The Molecular Mechanisms Of DHA Inhibiting Non-small Cell Lung Cancer(NSCLC) Growth Through The MTOR/HIF-1α Signaling Pathway

Background Cancer is one of the leading causes of death in the world.The incidence of lung cancer ranks the second in 2020.Non-small cell lung cancer(NSCLC)is the most common type of lung cancer,which accounts for 80-85% of all lung cancer cases.Studies have found that Dihydroartemisinin(DHA),derivatived from the traditional Chinese herb artemisia(ART),exhibited anti-tumor activity.But the mechanism by which DHA effects NSCLC growth and progression has not yet been explored.Objective In this study,A549 and the mouse Lewis lung cancer cell line(LLC)were selected for cell experiments,and tumor-bearing mouse models were established through axillary inoculation of tumor cells for in vivo verification.Here we aimed to identify the molecular mechanism of DHA inhibiting the growth of non-small cell lung cancer by regulating the mTOR/HIF-1α signaling pathway,which can provide theoretical basis for developing new clinical treatment plans.Contents and Methods Part Ⅰ 1.DHA inhibited the growth of NSCLC in vitro.The effects of DHA(1 μM、5 μM、10 μM、25 μM、50 μM、100 μM)on the activity of A549 and LLC cells were detected by MTS assay.The effects of DHA(5 μM,10 μM and 50 μM)on the proliferation of these cells were detected by colony formation assay.The effects of DHA on cell cycle regulation and apoptosis were detected by flow cytometry.Western Bolt was used to detect the effects of DHA on the expression of cell cycle and apoptosis-related proteins.2.To evaluate the tumor suppressive effect of DHA in vivo,a mouse model of Lewis lung cancer was established.LLC cells were implanted subcutaneously into female C57BL/6 mice.Mice were randomly divided into five groups(n = 6),which were respectively intraperitoneal injection of cisplatin 2 mg/kg(positive control),dimethyl sulfoxide(DMSO)(negative control),and dosage groups(12.5,25 and 50 mg/kg of DHA)continuously for 14 d.The tumor volume and the body weight of mice were monitored every 3 days.Twentyfour hours after the last therapy,the tumor was harvested,fixed in 4% paraformaldehyde and embedded in paraffin.Then the tissue sections were subsequently immunohistochemical staining for Ki-67 and the changes were statistically analyzed.Part Ⅱ 1.To explore the potential molecular mechanism of DHA in inhibiting progression of NSCLC.GO and KEGG analyses were performed to predict the potential molecular mechanism of DHA in inhibiting progression of NSCLC via network pharmacology techniques.2.The validation of DHA inhibiting the growth of NSCLC by mTOR/HIF-1α signaling pathway in both forward and reverse pathways.The expression levels of p-mTOR and HIF-1α in A549 and LLC cells,treated with 10 μM DHA for 6 h,12 h,24 h and 48 h,were detected by Western Bolt.Meanwhile,C57BL/6 tumor bearing mice were treated with 12.5 mg/kg DHA,and the protein expression levels of p-mTOR and HIF-1α in tumor tissues were detected by Western Bolt.At the same time,the protein expression levels of p-mTOR and HIF-1α in A549 and LLC cell lines were detected after treatment with 20 μM mTOR activator,MHY1485,which can abolish the cytotoxic effects of 10 μM DHA in NSCLC.It was further confirmed that DHA inhibits the growth of NSCLC by regulating the mTOR/HIF-1α signaling pathway Results Part Ⅰ1.The results of MTS assay showed that DHA inhibited cell proliferation in a concentration-dependent manner.With the increase of drug concentration,the inhibitory effect of DHA on cells was gradually enhanced.The IC50 values of DHA for A549 and LLC cells were 23.89 μM and 8.15 μM,respectively.2.After treated with DHA(5 μM,10 μM,50 μM)for 10 days,the numbers of colonies formed by A549 and LLC cells were significantly decreased when compared to mock group(P < 0.05)3.DHA induced G0/G1 arrest in A549 cells in a time and dose-dependent manner compared with the mock group.Irrespectively,no obvious cell cycle distribution difference between DHA and Mock treatments were detected in LLC cells.Simultaneously,the expression levels of key G1/S phase-inducing molecules in A549 and LLC cells were detected by Western blot assay,including CDK2,CDK4,Cyclin D1 and Cyclin E1(P < 0.05).These results suggest that DHA may inhibit the growth cycle of tumor cells by inhibiting the expression of cell cycle-associated proteins.4.Compared with mock control,treatment with DHA significantly increased the ratio of apoptotic cells in LLC cells.Similarity,DHA significantly down-regulated the expression of Bcl-2 and Bcl-xl in A549 and LLC cells(P<0.05).These results suggest that DHA may promote tumor apoptosis by inhibiting the expression of anti-apoptosisrelated proteins.5.Compared to the DMSO and cisplatin groups,DHA significantly decreased tumor volume and weight at different dose: 12.5 mg/kg,25 mg/kg or 50 mg/kg(P < 0.05).6.Compared with the DMSO group,the body weight of mice decreased significantly in the both cisplatin-treated group and dosage groups(25 and 50 mg/kg of DHA,but not 12.5 mg/kg),which indicated that DHA at dosage of 12.5 mg/kg is well-tolerated in C57BL/6 mice.7.Compared with the DMSO group,Ki-67 protein level was significantly decreased in tumor tissue after DHA(12.5 mg/kg)treatment which coincided with the growthinhibiting role of DHA in NSCLC cell lines(P < 0.05).Part Ⅱ1.Network pharmacology studies have shown that the main mechanisms of DHA exhibits its antitumor effects in NSCLC involving cell apoptosis,smooth muscle cell proliferation,reactive oxygen species and injury response,and affect several important cellular signaling pathways,including HIF-1 signaling pathway,p53 signaling pathway,NF-kappa B signaling pathway,PPAR signaling pathway,Calcium signaling pathway,and TGF-beta signaling pathway,which lay a foundation for subsequent selecting key signaling pathways induced by DHA.2.After DHA treatment,the expression level of both HIF-1α and p-mTOR were significantly reduced in LLC cells,especially HIF-1α in A549 cells.Compared with tumor tissues from mice treated with DMSO,the expression level of p-mTOR and HIF-1α was significantly reduced in those mice treated with 12.5 mg/kg.To further confirm the role of mTOR/HIF-1α signaling pathway in DHA inhibition of tumor,we applied the mTOR agonist MYH1485 against DHA and found that DHA inhibition of p-mTOR and HIF-1α activation in A549 and LLC cell lines was alleviated to a certain extent.These results suggest that the antitumor effect of DHA is indeed mediated by mTOR /HIF-1α signaling pathway.Conclusion Our study showed that DHA can inhibit the growth of NSCLC by induction the cell cycle arrest and apoptosis-related proteins through the mTOR/HIF-1α signaling pathway.