Antitumor Effect And Mechanism Of Lignans From Arctium Lappa L.

Cancer continues to be a major health problem of world wide.The development and discovery of newer anticancer therapeutics are still urgently needed because some cancer patients die due to the insensitivity to the existing anticancer drugs or developing drug-resistance after a period of treatment.The purpose of this work is to discover natural anticancer lead compound from Chinese medicine plants.Background and ObjectiveAccording to literatures and our previous experiments,arctigenin and arctiin exhibited interesting anticancer effects.Arctigenin,an active compound found in the seeds of burdock,has the ability to eradicate nutrient-deprived cancer cells.In addition to its broad spectrum of activities on different cancer cell lines,e.g.,PANC-1 and AsPC-1,arctigenin seems to exhibit a highly preferential cytotoxicity to cancer cells that are bathed in glucose-deprived conditions[91].The IC50 of K562 cell line with Lappaol B,Lappaol A,isolappaol A were 38.5 ?g/ml,53.5?g/ml,51.1 ?g/ml,respectively[137].Comprehensive anticancer activity and mechanism of lignans from Arctium lappa L.are still of significance for new lead discovery for active and secure anticancer agent.Method(1)Lignans were isolated from the chloroform extract of Arctium lappa L.by silica gel,Sephadex LH-20 column chromatography and ODS column chromatographies.Their structures were elucidated on the basis of spectroscopic analysis.For structural identification,spectroscopic data for all isolated compounds were measured;UV absorption spectra were run on a TU-1901 UV spectrometer(Purkinje General,China);Electrospray ionization mass spectrometry(ESI-MS)were measured on an API 2000 LC/MS/MS apparatus;1H and 13C nuclear magnetic resonance(NMR)were recorded on a Bruker DRX-400 instrument using tetramethylsilane(TMS)as an internal standard.(2)Effects of Arctigenin,Arctiin,and Lappaol F on cancer cell lines were examined by MTT assay and colony formation assay.Cell cycle was also determined by Flow Cytometry.Western blot analysis were also used.(3)Nude mice bearing HeLa xenograft tumor was rebuilt and in vivo anticancer effects of Lappaol F,Arctiin,and Arctigenin were observed.Results1.Manufacture for the lignans used in the study.Compound 1-3 were isolated from the seeds of Arctium lappa L.by means of chromatography.Compound 1-3 was identified as Arctigenin,Lappaol F and Arctiin for the TLC results,UV spectrum,1H and 13C NMR data were and identical to the literature.Purity Examination of Lappaol F by HPLC:Lappaol F was assayed by a Dinoex P680 HPLC with PDA monitoring absorbance at a wavelength of 280 nm.The HPLC was equipped with a Phenomenex Luna 4.6 x 150 mm,5 ?m column maintained at a temperature of 35?.The Mobile phase was a mixture of 47%methanol and 53%water(v/v)with a flow rate at 1.0 mL/min.The peak for the Lappaol F appears at ca.15.93 minutes.2.In vitro Antitumor Effect of the lignans of Arctium Lappa L.Lappaol F-mediated cell growth suppression was time-and dose-dependent and the estimated absolute IC50 was 13.3,16.8 and 25.2 ?M for MCF-7,MDA-MB-231 and RKO cells respectively.It is also found that Lappaol F exhibited minimal cytotoxicity to MCF-10A non-cancerous breast epithelial cells when treated under similar condition,3 days or with prolonged treatment to six days.3.Mechanism for Antitumor Effect of Lappaol FResults of flow cytometry analysis indicated that Lappaol F significantly increased the 2N G1 phase population or the 4N G2(or M)phase population in cells.It appears that there is some variation in the Lappaol F elicited response.MCF-7 and HeLa cells were predominantly arrested in 2N G1 phase.It is showed that significant increase(from 65%to 87%)in the G1 was observed in Lappaol F treated MCF-7 cells.It is showed that significant increase(from 56%to 67%)in