Login Register Cart 0
Generic placeholder image

Anti-Cancer Agents in Medicinal Chemistry

Editor-in-Chief

ISSN (Print): 1871-5206
ISSN (Online): 1875-5992

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
Research Article

Induction of Apoptosis by Pierisin-6 in HPV Positive HeLa and HepG2 Cancer Cells is Mediated by the Caspase-3 Dependent Mitochondrial Pathway

Author(s): Subbarayan Sarathbabu, Satheesh K. Marimuthu, Souvik Ghatak, Subramanian Vidyalakshmi, Guruswami Gurusubramanian, Sankar K. Ghosh, Selvi Subramanian*, Wenqing Zhang and Nachimuthu S. Kumar*

Volume 19, Issue 3, 2019

Page: [337 - 346] Pages: 10

DOI: 10.2174/1871520619666181127113848

Price: $65

Abstract

Background: To explore the cytotoxic and apoptotic activity of the pierisin-6 protein in HPV HeLa and HepG2 cell lines.

Methods: In this study, isolation, and purification of cytotoxic Prierisin-6 from the larvae of Pieris napi by affinity column chromatography techniques. Characterization of full-length mRNA of pierisin-6 gene was performed using 3’/5’ RACE PCR. The quantitative RT-PCR used to study the developmental stage-specific expression of pierisin-6 mRNA. The most effective concentration of Pierisin-6 protein was determined by measuring cell proliferation. Apoptosis was assessed using AO/Et-Br, Propidium Iodide, and Rhodamine 123 assays, whereas protein levels of caspase 3, cytochrome C were evaluated by ELISA method. Pierisin-6 induced cell cycle arrest was determined using Propidium iodide by FACS.

Results: In this study, Pierisin-6, a novel apoptotic protein was found to have cytotoxicity against HeLa, HepG2 human cancer cell lines and L-132 human lung epithelial cell line. Among the target cells, HeLa was the most sensitive to Pierisin-6. Flow cytometry analysis confirms an increased percentage of apoptotic cells in sub G1 phase and cell cycle arrest at S phase. Alteration in the transmembrane potential of mitochondria, Cytochrome c released from the mitochondrial membrane, and caspase substrate assay demonstrated the cleavage of Ac- DEVD-pNA signifying the activation of Caspase-3. These findings suggested that Pierisin-6 significantly induce apoptosis in HeLa and HepG2 cells and is attributed mainly through a mitochondrial pathway by activation of caspases. The developmental and stage-specific expression of pierisin-6 mRNA was one thousand-fold increased from second to third instar larvae and gradually declined before pupation.

Conclusion: Pierisin-6 represents a promising therapeutic approach for liver cancer patients.

Keywords: Pierisin-6, cytotoxicity, flow cytometry, cytochrome c, caspase-3, transmembrane potential.

