Mechanism Of Effective Components β,β-dimethylacrylakannion And Acetylshikonin Derived From Lithospermum Erythrorhizon Root In Colorectal Cancer Inhibition

Background and PurposeColorectal cancer(CRC)is a common gastrointestinal malignancy in clinical,and is one of the most common cancers with high incidence and mortality worldwide.The incidence of colorectal cancer in China is increasing every year.The clinical treatment of patients with advanced colorectal cancer is mainly chemotherapy or postoperative adjuvant chemotherapy.However,colorectal patients have showed drug resistance to the clinical treatment regimen.And chemotherapy can cause adverse reactions to petients.It is urgent to reduce the side effects of chemotherapy drugs and improve the quality of patients for life.Recent years,targets therapy has been favored by people.Targets therapy could directly target to oncogene in tumor cell and inhibit the proliferation of cancer cell,and decrease the damage to normal cell.Therefore,the development of new colorectal cancer treatment plans will provide the basis for the clinical treatment of colorectal cancer.Based on the advantages of targeted therapy,study the inhibition mechanism to colorectal cancer of small molecular will prodive scientific,reasonable and efficient treatment strategies for colorectal cancer patients.Lithospermum erythrorhizoninhibits is traditional Chinese medicine in China,with the function such as antibacterial,anti-inflammatory and anticancer.The active components are mainly distributed from root.The mainly compounds purified from L.erythrorhizoninhibits are shikonin,isoamylacylalkannin,acetylshikonin,β,β-dimeth-ylacrylalkannin,β,β-dimethylacrylshikonin etc.We have mainly purified two active compounds,acetylshikonin and β,β-dimethylacrylalkannin,from this root.From the first step of purify method,we have got the mixture of β,β-dimethylacrylalkannin(β,β-DMAA)and acetylshikonin,the ratio of the two compound is two to one.In this study,we found β,β-DMAA and acetylsikonin can inhibit the kinase activity of FGFR1 and TOPK in an ATP-dependent manner,respectively.Furthermore,β,β-DMAA and acetylshikonin could inhibit cell proliferation of colorectal cancer cells(HCT 116,HCT-15,SW 620 and DLD-1)and induces cell cycle arrest at G0/G1 phase and apoptosis.In addition,β,β-DMAA and acetylshikonin could arrest the phosphorylation levels in FGFR1 and TOPK signaling pathways,respectively,and result in inhibition the growth of colorectal cancer.Fibroblast growth factor receptor 1(FGFR1)is a tyrosine kinase,and T-LAK cell-originated protein kinase(TOPK)is a sernine/threonine kinase.FGFR1 and TOPK both involved in cell proliferation,differentiation and migration.β,β-DMAA and acetylshikonin could inhibition of tumor growth in a patient-derived xenograft(PDX)model of human colorectal cancer with FGFR1 or TOPK high expression.In addition,the present study showed that the combination of β,β-DMAA and acetylsikonin could inhibit colorectal cancer significantly compared to treated β,β-DMAA and acetylshikonin sigle in vivo and in vitro.In summary,this study demonstrated that the components of Lithospermum erythrorhizoninhibits,β,β-DMAA and acetylsikonin could target FGFR1 and TOPK,respectively,and the mixture of the two compound could combination inhibit colorectal cancer proliferation and tumor growth in vivo and in vitro.This study revealed the inhibitory effects of β,β-DMAA and acetylsikonin on colorectal cancer in vitro and in vivo and elucidated the regulatory mechanism of two copound in colorectal cancer.This study will provide a theoretical basis for the clinical application of two active componets in colorectal cancer.Methods1.Cytotoxicity assay was performed to check the effect of β,β-DMAA and acetylshikonin in colon normal cell CCD-18Co,and choose the suitable concentration for research.2.Cell proliferation assay was used to determine the effect of β,β-DMAA,acetylshikonin and the mixture of two compounds on the colorectal cancer cells.3.Soft agar assay was used to check the inhibition effect of β,β-DMAA and actylshikonin on the colony formation in colorectal cancer cells.4.Flow cytometry assay was used to detect the regulatory effects of β,β-DMAA,acetylshikonin and the mixture of two compounds on the cell cycle and cell apoptosis of colorectal cancer cells.5.Kinase profiling assay and supercomputer technology was used to predict the targets of β,β-DMAA and acetylshikonin.6.Western blot was used to detect the expression level of p-FGFR1/FGFR1 or pTOPK/TOPK in colorectal cancer cell lines with human colorectal cancer cells CCD-18Co as a control.7.The expression of p-FGFR1 and FGFR1 in colorectal cancer tissue microarray was determined by immunohistochemistry assay.8.In vitro kinase assay was used to detect the inhibition effect of