| BackgroundNowadays,lung cancer is the most common malignant tumor with the highest morbidity and mortality.Non-small cell lung cancer(NSCLC)occupies the largest proportion of lung cancer.The main treatments of NSCLC include surgery,radiotherapy,chemotherapy,targeted therapy and immunotherapy.Although sugery resulted in excellent prognosis,amounts of patients were seen in advanced stage and lost the optimal time for operation.These patiens were treated by radiotherapy or chemotherapy,but we were dissatisfied with the prognosis.Targeted drugs could save only part of the patients with susceptibility to resistance and high expense.Curative effect of immunotherapy was unclear.Rencent research had confirmed that monomers of natural Chinese drugs and their analogues had anti-tumor effects and they were one of important sources of antineoplastic agents.Breviscapine was the main extraction components of Erigeron breviscapus,which had a wide range of pharmacological functions,such as antioxidance,anti-inflammatory and vascular protection.Rencently,studies proved that breviscapine had anti-tumor activity.But the role and molecular mechanism of breviscapine in lung cancer were not yet clear,which warrants further study.ObjectiveVia cell lines in vitro and xenograft model in vivo,we analysed the effects of breviscapine on proliferation,apoptosis and tumor growth of NSCLC cells to clarify their antitumor effects.We focused on how the expression of miRNA-7 and IGFBP-4 were changed by breviscapine,while we observed the function of over-expressed or knock-down miRNA-7 and IGFBP-4 in NSCLC to elucidate the molecular mechanism of breviscapine against NSCLC.Methods(1)Using MTT cell proliferation assay and Trypan blue dying cell staining method to detect the the proliferation of A549,NCL-H460 and normal lung fibroblast MRC-5 cell lines and analyzd the selective killing effect of breviscapine in NSCLC.(2)Applying Annexin V/PI dual staining,Caspase-3 activity kit,Western blot and cell cycle detection kit to detect the apoptosis,caspase-3 activity,pro-apoptotic,anti-apoptotic protein expression and cell cycle of A549 cells and analyze the effects of breviscapine on cell cycle arrest and apoptosis.(3)A549 cells were incubated with 0、25、5 0、100 μM breviscapine for 48 h;using JC-1 and ROS detection kit to detect mitochondrial potential and intracellular ROS,analyze the effects of breviscapine on mitochondrial membrane potential and ROS level.A549 cells were incubated with ImM NAC,while conbined with 0,25,50,100μM breviscapine for 48 h;MTT cell proliferation and ROS detection kit were used to detect the proliferation and ROS level;then analyzed the role of ROS in breviscapine inducing cell proliferation inhibition in A549 cells.A549 cells were co-incubated with 100 μM breviscapine and 5 μM CsA for 48 h;the mitochondrial membrane potential was analyzed by JC-1;then analyzed the role of CsA in breviscapine inducion leading to the decrease of mitochondrial membrane potential.The cells were divided into four groups:control group,100 μM breviscapine,5 μM CsA+ 100 μM breviscapine group and 1 mM NAC+100 μM breviscapine group.ROS detection kit were used to detect the intracellular contents of ROS;then analyzd the effects of NAC and CsA on increase of ROS level induced by breviscapine in A549 cells.(4)A549 cells were used to establish subcutaneous Xenograft model in nude mice.The nude mice received lOmg/kg and 20mg/kg breviscapine by intraperitoneal injection everyday.Tumor size and weight of nude mice were recorded weekly and the mice were sacrificed to obtain fresh tumor tissue after twenty-one days.Western blot was used to detect the expression of Bax and Bcl-2,and the ratio of Bax/Bcl-2 in fresh tumor tissue was analyzed.We made paraffin slices immediately after the fresh tumor tissue were extracted.Then applied immunohistochemistry to analyze the expression of Ki67 and caspase-3 in the slices.TUNEL staining was used to detect the apoptosis in tumor tissue,then we confirmed the proliferation inhibition and apoptosis induced by breviscapine in vivo.(5)Using qRT-PCR to detect the expression of miRNA-7 after treatment with 0,25,50 and 100 μM breviscapine for 48 h;then to analyze the expression of miRNA-7 in A549 cell line.