Immune modulatory potentials of antineoplaston A-10 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.
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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].
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These data were partially presented at the 10th International Congress on Anti-Cancer Treatment, Paris, France, 2000.