| Purpose:Lung Cancer is a malignant tumor with a very high incidence in our country.Among them,Non Small Cell Lung Cancer（NSCLC）accounts for about 85%of the cases.Despite the widely application of molecular targeted drugs and other interventions,platinum-based chemotherapy is still the first choice for the treatment of NSCLC,but its multiple drug resistance seriously affects clinical treatment.Drug-resistant cells are often resistant to apoptosis.Exploring the induction of cisplatin-resistant cells in NSCLC to trigger non-apoptosis-dependent cell death can provide a new strategy for its treatment.Sijunzi Decoction has the effect of anti-lung cancer and drug resistance.It is extremely important to further study its mechanism of action and explore how to increase the sensitivity of NSCLC to platinum drugs.Therefore,we first induced the adaptive response of tumor cells by glutamine deprivation,analyzed and compared the differences in metabolic phenotype of cisplatin sensitive and cisplatin resistant cells in NSCLC,attempted to clarify the metabolic characteristics of cisplatin resistant cells in NSCLC,and provided new ideas for the prevention and treatment of cisplatin resistance in NSCLC from the perspective of metabolism.Secondly,based on the in vivo/in vitro studies,the mechanism of Sijunzi Decoction sensitization to cisplatin resistant NSCLC cells was clarified from the perspective of metabolic reprogramming of tumor cells and induction of ferroptosis,in order to provide laboratory basis for the clinical application of Sijunzi Decoction in cisplatin resistant NSCLC.Material and method:Thesis 1:（1）The morphologic and survival rates of A549 and A549/DDP cells during glutamine deprivation for 48h were compared by optical microscope and CCK-8 method.（2）Firstly,the effects of glutamine deprivation on autophagy and apoptosis of A549 and A549/DDP cells were observed by transmission electron microscopy.Secondly,Annexin V-FITC/PI staining and Z-VAD-fmk were used to quantify the apoptosis rates of A549 and A549/DDP cells during glutamine deprivation for 48h.Differences in autophagy flux between A549 and A549/DDP cells treated with glutamine deprivation for 48h were compared using Bafilomycin A1.（3）Firstly,the m RNA levels of aerobic glycolysis and mitochondrial respiration related proteins were identified between A549 and A549/DDP cells by RNA-Seq,and the protein levels were verified by western blot.Secondly,Seahorse assay was undertook to compare the mitochondrial metabolic dependence of A549 and A549/DDP cells on different substrates（glucose and glutamine）,and we analyzed the differences in OCR and ATP levels between A549 and A549/DDP cells under glutamine deprivation.We measured the differences in the expression of mitochondrial respiratory chain complex protein between A549 and A549/DDP cells under glutamine deprivation by western blot,and the Si-SDHB was used to observe whether Si-SDHB could enhance the inhibitory effect of glutamine deprivation on cell survival in A549/DDP cells.（4）Western blot assay was performed to detect the difference of iron metabolism-related protein expression between A549 and A549/DDP cells under glutamine deprivation.We assessed the oxidation-antioxidant levels of A549 and A549/DDP cells by C11-BODIPY581/591,ROS and GSH under glutamine deprivation.We calculated the effect of deferoxamine（DFO）combined with glutamine deprivation on morphology and cell viability in A549 and A549/DDP cells.（5）The effects of DFO combined with glutamine deprivation on the expression of iron metabolism-related proteins and the level of total iron ions in A549/DDP cells were assessed by western blot.The effects of DFO combined with glutamine deprivation on OCR and ATP levels were obtained by Seahorse.The effects of DFO combined with glutamine deprivation on the expression of mitochondrial respiratory chain complex protein in A549/DDP cells were detected by western blot and the effects on lipid peroxidation levels were calculated by C11-BODIPY581/591.CCK-8 method was used to observe whether NAC could rescue the decreased cell survival rate induced by DFO combined with glutamine deprivation.