Cancer Letters

Cancer Letters

Volume 157, Issue 1, 31 August 2000, Pages 57-63
Cancer Letters

Immune modulatory potentials of antineoplaston A-10 in breast cancer patients

https://doi.org/10.1016/S0304-3835(00)00472-9Get rights and content

Abstract

Antineoplastons are naturally occurring cytodifferentiating agents. Chemically, they are medium and small sized peptides, amino acid derivatives and organic acids, which exist in blood, tissues and urine. Antineoplaston A-10 (3-phenylacetylamino-2,6-piperidinedione) is the first chemically identified antineoplaston. Previously we have shown a strong inverse association of urinary antineoplaston A-10 with breast cancer. This study is designed to evaluate neutrophil apoptosis in patients with breast cancer at time of diagnosis and to correlate urinary antineoplaston A-10 levels with neutrophil apoptosis and to describe the direct effect of A-10 in vitro on neutrophil apoptosis in breast cancer patients. The participants were patients with a histologically confirmed diagnosis of breast cancer. Only those cases without previous treatment for breast cancer were included. Neutrophil apoptosis was assessed in breast cancer patients both morphologically and by DNA fragmentation and studied relative to healthy controls. Antineoplaston A-10 was measured using high performance liquid chromatography in urine samples collected from the patients. Urine samples from normal women served as controls. Direct effect of antineoplaston A-10 on neutrophil apoptosis was tested in vitro after adding A-10 at a concentration of 10 ng/ml to the cellular suspensions of breast cancer patients. Non-treated samples served as controls. Significantly higher neutrophil apoptosis levels were detected among patients with breast cancer with a P value <0.001. Urinary antineoplaston A-10 level is significantly negatively correlated with high apoptosis levels (P<0.0001). In vitro, antineoplaston A-10 was found to inhibit significantly the neutrophil apoptosis with a P value <0.0001. These findings confirm the presence of immune defects among patients with breast cancer and such results should stimulate the development of new strategies to induce and augment immunity for the treatment of breast cancer. Antineoplaston A-10 may provide rational basis for designing trials to employ its immune modulatory potentials as adjuvant therapy in breast cancer patients.

Introduction

Cell death in a multicellular organism can occur by two distinct mechanisms, apoptosis or necrosis [1]. Apoptosis, a form of programmed cell death, is becoming recognized as an important mechanism by which many cell populations and their functions are controlled [2]. Apoptosis has distinct features which include compaction of chromatin against the nuclear membrane, cell shrinkage with preservation of organelles, detachment from surrounding cells and nuclear and cytoplasmic budding to form membrane-bound fragments, known as apoptotic bodies, which are rapidly phagocytosed by adjacent parenchymal cells or macrophages [3]. Apoptosis takes place during emberogensis, in the course of normal tissue turnover and after withdrawal of atrophic hormone from its target tissue. Examples of apoptosis in adult somatic cells include formation of keratinocytes, shedding of the intestinal lining, atrophy of the prostate after castration, regression of lactating breast after weaning and the death of mature neutrophils [4]. Apoptosis is essential in the homeostasis of normal tissues of the body, especially those of the gastrointestinal tract, immune system and skin. It has become clear that regulatory mechanisms controlling programmed cell death are as fundamental and as complex as those regulating cell proliferation. Perturbation of signaling cascade regulating apoptosis can result in a wide variety of human diseases, notably cancers. There is increasing evidence that the processes of neoplastic transformation, progression and metastasis involve alterations in the normal apoptotic pathways. Failure of cells to die in response to premalignant damage allows the progression of the disease and maintains the resistance of cancer cells to cytotoxic therapy [5].

Agents that promote or suppress apoptosis can manipulate the disrupted balanced rates between cell generation and elimination that occur in cancer. Similarly, they can control cell survival and cell death pathways during a productive immune response against cancer. Neutrophils play a pivotal role in tumor immunology [6]. Neutrophil apoptosis seems to be a critical event in cancer development [7]. Therefore, a potential target in cancer treatment is to manipulate this event.

Antineoplastons are naturally occurring cytodifferentiating agents. Chemically, they are medium and small sized peptides, amino acid derivatives and organic acids, which exist in blood, tissues and urine. Antineoplaston A-10 (3-phenylacetylamino-2,6-piperidinedione) is the first chemically identified antineoplaston [8].

