Celastrus Orbiculatus Extraction Inhibits Vasculogenic Mimicry In Hepatocellular Carcinoma Via Modulating Notch1 Signalling

IntroductionHepatocellular carcinoma(HCC)is a common manignant tumor with high heterogeneity and full vasculature.Among the new diagnosed,few patients identified at early stage can be adapted to radical resection or liver plantation.However,many patients in the subpopulation received resection will be found intrahepatic reccurence or distant metastasis thus have poor 5-year surival.For those diagnosed with advanced stage,it is dismal to select some effective and safe treatments.Therefore,it is urgent to develop comprehensively therapeutic stategies.The failure of antiangiogenesis in manignantly solid tumors indicates another potential micro-vessel participating in tumor supplement.Vasculogenic mimicry(VM)lined with tumor cells independently is a type of functional vasculature involved in tumor nourishment and metastasis.The occurence of VM is a result of the trasforming of cellular phenotype and the cooperation between cells and microenvironment.It is now suggested that abundant oncogenes and angiogenesis genes are involved in VM formation.During the disease progression,VM indicates poor prognosis.Notch,divided into 4 isomers(Notch,2,3 and 4),is an important gene during embryonic development.In adult,Notch contributes to oncogenesis,tissue fibrosis and angiogenesis.Presently,several researches discover that Notch plays important roles in VM formation in melanoma and breast caner.However,the function of Notchl in HCC VM formation and the underlying mechanism are still unknown.HCC is termed "Gan-Ji" in Traditional Chinese Medicine(TCM)and belonged to the category of"collateral disease" in combination of Traditional and Western medicine.Cancerous angiogenesis is the incarnation of abnormal collaterals whose pathonogic characteristics are "weakness,blood stasis and toxin" Abnormal collaterals are the main pathways needed in tumor nourishment and metastasis.Theoretically,VM is another abnormal collateral occurring in HCC.Based on the law"cleaning the disease collateral with pungent agents",pungent herbs with the functions such as promoting blood stasis,clearning toxin and eliminating stagnation can cure abnormal collaterals.Some herb derived agents are now demonstrated to be effective in prevention of VM development.Celastrus orbiculatus thunb(Celastraceae)tastes pungent,belongs to the collateral channels of liver and bladder,functions as promoting qi to activate blood and cleaning toxin to reduce swelling.Our team has demonstrated that celastrus orbiculatus extraction(COE)can inhibit angiogenesis to reduce the growth of HCC in animal model,inhibit epithelial to mesenchymal transition(EMT)to reduce metastasis in nude mice bearing huaman gastric cancer.However,whether COE can modulate Notch1 to inhibit VM and prevent HCC progression has not been elucidated so far.As a result,we carried out the following 4 research parts to elucidate:1)the function of Notchl in VM formation and the potential mechanisms involved in;2)the bioactiveness of COE in inhibiting VM formation by modulating Notchl.Part I Expression of Notchl and its relationship with VM in HCC tissuesAims:This part research was conducted to detect the expression of Notchl and its downstream Hesl as well as EMT related proteins such as vimentin and E-cadherin in both HCC tissues and corresponding adjacent hepatic tissues.Relationships between Notchl and VM.EMT and VM were analized.Pathological indications of VM in HCC prognosis were elucidated.Methods:Matched HCC tissues were collected and corresponding patients informations were followed up.Notch1,Hes1,vimentin as well as E-cadherin were detected using immunohistochemical staining(IHC).RT-PCR was performed to confirm the IHC results.PAS-CD34 trial staining was used to shown VM.Kaplan-Meire survival curve was used to analyzed relation between VM and the time to postoperative reccurence.Results:A total of 44 pair of HCC tissues and adjacent non-cancer tissues were collected.Notchl and Hesl were found increase in HCC tissues compared with non-cancer tissues(P<0.001).In the 44 cases,17 specimens(38.64%,17/44)were identified with VM,the incidence of VM was closely correlated with Notchl and Hesl.Vimentin was observed positively stained in HCC cells in a subpopulation with 12 cases(27.27%,12/44).A positive correlation was found present between vimentin and VM.In the 12 specimens with positive vimentin staining,Notchl and Hes1 were significantly increased compared with those with negative vimentin staining.Survival curves showed that high Notch1,Hes1 and positive VM indicated shorter time to postoperative reccurence.Conclusion:Presence of VM in HCC tissues implies ealier postoperative reccurence.Increase of Notchl in HCC tissues correlates with EMT and VM formation.Part Ⅱ Notch1 contributes to the VM lined with HCC cells in vitroAims:To investigate the function of Notchl in VM formation and explore the underlying mechanisms.Methods:Using 3-dimensional culture,the capacity of forming VM in poor invasive HepG2 and high invasive MHCC97-H was mesured.RT-PCR and Western Blot were used to mesure expression of Notch1.After validating target cell,lentiviral vectors were employed to mediate overexpression or/and lowexpression in both cell lines.Capacities to form VM were observed in transforming cell lines using tube formation assay.Fluorescence conjugated phalloidin was used to label the cytoskeleton F-actin.RT-PCR and Western Blot were used to validate the increase or decrease of EMT related biomarkers such as E-cadherin