(6)miRNA-7-minics(over-expression)and miRNA-7-inhibtor(low-expression)was transfected into A549 cells and then treated with 100 μM breviscapine for 48 h.MTT cell proliferation assay was used to detect cell proliferation and analyze the role of miRNA-7 on the proliferation inhibited by breviscapine.The expression of Bax and Bcl-2 were dectected by western blot and the ratio of Bax/Bcl-2 was analyzed,so we could study the role of miRNA-7 on apoptosis in A549 cell via breviscapine induction.The luciferase reporter gene containing mutant and wild type 3’-UTR Bcl-2 gene were constructed.After miRNA-7-inhibitor transfecting into the A549 cell,the luciferase activity was observed to clarify the regulatory effect of miRNA-7 on Bcl-2 3’-UTR.(7)The expression of IGFBP-4 were detected by ELISA,Western blot and qRT-PCR after 0,25,50,100 μM breviscapine treatment for 48 h in A549 cells;then analyzed the expression of IGFBP-4 induced by breviscapine.A549 cells were divided into control group,hydrogen peroxide group(positive control),100 μM breviscapine group and 100 μM breviscapine +1 mM NAC group.Western blot,ELISA and qRT-PCR were used to analyze the expression of IGFBP-4 in medium and cells,which could clearify the role of ROS on expression of IGFBP-4 induced by breviscapine.(8)IGFBP-4-vector(over-expression)and IGFBP-4-siRNA(low-expression)were transfected into A549 cells.Using MTT cell proliferation assay to detect cell proliferation after 100 μM of breviscapine treatment for 48 h,which could clarify the role of IGFBP-4 on the proliferation inhibited by breviscapine.The expression of Bax and Bcl-2 were detected by western blot,then we could conclude the role of IGFBP-4 on apoptosis in A549 cell via breviscapine induction.Results(1)Breviscapine inhibited the proliferation of NSCLC cells but could not inhibit the proliferation of normal lung fibroblasts.MTT cell proliferation assay showed that the proliferation of A549 cells and NCL-H460 cells was significantly inhibited after the treatment with different concentrations of breviscapinet for 24,48 and 96 h.The 50%inhibiting concentration(IC50)of A549 and NCL-H460 cells in 24,48,96 h were 586.53±36.3 μM,174.36±10.41 μM,70.22±5.12μM and 211.45±21.47 μM,94.17±8.67 μM,46.28±3.89μM.When the concentration of breviscapine was lower than 100 μM,survival rate of A549 cells and NCL-H460 cells wes significantly inhibited,but it could not inhibit the survival rate of normal lung fibroblasts MRC-5.Trypan blue staining showed that in A549 cells the rate of death cells was increased after 48 h of 25,50,100 μM breviscapine treatment.(2)Breviscapine induced cell cycle arrest and apoptosisAnnexin V/PI dual straining analysis showed that cell apoptosis rate,especially early apoptosis rate was increased after 0,25,50,100 μM breviscapine treatment for 48 h.The cell cycle were arrest in G2/M phase in A549 cells which were received 0,25,50,100 μM breviscapine treatment for 48 h.Microplate Reader analysis showed that Caspase-3 activity increased significantly comparing with the control group.Western blot showed that the expression of anti-apoptotic protein Bcl-2 decreased significantly while the expression of pro-apoptotic protein Bax increased significantly,the Bax/Bcl-2 ratio was increased.(3)Breviscapine could upregulate ROS content and downregulate mitochondrial membrane potential in A549 cellsFlow cytometry analysis showed that DCFH-DA positive cells(intracellular ROS content)increased significantly after 0,25,50,100 μM of breviscapine treatment for 48 h,which were reversed by1 mM of breviscapine.Microplate reader analysis showed that the number of JC1 green fluorescence positive cells significantly increased(indicated the downregulating of the mitochondrial membrane potential).5 μM CsA could significantly reduce the number of green fluorescent positive cells in A549 cell treated with 100 μM breviscapine.Comparing with the control group,the ROS content in breviscapine group increased significantly,while the ROS content in NAC+breviscapine group and CsA+ breviscapine group did not increase significantly.This indicated that NAC and CsA could inhibit significantly the increasing ROS content induced by 100 μM breviscapine.