the G1 was observed in Lappaol F treated HeLa cells.MDA-MB-231 and RKO cells were mainly arrested in 4N G2(or M)phase.It is showed that significant increase(from 21%to 45%)in the 4N G2(or M)-phase cell death population(G2/M)was observed in Lappaol F treated MDA-MB-231 cells.It is showed that significant increase(from 19%to 71%)in the 4N G2(or M)phase cell death population(G2/M)was observed in Lappaol F treated RKO cells.We investigated the molecular mechanisms via which Lappaol F induces cell cycle arrest.It is showed that the expression levels of CDK inhibitors p21 and p27 were strikingly elevated whereas the levels of CDK2,cyclin B and CDK1 were drastically reduced in Lappaol F-treated MCF-7 cells.It is showed that the expression levels of CDK inhibitors p21 and p27 were strikingly elevated whereas the levels of cyclin B and CDK1 were drastically reduced in Lappaol F-treated HeLa cells.It is showed that the expression levels of CDK inhibitors p21 and p27 were strikingly elevated whereas the levels of cyclin B and CDK1 were drastically reduced in Lappaol F-treated MDA-MB-231 cells.It is showed that the expression levels of CDK inhibitors p21 and p27 were strikingly elevated whereas the levels of cyclin B and CDK1 were drastically reduced in Lappaol F treated RKO cells.Mechanistic studies into insight of the action of Lappaol F indicate that Lappaol F effectively arrested cell cycle at G1 and G2 phases.Lappaol F also altered the function and expression of a numbers of key regulators of cell cycle.Our studies thus indicate that Lappaol F has excellent potential to be developed as anticancer therapeutics.4.In vivo Antitumor Effect of Lappaol FNatural products active ingredient in vitro screening is the first step to provide the basis for it to become a drug candidate.In last chapter we have identified a plant-derived antitumor agent Lappaol F and revealed its tumor inhibitory potentials in cell lines.Followed that,the experiment of in vivo antitumor effect of Lappaol F was done.Nude mice were skin disinfected with 75%medical ethanol,subcutaneously injected with 100?L of cell suspension(5×107 HeLa cells/mL)with 1ml syringe with 5 gauge needle on the right back to establish tumor xenograft.Observe whether there is leakage,bleeding,hematoma,breathing vitality of nude mice.Nine-days after tumor cell injection,20 mice with tumor volume of 90 mm3-290 mm3 were simple randomized into 3 groups;the average initial tumor volumes in each treatment group of mice were 161.28 ± 23.9 mm3(for vehicle control),160.7 ± 17.7 mm3(for mice treated with Lappaol F 5 mg/kg)and 144.5 ±20.5 mm3(for mice treated with Lappaol F 10 mg/kg),respectively.Vehicle group:equal volume DMSO plus Tween 80 in 5%glucose solution,5 mg/kg/d,N = 7,by intravenous injection for 15 d.Lappaol F prevention dose group:5 mg/kg/d,N = 7,by intravenous injection for 15 d.Lappaol F group:10 mg/kg/d,N = 6,by intravenous injection for 15 d.In the experiment,HeLa tumors in mice treated with Lappaol F grew much slower than those in mice treated with vehicle.As shown in Fig 5.1,average initial tumor volumes in each treatment group of mice were 144.5 ± 20.4(Lappaol F 10 mg/kg),160.7 ± 17.7(Lappaol F 5 mg/kg)and 161.3 ± 23.9 mm3(vehicle),separately.After a 15 days treatment period,tumor volumes in Lappaol F treated mice were 634.8 ± 54.9(Lappaol F 10 mg/kg)and 820.3 ± 86.0 mm3(Lappaol F 5 mg/kg)respectively,and tumor volume in vehicle treated mice was 1765.3 ± 91.8 mm3.At one-way ANOVA resulted in F= 55.63,P<0.001.Further Bonferroni Test revealed that the differences in tumor volumes between the Lappaol F 10 mg/kg group and vehicle group and between the Lappaol F 5 mg/kg group and vehicle group were statistically significant P<0.05,expect that between the Lappaol F 10 mg/kg group and Lappaol F 5 mg/kg group.It is showed the pictures and mean weights of tumors