The Effect And Mechanism Of 8-Cetylberberine On Lung Cancer

Objective:Lung cancer is one of the most common malignancies facing mankind.In recent years,the incidence of lung cancer has risen rapidly,accounting for more than one-tenth of all cancer cases in the world.Its mortality rate ranks first in the world as it causes a serious threat to human health.According to histological classification,lung cancer is either small cell lung cancer(SCLC)or non-small cell lung cancer(NSCLC)with 85% of patients diagnosed of the latter.As a result of the toxicity and resistance of commonly used anti-cancer agents,surgical resection remained the best treatment option only in cases of early diagnosis.However,with the challenges of early diagnosis and comprehensive understanding of the molecular mechanism of NSCLC,it has become imperative to explore natural medicinal agents and their derivative in the effective treatment of this cancer.Berberine(BBR),one of the active ingredients found in plants family Pedicelidae,Pesticides,Papaveraceae,has long been used to treat gastrointestinal disorders in traditional Chinese medicine.In recent years,not only has BBR been shown to have anti-tumor activity and low toxicity,its derivatives 8-Cetylberberine(8-BBR-C16,HBBR),produced by introducing a 16 C linear alkyl modification to the C8 position of BBR,was found to be more bio-available with obvious pulmonary tendency.But,HBBR anti-lung cancer and tumor activities are yet to be explored.Therefore,this study attempts to explore possible molecular mechanisms of NSCLC pathogenesis through protein-protein interaction network relationship prediction,core gene screening,and prognostic analysis.It also intends to evaluate the inhibitory effect of HBBR on NSCLC as well as its possible In vitro and In vivo molecular mechanism of action.Methods and results:(1)Using bioinformatics methods,we screened the differentially expressed genes(DEGs)between NSCLC samples and their matched normal lung tissues by analyzing 4 different mRNA microarray datasets downloaded from the Gene Expression Omnibus database(GEO),and obtained 676,865,274 and 1797 up-regulated DEGs while 761,1262,689,2350 down-regulated ones in GSE21933,GSE33532,GSE44077 and GSE74706,respectively.In order to obtain the most reliable DEGs,we isolated the DEGs presented in all four datasets and finally got a total of 195 DEGs,consisting of 57 up-regulated and 138 down-regulated DEGs.Through GO(Gene Ontology)annotation of the DEGs,we found that the up-regulated DEGs were significantly enriched in biological processes(BP)like cell cycle,mitosis and cell proliferation while the down-regulated DEGs were significantly enriched in angiogenesis,cell apoptosis and cell adhesion.By constructing the protein-protein interaction(PPI)network of these 195 DEGs,25 Hub genes with a degree score ? 19 were excised,of which CCNB1,CCNA2,CEP55,PBK and HMMR genes had the highest scores.Hou Lung Statistics data from Oncomine further confirmed the upregulated mRNA expression level of these top 5 hub genes in NSCLC specimens.The Kaplan-Meier survival analysis showed that the high expression level of each of these hub genes correlates with worse overall survival(OS)in all NSCLC patients,which indicates that they might play important roles in the progression of NSCLC.(2)In studying its anti-lung cancer effect,HBBR was synthesized in our laboratory by a format reagent method and identified by 13C-NMR,1H-NMR and Mass spectrometry.(3)In the In vitro studies,MTT assay showed significantly higher inhibitory effect of HBBR on A549 cell viability than BBR with IC50 values of 2.325 and 27.398 ?g/mL respectively.With Flow Cytometry,EdU proliferation assay and Western blotting,HBBR could inhibit the proliferation of A549 cells and induce G0/G1 cell cycle arrest.With the addition of TUNEL apoptosis detection,its anti-tumor effect can be by apoptotic induction of A549.While HBBR inhibits A549 cell growth by inhibiting the activity of PI3K-Akt pathway,Scratch wound healing and Matrigel invasion assays,also show its impact on the migration and invasive potentials of A549 cells.(4)The In vivo studies of xenograft model on nude mice revealed a reduction in average tumor weight after HBBR administration at lower dosage(5,10mg/kg)compared to BBR(120mg/kg).There was also a significant reduction in the serum levels of NSE,CYFRA21-1 and CA125 tumor marker proteins 21 days after the administration of HBBR.Long-term administration of HBBR equally showed no obvious toxic or side effects on the growth of mice.The above results show that HBBR has a good potential to inhibit the growth of lung cancer.(5)The stability of the microflora in the intestine is closely related to the health of the body and its resistance ability to diseases.To investigate the possible relationship between the anti-tumor ability of HBBR and the stability of gut microbiota,the excreted feces of mice in the normal control group(NC),tumor model group(TC),and HBBR(10 mg/kg)group on the 20 th day of the animal experiment were collected.After extracting the genomic DNA from the feces,the 16 S rRNA gene sequencing method was used to detect the difference in intestinal flora between the groups.Totally,685707 effective pyrosequencing reads and 198 microbe species were identified from 9 stool samples.The results showed that the diversity and abundance of gut bacterial detected in TC group were lower than the NC and HBBR group.That implied that the treatment of HBBR is beneficial to maintain a stable and healthy microecology in mice.At the species level,HBBR administration is benefit for the abundance maintain of Clostridium clostridioforme,Bacteroides acidifaciens,Parabacteroides distasonis,Lactobacillus animalis,Lactobacillus gasseri,Lactococcus lactis,Erysipelatoclostridium ramosum and Mucispirillum schaedleri in the intestine tumor-bearing mice.(6)We evaluated the synergistic effect of HBBR and Paclitaxel in lung cancer treatment using two different subcutaneous tumor models-the A549 xenograft and Lewis lung carcinoma mice.Our work showed that the combined dose of 10 mg/kg HBBR(orally and daily)and 12 mg/kg Paclitaxel(intraperitoneally and weekly)could effectively increase the tumor inhibition rate in both mice models.It also indicates that the efficacy of the combination of HBBR and paclitaxel is an improvement over their respective individual efficacies.Conclusion:By showing the involvement and connection of a series of DEGs in the occurrence and progression of NSCLC through the analysis of network chip data,this work in a way,contributes to the understanding of the molecular mechanism of NSCLC.Through several In vitro and In vivo experiments,this work also unveils the potential of HBBR,as an anti-lung cancer agent that inhibit the growth of lung cancer without any deleterious but beneficial effect on certain intestinal bacterial microflora.With the increased combined efficacy of HBBR and Paclitaxel observed,this work has not only provided information on the possible mechanism of HBBR anti-cancer activity but also a potential possibility in the effective treatment of NSCLC.