| ?Background and Objective?Liver cancer,including hepatocellular carcinoma(HCC),is the third cause of cancer death worldwide.The chemo-and radiotherapies are only effective to late-stage patients in the initial stage,then it occurs many side effects,such as toxicity and drug-resistance,tumor relapse and metastasis.Although substantial efforts have been made to reduce the mortality of HCC and improve the life quality of patients,there is still no satisfying therapeutic method until today.Increasing evidence has shown that a small subpopulation of cells within tumors,termed cancer stem cells(CSCs),are not only the roos of tumor initiation,growth,metastasis,recurrence and drug-resistance,but also the promising targeted cells for noval cancer therapies.HCC CSCs can be isolated and characterized using various stem cell markers such as Ep CAM,CD133,CD90,and CD44.They exhibit stem/progenitor cell-like characteristics such as easy tumorigenesis,in vitro spherical colony formation,self-renewal and differentiation,and resistance to chemo-and radiotherapies.The self-renewal and differentiation of CSC is regulated by various signaling pathways,such as Wnt/?-catenin,Hedgehog and Notch pathways.Wnt/?-catenin signaling pathway plays important roles in liver metabolism and regeneration.However,the aberration of Wnt/?-catenin pathway could promote tumor occurrence,invasiveness and therapeutic resistance.For example,neutralizing antibodies and RNA interference against CSC markers or small molecule CSC inhibitors alone or combination with chemotherapies have been shown to reduce tumor growth,incidence,and metastasis in animals.Matrine,a major active alkaloid of the Chinese herbal medicine Sophora flavescens Ait,possesses significant anti-neoplastic,anti-fibrotic,anti-inflammatory,and anti-viral properties.Because of its low potency and short half-life,we have recently semi-synthesized a series of matrine derivatives by replacing the carbonyl oxygen atom with a sulfur atom and introducing various amino groups to the keto-beta position.The anti-inflammatory,anti-fibrotic and anti-tumor activities of these derivatives are significantly higher than those of matrine.Matrine derivatives can inhibit AKT/GSK3?,MAPK and NF?B signaling pathways in hepatoma cells,hepatic stellate cells,macrophagus and dendritic cells.Interestingly,we have previously demonstrated that matrine derivative M19 can treat liver fibrosis through its target ribosomal protein S5(RPS5).The present results indicate that matrine derivatives can be specifically bind to 67 KD laminin receptor(67LR),another member of ribosomal proteins.67 LR is encoded by ribosomal protein SA(RPSA)and considered to be a nonintegrin cell-surface receptor for laminin with high affinity.67 LR governs critical cellular processes including cell adhesion,migration,homing,proliferation,differentiation and polarization,which has been associated with metastatic cancer,neurodegenerative disease and developmental abnormalities.An increase in the expression of 67 LR as compared with the corresponding normal tissue has been found in a variety of common cancers.The green-tea-derived polyphenol,(-)-epigallocatechin-3-gallate(EGCG),is the only small molecule that has been reported to be the ligand of 67 LR.EGCG may be an agonist or allosteric agent of 67 LR.Because of the existence of many targets in cells,67 LR possessses extensive pharmacological activities but low specitivity.It has been reported that 67 LR antibody can block the anti-tumor effects of EGCG mainly acting on cell-membrane 67 LR.However,the downstream signaling pathways of 67LR-active EGCG are still unclear.The bind of EGCG and 67 LR improved protein phosphatase 2A(PP2A)activity or activated Akt pathway,which phosphorylated e NOS to release NO and enhanced c GMP in cells.WM130 is one of the novel matrine derivatives and exhibits better pharmacological activities and lower toxicity.We chose WM130 to study its anti-tumor effects and mechanisms on HCC.So far,the effects of matrine derivatives on hepatic stem cells and its anti-tumor effects on HCC associated with 67 LR have not been reported at home and abroad.The present study found that matrine derivative WM130 inhibited the proliferation and clone formation of hepatoma cells in vitro and HCC xenograft growth in vivo.WM130 can also inhibit hepatic cancer stem-like cells in both HCC cell lines and xenografts through Wnt/?