stripped from each group of mice at the end of experiment.In general,tumors in Lappaol F groups looked smaller than that in vehicle group.Tumor weights in Lappaol F groups were 0.40 ± 0.07g(Lappaol F 10 mg/kg)and 0.53 ± 0.07g(Lappaol F 5 mg/kg)respectively,and tumor weight in vehicle group was 1.21 ± 0.04g.At one-way ANOVA resulted in F= 56.21,P<0.001.Further Bonferroni Test revealed that the differences in tumor weight between the Lappaol F 10 mg/kg group and vehicle group and between the Lappaol F 5 mg/kg group and vehicle group were statistically significant P<0.05,expect that between the Lappaol F 10 mg/kg group and Lappaol F 5 mg/kg group.Animals appeared to tolerate the treatment of Lappaol F without significant body weight changes during treatment.In this experiment,Lappaol F significantly inhibited tumor growth without any observable toxic or side-effect to mice.Lappaol F-treated mice had their time-body weight curves similar to vehicle-treated mice.15 d after administration,Lappaol F,the weight gain of the two doses groups compared with the vehicle group,the differences were not statistically significant(P>0.05).The 15-day treatments with Lappaol F inhibited tumor growth by 54%(5 mg/kg/day,p<0.001,N = 7)and 64%(10 mg/kg/day,p<0.001,N = 6)relative to the vehicle-treated cohorts.It is also shows that increased Lappaol F dose from 5 mg/kg/day to 10 mg/kg/day further decreased tumor volume and weight.Importantly,we do not observe lethality or weight loss in mice given Lappaol F(5 mg/kg/day and 10 mg/kg/day)spanning 15 days of treatment;these results indicate that the Lappaol F given to the mice was well-tolerated and Lappaol F inhibits tumor growth in vivo.5.In vivo Antitumor Effect of Arctiin and ArctigeninAfter the antitumor effect and mechanism research of Lappaol F in vitro and vivo,as there are similar chemical structures,it is necessary to understand the antitumor effects of two main ingredients in Arctium lappa L,Arctiin,and Arctigenin.In the experiment,Nude mice were skin disinfected with 75%medical ethanol,subcutaneously injected with 100?L of cell suspension(5×107 HeLa cells/mL)with 1ml syringe with 5 gauge needle on the right back to establish tumor xenograft.Observe whether there is leakage,bleeding,hematoma,breathing vitality of nude mice.HeLa tumors in mice treated with Arctiin and Arctigenin grew much slower than those in mice treated with vehicle.It is showed that average initial tumor volumes in each treatment group of mice were 141.1 ± 17.6(Arctiin 30 mg/kg),139.3 ± 19.7(Arctiin 15 mg/kg)and 135.4 ± 28.3 mm3(Arctigenin 15 mg/kg),143.2 ± 21.8 mg/kg(vehicle),separately.Vehicle group:equal volume DMSO plus Tween 80 in 5%glucose solution,5mg mL/kg/d,N = 9,by intraperitoneal injection for 15 d.Arctiin 30 mg/kg group:30 mg/kg/d,N = 9,by intraperitoneal injection for 14 d.Arctiin 15 mg/kg group:15 mg/kg/d,N = 9,by intraperitoneal injection for 14 d.Arctigenin 15 mg/kg group:15 mg/kg/d,N = 9,by intraperitoneal injection for 14 d.In the experiment,HeLa tumors in mice treated with Arctiin and Arctigenin grew much slower than those in mice treated with vehicle.As shown in figure 6.1,average initial tumor volumes in each treatment group of mice were 141.1 ± 17.6(Arctiin 30mg/kg),139.3 ± 19.7(Arctiin 15 mg/kg)and 135.4 ± 28.3 mm3(Arctigenin 15 mg/kg),143.2 ±21.8 mm3(vehicle),separately.After a 14 days treatment period,tumor volumes in nude mice were 733.3 ± 88.4 mm3(Arctiin 30 mg/kg)and 795.6 ± 82,9 mm3(Arctiin 15 mg/kg)and 711.7 ± 104.9 mm3(Arctigenin 15mg/kg)respectively,which were much smaller than the tumor volume of 1351.7 ± 122.1 mm3 in mice treated with vehicle.At one-way ANOVA resulted in F = 9.14,P<0.001.Further Bonferroni Test revealed that the differences in tumor volumes between the Arctiin 30 mg/kg group and vehicle group,between the Arctiin 15 mg/kg group and vehicle group