ANTIPROLIFERATIVE ACTIVITY OF RECOMBINANT HUMAN INTERFERON ALPHA2B ON ESTROGEN POSITIVE HUMAN BREAST CANCER MCF-7 CELL LINE

Ratih Asmana Ningrum; Popi Hadi Wisnuwardhani; Adi Santoso; Neng Herawati

doi:10.14499/indonesianjpharm26iss2pp86

ANTIPROLIFERATIVE ACTIVITY OF RECOMBINANT HUMAN INTERFERON ALPHA2B ON ESTROGEN POSITIVE HUMAN BREAST CANCER MCF-7 CELL LINE

Ratih Asmana Ningrum, Popi Hadi Wisnuwardhani, Adi Santoso, Neng Herawati

Abstract

Indonesia is the top three countries with hepatitis and moreover has 40.000 cancer mortalities per year. Interferon alpha 2b (IFNα-2b) is a therapeutic standard for cancer and hepatitis B/C treatments. We developed recombinant human interferon alpha 2b (rhIFNα-2b) in methilotropic yeast Pichia pastoris X-33. The protein was produced as extracellular protein with 24 kDa in size. This research was aimed to characterize the protein based on its amino acid sequence and to study its antiproliferative activity on MCF-7 cell line. Amino acid sequencing by using MALDI TOF TOF mass spectrometry with trypsin as proteolytic enzyme identified protein as hIFNα-2b with 33% of amino acid coverage. The antiproliferative activity was determined by 3-[4.5-dimethyltiazol-2il]-2.5-diphenylltetrazolium bromide (MTT) assay. Based on several studies about the synergistic activity of rhIFNα-2b with other anticancer drugs, we combined our rhIFNα-2b with tamoxifen (tmx). The growth percentage of the cells after being treated with 1µM of tmx at various concentrations of rhIFNα-2b was compared with that of untreated cell. This study showed that the antiproliferative activity was dose-dependently. Cell viability assay with calcein and ethidium bromide-based staining by fluorescence microscope confirmed that the rhIFNα-2b had ability to inhibit proliferation of human breast cancer cell line MCF-7.

Keywords: human interferon alpha 2b, Pichia pastoris, antiproliferative and MCF-7

Full Text:

PDF(PP.86-93)

References

Bekisz J., Baron S., Balinsky C., Morrow A., Zoon KC. 2010. Antiproliferative properties of type I and type II interferon. Pharmaceuticals, 3, 994-1015.

Bernhard H., Jäger-Arand E., Bernhard G., Heike M., Klein O., Riemann JF., Meyer zum Büschenfelde KH., Dippold W., Knuth A. 1995. Treatment of advanced pancreatic cancer with 5-fluorouracil, folinic acid and interferon alpha-2A: results of a phase II trial, Brit J. Cancer. 71(1): 102–105.

Bornhorst JA., Falke JJ. 2000. Purification of Proteins Using Polyhistidine Affinity Tags. Methods in Enzymol. 326: 245–254.

Chiariello M., Gomez E., Eliana, Gutkind JS., (2000. Regulation of cyclin-dependent kinase (Cdk) 2 Thr-160 phosphorylation and activity by mitogen-activated protein kinase in late G1 phase, Biochemistry Journal, 349, 869-876.

Essafi IR., Sadok A., Khalaf N., Fathallah DM. 2007. A strategy for high-level expression of soluble and functional human interferon a as a GST-fusion protein in E.coli, Protein Eng, Des and Sel vol. 20 no. 5 pp. 201–209, 2007.

Ghosalkar A., Sahai V., Srivasta A., 2008. Secretory expression of interferon-alpha2b in recombinant Pichia pastoris using three different secretion signals, Protein Expr. Purif.60: 103-109.

Gonnet F., Lemaitre G., Waksman G. Tortajada. 2003. MALDI/MS peptide mass fingerprinting for proteome analysis: identification of hydrophobic proteins attached to eucaryote keratinocyte cytoplasmic membrane using different matrices in concert, Proteome Science. 1:2, 1-7. Clinical Applications, and Toxicities, The Oncologist. 6: 34-55.

Herawati N., Santoso A., Ningrum RA. 2014. The Effect of non-nutritional factors of culture condition on Human Interferon alpha-2b Production in Methilotropic Yeast Pichia pastoris, J.Pharm. Bioanalitical Science. 3 (1): 1-5.

Kolch W. 2000. Review article: Meaningful relationship the regulation of the Ras/Raf/MEK/ERK pathway by protein interaction, Biochem. J., 351, 289-305.

Lee KH., Lee JS., Suh C., Lee YS., Min YI., et al., 1992. Combination of 5-fluorouracil and recombinant interferon alpha-2B in advanced gastric cancer. A phase I study, Am. J. Clin. Oncology. 15(2) : 141-145.

Li P., Anumanthan A., Gao XG., Ilangovan K., Suzara V., Diizgiines, Renugopalakrishnan R., 2007, Appl. Biochem Biottechnol. 142: 105-124.

Lindner DJ., Borden EC. Alvakolanu DV. 1997. Synergistic antitumor effects of a combinantion of interferons and retinoic acid on human tumor cells in vitro and in vivo, Cin. Can. Res. 3: 931-937.

Lindner DJ., Borden EC. 1997. Synergistic Antitumor Effects of a Combination of Interferon and Tamoxifen on Estrogen Receptor-Positive and Receptor-Negative Human Tumor Cell Lines In Vivo and In Vitro. J. Interferon Cytokine Res. 17: 681-693.

