The Activity Of Artemisinin Against Lymphangiogenesis In Mice Bearing Lewis Lung Carcinoma

The lymphatic vasculature system is involved in transportation of tissue fluids, extravasated plasma proteins and cells back into blood circulation.Lymphatics also serve as an important part of the body's immunological surveillance system.Formation of new lymphatics(lymphangiogenesis) occurs in both normal developing tissues as well as in pathological processes such as inflammation,wound healing,and lymphedema.The lymphatic vessel system plays a major role in cancer biology,as malignant cells spread from their primary site to regional lymph nodes via the lymphatics.Lymph node involvement is clinically identified as a key factor in staging of cancers and considered as an important prognostic indicator in a variety of human solid cancers.Recent studies have indicated that tumor induced-lymphangiogenesis can promote metastatic spread of cancer cells and influence the prognosis and overall survival of cancer patients.Vvascular endothelial growth factor(VEGF) -C and -D has been identified as the most concerned lymphangiogenic growth factors.It is demonstrated that they can stimulate growth, migration and tube-like formation of lymphatcs endothelial cells(LEC) and induce lymphangiogenesis in tumor models by activating the VEGF receptor -3(VEGFR-3) tyrosine kinase signals.Therefore,suppression of these lymphangiogenesis-related properties of LEC and inhibition of the signals of lymphangiogenic factors may result in potential therapeutic values for cancer metastasis and other lymphangiogenesis-related diseases.Artemisinin(ART),a more water-soluble active metabolite of artemisinin isolated from the Chinese herb Artemisia annua L(qinghaosu,sweet wormwood),is a new generation of drugs against fever and chloroquine- and mefloquine-resistant strains of Plasmodium falciparum.Despite their pharmacological action on malaria,ART inhibits cellular proliferation in a selective way and induces cell apoptosis in breast,colon and many other cancers,with a mechanism involving production of carbon-centred free radicals by reacting with ferrous.Alternatively,artemisinin derivates can impair tumor-induced angiogenesis by influencing the properties of human umbilical vein endothelial cells such as cell migration and tube-like formation,which can be associated with decreased expressions of VEGF-A and VEGFR-1.However,whether artemisinin drugs affect lymphangiogenesis-related properties of LECs and attenuate tumor-induced lymphangiogenesis remains unclear.ObjectiveIn an effort to investigate whether antimalarial ART has a potent ability to inhibit lymphangiogenesis-related properties of LEC,including cell growth,apoptosis,cell migration and tube-like formation,and further its in vivo biological activities on lymphangiogenesis,lymph node metastasis and lung metastasis in Lewis lung carcinoma (LLC) models.Methods1.The models of LLC were established via subcutaneous injection of LLC cells in the dorsal site of the right ear of C57BL/6 mice.2.For in vivo experiments,mice were divided into control group and ART group(n = 25 for each group).The mice were orally administered with ART(50 mg/kg) daily for two weeks on the second day of tumor cell injection.Mice were sacrificed 30 days after tumor cell inoculation.Body weights,tumor volumes and weights were examined throughout the experimental period from injection of cancer cells until evaluation(n = 10 for each group). To analyze nodal and lung metastasis,numerous lymph nodes throughout the body of the animal as well as visceral organs were examined,including supracervical nodes, contralateral supracervical nodes,brachial nodes,axial nodes,deep cervical nodes, mediastinal nodes,suprarenal nodes,livers and lungs.The rest of animals were analyzed for survival rate 60 days after tumor implantation(n = 15 for each group).Tumor lymphatic microvessel density(LMVD) was determined by lymphatic vessel endothelial hyaluronan receptor-1(LYVE-1) immnohistochemistry.The mRNA and protein expression of VEGF-C in LLC tumors was determined by RT-PCR and Western blotting,respectively.3.LLC cells were incubated with ART at different concentrations for 24 h,total protein and RNA were isolated,and levels of VEGF-C protein and mRNA were analyzed by Western blotting and RT-PCR,respectively.4.Benign lymphangiomas were induced for 2 monthes by intraperitoneal injection of incomplete Freund's adjuvant(IFA).Murine LEC were isolated from the induced benign lymphangiomas and identified by indirect immunofluorence assay using a polyclonal anti-LYVE-1 antibody and fluorescence-activated cell sorting analysis using the Annexin V/PI binding assay.5.Mouse LECs were incubated with different doses of ART for 24 h,cell geowth,cell migration and tube-like formation was determined by MTT assay,Transwell saaay and Matrigel assay,respectively.Results1.At 30 days after tumor inoculation,the presence of supracervical node and contralateral supracervical node metastases in control mice was 100%and 90%, respectively,compared with 60%and 40%in ART-treated mice.In addition,the metastatic ratio of brachial nodes,axial nodes and suprarenal node but not brachial nodes and deep cervical nodes in control group was significantly less than those in ART group.Furthermore, 100%of mice in the control group developed lung metastasis compared with 50%in the ART group.Orally administrated ART reduced the numbers of lung metastastic nodules from 16.5±2.1 to 5.6±1.8.The average weight of LLC tumors in control mice and ART-treated mice was(1.96±0.32) g and(1.78±0.18) g,respectively.Immunochemical results showed that LMVD in control mice was 29.9±3.4 compared with 11.0±2.7 in ART-treated mice.2.Survival analysis showed that about 26.7%of mice in the control group died within the first 30 days.In contrast,only 33.3%of mice in the groups treated with 50 mg/kg of ART died within 45 days.Mean survival was prolonged to 54 days in ART-treated mice compared with 38 days in control animals(n = 15 for each group).3.Realtime RT-PCR and Western blotting assay showed that there was a significant difference of VEGF-C protein and mRNA expression between control LLC tumors and ART-treated tumors.Furthermore,ART with different concentrations significantly suppressed the expression of VEGF-C protein as well as expression of mRNA in LLC cells.4.Mouse LEC was successfully obtained form the benign lymphangiomas induced by IFA with a LYVE-1 positive ratio of 60%-86%by fluorescence-activated cell sorting analysis.Isolated cells expressed cytopasmic and membrane LYVE-1 by immunofluorence assay. 5.After LEC was treated with ART for 24 h,the growth of LEC was significantly inhibited and LEC showed typical apoptotic morphological features,with the inhibitory rate of growth from 7.78%to 62.72%,and apoptotic rates from 42.36%to 53.37%.In addition, ART also exerted a significantly inhibitory effect on cell migration and tube-like formation of LEC in a dose-dependent manner.Inhibitory ratio of cell migration and tube-like formation was 35.2%and 54.5%,respectively,and inhibitory ratio of cell tube-like formation was 77.5%and 71.7%,respectively.Conclusions In summary,our results indicate that ART may be useful as a potential promising chemotherapeutic agent for prevention of tumor metastasis.ART-mediated inhibition of lymphangiogenesis is associated with induction of cell apoptosis,inhibition of the migration and formation of tube-like structures of LEC and down-regulated expression of VEGF-C in tumor cells.