（6）We calculated the effect of DFO combined with glutamine deprivation on mitochondrial membrane potential（MMP）of A549/DDP cells by JC-1 method.Annexin V-FITC/PI flow cytometry was used to observe the effect of DFO combined with glutamine deprivation on apoptosis rate of A549/DDP cells.CCK-8 method was used to observe whether Z-VAD-fmk could rescue apoptosis induced by DFO combined with glutamine deprivation.The effects of DFO combined with glutamine deprivation on the expression of apoptosis-related proteins in A549/DDP cells were detected by western blot.The effects of DFO combined with glutamine deprivation on autophagy levels of A549/DDP cells were observed by Bafilomycin A1.The effect of DFO combined with glutamine deprivation on the expression of MAPK signaling pathway protein in A549/DDP cells was determined by western blot.（7）BALB/c-nu mice were injected subcutaneously with A549/DDP cells to observe the inhibitory effect of DFO on tumor cell growth in vivo.Thesis 2:（1）The effect of different concentrations of Sijunzi Decoction medicated serum on the survival rate of glutamine-deprived A549/DDP cells was detected by CCK-8 method;Calcein/PI double staining was used to observe the effects of different concentrations of Sijunzi Decoction medicated serum on the cell death of glutamine-deprived A549/DDP cells.（2）Colorimetric method was used to determine the effect of Sijunzi Decoction serum combined with cisplatin on lactate production and ATP level of glutamine-deprived A549/DDP cells.The effects of Sijunzi Decoction serum combined with cisplatin on aerobic glycolysis and mitochondrial respiratory chain related protein expression in glutamine-deprived A549/DDP cells were detected by western blot.Mitotracker was used to observe the effects of Sijunzi Decoction medicated serum combined with cisplatin on mitochondrial morphology of glutamine-deprived A549/DDP cells.（3）The effect of Sijunzi Decoction combined with cisplatin on MMP of glutamine-deprived A549/DDP cells was determined by JC-1 method.DCFH-DA/Mitotracker/Hochst33342 were used to compare the effects of Sijunzi Decoction medicated serum combined with cisplatin on mitochondrial ROS levels in glutamine-deprived A549/DDP cells.CCK-8 assay was used to determine whether NAC could rescue the inhibitory effect of Sijunzi Decoction serum combined with cisplatin on the survival rate of A549/DDP cells deprived by glutamine.（4）The effect of Sijunzi Decoction combined with cisplatin on the expression of iron metabolizing protein in glutamine-deprived A549/DDP cells was detected by western blot.The effects of Sijunzi Decoction medicated serum combined with cisplatin on the content of total and intra-cellular ferrous ions in glutamine-deprived A549/DDP cell homogenate/culture were determined by colorimetric method and Ferro Orange.C11-BODIPY581/591 was used to calculated the effects of Sijunzi Decoction combined with cisplatin on lipid peroxidation levels of glutamine-deprived A549/DDP cells.In addition,colorimetric method was used to determine the effects of Sijunzi Decoction combined with cisplatin on GSH levels in glutamine-deprived A549/DDP cells.（5）Firstly,TEM was used to observe whether Ferrostatin-1 could rescue ferroptosis of glutamine-deprived A549/DDP cells induced by Sijunzi Decoction combined with cisplatin-containing serum;Secondly,DCFH-DA/Mitotracker/Hochst33342 was used to observe whether Ferrostatin-1 could rescue the ROS accumulation of glutamine deprived A549/DDP cells induced by Sijunzi Decoction combined with cisplatin-containing serum.In addition,C11-BODIPY581/591was used to observe whether Ferrostatin-1 could rescue lipid peroxidation of glutamine deprived A549/DDP cells induced by Sijunzi Decoction combined with cisplatin-containing serum.Finally,CCK-8 method was used to determine whether Z-VAD-fmk and Ferrostatin 1 could rescue the glutamine-deprived A549/DDP cell death induced by Sijunzi Decoction with cisplatin-containing serum.（6）The effect of Sijunzi Decoction serum combined with cisplatin on Keap1/Nrf2 pathway in A549/DDP cells under glutamine deprivation was detected by western blot.The effect of Sijunzi Decoction serum combined with cisplatin on the nuclear localization of Nrf2 protein in glutamine-deprived A549/DDP cells was determined by immunofluorescence method.Western blot and CCK-8were used to observe the effect of Si-Keap1 on glutamine-deprived A549/DDP cell death induced by Sijunzi Decoction serum combined with cisplatin.The effects of Sijunzi Decoction serum combined with cisplatin on the protein expressions of p62 and LC3B in glutamine deprived A549/DDP cells were detected by western blot.Finally,CCK8 method was used to observe the effect of 3-MA on A549/DDP cell death under the condition of glutamine deprivation induced by cisplatin-containing serum of Sijunzi Decoction.Thesis 3:（1）Firstly,we observed the effects of Sijunzi Decoction combined with cisplatin on C57BL/nu mice tumor growth;HE and immunohistochemical Ki67 staining were used to observe the inhibitory effect of Sijunzi Decoction combined with cisplatin on tumor cell growth in nude mice.