| Background:Lung cancer is the most common malignant tumor in clinical practice,ranking first in terms of incidence and mortality.Small cell lung cancer(SCLC)is a special type of lung cancer,with about 323,000 new cases per year worldwide,accounting for about 15%of all lung cancers.About 60%-70%of small cell lung cancer patients are already in Extensive Disease(ED)at the time of diagnosis,losing the opportunity for surgical treatment.Unlike non-small cell lung cancer,small cell lung cancer currently has no targeted drugs that explicitly target the driver gene available,and clinical choices are limited to anti-vascular targeted drugs such as Bevacizumab injection and Anlotinib Hydrochloride Capsules,which have large side effects and are highly restricted in application.With the advent of tumor immunotherapy era,immunotherapy brings great hope to patients,but because the predictive index of immune efficacy is unclear,only a small number of clinical trials have seen the exact efficacy,and at the same time,immune drugs are expensive and have more potential adverse drug reactions,making clinical promotion more difficult.In summary,chemotherapy remains the most important medical treatment for small cell lung cancer,and etoposide combined with platinum or irinotecan combined with platinum remains the first choice for small cell lung cancer,with a response rate of 70-80%.However,the difficulties often faced in clinical treatment are:patients are easily resistant to chemotherapy,and most patients will relapse within a few months;on the other hand,patients are less tolerant to chemotherapy,and their physical status decreases dramatically during chemotherapy,and although the initial treatment may have better chemotherapy effect,the disease will relapse and progress quickly when treatment is discontinued due to intolerance of chemotherapy.The mechanism of chemoresistance mainly involves the alteration of genetic or epigenetic regulation of key genes,and the process of drug resistance due to epigenetic regulation is reversible,therefore,it has also been suggested that the alteration of epigenetic regulation may become a research direction to reverse chemoresistance and enhance the efficacy of chemotherapy.In recent years,Cyclin-dependent kinase inhibitors(CDKIs),a large class of proteins,have been proposed to exert tumor suppressive effects by regulating cell proliferation and differentiation,cell cycle progression,and through the histone methyltransferase gene EZH2(Enhancer of The epigenetic pathway in which the histone methyltransferase gene EZH2(Enhancer of zeste homolog 2,EZH2)is located regulates tumor growth,but the exact mechanism remains to be explored.The advantages of Traditional Chinese Medicine(TCM)in the treatment of malignant tumors are mainly in prolonging patient survival,enhancing the efficacy of radiotherapy and chemotherapy,improving patients’ tolerance,and improving patients’ quality of life.According to Chinese medicine,"Buhe" is the cause of disease.Therefore,this study aims to summarize and review the literature on the treatment of malignant tumors by "He Fa",and to conduct a theoretical discussion on the treatment of small cell lung cancer by"Hehuan xiaoji".Based on the theoretical discussion of the treatment of small cell lung cancer by "Hehuan xiaoji ",we conducted this study with the direction of epigenetic regulation changes.Objectives:1.This study intends to explore whether the patients with small cell lung cancer who use the maintenance therapy of "Hehuan xiaoji" can obtain prolonged PFS,OS and its influencing factors through a retrospective cohort study.2.By establishing a mouse subcutaneously transplanted tumor model,from the perspectives of survival,nutritional status,organ function,and inflammatory status of Lewis lung cancer tumor-bearing mice,to explore the effects of Xiaoji Fang on survival and overall status.3.Through the in vivo experiments of Lewis lung cancer tumor-bearing mice and the in vitro experiments of human small cell lung cancer cells NCI-H446,to explore whether Xiaoji Fang can enhance the efficacy of chemotherapy and its mechanism of action.Methods:1.In this study,we investigated the general study data,each clinical characteristic,and treatment status of patients with extensive-stage small cell lung cancer admitted to wangjing hospital and China-Japan Friendship Hospital from January 1,2010 to December 31,2020,analyzing their clinical data to calculate progression-free survival,overall