Apoptosis

, Volume 20, Issue 10, pp 1388–1409 | Cite as

In situ allicin generation using targeted alliinase delivery for inhibition of MIA PaCa-2 cells via epigenetic changes, oxidative stress and cyclin-dependent kinase inhibitor (CDKI) expression

  • Sagar V. Chhabria
  • Mohammad A. Akbarsha
  • Albert P. Li
  • Prashant S. Kharkar
  • Krutika B. Desai
Original Paper

Abstract

Allicin, an extremely active constituent of freshly crushed garlic, is produced upon reaction of substrate alliin with the enzyme alliinase (EC 4.4.1.4). Allicin has been shown to be toxic to several mammalian cells in vitro in a dose-dependent manner. In the present study this cytotoxicity was taken to advantage to develop a novel approach to cancer treatment, based on site directed generation of allicin. Alliinase was chemically conjugated to a monoclonal antibody (mAb) which was directed against a specific pancreatic cancer marker, CA19-9. After the CA19-9 mAb-alliinase conjugate was bound to targeted pancreatic cancer cells (MIA PaCa-2 cells), on addition of alliin, the cancer cell-localized alliinase produced allicin, which effectively induced apoptosis in MIA PaCa-2 cells. Specificity of anticancer activity of in situ generated allicin was demonstrated using a novel in vitro system—integrated discrete multiple organ co-culture technique. Further, allicin-induced caspase-3 expression, DNA fragmentation, cell cycle arrest, p21Waf1/Cip1 cyclin-dependent kinase inhibitor expression, ROS generation, GSH depletion, and led to various epigenetic modifications which resulted in stimulation of apoptosis. This approach offers a new therapeutic strategy, wherein alliin and alliinase-bound antibody work together to produce allicin at targeted locations which would reverse gene silencing and suppress cancer cell growth, suggesting that combination of these targeted agents may improve pancreatic cancer therapy.

Keywords

Allicin Apoptosis CA19-9 Cyclin-dependent kinase inhibitor (CDKI) Integrated discrete multiple organ co-culture (IdMOC) technique MIA PaCa-2 cells 

