Exploring The Mechanism And Clinical Study Of FuZheng Kang-Ai Decoction Combined With Gefitinib In The Treatment Of Non-small Cell Lung Cancer Based On FBW7/MCL-1 Signal Axis

Objective:1.The effects of FuZheng Kang-Ai decoction combined with gefitinib on the proliferation,apoptosis,invasion and metastasis of A549 cells and the expression of F-box and WD repeat domain containing protein 7(FBW7)and myeloid cell leukemia-1(MCL-1)were observed in vitro;2.To further explore the anti-tumor mechanism of FuZheng Kang-Ai decoction combined with gefitinib on A549 transplanted tumor model mice through FBW7/MCL-1 signal axis in vivo;3.To investigate the clinical significance of tumor suppressor FBW7 and cancer-promoting factor MCL-1 in non-small cell lung cancer(NSCLC)in order to further clarify the role of FBW7/MCL-1 signal axis in invasion and metastasis of non-small cell lung cancer and its impact on prognosis of patients;4.To observe the clinical efficacy and adverse reactions of Fuzheng Anti-cancer Prescription combined with gefitinib in the treatment of advanced non-small cell lung cancer with EGFR gene mutation.Method:1.In vitro experiment:The A549 cell proliferation of the control group,FuZheng Kang-Ai decoction groups(0.2,0.4,0.8,1.6,3.2g·L-1),Gefitinib groups(10,20,40,60,80,100 μ M·L-1)for 24,48,72 hours,and FuZheng Kang-Ai decoction(2g·L-1)combined with Gefitinib(40 μ M·L-1)groups for 24 hours was detected by MTT assay;The changes of cell apoptosis,invasion and metastasis ability on A549 cells were analyzed by flow cytometry,Wound Healing,transwell invasion assay.Western blot assay was used to examine the protein expression of Cleaved-Caspase-3,B-cell lymphoma-2(Bcl-2),B-cell lymphoma-2 associated X(Bax),F-box and WD repeat domain-containing(FBW7)and myeloid cell leukemia-1(MCL-1)in vitro.2.In vivo experiment:The model of axillary implantation of non-small cell lung cancer in nude mice was established and divided into different groups according to the administration mode(blank control group,FuZheng Kang-Ai decoction group 7.4g/kg,gefitinib group 51.3mg/kg,FuZheng Kang-Ai decoction 7.4g/kg combined with gefitinib group 51.3mg/kg).In vivo imaging was used to observe the growth of tumors in mice.The volume and weight of transplanted tumors in each group were compared.The apoptotic index was detected by TUNEL method.The expression of Bcl-2,Bax,Cleaved-Caspase-3,FBW7 and MCL-1 in transplanted tumors was detected by Western blot.3.Retrospective analysis of 30 cases of non-small cell lung cancer tissue and adjacent tissue specimens,detection of FBW7 and MCL-1 expression in non-small cell lung cancer and adjacent tissues by immunohistochemistry,the patients with NSCLC were grouped according to the expression of protein,and the main clinicopathological characteristics and prognosis of the patients with high expression and low expression were analyzed.4.A randomized controlled study was conducted,Forty-three patients with advanced non-small cell lung cancer who met the inclusion and exclusion criteria were randomly divided into treatment group(21 cases)and control group(22 cases).The treatment group was given gefitinib+Fuzheng Anticancer Decoction,while the control group was given gefitinib only.Treatment to cancer progression or death or intolerable adverse reactions.The progression-free survival(PFS),overall survival(OS),objective response rate(ORR),disease control rate(DCR),PS score,quality of life score(KPS score)and adverse reactions were observed.Result:1.The mechanism of Fuzheng Anticancer Prescription combined with gefitinib inhibiting NSCLC proliferation through FBW7/MCL-1 signal axis:1.1 In vitro experiment:Compared with the blank group,FuZheng Kang-Ai decoction and gefitinib significantly inhibited the proliferation of A549 cells in a dose-dependent and time-dependent manner(P<0.05).Compared with the blank group,FuZheng Kang-Ai decoction group and Gefitinib group could reduce the wound healing ability and invasive ability of cells,promote cell apoptosis(P<0.05),increase the expression of Bax,Cleaved-Caspase-3,FBW7,and down-regulate the expression of Bcl-2 and MCL-1(P<0.05).Compared with gefitinib alone,FuZheng Kang-Ai decoction combined with gefitinib inhibited the proliferation and induced apoptosis of A549 cells more significantly(P<0.05).Compared with gefitinib alone,the healing and invasiveness of scratches were significantly weakened(P<0.05);the