Β-Elemene Inhibits The Proliferation Of Non-Small-Cell Lung Cancer By Targeting ALDH3A1 Through Regulating Energy Metabolism Reprogramming

Objective:According to the recent cancer statistics of the International Agency for Cancer Research,lung cancer ranks the second among the common types of cancers,and the death rate ranks the first in the world.Non-small cell lung cancer(NSCLC)accounts for 80～85%of lung cancers,and adenocarcinoma is the most common histological subtype.In the elderly population,the incidence rate of lung cancer is higher and the prognosis is worse.Although great progress has been made in the diagnosis and therapy of lung cancer in recent decades,the five-year survival rate of NSCLC is still very poor,which is mainly due to tumor recurrence and metastasis.Tumor heterogeneity is the root cause of tumor cells escaping from immune surveillance,producing chemotherapy resistance,and forming recurrence and metastasis.The important aspect of tumor cell heterogeneity is that its energy metabolism is different from that of normal cells,which is the molecular biological basis for its rapid proliferation and metastasis.Some studies have confirmed that tumor cells will eventually acquire strong proliferation and metastasis ability after obtaining higher energy storage through metabolic reprogramming.Therefore,the concept of energy metabolic reprogramming provides a new idea and treatment direction for overcoming the recurrence and metastasis of NSCLC.β-elemene is an effective anti-tumor monomer component isolated from the volatile oil of Curcuma wenzedoary which is a kind of traditional Chinese medicine.Its anti-tumor spectrum is wide,efficient,safe,non-toxic and small side effects which is different from general cytotoxic chemotherapy drugs.For the elderly population with multiple chronic diseases,β-elemene is more secure.Sinceβ-elemene was applied in clinic,some patients with NSCLC have benefited from both single and combined chemotherapy.However,due to the lack of large-scale clinical trial research,it is not possible to clearly identify the beneficiaries ofβ-elemene,which limits its popularization and application.The anti-tumor mechanism ofβ-elemene mainly includes inducing cell cycle arrest and apoptosis(calcium homeostasis imbalance),reversing chemotherapy resistance,regulating tumor immunity,inhibiting invasion and metastasis,and anti-angiogenesis.Recent research suggests thatβ-elemene can regulate the energy metabolism reprogramming of thyroid cancer,but there is still a lack of relevant study aboutβ-elemene in energy metabolism reprogramming of NSCLC.Therefore,to explore whetherβ-elemene can affect the reprogramming of tumor energy metabolism,especially mining the target ofβ-elemene and potential mechanism is necessary and significant.In this study,in order to explore the potential target and mechanism ofβ-elemene in NSCLC,we first proved that the anti-tumor effect ofβ-elemene in NSCLC in vivo and in vitro,and then screen out possible drug targets based on network pharmacology,and finally screen out target molecules by PPI network mapping and survival prognosis analysis.We explore and verify the function and mechanism of the drug target through molecular biology experiments which provides a new theoretical basis for the clinical application ofβ-elemene.Methods:1.MTT is use for drug sensitivity test.2.The colony formation ability of tumor cells was tested by Cell colony test.3.Edu experiment was used to detect cell proliferation activity and DNA replication.4.Cell cycle changes were detected by flow cytometry.5.BATMAN-TCM database is used to predict the drug target ofβ-elemene.String database is used to analyze the protein-protein interaction of the selected target.Cytoscape software is used to build PPI network.6.GEPIA online database is used for analyzing the prognosis of target molecules.7.Molecular docking is used for the verification of the combination ofβ-elemene and ALDH3A1.8.Limma package of R software is used for analyzing the expression of target molecules.9.si RNA is used to transiently knock down target molecules.10.The lentivirus infected cells are used to establish stable cell lines for animal and cell experiments.11.MTS assay was used to detect cell proliferation.12.GSEA database analyzes the functional enrichment pathway of target molecules.13.Immunohistochemistry and q RT-PCR were used to verify the protein and m RNA expression level of related molecules.14.Western blot technique was used to detect the expression level of associative protein about cell cycle and glycolysis.15.Unilateral and bilateral nude mice subcutaneous tumorigenesis model were use for the validation ofβ-elemene and ALDH3A1 on the proliferation of NSCLC in vivo.16.Animal micro-PET-CT is used to analyze the SUV value in the tumor tissue of mice before and after the drug application.17.Statistical analysis:all experiments except animal experiments were replicated three times independently,and the expresimental results were expressed with mean±standard deviation.SPSS 16.0 was used for statistical analysis and t-test was used for inter-groups comparison,P<0.05 was was defined as statistically significant.Results:1.