Apoptosis of RKO induced by catechins and GA through Ca2+ and LIP


The anti-tumor effects of catechins and gallic acid (GA) in-vitro was investigated in this paper. Fluo-3AM, Calcium-AM (Ca-AM), 2′, 7′ -dichlorofluorescein-diacetate(DCFH-DA), 4′, 6-diamidino-2- phenylindole (DAPI) and Ca-AM plus colbat were used to characterize intracellular calcium, labile iron pool (LIP), reactive oxygen species (ROS), nuclei morphology and mitochondrial permeability transition pore (mPTP) opening, respectively. High performance liquid chromatography (HPLC) was used to quantitate catechins and GA in the cultural medium. The results indicated that each of them showed dose response inhibition of cell growth, provoking nuclei condensation, intracellular calcium elevation, mPTP opening, LIP reduction, and cytochrome c (Cyt-C) to release into cytosol. The caspase inhibitors, 2-aminoethoxydiphenol borate (APB) or Fe3+ could inhibit lethal effects of GA and (-)-epigallocatechin (EGC), but failed to affect (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG). Level of ROS presented negative growth while their concentration decreased in the medium. In conclusion, our findings suggest that viability of RKO decreased because of their good correlation with elevation of calcium and loss of LIP and ROS in cytosol.

This is a preview of subscription content, access via your institution.


  1. [1]

    Ahmedin J, Freddie B, Melissa M C, et al. Global Cancer statistics [J]. CA: A Cancer Journal for Clinicians, 2011, 61(2): 69–90.

    Google Scholar 

  2. [2]

    Rebecca S, Elizabeth W, Otis B, et al. Cancer Statistics, 2011: The impact of eliminating socioeconomic and racial disparities on premature cancer deaths [J]. CA: A Cancer Journal for Clinicians, 2011, 61(4): 212–236.

    Google Scholar 

  3. [3]

    Watson A J M, Collins P D. Colon Cancer: A civilization disorder [J]. Digestive Diseases, 2011, 29(2):222–228.

    Article  PubMed  Google Scholar 

  4. [4]

    Cunningham D, Atkin W, Lenz H J, et al. Colorectal cancer [J]. The Lancet, 2010, 375(9719):1030–1047.

    Article  Google Scholar 

  5. [5]

    Ravishankar D, Rajora A K, Greco F, et al. Flavonoids as prospective compounds for anti-cancer therapy [J]. The International Journal of Biochemistry & Cell Biology, 2013, 45(12):2821–2831.

    Article  CAS  Google Scholar 

  6. [6]

    Mascitelli L, Goldstein M R. Inhibition of iron absorption by polyphenols as an anti-cancer mechanism [J]. The Quarterly journal of medicine, 2011, 104(5):459–461.

    Article  CAS  Google Scholar 

  7. [7]

    Lin J K, Liang Y C, Lin-Shiau S Y. Cancer chemoprevention by tea polyphenols through mitotic signal transduction blockade [J]. Biochemical Pharmacology, 1999, 58(6):911–915.

    Article  CAS  PubMed  Google Scholar 

  8. [8]

    Lea M A, Xiao Q, Sadhukhan A K, et al. Inhibitory effects of tea extracts and (-)-epigallocatechin gallate on DNA synthesis and proliferation of hepatoma and erythroleukemia cells [J]. Cancer Letters, 1993, 68(2–3): 231–236.

    Article  CAS  PubMed  Google Scholar 

  9. [9]

    Valcic S, Timmermann B N, Alberts D S, et al. Ihibitory effect of six green tea catechins and caffeine on the growth of four selected human tumor cell lines [J]. Anti-Cancer Drugs, 1996, 7(4):461–468.

    Article  CAS  PubMed  Google Scholar 

  10. [10]

    Yang C S, Wang Z Y. Tea and Cancer [J]. Journal of the National Cancer Institute, 1993, 85(13): 1038–1049.

    Article  CAS  PubMed  Google Scholar 

  11. [11]

    Yuan J M. Cancer prevention by green tea: Evidence from epidemiologic studies [J]. The American Journal of Clinical Nutrition, 2013, 98(6):1676S–1681S.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  12. [12]

    Lambert J D. Does tea prevent cancer? Evidence from laboratory and human intervention studies [J]. The American Journal of Clinical Nutrition, 2013, 98(6):1667S–1675S.

