,and reduced tumor weight and tumor volume.Relative to control group,HE staining revealed the relatively less tumor cells in lidocaine intervention group,which were loosely arranged and showed large necrosis.Meanwhile,IHC results revealed that the expression of PTEN and Cleaved caspase3 proteins increased in lidocaine intervention group.Conclusion: Local injection of lidocaine can effectively inhibit the growth of BC xenograft tumors,which may be associated with the regulation of the PTEN pathway. |
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