β,β-DMAA and acetylshikonin on the kinase activity of FGFR or TOPK.9.ex vivo pull-down assay was used to demonstrate the interactive between β,β-DMAA and FGFR1.10.ATP compepation assay and kinetic assay were performed to check the interactive effect between acetylshikonin and TOPK by ATP competation method or not.11.Cell proliferation and anchorage-indenpent growth were arrested in colorectal cancer cells which were knocked down the expression of FGFR1 or TOPK.12.Soft agar assay to check the effect of β,β-DMAA or acetylshikonin on anchorage-independent growth in in colorectal cancer cells which were knocked down the expression of FGFR 1 or TOPK.13.Western blot assay was used to detect the effect of β,β-DMAA or acetylshikonin on FGFR1 or TOPK signaling pathway.14.Luciferase assay was used to check the effect of acetylshikonin on the activity transcript factor NF-κB.15.The effect of cell proliferation and anchorage-independent growth in FGFR1 and TOPK both silenced cells was determined.16.The inhibitory effect of combination treatment of FGFR1 inhibitor(AZD4547)and TOPK inhibitor(HI-TOPK-032)in colorectal cancer cell was checked by cell proliferation aasay.17.The inhibitory effect of combination treatment β,β-DMAA and acetylshikonin in colorectal cancer cell was checked by cell proliferation assay.18.The expression level of FGFR1 or TOPK in the colorectal tissues of different patients was detected by western blot.And choose the suitable case of PDX model for in vivo study.19.In vivo experiment was performed to check the activity of β,β-DMAA,acetylshikonin or mixture of these two compounds on highly expression PDX mouse model.20.Immunohistochemial assay was used to detect the expression of FGFR1 or TOPK signaling pathway in PDX mice model treated with β,β-DMAA or acetylshikonin.Results1.β,β-DMAA and acetylshikonin showed no obvious inhibitory effect on the activity of human normal colon cells.The results of cell toxicity showed:The concentrations of β,β-DMAA and acetylshikonin less than 10 μM showed no cytotoxcity effect to human normal colon cell CCD-18Co,and the concentration less than 10 could be used for subsequent experiments.2.β,β-DMAA,acetylshikonin or mixture(β,β-DMAA and acetylshikonin)significantly inhibited proliferation of colorectal cancer cells.Cell proliferation results showed that β,β-DMAA with concentration at 1.25,2.5,5 or 10 μM,acetylshikonin with concentration at 2.5,5 or 10 μM,mixture of β,β-DMAA and acetylshikonin with the concentration of 0.38,0.75 or 1.5 μg/μL could significantly inhibited the colorectal cell growth by time-and dose-dependent manner(p<0.05).3.β,β-DMAA and acetylshikonin could significantly inhibit anchorage-inde-pendent growth of colorectal cancer cellsSoft agar assay showed that the colony formation ability of HCT 116,HCT-15,SW 620 and DLD-1was inhibited by β,β-DMAA or acetylshikonin in colorectal cancer cells,and the inhibitory effect of β,β-DMAA or acetylshikonin on the anchorage-independent growth of cancer cells,and was positively correlated with the drug dose(p<0.05).4.β,β-DMAA,acetylshikonin and mixture of these two compounds could induce colorectal cancer cells cycle arrest at G0/G1 phase and apoptosis.Cell cycle assay showed that β,β-DMAA or acetylshikonin at 5 and 10 μM,mixture at 0.38,0.75 and 1.5 μg/μL could induce cell cycle arrested of colorectal cancer cells at G0/G1 phase,as well as induced apoptosis.The expression of cell cycle markers cyclinDl and cyclinD3 were down-regulated.The expression level of cell apoptosis markers,PARP,caspase-3 and caspase-7 were down-regulated by β,β-DMAA or acetylshikonin.The expression of p21,cleaved PARP,cleaved caspase-3 and cleaved caspase-7 were up-regulated when treated with β,β-DMAA or acetylshikonin.5.β,β-DMAA is combined with FGFR1,and acetylshikonin is combined with TOPK.Kinase profiling assay showed the effect of β,β-DMAA on the kinase activity of 50 kinases and found the inhibition effect of β,β-DMAA to FGFR1 kinase is 66%.Supercomputional docking model simulations have shown that β,β-DMAA interacts with ATP pocket of FGFR1,and formed a complex.The binding site between β,β-DMAA and FGFR1 are Glu531,Val559,Ala564 and Asp641.Acetylshikonin interacts with ATP pocket of TOPK and formed a complex.The binding site between acetylshikonin and TOPK are Lys64,Gly118 and Asn172.6.p-FGFR1/FGFR1,pTOPK/TOPK were highly expression in colorectal cancer cells compared to normal colorectal cancer cells CCD-18Co.Western blot results showed that p-FGFR1 and FGFR1 were highly expressed in HCT-15,HCT 116,DLD-1,SW 620 and HT-29 cell lines compared to human colorecatal normal cell CCD-18Co.p-TOPK and TOPK were highly expressed in HCT-15,HCT 116,DLD-1 and SW 620 cell lines compared to human colorecatal normal cell CCD-18Co.7.p-FGFR1 and FGFR1 were highly expression in colorectal cancer tissue when compared to adjacent tissue.Immunohistochemistry assay showed p-FGFR1 and FGFR1 in colorectal cancer tissues were statistically significant(p<0.05),suggesting that p-FGFR1 and FGFR1 may play as an oncogene in the development of colorectal cancer.8.