（2）The effects of Sijunzi Decoction combined with cisplatin on glutamine metabolism,aerobic glycolysis and mitochondrial respiration related protein expression in nude mice were detected by western blot.The effects of Sijunzi Decoction combined with cisplatin on the levels of ATP and H2O2 in tumor were determined by colorimetric method.（3）Colorimetric method was also used to assess the effect of Sijunzi Decoction combined with cisplatin on total iron ion level in tumor tissues.Western blot was used to observe the effect of Sijunzi Decoction combined with cisplatin on iron metabolism related proteins in tumors.The effect of Sijunzi Decoction combined with cisplatin on the characteristic morphology of tumor tissue was observed by TEM.TUNEL staining/western blot method was used to observe the effect of Sijunzi Decoction combined with cisplatin on apoptosis of tumors.Finally,colorimetric method was used to detect the effects of Sijunzi Decoction combined with cisplatin on GSH and MDA in tumors.（4）The effects of Sijunzi Decoction combined with cisplatin on autophagy and Keap1/Nrf2 pathway related protein expression in tumors were detected by western blot.Immunofluorescence method was used to observe the effects of Sijunzi Decoction combined with cisplatin on the expression and nuclear localization of x CT,Keap1,Nrf2 proteins in tumors.Results:Thesis 1:1.Cisplatin-resistant NSCLC cells not only acquire cisplatin-resistant phenotype characteristics,but also acquire adaptation to glutamine deprivationUnder glutamine deprivation,A549 cells showed fragmented and irregular morphology,while A549/DDP cells were elongated.In addition,compared with A549 cells,A549/DDP cells showed higher cell survival rate during glutamine deprivation.2.Cisplatin-resistant NSCLC cells up-regulated protective autophagy and apoptotic resistance under glutamine deprivationFirstly,TEM showed that glutamine deprivation induced significant autophagosomes and fewer apoptotic bodies in A549/DDP cells compared with A549 cells.Secondly,flow cytometry with Annexin V-FITC/PI staining showed that glutamine deprivation significantly increased the apoptosis rate of A549 cells,but not A549/DDP cells.CCK8 method also observed that apoptosis inhibitor Z-VAD-fmk rescued glutamine deprivation induced A549cell death,but not A549/DDP cells.Moreover,glutamine deprivation inhibited the expression of anti-apoptotic protein Bcl-2 and promoted the activation of pro-apoptotic protein Bax and Caspase-3 in A549 cells,while A549/DDP cells showed significantly reduced Bax and Caspase-3,accompanied by a slight decrease in Bcl-2.Finally,protein expressions of LC3B-II and p62 in A549/DDP cells were significantly higher than those in A549 cells using the autophagy inhibitor Bafilomycin A1.3.Cisplatin-resistant NSCLC cells are more dependent on the glucose-driven mitochondrial subrespiratory chain under glutamine deprivationFirst,RNA Seq was used to verify m RNA levels associated with aerobic glycolysis and mitochondrial respiration between A549 and A549/DDP cells and confirmed the protein levels by western blot.The results demonstrated that the expressions of HK2,PKM2 and SDHB proteins in A549/DDP cells were up-regulated compared with those in A549 cells.Second,OCR measurement using Seahorse showed no difference in basal respiration and maximum respiration between the two cell lines in glutamine-containing medium,but A549/DDP cells showed higher levels of basal respiration and maximum respiration after glutamine deprivation compared to A549 cells.A549/DDP cells also had higher ATP levels.Finally,western blot confirmed that A549/DDP cells had higher expression level of ISC subunit SDHB protein under glutamine deprivation than A549 cells,and glutamine deprivation enhanced the inhibitory effect of Si-SDHB on cell survival.In addition,a survival curve（Kaplan-Meier,KM）analysis of 718 patients with lung adenocarcinoma found that high SDHB expression was associated with shorter five-year survival.4.Glutamine deprivation up-regulates the iron storage