    Article  CAS  PubMed  Google Scholar 

  13. [13]

    Khan N, Mukhtar H. Multitargeted therapy of cancer by green tea polyphenols [J]. Cancer Letters, 2008, 269(2): 269–280.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  14. [14]

    Ramos S. Effects of dietary flavonoids on apoptotic pathways related to cancer chemoprevention [J]. The Journal of Nutritional Biochemistry, 2007, 18(7):427–442.

    Article  CAS  PubMed  Google Scholar 

  15. [15]

    Baek S J, Kim J S, Jackson F R, et al. Epicatechin gallate-induced expression of NAG-1 is associated with growth inhibition and apoptosis in colon cancer cells [J]. Carcinogenesis, 2004, 25(12):2425–2432.

    Article  CAS  PubMed  Google Scholar 

  16. [16]

    Hong J, Smith T J, Ho C T, et al. Effects of purified green and black tea polyphenols on cyclooxygenase and lipoxygenase–dependent metabolism of arachidonic acid in human colon mucosa and colon tumor tissues [J]. Biochemical Pharmacology, 2001, 62(9):1175–1183.

    Article  CAS  PubMed  Google Scholar 

  17. [17]

    Larsen C A, Bisson W H, Dashwood R H. Tea catechins inhibit hepatocyte growth factor receptor (MET kinase) activity in human colon cancer cells: Kinetic and molecular docking studies [J]. Journal of Medicinal Chemistry, 2009, 52(21):6543–6545.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  18. [18]

    Berger N A, Petzold S J. Identification of minimal size requirements of DNA for activation of poly(ADP-ribose) polymerase [J]. Biochemistry, 1985, 24(16):4352–4355.

    Article  CAS  PubMed  Google Scholar 

  19. [19]

    Hou Z, Lambert J D, Chin K V, et al. Effects of tea polyphenols on signal transduction pathways related to cancer chemoprevention [J]. Mutation Research, 2004, 555(1–2):3–19.

    Article  CAS  PubMed  Google Scholar 

  20. [20]

    Baumgartner H K, Gerasimenko J V, Thorne C, et al. Calcium elevation in mitochondria is the main Ca2+ requirement for mitochondrial permeability transition pore (mPTP) opening [J]. The Journal of Biological Chemistry, 2009, 284(31):20796–20803.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  21. [21]

    Xiao J X, Huang G Q, Chi Y S, et al. Soy isoflavones induced apoptosis, cell cycle arrest and [Ca2+]i elevation in hela cells [J]. Acta Nutrimenta Sinica, 2012, 34(4):373–378 (Ch).

    CAS  Google Scholar 

  22. [22]

    Liu X P, Wen X L, Zou S N, et al. Induction of apoptosis by Epigallocatechin-3-gallate via activating mitochondrial signaling in human gastric cancer cells [J]. Journal of Nanhua University (Medical Edition), 2007, 35(4): 499–502 (Ch).

    CAS  Google Scholar 

  23. [23]

    Chitambar C R, Narasimhan J. Targeting iron-dependent DNA synthesis with gallium and transferrin-gallium [J]. Pathobiology, 1991, 59(1):3–10.

    Article  CAS  PubMed  Google Scholar 

  24. [24]

    Muñoz M, Villar I, García-Erce J A. An update on iron physiology [J]. World Journal of Gastroenterology, 2009, 15(37):4617–4626.

    PubMed Central  Article  PubMed  Google Scholar 

  25. [25]

    Yang S F, Xia H Y, Zhou D, et al. Recent progress on mitochondrial iron metabolism and human diseases [J]. Chinese Bulletin of Life Sciences, 2012, 24(8): 742–752 (Ch).

    Google Scholar 

  26. [26]

    Ramiro-Cortés Y, Morán J. Role of oxidative stress and JNK pathway in apoptotic death induced by potassium deprivation and staurosporine in cerebellar granule neurons [J]. Neuro–chemistry International, 2009, 55(7):581–592.

    Article  Google Scholar 

  27. [27]

    Lipinski P, Drapier J C, Oliveira L, et al. Intracellular iron status as a hallmark of mammalian cell susceptibility to oxidative stress: a study of L5178Y mouse lymphoma cell lines differentially sensitive to H2O2 [J]. Blood, 2000, 95(9):2960–2966.

    CAS  PubMed  Google Scholar 

  28. [28]

    Epsztejn S, Kakhlon O, Glickstein H, et al. Fluorescence analysis of the Labile Iron Pool of mammalian cells [J]. Analytical Biochemistry, 1997, 248(1):31–40.

    Article  CAS  PubMed  Google Scholar 

  29. [29]

    Berridge M J. Inositol trisphosphate and calcium signalling mechanisms [J]. Biochimica et Biophysica Acta, 2009, 1793(6):933–940.