and vimentin.Ultimately,TGF-β1 was used to induce EMT in different cell lines and VM formation,EMT expressions of related proteins were checked.Results:Poor invasive HepG2 cells with low Notch1 expression were not capable of forming VM on matrigel.Aferter overexpression of Notchl,cell invasion was enhanced and the capacity of VM was acquired.Cell phenotype transformed to mesenchymal type such as:1)F-actin showing a compact and irectional alignment with obvious fibrous tension;2)EMT markers vimentin increasing and E-cadherin decreasing.Instead of infection of lentivirus,TGF-β1 could also increase Notchl in HepG2 and cause the EMT,ultimately induced the invasion enhancced and VM formation.As the contrary,kncocdown of Notchl in MHCC97-Hcells blocked the EMT,inhibited invasion and VM formation.Additionally,downregulation of Notchl in MHCC97-H attenuated the response to TGF-β1 stimulation.Conclusion:Notchl promotes VM formation in HCC cells by inducing the EMT signalling.Interruption of Notchl can inhibit EMT in HCC cells and prevent VM formation ultimately.Part Ⅱ Notchl promotes VM formation in human derived subcutaneous xenograft in nude miceAims:This part was carried out to explore the roles of Notchl in VM formation in nude mice model bearing human HCC.Methods:In part 2 we have set up 4 transforming HCC cell lines,HepG2/vector、HepG2/Notchl +、MHCC97-H/vector and MHCC97-H/Notch1-.In this part,the 4 cell lines were utilized to construct animal model.Favourably proliferative cells(1×106)were respectively inoculated into the left flank of nude mice to develop subcutaneous xenograft.After the nude mice models were established,the tumor dimensions were measured every three days by caliper and tumor volume was calculated using the formula:V = length×width2×0.5.At the 28th days after establishment,the subcutaneous xenografts were scaned with an in vivo fluorescence imaging system.Then all the mice were sacrificed and tumors were excised and weighed.Haematoxylin and eosin(H&E)staining was used for the pathological observation.PAS-CD34 double staining and transmission electron microscope(TEM)was used to detect VM.EMT related biomarkers,vimentin and E-cadherin were mesured by IHC.Results:HepG2/Notch1 + transplanted tumors were accelerated on the 20th day after modeling,the tumor volume and mass were higher than that of HepG2/vector at the end of the experiment.On the contrary,MHCC97-H/Notchl-transplanted tumors significantly slow than MHCC97-H/vector on the 20th day after modeling,to the end the tumor volume and quality were less than MHCC97-H/vector.Apoptosis and necrosis were hardly found in HepG2/Notchl+ and MHCC97-H/vector while it was obvious in their counterparts respectively.In HepG2/vector and MHCC97-H/Notchl-subcutaneous xenografts with low Notchl typical VM structure was fewer compared with their counterparts respectively.Immunohistochemical staining showed that the vimentin located in tumor cells was more obvious in HepG2/Notchl+and MHCC97-H/vector tumors than it in their counterparts respectively.Correspondingly,E-cadherin was lower in HepG2/Notchl+and MHCC97-H/vector subcutaneous xenografts.Conclusion:Notchl promotes VM formation in human derived subcutaneous xenografts in nude mice while downregulation of Notchl can attenuate VM and inhibit tumor growth ultimately.Part IVCOE inhibits vasculogenic mimicry in hepatocellular carcinoma via suppressing Notchl signallingAims:In the current study,experiments in vitro and in vivo were carried out to examine the effects of COE on VM formation and HCC tumor growth via suppressing Notchl signalling.Methods:In the part of experiments in vitro,viability of various tumor cell lines exposured to COE was firstly analyzed using CCK-8.Then biostation dynamic observation were used to examine cellular confluence in MHCC97-H.In the following researches concentrations of COE(20,40,80 μg/mL)were set below the IC50.Cell invasion was examined using Transwell assay.Matrigel was used to establish a 3-D culture condition for VM formation.Changes of mRNA and protein were examined by RT-PCR and Western Blot respectively.Cytoskeleton was analyzed using fluorescence-conjugated phalloidin to label F-actin.In the research in vivo,subcutaneous xenograft was established using GFP conjugated MHCC97-H cells.After modelling,animals were randomly distributed into control group(saline),treating groups with COE(20,40,80 mg/kg,intragastricly,daily)or DAPT(20mg/kg,intraperitoneally,daily).Tumor growth was monitored using caliper and an in vivo fluorescence imaging system.PAS-CD34 double staining and TEM were used to observe VM formation.IHC was used to examine Notchl,Hesl,vimentin and E-cadherin respectively.Results:COE could inhibit viability of various tumor cell lines such as HCC cell line,lung cancer cell line,melanoma cell line and gastric cell line.In MHCC97-H,COE inhibited the viability with concentration and time-dependent manners,the IC50 in 24 h was 145.88μg/mL.COE(20,40,80μg/mL)and DAPT(5μM)for 24 h could effectively inhibit the invasion and VM formation in MHCC97-H or in HepG2 induced by TGF-β1(15ng/mL).Notch1 and Hesl could significantly modulated by COE or DAPT.The distribution and tension of F-actin were significantly disturbed by COE.In mouse xenograft model,COE or DAPT could inhibit tumor growth and VM formation,downregulat Notch1 and Hes1.Conclusion:Our results indicated that COE can inhibit VM formation and HCC tumor growth by downregulating Notchl signalling.This study demonstrated that COE is superior to other anti-angiogenesis agents and can be considered as a promising candidate in HCC treatment.