« Previous Next »
Graphical Abstract

[1]
Slocinska, M.; Marciniak, P.; Rosinski, G. Insects antiviral and anticancer peptides: New leads for the future. Protein Pept. Lett., 2008, 15, 575-585.
[2]
Koyama, K.; Wakabayashi, K.; Masutani, M.; Koiwai, K.; Watanabe, M.; Yamazaki, S.; Kono, T.; Miki, K.; Sugimura, T. Presence in Pieris rapae of cytotoxic activity against human carcinoma cells. Jpn. J. Cancer Res., 1996, 87, 1259-1262.
[3]
Takamura-Enya, T.; Watanabe, M.; Totsuka, Y.; Kanazawa, T.; Matsushima-Hibiya, Y.; Koyama, K.; Sugimura, T.; Wakabayashi, K. Mono(ADP-Ribosyl)ation of 2′-deoxyguanosine residue in DNA by an apoptosis-inducing protein, pierisin-1, from cabbage butterfly. Proc. Natl. Acad. Sci. USA, 2001, 98, 12414-12419.
[4]
Watanabe, M.; Kono, T.; Koyama, K.; Sugimura, T.; Wakabayashi, K. Purification of pierisin, an inducer of apoptosis in human gastric carcinoma cells, from cabbage butterfly, Pieris rapae. Jpn. J. Cancer Res., 1998, 89, 556-561.
[5]
Orth, J.H.C.; Schorch, B.; Boundy, S.; Ffrench-constant, R.; Kubick, S.; Aktories, K. Cell-free synthesis and characterization of a novel cytotoxic pierisin-like protein from the cabbage butterfly Pieris rapae. Toxicon, 2011, 57, 199-207.
[6]
Matsushima-Hibiya, Y.; Watanabe, M.; Kono, T.; Kanazawa, T.; Koyama, K.; Sugimura, T.; Wakabayashi, K. Purification and cloning of pierisin-2, an apoptosis-inducing protein from the cabbage butterfly, Pieris brassicae. Eur. J. Biochem., 2000, 267, 5742-5750.
[7]
Yamamoto, M.; Nakano, T.; Matsushima-Hibiya, Y.; Totsuka, Y.; Takahashi-Nakaguchi, A.; Matsumoto, Y.; Sugimura, T.; Wakabayashi, K. Molecular cloning of apoptosis-inducing pierisin-like proteins, from two species of white butterfly, Pieris melete and Aporia crataegi. Mol. Biol., 2009, 154, 326-333.
[8]
Subbarayan, S.; Marimuthu, S.K.; Nachimuthu, S.K.; Zhang, W.; Subramanian, S. Characterization and cytotoxic activity of apoptosis-inducing Pierisin-5 protein from white cabbage butterfly. Int. J. Biol. Macromol., 2016, 87, 16-27.
[9]
Matsumoto, Y.; Nakano, T.; Yamamoto, M.; Matsushima-Hibiya, Y.; Odagiri, K.I.; Yata, O.; Koyama, K.; Sugimura, T.; Wakabayashi, K. Distribution of cytotoxic and DNA ADP-Ribosylating activity in crude extracts from butterflies among the family Pieridae. Proc. Natl. Acad. Sci. USA, 2008, 105, 2516-2520.
[10]
Watanabe, M.; Nakano, T.; Shiotani, B.; Matsushima-Hibiya, Y.; Kiuchi, M.; Yukuhiro, F.; Kanazawa, T.; Koyama, K.; Sugimura, T.; Wakabayashi, K. Developmental stage-specific expression and tissue distribution of Pierisin-1, a Guanine-Specific ADP-Ribosylating toxin in Pieris rapae. Mol. Integr. Physiol; , 2004, 139, pp. 125-131.
[11]
Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic local alignment search tool. J. Mol. Biol., 1990, 215, 403-410.
[12]
Gasteiger, E.; Hoogland, C.; Gattiker, A.; Duvaud, S.; Wilkins, M.R.; Appel, R.D.; Bairoch, A. Protein identification and analysis tools on the ExPASy server. In: The Proteomics Protocols Handbook; Humana Press, 2005; pp. 571-607.
[13]
Ponting, C.P.; Schultz, J.; Milpetz, F.; Bork, P. SMART: Identification and annotation of domains from signalling and extracellular protein sequences. Nucleic Acids Res., 1999, 27, 229-232.
[14]
Chang, J.C.; Ramasamy, S. Identification and expression analysis of Diapause Hormone and Pheromone Biosynthesis Activating Neuropeptide (DH-PBAN) in the legume pod borer, Maruca vitrata Fabricius. PLoS One, 2014, 9, 1-11.
[15]
Pfaffl, M.W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res., 2001, 29, e45-e45.
[16]
Bradford, M.M. A Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 1976, 72, 248-254.
[17]
Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227, 680-685.
[18]
Kasibhatla, S.; Amarante-Mendes, G.P.; Finucane, D.; Brunner, T.; Bossy-Wetzel, E.; Green, D.R. Analysis of DNA fragmentation using agarose gel electrophoresis. CSH Protoc, 2006, 1, 4429-4438.
[19]
Afsar, T.; Trembley, J.H.; Salomon, C.E.; Razak, S.; Khan, M.R.; Ahmed, K. Growth inhibition and apoptosis in cancer cells induced by polyphenolic compounds of Acacia hydaspica: Involvement of multiple signal transduction pathways. Nat. Publ. Gr, 2016, 6, 1-12.
[20]
