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

Zu,Guohong; Wang,Houwei; Wang,Jie; Dou,Yan; Zhao,Weichong; Sun,Yuping

doi:10.3892/etm.2014.1701

July-2014 Volume 8 Issue 1

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July-2014 Volume 8 Issue 1

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Article

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

Authors:
- Guohong Zu
- Houwei Wang
- Jie Wang
- Yan Dou
- Weichong Zhao
- Yuping Sun
View Affiliations / Copyright

Affiliations: Department of Radiation Oncology, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong 250013, P.R. China, Department of Chinese Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan, Shandong 250013, P.R. China, Department of Surgery, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong 250013, P.R. China
Pages: 248-254
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Published online on: May 8, 2014

https://doi.org/10.3892/etm.2014.1701
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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.

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1	Zhao GP, Dai S and Chen RS: Dictionary of traditional Chinese medicine. Shanghai Scientific and Technical Publishers; Second Edition. pp. 1071–1072. 2005
2	Han MH, Yang XW, Zhong GY and Zhang M: Bioactive constituents inhibiting TNF-alpha production in fresh rhizome of Pinellia ternata. Zhongguo Zhong Yao Za Zhi. 32:1755–1759. 2007.(In Chinese).
3	He P, Li S, Wang SJ, Yang YC and Shi JG: Study on chemical constituents in rhizome of Pinellia ternata. Zhongguo Zhong Yao Za Zhi. 30:671–674. 2005.(In Chinese).
4	Lee MY, Shin IS, Jeon WY, Lim HS, Kim JH and Ha H: Pinellia ternata Breitenbach attenuates ovalbumin-induced allergic airway inflammation and mucus secretion in a murine model of asthma. Immunopharmacol Immunotoxicol. 35:410–418. 2013. View Article : Google Scholar
5	Li GL, Gui SQ and Wang L: Effect of pinellia extract on HeLa cell line in vitro and associated mechanism. Zhongguo Zhong Xi Yi Jie He Za Zhi. 30:303–307. 2010.(In Chinese).
6	Lin J, Yao J, Zhou X, Sun X and Tang K: Expression and purification of a novel mannose-binding lectin from Pinellia ternata. Mol Biotechnol. 25:215–222. 2003. View Article : Google Scholar : PubMed/NCBI
7	Xu T, Wang B, Wang L, Zhang Y and Lv Z: Pinellia ternata agglutinin produced in Bombyx mori cells exhibits bioactivity. Acta Biochim Pol. 59:231–236. 2012.
8	Lu Q, Li N, Luo J, et al: Pinellia pedatisecta agglutinin interacts with the methylosome and induces cancer cell death. Oncogenesis. 1:e292012. View Article : Google Scholar
9	Yoon JS, Seo JC and Han SW: Pinelliae Rhizoma herbal-acupuncture solution induced apoptosis in human cervical cancer cells, SNU-17. Am J Chin Med. 34:401–408. 2006. View Article : Google Scholar : PubMed/NCBI
10	Li GL, Jiang W, Xia Q, et al: HPV E6 down-regulation and apoptosis induction of human cervical cancer cells by a novel lipid-soluble extract (PE) from Pinellia pedatisecta Schott in vitro. J Ethnopharmacol. 132:56–64. 2010. View Article : Google Scholar : PubMed/NCBI
11	Chen XY, Zhou L and Zheng FY: Proteomica study of total protein of Pinellia pedactisecta Schott effect on human ovarian cancer SKOV3 cells. Zhongguo Zhong Xi Yi Jie He Za Zhi. 31:1651–1656. 2011.(In Chinese).
12	Zhou W, Huang Y, Xu S, et al: Prokaryotic expression and bioactivity analysis of N-terminus domain of Pinellia ternata agglutinin using alkaline phosphatase signal peptide. Protein Expr Purif. 89:84–91. 2013. View Article : Google Scholar : PubMed/NCBI
13	Sierko E, Wojtukiewicz MZ, Zimnoch L, et al: Protein Z/protein Z-dependent protease inhibitor system in human non-small-cell lung cancer tissue. Thromb Res. 129:e92–e96. 2012. View Article : Google Scholar : PubMed/NCBI
