Investigational New Drugs

, Volume 30, Issue 3, pp 1012–1027 | Cite as

A novel synthetic C-1 analogue of 7-deoxypancratistatin induces apoptosis in p53 positive and negative human colorectal cancer cells by targeting the mitochondria: enhancement of activity by tamoxifen

  • Dennis Ma
  • Phillip Tremblay
  • Kevinjeet Mahngar
  • Pardis Akbari-Asl
  • Jonathan Collins
  • Tomas Hudlicky
  • James McNulty
  • Siyaram Pandey


The natural compound pancratistatin (PST), isolated from the Hymenocallis littoralis plant, specifically induces apoptosis in many cancer cell lines. Unlike many other chemotherapeutics, PST is not genotoxic and has minimal adverse effects on non-cancerous cells. However, its availability for preclinical and clinical work is limited due to its low availability in its natural source and difficulties in its chemical synthesis. Several synthetic analogues of 7-deoxypancratistatin with different modifications at C-1 were synthesized and screened for apoptosis inducing activity in human colorectal cancer (CRC) cells. We found that a C-1 acetoxymethyl derivative of 7-deoxypancratistatin, JC-TH-acetate-4 (JCTH-4), was effective in inducing apoptosis in both p53 positive (HCT 116) and p53 negative (HT-29) human CRC cell lines, demonstrating similar efficacy to that of natural PST. JCTH-4 was able to decrease mitochondrial membrane potential (MMP), increase levels of reactive oxygen species in isolated mitochondria, cause release of the apoptogenic factor cytochrome c (Cyto c) from isolated mitochondria, and induce autophagy in HCT 116 and HT-29 cells. Interestingly, when JCTH-4 was administered with tamoxifen (TAM), there was an enhanced effect in apoptosis induction, reactive oxygen species (ROS) production and Cyto c release by isolated mitochondria, and autophagic induction by CRC cells. Minimal toxicity was exhibited by a normal human fetal fibroblast (NFF) and a normal colon fibroblast (CCD-18Co) cell line. Hence, JCTH-4 is a novel compound capable of selectively inducing apoptosis and autophagy in CRC cells alone and in combination with TAM and may serve as a safer and more effective alternative to current cancer therapies.


Colorectal cancer Tamoxifen Combination therapy Apoptosis Autophagy 



adenine nucleotide translocase


creatine kinase


colorectal cancer


cyclophilin D

Cyto c

cytochrome c


estrogen receptor


half-maximal inhibitory concentration








microtubule-associated protein 1 light chain 3




mitochondrial membrane potential


mitochondrial respiratory chain


mitochondrial DNA


normal human fetal fibroblast


peripheral benzodiazepine receptor






permeability transition pore


relative fluorescence units


reactive oxygen species


succinate dehydrogenase subunit A




tetramethylrhodamine methyl ester


voltage-dependent anion channel



This work has been supported by the Knights of Columbus Chapter 9671 (Windsor, Ontario), NSERC, and a CIHR Frederick Banting and Charles Best Canada Graduate Scholarship awarded to Dennis Ma. Thank you to Carly Griffin for providing the pancratistatin results presented in this manuscript. We would also like to thank Colleen Mailloux for the critical review of this manuscript.