References

  1. 1.
    Burris HA, Moore MJ, Andersen J et al (1997) Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial. J Clin Oncol 15:2403–2413PubMedGoogle Scholar
  2. 2.
    Keleg S, Büchler P, Ludwig R et al (2003) Invasion and metastasis in pancreatic cancer. Mol Cancer 2:14. doi:10.1186/1476-4598-2-14 PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Shore S, Raraty MGT, Ghaneh P, Neoptolemos JP (2003) Chemotherapy for pancreatic cancer. Aliment Pharmacol Ther 18:1049–1069. doi:10.1046/j.1365-2036.2003.01781.x CrossRefPubMedGoogle Scholar
  4. 4.
    Tawada K, Yamaguchi T, Kobayashi A et al (2009) Changes in tumor vascularity depicted by contrast-enhanced ultrasonography as a predictor of chemotherapeutic effect in patients with unresectable pancreatic cancer. Pancreas 38:30–35. doi:10.1097/MPA.0b013e318183ff73 CrossRefPubMedGoogle Scholar
  5. 5.
    Ilic D, Nikolic V, Nikolic L et al (2011) Allicin and related compounds: biosynthesis, synthesis and pharmacological activity. Facta Univ 9:9–20. doi:10.2298/FUPCT1101009I CrossRefGoogle Scholar
  6. 6.
    Nakao A, Oshima K, Nomoto S et al (1998) Clinical usefulness of CA-19-9 in pancreatic carcinoma. Semin Surg Oncol 15:15–22. doi:10.1002/(SICI)1098-2388(199807/08)15:1<15:AID-SSU4>3.0.CO;2-Z CrossRefPubMedGoogle Scholar
  7. 7.
    Makovitzky J (1986) The distribution and localization of the monoclonal antibody-defined antigen 19-9 (CA19-9) in chronic pancreatitis and pancreatic carcinoma. Virchows Arch B 51:535–544. doi:10.1007/BF02899058 CrossRefPubMedGoogle Scholar
  8. 8.
    Girgis MD, Olafsen T, Kenanova V et al (2011) CA19-9 as a potential target for radiolabeled antibody-based positron emission tomography of pancreas cancer. Int J Mol Imaging 2011:1–9. doi:10.1155/2011/834515 CrossRefGoogle Scholar
  9. 9.
    Miron T, Mironchik M, Mirelman D et al (2003) Inhibition of tumor growth by a novel approach: in situ allicin generation using targeted alliinase delivery. Mol Cancer Ther 2:1295–1301PubMedGoogle Scholar
  10. 10.
    Arditti F, Rabinkov A, Miron T et al (2005) Apoptotic killing of B-chronic lymphocytic leukemia tumor cells by allicin generated in situ using a rituximab-alliinase conjugate. Mol Cancer Ther 4:325–331PubMedGoogle Scholar
  11. 11.
    Carlsson J, Drevin H, Axen R (1978) Protein thiolation and reversible protein–protein conjugation. J Biochem 173:723–737CrossRefGoogle Scholar
  12. 12.
    Miron T, Shin I, Feigenblat G et al (2002) A spectrophotometric assay for allicin, alliin, and alliinase (alliin lyase) with a chromogenic thiol: reaction of 4-mercaptopyridine with thiosulfinates. Anal Biochem 307:76–83. doi:10.1016/S0003-2697(02)00010-6 CrossRefPubMedGoogle Scholar
  13. 13.
    Yuen SH, Pollard GA (1954) Determination of nitrogen in agricultural materials by the nessler reagent. II micro-determinations in plant tissue and in soil extracts. J Sci Food Agric 5:364–369CrossRefGoogle Scholar
  14. 14.
    Anthon GE, Barrett DM (2003) Modified method for the determination of pyruvic acid with dinitrophenylhydrazine in the assessment of onion pungency. J Sci Food Agric 83:1210–1213. doi:10.1002/jsfa.1525 CrossRefGoogle Scholar
  15. 15.
    Suman S, Pandey A, Chandna S (2012) An improved non-enzymatic “DNA ladder assay” for more sensitive and early detection of apoptosis. Cytotechnology 64:9–14. doi:10.1007/s10616-011-9395-0 PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Olive PL, Banáth JP (2006) The comet assay: a method to measure DNA damage in individual cells. Nat Protoc 1:23–29. doi:10.1038/nprot.2006.5 CrossRefPubMedGoogle Scholar
  17. 17.
    Lowry OH, Rosebrough NJ, Farr L, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  18. 18.