capacity of cisplatin-resistant NSCLC cells,but renders them with phenotype sensitive to iron chelating agents DFOFirst,western blot analysis showed that,compared with A549 cells,there were no difference in the expression of Tfr-1,FTH1 and Fpn protein expression in A549/DDP cells in glutamine-containing medium.Glutamine deprivation decreased the expression of FTH1protein expression in A549 cells,but not other protein related to iron metabolism.Conversely,glutamine deprivation up-regulated FTH1 and down-regulated DMT1 protein expression in A549/DDP cells.Secondly,the lipid peroxidation probe C11-BODIPY581/591 was performed to detect the difference in lipid peroxidation levels.It was found that A549/DDP cells had a lower degree of lipid peroxidation than A549 cells under glutamine deprivation.Meanwhile,under glutamine deprivation,ROS level of A549/DDP cells was significantly lower than that of A549 cells,while GSH level of A549/DDP cells was significantly higher than that of A549cells.In addition,DFO combined with glutamine deprivation significantly damaged the morphology of A549/DDP cells.In addition,CCK8 results showed that DFO did not enhance glutamine deprivation induced growth inhibition in A549 cells but significantly enhanced that in A549/DDP cells.5.DFO disrupts iron homeostasis and promote cisplatin-resistant cell death in glutamine-deprived NSCLC by inducing iron starvation response and oxidative stressFirstly,western blot showed that DFO combined with glutamine deprivation significantly up-regulated the expression of Tfr-1 and DMT1 in A549/DDP cells,while down-regulated the expression of FTH1,but not Fpn.At the same time,DFO combined with glutamine deprivation significantly decreased the concentration of total iron in A549/DDP cells.Secondly,we found that DFO combined with glutamine deprivation significantly down-regulated basal respiration and maximum respiration by Seahorse assay,as well as the ATP levels in A549/DDP cells.Meanwhile,DFO combined with glutamine deprivation significantly down-regulated C-I/II/III protein expression in A549/DDP cells.Finally,we observed that DFO combined with glutamine deprivation significantly induced lipid peroxidation in A549/DDP cells by C11-BODIPY581/591.Moreover,CCK-8 showed that NAC could completely rescue the cell death induced by DFO combined with glutamine deprivation.6.DFO promotes cisplatin-resistant cell apoptosis and autophagic cell death in glutamine-deprived NSCLC through ROS-dependent JNK signalingFirstly,the JC-1 and Annexin V-FITC/PI fluorescent probes were used.We found that DFO combined with glutamine deprivation significantly down-regulated MMP and up-regulated the apoptosis rate of A549/DDP cells.In addition,Z-VAD-fmk completely rescued the apoptosis of A549/DDP cells induced by DFO combined with glutamine deprivation by CCK-8 assay.Moreover,DFO combined with glutamine deprivation significantly induced the activation of Bax and cleaved Caspase-3 while inhibited Bcl-2 in A549/DDP cells.Secondly,DFO combined with glutamine deprivation significantly induced autophagy in A549/DDP cells using the autophagy inhibitor Bafilomycin A1.Finally,CCK-8assay also confirmed that DFO combined with glutamine deprivation up-regulated the phosphorylation of JNK protein in A549/DDP cells,but not p-p38 and p-ERK,and the JNK1-specific inhibitor JNK-in-8 completely rescue cell death induced by DFO combined with glutamine deprivation in A549/DDP cells.7.DFO inhibit the growth of cisplatin-resistant lung adenocarcinoma in NSCLCWhen BALB/c-nu mice were subcutaneously injected with A549/DDP cells,it was observed that DFO did not affect overall body weight and tumor weight,but DFO significantly slowed the growth of tumors in male nude mice,but not female.Thesis 2:1.Sijunzi Decoction medicated serum enhanced cell death in cisplatin-resistant NSCLC under glutamine deprivationFirstly,CCK-8 assay was used to assess the effect of different concentrations of Sijunzi Decoction medicated serum on the survival rate of A549/DDP cells under glutamine deprivation.It was found that Sijunzi Decoction medicated serum combined with cisplatin could significantly inhibit cell survival,and the effect was time-dependent and concentration-gradient dependent.That is,high-dose Sijunzi Decoction medicated serum combined with cisplatin（combination group）continuously interfered with A549/DDP cells for 48h and showed the greatest tumor killing effect.Secondly,Calcein/PI