Effect And Mechanism Of Clearing-heat Chinese Medicine Herbs On Reversing Gefitinib Resistance Induced By PI3K-pathway Activation In Non-small Cell Lung Cancer

ObjectiveEpidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI)is the first choice for advanced non-small cell lung cancer(NSCLC)patients harboring EGFR sensitive mutation.However,there occurs high resistance rate,which limits the clinical efficacy of EGFR-TKI.The abnormal activation on PI3K/AKT/mTOR pathway(PI3K pathway,the same below)is an important EGFR-TKI resistance mechanism,without effective treatments.In order to find out traditional Chinese medicine(TCM)methods to reverse EGFR-TKI resistance,a series of studies will be needed,to clarify the TCM theory,treatment principle,appropriate decoction,relevant drug,and treatment targets.In this paper,we focused on the TCM theory and treatment principle,and carried out two parts of researches.Part Ⅰ:Clinical investigation.To explore the correlation between Traditional Chinese Medicine(TCM)syndrome types,PI3K pathway related genotypes,and EGFR-TKI efficacy in NSCLC patients,so as to clarify the TCM key pathogenesis for EGFR-TKI resistance induced by abnormal activation on PI3K pathway.Part Ⅱ:In vitro experiments.Based on the key TCM pathogenesis revealed in clinical investigation for EGFR-TKI resistance(theory),the purpose of the in vitro experiment was to clarify the appropriate TCM treatment principles to reverse EGFR-TKI resistance.Effect of herbs with different TCM therapeutic principles,clearing-heat or warming-yang,were compared,when combining with gefitinib,on EGFR-TKI resistant NSCLC models with abnormal activation on PI3K pathway.Mechanism of these TCM herbs in reversing EGFR-TKI resistance were also revealed.MethodsPart Ⅰ:Clinical investigationIn this case series analysis study,patients with NSCLC were consecutively enrolled.TCM syndrome types were diagnosed respectively by three TCM experts,to decide whether the patient was with qi deficiency,yin deficiency,heat-toxicity,blood stasis or phlegm dampness TCM syndromes.Using a next generation sequencing panel with 168 genes,matched tumor biopsy and plasma samples prior to any therapies from NSCLC patients were detected.The correlation of TCM patterns with the genetic alterations was analyzed.Patients treated with EGFR-TKI were followed up regularly by imaging examination,and the correlation between TCM syndrome types,genetic types,and progression free survival(PFS)were analyzed.Part Ⅱ:In vitro experiments1.Drug sensitivity study:human lung cancer cell line PC-9 cells(deletion of exon 19 of EGFR gene)were transfected with PIK3CA mutant plasmid lentivirus to construct EGFR/PIK3CA concurrent mutant PC-9-PIK3CA-M cell lines.Using PC-9-PIK3CA-M cells and H1650 cells(exon 19 deletion of EGFR gene combined with PTEN deletion)as the gefitinib resistance model caused by abnormal activation on PI3K pathway,freeze-dried powder of four herbs,which are all with clearing-heat TCM treatment principle but have different characteristics,Scutellaria Baicalensis(Huang-qin,drying dampness and clearing heat),Radix Trichosanthis(Tian-hua-fen,nourishing yin and clearing heat),Rhizoma Fzgopyri Dibotryis(Jin-qiao-mai,clearing heat and detoxification),and Anemarrhena Asphodeloides(Zhi-mu,clearing deficient heat)were selected to represent the clearing-heat treatment principles;The freeze-dried powder of dried Ginger(Gan-jiang),with warming-yang TCM treatment principle,was selected as the positive control.All of these 5 TCM herbs affect on the lung meridian.The 3D cultured PC-9-PIK3CA-M and H1650 cell clusters were treated with clearing-heat herbs,warming-yang herbs,gefitinib,clearing-heat herbs combining with gefitinib,and warming-yang herbs combining with gefitinib,respectively.The cell proliferation rate was detected using ATP method;the cell activity was detected by Edu method;the cell apoptosis rate was