and between the Arctigenin 15 mg/kg group and vehicle group were statistically significant P<0.05,expect that between the Arctiin 30 mg/kg group and Arctiin 15 mg/kg group,between the Arctiin 30 mg/kg group and Arctigenin 15 mg/kg group,and between the Arctiin 15 mg/kg group and Arctigenin 15 mg/kg group.It is showed that the pictures and mean weights of tumors stripped from each group of mice at the end of experiment.In general,tumors in Arctiin and Arctigenin groups looked smaller than that in vehicle group.Tumor weights in Arctiin and Arctigenin groups were 0.49 ± 0.07g(Arctiin 30mg/kg)and 0.63 ± 0.12g(Arctiin 15mg/kg)and 0.56 ± 0.10 g(Arctigenin 15mg/kg)respectively,and tumor weight in vehicle group was 0.94 ± 0.12g.At one-way ANOVA resulted in F = 3.95,P<0.05.Further Bonferroni Test revealed that the differences in tumor volumes between the Arctiin 30 mg/kg group and vehicle group,was statistically significant P<0.05,expect that between the Arctiin 15 mg/kg group and vehicle group and between the Arctigenin 15 mg/kg group and vehicle group,between the Arctiin 30 mg/kg group and Arctiin 15 mg/kg group,between the Arctiin 30 mg/kg group and Arctigenin 15 mg/kg group,and between the Arctiin 15 mg/kg group and Arctigenin 15 mg/kg group,and see Table 6.6 and 6.8.It is showed that the pictures and mean weights of tumors stripped from each group of mice at the end of experiment.In general,tumors in Arctiin and Arctigenin groups looked smaller than that in vehicle group.Tumor weights in Arctiin and Arctigenin groups were 0.49 ± 0.07g(Arctiin 30mg/kg)and 0.63 ± 0.12g(Arctiin 15mg/kg)and 0.56 ± 0.10 g(Arctigenin 15mg/kg)respectively,which were much lighter than that in vehicle group(0.94 ± 0.12g,P<0.05).It is showed that the tumor weight of the Arctiin 30mg/kg/d and 15mg/kg/d,Arctigenin 15mg/kg/d group compared with the vehicle group,the differences have been statistically significant(P<0.05).Conclusion and Innovation1.Lappaol F,a novel anticancer lead compound was identified from the isolated lignan constituents by this study.It is showed that Lappaol F suppressed cancer cell growth in time-and dose-dependent manners in human cancer cell lines of various tissue types.The IC50 values of Lappaol F to cell lines MCF-7,MDA-MB-231 and RKO were measured as 13.3,16.8,25.2 ?M for the first time.2.It is showed that Lappaol F induced G1 and G2 cell cycle arrest which was associated with strong induction of p21 and p27 and reduction of CDK2,cyclin B1 and CDKl for the first time.p21 and p27 played crucial roles in Lappaol F mediated regulation of CDK1 and cyclin B.3.It is showed that Lappaol F exhibited strong growth inhibitory action on the growth of HeLa tumors in nude mice model,as well as a well tolerance and safety to the treated animals for the first time.Average initial tumor volumes in each treatment group of mice were 144.5 ± 22.4(Lappaol F 10 mg/kg),160.7 ± 19.1(Lappaol F 5 mg/kg)and 161.28 ±25.8 mm3(vehicle),separately.After a 15-d treatment period,tumor volumes in lappaol F-treated mice were 634.8 ± 60.1((Lappaol F 10 mg/kg)and 820.3 ± 92.9 mm3((Lappaol F 5 mg/kg)respectively,which were much smaller than the tumor volume of 1765.3 ± 99.2 mm3 in mice treated with vehicle(P<0.01).4.Arctigenin and arctiin exhibited their in vivo growth-inhibiting effects against HeLa tumors in nude mice for the first time.Average initial tumor volumes in each treatment group of mice were 141.1 ± 17.6(Arctiin 30 mg/kg),139.3 ± 19.7(Arctiin 15 mg/kg)and 135.4 ± 28.3 mm3(Arctigenin 15 mg/kg),143.2 ± 21.8 mm3(vehicle),separately.After a 14 day treatment period,tumor volumes in nude mice were 733.3 ± 88.4 mm3(arctiin 30 mg/kg)and 795.6 ± 82.9 mm3(Arctigenin 15 mg/kg)and 711.7 ± 104.9 mm3(Arctigenin 15mg/kg)respectively,which were much smaller than the tumor volume of 1351.7 ± 122.1 mm3 in mice treated with vehicle(P<0.05).