| Background:Dopamine receptor agonist(DA)is the first choice for the treatment of prolactinoma while some patients are resistant to DA.Our previous study confirmed that the sensitivity and mechanism of two prolactinoma cell lines(GH3 and MMQ)responding to bromocriptine(BRC)and cabergoline(CAB)were different.GH3 cell line is relatively resistant to CAB which mainly leads to autophagy of tumor cells by inhibiting mammalian target of rapamycin(m TOR)pathway.MMQ cell line is relatively resistant to BRC which mainly leads to apoptosis of tumor cells by activating early growth response protein 1(EGR1)pathway.As an upstream signal,AMPK can regulate both EGR1 and m TOR signal pathways.Theoretically,AMPK can be used as a target to activate EGR1 and inhibit m TOR,which plays a therapeutic role in reversing drug resistance.As a derivative of parthenolide analogues,ACT001 is capable of activating AMPK by increasing intracellular reactive oxygen species(ROS).The purpose of this study is to verify that ACT001 can reverse drug resistance of prolactinomas to DA through AMPK-mediated EGR1 and m TOR pathways.Part one:ACT001 induced death of DA-resistant prolactinoma cells through ROS-AMPK pathwayObjective:The aim was to verify that ACT001 can induce death of DA-resistant prolactinoma cells by increasing the level of intracellular ROS level and the AMP/ATP ratio and activating AMPK in combination with DA.Methods:Five groups were divided for GH3 cells:the control group,the CAB treatment group,the ACT001 treatment group,the CAB+ACT001 treatment group and the CAB+ACT001+N-Acetyl-L-cysteine(NAC)treatment group.Five groups were divided for MMQ cells:the control group,the BRC treatment group,the ACT001treatment group,the BRC+ACT001 treatment group and the BRC+ACT001+NAC treatment group.CCK8 and clone formation assay were used to evaluate the proliferation ability of cells in each group and fluorescence probe was used to test intracellular level of ROS.The AMP/ATP ratio was detected by high performance liquid chromatography-mass spectrometry.We used Western Blot to compare the expression levels of AMPK in each group.Results:For GH3 cells,the CAB+ACT001 group showed the lower cell survival rate(36.80±6.40%vs 52.07±5.61%,P<0.05;36.80±6.40%vs 84.10±2.95%,P<0.001),the higher intracellular level of ROS(P<0.001,P<0.01),the higher ratio of AMP/ATP(P<0.001,P<0.001)and the higher expression level of AMPK(p Thr172)(P<0.01,P<0.01)than those of CAB group and ACT001 group.We found no significant difference after adding antioxidant NAC in cell survival rate,intracellular ROS level,AMP/ATP ratio and AMPK(p Thr172)expression level between CAB+ACT001+NAC group and CAB group.For MMQ cells,the BRC+ACT001 group showed the lower cell survival rate(52.53±5.05%vs 69.83±1.17%,P<0.01;52.53±5.05%vs81.80±1.10%,P<0.01),the higher intracellular level of ROS(P<0.001,P<0.01),the higher ratio of AMP/ATP(P<0.01,P<0.05)and the higher expression level of AMPK(p Thr172)(P<0.01,P<0.01)than those of BRC group and ACT001 group.We found no significant difference after adding antioxidant NAC in cell survival rate,intracellular ROS level,AMP/ATP ratio and p-AMPK expression level between CAB+ACT001+NAC group and BRC group.Conclusion:ACT001 can induce death of DA-resistant prolactinoma cells by increasing the level of intracellular ROS and the AMP/ATP ratio and activating AMPK in combination with DA.Part two:The mechanism of ACT001 reversing CAB resistance in GH3 cells by activating AMPK to inhibit m TOR pathwayObjective:The aim was to verify that ACT001 could activate AMPK and induce autophagy of CAB-resistant GH3 