expression of Bax,Cleaved-Caspase-3 and FBW7 protein was up-regulated by FuZheng Kang-Ai decoction combined with gefitinib,and the expression of Bcl-2 and MCL-1 protein was down-regulated significantly better than that of gefitinib alone(P<0.05).1.2 In vivo experiment:The weight,volume and fluorescence intensity of transplanted tumor were significantly inhibited in FuZheng Kang-Ai decoction group,gefitinib group and combined administration group,and the inhibition effect of combined administration group was significantly better than that of gefitinib alone group(P<0.05),the results were consistent with the in vitro experiment.The expression of FBW7 and MCL-1 in transplanted tumors of nude mice was detected by Western blot,The results showed that the expression of FBW7 and the expression of MCL-1 were significantly down-regulated in FuZheng Kang-Ai decoction group,Gefitinib group and combined administration group(P<0.05).Moreover,the combination group was significantly better than gefitinib alone group(P<0.05),and the results were consistent with in vitro experiments.2.Expression of FBW7/MCL-1 in human non-small cell lung cancer and its relationship with biological behavior:The expression of FBW7/MCL-1 in tumor tissue(T)and adjacent 5 cm lung tissue(N)was detected by immunohistochemical staining.The results showed that MCL-1 was expressed in both cytoplasm and nucleus,but the expression level in cytoplasm was higher.There was no significant expression of FBW7 in tumors and adjacent tissues.Compared with 5 cm adjacent lung tissues,MCL-1 protein was significantly higher in lung cancer tissues(P<0.05).The relationship between MCL-1 expression and clinicopathological characteristics and prognosis of 30 patients with non-small cell lung cancer was analyzed.The high expression of MCL-1 had no significant effect on the prognosis of patients with non-small cell lung cancer(P<0.05).The prognosis of NSCLC patients was significantly affected by tumor stage(P<0.05).There was no significant difference in the prognosis of non-small cell lung cancer in terms of sex,age,smoking or pathological type(P<0.05).3.Clinical study of FuZheng Kang-Ai decoction combined with gefitinib in the treatment of advanced non-small cell lung cancer:3.1 Analysis of general clinical features There was no significant difference in general clinical characteristics between the two groups before treatment(P<0.05),and the two groups were comparable.3.2 Short-term efficacy3.2.1 Comparison of short-term efficacy between two groupsThe ORR of treatment group and control group were 76.2%and 54.5%respectively,and the DCR of treatment group and control group were 95.2%and 72.7%respectively.There was no significant difference in ORR(P<0.05)and DCR(P=0.046)between the two groups.3.2.2 Stratified analysis of short-term efficacy in treatment groupDCR was 100%in adenocarcinoma patients and 0%in non-adenocarcinoma patients(P<0.05).There was no significant difference in ORR and DCR in other patients with different clinical characteristics(P<0.05).3.2.3 Stratified analysis of short-term efficacy in control groupAmong the patients with different mutation types,the DCR of exon 18 mutation,exon 19 deletion,exon 20 mutation and exon 21 mutation were 0%,90%,0%and 87.5%,respectively,with significant difference(P<0.05);the ORR and DCR of the other patients with different clinical characteristics had no significant difference(P<0.05).3.2.4 Stratified analysis of short-term efficacy between two groupsThe ORR of patients under 65 years old was 86.7%in the treatment group and 47.1%in the control group(P<0.05).Among the patients with smoking history,DCR was 100%in the treatment group and 50%in the control group(P<0.05).Among the patients with adenocarcinoma,DCR was 100%in the treatment group and 70%in the control group(P<0.05).DCR was 100%in the treatment group and 64.3%in the control group(P<0.05).Among the patients with IIIB,DCR was 100%in the treatment group and 64.3%in the control group(P<0.05).There was no significant difference between ORR and DCR groups in other patients with different clinical characteristics(P<0.05).3.3 Long-term efficacy3.3.1 Comparison of disease progression-free survival between two groupsThe median PFS was 13 months in the treatment group and 10 months in the control group.There was significant difference in progression-free survival between the two groups(P<0.05).3.3.2 Stratified analysis of