Journal of Molecular Medicine

, Volume 81, Issue 10, pp 664–673 | Cite as

Antioxidant agents are effective in inducing lymphocyte progression through cell cycle in advanced cancer patients: assessment of the most important laboratory indexes of cachexia and oxidative stress

  • Giovanni MantovaniEmail author
  • Antonio Macciò
  • Clelia Madeddu
  • Loredana Mura
  • Giulia Gramignano
  • Maria Rita Lusso
  • Elena Massa
  • Miria Mocci
  • Roberto Serpe
Original Article


This study assessed in a wide population of advanced cancer patients the biological parameters relevant to cancer cachexia, such as serum levels of proinflammatory cytokines (IL-1β, IL-6, TNFα), IL-2, acute-phase proteins (C-reactive protein and fibrinogen), leptin, and relevant to oxidative stress (OS), such as ROS, body antioxidant enzymes GPx and SOD. We also studied the ability of effective antioxidant agents α-lipoic acid (ALA), N-acetyl cysteine (NAC), and amifostine (AMI) added into culture to induce lymphocyte progression through the cell cycle, namely to enter into S phase. Additionally, we assessed the most significant clinical indexes of nutritional status such as body mass index and disease progression such as stage and ECOG-PS in the same cancer patient population. Cell cycle analysis of cultured unstimulated or PHA-stimulated PBMCs isolated from 120 cancer patients and 60 controls, with or without ALA, NAC, or AMI, was studied. The biological parameters relevant to cancer cachexia and OS were also studied. The addition of antioxidants ALA, NAC and AMI, enhanced significantly the progression through the cell cycle, namely from G0/G1 to S phase, of PBMCs isolated from cancer patients (+132%, +150% and +141%, respectively). The percentage of PHA-stimulated PBMCs of cancer patients entering S phase, which was significantly lower than that of controls, increased significantly to more than physiological level after coculture with antioxidants. ROS levels were significantly higher and GPx and SOD activities significantly lower in cancer patients than controls. Serum levels of IL-1 β, IL-6, and TNFα were significantly higher and serum levels of IL-2 and leptin significantly lower in cancer patients than controls. Serum levels of C-reactive protein and fibrinogen were significantly higher in cancer patients than controls. A significant correlation was found in laboratory parameters only between serum levels of leptin and body mass index. Patients with advanced cancer thus exhibit both a high-grade OS and a chronic inflammatory condition. Antioxidant agents ALA, NAC, and AMI enhanced significantly the PBMCs progression through the cell cycle, thus providing evidence of their potential role in the functional restoration of the immune system in advanced cancer patients. Our data warrant further investigation with adequate clinical trials.


Antioxidant agents Cell cycle progression Proinflammatory cytokines Oxidative stress Advanced cancer patients 



α-Lipoic acid




Body mass index


C-reactive protein


Performance status as assessed by the Eastern Cooperative Oncology Group


Glutathione peroxidase




N-Acetyl cysteine


Oxidative stress


Peripheral blood mononuclear cell




Reactive oxygen species


Superoxide dismutase


T cell receptor


Tumor necrosis factor



This research was supported by a grant from Ministry of University, Scientific Research and Technology, Rome, Italy (National Research Project No. 9906041835). We thank Dr. Roberto Serpe for his excellent technical assistance and Ms. Anna Rita Succa for her helpful linguistic assistance.