β,β-DMAA inhibited kinase activity of FGFR1,acetylshikonin inhibited TOPK kinase activity.In vitro kinase assay showed β,β-DMAA at 0.6 or 1.25 μM could significantly inhibited FGFR1 kinase activity.That indicated that FGFR1 was the target of β,β-DMAA.Acetylshikonin at 0.6,1.25 and 2.5 μM could significantly inhibited TOPK kinase activity.This indicated TOPK was the target of acetylshikonin.9.β,β-DMAA could interact with FGFR1,and acetylshikonin could interact with TOPK by pull-down assay.10.ATP competation assay and kinetic assay showed acetylshikonin could bind with TOPK by ATP competation method with the increase of ATP concentration,the binding between acetylshikonin and TOPK has decreased.Kinetic assay showed the Km value of TOPK has increase from 15 to 20 when treated with acetylshikonin at 0.3 μM.11.Downregulation the expression of FGFR1 or TOPK by shRNA knockdown assay could significantly inhibited the cell proliferation and colony formation of colorectal cancer cell lines.12.β,β-DMAA or acetylshikonin showed no significant effect on the anchorage-independent growth of colorectal cells which were silenced the expression of FGFR1.13.β,β-DMAA and acetylshikonin inhibited the phosphorylation expression of FGFR1 or TOPK downstream signaling pathway in colorectal cancer cell lines.14.Acetylshikonin inhibited the transcript activity of NF-κB.Luciferase assay showed acetylshikonin inhibited the transcript activity of NF-κB by a dose-dependent manner compared with control(p<0.05).15.Knock down both the expression of FGFR1 and TOPK could combination inhibit cell proliferation and anchorage-independent growth.Knock down assay showed silence the expression of FGFR1,TOPK and FGFR1 and TOPK could significantly inhibit cell proliferation and colony formation.Silence the expression of FGFR1 and TOPK together could significantly inhibit cell proliferation and colony information compared to silence the expression FGFR1 or TOPK single(p<0.05).16.FGFR1 inhibitor(AZD4547)and TOPK inhibitor(HI-TOPK-032)were treated to colorectal cancer cell together showed combination effect to the cell proliferation of HCT 116.Cell proliferation assay showed treatment 5 μM FGFR1 inhibitor(AZD4547),0.6 μM TOPK inhibitor(HI-TOPK-032)or treatment AZD4547 and 0.6 HI-TOPK-032 together,all could significantly inhibit cell proliferation.Combination treatment AZD4547 with HI-TOPK-032 could additive significantly inhibit cell proliferation compared to treatment AZD4547 and HI-TOPK-032 single(p<0.05).17.Combination treatment of β,β-DMAA and acetylshikonin showed additive effect to the cell proliferation of DLD-1.Cell proliferation assay showed treatment 0.6 μM β,β-DMAA,2.5 μM acetylshikonin or combination treatment β,β-DMAA with acetylshikonin all could significantly inhibit cell proliferation for DLD-1.Combination treatment β,β-DMAA with acetylshikonin could significant inhibit cell proliferation compared to treatmentβ,β-DMAA or acetylshikonin single(p<0.05).18.The expression level of FGFR1 or TOPK showed significant difference in colorectal patients tissues and choose the case of HJG101,HJG41 and HJG86 which is highly expression of FGFR1 or TOPK for in vivo study.19.β,β-DMAA,acetylshikonin or mixture of these two compounds arrested tumor growth in FGFR1-positive or TOPK-positive PDX mice model.In vivo study showed β,β-DMAA,acetylshikonin,mixture of these two compounds significantly inhibited tumor growth on FGFR1-positive PDX mice model with no significant loss in body weitht,liver weight,spleen weight and the number of WBC(p<0.05).20.β,β-DMAA or acetylshikonin inhibit the expression level of Ki-67 and phosphorylation level of FGFR1 and TOPK signaling pathways.Immunohistochemical results show that β,β-DMAA or acetylshikonin significant inhibited the expression of Ki-67,and phosphorylation expression level of FGFR1 or TOPK signaling pathway compared with vehicle group(p<0.05).Conclusions1.β,β-DMAA inhibits the proliferation,colony formation of colorectal cancer cells,induces cell cycle arrest at G0/G1 phase and apoptosis,inhibits tumor growth in PDX mouse model of colorectal cancer by targeted FGFR1.2.Acetylshikonin inhibits the proliferation,colony formation of colorectal cancer cells,induces cell cycle arrest at G0/G1 phase and apoptosis,and inhibits tumor growth in PDX mouse model of colorectal cancer by targeted TOPK.3.β,β-DMAA and acetylshikonin combination inhibits the proliferation,induces cell cycle arrest at G0/G1 phase and apoptosis,inhibits tumor growth in PDX mouse model of colorectal cancer.