This study is designed to evaluate neutrophil apoptosis in patients with breast cancer at time of diagnosis to determine if difference in immunity exists between breast cancer patients and control, to correlate urinary antineoplaston A-10 levels with neutrophil apoptosis and to describe the direct effect of A-10 in vitro on neutrophil apoptosis in breast cancer patients.

Section snippets

Patients and controls

The participants were women between 30 and 67 years of age who had a histologically confirmed diagnosis of breast cancer. Only those cases without previous treatment for breast cancer were included. All of the age-matched controls had no history of cancer or any other breast diseases.

Preparation of blood neutrophils

Neutrophils were isolated from peripheral blood of patients and healthy controls by a combination of dextran sedimentation and centrifugation through discontinuous plasma percol gradients as described by Haslett [9]

Morphological assessment of apoptosis

At time zero and after 72 h, cells were removed from each culture, harvested on slides, fixed in methanol, and stained with:

  • 1.

    May Grunwald Giemsa [10] and examined by oil immersion light microscope. Five hundred cells/ slide were examined. Neutrophils were considered apoptotic if they exhibited the highly characteristic morphological features of chromatin aggregation, condensed and fragmented nuclei, decrease cell size, loss of membrane integrity and cytoplasmic vaculation [11].

  • 2.

    Acridine orange

Agarose gel electrophoresis for DNA fragmentation assay

Assessment of chromatin fragmentation of neutrophils was done by modification of methods previously used for thymocytes [13]. Cells were lysed by adding 0.5 ml of cell suspension to lysis buffer [4.5 ml of Tris–HCl buffer (pH 8.0), 20 mmol of EDTA and 0.2% Triton X-100]. After standing for 1 h, the lysate was centrifuged at 3500 rev./min at 4°C for 20 min to separate high from low molecular weight chromatin. The supernatants were collected into tubes and precipitated with one volume of 5 m

Neutrophil apoptosis levels in breast cancer patients and controls

Apoptosis of neutrophils as assessed both morphologically and by DNA fragmentation of 28 breast cancer patients was studied relative to 28 healthy controls. Readings after 72 h of culture were recorded. Significantly higher neutrophil apoptosis levels were detected among patients with breast cancer with a P value <0.001 (Fig. 1).

Correlation between neutrophil apoptosis and urinary A-10 level in breast cancer patients

The correlation coefficients between urinary antineoplaston levels and neutrophil apoptosis levels in the same 28 breast cancer patients were estimated by Spearman's

Discussion

Neutrophils play a pivotal role in tumor immunology. Several reports indicate that these cells are capable of mediating the lysis of leukemic cells as well as solid tumor cells [6], [16], [17], [18], [19]. The neutrophil response to mediate tumor cell lysis includes the generation of reactive oxygen intermediates [20] and/or the release of lytic molecules prepacked in their granules [21], [22]. The lysis of tumor cells by neutrophils also was shown to be enhanced by various cytokines [23], [24].

References (35)

  • S.R. Bruzynski et al.

    Biologically active peptides in human urine

    Physiol. Chem. Phys.

    (1976)
  • C. Haslett et al.

    Modulation of multiple neutrophil functions by preparation methods

    Am. J. Pathol.

    (1985)
  • C.D. Gregory et al.

    Activation of Epstien–Barr virus latent genes protect human B cells from death by apoptosis

    Nature

    (1991)
  • A.H. Wyllie et al.

    Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis

    J. Pathol.

    (1984)
  • M. Whyte et al.

    Loss of functional responsiveness to surface stimuli in apoptotic neutrophils

    J. Leukocyte Bio

    (1990)
  • D.L. Green et al.

    Effects of cisplatin on the induction of apoptosis in proliferating hepatoma cells and non-proliferating immature thymocytes

    Cancer Res.

    (1993)
  • L. Abou-Zeid

    Antiestrogen and/or antiprogestin structurally related new drugs of chemotherapeutic value

    (1995)
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    These data were partially presented at the 10th International Congress on Anti-Cancer Treatment, Paris, France, 2000.

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