double staining showed enhanced cell death in combination group compared with cisplatin group under glutamine deprivation.2.Sijunzi Decoction medicated serum induced abnormal mitochondrial energy metabolism in cisplatin-resistant NSCLC under glutamine deprivationFirst of all,there was no difference in ATP level while lactate production down-regulated between the combination group and the cisplatin group.Secondly,western blot analysis showed that Glut1,HK2,PDH,TIGAR,NDUFB8 and SDHB were up-regulated in the combination group compared with those cisplatin group,while LDHA was down-regulated.In addition,we found that mitochondrial networks changed from linear and uniformly distributed to clustered by Mitotracker staining in the combination group.3.Sijunzi Decoction medicated serum induced oxidative stress in cisplatin-resistant NSCLC under glutamine deprivationFirstly,the MMP in the combination group was decreased compared with the cisplatin group by JC-1 assay.Secondly,we found an increase of mitochondrial ROS in the combination group by DCFH-DA/Mitotracker/Hochst33342 compared with cisplatin group,.In addition,CCK8 method observed that in the presence of NAC,the inhibitory effect of combination group on cell survival was eliminated compared with cisplatin group.4.Sijunzi Decoction medicated serum induced ferrous ion accumulation and lipid peroxidation in cisplatin-resistant NSCLC under glutamine deprivationFirstly,western blot analysis showed that compared with cisplatin group,the expression of Tfr-1 and DMT1 protein in the combination group were up-regulated,while the expressions of FPN and FTH1 protein were down-regulated.In addition,colorimetric and Ferro Orange assay confirmed that compared with cisplatin group,total iron in culture medium showed no difference,while in cell homogenate and intracellular ferro-bivalent ion content increased.Secondly,the lipid peroxidation probe C11-BODIPY581/591 was increased in the combination group compared with that in the cisplatin group.Meanwhile,GSH level was decreased in combination group compared with cisplatin group.5.Sijunzi Decoction medicated serum induced ferroptosis in cisplatin-resistant NSCLC under glutamine deprivationFirstly,transmission electron microscopy showed that compared with cisplatin group,mitochondria in combination group became smaller and membrane density became thicker.Compared with the combined group,Ferrostatin-1,an iron death inhibitor,restored mitochondrial morphology.Second,fluorescent probe DCFH-DA/Mitotracker/Hochst33342observation found that compared with joint group,iron death inhibitors can be lowered mitochondrial ROS accumulation Ferrostatin-1.In addition,lipid peroxidation probe C11-BODIPY581/591 observed that Ferrostatin-1 could reduce lipid peroxidation levels compared with the combined group.Finally,CCK8 compared the effects of Z-VAD-fmk,an inhibitor of apoptosis,and Ferrostatin-1,an iron death inhibitor,and found that,compared with the combined group,Ferrostatin 1 can completely save the A549/DDP cell death induced by cisplatin-induced glutamine deprivation in Sijunzi Decoction serum and Z-VAD-fmk（5m M;48 h）could not save the glutamine deprivation A549/DDP cell death induced by cisplatin-containing serum of Sijunzi Decoction;More importantly,in the presence of Ferrostatin-1,Sijunzi Decoction medicated serum could not up-regulate A549/DDP cell death under cisplatin-induced glutamine deprivation,while in the presence of Z-VAD-fmk,Sijunzi Decoction medicated serum could still up-regulate A549/DDP cell death under cisplatin-induced glutamine deprivation.6.Sijunzi Decoction medicated serum induced autophagy dependent ferroptosis in cisplatin-resistant NSCLC and inhibited p62/Keap1/Nrf2 signaling pathway under glutamine deprivationFirst,western blot observation showed that,compared with cisplatin group,Keap1protein expression was up-regulated while Nrf2 and x CT protein expression was down-regulated in combination group.In addition,immunofluorescence method determined that Nrf2 protein nuclear localization decreased in the combination group.Secondly,Si-Keap1 and western blot method were used to verify the efficiency of Si,and it was found that compared with the Si-NC group,the protein expressions of Nrf2,x CT and FTH in Si-Keap1 group were up-regulated.In addition,CCK8 method showed that compared with the Si-NC group,The survival rate of glutamine-deprived A549/DDP cells was