Riccardi, C.; Nicoletti, I. Analysis of apoptosis by Propidium Iodide staining and flow cytometry. Nat. Protoc., 2006, 1, 1458-1461.
[21]
Baracca, A.; Sgarbi, G.; Solaini, G.; Lenaz, G. Rhodamine 123 as a probe of mitochondrial membrane potential: Evaluation of proton flux through F0 during ATP synthesis. Biochim. Biophys. Acta Bioenerg., 2003, 1606, 137-146.
[22]
Aravind, L.; Zhang, D.; de Souza, R.F.; Anand, S.; Iyer, L.M. The natural history of ADP-Ribosyltransferases and the ADP-ribosylation system. Curr. Top. Microbiol. Immunol., 2015, 384, 3-32.
[23]
Takamura-Enya, T.; Watanabe, M.; Koyama, K.; Sugimura, T.; Wakabayashi, K. Mono(ADP-Ribosyl)ation of the N 2 amino groups of guanine residues in DNA by Pierisin-2, from the cabbage butterfly. Pieris Brassicae. Biochem. Biophys. Res. Commun, 2004, 323, 579-582.
[24]
Treiber, N.; Reinert, D.J.; Carpusca, I.; Aktories, K.; Schulz, G.E. Structure and mode of action of a mosquitocidal holotoxin. J. Mol. Biol., 2008, 381, 150-159.
[25]
Watanabe, M.; Enomoto, S.; Takamura-Enya, T.; Nakano, T.; Koyama, K.; Sugimura, T.; Wakabayashi, K. Enzymatic properties of pierisin-1 and its N-terminal domain, a guanine-specific ADP-Ribosyltransferase from the cabbage butterfly. J. Biochem., 2004, 135, 471-477.
[26]
Shin, I.S.; Ishii, S.; Shin, J.S.; Sung, K.I.; Park, B.S.; Jang, H.Y.; Kim, B.W. Globotriaosylceramide (Gb3) content in HeLa cells is correlated to shiga toxin-induced cytotoxicity and Gb3 synthase expression. BMB Rep., 2009, 42, 310-314.
[27]
Krueger, K.M.; Barbieri, J.T. The Family of bacterial ADPribosylating the family of bacterial ADP-ribosylating exotoxins. 1995, 8, 34-47.
[28]
Takahashi-Nakaguchi, A.; Matsumoto, Y.; Yamamoto, M.
Iwabuchi, K.; Totsuka, Y.; Sugimura, T.; Wakabayashi, K. Demonstration of cytotoxicity against wasps by pierisin-1: A possible defense factor in the cabbage white butterfly. PLoS One, 2013, 8e60539
[29]
Ribble, D.; Goldstein, N.B.; Norris, D.A.; Shellman, Y.G. A simple technique for quantifying apoptosis in 96-well plates. BMC Biotechnol., 2005, 5, 12.
[30]
Snigdha, S.; Smith, E.D.; Prieto, G.A.; Cotman, C.W. Caspase-3 activation as a bifurcation point between plasticity and cell death. Neurosci. Bull., 2012, 28(1), 14-24.
[31]
Heibein, J.A.; Barry, M.; Motyka, B.; Bleackley, R.C. Granzyme B-induced loss of mitochondrial inner membrane potential (Delta Psi M) and cytochrome c release are caspase independent. J. Immunol., 1999, 163, 4683-4693.
[32]
Riedl, S.J.; Shi, Y. Molecular mechanisms of caspase regulation during apoptosis. Nat. Rev. Mol. Cell Biol., 2004, 5, 897-907.
[33]
Kanazawa, T.; Kono, T.; Watanabe, M.; Matsushima-hibiya, Y.; Nakano, T.; Koyama, K.; Tanaka, N.; Sugimura, T.; Wakabayashi, K. Bcl-2 blocks apoptosis caused by pierisin-1, a guanine-specific ADP-ribosylating toxin from the cabbage butterfly. Biochem. Biophys. Res. Commun., 2002, 296, 20-25.
[34]
Cullen, S.P.; Martin, S.J. Caspase activation pathways: Some recent progress. Cell Death Differ., 2009, 16, 935-938.
[35]
Elmore, S. Apoptosis: A review of programmed cell death. Toxicol. Pathol., 2007, 35, 495-516.
[36]
Brown, C. An elusive cancer target. Nature, 2016, 537, S106-S108.
[37]
Jacobs, S.A. Yttrium ibritumomab tiuxetan in the treatment of non-hodgkin’s lymphoma. Curr. Stat. Fut. Prospects, 2007, 1, 215-227.
[38]
Senter, P.D.; Sievers, E.L. Perspective the discovery and development of brentuximab vedotin for use in relapsed hodgkin lymphoma and systemic anaplastic large cell lymphoma. Nat. Biotechnol., 2012, 30, 631-637.
[39]
Macrae, E.R. Role of trastuzumab emtansine in the treatment of HER2-positive breast cancer. Breast Cancer Targets Ther, 2014, 39, 103-113.
[40]
Ferrara, N.; Hillan, K.J.; Gerber, H.P.; Novotny, W. Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat. Rev. Drug Discov., 2004, 3, 391-400.
[41]
Pozzi, C.; Cuomo, A.; Spadoni, I.; Magni, E.; Silvola, A.; Conte, A.; Sigismund, S.; Ravenda, P.S.; Bonaldi, T.; Zampino, M.G.; Cancelliere, C.; Di Fiore, P.P.; Bardelli, A.; Penna, G.; Rescigno, M. The EGFR-specific antibody cetuximab combined with chemotherapy triggers immunogenic cell death. Nat. Med., 2016, 22, 1-11.

Rights & Permissions Print Cite
Article Metrics
41
7
1
1
© 2024 Bentham Science Publishers | Privacy Policy