detected by Annexin V-FITC/PI method;so as to evaluate whether the herbs with clearing-heat TCM treatment principle were with synergistic effect with gefitinib.Lung cancer organoids derived from NSCLC patients harboring EGFR/PIK3CA concurrent mutation were also studied as a gefitinib resistance model caused by abnormal activation on PI3K pathway,and were treated with the above drugs.The cell proliferation rate was detected by ATP method to verify the synergistic effect of clearing-heat TCM herbs with gefitinib.With these resear-ches,we want to verify whether "clearing-heat"TCM treatment principle should be used to reverse gefitinib resistance caused by abnormal activation on PI3K pathway.2.Mechanism study:3D cultured PC-9-PIK3CA-M cell clusters and H1650 cell clusters were used as gefitinib resistance models caused by abnormal activation on PI3K pathway,and were treated with the above drugs.The cell optical redox ratio was detected by femtosecond label-free imaging microscopy,and the cell reactive oxygen species(ROS)level was detected by confocal micro fluorescence technology.The mRNA and protein expression levels of sex-determining region Y[SRY]-box(SOX2)and aldehyde dehydrogenase 1A1(ALDH1A1)were detected by qRT-PCR and Western blot,respectively.With these researches,we want to reveal the mechanism of clearing-heat TCM herbs on reversing gefitinib resistance,from the perspective of cell metabolism and cancer stem cells.ResultsPart Ⅰ:Clinical investigation1.A total of 109 NSCLC patients were enrolled.Of the 5 TCM syndrome types recorded,only TCM heat-toxicity syndrome type was significantly associated with genetic alterations.A total of 26 patients(23.85%)were diagnosed with TCM heat-toxicity syndrome type.There was a significant enrichment of PIK3CA(23.08%vs.8.43%,P=0.044),MYC,PMS2,and NTRK1 genetic alterations in heat-toxicity patients.There was no significant correlation between genetic types and other TCM syndrome types.Multivariate logistic regression model indicated that PIK3CA gene mutation was independently associated with heat-toxicity TCM syndrome type(OR=3.72,95%CI:1.0213.55,P=0.047).2.A total of 48 NSCLC patients harboring sensitive EGFR mutation were treated with EGFR-TKI.In these patients,only TCM heat-toxicity syndrome type was significantly associated with genetic alterations.A total of 13 patients(27.08%)were diagnosed with TCM heat-toxicity syndrome type.There was a significant enrichment of PIK3CA(38.46%vs.8.57%,P=0.025),MYC,and PMS2 genetic alterations in heat-toxicity patients.NTRK1 genetic alteration was also enriched,although not significantly,in heat-toxicity patients.There was no significant correlation between genetic types and other TCM syndrome types.Multivariate logistic regression model indicated that PIK3CA gene mutation was independently associated with heat-toxicity TCM syndrome type(OR=7.90,95%CI:1.15-54.00,P=0.035).Patients with heat-toxicity TCM syndrome type achieved significantly worse median PFS after EGFR-TKI treatment than those without heat-toxicity TCM syndrome type(5.13 vs.10.20 months,P=0.0045).There was no significant correlation between PFS and other TCM syndrome types or clinical characteristics.3.In the 48 NSCLC patients treated with EGFR-TKI,RBI(4.70 vs.10.20 months,P=0.0005),ERBB2(3.20 vs.10.20 months,P<0.0001),and PIK3CA(4.57 vs.10.20 months,P=0.0002)co-alterations beyond EGFR sensitive mutation,as well as more concurrent mutated gene number and co-alterated BRCA1,SMAD4 and NFE2L2 genes,were significantly associated with worse PFS.There was no significant correlation between PFS and other co-alterated genes.4.Multivariate Cox regression model indicated that the clearing-heat TCM syndrome type(HR=2.56,95%CI:1.02-6.47,P=0.046),concurrent RBI(HR=3.69,95%CI:1.33-10.25,P=0.012),ERBB2(HR=4.73,95%CI:1.07-20.81,P=0.040),and PIK3CA(HR=3.32,95%CI:1.09-10.15,P=0.035)genetic alterations were independently