(4)Breviscapine inhibited the growth of A549 cells subcutaneous Xenograft in vivoThe A549 cells subcutaneous Xenograft receiving 10mg/kg and 20mg/kg breviscapine via intraperitoneal injection everyday.Tumor size in mouse which received 10mg/kg and 20mg/kg breviscapine decreased significantly comparing with the control group in the fourteenth and twenty-first days and the difference was statistically significant.However,the body weight of nude mice did not reduce significantly.Western blot showed that 10mg/kg and 20mg/kg breviscapine treatment for twenty-one days could significantly upregulate the expression of Bax and downregulate the expression of Bcl-2.Immunohistochemistry showed that the expression of Ki67 significantly decreased while the expression caspase-3 increased significantly.The TUNEL staining showed that the percentage of apoptosis in experiment group was significantly higher than in control group after 10mg/kg and 20mg/kg treatment.(5)Breviscapine upregulated the miRNA-7 expression in A549 cellsqRT-PCR showed that 25,50,100 μM breviscapine treatment significantly increased miRNA-7 expression in A549 cells,which indicated breviscapine could promote the miRNA-7 expression in A549 cells.(6)miRNA-7 mediated the proliferation inhibition induced by breviscapine in A549 cellsMTT cell proliferation assay showed that miRNA-7-minic significantly enhanced the proliferation inhibition,whereas miRNA-7-inhibitor significantly alleviated the proliferation inhibition induced by breviscapine in A549 cells.Western blot showed that the expression of Bax increased while the expression of Bcl-2 decreased.And the ratio of Bax/Bcl-2 increased in miRNA-7-minic cells.After 100 μM breviscapine treatment for 48 h,comparing with miRNA-NC cells,the expression of Bax miRNA-7-minic in cells increased,Bcl-2 decreased and Bax/Bcl-2 ratio increased.In contract,the expression of Bax decreased significantly,the expression of Bcl-2 increased,Bax/Bcl-2 ratio decreased in miRNA-7-inhibitor cells.After 100 mol/L breviscapine treatment for 48 h.Comparing with Control-siRNA cells,the expression of Bax decreased,Bcl-2 expression increased,and Bax/Bcl-2 ratio decreased in miRNA-7-inhibitor cells.In miRNA-7-inhibitor cells,luciferase reporter assay showed that miRNA-7-inhibitor could increase luciferase activity in the wild 3 ’-UTR Bcl-2 but could not increase 3’-UTR Bcl-2 luciferase activity in the mutation 3 ’-UTR Bcl-2.(7)ELISA detection results showed that 25,50 and 100 μM breviscapine significantly increased IGFBP-4 content in culture medium.Western blot and RT-qPCR results showed that breviscapine could increase the protein and mRNA expression of IGFBP-4 in A549 cells.Comparing with treatment 100 μM breviscapine,IGFBP-4 expression decreased significantly in A549 cells recieving combination treatment 100 μM breviscapine with 1 mM detected by ELISA,Western blot and RT-qPCR,suggesting that ROS mediated the expression of IGFBP-4 in A549 cells receiving breviscapine treatment.(8)IGFBP-4 mediated the proliferation inhibition induced by breviscapine in A549 cells.MTT cell proliferation assay showed that IGFBP-4-vector significantly enhanced the proliferation inhibition while IGFBP-4-siRNA significantly alleviated proliferation inhibition induced by breviscapine in A549 cells.Western blot showed that the expression of Bax increased while the expression of Bcl-2 decreased in IGFBP-4-vector cells after 100 μM breviscapine treatment for 48 h.Comparing with NC-vector,the expression of Bax increased and the expression of Bcl-2 decreased in IGFBP-4-vector cells.The expression of Bax decreased significantly and the expression of Bcl-2 increased in IGFBP-4-siRNA cells after 100 μM breviscapine treatment for 48 h.Comparing with NC-siRNA,the expression of Bax decreased and the expression of Bcl-2 increased in IGFBP-4-siRNA cells.Conclusion(1)Breviscapine had a selective inhibition effect in NSCLC cells,and it could increase apoptosis and cell cycle arrest.(2)Breviscapine could downregulate the mitochondrial membrane potential,while upregulating intracellular ROS levels.Importantly,breviscapine could upregulate the expression of miRNA-7 and IGFBP-4.(3)miRNA-7 binding to 3,untranslated region(3-UTR)of Bcl-2 could downregulate the expression of Bcl-2 and increase Bax/Bcl-2 ratio,which resulted in apoptosis.(4)Breviscapine could promote the expression of IGFBP-4 via upregulating the intracellular ROS levels;IGFBP-4 paticipated in the increasing of Bax/Bcl-2 ratio,which resulted in cell proliferation inhibition in NSCLC.In brief,breviscapine inhibited the proliferation via upregulating the expression of miRNA-7 and IGFBP-4 in non-small cell lung cancer. |
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