14	Sierko E, Wojtukiewicz MZ, Zimnoch L, Tokajuk P, Ostrowska-Cichocka K and Kisiel W: Co-localization of Protein Z, Protein Z-Dependent protease inhibitor and coagulation factor X in human colon cancer tissue: implications for coagulation regulation on tumor cells. Thromb Res. 129:e112–e118. 2012. View Article : Google Scholar
15	Sun L, Niu L, Zhu X, Hao J, Wang P and Wang H: Antitumour effects of a protease inhibitor, nelfinavir, in hepatocellular carcinoma cancer cells. J Chemother. 24:161–166. 2012. View Article : Google Scholar : PubMed/NCBI
16	Tamburino R, Pizzo E, Sarcinelli C, et al: Enhanced cytotoxic activity of a bifunctional chimeric protein containing a type 1 ribosome-inactivating protein and a serine protease inhibitor. Biochimie. 94:1990–1996. 2012. View Article : Google Scholar
17	Gocho T, Uwagawa T, Furukawa K, et al: Combination chemotherapy of serine protease inhibitor nafamostat mesilate with oxaliplatin targeting NF-κB activation for pancreatic cancer. Cancer Lett. 333:89–95. 2013.PubMed/NCBI
18	Morjen M, Kallech-Ziri O, Bazaa A, et al: PIVL, a new serine protease inhibitor from Macrovipera lebetina transmediterranea venom, impairs motility of human glioblastoma cells. Matrix Biol. 32:52–62. 2013. View Article : Google Scholar : PubMed/NCBI
19	Kobayashi H, Gotoh J, Hirashima Y and Terao T: Inter-alpha-trypsin inhibitor bound to tumor cells is cleaved into the heavy chains and the light chain on the cell surface. J Biol Chem. 271:11362–11367. 1996. View Article : Google Scholar : PubMed/NCBI
20	Baba T, Kawaguchi M, Fukushima T, et al: Loss of membrane-bound serine protease inhibitor HAI-1 induces oral squamous cell carcinoma cells’ invasiveness. J Pathol. 228:181–192. 2012.PubMed/NCBI
21	Brandi G, Tavolari S, De Rosa F, et al: Antitumoral efficacy of the protease inhibitor gabexate mesilate in colon cancer cells harbouring KRAS, BRAF and PIK3CA mutations. PLoS One. 7:e413472012. View Article : Google Scholar : PubMed/NCBI
22	Chen DY, Lee Y, Van Tine BA, et al: A pharmacologic inhibitor of the protease Taspase1 effectively inhibits breast and brain tumor growth. Cancer Res. 72:736–746. 2012. View Article : Google Scholar : PubMed/NCBI
23	Magee PJ, Owusu-Apenten R, McCann MJ, Gill CI and Rowland IR: Chickpea (Cicer arietinum) and other plant-derived protease inhibitor concentrates inhibit breast and prostate cancer cell proliferation in vitro. Nutr Cancer. 64:741–748. 2012.
24	Palavalli MH, Natarajan SS, Wang TT and Krishnan HB: Imbibition of soybean seeds in warm water results in the release of copious amounts of Bowman-Birk protease inhibitor, a putative anticarcinogenic agent. J Agric Food Chem. 60:3135–3143. 2012. View Article : Google Scholar : PubMed/NCBI
25	Rengan R, Mick R, Pryma D, et al: A phase I trial of the HIV protease inhibitor nelfinavir with concurrent chemoradiotherapy for unresectable stage IIIA/IIIB non-small cell lung cancer: a report of toxicities and clinical response. J Thorac Oncol. 7:709–715. 2012. View Article : Google Scholar : PubMed/NCBI
26	Shim JS, Rao R, Beebe K, et al: Selective inhibition of HER2-positive breast cancer cells by the HIV protease inhibitor nelfinavir. J Natl Cancer Inst. 104:1576–1590. 2012. View Article : Google Scholar : PubMed/NCBI
27	Loewenberg JR: Cyanide and the determination of protein with the Folin phenol reagent. Anal Biochem. 19:95–97. 1967. View Article : Google Scholar : PubMed/NCBI
28	Al-Tubuly AA: SDS-PAGE and western blotting. Methods Mol Med. 40:391–405. 2000.
29	Edman P, Högfeldt E, Sillén LR and Kinell P-O: Method for determination of the amino acid sequence in peptides. Acta Chemica Scandinavica. 4:283–293. 1950. View Article : Google Scholar
30	Dudai M, Mayer M and Kidron M: Protease inhibitor activity in rat skeletal muscle. Hoppe Seylers Z Physiol Chem. 363:651–654. 1982.PubMed/NCBI
31	Berg K, Hansen MB and Nielsen SE: A new sensitive bioassay for precise quantification of interferon activity as measured via the mitochondrial dehydrogenase function in cells (MTT-method). APMIS. 98:156–162. 1990. View Article : Google Scholar