-catenin signaling pathway.The further study showed that WM130 displayed its anti-tumor effects on HCC partly through its target 67 LR.?Method?1.The effects of matrine derivative WM130 on the proliferation and clone formation of hepatoma cells1)HCC MHCC-LM3,Hep3 B and MHCC-97 H cells were cultured at a density of 3×103 cells per well in 96-well plates overnight and then exposed to WM130(0,2,10,20?mol/L).After 72 h of culture,cell proliferation was assessed using a Cell Counting Kit-8(CCK8),and the 50% inhibition concentration(IC50)was calculated.2)HCC MHCC-LM3,Hep3 B and MHCC-97 H cells were seeded at a density of 1×103 cells per well in 6-well plates and treated with WM130(0,2,10,20?mol/L).Culture medium was changed every 3–5 days.After 17–21 days,the colonies were stained with coomassie brilliant blue and photographed.The total number of colonies(>50 cells/colony)was counted.2.The effects of matrine derivative WM130 on hepatic cancer stem-like cells(1)The effects of WM130 on DOX-resistant hepatoma cellsHep3B and MHCC-LM3 cells were treated with DOX(2?mol/L)for 5 days(Hep3B-DOXR and LM3-DOXR cells)and then exposed to WM130(0,2,10,20?mol/L)or 2?mol/L DOX for 72 hours.CCK8 assay was used to test the proliferation of cells and the number of cells that expressed the marker of hepatic stem cells(Ep CAM)was assessed by flow cytometric analysis.(2)The effects of WM130 on the expression of stemness-related and liver-specific genesAfter 24 h treatment of WM130(0,2,10,20?mol/L),Hep3 B and MHCC-LM3 cells were identified by RT-PCR for the expression of stemness-related genes Ep CAM,CD133,CD90,Oct3/4,Sox2,NANOG and liver-specific genes CYP1A3,G-6-P,ALB and ALDOB.(3)The effects of WM130 on spheroid formation ability of hepatoma cells1)We used spheroid formation method to enrich sphere cells in hepatoma cells(hepatic cancer stem-like cells).Sphere MHCC-LM3,Hep3 B and MHCC-97 H cells were collected and digested by 0.25% trypsogen for 3 minutes,and stoped by 10% FBS DMEM.Cell suspensions(primary spheres)were gathered,added with flow antibodies and mixed away from light at the same time.A flow cytometer was used to analyse the number of cells that expressed Ep CAM and CD133.2)The primary spheres were cultured at a density of 1×103 cells per well in 96-well ultra-low attachment plates overnight and then exposed to 0-20?mol/L WM130 for 7 days and photographed under a phase contrast microscope.Primary sphere MHCC-LM3,Hep3 B and MHCC-97 H cells were dissociated with trypsin to single cell suspensions and seeded in 96-well ultra-low attachment plates at a density of 1×103 cells/well.The second and third passages of cells were grown for 7 days in the absence of WM130.The number of spheres(>30?m in diameter)was counted.3)Hep3B spheres were treated with or without 10?mol/L WM130 for 4 days and then allowed to recover and expand for another 4 days without treatment prior to injection in vivo.The WM130-treated cells(1×105)were subcutaneously injected into the flank of each mouse.To avoid individual differences,an equal number of vehicle-treated control cells were subcutaneously injected into the opposite flank of the same mouse.Tumor growth was monitored for 45 days after cell injection.Immunohistochemical(IHC)assay was applied to test the expression of Ep CAM in the tumor tissue.3.The preferential effects of matrine derivative WM130 on HCC spheresHepatoma and sphere cells were treated with WM130(0,2,10,20?mol/L)or DOX(0,1,2,5?mol/L).CCK8 and clone formation assays were used to compare the distinction of WM130 and DOX on these two species of cells.Hepatoma and sphere cells were treated with 10?mol/L WM130,and RT-PCR analysis was used to test the expression of stemness-related genes Ep CAM,CD133 and Oct3/4.4.The effect of matrine derivative WM130 on HCC growth in vivo(1)The effects of WM130 on HCC xenograft growth1×106 MHCC-LM3 cells were subcutaneously injected into nude mice.Animals were administered either saline,WM130(20mg/kg,orally by gavage daily),DOX(6mg/kg,intraperitoneal injection,once a week),or WM130 in combination with DOX for 3 weeks.The tumor size was measured with a caliper every 2-3 days and the weight of mice was observed.All mice were sacrificed,and tumor tissues were excised and weighted at the end of experiments.