    Shechter D, Dormann HL, Allis CD, Hake SB (2007) Extraction, purification and analysis of histones. Nat Protoc 2:1445–1457. doi:10.1038/nprot.2007.202 CrossRefPubMedGoogle Scholar
  19. 19.
    Li AP (2007) In vitro evaluation of human xenobiotic toxicity : scientific concepts and the novel integrated discrete multiple cell co-culture (IdMOC) technology. ALTEX 25:43–49Google Scholar
  20. 20.
    Löwe J, Li H, Downing KH, Nogales E (2001) Refined structure of alpha beta-tubulin at 3.5 A resolution. J Mol Biol 313:1045–1057. doi:10.1006/jmbi.2001.5077 CrossRefPubMedGoogle Scholar
  21. 21.
    Gigant B, Wang C, Ravelli RBG et al (2005) Structural basis for the regulation of tubulin by vinblastine. Nat Lett 435:519–522. doi:10.1038/nature03566 CrossRefGoogle Scholar
  22. 22.
    Wu CC, Bratton SB (2013) Regulation of the intrinsic apoptosis pathway by reactive oxygen species. Antioxid Redox Signal 19:546–558. doi:10.1089/ars.2012.4905 PubMedCentralCrossRefPubMedGoogle Scholar
  23. 23.
    Azadi HG, Riazi HG, Ghaffari SM et al (2009) Effects of Allium hirtifolium (Iranian shallot) and its allicin on microtubule and cancer cell lines. Afr J Biotechnol 8:5030–5037Google Scholar
  24. 24.
    Fuchs Y, Steller H (2011) Programmed cell death in animal development and disease. Cell 147:742–758. doi:10.1016/j.cell.2011.10.033 PubMedCentralCrossRefPubMedGoogle Scholar
  25. 25.
    McElroy M, Kaushal S, Luiken GA et al (2008) Imaging of primary and metastatic pancreatic cancer using a fluorophore-conjugated anti-CA19-9 antibody for surgical navigation. World J Surg 32:1057–1066. doi:10.1007/s00268-007-9452-1 PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Kondo T, Yamauchi M, Tominaga S (2000) Evaluation of usefulness of in vitro drug sensitivity testing for adjuvant chemotherapy of stomach cancer. Int J Clin Oncol 5:174–182CrossRefGoogle Scholar
  27. 27.
    Ostling O, Johnason K (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123:291–298CrossRefPubMedGoogle Scholar
  28. 28.
    Barbouti A, Doulias T, Nousis L et al (2002) DNA damage and apoptosis in hydrogen peroxide-exposed jurkat cells: bolus addition versus continuous generation of H2O2. Free Radic Biol Med 33:691–702CrossRefPubMedGoogle Scholar
  29. 29.
    Godard T, Deslandes E, Lebailly P et al (1999) Early detection of staurosporine-induced apoptosis by comet and annexin V assays. Histochem Cell Biol 112:155–161. doi:10.1007/s004180050402 CrossRefPubMedGoogle Scholar
  30. 30.
    Kindzelskii A, Petty H (1999) Ultrasensitive detection of hydrogen peroxide-mediated DNA damage after alkaline single cell gel electrophoresis using occultation microscopy and TUNEL labeling. Mutat Res Mol Mech Mutagen 426:11–22CrossRefGoogle Scholar
  31. 31.
    Gupta J, Bhargava P, Bahadur D (2015) Fluorescent ZnO for imaging and induction of DNA fragmentation and ROS-mediated apoptosis in cancer cells. J Mater Chem B 3:1968–1978. doi:10.1039/C4TB01661K CrossRefGoogle Scholar
  32. 32.
    Pelicano H, Carney D, Huang P (2004) ROS stress in cancer cells and therapeutic implications. Drug Resist Updat 7:97–110. doi:10.1016/j.drup.2004.01.004 CrossRefPubMedGoogle Scholar
  33. 33.
    Zhou Y, Hileman E (2003) Free radical stress in chronic lymphocytic leukemia cells and its role in cellular sensitivity to ROS-generating anticancer agents. Blood 101:4098–4104. doi:10.1182/blood-2002-08-2512.Supported CrossRefPubMedGoogle Scholar
  34. 34.
    Senthil K, Aranganathan S, Nalini N (2004) Evidence of oxidative stress in the circulation of ovarian cancer patients. Clin Chim Acta 339:27–32. doi:10.1016/j.cccn.2003.08.017 CrossRefPubMedGoogle Scholar
