BACKGROUND Colorectal cancer(CRC)is the third most common malignancy of the digestive tract and the fifth leading cause of cancer-related mortality in China.Sporamin,a Kunitz-type trypsin inhibitor isolated from sweet...BACKGROUND Colorectal cancer(CRC)is the third most common malignancy of the digestive tract and the fifth leading cause of cancer-related mortality in China.Sporamin,a Kunitz-type trypsin inhibitor isolated from sweet potato,is a potential anti-cancer agent with activities against a number of malignant tumor cells in vitro.The liver secretes a myriad of endocrine factors that may facilitate the growth and transformation of tumors in the development of CRC.AIM To investigate the effects of sporamin on liver morphology and biomarkers of xenografted CRC in the liver of athymic BALB/c mice.METHODS Twenty-seven male BALB/c nude mice were randomly divided into control,vehicle,and sporamin groups.Mice in the latter two groups were intraperitoneally xenografted with LoVo colorectal carcinoma cells and intragastrically infused with saline or sporamin(0.5 g/kg body weight/d),respectively,for 3 wk.Hematoxylin and eosin(HE)staining of the sections was performed to observe morphological changes in hepatic tissue and real-time fluorescent quantitative PCR(qPCR)and enzyme-linked immunosorbent assay(ELISA)were used to measure the expression ofβ-catenin and vascular endothelial growth factor(VEGF)in the liver.RESULTS Sporamin significantly reduced the number and weight of tumor nodules formed in the abdominal cavity.Compared with the vehicle group,the mean tumor weight(±SD)in the sporamin group was significantly reduced(0.44±0.10 g vs 0.26±0.15 g)and the total number of tumors decreased from 93 to 55.HE staining showed that enlargement of the nucleus and synthesis of proteins within hepatocytes,as well as infiltration of inflammatory cells into the liver,were attenuated by sporamin.Immunohistochemical staining and ELISA showed that the concentrations ofβ-catenin and VEGF in the liver were significantly reduced by sporamin.Compared with the vehicle group,the expression ofβ-catenin measured in integrated optical density units per area was reduced in the sporamin group(47.29±9.10 vs 26.14±1.72;P=0.003).Expression of VEGF was also reduced after sporamin intervention from 20.78±2.06 in the vehicle group to 15.80±1.09 in the sporamin group(P=0.021).Compared with the vehicle group,the concentration ofβ-catenin decreased from 134.42±22.04 pg/mL to 109.07±9.65 pg/mL after sporamin intervention(P=0.00002).qPCR indicated that compared to the vehicle group,relative mRNA expression ofβ-catenin and VEGF in the liver of mice in the sporamin-treated group was significantly reduced to 71%±1%(P=0.000001)and 23%±7%(P=0.00002),respectively,of the vehicle group levels.CONCLUSION Sporamin down-regulates the expression and secretion ofβ-catenin and VEGF in the liver,which subsequently inhibits the transcription of downstream genes involved in cancer progression and angiogenesis.展开更多
The aim of this study was to investigate the effect of different concentrations of sporamin on the differentiation and proliferation of 3T3-LI preadipocytes, providing the theoretical basis for the development of food...The aim of this study was to investigate the effect of different concentrations of sporamin on the differentiation and proliferation of 3T3-LI preadipocytes, providing the theoretical basis for the development of food to treat obesity and diabetes, The isolation and purification of sporamin from sweet potato species 55-2 were performed by ammonium sulphate precipitation in combination with ion-exchange and gel filtration chromatography. With berberine as a positive control, different concentrations ofsporamin （0.000, 0.125, 0.025, 0.250, 0.500, and 1.000 mg·mL^-1 were used to treat 3T3-L1 preadipocytes. Intracellular fat accumulation and the degree of adipogenesis were quantified using Oil Red O staining and colorimetry, Preadipocytes differentiation was measured by 3（4,5-dimethylthiazolyl-2-yl）-2,5-diphenyltetrazolium bromide （MTT） spectrophotometric assay. Two sporamin proteins, which were separated into sporamin A （31 kD） and sporamin B （22 kD）, could be purified by ion-exchange and gel filtration chromatography. After being treated by different concentrations of sporamin, the differentiation of 3T3-L1 preadipocytes was significantly inhibited, compared with the positive control. When the sporamin solution concentration was 0.500 mg mL-1, the accumulation of lipid droplets within the cells was significantly decreased and the optical density （OD） value of the solution from destained Oil Red O reached to 0.35, which was the lowest value （P〈 0.05）. The proliferation of 3T3-L1 preadipocytes was significantly inhibited by treating at higher sporamin concentrations. In addition, the inhibitory effect was more obvious with the prolonged treatment time （P〈 0.05）. The differentiation and proliferation of 3T3-L1 preadipocytes could be inhibited significantly by the addition of higher concentration sporamin. It was, therefore, suggested that the sporamin was potentially effective for weight loss.展开更多