    Article  CAS  PubMed  Google Scholar 

  30. [30]

    Berridge M J, Bootman M D, Lipp P. Calcium—A life and death signal [J]. Nature, 1998, 395(6703): 645–648.

    Article  CAS  PubMed  Google Scholar 

  31. [31]

    Gaido M L, Cidlowski J A. Identification, purification, and characterization of a calcium-dependent endonuclease (NUC18) from apoptotic rat thymocytes. NUC18 is not histone H2B [J]. The Journal of Biological Chemistry, 1991, 266(28):18580–18585.

    CAS  PubMed  Google Scholar 

  32. [32]

    Cohen J J, Duke R C. Glucocorticoid activation of a calciumdependent endonuclease in thymocyte nuclei leads to cell death [J]. The Journal of Immunology, 1984, 132(1): 38–42.

    CAS  PubMed  Google Scholar 

  33. [33]

    Zhivotovsky B, Nicotera P, Bellomo G, et al. Ca2+ and Endonuclease Activation in Radiation-Induced Lymphoid Cell Death [J]. Experimental Cell Research, 1993, 207(1): 163–170.

    Article  CAS  PubMed  Google Scholar 

  34. [34]

    Bootman M D, Collins T J, Mackenzie L, et al. 2-aminoethoxydiphenyl borate (2-APB) is a reliable blocker of store-operated Ca2+ entry but an inconsistent inhibitor of InsP3-induced Ca2+ release [J]. The FASEB Journal, 2002, 16(10):1145–1150.

    Article  CAS  PubMed  Google Scholar 

  35. [35]

    Nicholson D W, Ali A, Thornberry N A, et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis [J]. Nature, 1995, 376(6535):37–43.

    Article  CAS  PubMed  Google Scholar 

  36. [36]

    Tewari M, Quan L T, O'Rourke K, et al. Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase [J]. Cell, 1995, 81(5):801–809.

    Article  CAS  PubMed  Google Scholar 

  37. [37]

    Le R Y, Kirkland J B, Shah G M. Cellular responses to DNA damage in the absence of poly(ADP-ribose) polymerase [J]. Biochemical and Biophysical Research Communications, 1998, 245(1):1–10.

    Article  Google Scholar 

  38. [38]

    Nie G, Chen G, Sheftel A D, et al. In vivo tumor growth is inhibited by cytosolic iron deprivation caused by the expression of mitochondrial ferritin [J]. Blood, 2006, 108(7):2428–2434.

    Article  CAS  PubMed  Google Scholar 

  39. [39]

    López-Lázaro M. Dual role of hydrogen peroxide in cancer: possible relevance to cancer chemoprevention and therapy [J]. Cancer Letters, 2007, 252(1):1–8.

    Article  PubMed  Google Scholar 

  40. [40]

    Elizabeth A V, Alison M D, Brain A M. Hydrogen peroxide sensing and Signaling [J]. Molecular Cell, 2007, 26(1): 1–14.

    Article  Google Scholar 

  41. [41]

    Hancock J T, Desikan R, Neill S J. Role of reactive oxygen species in cell signalling pathways [J]. Biochemical Society Transactions, 2001, 29(2):345–350.

    Article  CAS  PubMed  Google Scholar 

  42. [42]

    Mitchell C A. The colorimetric estimation of pyrogallol, gallotannin and gallic acid[J]. Analyst, 1923, 562(48): 2–15.

    Article  Google Scholar 

  43. [43]

    Srichairatanakool S, Kulprachakarn K, Pangjit K, et al. Green tea extract and epigallocatechin 3-gallate reduced Labile Iron Pool and protected oxidative stress in iron-loaded cultured hepatocytes [J]. Advances in Bioscience and Biotechnology, 2012, 3(8): 1140–1150.

    Article  Google Scholar 

Download references

Author information



Corresponding author

Correspondence to Yunfei Tu.

Additional information

Foundation item: Supported by Zhejiang Provincial Natural Science Foundation of China (Y3100683)

Biography: TU Yunfei, male, M.D., research direction: active components of tea and their anti-cancer activity.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Tu, Y. Apoptosis of RKO induced by catechins and GA through Ca2+ and LIP. Wuhan Univ. J. Nat. Sci. 19, 341–349 (2014). https://doi.org/10.1007/s11859-014-1023-3

Download citation

Key words

  • tea polyphenols
  • apoptosis
  • labile iron pool
  • reactive oxygen species

CLC number

  • R73-34
  • R735.3