  1. 1.
    Mantovani G, Macciò A, Lai P, Massa E, Ghiani M, Santona MC (1998) Cytokine activity in cancer-related anorexia/cachexia: role of megestrol acetate and medroxyprogesterone acetate. Semin Oncol 25:45–52Google Scholar
  2. 2.
    Malmberg KJ, Lenkei R, Petersson M, Ohlum T, Ichihara F, Glimelius B, Frodin JE, Masucci G, Kiessling R (2002) A short-term dietary supplementation of high doses of vitamin E increases T helper 1 cytokine production in patients with advanced colorectal cancer. Clin Cancer Res 8:1772–1778PubMedGoogle Scholar
  3. 3.
    Kiessling R, Wasserman K, Horiguchi S, Kono K, Sjoberg J, Pisa P, Petersson M (1999) Tumor-induced immune dysfunction. Cancer Immunol Immunother 48:353–362CrossRefPubMedGoogle Scholar
  4. 4.
    Kiessling R, Kono K, Petersson M, Wasserman K (1996) Immunosuppression in human tumor-host interaction: role of cytokines and alterations in signal-transducting molecules. Springer Semin Immunopathol 8:227–242Google Scholar
  5. 5.
    Young RC, Corder MP, Haynes HA, DeVita VT (1972) Delayed hypersensitivity in Hodgkin's disease: a study of 103 untreated patients. Am J Med 52:63–72PubMedGoogle Scholar
  6. 6.
    Alexander JP, Udoh S, Melsop KA, Hamilton TA, Edinger MG, Tubbs RR, Sica D, Tuason L, Klein E, Bukowski RM, et al (1993) T cells infiltrating renal cell carcinoma display a poor proliferative response even though they can produce interleukin 2 and express interleukin 2 receptors. Cancer Res 53:1380–1387PubMedGoogle Scholar
  7. 7.
    Miescher S, Stoeck M, Qiao L, Barras C, Barrelet L, von Fliedner V (1998) Preferential clonogenic deficit of CD8-positive T lymphocytes infiltrating human solid tumors. Cancer Res 48:6992–6998Google Scholar
  8. 8.
    Mantovani G, Macciò A, Melis G, Mura L, Massa E, Mudu MC (2000) Restoration of functional defects in peripheral blood mononuclear cells isolated from cancer patients by thiol antioxidants alpha-lipoic acid and N-acetyl cysteine. Int J Cancer 86:842–847CrossRefPubMedGoogle Scholar
  9. 9.
    Matsuda M, Peterson M, Lenkei R, Taupin JL, Magnusson I, Mellstedt H, Anderson P, Kiessling R (1995) Alterations in the signal-transducing molecules of T cells and NK cells in colorectal tumor-infiltrating, gut mucosal, and peripheral lymphocytes: correlation with the stage of the disease. Int J Cancer 61:765–772PubMedGoogle Scholar
  10. 10.
    Bukowski RM, Rayman P, Uzzo R, Bloom T, Sandstrom K, Peereboom D, Olencki T, Budd GT, McLain D, Elson P, Novick A, Finke JH (1998) Signal transduction abnormalities in T lymphocytes from patients with advanced renal carcinoma: clinical relevance and effects of cytokine therapy. Clin Cancer Res 4:2337–2347PubMedGoogle Scholar
  11. 11.
    Gunji Y, Hori S, Aoe T, Asano T, Ochiai T, Isono K, Saito T (1994) High frequency of cancer patients with abnormal assembly of the T cell receptor-CD3 complex in peripheral blood T lymphocytes. Jpn J Cancer Res 85:1189–1192PubMedGoogle Scholar
  12. 12.
    Kono K, Ressing ME, Brandt RM, Melief CJ, Potkul RK, Andersson B, Peterson M, Kast WM, Kiessling R (1996) Decreased expression of signal-transducing ζ chain in peripheral T cells and natural killer cells in patients with cervical cancer. Clin Cancer Res 2:1825–1828PubMedGoogle Scholar
  13. 13.
    Uzzo RG, Clark PE, Rayman P, Bloom T, Rybicki L, Novick AC, Bukowski RM, Finke JH (1999) Alterations in NFκB activation in T lymphocytes of patients with renal cell carcinoma. J Natl Cancer Inst 91:718–721CrossRefPubMedGoogle Scholar
  14. 14.
    Ling W, Rayman P, Uzzo R, Clark P, Kim HJ, Tubbs R, Novick A, Bukowski R, Hamilton T, Finke J (1998) Impaired activation of NFκB in T cells from a subset of renal cell carcinoma patients is mediated by inhibition of phosphorylation and degradation of the inhibitor IκBα. Blood 92:1334–1341PubMedGoogle Scholar