Sporamin is a soluble protein in sweet potato, and falls into two distinct homology groups, subfamilies A and B. In this research, a sporamin B was purified and its amino acid sequences, trypsin inhibitor activity （T...Sporamin is a soluble protein in sweet potato, and falls into two distinct homology groups, subfamilies A and B. In this research, a sporamin B was purified and its amino acid sequences, trypsin inhibitor activity （Ti activity） were analyzed. This sporamin B was isolated from sweet potato tubers [Ipomoea batatas （L.） Lam cv. 55-2] through extraction of the water-soluble fraction, dialysis, ultrafiltration and ion-exchange chromatography. Homology determined by polyacrylamide gel electrophoresis showed that mainly one bond appeared in gel after being reduced by SDS （sodium dodecyl sulfate）, or by SDS and 2-mercaptoethanol, or in native situation. By comparing the data of the polypeptide mass Matrix-assisted laser desorption/ionization time-of-flight （MALDI-TOF） mass spectrometry with those of the mass of the theoretical amino acid sequences from NCBI protein database, it was revealed that it was Q40091｜Q40091_IPOBA, sweet potato sporamin B - Ipomoea Batatas （sweet potato） （Batate）. The sequence coverage was 70.6%. N-terminal sequence was SETPV （Ser-Glu-Thr-Pro-Val）. There is a linear relationship between trypsin inhibitor activity （Ti activity） and amounts of this sporamin B （3-18 μg mL-1）. The equation of linear regression was y = 2.5809x ＋ 17.049 （r2 = 0.9966）. There was a curvilinear relationship between Ti activity and amounts of this sporamin B （21-150 μg mL-1）. The equation of curvilinear regression is y = 14.417ln（x） ＋ 23.26 （r2 = 0.9924）. The concentration of sporamin B with Ti activity after heating at 40°C may induce part denature of this sporamin B, and there was no statistic difference after heating at 40, 50, 60°C for 20 min. Heat treatment at more than 90°C leads to a dramatic decrease of trypsin inhibitor efficiency. The results suggested that Q40091｜Q40091_IPOBA was the major sporamin B in sweet potato tubers [Ipomoea Batatas （L.） Lam cv. 55-2], which had strong Ti activity, and was stable to both thermal and DTT （DL-dithiothreitol） relatively.展开更多
基金Supported by the National Natural Science Foundation of China,No.81573128 and No.81703216
文摘BACKGROUND Colorectal cancer(CRC)is the third most common malignancy of the digestive tract and the fifth leading cause of cancer-related mortality in China.Sporamin,a Kunitz-type trypsin inhibitor isolated from sweet potato,is a potential anti-cancer agent with activities against a number of malignant tumor cells in vitro.The liver secretes a myriad of endocrine factors that may facilitate the growth and transformation of tumors in the development of CRC.AIM To investigate the effects of sporamin on liver morphology and biomarkers of xenografted CRC in the liver of athymic BALB/c mice.METHODS Twenty-seven male BALB/c nude mice were randomly divided into control,vehicle,and sporamin groups.Mice in the latter two groups were intraperitoneally xenografted with LoVo colorectal carcinoma cells and intragastrically infused with saline or sporamin(0.5 g/kg body weight/d),respectively,for 3 wk.Hematoxylin and eosin(HE)staining of the sections was performed to observe morphological changes in hepatic tissue and real-time fluorescent quantitative PCR(qPCR)and enzyme-linked immunosorbent assay(ELISA)were used to measure the expression ofβ-catenin and vascular endothelial growth factor(VEGF)in the liver.RESULTS Sporamin significantly reduced the number and weight of tumor nodules formed in the abdominal cavity.Compared with the vehicle group,the mean tumor weight(±SD)in the sporamin group was significantly reduced(0.44±0.10 g vs 0.26±0.15 g)and the total number of tumors decreased from 93 to 55.HE staining showed that enlargement of the nucleus and synthesis of proteins within hepatocytes,as well as infiltration of inflammatory cells into the liver,were attenuated by sporamin.Immunohistochemical staining and ELISA showed that the concentrations ofβ-catenin and VEGF in the liver were significantly reduced by sporamin.Compared with the vehicle group,the expression ofβ-catenin measured in integrated optical density units per area was reduced in the sporamin group(47.29±9.10 vs 26.14±1.72;P=0.003).Expression of VEGF was also reduced after sporamin intervention from 20.78±2.06 in the vehicle group to 15.80±1.09 in the sporamin group(P=0.021).Compared with the vehicle group,the concentration ofβ-catenin decreased from 134.42±22.04 pg/mL to 109.07±9.65 pg/mL after sporamin intervention(P=0.00002).qPCR indicated that compared to the vehicle group,relative mRNA expression ofβ-catenin and VEGF in the liver of mice in the sporamin-treated group was significantly reduced to 71%±1%(P=0.000001)and 23%±7%(P=0.00002),respectively,of the vehicle group levels.CONCLUSION Sporamin down-regulates the expression and secretion ofβ-catenin and VEGF in the liver,which subsequently inhibits the transcription of downstream genes involved in cancer progression and angiogenesis.