survival,and 1-,2-,and 3-year survival rates,compiling their general study data,each clinical characteristic,and treatment status.To find the influencing factors associated with progression-free survival.2.To evaluate the overall status of the mice after the application of Xiaoji Fang,as well as the survival period after treatment,by using the subcutaneous tumorigenic model of Lewis lung cancer cells,from the organ index,biochemical indexes,and the expression levels of serum pro-inflammatory factors,immune-related factors,and vascular endothelial growth factor detected by enzyme-linked immunosorbent assay(ELISA).3.To investigate the pharmacological effects of the drug-containing serum of the Xiaoji Fang on human small cell lung cancer NCI-H446 cells using cell proliferation assay,cell scratch assay,Transwell assay and cell flow assay.4.In vivo experiments on Lewis lung cancer tumor-bearing mice and in vivo experiments on human small cell lung cancer NCI-H446 cells,immunoblotting,real-time fluorescence quantitative PCR and immunohistochemical staining were used to investigate whether the Xiaoji Fang has synergistic effects on cisplatin and its mechanism of action.Results:1.The results of a retrospective cohort study of patients with small cell lung cancer treated with maintenance therapy with Hehuan xiaoji showed that a total of 120 patients with extensive-stage small cell lung cancer were finally enrolled,including 61 in the palliative elimination cohort and 59 in the control cohort.①The median PFS was 4.000(95%CI:3.399,4.601)months in the control cohort and 6.300(95%CI:5.477,7.123)months in the palliative cohort,with a reduced risk of progression in the palliative cohort compared with the control cohort(HR=0.517,95%CI:0.3 57-0.748).The median OS of the control cohort was 11.133(95%CI:9.485,12.782)months;the median OS of the palliative cohort was 16.000(95%CI:13.303,18.697)months,and the risk of death was significantly lower in the palliative cohort compared with the control cohort(HR=0.583,95%CI:0.405-0.839)② The results of the correlation analysis between palliative treatment and other treatments showed that the number and proportion of patients with>4 cycles of first-line chemotherapy was significantly higher in the palliative cohort compared with the control cohort(P<0.05).③The results of the analysis of factors influencing PFS showed that the number of first-line chemotherapy cycles≥4 and the use of maintenance therapy with palliative care were independent factors influencing PFS.2.Experiments on the effects of elimination formula on the overall status and survival of Lewis lung carcinoma-bearing mice.① Cisplatin,cisplatin+Xiaoji Fang,and cisplatin+Xiaoji Fang maintenance treatment all prolonged the survival of Lewis lung carcinoma-bearing mice(P<0.05),and mice in the extirpated formula maintenance treatment group could obtain a longer survival period.② Weighing the subcutaneous white fat in the groin,white fat around the gonads and mesenteric fat of mice separately,the fat weight of mice in the cisplatin+Xiaoji Fang group increased compared with the control and cisplatin groups(P<0.05).③ Organ index:compared with cisplatin group and control group,heart index was higher in cisplatin+Xiaoji Fang group(P<0.05),and spleen index and liver index were higher in cisplatin+Xiaoji Fang group compared with control group(P<0.05).④ Comparing the weights of gastrocnemius,tibialis anterior and quadriceps muscles in mice,there was no statistically significant difference in muscle weights of both lower limbs in the three groups.⑤ Biochemical results:compared with the control group,the serum albumin and total protein levels increased in the cisplatin+Xiaoji Fang group(P<0.05).Compared with the mice in the control group,serum urea was decreased(P<0.05)and creatinine was decreased(P>0.05)in the cisplatin+Xiaoji Fang group.⑥ Inflammatory factors:Compared with the control group,the expression of pro-inflammatory factors IL-1α,IL-1β,IL-6,IL-9 and TNF-α were down-regulated in the serum of mice in the cisplatin group(P<0.05);compared with the control group,the expression of pro-inflammatory factors in the serum of mice in the cisplatin+Xiaoji Fang group was down-regulated(P<0.05).Immune-related factors:IL-3 expression was down-regulated and IFN-γ expression was up-regulated in the serum of mice in the cisplatin group compared with the control group(P<0.05);IL-3 and IFN-y expression were up-regulated in the serum of mice in the cisplatin+Xiaoji Fang group compared with the blank group and the cisplatin group(P<0.05).Vascular endothelial growth factor:compared with the control group,the expression of VEGF was down-regulated in the serum of mice in the cisplatin group(P<0.05);compared with the blank group and cisplatin group,the expression of VEGF was down-regulated in the serum of mice in the cisplatin+Xiaoji Fang group(P<0.05).3.In vivo experiments on the inhibition of Lewis lung cancer through CDYL-EZH2-CDKN1C pathway by Xiaoji Fang.