  35. 35.
    Schumacker PT (2006) Reactive oxygen species in cancer cells: live by the sword, die by the sword. Cancer Cell 10:175–176. doi:10.1016/j.ccr.2006.08.015 CrossRefPubMedGoogle Scholar
  36. 36.
    Fruehauf JP, Meyskens FL (2007) Reactive oxygen species: a breath of life or death? Clin Cancer Res 13:789–794. doi:10.1158/1078-0432.CCR-06-2082 CrossRefPubMedGoogle Scholar
  37. 37.
    Ho B, Wu Y, Chang K, Pan T (2011) Dimerumic acid inhibits SW620 cell invasion by attenuating H2O2-mediated MMP-7 expression via JNK/C-Jun and ERK/C-Fos activation in an AP-1-dependent manner. Int J Biol Sci 7:869–880PubMedCentralCrossRefPubMedGoogle Scholar
  38. 38.
    Ding H, Han C, Guo D et al (2009) Selective induction of apoptosis of human oral cancer cell lines by avocado extracts via a ROS-mediated mechanism. Nutr Cancer 61:348–356. doi:10.1080/01635580802567158 CrossRefPubMedGoogle Scholar
  39. 39.
    Ryter W, Kim P, Hoetzel A et al (2007) Mechanism of cell death in oxidative stress. Antioxid Redox Signal 9:49–89CrossRefPubMedGoogle Scholar
  40. 40.
    Oommen S, Anto RJ, Srinivas G, Karunagaran D (2004) Allicin (from garlic) induces caspase-mediated apoptosis in cancer cells. Eur J Pharmacol 485:97–103. doi:10.1016/j.ejphar.2003.11.059 CrossRefPubMedGoogle Scholar
  41. 41.
    Park SY, Cho SJ, Kwon HC et al (2005) Caspase-independent cell death by allicin in human epithelial carcinoma cells: involvement of PKA. Cancer Lett 224:123–132. doi:10.1016/j.canlet.2004.10.009 CrossRefPubMedGoogle Scholar
  42. 42.
    Belmokhtar C, Hillion J, Bendirdjian SE (2001) Staurosporine induces apoptosis through both caspase-dependent and caspase-independent mechanisms. Oncogene 20:3354–3362CrossRefPubMedGoogle Scholar
  43. 43.
    Mooney LM, Al-Sakkaf KA, Brown BL, Dobson PRM (2002) Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells. Br J Cancer 87:909–917. doi:10.1038/sj.bjc.6600541 PubMedCentralCrossRefPubMedGoogle Scholar
  44. 44.
    Armstrong J, Steinauer K, Hornung B et al (2002) Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line. Cell Death Differ 9:252–263. doi:10.1038/sj.cdd.4400959 CrossRefPubMedGoogle Scholar
  45. 45.
    Franco R, Cidlowski JA (2009) Apoptosis and glutathione: beyond an antioxidant. Cell Death Differ 16:1303–1314. doi:10.1038/cdd.2009.107 CrossRefPubMedGoogle Scholar
  46. 46.
    Rahman I, Marwick J, Kirkham P (2004) Redox modulation of chromatin remodeling: impact on histone acetylation and deacetylation, NF-κB and pro-inflammatory gene expression. Biochem Pharmacol 68:1255–1267. doi:10.1016/j.bcp.2004.05.042 CrossRefPubMedGoogle Scholar
  47. 47.
    Simboeck E, Sawicka A, Zupkovitz G et al (2010) A phosphorylation switch regulates the transcriptional activation of cell cycle regulator p21 by histone deacetylase inhibitors. J Biol Chem 285:41062–41073. doi:10.1074/jbc.M110.184481 PubMedCentralCrossRefPubMedGoogle Scholar
  48. 48.
    Su B, Xiang L, Su J et al (2012) Diallyl disulfide increases histone acetylation and P21WAF1 expression in human gastric cancer cells in vivo and in vitro. Biochem Pharmacol 1:1–10. doi:10.4172/2167-0501.1000106 CrossRefGoogle Scholar
  49. 49.
    Xu WS, Parmigiani RB, Marks PA (2007) Histone deacetylase inhibitors: molecular mechanisms of action. Oncogene 26:5541–5552. doi:10.1038/sj.onc.1210620 CrossRefPubMedGoogle Scholar
  50. 50.
    Mitani Y, Oue N, Hamai Y (2005) Histone H3 acetylation is associated with reduced p21WAF1/CIP1 expression by gastric carcinoma. J Pathol 205:65–73. doi:10.1002/path.1684 CrossRefPubMedGoogle Scholar
  51. 51.