(2)The effects of WM130 on hepatic stem-like cells in vivoA portion of the tumor tissues was dissociated enzymatically to obtain a single cell suspension for the spheroid formation assay,colony formation assay and drug-resistant analysis,and the remaining tumor tissues were used for IHC,RT-PCR and Western blot.5.The effects of matrine derivative WM130 on Wnt/?-catenin signaling pathway(1)The effects of WM130 on Wnt/?-catenin signaling pathway in vitroAfter treatment with WM130(0,2,10,20?mol/L)for 4 days,hepatoma Hep3 B and MHCC-LM3 cells were evaluated by western blot for the expression of ?-catenin,cyclin D1,GSK3? and p-GSK3?(ser9).(2)The effects of WM130 on Wnt/?-catenin signaling pathway in vivoIHC and western blot assays were used to test the expression of ?-catenin,Ep CAM,GSK3? and p-GSK3?(ser9)in tumor tissues of MHCC-LM3 xenograft.6.The anti-tumor effects of matrine derivative WM130 on HCC associated with 67LR(1)The effect of 67 LR antibody on the inhibition of WM130 to the proliferation of hepatoma cellsHepatoma Hep3 B and MHCC-LM3 cells were incubated with anti-67 LR Ab(Mlu C5),or isotypematched control mouse Ig M(20mg/m L)at 37°C in 5%CO2 for 1hour before the addition of EGCG(40?mol/L)or WM130(0,10,20,40?mol/L).After 72 h of culture,CCK8 assay was used to test the proliferation of cells.(2)The effect of 67 LR knockdown on the inhibition of WM130 to the prolifer--ation of hepatoma cellsHepatoma Hep3 B and MHCC-LM3 cells were interfered by 67 LR si RNA for 48 ho--urs or formed sh67 LR stable-transfected hepatoma cells before the addition of WM130(0,10,20,40?mol/L).After 72 h of culture,CCK8 assay was used to explore the proliferation of cells.(3)The effect of EGCG on the inhibition of WM130 to the proliferation of hepatoma cellsHepatoma Hep3 B and MHCC-LM3 cells were treated with EGCG(20?mol/L)and WM130(0,10,20,40?mol/L)for 72 hours,and CCK8 assay was used to assess the proliferation of cells.(4)The effects of PDE5 inhibitor on the inhibition of WM130 to the proliferation of hepatoma cellsHepatoma MHCC-LM3 cells were treated with PDE5 inhibitor sildenafil(20?mol/L)and EGCG(20?mol/L)or WM130(0,10,20,40?mol/L)for 72 hours,and CCK8 assay was used to assess the proliferation of cells.(5)The effects of PP2 A inhibitor on the inhibition of WM130 to the proliferation of hepatoma cells1)Hepatoma Hep3 B and MHCC-LM3 cells were treated with OA(0,5,10,20,40,100 n M)for 3 hours before the addition of 20?mol/L WM130 or 40?mol/L EGCG.After 72 h of culture,CCK8 assay was used to explore the proliferation of cells.2)Hepatoma cells were treated with 10 n M OA for 3 hours before the addition of WM130(0,10,20,40?mol/L)or EGCG(0,10,20,40?mol/L).After 72 h of culture,CCK8 assay was used to assess the proliferation of cells.?Results?1.Matrine derivative WM130 inhibits the proliferation and clone formation of hepatoma cellsCCK8 assay showed that WM130(0,2,10,20?mol/L)suppressed the proliferation of all three HCC cell lines in a concentration-dependent manner with IC50 values of 8.2,10.6 and 12.5?mol/L for Hep3 B,MHCC-LM3 and MHCC-97 H cells,respectively.Moreover,the anti-proliferative effect of WM130 was exerted in a time-dependent manner(0,24,48,72,96h).Clonogenic assays showed that WM130 significantly reduced the number of colonies after the long time(17-21 days)treatment of WM130.2.Matrine derivative WM130 inhibits hepatic cancer stem-like cells(1)The effects of WM130 on DOX-resistant hepatoma cellsSingle drug-resistant assay showed that DOX had no inhibitory effect on the proliferation of Hep3B-DOXR and LM3-DOXR cells but WM130 remained to inhibit their proliferation in a concentration-dependent manner.Flow cytometric analysis indicated that the number of Ep CAM+ cells was increased 9 and 20 times among Hep3B-DOXR and LM3-DOXR cells in comparison with parental Hep3 B and MHCC-LM3 cells that had not been pretreated with DOX.The enriched Ep CAM+ fraction in Hep3B-DOXR and LM3-DOXR cells significantly reduced after 10?mol/L WM130 treatment.(2)The effects of WM130 on the expression of stemness-related and liver-specific genesRT-PCR showed that WM130 treatment reduced the expression of Ep CAM,CD133 and CD90,cell surface markers of HCC CSCs.Furthermore,the expression of genes that abundantly expressed in human embryonic stem cells and involved in the maintenance of pluripotency,including Oct3/4,Sox2 and NANOG was decreased markedly.WM130 treatment also increased the expression of a cluster of liver-specific genes,including CYP1A3,G-6-P,ALB and ALDOB,which was accompanied by down-regulation of hepatocyte malignance gene AFP,while BR expression remained unchanged.These results suggest that WM130 could promote hepatic stem cells to differentiate into hepatic cells.