  15. 15.
    Kuss I, Saito T, Johnson JT, Whiteside TL (1999) Clinical significance of decreased ζ chain expression in peripheral blood lymphocytes of patients with head and neck cancer. Clin Cancer Res 5:329–334PubMedGoogle Scholar
  16. 16.
    Reichbert TE, Day R, Wagner EM, Whiteside TL (1998) Absent or low expression of the ζ chain in T cells at the tumor site corrrelates with poor survival in patients with oral carcinoma. Cancer Res 58:5344–5347PubMedGoogle Scholar
  17. 17.
    Kono K, Salazar-Onfray F, Petersson M, Hansson J, Masucci G, Wasserman K, Nakazawa T, Anderson P, Kiessling R (1996) Hydrogen peroxide secreted by tumor-derived macrophages down-modulates signal-transducing ζ molecules and inhibits tumor-specific T-cell and natural killer cell-mediated cytotoxicity. Eur J Immunol 26:1308–1313PubMedGoogle Scholar
  18. 18.
    Aoe T, Okamoto Y, Saito T (1995) Activated macrophages induce structural abnormalities of the T cell receptor-CD3 complex. J Exp Med 181:1881–1886PubMedGoogle Scholar
  19. 19.
    Otjuji M, Kimura Y, Aoe T, Okamoto Y, Saito T (1996) Oxidative stress by tumor-derived macrophages suppresses the expression of CD3 ζ chain of T-cell receptor complex and antigen-specific T-cell responses. Proc Natl Acad Sci USA 93:13119–13124CrossRefPubMedGoogle Scholar
  20. 20.
    Bingisser RM, Tilbrook PA, Holt PG, Kees UR (1988) Macrophage-derived nitric oxide regulates T-cell activation via reversible disruption of the Jak3/STAT5 signaling pathway. J Immunol 160:5729–5734Google Scholar
  21. 21.
    Weijl NI, Cleton FJ, Osanto S (1997) Free radicals and antioxidants in chemotherapy-induced toxicity. Cancer Treat Rev 23:209–240PubMedGoogle Scholar
  22. 22.
    Miescher S, Stoeck M, Qiao L, Barras C, Barrelet L, von Fliedner V (1988) Int J Cancer 42:659–666PubMedGoogle Scholar
  23. 23.
    Salmon M, Bacon PA (1988) A cellular deficiency in the rheumatoid one-way mixed lymphocyte reaction. Clin Exp Immunol 71:79–84PubMedGoogle Scholar
  24. 24.
    Clerici M, Stocks NI, Zajac RA, Boswell RN, Lucey DR, Via CS, Shearer GM (1989) Detection of three distinct patterns of T helper cell dysfunction in asymptomatic, human immunodeficiency virus-seropositive patients. Independence of CD4+ cell numbers and clinical staging. J Clin Invest 84:1892–1899PubMedGoogle Scholar
  25. 25.
    Ottenhoff TH (1994) Immunology of Leprosy: lessons from and for leprosy. Int J Lepr Other Mycobact Dis 62:108–121PubMedGoogle Scholar
  26. 26.
    Stefanova I, Saville MW, Peters C, Cleghorn FR, Schwartz D, Venzon DJ, Weinhold KJ, Jack N, Bartholomew C, Blattner WA, Yarchoan R, Bolen JB, Horak ID (1996) HIV infection-induced posttranslational modification of T cell signaling molecules associated with disease progression. J Clin Invest 98:1290–1297PubMedGoogle Scholar
  27. 27.
    Otsuji M, Kimura Y, Aoe T, Okamoto Y, Saito T (1996) Oxidative stress by tumor-derived macrophages suppresses the expression of CD3 ζ chain of T-cell receptor complex and antigen-specific T-cell responses. Proc Natl Acad Sci USA 93:13119–13124CrossRefPubMedGoogle Scholar
  28. 28.
    Maurice MM, Nakamura H, van der Voort EA, van Vliet AI, Staal FJ, Tak PP, Breedveld FC, Verweij CL (1997) Evidence for the role of an altered redox state in hyporesponsiveness of synovial T cells in rheumatoid arthritis. J Immunol 158:1458–1465PubMedGoogle Scholar
  29. 29.
    Schmielau J, Finn OJ (2001) Activated granulocytes and granulocyte-derived hydrogen peroxide are the underlying mechanism of suppression of T-cell function in advanced cancer patients. Cancer Res 61:4756–4760PubMedGoogle Scholar
