Pharmacodynamic Mechanism Research Of Apigenin Inhibiting HK1 Against Lung Cancer

Objective: Lung cancer is one of the most common malignant tumors,which occurs in China with a high incidence.At present,the treatment of lung cancer mainly includes surgical treatment and chemotherapy drugs.However surgical treatment is easy to cause complications,and commonly used chemotherapy drugs have large side effects.Therefore,it is necessary to find new high-efficiency and low-toxicity drugs,for that natural products may be a breakthrough point.Based on this background,the anti-lung cancer activity of small molecule compounds in the natural product library of our group was screened in this study.It was found that apigenin,a flavonoid,has an excellent selective anti-lung cancer effect under a hypoxia environment.This study confirmed the efficacy of apigenin in vivo and in vitro anti-lung cancer effect,and revealed its mechanism by combining quantitative proteomics and quantitative metabolomics.And we further explored the direct target through chemical proteomics,to provide new ideas for the treatment of lung cancer and the development of new anticancer drugs.Methods: Firstly,determine the anti-lung cancer efficacy of apigenin at the cellular level.We conducted CCK-8 assay,colony forming assay,giemsa staining,flow cytometry and western blot experiments to investigate the effect of apigenin on A549 cells under hypoxic conditions.Secondly,clarify the pharmacodynamic mechanism network of apigenin.We analyzed the changes of the whole proteome and metabolites after apigenin acting on A549 cells under a hypoxic environment by quantitative proteomics based on stable isotope dimethylation labeling and quantitative metabolomics to explore the key pathways.Thirdly,identify the direct action target of apigenin.We further identified direct targets of apigenin by using active probe-based chemical proteomics techniques.Then we knocked down the target protein with RNA interference technology.The effect of the target protein on apigenin inhibition of A549 cells was detected by CCK-8 assay and flow cytometry.Next,we verified the interaction between apigenin and the target protein by pull-down experiment.Fourthly,clarify the anti-lung cancer efficacy of apigenin at the animal level.We constructed a mouse lung cancer model with the help of Urethane to evaluate the anti-lung cancer activity of apigenin in vivo from the aspects of body weight,growth,and pathological sections of mice.Results: Firstly,the results of CCK-8,giemsa staining,and colony forming assay showed that apigenin could inhibit the cell viability and clonal formation ability of A549 cells under hypoxic conditions.And the flow cytometry results showed that apigenin could induce apoptosis of A549 cells time-dependently at 25 μM under hypoxia conditions.We carried out western blot experiments,and the results showed that apigenin could downregulate the ratio of Bcl-2 to Bax in A549 cells and the expression of Caspase 9 and PARP.At the same time,the expression of Cytochrome C,Caspase 3,Cleaved Caspase 3,Cleaved Caspase 9,and Cleaved PARP was upregulated.Thereby apigenin promoted apoptosis in A549 cells.Secondly,we analyzed the results of quantitative proteome and metabolome experiments and found that apigenin mainly interfered with glycolysis of A549 cells under hypoxic conditions.Further,focusing on glycolysis,we designed and synthesized an apigenin probe.Using chemical proteomics techniques,we found six key target proteins of glycolysis pathway,namely HK1,LDHA,MARS1,ACOT7,GSTO1,KPNA2.We performed gene knockdown on the target by RNA interference technology and then administered apigenin for efficacy test.The results showed that the key target of apigenin inhibiting A549 cells was HK1(Hexokinase 1).The interaction between apigenin and HK1 was confirmed by pull-down experiment.Finally,results at the animal level also confirmed that 5-10 mg/kg apigenin has anti-lung cancer activity in vivo,and can improve the growth and lung tissue status of mice with Urethane-induced lung cancer.Conclusion: In conclusion,apigenin can regulate glycolysis and induce apoptosis pathway by inhibiting HK1 under hypoxia conditions,thus exerting its inhibitory effect on A549 cells.