scholarly journals Expression and bioactivity of human α-fetoprotein in a Bac-to-Bac system

2017 ◽  
Vol 37 (1) ◽  
Author(s):  
Bo Lin ◽  
Kun Liu ◽  
Wenting Wang ◽  
Wei Li ◽  
Xu Dong ◽  
...  

α-fetoprotein (AFP) is an early serum growth factor in foetal embryonic development and hepatic oncogenesis. A growing number of investigations of AFP as a tumour-specific biomarker have concluded that AFP is an important target for cancer treatment. AFP also plays an immunomodulatory role in the treatment of several autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis and thyroiditis. In an effort to support biochemical screening and drug design and discovery, we attempted to express and purify human AFP in a Bac-to-Bac system. Two key factors affecting the expression of recombinant human AFP (R-AFP), namely the infectious baculovirus inoculum volume and the culturing time post-infection, were optimized to maximize the yield. We achieved a high yield of approximately 1.5 mg/l of harvested medium with a 72–96 h incubation period after infection and an inoculum volume ratio of 1:100. We also assessed the role of R-AFP in the proliferation of the human liver cancer cell line Bel 7402, and the results indicated that R-AFP promoted the growth of hepatoma cells. We concluded that this method can produce high yields of R-AFP, which can be used for studies related to AFP.

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Bo Lin ◽  
Hailing Meng ◽  
Hui Bing ◽  
Dongting Zhangsun ◽  
Sulan Luo

The Bac-to-Bac baculovirus expression system can efficiently produce recombinant proteins, but the system may have to be optimized to achieve high-level expression for different candidate proteins. We reported here the efficient expression of acetylcholine-binding proteins from sea haresAplysia californica(Ac-AChBP) and a convenient method to monitor protein expression level in this expression system. Three key factors affecting expression of Ac-AChBP were optimized for maximizing the yield, which included the cell density, volume of the infecting baculovirus inoculums, and the culturing time of postinfection. We have found it to reach a high yield of ∼5 mg/L, which needs 55 h incubation after infection at the cell density of 2 × 106 cells/mL with an inoculum volume ratio of 1 : 100. The optimized expression system in this study was also applied for expressing another protein Ls-AChBP fromLymnaea stagnalissuccessfully. Therefore, this established method is helpful to produce high yields of AChBP proteins for X-ray crystallographic structural and functional studies.