Lippmann ME., Bolan G. 1975. Oestrogen-responsive human breast cancer in long-term tissue culture. Nature, 256,593-595.

Maemura M., Iino Y., Horiguchi J., Takei, H., et al., 1999. Effects of interferon-alpha on cellular proliferation and adhesion of breast carcinoma cells, Oncology reports. 6(3): 557-561

Neves FO., Ho PL., Raw I., Pereira CA., Moreira C., Nascimento ALTO. 2004. Overexpression of A Synthetic Gene Encoding Human Alpha Interferon in Escherichia coli, Protein Expression and Purif. 35: 353–359.

Ningrum RA. 2014. Interferon alpha2b: a therapeutic protein for cancer treatment. scientifica,1-8.

Ningrum RA., Santoso A., Herawati N. 2013. Secretory Expression of Recombinant Human Interferon-alpha2b in methilotropic yeast Pichia pastoris, Int. J.Res. in Pharm. Scie.. 4(2): 207-210.

Ningrum RA., Rahmatika DE., Retnoningrum DS., Wangsaatmadja AH., Sumirtapura YC., Rachmawati H. 2012. Development of Novel Interferon Alpha2b Muteins and Study the Pharmacokinetic and Biodistribution Profiles in Animal Model, J. Biomed. Sci. Eng. 5: 104-112.

Papadopoulos NG., Dedoussis GV., Spanakos G., Gritzapis AD., Baxevanis CN., Papamichail M. 1994. An improved fluorescence assay for the determintion of lymphocyte-mediated cytotoxicity using flocitomety, J.Immunology methods.

Robinson SP., Goldstein D., Witt PL., Borden EC., Jordan VC. 1990. Inhibition of hormone-dependent and independent breast cancer cell growth in vivo and in vitro with the antiestrogen toremifene and recombinant human interferon-alpha 2, Breast Cancer Research Treament, 15(2):95-101.

Romerio F., Zella D., 2002. MEK and ERK inhibitors enhance the antiproliferative effect of interferon-α2b, FASEB Journal, USA.

Romerio F., Riva A., Zella D. 2000. Interferon alpha2b reduces phosphorylation and activity of MEK and ERK throgh a Ras/Raf-independent mechanism, Brit. J.Cancer, 83, 532-538.

Porta C., Slimane RH., Nejmeddine M., Pampin M., Tovey MG., Lucile Espert., Alvarez S., Chelbi-Alix MK. 2005. Interferons α and β induce p53-dependent and p53-independent apoptosis, respectively. 24: 605–615

Santoso A., Ningrum RA., Herawati N. 2013. Purification and Characterization of Recombinant Human Interferon-alpha2b in methilotropic yeast Pichia pastoris, Int. J.Pharm.Sci. Health Care. 3(3): 13-19. ISSN: 2249-5378.

Sarkar MC., Lindner DJ., Liu YF., Williams BR., Sen GC., Silverman RH., Borden EC. 2003. Apoptosis and interferons: role of interferon-stimulated genes as mediators of apoptosis, Apoptosis. 8: 237–249.

Septisetyani EP., Ningrum RA., Romadhani Y., Wisnuwardhani PH., Santoso A. 2014. Optimization of sodium dodecyl sulphate as a formazan solvent and comparison of MTT assay with WST-1 assy in MCF-7 cells, Indonesian J. Pharm. 25(4): 245-254.

Shi L., Wang D., Chan W., Cheng L. 2007. Efficient expression and purification of human interferon alpha2b in the methilotropic yeast, Pichia pastoris, Protein Expr. Purif. 54: 220-226.

Srivastava, P., Bhattacharaya, P., Pandey, G. And Mukherjee, K.J. 2005. Overexpression and purification of recombinant human interferon alpha2b in Escherichia coli, Protein Expression Purif. 41: 313–322.

Steelman LS., Pohnert SC., Shelton JG., Franklin RA., Bertrand FE. McCubrey, JA., 2004. JAK/STAT, Raf/MEK/ERK, P13/Akt and BCR-ABL in cell cycle progression and leukemogenesis, Leukemia, 18, 189-218.

Taylor CW., Modiano MR., Woodson ME., Marcus SG., Alberts DS. Hersh, EM. 1992. A phase I trial of fluorouracil, leucovorin, and recombinant interferon alpha-2b in patients with advanced malignancy, Seminanr in Oncology. 19 (2 Suppl 3):185-190.

Tiwari RK., Wong GY., Liu J., Miller D., Osborne MP. 1991. Augmentation of cytotoxicity using combinations of interferons (types I and II), tumor necrosis factor-alpha, and tamoxifen in MCF-7 cells, Cancer Letter. 61(1):45-52.

Valente CA., Monteiro GA., Cabral JMS., Fevereiro M., Prazeres DMF. 2006. Optimization of the primary recovery of human interferon2b from Escherichia coli inclusion bodies, Protein Expression Purif. 45: 226–234.

Wang YS.,Youngster S., Grace M., Bausch J., Borden R., Wyss DF. 2002. Structural and biological characterization of pegylated recombinant interferon alpha-2b and its therapeutic implications, Adv. Drug Delivery Rev. 54: 547–570.

DOI: http://dx.doi.org/10.14499/indonesianjpharm26iss2pp86