  1. 1.
    Parkin DM, Bray F, Ferlay J, Pisani P (2005) Global cancer statistics, 2002. CA Cancer J Clin 55:74–108PubMedCrossRefGoogle Scholar
  2. 2.
    Murphy JE, Ryan DP (2010) American Society of Clinical Oncology 2010 colorectal update. Expert Rev Anticancer Ther 10(9):1371–1373PubMedCrossRefGoogle Scholar
  3. 3.
    de Gramont A, Figer A, Seymour M, Homerin M, Hmissi A, Cassidy J, Boni C, Cortes-Funes H, Cervantes A, Freyer G, Papamichael D, Le Bail N, Louvet C, Hendler D, de Braud F, Wilson C, Morvan F, Bonetti A (2000) Leucovorin and fluorouracil with or without oxaliplatin as first-line treatment in advanced colorectal cancer. J Clin Oncol 18:2938–2947PubMedGoogle Scholar
  4. 4.
    Douillard JY, Cunningham D, Roth AD, Navarro M, James RD, Karasek P, Jandik P, Iveson T, Carmichael J, Alakl M, Gruia G, Awad L, Rougier P (2000) Irinotecan combined with fluorouracil compared with fluorouracil alone as first-line treatment for metastatic colorectal cancer: a multicentre randomised trial. Lancet 355:1041–1047PubMedCrossRefGoogle Scholar
  5. 5.
    Maindrault-Goebel F, Louvet C, André T, Carola E, Lotz JP, Molitor JL, Garcia ML, Gilles-Amar V, Izrael V, Krulik M, de Gramont A (1999) Oxaliplatin added to the simplified bimonthly leucovorin and 5-fluorouracil regimen as second-line therapy for metastatic colorectal cancer (FOLFOX6): GERCOR. Eur J Cancer 35:1338–1342PubMedCrossRefGoogle Scholar
  6. 6.
    André T, Louvet C, Maindrault-Goebel F, Couteau C, Mabro M, Lotz JP, Gilles-Amar V, Krulik M, Carola E, Izrael V, de Gramont A (1999) CPT-11 (irinotecan) addition to bimonthly, high-dose leucovorin and bolus and continuous-infusion 5-fluorouracil (FOLFIRI) for pretreated metastatic colorectal cancer: GERCOR. Eur J Cancer 35:1343–1347PubMedCrossRefGoogle Scholar
  7. 7.
    Borst P, Rottenberg S (2004) Cancer cell death by programmed necrosis? Drug Resist Updat 7(6):321–324 [Epub 2005 Jan 11]PubMedCrossRefGoogle Scholar
  8. 8.
    Earnshaw WC (1999) Apoptosis. A cellular poison cupboard. Nature 397(6718):387–389PubMedCrossRefGoogle Scholar
  9. 9.
    Kekre N, Griffin C, McNulty J, Pandey S (2005) Pancratistatin causes early activation of caspase-3 and the flipping of phosphatidyl serine followed by rapid apoptosis specifically in human lymphoma cells. Cancer Chemother Pharmacol 56(1):29–38PubMedCrossRefGoogle Scholar
  10. 10.
    McLachlan A, Kekre N, McNulty J, Pandey S (2005) Pancratistatin: a natural anti-cancer compound that targets mitochondria specifically in cancer cells to induce apoptosis. Apoptosis 10(3):619–630PubMedCrossRefGoogle Scholar
  11. 11.
    Collins J, Rinner U, Moser M, Hudlicky T, Ghiviriga I, Romero AE, Kornienko A, Ma D, Griffin C, Pandey S (2010) Chemoenzymatic synthesis of Amaryllidaceae constituents and biological evaluation of their C-1 analogues. The next generation synthesis of 7-deoxypancratistatin and trans-dihydrolycoricidine. J Org Chem 75(9):3069–3084PubMedCrossRefGoogle Scholar
  12. 12.
    Baum M (2005) Adjuvant endocrine therapy in postmenopausal women with early breast cancer: where are we now? Eur J Cancer 41:1667–1677PubMedCrossRefGoogle Scholar
  13. 13.