① Tumor volume:a statistically significant difference in tumor volume appeared on day 7,with larger tumor volume in the control group compared to the cisplatin+Xiaoji Fang group(P<0.05).On day 15,it appeared that the mean value of tumor volume in the cisplatin+Xiaoji Fang group was smaller than that in the cisplatin group(P<0.05).② Immunohistochemistry:compared with the control group,the expression of H3,H3K27me3 and CDYL in tumor tissues of mice in the cisplatin group was down-regulated(P<0.05),and the expression of CDKN1C was up-regulated(P<0.05);compared with the blank control group,the expression of EZH2,H3,H3K27me3 and CDYL in tumor tissues of mice in the cisplatin+Xiaoji Fang group was down-regulated(P<0.05),and the expression of CDKN1C was up-regulated(P<0.05).The expression of CDKN1C was upregulated(P<0.05).③ Real-time fluorescence quantitative PCR:cisplatin up-regulated CDKN1C,down-regulated CDYL(P<0.05),EZH2,H3(P<0.05)mRNA expression levels compared with the blank control group;the down-regulation of CDYL,H3,EZH2 mRNA expression levels and up-regulation of CDKN1C mRNA expression levels in the cisplatin+Xiaoji Fang group were higher than those in the blank control group(P<0.05).4.The efficacy experiments of elimination formula on human-derived small cell lung cancer NCI-H446 cells.① Cell proliferation assay:compared with the blank serum group,the cell proliferation rate was reduced in the Xiaoji Fang group,cisplatin group,and cisplatin+Xiaoji Fang group(P<0.05).② Cell scratching experiment:the cell fusion rate of cells in the cisplatin+Xiaoji Fang group was reduced at 24h and 48h compared with the blank serum group(P<0.05).③ Transwell assay:the number of cell invasion was reduced in the cisplatin group and cisplatin+Xiaoji Fang group compared with the blank serum group(P<0.05).④ Flow cytometry:the number of early and late apoptosis was increased in the cisplatin+Xiaoji Fang group compared with the blank serum group and the cisplatin group(P<0.05);the proportion of G0/G1 phase cells was increased and the proportion of G2/M phase was decreased in the cisplatin+Xiaoji Fang group compared with the blank serum group(P<0.05).5.Experimental study on the inhibition of human-derived small cell lung cancer NCI-H446 through CDYL-EZH2-CDKN1C pathway by elimination formula:Western blotting assay and RT-PCR assay results showed that cisplatin+Xiaoji Fang acting on NCI-H446 cells could down-regulate the expression of CDYL,H3,H3K27me3,and CDKN 1C compared with the control group(P<0.05).EZH2 expression(P<0.05)and up-regulated CDKN1C expression(P<0.05)compared with control.Conclusion:1.The maintenance therapy of Hehuan xiaoji can prolong the PFS and OS of patients with small cell lung cancer,and at the same time,it can prolong the number of cycles of first-line chemotherapy in patients with small cell lung cancer.Moderate and elimination maintenance therapy and the number of first-line chemotherapy cycles were independent influencing factors of PFS in small cell lung cancer.2.The application of Xiaoji Fang can improve the nutritional status of Lewis lung cancer tumor-bearing mice,improve their liver,kidney,and heart functions,and slow down the in vivo inflammatory state of tumor-bearing mice,improve the immune function of mice,and prolong the survival of tumor-bearing mice.It plays a role in regulating the overall functional state and prolonging the survival period.3.The effect of Xiaoji Fang on Lewis lung cancer tumor-bearing mice and NCI-H446 cells may be by down-regulating the recruitment of chromatin domain Y-like protein(CDYL)to histone methyltransferase(EZH2),thereby reducing the Trimethylation of histone H3 lysine 27 mediated by the histone methyltransferase EZH2 at the CDKN1C promoter.It induces tumor cells to block in G0/G1 phase,accelerates their G2/M phase process,induces tumor cell apoptosis,and synergistically enhances the effect of cisplatin chemotherapy. |
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