  30. 30.
    Zoschke DC, Staite ND (1987) Suppression of human lymphocyte proliferation by activated neutrophils or H2O2: surviving cells have an altered T helper/T suppressor ratio and an increased resistance to secondary oxidant exposure. Clin Immunol Immunopathol 42:160–170PubMedGoogle Scholar
  31. 31.
    Finke JH, Zea AH, Stanley J, Longo DL, Mizoguchi H, Tubbs RR, Wiltrout RH, O'Shea JJ, Kudoh S, Klein E (1993) Loss of T-cell receptor ζ chain and p56lck in T-cells infiltrating human renal cell carcinoma. Cancer Res 53:5613–5616PubMedGoogle Scholar
  32. 32.
    Zea AH, Curti BD, Longo DL, Alvord WG, Strobl SL, Mizoguchi H, Creekmore SP, O'Shea JJ, Powers GC, Urba WJ (1995) Alterations in T cell receptor and signal transduction molecules in melanoma patients. Clin Cancer Res 1:1327–1335PubMedGoogle Scholar
  33. 33.
    Mizoguchi H, O'Shea JJ, Longo DL, Loeffler CM, McVicar DW, Ochoa AC (1992) Alterations in signal transduction molecules in T lymphocytes from tumor-bearing mice. Science 258:1795–1798PubMedGoogle Scholar
  34. 34.
    Cemerski S, Cantagrel A, van Meerwijk Joost PM, Romagnoli P (2002) Reactive oxygen species differentially affect T cell receptor-signaling pathways. J Biol Chem 277:19585–19593CrossRefPubMedGoogle Scholar
  35. 35.
    Han D, Handelman G, Marcocci L, Sen CK, Roy S, Kobuchi H, Tritschler HJ, Flohe L, Packer L (1997) Lipoic acid increases de novo synthesis of cellular glutathione by improving cysteine utilization. Biofactors 6:321–338PubMedGoogle Scholar
  36. 36.
    Beher J, Maier K, Degenkolb B, Krombach F, Vogelmeier C (1997) Antioxidative and clinical effects of high-dose N-acetylcysteine in fibrosing alveolitis. Adjunctive therapy to maintenance immunosuppression. Am J Respir Crit Care Med 156:1897–1901Google Scholar
  37. 37.
    Mantovani G, Macciò A, Madeddu C, Mura L, Gramignano G, Lusso MR, Mulas C, Mudu MC, Murgia V, Camboni P, Massa E, Contu P, Rinaldi A, Sanjust E, Atzei D, Elsener B (2002) Quantitative evaluation of oxidative stress, chronic inflammatory indexes and leptin in cancer patients: correlation with stage and performance status. Int J Cancer 98:84–91CrossRefPubMedGoogle Scholar
  38. 38.
    Mantovani G, Macciò A, Madeddu C, Mura L, Massa E, Gramignano G, Lusso MR, Murgia V, Camboni P, Ferreli L, Mocci M (2003) The impact of different antioxidant agents alone or in combination on reactive oxygen species, antioxidant enzymes and cytokines in a series of advanced cancer patients at different sites: correlation with disease progression. Free Radic Res 37:213–233PubMedGoogle Scholar
  39. 39.
    WHO (1995) Report on physical status. The use and interpretation of anthropometry. WHO Technical Report Series no 854. WHO, GenevaGoogle Scholar
  40. 40.
    Mantovani G, Macciò A, Mura L, Massa E, Mudu MC, Mulas C, Lusso MR, Madeddu C, Dessì A (2000) Serum levels of leptin and proinflammatory cytokines in patients with advanced-stage cancer at different sites. J Mol Med 78:554–561PubMedGoogle Scholar
  41. 41.
    Mantovani G, Macciò A, Mura L, Massa E, Mudu MC, Mulas C, Lusso MR, Madeddu C, Gramignano G, Piras MB, Massa D (2001) Serum values of proinflammatory cytokines inversely correlate with serum leptin levels in patients with advanced stage cancer at different sites. J Mol Med 79:406–414PubMedGoogle Scholar
  42. 42.
    Oken MM, Creech RH, Tormey DC, Horton J, Davis TE, McFadden ET, Carbone PP (1982) Toxicity and response criteria of the Eastern Cooperative Oncology Group. J Clin Oncol 5:649–655PubMedGoogle Scholar
  43. 43.