    Mandlekar S, Kong ANT (2001) Mechanisms of Ttamoxifen‑induced apoptosis. Apoptosis 6:469–477PubMedCrossRefGoogle Scholar
  14. 14.
    Moreira P, Custodio J, Morena A, Oliveira C, Santos M (2006) Tamoxifen and estradiol interact with the flavin mononucleotide site of complex I leading to mitochondrial failure. J Biol Chem 281:10143–10152PubMedCrossRefGoogle Scholar
  15. 15.
    Siedlakowski P, McLachlan-Burgess A, Griffin C, Tirumalai SS, McNulty J, Pandey S (2007) Synergy of pancratistatin and tamoxifen on breast cancer cells in inducing apoptosis by targeting mitochondria. Cancer Biol Ther 7(3):376–384PubMedGoogle Scholar
  16. 16.
    Chatterjee SJ, McNulty J, Pandey S (2010) Sensitization of human melanoma cells by tamoxifen to apoptosis induction by pancratistatin, a nongenotoxic natural compound. Melanoma Res 2010 Mar 17. [Epub ahead of print]Google Scholar
  17. 17.
    Bruning A, Friese K, Burges A, Mylonas I (2010) Tamoxifen enhances the cytotoxic effects of nelfinavir in breast cancer cells. Breast Cancer Res 12:R45PubMedCrossRefGoogle Scholar
  18. 18.
    Griffin C, Karnik A, McNulty J, Pandey S (2011) Pancratistatin selectively targets cancer cell mitochondria and reduces growth of human colon tumor xenografts. Mol Cancer Ther 10(1):57–68PubMedCrossRefGoogle Scholar
  19. 19.
    Madesh M, Hajnóczky G (2001) VDAC-dependent permeabilization of the outer mitochondrial membrane by superoxide induces rapid and massive cytochrome c release. J Cell Biol 155(6):1003–1015 [Epub 2001 Dec 10]PubMedCrossRefGoogle Scholar
  20. 20.
    Simon HU, Haj-Yehia A, Levi-Schaffer F (2000) Role of reactive oxygen species (ROS) in apoptosis induction. Apoptosis 5(5):415–418PubMedCrossRefGoogle Scholar
  21. 21.
    Batandier C, Leverve X, Fontaine E (2004) Opening of the mitochondrial permeability transition pore induces reactive oxygen species production at the level of the respiratory chain complex I. J Biol Chem 279(17):17197–17204 [Epub 2004 Feb 11]PubMedCrossRefGoogle Scholar
  22. 22.
    Cochemé HM, Murphy MP (2008) Complex I is the major site of mitochondrial superoxide production by paraquat. J Biol Chem 283(4):1786–1798 [Epub 2007 Nov 26]PubMedCrossRefGoogle Scholar
  23. 23.
    Green DR, Reed JC (1998) Mitochondria and apoptosis. Science 281:1309–1312PubMedCrossRefGoogle Scholar
  24. 24.
    Stennicke HR, Salvesen GS (1999) Catalytic properties of the caspases. Cell Death Differ 6(11):1054–1059PubMedCrossRefGoogle Scholar
  25. 25.
    Kroemer G, Mariño G, Levine B (2010) Autophagy and the integrated stress response. Mol Cell 40(2):280–293PubMedCrossRefGoogle Scholar
  26. 26.
    McNulty J, Nair JJ, Griffin C, Pandey S (2008) Synthesis and biological evaluation of fully functionalized seco-pancratistatin analogues. J Nat Prod 71(3):357–363PubMedCrossRefGoogle Scholar
  27. 27.
    McNulty J, Larichev L, Pandey S (2005) A synthesis of 3-deoxy-dihydrocoricidine: refinement of a structurally minimum Pancratistatin pharmacophore. Bioorg Med Chem Lett 15:5315–5318PubMedCrossRefGoogle Scholar
  28. 28.
    Chen G, Wang F, Trachootham D, Huang P (2010) Preferential killing of cancer cells with mitochondrial dysfunction by natural compounds. Mitochondrion 2010 Aug. 14 [Epub ahead of print]Google Scholar
  29. 29.
