Experimental Study Of Rottlerin's Inhibition Of Adrenocortical Carcinoma

BackgroundAdrenal cortical carcinoma(ACC)is a kind of malignancy originated from adrenal cortex,account for 0.01%-0.02% of all malignancies.It is a rare,insidious onset but aggressive endocrine malignancy with a generally poor clinical outcome.Most of the patients had local invasion and distant metastasis when they were diagnosied.The survival rates of five years is lower than 30%.Until recently,radical surgical resection has been the only potentially curative option for ACC,but the radical surgical resection is not applicable to the advanced and metastatic ACC.There are no more effective treatment for advanced and metastatic ACC.Mitotane is a key agent for the current therapeutic,which is with limited efficacy and significant toxicity.Therefore,new agents for ACC treatment are urgently required.Rottlerin,a natural plant polyphenol,is a traditional Indian medicine previously used as a agent treatment for cestode and trematode infections.In this study,we attempted to identify a novel agent,rottlerin,could exhibits its antineoplastic activities for ACC in vivo and in vitro,and safer than traditional therapy.MATERIALS AND METHODS1.Experiments in vitroCell Counting Kit 8(cck-8)tests were performed to verifiy that rottlerin could inhibit ACC cell(NCI-H295 R and SW-13)proliferation in vitro,and in a time-and dose-dependent manner.Annexin-V /PI staining and AO/EB staining were performed to verifiy that rottlerin could induce ACC cell apoptosis in vitro.Rottlerin induces apoptosis in a dose-dependent manner.We investigated the effect of rottlerin on the cell cycle of ACC cells using flow cytometry analyses.To determine if rottlerin inhibits cell invasion and migration of ACC cells,we performed transwell invasion and migration assays.To determine if rottlerin suppresses the Wnt/?catenin signaling pathway in ACC,and to elucidate the mechanism by which rottlerin mediates ACC cell apoptosis,some signaling molecules in the Wnt/?-catenin pathway were examined.Animal experiments were performed using 4-week old male nude mice(n=20,athymic,BALB/C nu/nu).SW-13 cells were(3 × 106 cells)suspended in 200 ?l of serum-free culture medium and inoculated subcutaneously in the flank region of nude mice.Tumors were permitted to grow to approximately 4mm,and mice were randomized into 4 treatment groups(control,DMSO,rottlerin 4 mg/kg,and rottlerin 2 mg/kg)with 5 mice per group.All the mice were injected intraperitoneally daily for 4 weeks.Rottlerin was dissolved in DMSO,and diluted with normal saline(NS).The control group was injected with 0.5 ml of NS and the DMSO group was injected with 0.45 ml of NS mixed with 50 ?l of DSMO.The rottlerin groups were injected with rottlerin(4 mg or 2 mg per kilogram).The mice weights and tumor volumes were monitored every 4 days.After 4 weeks,the mice were euthanized and the xenografts were removed from the animals.Tumor tissues were fixed in 10% formalin solution and paraffin-embedded.The sample sections were prepared for hematoxylin and eosin(HE)staining,immunohistochemistry staining of?-catenin?LRP6 and p-LRP6,and terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assays to verify rottlerin could induce ACC cell apoptosis in vivoResults1.Rottlerin inhibits ACC cell proliferation in vitro,the results of cck-8 assay was shown that rottlerin inhibited proliferation of ACC cells in a time-and dose-dependent manner.The 48-h IC50 was 5.02 ?M in NCI-H295 R cells and 4.87 ?M in SW-13 cells.Rottlerin induced ACC cell apoptosis in vitro.Rottlerin induced ACC cell apoptosis in vitro.The results showed an increase in the percentage of apoptotic cells in the rottlerin-treated group compared with the control and DMSO groups.After treatment with 2.5 ?M rottlerin for 48 h,the percentage of apoptotic cells reached 13.0% in the NCI-H295 R cells and 10.9% in the SW-13 cells.After treatment with 5 ?M rottlerin for 48 h,the percentage of apoptotic cells reached 43.3% in the NCI-H295 R cells and 38.3% in the SW-13 cells.The results suggest that rottlerin induces apoptosis in a dose-dependent manner.The results of AO/EB staining support these conclusions regarding morphology changes.2.Rottlerin caused cell cycle arrest of ACC cells in vitro.We investigated the effect of rottlerin on the cell cycle of ACC cells using flow cytometry analyses.After treatment with 5 ?M rottlerin for 48 h,G0/G1 cell cycle arrest occurred in ACC cells.The percentage of cells in the G1 phase was 85.14% in NCI-H295 R cells and 81.88% in SW-13 cells,2.Animal experiment which was significantly increased compared with the control group.Rottlerin inhibited cell invasion and migration of ACC cells.In the rottlerin treatment group,the invasion of ACC cells was impaired by rottlerin(5?M);significantly fewer cells migrated to the lower side of the transwell chamber membrane than the control group.The results of wound healing assay demonstrated that the migration rate of SW-13 cells was significantly reduced by rottlerin(5 ?M)compared with the control group.The 24 h migration rate was significantly decreased in SW-13 cells3.Based on the results of Western blotting,we can conclude that rottlerin reduced the expression of ?-catenin,LRP6,and p-LRP in ACC cells in a dose-dependent manner.4.Rottlerin inhibited tumor growth in vivo.With the administration of rottlerin for 4 weeks,tumor sizes and body weight were measured every 4 days.Assessment of tumor volume showed that the rottlerin-treated groups had delayed tumor growth compared to the control group and the group treated with DSMO.Here was no significant weight differences among the groups and there was no toxicity or side effects in the mice after 4 weeks of treatment.Tumors were smaller in the experimental groups than the control and DSMO groups,and tumors were smaller in the rottlerin 4 mg/kg group than the rottlerin 2 mg/kg group.The mean volume of the tumors decreased from 3.23 cm3(control)and 3.35 cm3(DMSO)to 1.56 cm3(rottlerin 4 mg/kg)and 1.94 cm3(rottlerin 2 mg/kg);the difference was statistically significant).TUNEL assays were performed to assess the anti-tumor activity of rottlerin in vivo(Figure 4C).The TUNEL assays indicated that the rottlerin-treated groups had higher apoptotic rates compared with the control and DMSO-treated groups.Immunohistochemistry staining in xenograft tissues,and the results showed that rottlerin also down-regulated the expression of ?-catenin,LRP6,and p-LRP compared with the control and DMSO-treated groups in vivo.Moreover,in the higher dose-treated group(rottlerin 4 mg/kg),the expression of ?-catenin,LRP6,and p-LRP was lower than the lower dose-treated group(rottlerin 2 mg/kg).ConclusionThe results showed that rottlerin inhibited proliferation,induced apoptosis and G0/G1 cell cycle arrest,and decreased invasion and mobility of ACC cell lines.Furthermore,the results of animal experiments showed that rottlerin inhibited tumor growth in vivo,and there was no obvious toxic effect in nude mice.The results showed that rottlerin has anti-neoplastic activity in ACC.Based on Western blot and immunohistochemistry staining,the expression of ?-catenin,LRP6,and p-LRP6 was downregulated after treatment with rottlerin.We deduced that rottlerin plays a role as an anti-tumor agent in ACC related to Wnt/?-catenin signaling pathway,likely via suppression of the Wnt/?-catenin signaling pathway.All these avidences indicate that rottlerin could serve as a novel agent for the treatment of ACC for its low toxicity or side effects.