Author(s):  
Do Huu Nghi ◽  
Vo Thi Ngoc Hao ◽  
Nguyen Thi Hong Nhung

This study discusses the results of the experimental application of high-content screening (HCS) techniques in evaluating the induction of cell-cycle arrest and apoptosis on human liver cancer cell line, Hep-G2. Accordingly, the bisbenzimide-stained cells (Hoechst 33342; 350 to 500 nM) were analyzed by using an Olympus scanˆR HCS-system to determine the cell-cycle phases (G1, S, and G2/M) and apoptosis as well. As a result, the cell-cycle arrest could be indicated by an increase in G2/M population of Hep-G2 cells after 24h exposure to zerumbone (Zer4; 9 µg/mL) and a similar observation could be made for paclitaxel (Pac; 4 µg/mL) as a reference substance. Keywords Apoptosis, cell-cycle arrest, high-content screening, human liver cancer cell line Hep-G2. References [1] D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation, Cell 144 (2011) 646–674.[2] M. Malumbres, M. Barbacid, Cell cycle, CDKs and cancer: a changing paradigm, Nat. Rev. Cancer 9 (2009) 153–166.[3] S. Diermeier-Daucher, et al., Cell type specific applicability of 5-ethynyl-2'-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry, Cytometry A 75 (2009) 535-546.[4] J. Essers, et al., Nuclear dynamics of PCNA in DNA replication and repair, Mol. Cell Biol 25 (2005) 9350- 9359. [5] V. Roukos, et al., Dynamic recruitment of licensing factor Cdt1 to sites of DNA damage. J. Cell Sci. 124 (2011) 422-434.[6] M. Hesse, et al., Direct visualization of cell division using high-resolution imaging of M-phase of the cell cycle, Nat. Commun 3 (2012) 1076. doi: 10.1038/ncomms2089.[7] P. Cappella, F. Gasparri, M. Pulici, J. Moll, A novel method based on click chemistry, which overcomes limitations of cell cycle analysis by classical determination of BrdU incorporation, allowing multiplex antibody staining, Cytometry A 73 (2008) 626–636. [8] S. Diermeier-Daucher, et al., Cell type specific applicability of 5-ethynyl-2’-deoxyundine (EdU) for dynamic proliferation assessment in flow cytometry, Cytometry A 75 (2009) 535–546.[9] T. Yokochi, D.M. Gilbert, Replication labeling with halogenated thymidine analogs, Curr. Protoc. Cell Biol, 35 (2007) 22.10.1–22.10.14. [10] T.J. McGarry, M.W. Kirschner, Geminin, an inhibitor of DNA replication, is degraded during mitosis, Cell 93 (1998) 1043–1053. [11] H. Nishitani, S. Taraviras, Z. Lygerou, T. Nishimoto, The human licensing factor for DNA replication Cdt1 accumulates in G1 and is destabilized after initiation of S-phase. J. Biol. Chem 276 (2001) 44905–44911.[12] J. Pines, T. Hunter, Human cyclin A is adenovirus E1A-associated protein p60 and behaves differently from cyclin B, Nature 346 (1990) 760–763. [13] A. Stathopoulou, et al., Cdt1 is differentially targeted for degradation by anticancer chemotherapeutic drugs. PLoS ONE 7, e34621 (2012). [14] M. Hesse, A. Raulf, G.A. Pilz, C. Haberlandt, A.M. Klein, R. Jabs, H. Zaehres, C.J. Fügemann, K. Zimmermann, J. Trebicka, A. Welz, A. Pfeifer, W. Röll, M.I. Kotlikoff, C. Steinhäuser, M. Götz, H.R. Schöler, B.K. Fleischmann, Direct visualization of cell division using high-resolution imaging of M-phase of the cell cycle, Nat. Commun 3 (2012): 1076.[15] D.A. Ridenour, M.C. McKinney, C.M. Bailey, P.M. Kulesa, CycleTrak: a novel system for the semiautomated analysis of cell cycle dynamics. Dev. Biol 365 (2012) 189–195. [16] A. Roukos, et al., Cell cycle staging of individual cells by fluorescence microscopy, Nat. Protoc 10 (2015) 334-348.[17] E. Harlow, D. Lane, Fixing attached cells in paraformaldehyde, CSH Protoc 3 (2006) doi: 10.1101/pdb.prot4294.[18] G. Mazzini, M. Danova, Fluorochromes for DNA staining and quantitation, Method. Mol. Biol 1560 (2017) 239-259.[19] A. Gottfried, E. Weinhold, Sequence-specific covalent labelling of DNA, Biochem. Soc. Trans 39 (2011) 623-628.[20] J. Bucevičius, G. Lukinavičius, R. Gerasimaitė, The use of Hoechst dyes for DNA staining and beyond, Chemosensor 6 (2018) 1-18.[21] V. Kumar, A.K. Abbas, J.C. Aster, Robbins and Cotran Pathologic Basis of Disease, Ninth ed., Elsevier/Saunders, Philadelphia (2015).[22] N.A. Jensen et al., Establishment of a high content assay for the identification and characterisation of bioactivities in crude bacterial extracts that interfere with the eukaryotic cell cycle, J. Biotechnol 140 (2009) 124-134.[23] H.S. Rahman, et al., Zerumbone induces G2/M cell cycle arrest and apoptosis via mitochondrial pathway in Jurkat cell line, Nat. Prod. Commun 9 (2014) 1237-1242.[24] S.I. Abdelwahab, et al., Zerumbone inhibits interleukin-6 and induces apoptosis and cell cycle arrest in ovarian and cervical cancer cells, Intern. Immunopharm 12 (2012) 594-602.[25] M. Xian, et al., Zerumbone, A bioactive sesquiterpene, induces G2/M cell cycle arrest and apoptosis in leukemia cells via a Fas- and mitochondria-mediated pathway, Cancer Sci 98 (2007) 118-126.[26] A. Sehrawat, et al., Zerumbone causes Bax-and Bak-mediated apoptosis in human breast cancer cells and inhibits orthotopic xenograft growth in vivo, Breast Cancer Res. Treat. 136 (2012) 429-441.[27] Y.Z. Zhou, et al., Zerumbone induces G1 cell cycle arrest and apoptosis in cervical carcinoma cells, Int. J. Clin. Exp. Med. 10 (2017) 6640-6647.


2021 ◽  
Vol 2021 ◽  
pp. 1-7
Author(s):  
Ali Sahragard ◽  
Zohreh Alavi ◽  
Zohreh Abolhassanzadeh ◽  
Mahmoodreza Moein ◽  
Afshin Mohammadi-Bardbori ◽  
...  

Cancer chemotherapies may result in resistance, and therefore, contemporary treatments including natural products may find an increasing consideration. As per Persian medicine (PM), many natural products have been used for malignant and chronic diseases. Triphala, with a combination of Terminalia chebula Retz., Terminalia bellirica Retz., Phyllanthus emblica L., and honey, is a multi-ingredient traditional formulation attributed to anticancer activities in PM. This study is aimed at evaluating the cytotoxic activity of this preparation on HepG2, the human liver cancer cell line. Hydroalcoholic extracts were prepared from the formulation and its components. Compared with the control and Cisplatin, the extracts were tested using MTT assay at different concentrations. All concentrations of the preparation, as well as Cisplatin, were effective significantly against HepG2 cells. All extract preparations at multiple concentrations were significantly effective as evidenced by MTT assay when compared to the control group. The IC50 level for Triphala extract was 77.63 ± 4.3   μ g / ml . Based on the results, Triphala and its components have cytotoxic activity on the HepG2 cancer cell line and they can reduce the survival rate significantly.


2016 ◽  
Vol 11 (1) ◽  
pp. 1-8
Author(s):  
Manal M. Ramadan ◽  
Amr F. Mansour ◽  
Reda M. Fekry ◽  
Marwa T. Salem ◽  
Fathy M. Mahaya ◽  
...  

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