The Mechanisms Of The Effect Of ASP On Tumor Growth In Tumor-bearing Mice

BackgroundAngelica sinensis polysaccharide(ASP)has many biological activities,such as anti-tumor,immunoregulation,anti-radiation,anti-oxidation and so on.Hepcidin is a key negative regulator of body iron homeostasis.The research showed that:(1)increased serum Hepcidin was associated with the development of breast cancer,multiple myeloma,renal cell carcinoma and prostate cancer.(2)the inflammatory pathway,the BMP/SMAD pathway and the erythrocytogenetic pathway could regulate the transcription of the hepcidin gene in the liver cells.(3)the breast cancer MCF-7 cells could synthesize Tf.(4)compared with the corresponding normal cells,the cancer cells expressed higher TfR1 and TfR2.The results of our previous study indicated that ASP could inhibit the growth of tumor in tumor bearing mice and regulate iron metabolism.ObjectivesThe mechanism of the effect of ASP on tumor growth in tumor-bearing mice was studyed by immunohistochemistry(IHC),Western blot and enzyme-linked immunosorbent assay(ELISA).Methods1.Human hepatoma HepG2 cells and human normal liver L-02 cells were taken as subjects.The cells were treated with different concentrations of APS.The effects of ASP on Hepcidin expression in HepG2 and L-O2 cells were analyzed by reverse transcription-polymerase chain reaction(RT-PCR)and Western blot assay.2.The liver tissue and serum from tumor bearing mice prepared by breast cancer 4T1 cells and mouse hepatoma H22 cells were studied.The effects of ASP on the expression of hepcidin were detected by Western blot and ELISA assay in vivo.3.The levels of ferritin,Tf,TfR1 and TfR2 in serum from 4T1 and H22 tumor bearing mice were detected by ELISA.4.The expression of IL-6 in the liver tissues and the level of IL-6 in serum from 4T1 and H22 tumor bearing mice were detected by Western blot and ELISA assy.5.The expression of JAK2,phosphorylated STAT3 and phosphorylated Smad1 / 5/8 in liver tissues from 4T1 and H22 bearing tumor bearing mice were analyzed by Western blot assay.Results1.The effects of ASP on the expression of hepcidin in vitro and in vivo(1)The effects of ASP on Hepcidin gene expression in HepG2 and L-O2 cellsAfter,the cells were treated with 0 g / L,0.10 g / L,0.20 g / L,0.40 g / L ASP for 48 h,respectively.The results from RT-PCR displayed that,0.10g/L of ASP group,the hepcidin expression was increased(P <0.05).However,0.20 g/L and 0.40g/L of ASP groups,the expression of hepcidin were decreased in a concentration dependent manner(P <0.01 or 0.05).After,the cells were treated with 0.40 g / L ASP for 0h,24 h,48h,72 h,96h,respectively.The results from RT-PCR revealed that the hepcidin expression was decreased in a temporal dependent manner after 48 hours administration(P <0.01).The HepG2 and L-02 cells treated or untreated with 0.40 g/L of ASP for 96 h were harvested and subjected to western blot assay.Western blot results showed that the expression of hepcidin was dramatically down-regulated in HepG2 and L-02 cells treated with ASP(P <0.01).(2)The effects of ASP on the expression of hepcidin in vivoThe hepcidin expression in liver tissue of 4T1 and H22 tumor-bearing mice was investigated by western blot.The results showed that the expression of hepcidin protein in each control group was up-regulated compared to the blank group(P <0.01).While in each ASP group the hepcidin protein level decreased significantly compared to the control group(P <0.01).We next used the IHC method to detect the expression of hepcidin in liver.Consistent with our western blot data,IHC results revealed that hepcidin was dramatically expressed in the control group compared with the blank group(P <0.01),where ASP therapies significantly decreased its amount both in tumor-bearing mice(P <0.01).We determined the impact of ASP on hepcidin in serum of each tumor-bearing mice by ELISA.The results from ELISA showed that hepcidin level in the control group of 4T1 and H22 tumor-bearing mice increased 1.78-fold and 1.82-fold compared with the blank group,respectively(P < 0.01),and compared to the control group,the levels of hepcidin in the ASP group of 4T1 and H22 tumor-bearing mice were markedly reduced by 54.55% and 47.29%,respectively(P < 0.01).2.The effect of ASP on iron metabolism related factorsWe examined ferritin,Tf,TfR1,and TfR2 levels in serum of 4T1 and H22 tumor-bearing mice by ELISA.The results from ELISA showed that,in 4T1 tumor-bearing mice,compared to the blank control,serum ferritin,Tf,TfR1,and TfR2 levels in the control group increased 4.27-fold(P< 0.001),3.45-fold(P<0.001),3.83-fold(P<0.001),and 1.43-fold(P<0.05),respectively.In H22 tumor-bearing mice compared to the blank control,serum ferritin,Tf,TfR1,TfR2 and IL-6 levels in the control group increased 3.30-fold(P<0.001),3.37-fold(P< 0.001),3.09-fold(P< 0.001),and 1.50-fold(P< 0.001),respectively.However,after ASP therapies,the level of these factors in serum were significantly reduced compared to the control group.In 4T1 tumor-bearing mice compared to the control group,the levels of serum ferritin,Tf,TfR1,and TfR2 in the ASP group were reduced by 50.73%(P< 0.001),12.20%(P>0.05),69.36%(P< 0.001)and 30.90%(P< 0.01),respectively.In H22 tumor-bearing mice,compared to the control group,the levels of serum ferritin,Tf,TfR1,and TfR2 in the ASP group were reduced by 34.31%(P< 0.01),18.00%(P<0.05),39.74%(P< 0.01)and 28.16%(P< 0.001),respectively.3.The effects of ASP on the signal proteins involved in Hepcidin regulation(1)The effects of ASP on IL-6 in serum and liver tissue from tumor bearing miceWe first determined the effects of ASP on IL-6 in serum and liver tissues of tumor-bearing mice by ELISA and western blot,respectively.The results from ELISA showed that,in the control group of 4T1 and H22 tumor-bearing mice,IL-6 level of serum increased 2.21-fold and 2.10-fold compared with the blank control,respectively(P<0.001).And compared to the control group,the levels of IL-6 in the ASP group of each tumor-bearing mice were markedly reduced by 51.09% and 33.83%,respectively(P<0.001).Next,the expression of IL-6 was also examined at the protein level in liver tissues of tumor-bearing mice.Consistent with ELISA data,Western blot results showed that the protein level of IL-6 was dramatically expressed in the control group compared with the blank group(P <0.01),where ASP therapies significantly decreased its amount both in 4T1 and H22 tumor-bearing mice(P <0.01).(2)The effects of ASP on expression of JAK2,phosphorylated STAT3 and phosphorylated Smad1 / 5/8 in liver tissue of tumor bearing miceThe expressions of JAK2,phosphorylated STAT3 and phosphorylated Smad1 / 5/8 wwere examined in liver tissues from 4T1 and H22 tumor-bearing mice.Our results showed that the expressions of JAK2,phospho-STAT3,and phosphor-Smad1/5/8 wwere significantly increased in the control group compared with the blank group(P <0.01).Nevertheless,ASP administration significantly decreased the mean levels of hepatic JAK2,phospho-STAT3,and phosphor-Smad1/5/8(P <0.01).Conclusions1.ASP can significantly inhibit the expression of Hepcidin in vitro and in vivo.2.ASP can remarkably reduce the level of iron metabolism related factors(ferritin,Tf,TfR1,and TfR2).3.ASP observably inhibit the signal proteins involved in Hepcidin regulation.