基金supported by the National 863 Program of China (2006AA10Z332)the Ear Marked Fund for Modern Agro-Industry Technology Research System, China
文摘The aim of this study was to investigate the effect of different concentrations of sporamin on the differentiation and proliferation of 3T3-LI preadipocytes, providing the theoretical basis for the development of food to treat obesity and diabetes, The isolation and purification of sporamin from sweet potato species 55-2 were performed by ammonium sulphate precipitation in combination with ion-exchange and gel filtration chromatography. With berberine as a positive control, different concentrations ofsporamin （0.000, 0.125, 0.025, 0.250, 0.500, and 1.000 mg·mL^-1 were used to treat 3T3-L1 preadipocytes. Intracellular fat accumulation and the degree of adipogenesis were quantified using Oil Red O staining and colorimetry, Preadipocytes differentiation was measured by 3（4,5-dimethylthiazolyl-2-yl）-2,5-diphenyltetrazolium bromide （MTT） spectrophotometric assay. Two sporamin proteins, which were separated into sporamin A （31 kD） and sporamin B （22 kD）, could be purified by ion-exchange and gel filtration chromatography. After being treated by different concentrations of sporamin, the differentiation of 3T3-L1 preadipocytes was significantly inhibited, compared with the positive control. When the sporamin solution concentration was 0.500 mg mL-1, the accumulation of lipid droplets within the cells was significantly decreased and the optical density （OD） value of the solution from destained Oil Red O reached to 0.35, which was the lowest value （P〈 0.05）. The proliferation of 3T3-L1 preadipocytes was significantly inhibited by treating at higher sporamin concentrations. In addition, the inhibitory effect was more obvious with the prolonged treatment time （P〈 0.05）. The differentiation and proliferation of 3T3-L1 preadipocytes could be inhibited significantly by the addition of higher concentration sporamin. It was, therefore, suggested that the sporamin was potentially effective for weight loss.
基金supported by the Green-Agricultural Science Research and Demonstration Project (2007-1-2)the National High Technology Research andDevelopment Program of China (863 Program,20060110Z3051)
文摘Sporamin is a soluble protein in sweet potato, and falls into two distinct homology groups, subfamilies A and B. In this research, a sporamin B was purified and its amino acid sequences, trypsin inhibitor activity （Ti activity） were analyzed. This sporamin B was isolated from sweet potato tubers [Ipomoea batatas （L.） Lam cv. 55-2] through extraction of the water-soluble fraction, dialysis, ultrafiltration and ion-exchange chromatography. Homology determined by polyacrylamide gel electrophoresis showed that mainly one bond appeared in gel after being reduced by SDS （sodium dodecyl sulfate）, or by SDS and 2-mercaptoethanol, or in native situation. By comparing the data of the polypeptide mass Matrix-assisted laser desorption/ionization time-of-flight （MALDI-TOF） mass spectrometry with those of the mass of the theoretical amino acid sequences from NCBI protein database, it was revealed that it was Q40091｜Q40091_IPOBA, sweet potato sporamin B - Ipomoea Batatas （sweet potato） （Batate）. The sequence coverage was 70.6%. N-terminal sequence was SETPV （Ser-Glu-Thr-Pro-Val）. There is a linear relationship between trypsin inhibitor activity （Ti activity） and amounts of this sporamin B （3-18 μg mL-1）. The equation of linear regression was y = 2.5809x ＋ 17.049 （r2 = 0.9966）. There was a curvilinear relationship between Ti activity and amounts of this sporamin B （21-150 μg mL-1）. The equation of curvilinear regression is y = 14.417ln（x） ＋ 23.26 （r2 = 0.9924）. The concentration of sporamin B with Ti activity after heating at 40°C may induce part denature of this sporamin B, and there was no statistic difference after heating at 40, 50, 60°C for 20 min. Heat treatment at more than 90°C leads to a dramatic decrease of trypsin inhibitor efficiency. The results suggested that Q40091｜Q40091_IPOBA was the major sporamin B in sweet potato tubers [Ipomoea Batatas （L.） Lam cv. 55-2], which had strong Ti activity, and was stable to both thermal and DTT （DL-dithiothreitol） relatively.
