| Objective:Lung cancer is one of the most common malignant cancers that has the highest mortality worldwide.Non-small cell lung cancer(NSCLC)accounts for nearly 80%among lung cancers.Abnormal proliferation and metastasis of NSCLC cells usually cause poor prognosis of cancer patients.Although therapeutic treatments,such as surgery,radiotherapy and chemotherapy,targeted therapy and immunotherapy,have achieved great success,promising therapeutic strategies are still lack.In traditional Chinese medicine,non-small cell lung cancer can be classified as "pneumothorax","hemoptysis","lung carbuncle","phlegm",et al.Many clinical and basic studies have found that the drugs with ability for clearing away heat and toxin,clearing away heat and drying dampness are commonly used to treat lung cancer.Silybin is a Chinese medicine with bitter and cool properties.It can clear away heat and toxin,soothe liver and gallbladder.Silibinin extract from silybin fruit is one of the main active components,many studies have found that silibinin has good anti-tumor activities.Although studies on anti-NSCLC effect of silibinin have been reported,the underlying mechanism by which silibinin inhibits the proliferation of NSCLC is still unclear.In human cancers,overexpression of Skp2 protein is usually associated to the degree of malignancy.In our previous study,we found that silibinin may have a targeting effect on Skpl-Skp2 complex through molecular docking.Based on this,we explored the mechanism underlying the anticancer effect of silibinin on NSCLC,and provide a theoretical basis for silibinin anti-NSCLC.Method:In this study,the effects of silibinin on the proliferation of NSCLC cells were investigated by MTT assay and cell colonies experiments;Flow cytometry was used to detect the effect of silibinin on cell cycle of NSCLC cells(A549,H1299);The wound-healing experiment and Transwell invasion assay was used to analyze the inhibitory effects of silibinin on metastasis and invasion of NSCLC cells,including H1299,A549,LLC;The effects of silibinin on Skp1-Skp2 complex were evaluated by molecular docking and co-immunoprecipitation;The targeting of silibinin to Skp2 was evaluated by CETSA;Western blot analysis was used to examined the effects of silibinin on Skp2 and its substrate P27;The effects of silibinin on Skp2 protein stability in A549 cells was evaluated by CHX assay;Skp2 overexpression was used to validate the effects of silibinin on Skp2 and to prove the relationship between Skp2 and P27 in NSCLC cells;The anti-tumor effect and potential mechanism of silibinin in vivo were examined by establishing an orthotopic lung cancer model in C57BL/6 mice.Results:The results of MTT experiment and cell colonies experiments showed that silibinin could selectively inhibit proliferation of NSCLC cells;Silibinin has no toxicity to normal human lung epidermal cells Beas-2B at higher concentrations(200μM).Cell cycle analysis showed that silibinin could induce G1 phase arrest of NSCLC cells(A549,H1299);Wound-healing and Transwell experiments showed that silibinin inhibited the metastasis and invasion of NSCLC cells in a dose-dependent manner;Molecular docking and co-immunoprecipitation results confirmed that silibinin can bind to Skpl-Skp2 complex binding interface and interrupt Skp1-Skp2 complex stability;CETSA experimentprovedthatsilibinin targetto Skp2;The CHX test expressed that silibinin can reduce the stability of Skp2 protein and promote its degradation;Silibinin could significantly decrease the protein level of Skp2 and up-regulate the expression of P27 in NSCLC cells,providing a key evidence that silibinin inhibits the proliferation of NSCLC cells by inhibiting Skp2 signaling pathway.It was found that silibinin has a good anti-tumor activity under a dose of 400 mg/kg in vivo.Compared with cisplatin treatment,silibinin did not exhibit any damage to liver or kidney in mice.Conclusion:We investigated the underlying molecular mechanism by which silibinin inhibited the proliferation of NSCLC cells(A549,H1299)by targeting Skp2-SCF E3 ligase,and explored its antitumor effect in vivo.Molecular docking experiments showed that silibinin could bind to the protein interaction pocket of Skp1-Skp2 complex.Co-immunoprecipitation showed that silibinin could inhibit the protein interaction between Skp1 and Skp2.CETSA experiment confirmed that silibinin binds to Skp2.Western blotting further confirmed that silibinin could inhibit the expression of Skp2 and upregulate the expression of substrate protein P27,suggesting that silibinin may inhibit cell proliferation by targeting Skp2/P27 pathway.Animal experiments showed that silibinin had promising antitumor effects at a high dose(400mg/kg),and showed no obvious toxicity towards liver and kidney compared with cisplatin.Therefore,silibinin serves as a novel Skp2 inhibitor in NSCLC.Next,we will further explore the detailed molecular mechanism of silibinin targeting Skp2 in non-small cell lung cancer as well as the effect on anti-tumor metastasis. |