    Mantovani G, Macciò A, Madeddu C, Mura L, Gramignano G, Lusso MR, Mulas C, Murgia V, Camboni P, Massa E, Ferreli L (2003) Correlation between reactive oxygen species, antioxidant mechanisms and disease progression in cancer patients: impact of an antioxidant treatment. J Environ Pathol Toxicol Oncol 22:17–27PubMedGoogle Scholar
  44. 44.
    Capizzi RL (1996) Amifostine: the preclinical basis for broad-spectrum selective cytoprotection of normal tissues from cytotoxic therapies. Semin Oncol 23:2–17Google Scholar
  45. 45.
    Moroni M, Porta C, Finotti N, Borasio E, Greco R, Bertorelli L, Regazzi-Bonora M, Marzatico F (1997) Amifostine (WR-2721, Ethyol) can protect human endothelial but not tumor cells from cisplatin-induced cytotoxicity. An in vitro study. Oncol Rep 4:729–732Google Scholar
  46. 46.
    Wersto RP, Chrest FJ, Leary JF, Morris C, Stetler-Stevenson MA, Gabrielson E (2001) Double discrimination in DNA cell-cycle analysis. Cytometry 46:296–306CrossRefPubMedGoogle Scholar
  47. 47.
    Vindelov Lars L, Christensen Ib J, Keiding N, Spang-Thomsen M, Nissen Nis I (1982) Long-term storage of samples of flow cytometric DNA analysis. Cytometry 3:317–322Google Scholar
  48. 48.
    Alberti A, Bolognini L, Macciantelli D, Caratelli M (2000) The radical cation of N,N-diethyl-para-phenylendiamine: a possible indicator of oxidative stress in biological samples. Res Chem Intermed 26:253–267Google Scholar
  49. 49.
    Cornelli U, Terranova R, Luca S, Cornelli M, Alberti A (2001) Bioavailability and antioxidant activity of some food supplements in men and women using the D-Roms test as a marker of oxidative stress. J Nutr 131:3208–3211PubMedGoogle Scholar
  50. 50.
    Balomenos D, Martinez-AC (2000) Cell-cycle regulation in immunity, tolerance and autoimmunity. Immunol Today 21:551–555CrossRefPubMedGoogle Scholar
  51. 51.
    Lai P, Rabinowich H, Crowley-Nowick PA, Bell MC, Mantovani G, Whiteside TL (1996) Alterations in expression and function of signal transducing proteins in tumor-associated T and NK cells in patients with ovarian carcinoma. Clin Cancer Res 2:161–173PubMedGoogle Scholar
  52. 52.
    Sieg SF, Harding CV, Lederman MM (2001) HIV-1 infection impairs cells cycle progression of CD4+ T cells without affecting early activation responses. J Clin Invest 108:757–764CrossRefPubMedGoogle Scholar
  53. 53.
    Mantovani G, Macciò A, Bianchi A, Curreli L, Ghiani M, Santona MC, Del Giacco GS (1995) Megestrol acetate in neoplastic anorexia/cachexia: clinical evaluation and comparison with cytokine levels in patients with head and neck carcinoma treated with neoadjuvant chemotherapy. Int J Clin Lab Res 25:135–141PubMedGoogle Scholar
  54. 54.
    Mantovani G, Macciò A, Esu S, Lai P, Santona MC, Massa E, Dessì D, Melis GB, Del Giacco GS (1997) Medroxyprogesterone acetate reduces the in vitro production of cytokines and serotonin involved in anorexia/cachexia and emesis by peripheral blood mononuclear cells of cancer patients. Eur J Cancer 33:602–607PubMedGoogle Scholar
  55. 55.
    Weijl NI, Hopman GD, Wipkink-Bakker A, Lentjes EGWM, Berger HM, Cleton FJ, Osanto S (1998) Cisplatin combination chemotherapy induces a fall in plasma antioxidants of cancer patients. Ann Oncol 9:1331–1337Google Scholar
  56. 56.
    Kong Q, Lillehei O (1998) Antioxidant inhibitors for cancer therapy. Med Hypotheses 51:405–409PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • Giovanni Mantovani
    • 1
    Email author
  • Antonio Macciò
    • 1
  • Clelia Madeddu
    • 1
  • Loredana Mura
    • 1
  • Giulia Gramignano
    • 1
  • Maria Rita Lusso
    • 1
  • Elena Massa
    • 1
  • Miria Mocci
    • 1
  • Roberto Serpe
    • 1
  1. 1.Department of Medical Oncology, Policlinico UniversitarioUniversity of CagliariMonserratoItaly

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