cells by inhibiting m TOR pathway.Methods:Six groups were divided for GH3 cells:the control group,the CAB treatment group,the ACT001 treatment group,the CAB+ACT001 treatment group,the CAB+A-769662 treatment group and the CAB+ACT001+Compound C treatment group.Clone formation assay and CCK8 were used to evaluate the cell proliferation ability in each group.The expression levels of AMPK,m TOR regulatory protein(Raptor,Tuberous sclerosis complex(TSC2)),autophagy related protein(Beclin-1,UNC51 like kinase 1(ULK1))and autophagy markers light chain(LC3)Ⅰ/Ⅱin each group were evaluated by Western Blot.We observed and counted the numbers of autophagosomes in different groups under transmission electron microscope.The AMPK silenced GH3 cell line was constructed by lentivirus,and the effect of drugs on the cell line was detected.Results:(1)The cell survival rate of CAB+ACT001 group was obviously lower than that of CAB group and ACT001 group(35.30±3.33%vs 59.63±1.76%,P<0.001;35.30±3.33%vs 84.10±3.90%,P<0.001).After adding A-769662,the cell survival rate of CAB+A-769662 group was similar to that of CAB+ACT001 group(35.70±3.86%vs 35.30±3.33%,P>0.05).After adding Compound C,the cell survival rate of CAB+ACT001+Compound C group was similar to that of CAB group(57.23±9.86%vs 59.63±1.76%,P>0.05).(2)The protein expression levels of AMPK(p Thr172)and m TOR negative regulatory proteins Raptor(p Ser792)and TSC2(p Ser1387)in CAB+ACT001 group were significantly higher than those in CAB group and ACT001group.The expression levels of Beclin-1(p Ser14)and ULK1(p Ser317)proteins which promote autophagy were higher and the expression level of ULK1(p Ser757)protein which inhibits autophagy was lower in CAB+ACT001 group compared with CAB group and ACT001 group.The LC3Ⅱ/LC3Ⅰratio was also higher in CAB+ACT001group compared with CAB group and ACT001 group.After adding A-769662,the protein expression levels in CAB+A-769662 group were similar to those in CAB+ACT001 group.After adding Compound C,the protein expression levels in CAB+ACT001+Compound C group were similar to those in CAB group.(3)The number of autophagosomes in CAB+ACT001 group was significantly higher than that in CAB group and ACT001 group(P<0.001,P<0.001).After adding A-769662,the number of autophagosomes in CAB+A-769662 group was similar to that in CAB+ACT001 group(P>0.05).After adding Compound C,the number of autophagosomes in CAB+ACT001+Compound C group was similar to that in CAB group(P>0.05).(4)For AMPK silenced GH3 cells,the cell survival rate of CAB+ACT001+A-769662 group was lower than that of CAB+ACT001 group(40.53±3.56%vs 71.77±3.35%,P<0.001).The cell survival rate of AMPK silenced GH3 cells which were treated with CAB+ACT001 was higher than that of normal GH3cells which were treated with CAB and ACT001(71.77±3.35%vs 35.20±2.98%,P<0.001).Conclusion:ACT001 can inhibit m TOR pathway by activating AMPK and induce autophagy of CAB-resistant GH3 cells in combination with CAB.Part three:The mechanism of ACT001 reversing BRC resistance in MMQ cells by activating AMPK to activate EGR1 pathwayObjective:The aim was to verify that ACT001 could activate AMPK and induce apoptosis of BRC-resistant MMQ cells by activating EGR1 pathway.Methods:Six groups were divided for MMQ cells:the control group,the BRC treatment group,the ACT001 treatment group,the BRC+ACT001 treatment group,the BRC+A-769662 treatment group and the BRC+ACT001+Compound C treatment group.CCK8 was used to measure the cell proliferation ability in each group.The intracellular expression levels of AMPK,EGR1,cell apoptosis associated protein(BAX,Bcl-2)and apoptosis