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Rhizoma Pinelliae trypsin inhibitor separation, purification and inhibitory activity on the proliferation of BGC-823 gastric adenocarcinoma cells.

Zu G, Wang H, Wang J, Dou Y, Zhao W, Sun Y - Exp Ther Med (2014)

Bottom Line: The tumour inhibitory effect was concentration- and dose-dependent.RPTI did not significantly influence the spleen coefficient of the mice.In conclusion, RPTI is a serine proteinase inhibitor with antitumour activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong 250013, P.R. China.

ABSTRACT
The aim of this study was to isolate and purify Rhizoma Pinelliae trypsin inhibitor (RPTI), determine its N-terminal amino acid sequence and evaluate its inhibitory effect on the proliferation of poorly differentiated BGC-823 human gastric adenocarcinoma cells. RPTI was separated and purified from a 40% (NH4)2SO4 precipitate of crude protein extract of Pinellia ternata tuber using affinity chromatography with trypsin as the ligand. The N-terminal amino acid sequence of RPTI was determined using the Edman degradation method. The inhibitory effect of RPTI on BGC-823 cell proliferation was detected in vitro using the MTT method and in vivo in tumour-bearing mice. The purified RPTI showed a single band under SDS-PAGE, its molecular weight was 14 kDa and its N-terminal amino acid sequence was DPVVDG. RPTI inhibited trypsin activity, with an inhibition ratio of 1:6.78 (mass). RPTI significantly inhibited the proliferation of BGC-823 cells in vitro. The IC50 of RPTI was 16.96 μg/ml within 48 h after treatment and 9.61 μg/ml within 72 h after treatment. Subcutaneous injection of RPTI around the tumour significantly inhibited BGC-823 tumour growth in mice. The tumour inhibitory effect was concentration- and dose-dependent. RPTI did not significantly influence the spleen coefficient of the mice. In conclusion, RPTI is a serine proteinase inhibitor with antitumour activity.

No MeSH data available.


Related in: MedlinePlus

Separation, purification and electrophoresis of RPTI. (A) Sepharose 4B trypsin-affinity chromatography. (B) Sephadex G-50 gel filtration chromatography. (C) SDS-PAGE of RPTI: Lane 1, 40% (NH4)2SO4 precipitation; 2, affinity chromatography; 3, molecular weight markers (from top to bottom: 97.4, 66.2, 43.0, 31.0, 20.1 and 14.4 kDa); and 4, gel filtration. OD, optical density; RPTI, Rhizoma Pinelliae trypsin inhibitor.
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f1-etm-08-01-0248: Separation, purification and electrophoresis of RPTI. (A) Sepharose 4B trypsin-affinity chromatography. (B) Sephadex G-50 gel filtration chromatography. (C) SDS-PAGE of RPTI: Lane 1, 40% (NH4)2SO4 precipitation; 2, affinity chromatography; 3, molecular weight markers (from top to bottom: 97.4, 66.2, 43.0, 31.0, 20.1 and 14.4 kDa); and 4, gel filtration. OD, optical density; RPTI, Rhizoma Pinelliae trypsin inhibitor.

Mentions: The 40% (NH4)2SO4 precipitate was designated as the crude RPTI product and subjected to trypsin-Sepharose 4B affinity chromatography to form two protein peaks (A280 nm; Fig. 1A). P1 was a saliferous elution peak that exhibited no TI activity and P2 was an affinity adsorption activity peak, which was further separated using Sephadex G-50 to form four protein peaks (Fig. 1B). The P1 and P2 formed by Sephadex G-50 separation were TI activity peaks, which were mainly concentrated in tubes 10–16. The 12% SDS-PAGE purity detection indicated that only the components in tubes 10–12 (the first half of P1) exhibited a single protein band and were estimated to have >90% purity. The eluents of the samples in tubes 10–12 following repeated sample loading were collected and combined to obtain purified RPTI. Fig. 1C shows that the crude RPTI protein had numerous bands and a complex composition. Further affinity chromatography removed the majority of the hybrid proteins and only left two clear main bands. More uniform main bands of RPTI were obtained following the gel filtration, with an apparent relative molecular weight of ~14 kDa. The activity recovery during each purification step is listed in Table II. Following the affinity chromatography, the specific inhibitory activity increased by 3.45-fold. The Sephadex G-50 separation produced a homogenous component. The activity recovery was 24.40% and 6.02-fold purified RPTI was obtained.


