| [Objective]:A number of studies have found that artemisinin and its derivatives possess antitumor activity, which has close relationship with tumor proliferation, invasion, metastasis and apoptosis. In recent years, studies have confirmed that the peroxisome proliferator-activated receptors-γ(PPARy) is closely related to the proliferation, invasion and metastasis of the tumor too. For more extensive exploration of artemisinin and its derivatives possible anti-tumor mechanism, this research through the establishment of Lewis lung cancer mouse model, to explore the effects of Artemether on the growth and the inhibition of invasion and metastasis of Lewis lung cancer xenograft in mice, to investigate the mechanism of the therapeutic whether is involved in activating PPARy, regulating NF-κB and Caspase-3 expression, in order to inhibition the tumor angiogenesis, promotion the cell cycle arrest and apoptosis.So,provideing target for explore new differentiation agents and experimental data for clinic utilization.[Methods]:1. To establish a model of Lewis lung cancer:Lewis lung cancer model was established by LLC cells and C57BL/6 mice, and the drug was given by different groups. ①NS group(injected with normal saline 90mg/kg)、②ARE group(injection of artemether 50mg/kg)、③GW9662group(injection of PPAR gamma specific inhibitor GW96621mg/kg)、④GW9662+AREcombined group (injection of GW9662 30min after then injection ARE,all drug concentration are the same with before).GW9662 is a specific inhibitor of PPARy, which can specifically blocking the expression of PPARy.2. observe the survival state of each group of mice, weight change, tumor volume change;3. the inhibition rate was calculated:the tumor inhibition rate= tumor weight of control group-administration group]/[the tumor weight of control group];4. pathological changes:take 10% formalin fixed the tumor tissue, paraffin embedded,sections, HE staining, light microscope observation;5. Immunohistochemistry:Immunohistochemical staining for PPARy, NF-κB, Caspase-3, CD31 and VEGF was performed in tumor tissues collected on d14 after treatment initiation6. Flow cytometry:flow cytometry detected each group of tumor cell cycle and apoptosis rate.7. RT-PCR method was used to detect the levels of PPARy,NF-κB, Caspase-3, mRNA in the tumor tissues of each group.8. Western Blot method was used to detect the expression levels of PPARy,NF-κB, Caspase-3 protein in tumor tissues of each group.9. All data were analyzed by SPSS17.0 software and statistics were expressed as mean±standard deviation (x±s), and single factor analysis of variance (ONE WAY ANOVA) and t test were used to get the statistical significance.[Results]:1.(1) ARE group could significantly inhibit the growth of Lewis lung cancer xenograft (the inhibition rate was 64.51%);(2) the ARE+GW9662 combined group had inhibitory effect on the Lewis lung cancer xenograft (the inhibition rate was 45.14%);(3) GW9662 didn’t display obvious inhibitory effect in Lewis lung cancer xenograft, there was no significant difference in tumor tissue size between GW9662 group and normal saline group;2. Pathological changes:Hematoxylin and eosin (HE) staining were visible tumor cell morphology and size are not consistent, nuclei enlarged, and we can noticeed binuclear,polynuclear and heteromorphic nucleus, chromatin was coarse, uneven distribution, the nuclear thickening has been observed, nucleolar hypertrophy, an increase in number, and the nucleocytoplasmic ratio disorders.NS group and GW9662 group showed rapid proliferation of tumor cells and in the tumor center we can visible hemorrhagic necrotic area due to the large tumor load, nearby a large number of blood cells infiltration, NS group and GW9662 group had distant metastasis, NS group showed obvious muscle invasion, and GW9662 group also visible skeletal muscle invasion. ARE group and ARE+GW9662 combined group showed no significant distant invasion, but all can see apoptotic tumor cells, especially in the ARE group;3. Immunohistochemical analysi:ARE group and combination group PPAR Y expression were significantly increased than that in NS group, and GW9662 group were negative; GW9662 group NF-κB expression significantly higher than the other groups, ARE group and combination group were reduced significantly compared with NS group;the CD31 expression were obvious in GW9662 group and NS group; VEGF in GW9662 group and NS group, positive expression was significantly higher than those in the other two groups;the differences expression of caspase-3 in each group were meaningless.4. Flow cytometry:ARE group and the combined group were significantly increased the proportion of G0/G1 phase, S phase ratio decreased, the rate of apoptosis were increased. Among them, ARE group was obvious.5. RT-PCR:PPARγ mRNA expression levels:ARE group> combined group> NS group> GW9662 group; NF-κB mRNA:GW9662 group> NS group> combined group>ARE group; caspase-3 mRNA in the expression levels of each group were no statistical significance.6. Blot Western results:PPARγ protein:ARE group> combined group> NS group> GW9662 group; NF-κB protein:GW9662 group>NS group> combined group> ARE group; Caspase-3 protein levels in each group were not statistically significant difference.[Conclusion]:1.ARE can inhibit the growth of transplanted tumor of Lewis lung cancer by inhibiting angiogenesis;2.ARE can promote tumor cell apoptosis and inhibit tumor growth through G0/G1 phase arrest, and reduce the proportion of S phase;3. ARE might restrain NF-κB through up-regulating PPARγ to inhibit the proliferation and invasive potential of Lewis lung cancer xenograft. |
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