3-Deoxysappanchalcone(3-DSC) Inhibits Skin Cancer Proliferation In Vitro And In Vivo By TOPK

Skin cancer is one of the most common cancers in the world.The latest research shows that the incidence of skin cancer in China has increased year by year over the past 30 years.Without a public health system that effectively prevents skin cancer,countries with a low incidence of skin cancer(especially China)will face huge health challenges.BRAF and MEK inhibitors are important target drugs for advanced skin cancer,but the accompanying resistance problem makes the development of new targeted drugs more urgent.TOPK is a novel anticancer target and a kinase of the MAPKK family,so we predict that TOPK inhibitors may have an inhibitory effect on skin cancer.In this article,we assess the inhibitory effect of 3-deoxychalcone(3-DSC),a compound extracted from the Chinese herbal medicine Sumu in the context of melanoma.We previously observed that this compound targets TOPK.Therefore,this article mainly studies the mechanism of 3-DSC in skin cancer prevention and inhibition,and provides new ideas for the development of skin cancer drugs.In the short-term solar simulated light(SSL)irradiation experiment of hairless mice SKH1,the topical application of 3-DSC on the dorsal skin can prevent skin thickening caused by ultraviolet irradiation and reduce the expression of p-TOPK and its related signaling pathway proteins.3-DSC can inhibit the proliferation of skin cancer cells(SK-MEL-2,SK-MEL-28,A3 75 and A431)and induce cell cycle arrest in the G2/M phase by regulating the cell cycle.At the same time,the expression of G2/M phase related protein cyclinB1 was down-regulated in western blot experiments,which confirmed the G2/M phase arrest result at the protein level.3-DSC can induce skin cancer cell apoptosis and increase the expression of cleaved caspase-3 and caspase-7,which are markers of apoptosis.3-DSC can down-regulate the phosphorylation level of the TOPK signal transduction pathway,thereby inhibiting the proliferation of skin cancer cells in SK-MEL-2 derived xenograft(CDX)mice model.Thus,this article demonstrates that 3-DSC can inhibit the growth of skin cancer in vitro and in vivo by inhibiting the activity of TOPK kinase.This study will provide theoretical basis for the clinical prevention and treatment of 3-DSC and other TOPK inhibitors in skin cancer.Methods1.Kinase assay:Check the effect of different concentrations of 3-DSC on TOPK kinase activity using MBP substrate and ATP.2.Protein pull-down assay:Cell lysate is incubated with affinity ligand beads or 3-DSC-affinity ligand beads and then eluted.Use its physical structure to determine if the target of 3-DSC is TOPK and then determine the binding sites.3.Western blot:Detect TOPK expression in skin cancer cell lines.4.Cell cytotoxicity and growth experiments:Active cells incubated with yellow tetrazolium salt(MTT)produce purple formazan crystals and at the same time the live cells containing NAD(P)H-dependent oxidoreductase reduce the formazan.The insoluble formazan is dissolved using dimethyl sulphoxide(DMSO)and the absorbance of the solution is measured at 470/590 nm wavelength using a 96 well spectrophotometer.The final figure extrapolates the live cell number according to the optical density(OD)value.5.Soft agar colony formation experiment:To detect the inhibitory effect of 3-DSC on the colony formation of skin cancer cell lines.6.Cell cycle experiment:Using propidium iodide(PI)dye to stain intracellular DNA to distinguish cells cycle G1(IN)/S(1N-2N)/G2/M(2N)by flow cytometry,and then explore the effect of 3-DSC on the cell cycle of skin cancer cells.7.Cell apoptosis experiment:The specific binding of negatively charged phospholipids on the inner side of cell membrane by Annexin V in cell flow cytometer accords with the characteristics of early apoptotics and the impermeability of PI dye to living cells.Effects of apoptosis and the proportion of viable,early,late,and necrotic cells.8.Knock-down experiment:Plasmids containing short hairpins that target TOPK were constructed and used to create lentivirus particles.Skin cancer cell lines that express high levels of TOPK were chosen for infection.Puromycine-selected skin cancer cells with knocked down TOPK were selected and compared with the control group with respects to proliferation and sensitivity to 3-DSC.9.Detection of cell signal transduction pathways TOPK/ERK/RSK/c-Jun:Apply 3DSC(0,5,10,20 μM)to skin cancer cell lines(SK-MEL-2,SK-MEL-28,A375 and A431)and assess the effects of different concentrations on phosphorylated TOPK and its downstream signaling pathway were investigated by western blot experiments.10.Ultraviolet short-term irradiation experiment(Solar simulated light(SSL)):Check skin thickness and pTOPK signal transduction pathway protein expression in the control group and 3-DSC treatment group with different concentrations after UV irradiation.11.Mouse in vivo cell xenograft(CDX)model experiment:The mouse skin cancer cell line SK-MEL-2 was transplanted into mice.Tumors were allowed to grow before the mice were injected intraperitoneally with 3-DSC to detect its inhibition of skin cancer growth.Results1.3-DSC inhibits TOPK kinase activity in vitro.2.3-DSC binds to TOPK at Thr42 and Asnl72 in vitro.3.TOPK protein is higher expressed in skin cancer cell line SK-MEL-2 and A375 comparing with SK-MEL-28 and A431 cell lines.4.3-DSC(0,5,10,20 μM)is not toxic to Normal Human Skin Fibroblasts(NHDF).The concentration of 3-DSC at 10 μM and 20 μM inhibits the growth and proliferation of skin cancer cell lines(SK-MEL-2,SK-MEL-28,A375 and A431)at 48 h,72h,and96h(p<0.01).5.3-DSC can inhibit the formation of soft agar clones of skin cancer cells(SK-MEL-2,SK-MEL-28,A375 and A431)at the concentration of 10 μM and 20μM.6.3-DSC induces skin cancer cells(SK-MEL-2,A375 and A431)cell cycle arrest at G2/M phase at 20 μM.7.3-DSC induces apoptosis of skin cancer cells(SK-MEL-2,A375 and A431)at the concentration of 10,20 μM.8.After knocking down TOPK in skin cancer cell lines(SK-MEL-2 and A3 75)with high expression of TOPK protein,cell proliferation was suppressed and soft agar colony number was reduced.The inhibitory effect on knock-down cells was reduced after applying 3-DSC.9.3-DSC inhibites the phosphorylation TOPK/ERK/RSK/c-Jun signaling pathway in a dose-dependent manner.10.At 3-DSC(500 nmole,1000 nmole)concentration,increased skin thickness caused by acute UV exposure can be reduced by inhibiting the TOPK signal pathway.11.3-DSC inhibits the tumor growth of SK-MEL-2 cells CDX model at a concentration of 10 mg/kg and 20 mg/kg by inhibiting the phosphorylation TOPK protein signal transduction pathway.And after high concentration 3-DSC(20 mg/kg)treatment to nu/nu mice for 43 days,3-DSC has no significant effect on the liver and spleen weight and body weight curve of mice.Conclusion1.3-DSC binds with TOPK at Thr42 and Asn172 and inhibits TOPK kinase activity in vitro.2.3-DSC inhibits skin cancer cells proliferation and reduces soft agar colony number by targeting TOPK.3-DSC induces cell cycle arrest at G2/M phase and cell apoptosis.3.3-DSC prevents mice skin damage from UV exposure by inhibiting TOPK signal pathway.4.3-DSC inhibits the growth of human skin cancer cell derived xenograft tumors(CDX)in mice...