(3)The effects of WM130 on spheroid formation ability of hepatoma cellsFlow cytometric analysis revealed that Hep3 B and MHCC-97 H spheres expressed more than 95% Ep CAM+CD133+ cells.MHCC-LM3 spheres expressed 65% Ep CAM+ cells and 43% CD133+ cells.WM130 treatment reduced the number of Ep CAM+ cells in the three spheroids in a concentration-dependent manner,while WM130 rendered no evident influence on CD133+ cells.Moreover,WM130 noticeably inhibited the formation of primary Hep3 B,MHCC-LM3 and MHCC-97 H spheres with significant decreases in the number and size.The number of spheres decreased when WM130-treated primary spheres were cultured for the subsequent two passages in the absence of WM130,indicating that WM130 inhibited the self-renewal ability of CSCs.In the Hep3 B sphere xenograft,we observed that palpable tumors(about 1mm in diameter)developed from vehicle-treated spheres within 2–3 weeks and WM130 treatment delayed palpable tumor formation by about 2 weeks.The tumor xenografts derived from WM130-treated spheres were significantly smaller than those derived from control cells.The number of Ep CAM+ cells was diminished around 35%(P<0.01)in the WM130 group compared with the control group as measured by IHC staining.3.Matrine derivative WM130 preferentially inhibits HCC spheresWM130(2,10,20?mol/L)preferentially inhibited Hep3 B and MHCC-LM3 sphere cell proliferation compared to their hepatoma cells.In contrast,DOX treatment(1,2,5?mol/L)had stronger inhibitory effects on the proliferation of Hep3 B and MHCC-LM3 cells than their spheres.Colony formation assay had the same results.Remarkably,WM130 treatment resulted in a greater decrease in the expression level of Ep CAM m RNA in the two types of spheres than in their parental cells.However,there was no significant difference in CD133 or Oct3/4 m RNA levels between spheres and their parental cells.These results show that WM130 can preferentially inhibit HCC spheres.4.Matrine derivative WM130 inhibits HCC growth in vivo(1)WM130 inhibits HCC xenograft growthThe growth of MHCC-LM3 xenografts in the WM130(10mg/kg)group was reduced compared with that in the control group.When combined with DOX(3mg/kg),WM130 exerted a stronger inhibitory effect on tumor growth than either WM130 or DOX alone.These results were in accordance with the weights of MHCC-LM3 xenografts at sacrifice.The average body weight of control and WM130-treated mice did not vary significantly throughout the experiment(21 days).Moreover,WM130 did not increase DOX-induced weight loss which indicated the safety of WM130 and the toxicity of DOX.(2)WM130 inhibits hepatic stem-like cells in vivoTumors were harvested from the MHCC-LM3 xenograft animals and analyzed for the presence of surviving cancer stem-like cells by in vitro tumor sphere formation assays.The results showed a considerable decrease in the number of tumor sphere-forming cells in WM130-treated tumors in contrast to an increase in DOX-treated tumors.Moreover,WM130 further reduced the number of tumor sphere-forming cells when administered in combination with DOX.In addition,the colony formation ability of MHCC-LM3 cells from WM130-treated mice and WM130 plus DOX-treated mice was reduced significantly.WM130 administration remarkably decreased the level of Ep CAM m RNA in tumor xenografts,which was accompanied by increased expression of CYP1A3,G-6-P and ALB.In contrast,DOX increased expression of Ep CAM,CD133 and Oct3/4,and decreased expression of CYP1A3 and ALB.Interestingly,MHCC-LM3 cells from DOX-treated mice displayed higher proliferation ability than those from control-treated mice,which were resistant to DOX but sensitive to WM130 upon in vitro treatment.5.WM130 downregulates the Wnt/?-catenin signaling pathway(1)WM130 inhibits Wnt/?-Catenin signaling pathway in vitroWM130 markedly decreased phosphorylation of glycogen synthase kinase 3?(GSK3?)(Ser9)in Hep3 B and MHCC-LM3 cells.Moreover,WM130 reduced the protein level of ?-catenin in parallel with decreased expression of cyclin D1,a Wnt/?-catenin target gene.(2)WM130 inhibits Wnt/?-Catenin signaling pathway in vivoAccordingly,WM130 administration noticeably decreased phosphorylation of GSK3?