disease progression-free survival in treatment groupIn the stratified analysis of the treatment group,the median PFS of women in different genders was 17 months,while the median PFS in men was 9 months,with a statistically significant difference(P<0.05).The median PFS of 18 exon mutation,19 exon deletion,20 exon mutation and 21 exon mutation in different mutation types were 5 months,15.59 months,3 months and 11 months respectively(P<0.05).There was no significant difference in the median PFS among the other patients with different clinical characteristics(P<0.05).3.3.3 Stratified analysis of disease progression-free surv-ival in treatment groupThe median PFS of 18 exon mutation,19 exon deletion,20 exon mutation and 21 exon mutation in different mutation types were 2,10,2 and 8 months respectively(P<0.05).There was no significant difference in the median PFS among the other patients with different clinical characteristics(P<0.05).3.3.4 Stratified analysis of progression-free survival between two groupsIn female patients,the median PFS was 17 months in the treatment group and 12 months in the control group(P<0.05).The median PFS was 17 months in the treatment group and 9 months in the control group(P<0.05).The median PFS was 15.59 months in the treatment group and 11.29 months in the control group(P<0.05).There was no significant difference in the other patients with different clinical characteristics between the median PFS group(P<0.05).3.4 KPS scoreThere was no significant difference between the two groups before treatment(P<0.05).There was significant difference between the two groups after treatment(P<0.01).There was significant difference in the treatment group before and after treatment(P<0.05).There was no significant difference in the control group before and after treatment(P<0.05).Before treatment,the KPS scores of the two groups were comparable;after treatment,the KPS scores of the treatment group were significantly higher than those of the control group.3.5 Side effectsIn the treatment group,there were 2 cases of skin rash and 10 cases of skin rash in the control group.There was significant difference between the two groups(P<0.05).No interstitial pneumonia occurred in all patients.There were 2 cases of diarrhea in the treatment group and 4 cases in the control group.There was no significant difference between the two groups(P<0.05).There were 2 cases of abnormal liver function in the treatment group and 3 cases in the control group.There was no significant difference between the two groups(P<0.05).There was no oral mucositis in the treatment group and 2 cases in the control group.There was no significant difference between the two groups(P<0.05).Conclusion:1.In vitro,FuZheng Kang-Ai decoction inhibited A549 cell invasion and migration in a concentration-dependent manner;Fuzheng Kang-Ai Decoction combined with gefitinib could induce A549 cell apoptosis,inhibit A549 cell proliferation and invasion and metastasis more effectively than gefitinib alone.Both of them had synergistic anti-cancer effect,and the mechanism might be related to the regulation of FBW7/MCL-1 pathway.2.In vivo experiments further confirmed that Fuzheng Kang-Ai Decoction combined with gefitinib may inhibit the proliferation of non-small cell lung cancer A549 cell line in vivo through FBW7/MCL-1 signaling pathway,and the combination of drugs has a stronger inhibitory effect on the proliferation of A549 cells.3.30 cases of human non-small cell lung cancer tissues showed no difference in FBW7 expression.There was positive expression of MCL-1.MCL-1 was localized in the cytoplasm of cells.The expression level of FBW7 in tumor tissues was significantly higher than that in adjacent tissues.The difference was statistically significant.4.The expression of MCL-1 has no significant relationship with the biological behavior of NSCLC and the prognosis of NSCLC patients.Multivariate analysis showed that tumor staging was an independent risk factor for the prognosis of NSCLC patients.5.FuZheng Kang-Ai decoction combined with gefitinib can can improve the disease control rate of non-small cell lung cancer patients,prolonging progression-free survival in patients with non-small cell lung disease.It has synergistic anti-cancer effect.FuZheng Kang-Ai decoction combined with gefitinib can reduce the incidence of skin rash and the side effects of Gefitinib,which has the effect of reducing toxicity.