Study On The Underlying Molecular Mechanism By Which Citrus Grandis ’Tomentosa’ Exerts Anti-Non-Small Cell Lung Cancer Effects By Regulating Skp2/p27 Axis

ObjectiveLung cancer is a malignant tumor with extremely high morbidity and mortality,among which non-small cell lung cancer(NSCLC)accounts for the majority of lung cancer pathologies,and targeted therapy is an important strategy for lung cancer treatment.Skp2,the oncogenic F-box protein overexpressed in many tumor types,mediates the ubiquitin-dependent degradation of its classical substrate p27 protein,thus allowing for the cell cycle transition from G1 to S phase and promoting proliferation of cancer cells.Skp2 has been considered as a target in the development of novel cancer drugs.Lung cancer can be classified as “phlegm” in traditional Chinese medicine(TCM),and phlegm is both the cause and the pathological product of lung cancer.The Chinese herb Citrus grandis ‘Tomentosa’(Citrus grandis ‘Tomentosa’,CGT)has a pungent and bitter taste,belongs to the lung meridian,and has the function of regulating qi and resolving phlegm,which has been used clinically to treat many respiratory diseases,but its direct anti-lung cancer efficacy,mechanism and active ingredients of the extract have not been reported in the literature.In this study,we investigated the anti-NSCLC effect and the underlying molecular mechanism of CGT based on Skp2/p27 signaling axis,and preliminarily investigated the active ingredients.In order to explore the prospects for the development of anti-NSCLC drugs of CGT and provide the theoretical basis for the development of efficient and low toxicity NSCLC treatment strategies,as well as to provide basic research support for the theory of lung cancer treatment by “phlegm removal” method in TCM.Methods1.To prepare aqueous and ethanol extracts of CGT,the dried powders of CGT were extracted by pure water with reflux or anhydrous ethanol with ultrasound.The ethanol extract of Citrus grandis ‘Tomentosa’(CGTE)was extracted by solvent extraction method to prepare the petroleum ether extract of Citrus grandis ‘Tomentosa’(CGTPE),the ethyl acetate extract of Citrus grandis ‘Tomentosa’(CGTEA)and the normal-butanol extract of Citrus grandis ‘Tomentosa’(CGTNB),respectively.The MTT method was used to compare the effects of different extracts on the viability of NSCLC cells and to screen the antiNSCLC activity of various extracts of CGT.2.The effects of CGTE and CGTPE on the proliferation of lung cancer cells were examined by MTT,Colony formation and Cell cycle assays.3.The effect of CGTE on Cyclin D1,p27 and p21 proteins was examined by Western blotting.4.The effects of CGTE and CGTPE on the Skp2/p27 signaling axis were examined by Western blotting、CHX and MG-132 assays.5.The effects of CGTE and CGTPE on Skp2-SCF E3 ubiquitin ligase activity were examined by cellular thermal shift assay(CETSA),MG-132,co-immunoprecipitation(co-IP)and in vivo ubiquitination assays.6.Construction of Skp2 overexpressing stable cell line to verify the underlying mechanism of CGTE and CGTPE through the Skp2/p27 signaling axis to exert anti-NSCLC effects.7.A subcutaneous allograft tumor model of LLC in C57BL/6 mice and a subcutaneous xenograft tumor model of A549 in BALB/c nude mice were established to evaluate the inhibition of NSCLC growth by CGTE and CGTPE in vivo,and using hematoxylin and eosin(H&E)staining,immunohistochemistry(IHC)and Western blotting assays and to verify their underlying mechanism.8.The chemical composition of CGTE was characterized by ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).The preliminary exploration of the active ingredients of CGTE against NSCLC was also carried out by MTT and protein immunoblotting assays.ResultsFirstly,we investigated the anti-NSCLC effect and mechanism of CGTA and CGTE.By screening the anti-NSCLC activity of CGTA and CGTE,we found that CGTE had better anti-lung cancer activity than CGTA in vitro.CGTE was chosen to investigate the anti-NSCLC efficacy,the underlying mechanism and material basis of CGT.The results of MTT assay showed that CGTE inhibited the proliferation of several NSCLC cell lines in a dose-dependent manner.We chose a relatively lower concentration of CGTE(100 、 200 and 300 μg/m L),and continued to test the effect of CGTE on the proliferation of NSCLC cells.The colony formation assay revealed that CGTE significantly inhibited the low-density proliferation ability of NSCLC cells,and the cell cycle assay further demonstrated that CGTE remarkably induced G0/G1 phase cycle arrest in lung cancer cells,indicating that CGTE could inhibit the proliferation of NSCLC cells by inducing G0/G1 phase arrest.Then,we examined the effect of CGTE on Cyclin D1,p27 and p21 protein expression by Western blotting assay,and the results showed that CGTE had no effect on G1 phase specific cyclin D1 and cell cycle protein-dependent kinase inhibitor p21 protein levels,but significantly increased p27 protein expression.p27 can strictly regulate the transition from G1 to S phase.The main mechanism of p27 protein degradation is the Skp2-dependent ubiquitin protein hydrolysis pathway.Given that the expression of Skp2 and its classical substrate protein p27 are closely related to cell cycle G1 phase arrest,we examined the effect of CGTE on the expression of these two proteins by Western blotting assay and found that CGTE could dramatically increase p27 expression and downregulate Skp2 expression in NSCLC cells.CHX assay further demonstrated that CGTE increased the