related with poorer PFS after EGFR-TKI treatment.In these genes,both ERBB2 and PIK3CA are PI3K pathway related genes.Part Ⅱ:In vitro experiments1.Effect on cell proliferation:In the 3D cultured PC-9-PIK3CA-M and H1650 cell clusters,gefitinib combining with Scutellaria Baicalensis(Huang-qin),Anemarrhena Asphodeloides(Zhi-mu),and Radix Trichosanthis(Tian-hua-fen)can significantly inhibit cell activity and proliferation,when compared with gefitinib alone.Especially,Scutellaria Baicalensis(Huang-qin)and Anemarrhena Asphodeloides(Zhi-mu)showed most obvious effect to reverse gefitinib resistance.By contrary,gefitinib combining with the warming-yang dried Ginger(Gan-jiang)even increased cell activity.2.Effect on cell apoptosis:In the 3D cultured PC-9-PIK3CA-M and H1650 cell clusters,all of the four clearing-heat TCM herbs,when combing with gefitinib,can significantly increase the cell apoptosis rate,when compared with gefitinib alone.By contrary,in the PC-9-PIK3CA-M cell clusters,gefitinib combining with the warming-yang dried Ginger(Gan-jiang)even significantly reduced the apoptosis rate.3.In the lung cancer organoids harboring EGFR/PIK3CA concurrent mutations,Scutellaria Baicalensis(Huang-qin)combining with gefitinib can significantly inhibit cell activity,when compared with gefitinib alone,while the other TCM herbs has no effect on cell activity.4.Effect on cell metabolism:In the 3D cultured PC-9-PIK3CA-M and H1650 cell clusters,gefitinib significantly decreased the cell redox ratio and ROS level.Gefitinib combining with Scutellaria Baicalensis(Huang-qin),and Radix Trichosanthis(Tian-hua-fen)can significantly increase the cell redox ratio and ROS level,when compared with gefitinib alone,while the other TCM herbs have no effect on cell metabolism.5.Effect on cancer stem cells:In the 3D cultured PC-9-PIK3CA-M cell clusters.gefitinib significantly increased the mRNA and protein expression of stem cell markers SOX2 and ALDH1A1.Gefitinib combining with Scutellaria Baicalensis(Huang-qin),Anemarrhena Asphodeloides(Zhi-mu),and Radix Trichosanthis(Tian-hua-fen)can significantly own-regulate the expression of SOX2 and ALDH1A1 mRNA and protein,when compared with gefitinib alone.The dried Ginger(Gan-jiang),with warming-yang effect,has no effect on expression of ALDH1A1 or SOX2 protein.Conclusions1.The hecat-toxicity TCM syndrome type was associated with the abnormal activation on PI3K pathway,both of which were independently associated with poorer prognosis after EGFR-TKI treatment.These results indicated that the heat-toxicity TCM syndrome type was one of the key pathogeneses of EGFR-TKI resistance caused by the abnormal activation on PI3K pathway in lung cancer patients.2.In the 3D cultured PC-9-PIK3CA-M cell clusters,H1650 cell clusters,and the lung cancer organoids harboring EGFR/PIK3CA concurrent mutations,representing gefitinib resistance models with abnormal activation on PI3K pathway,clearing-heat TCM herbs can reverse gefitinib resistance.Especially,Scutellaria Baicalensis(Huang-qin)and Anemarrhena Asphodeloides(Zhi-mu)showed synergistic effects with gefitinib in many models.On the contrary,dried Ginger(Gan-jiang),with warming-yang TCM effect,showed antagonistic effect.These results suggested that clearing-heat TCM treatment principle should be used to reverse EGFR-TKI resistance caused by abnormal activation on PI3K pathway in lung cancer.This finding also verified with the above key pathogenesis of heat-toxicity TCM syndrome type.3.The clearing-heat TCM herbs,Scutellaria Baicalensis(Huang-qin)and Radix Trichosanthis(Tian-hua-fen)can reverse gefitinib resistance by up-regulating redox ratio and ROS level and down-regulating cancer stem cell markers.Although the synergistic effect of Anemarrhena Asphodeloides(Zhi-mu)and gefitinib also affected on cancer stem cells,it has no effect on cell metabolism,and may reverse drug resistance through other mechanisms.