    Leber B, Geng F, Kale J, Andrews DW (2010) Drugs targeting Bcl-2 family members as an emerging strategy in cancer. Expert Rev Mol Med 12:e28PubMedCrossRefGoogle Scholar
  30. 30.
    Gueorguieva D, Li S, Walsh N, Mukerji A, Tanha J, Pandey S (2006) Identification of singledomain, Bax-specific intrabodies that confer resistance to mammalian cells against oxidative-stress-induced apoptosis. FASEB J 20(14):2636–2638PubMedCrossRefGoogle Scholar
  31. 31.
    Miyashita T, Reed JC (1995) Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80(2):293–299PubMedCrossRefGoogle Scholar
  32. 32.
    Oda E, Ohki R, Murasawa H, Nemoto J, Shibue T, Yamashita T, Tokino T, Taniguchi T, Tanaka N (2000) Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science 288:1053–1058PubMedCrossRefGoogle Scholar
  33. 33.
    Nakano K, Vousden KH (2001) PUMA, a novel proapoptotic gene, is induced by p53. Mol Cell 7:683–694PubMedCrossRefGoogle Scholar
  34. 34.
    Puthalakath H, Strasser A (2002) Keeping killers on a tight leash: transcriptional and post-translational control of the pro-apoptotic activity of BH3-only proteins. Cell Death Differ 9:505–512PubMedCrossRefGoogle Scholar
  35. 35.
    Fang YJ, Pan ZZ, Li LR, Lu ZH, Zhang LY, Wan DS (2009) MMP7 expression regulated by endocrine therapy in ERbeta-positive colon cancer cells. J Exp Clin Cancer Res 28:132PubMedCrossRefGoogle Scholar
  36. 36.
    Janakiram NB, Steele VE, Rao CV (2009) Estrogen receptor-beta as a potential target for colon cancer prevention: chemoprevention of azoxymethane-induced colon carcinogenesis by raloxifene in F344 rats. Cancer Prev Res Phila 2(1):52–59PubMedCrossRefGoogle Scholar
  37. 37.
    Warburg O (1956) On the origin of cancer cells. Science 123(3191):309–314PubMedCrossRefGoogle Scholar
  38. 38.
    Szatrowski TP, Nathan CF (1991) Production of large amounts of hydrogen peroxide by human tumor cells. Cancer Res 51(3):794–798PubMedGoogle Scholar
  39. 39.
    Carew JS, Zhou Y, Albitar M, Carew JD, Keating MJ, Huang P (2003) Mitochondrial DNA mutations in primary leukemia cells after chemotherapy: clinical significance and therapeutic implications. Leukemia 17(8):1437–1447PubMedCrossRefGoogle Scholar
  40. 40.
    Indo HP, Davidson M, Yen HC, Suenaga S, Tomita K, Nishii T, Higuchi M, Koga Y, Ozawa T, Majima HJ (2007) Evidence of ROS generation by mitochondria in cells with impaired electron transport chain and mitochondrial DNA damage. Mitochondrion 7(1–2):106–118PubMedCrossRefGoogle Scholar
  41. 41.
    Ishikawa K, Takenaga K, Akimoto M, Koshikawa N, Yamaguchi A, Imanishi H, Nakada K, Honma Y, Hayashi J (2008) ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis. Science 320(5876):661–664PubMedCrossRefGoogle Scholar
  42. 42.
    Adam-Vizi V, Chinopoulos C (2006) Bioenergetics and the formation of mitochondrial reactive oxygen species. Trends Pharmacol Sci 27(12):639–645PubMedCrossRefGoogle Scholar
  43. 43.
    Brandon M, Baldi P, Wallace DC (2006) Mitochondrial mutations in cancer. Oncogene 25(34):4647–4662PubMedCrossRefGoogle Scholar
  44. 44.
    Patel BP, Rawal UM, Dave TK, Rawal RM, Shukla SN, Shah PM, Patel PS (2007) Lipid peroxidation, total antioxidant status, and total thiol levels predict overall survival in patients with oral squamous cell carcinoma. Integr Cancer Ther 6(4):365–372PubMedCrossRefGoogle Scholar