markers Casepase-3 were measured by Western Blot.The condition of apoptosis in different groups was tested by flow cytometry.The AMPK silenced MMQ cell line was constructed by lentivirus,and the effect of drugs on the cell line was detected.Results:(1)The cell survival rate of BRC+ACT001 group was significantly lower than that of BRC group and ACT001 group(49.40±5.46%vs 69.50±3.30%,P<0.01;49.40±5.46%vs 81.00±2.09%,P<0.01).After adding A-769662,the cell survival rate of BRC+A-769662 group was similar to that of BRC+ACT001 group(49.10±4.52%vs49.40±5.46%,P>0.05).After adding Compound C,the cell survival rate of BRC+ACT001+Compound C group was similar to that of BRC group(73.83±3.97%vs 69.50±3.30%,P>0.05).(2)The expression levels of AMPK(p Thr172)and EGR1 in BRC+ACT001 group were higher than those in BRC group and ACT001 group.The expression level of BAX which promotes apoptosis and Casepase-3 were significantly higher,and the expression level of Bcl-2 which inhibits apoptosis was significantly lower in BRC+ACT001 group compared with BRC group and ACT001 group.The protein expression levels in BRC+A-769662 group were similar to that in BRC+ACT001 group after adding A-769662.After adding Compound C,the protein expression levels in BRC+ACT001+Compound C group were similar to that in BRC group.(3)The rate of apoptosis in BRC+ACT001 group was higher than that in BRC group and ACT001 group(P<0.001,P<0.001).After adding A-769662,the rate of apoptosis in BRC+A-769662 group was similar to that in BRC+ACT001 group(P>0.05).After adding Compound C,the rate of apoptosis in BRC+ACT001+Compound C group was similar to that in BRC group(P>0.05).(4)For AMPK silenced MMQ cells,the cell survival rate of BRC+ACT001+A-769662 group was lower than that of BRC+ACT001 group(43.13±1.40%vs 68.13±3.47%,P<0.001).The cell survival rate of AMPK silenced MMQ cells which were treated with BRC+ACT001 was higher than that of normal MMQ cells which were treated with BRC and ACT001(68.13±3.47%vs 35.13±2.00%,P<0.001).Conclusion:ACT001 can activate EGR1 pathway by activating AMPK and induce apoptosis of BRC-resistant MMQ cells in combination with BRC.Part four:Subcutaneous tumor-transplanted models demonstrated that ACT001 could activate AMPK and reverse the resistance of prolactinomas to CAB in combination with CABObjective:The aim was to verify that ACT001 can activate AMPK and reverse the resistance of GH3 cells to CAB in combination with CAB in vivo by subcutaneous tumor-transplanted models.Methods:The nude mouse model of prolactinoma transplanted with GH3 cells was established.The nude mice were divided into control group,CAB treatment group,ACT001 treatment group,CAB+ACT001 treatment group,and CAB+ACT001+NAC treatment group.Immunohistochemical staining was used to detect the expression levels of AMPK(p Thr172),Raptor(p Ser792)and Beclin(p Ser14)in xenografts tumors.Results:Tumor volume of nude mice in CAB+ACT001 group was smaller than that in CAB and ACT001 group(409.67±87.02mm~3 vs 1392.10±348.16mm~3,P<0.001;409.67±87.02mm~3 vs 1623.00±162.52mm~3,P<0.001),and tumor volume of nude mice in CAB+ACT001+NAC group was similar to that in CAB group(1429.00±429.64mm~3vs 1392.10±348.16mm~3,P>0.05).Results of immunohistochemical staining showed that the expression levels of AMPK(p Thr172),Raptor(p Ser792)and Beclin(p Ser14)in CAB+ACT001 group were higher than that in CAB group and ACT001 group,and the expression levels of CAB+ACT001+NAC group were similar to that in CAB group.Conclusion:ACT001 can reverse resistance of prolactinoma cells to CAB in vivo by activating AMPK in combination with CAB. |