    Clayton AL, Rose S, Barratt MJ, Mahadevan LC (2000) Phosphoacetylation of histone H3 on c-fos- and c-jun-associated nucleosomes upon gene activation. EMBO J 19:3714–3726PubMedCentralCrossRefPubMedGoogle Scholar
  52. 52.
    Lo WS, Trievel RC, Rojas JR et al (2000) Phosphorylation of serine 10 in histone H3 is functionally linked in vitro and in vivo to Gcn5-mediated acetylation at lysine 14. Mol Cell 5:917–926. doi:10.1016/S1097-2765(00)80257-9 CrossRefPubMedGoogle Scholar
  53. 53.
    Cheung P, Tanner KG, Cheung WL et al (2000) Synergistic coupling of histone H3 phosphorylation and acetylation in response to epidermal growth factor stimulation. Mol Cell 5:905–915CrossRefPubMedGoogle Scholar
  54. 54.
    Varier RA, Timmers HTM (2011) Histone lysine methylation and demethylation pathways in cancer. Biochim Biophys Acta 1815:75–89. doi:10.1016/j.bbcan.2010.10.002 PubMedGoogle Scholar
  55. 55.
    Hung SW, Mody H, Marrache S et al (2013) Pharmacological reversal of histone methylation presensitizes pancreatic cancer cells to nucleoside drugs: in vitro optimization and novel nanoparticle delivery studies. PLoS ONE 8:1–14. doi:10.1371/journal.pone.0071196 Google Scholar
  56. 56.
    Pavletich N (1999) Mechanisms of cyclin-dependent kinase regulation: structures of cdks, their cyclin activators, and cip and INK4 inhibitors 1, 2. J Mol Biol 287:821–828. doi:10.1006/jmbi.1999.2640 CrossRefPubMedGoogle Scholar
  57. 57.
    Molinari M (2000) Cell cycle checkpoints and their inactivation in human cancer. Cell Prolif 33:261–274. doi:10.1046/j.1365-2184.2000.00191.x CrossRefPubMedGoogle Scholar
  58. 58.
    Kuriyama R, Sakai H (1974) Role of tubulin-SH groups in polymerization to microtubules. J Biochem 76:651–654PubMedGoogle Scholar
  59. 59.
    Li M, Ciu J, Ye Y et al (2002) Antitumor activity of Z-ajoene, a natural compound purified from garlic : antimitotic and microtubule-interaction properties. Carcinogenesis 23:573–579CrossRefPubMedGoogle Scholar
  60. 60.
    Xiao D, Pinto JT, Soh J et al (2003) Induction of apoptosis by the garlic-derived compound S-allylmercaptocysteine (SAMC) is associated with microtubule depolymerization and c-Jun NH2-terminal kinase 1 activation. Cancer Res 63:6825–6837PubMedGoogle Scholar
  61. 61.
    Nishida E, Kobayashi T (1977) Relationship between tubulin SH groups and bound guanine nucleotides. J Biochem 81:343–347PubMedGoogle Scholar
  62. 62.
    Rabinkov A, Miron T, Konstantinovski L et al (1998) The mode of action of allicin: trapping of radicals and interaction with thiol containing proteins. Biochim Biophys Acta 1379:233–244. doi:10.1016/S0304-4165(97)00104-9 CrossRefPubMedGoogle Scholar
  63. 63.
    Cotter TG (2009) Apoptosis and cancer: the genesis of a research field. Nat Rev 9:501–507. doi:10.1038/nrc2663 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Sagar V. Chhabria
    • 1
  • Mohammad A. Akbarsha
    • 2
    • 3
  • Albert P. Li
    • 4
  • Prashant S. Kharkar
    • 5
  • Krutika B. Desai
    • 6
  1. 1.Department of Biological Sciences, School of ScienceSVKM’s NMIMS UniversityMumbaiIndia
  2. 2.Mahatma Gandhi-Doerenkamp Center (MGDC) for AlternativesBharathidasan UniversityTiruchirappalliIndia
  3. 3.Department of Food Science and Nutrition, College of Food and AgricultureKing Saud UniversityRiyadhKingdom of Saudi Arabia
  4. 4.In Vitro ADMET Laboratories LLCColumbiaUSA
  5. 5.Department of Pharmaceutical Chemistry, Shobhaben Pratapbhai Patel School of Pharmacy and Technology ManagementSVKM’s NMIMS UniversityMumbaiIndia
  6. 6.Department of MicrobiologySVKM’s Mithibai College of Arts, Chauhan Institute of Science & Amrutben Jivanlal College of Commerce & EconomicsMumbaiIndia

Personalised recommendations