  45. 45.
    Kumar B, Koul S, Khandrika L, Meacham RB, Koul HK (2008) Oxidative stress is inherent in prostate cancer cells and is required for aggressive phenotype. Cancer Res 68(6):1777–1785PubMedCrossRefGoogle Scholar
  46. 46.
    Fruehauf JP, Meyskens FL Jr (2007) Reactive oxygen species: a breath of life or death? Clin Cancer Res 13(3):789–794PubMedCrossRefGoogle Scholar
  47. 47.
    Berridge MV, Herst PM, Lawen A (2009) Targeting mitochondrial permeability in cancer drug development. Mol Nutr Food Res 53(1):76–86PubMedCrossRefGoogle Scholar
  48. 48.
    Halestrap AP (2009) What is the mitochondrial permeability transition pore? J Mol Cell Cardiol 46(6):821–831PubMedCrossRefGoogle Scholar
  49. 49.
    Corsi L, Geminiani E, Baraldi M (2008) Peripheral benzodiazepine receptor (PBR) new insight in cell proliferation and cell differentiation review. Curr Clin Pharmacol 3(1):38–45PubMedCrossRefGoogle Scholar
  50. 50.
    Meffert G, Gellerich FN, Margreiter R, Wyss M (2005) Elevated creatine kinase activity in primary hepatocellular carcinoma. BMC Gastroenterol 5:9PubMedCrossRefGoogle Scholar
  51. 51.
    Palmieri D, Fitzgerald D, Shreeve SM, Hua E, Bronder JL, Weil RJ, Davis S, Stark AM, Merino MJ, Kurek R, Mehdorn HM, Davis G, Steinberg SM, Meltzer PS, Aldape K, Steeg PS (2009) Analyses of resected human brain metastases of breast cancer reveal the association between up-regulation of hexokinase 2 and poor prognosis. Mol Cancer Res 7(9):1438–1445PubMedCrossRefGoogle Scholar
  52. 52.
    Kim GJ, Chandrasekaran K, Morgan WF (2006) Mitochondrial dysfunction, persistently elevated levels of reactive oxygen species and radiation-induced genomic instability: a review. Mutagenesis 21(6):361–367PubMedCrossRefGoogle Scholar
  53. 53.
    Pedersen PL (2008) Voltage dependent anion channels (VDACs): a brief introductionwith a focus on the outermitochondrial compartment’s roles togetherwith hexokinase-2 in the “Warburg effect” in cancer. J Bioenerg Biomembr 40(3):123–126PubMedCrossRefGoogle Scholar
  54. 54.
    Chen G, Izzo J, Demizu Y, Wang F, Guha S, Wu X, Hung MC, Ajani JA, Huang P (2009) Different redox states in malignant and nonmalignant esophageal epithelial cells and differential cytotoxic responses to bile acid and honokiol. Antioxid Redox Signal 11(5):1083–1095PubMedCrossRefGoogle Scholar
  55. 55.
    Dalby KN, Tekedereli I, Lopez-Berestein G, Ozpolat B (2010) Targeting the prodeath and prosurvival functions of autophagy as novel therapeutic strategies in cancer. Autophagy 6(3):322–329 [Epub 2010 Apr 26]PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Dennis Ma
    • 1
  • Phillip Tremblay
    • 1
  • Kevinjeet Mahngar
    • 1
  • Pardis Akbari-Asl
    • 1
  • Jonathan Collins
    • 2
  • Tomas Hudlicky
    • 2
  • James McNulty
    • 3
  • Siyaram Pandey
    • 1
  1. 1.Department of Chemistry and BiochemistryUniversity of WindsorWindsorCanada
  2. 2.Chemistry Department and Centre for BiotechnologyBrock UniversitySt. CatharinesCanada
  3. 3.Department of ChemistryMcMaster UniversityHamiltonCanada

Personalised recommendations