(Ser9)and expression of ?-catenin and its target Ep CAM in the MHCC-LM3 tumor xenografts as assessed by western blotting and immunostaining.6.The anti-tumor effects of matrine derivative WM130 on HCC associated with 67LR(1)The effect of 67 LR antibody on the inhibition of WM130 to the proliferation of hepatoma cells67LR antibody did not affect MHCC-LM3 and Hep3 B cell proliferation.MHCCLM3 and Hep3 B were pretreatment with Ig M antibody and cell proliferation was inhibited by EGCG and WM130.The pretreatment of 67 LR antibody made the inhibitory effect of 40?mol/L EGCG on the proliferation of hepatoma cells weaken 24% and 30%,respectively.The inhibitory effect of WM130(10,20,40?mol/L)on the proliferation of MHCC-LM3 cell weakened 21%,45% and 48%,respectively.The inhibitory effect of WM130(10,20,40?mol/L)on the proliferation of Hep3 B cell weakened 31%,36% and 56%,respectively.The result demonstrated that the inhibitory effect of WM130 on the proliferation of hepatoma cells is at least partly through cell-membrane 67 LR.(2)The effect of 67 LR knockdown on the inhibition of WM130 to the proliferation of hepatoma cells1)67LR si RNA(100n M and 200 n M)reduced the expression of 67 LR m RNA by 50% and 75%,respectively.The expression of 67 LR protein accordingly reduced.Compared to NC si RNA,67 LR si RNA significantly inhibited hepatoma cell proliferation.The inhibition rate to MHCC-LM3 and Hep3 B cells is 25% and 59%,repectively.In the case of NC si RNA,WM130(10,20,40?mol/L)could significantly inhibited cell proliferation in a concentration-dependent manner.However,67 LR si RNA in hepatoma cells eliminated the inhibitory effect of WM130 on the proliferation of MHCC-LM3 and Hep3 B cells.2)WM130(10,20,40?mol/L)could inhibit the proliferation of Hep3 B cell infected by Lenti-NC sh RNA.While the inhibitory effect on the proliferation of Hep3 B cell infected by Lenti-67 LR sh RNA eliminated.(3)The effect of EGCG on the inhibition of WM130 to the proliferation of hepatoma cellsHepatoma cells were co-treated with EGCG(20?mol/L)and WM130(10,20,40 ?mol/L).EGCG weakened the inhibitory effect of WM130(10?mol/L)on the proliferation of hepatoma cells,while rendered no evident influence on the effect of(20 and 40?mol/L)WM130.The result proposed that EGCG had a competitive relationship with low-concentration of WM130 and weakened the inhibitory effect of WM130 on the proliferation of hepatoma cells.The effect of high-concentration WM130 may not be related to 67 LR,while associated with cell apoptosis.(4)The effects of PDE5 inhibitor on the inhibition of WM130 to the proliferation of hepatoma cells20?mol/L EGCG significantly enhanced the inhibitory effect of sildenafil(20?mol/L)on the proliferation of cancer cells,which was in accordance with the results from the reference.On the contray,WM130 did not improve the inhibitory effect of sildenafil on the proliferation of hepatoma cells.The results demonstrated that this pathway is not the anti-tumor mechanism of WM130.(5)The effects of PP2 A inhibitor on the inhibition of WM130 to the proliferation of hepatoma cells1)5n M and 10 n M OA had no inhibitory effect on hepatoma cell growth.While 20-100 n M suppressed the proliferation of MHCC-LM3 and Hep3 B cells in a concentration-dependent manner.The pretreatment of OA(5-40 n M)weakened the inhibitory effect of 20?mol/L WM130 and 50?mol/L EGCG on the proliferation of hepatoma cells.2)10n M OA made the inhibitory effect of WM130(10,20,40?mol/L)and EGCG(40 ?mol/L)on the proliferation of MHCC-LM3 cell decrease by 26%,42%,15% and 15%,respectively.The inhibitory effect of WM130(10,20,40?mol/L)and EGCG(40 ?mol/L)on the proliferation of Hep3 B cell decrease by 25%,44%,22% and 15%,respectively.The results demonstrated that the inhibitory effect of WM130 on hepatoma cell proliferation is partly through 67LR-dependent active PP2 A pathway.?Conclusion?1.Matrine derivative WM130 inhibits proliferation and clone formation of hepatoma cells.2.Matrine derivative WM130 preferentially inhibits hepatic cancer stem-like cells.3.Matrine derivative WM130 inhibits the growth of HCC in vivo and reduces hepatic cancer stem-like cells.3.Matrine derivative WM130 downregulates the Wnt/?-catenin signaling pathway.4.Matrine derivative WM130 inhibits cell proliferation partly through its target 67 LR. |
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