β-elemene inhibits the proliferation of NSCLC cells.MTT results show thatβ-elemene inhibited the proliferation of NSCLC cells in a dose-dependent manner.Similar results are obtained from the colony formation experiment.Edu experiment confirmed thatβ-elemene can inhibit DNA synthesis of NSCLC.Flow cytometry cell cycle test showed thatβ-elemene can induce G1 phase arrest of tumor cells.These results confirm thatβ-elemene can inhibit the proliferation of NSCLC.2.β-elemene can inhibit the growth of transplanted tumor in nude mice.β-elemene can inhibit the growth of NSCLC transplanted tumor in nude mice.The animal model of subcutaneously transplanted tumor in nude mice was established with A549cells.It was observed that the tumor volume and weight of mice are significantly reduced after 2 weeks ofβ-elemene treatment.These results show thatβ-elemene can significantly inhibit the proliferation of NSCLC in vivo.3.β-elemene regulates the energy metabolism reprogramming of NSCLC in vivo and in vitro.Mass spectrometry and metabolite analysis of transplanted tumor in nude mice and NSCLC cells confirmed thatβ-elemene reduces glucose uptake,lactate and NAD~+/NADH ratio.These results show that:β-elemene can regulate the energy metabolism of NSCLC tumor.4.ALDH3A1 is the drug target ofβ-elemene.The results of network pharmacological analysis suggest that ALDH3A1 is the drug target ofβ-elemene.GSEA function enrichment analysis for ALDH3A1 suggests that ALDH3A1 regulates cell cycle and glycolysis in a positive way.The above results suggest:β-elemene may participate in the regulation of cell cycle progression and glycolysis of NSCLC by targeting ALDH3A1.5.β-elemene regulates the expression of ALDH3A1 in NSCLC.Molecular docking,Western blot and q RT-PCR suggest thatβ-elemene has high affinity binding sites for ALDH3A1.β-elemene can regulate the expression of ALDH3A1 at the level of transcription and translation.6.ALDH3A1 is highly expressed in NSCLC which is related to clinical stage.The results of GEO database,Kaplan–Meier database and immunohistochemical analysis showed that ALDH3A1 is highly expressed in NSCLC and correlated with clinical stage which is a poor prognostic factor.7.The knockdown of ALDH3A1 inhibits the proliferation of A549 and H1299 cells and induces cell cycle arrest.The results of MTS,Edu,colony test and flow cytometry showed that compared with NC,the proliferation,colony formation and DNA replication of A549 and H1299 cells transfected with ALDH3A1 si RNA(sequence1 and sequence 2)was significantly reduced,and G1/S phase arrest was induced.8.ALDH3A1 regulates the reprogramming of energy metabolism in NSCLC cells.Compared with NC,glucose uptake,lactate level and NAD~+/NADH ratio were significantly decreased after the knockdown of ALDH3A1.However,α-KG level increased.The use of glycolysis pathway inhibitor 2-DG can partially block the promotion on glycolysis of NSCLC of ALDH3A1.9.β-elemene inhibits NSCLC proliferation and induces cell cycle arrest by targeting ALDH3A1.The results of colony and Edu experiments show thatβ-elemene further enhance the effect of the down-regulation of ALDH3A1.The results of Western blot showed that the cell cycle-related proteins were further down-regulated.The above results strongly suggest thatβ-elemene inhibits the proliferation of NSCLC by targeting ALDH3A1.10.β-elemene blocks HIF-1α/LDHA axis and regulate the reprogramming of NSCLC energy metabolism.In order to further clarify the mechanism of ALDH3A1in the glycolysis of NSCLC,q RT-PCR and Western Blot were used to detect the expression level of downstream molecules after the knockdown and over-expression of ALDH3A1.The results showed that:The expression of important molecules in glycolysis such as PFKL and LDHA decreased,but HIF-1αhas no significant change at the m RNA level.Whenβ-elemene was used in sh-ALDH3A1 cells,the protein and m RNA levels of the above molecules are further significantly down-regulated,but HIF-1αhas not changed significantly at the m RNA level.The above results strongly suggest thatβ-elemene plays an important role in the regulation of NSCLC energy metabolism reprogramming by targeting ALDH3A1 through HIF-1α/LDHA signal pathway.11.β-elemene inhibits HIF-1α/LDHA pathway by targeting ALDH3A1 for inhibiting the growth of transplanted tumor in nude mice.Immunohistochemical staining and Western Blot used for the analysis of transplanted tumor tissue in nude mice.β-elemene reduces the expression of related proteins of cell cycle and glycolysis in tumor tissue.Conclusion:1.β-elemene inhibits NSCLC proliferation and reprogramming of energy metabolism in vivo and in vitro.2.The network pharmacology analysis and bioinformatics suggests that ALDH3A1 is the drug target ofβ-elemene.3.ALDH3A1 is highly expressed in NSCLC which is related to clinical stage.4.β-elemene interferes with the reprogramming of energy metabolism and inhibits the growth of NSCLC by targeting ALDH3A1.5.β-elemene inhibits the energy metabolism reprogramming of NSCLC by blocking HIF-1α/LDHA pathway.