Rhizoma Pinelliae trypsin inhibitor separation, purification and inhibitory activity on the proliferation of BGC-823 gastric adenocarcinoma cells.

Zu G, Wang H, Wang J, Dou Y, Zhao W, Sun Y - Exp Ther Med (2014)

Separation, purification and electrophoresis of RPTI. (A) Sepharose 4B trypsin-affinity chromatography. (B) Sephadex G-50 gel filtration chromatography. (C) SDS-PAGE of RPTI: Lane 1, 40% (NH4)2SO4 precipitation; 2, affinity chromatography; 3, molecular weight markers (from top to bottom: 97.4, 66.2, 43.0, 31.0, 20.1 and 14.4 kDa); and 4, gel filtration. OD, optical density; RPTI, Rhizoma Pinelliae trypsin inhibitor.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061196&req=5

f1-etm-08-01-0248: Separation, purification and electrophoresis of RPTI. (A) Sepharose 4B trypsin-affinity chromatography. (B) Sephadex G-50 gel filtration chromatography. (C) SDS-PAGE of RPTI: Lane 1, 40% (NH4)2SO4 precipitation; 2, affinity chromatography; 3, molecular weight markers (from top to bottom: 97.4, 66.2, 43.0, 31.0, 20.1 and 14.4 kDa); and 4, gel filtration. OD, optical density; RPTI, Rhizoma Pinelliae trypsin inhibitor.
Mentions: The 40% (NH4)2SO4 precipitate was designated as the crude RPTI product and subjected to trypsin-Sepharose 4B affinity chromatography to form two protein peaks (A280 nm; Fig. 1A). P1 was a saliferous elution peak that exhibited no TI activity and P2 was an affinity adsorption activity peak, which was further separated using Sephadex G-50 to form four protein peaks (Fig. 1B). The P1 and P2 formed by Sephadex G-50 separation were TI activity peaks, which were mainly concentrated in tubes 10–16. The 12% SDS-PAGE purity detection indicated that only the components in tubes 10–12 (the first half of P1) exhibited a single protein band and were estimated to have >90% purity. The eluents of the samples in tubes 10–12 following repeated sample loading were collected and combined to obtain purified RPTI. Fig. 1C shows that the crude RPTI protein had numerous bands and a complex composition. Further affinity chromatography removed the majority of the hybrid proteins and only left two clear main bands. More uniform main bands of RPTI were obtained following the gel filtration, with an apparent relative molecular weight of ~14 kDa. The activity recovery during each purification step is listed in Table II. Following the affinity chromatography, the specific inhibitory activity increased by 3.45-fold. The Sephadex G-50 separation produced a homogenous component. The activity recovery was 24.40% and 6.02-fold purified RPTI was obtained.

Bottom Line: The tumour inhibitory effect was concentration- and dose-dependent.RPTI did not significantly influence the spleen coefficient of the mice.In conclusion, RPTI is a serine proteinase inhibitor with antitumour activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong 250013, P.R. China.

ABSTRACT
The aim of this study was to isolate and purify Rhizoma Pinelliae trypsin inhibitor (RPTI), determine its N-terminal amino acid sequence and evaluate its inhibitory effect on the proliferation of poorly differentiated BGC-823 human gastric adenocarcinoma cells. RPTI was separated and purified from a 40% (NH4)2SO4 precipitate of crude protein extract of Pinellia ternata tuber using affinity chromatography with trypsin as the ligand. The N-terminal amino acid sequence of RPTI was determined using the Edman degradation method. The inhibitory effect of RPTI on BGC-823 cell proliferation was detected in vitro using the MTT method and in vivo in tumour-bearing mice. The purified RPTI showed a single band under SDS-PAGE, its molecular weight was 14 kDa and its N-terminal amino acid sequence was DPVVDG. RPTI inhibited trypsin activity, with an inhibition ratio of 1:6.78 (mass). RPTI significantly inhibited the proliferation of BGC-823 cells in vitro. The IC50 of RPTI was 16.96 μg/ml within 48 h after treatment and 9.61 μg/ml within 72 h after treatment. Subcutaneous injection of RPTI around the tumour significantly inhibited BGC-823 tumour growth in mice. The tumour inhibitory effect was concentration- and dose-dependent. RPTI did not significantly influence the spleen coefficient of the mice. In conclusion, RPTI is a serine proteinase inhibitor with antitumour activity.

No MeSH data available.


Related in: MedlinePlus