half-life of p27 in NSCLC cells,while shortened the half-life of Skp2 and destabilized Skp2.It is suggested that CGTE may promote p27 protein accumulation by inhibiting Skp2-SCF E3 ubiquitin ligase activity and induce G0/G1 phase arrest,thus inhibiting the proliferation of NSCLC cells.We then validated that CGTE inhibited the proliferation of NSCLC cells by targeting Skp2/p27 axis.The results of CETSA showed that CGTE enhanced the protein thermal stability of Skp2,indicating that the active ingredients of CGTE has the potential to bind Skp2.The results of co-IP assay showed that CGTE inhibited the binding of Skp2 to the backbone protein Skp1,thereby disrupting the integrity of the Skp2-SCF complex;CHX assay showed that CGTE significantly reduced Skp2 protein half-life,while the proteasome inhibitor MG-132 inhibited the pro-degradation effect of CGTE on Skp2 protein,demonstrating that CGTE promotes Skp2 protein degradation by interfering with the integrity of the Skp2-SCF complex.The results of in vivo ubiquitination assay showed that CGTE inhibited the ubiquitination level of p27.The above experimental findings confirmed that CGTE inhibited the activity of Skp2-SCF E3 ubiquitin ligase,which reduced the level of p27 ubiquitination and thus promoted the accumulation of p27.Overexpression of Skp2 by lentiviral infection in non-small cell cells significantly attenuated the inhibitory effect of CGTE on the Skp2/p27 signaling axis as well as the proliferation of NSCLC cells,further verifying that CGTE inhibited non-small cell cell proliferation through the Skp2/p27 signaling axis.In addition,we established a subcutaneous allograft tumor model of LLC in C57BL/6mice and a subcutaneous xenograft tumor model of A549 in BALB/c nude mice,and confirmed that CGTE(1 g/kg and 2 g/kg)inhibited the growth of lung cancer in vivo.CGTE was able to downregulate Skp2 protein expression and increase p27 protein expression in lung cancer tissues,and down-regulate the expression level of Ki67,a nuclear antigen associated with cell proliferation,in lung cancer tissues,thus verifying the mechanism of CGTE to inhibit the proliferation of lung cancer by targeting Skp2/p27 axis in vivo.CGTE had no obvious side effects on the serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),creatinine(CRE)and uric acid(UA)in C57BL/6 mice and BALB/c nude mice,nor did they cause apparent histopathological lesions.However,the CGTE high dose group(2 g/kg)of C57BL/6 mice showed a decrease in body weight.Moreover,CGTE was able to downregulate Skp2 expression and increase p27 expression in lung cancer tissues.The results of UPLC-MS/MS analysis identified 390 compounds in CGTE,mainly including flavonoids,coumarins,lipids,phenolic acids,alkaloids and terpenoids.The matching and relatively high contents of CGTE compounds were naringin,naringenin,rhoifolin,apigenin,neoeriocitrin,meranzin,bergaptol,osthole,aurapten,obacunone and limonin.These results have laid the foundation for the research on the anti-NSCLC active ingredients of CGT.Based on the efficacy and mechanism of CGTE against NSCLC,we conducted a preliminary exploration of the active ingredients in CGTE that exert anti-NSCLC activity.Based on the UPLC-MS/MS identification results and literature reports,we selected five active compounds,namely Naringin、Rhoifolin、Apigenin、Naringenin and Limonin.The anti-NSCLC effects of these five active compounds were compared by cell activity assay.The results showed that limonin could significantly reduce the cellular activity of NSCLC cells compared with the other four compounds.We also found that limonin significantly increased p27 expression and downregulated Skp2 protein expression in NSCLC cells.It is suggested that limonin may promote the accumulation of p27 protein by inhibiting Skp2-SCF E3 ubiquitin ligase activity.It was shown that limonin was an active ingredient with higher anti-NSCLC activity in CGTE.Given the high dose of CGTE administration and the impact on mouse body weight when administered at higher doses,it is suggested that the direct use of CGTE for drug development is limited.Therefore,based on the results of the CGTE study,the effective site in CGT with better anti-NSCLC effect was explored.The anti-lung cancer activity of CGTPE,CGTEA and CGTNB was examined.We found that CGTPE had a better effect on lung cancer cells than CGTEA and CGTNB,and it had the same inhibitory effect on lung cancer cell viability at lower dosages as compared to CGTE.Therefore,we selected CGTPE(50、75 and 100 μg/m L)to continue to investigate the antitumor effect and underlying mechanism of CGT.In vitro cellular assays revealed that CGTPE,similar to CGTE,can inhibit the proliferation of lung cancer cells by inducing G0/G1 phase cycle arrest through targeting Skp2/p27 axis.In vivo subcutaneous A549 xenograft assay also showed that CGTPE(200mg/kg、400 mg/kg 和 800 mg/kg)had the same anti-lung cancer effect.CGTPE could also decrease the protein level of Skp2 while increase p27 expression,and downregulated Ki67 expression in lung cancer tissues.CGTPE showed no significant decrease in body weight and organ toxicity in nude mice at all doses.CGTPE dose groups did not show a significant body weight loss and organ toxicity after administration,and the dose were lower compared to CGTE.ConclusionCGTE and CGTPE were capable of inhibiting the proliferation of lung cancer cells in vitro and in vivo,and the underlying mechanism was related to their regulation of Skp2/p27 axis,which confirmed the anti-NSCLC effect of CGT.