significantly decreased in Si-Keap1 group whether cisplatin alone or Sijunzi Decoction medicated serum combined with cisplatin.Finally,western blot analysis showed that compared with cisplatin group,LC3B-II protein expression was up-regulated and p62 protein expression was down-regulated in combination group.Moreover,the effect of 3-MA,an autophagy inhibitor,was observed by CCK8 method,and it was found that after the use of 3-MA to inhibit autophagy,compared with cisplatin group,the combined group could not up-regulate the death of A549/DDP cells under glutamine deprivation.Thesis 3:1.Sijunzi Decoction combined with cisplatin significantly inhibited the growth of cisplatin resistant cells in NSCLCC57BL/nu mice inoculated with A549/DDP cells by continuous gavage of high-dose Sijunzi decoction at 21 days showed no difference in body weight and tumor rereduction compared with cisplatin group.In addition,HE and immunohistochemical Ki67 staining showed that compared with the cisplatin group,the cells in the combination group were more loosely arranged and the expression of Ki67 protein was down-regulated.2.Sijunzi Decoction inhibited glutamine decomposition in cisplatin-resistant NSCLC cells and affected mitochondrial energy metabolismFirst of all,Western blot detection of the expression of glutamine metabolizing enzyme and transporter protein found that,compared with cisplatin group,GLS1 protein expression decreased in the combined group,but there was no difference in the expression of GS and SLC1A5 protein.Compared with cisplatin group,no difference was found in ATP level in combination group.Western blot analysis showed that Glut1,HK2,PDH,TIGAR,PGC-1α,NDUFB8 and SDHB in the combination group were up-regulated compared with cisplatin group.In addition,the H2O2 level detected in the combination group was significantly higher than that in the cisplatin group.3.Sijunzi Decoction induced lipid peroxidation and ferroptosis in cisplatin-resistant NSCLC cellsFirst,colorimetric method was used to detect total iron ion level,and it was found that compared with cisplatin group,total iron ion level in combination group was up-regulated.western blot also observed that compared with cisplatin group,the expressions of Tfr-1,DMT-1 and FTH1 proteins in combination group were up-regulated,while there was no difference in the expression of Fpn proteins.Secondly,transmission electron microscopy showed that compared with cisplatin group,mitochondria in combination group became smaller and membrane shrank more.TUNEL staining showed no difference in apoptosis rate between the combination group and cisplatin group.western blot also verified the changes of apoptotic proteins,that is,there were no differences in cleaved-PARP,cleaved-Caspase3,Bcl2 and Bax in the combined group compared with the cisplatin group.Finally,colorimetry was used to detect lipid peroxidation end products MDA and antioxidant GSH.Compared with cisplatin group,GSH level was down-regulated and MDA level was up-regulated in combination group.4.Sijunzi Decoction induced autophagy of cisplatin-resistant NSCLC cells and inhibited Keap1/Nrf2 pathwayWestern blot showed that compared with cisplatin group,LC3B-II and Keap1 protein expressions were up-regulated,while p62,Nrf2 and x CT protein expressions were down-regulated in combination group.In addition,immunofluorescence method showed that compared with cisplatin group,Keap1 protein expression was up-regulated,x CT and Nrf2protein expression was down-regulated and Nrf2 nuclear localization was down-regulated in combination group.Conclusion:1.Firstly,Cisplatin-resistant NSCLC cells show a Hybrid Metabolic Phenotype,that is,both up-regulated glycolysis and OXPHOS;Secondly,Cisplatin-resistant NSCLC cells acquire cisplatin-resistant phenotype while enhancing their adaptation to metabolic stress such as glutamine deprivation,and exhibit metabolic characteristic that are more dependent on glucose driven mitochondrial sub-respiratory chain.Finally,Adaptive response in Cisplatin-resistant NSCLC cells confer a greater dependence on iron metabolism and a phenotype sensitive to iron chelators such as DFO.2.Sijunzi Decoction enhances glucose-driven mitochondrial respiration in tumor cells and sensitises Cisplatin-resistant NSCLC cells by inhibiting the p62/Keap1/Nrf2 pathway and inducing lipid peroxidation and autophagy dependent ferroptosis. |
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