Disturbances in lipid second messengers generation by stimulated blood lymphocytes in breast cancer

Aim. The main objective of this study was the comparative investigation of diverse lipid second messenger (LSM) generation by human peripheral blood lymphocytes (HPBL) at different (5, 10, 30 and 60 s) time points of cell co-stimulation by anti-CD3 and anti-CD28 monoclonal antibodies in norm and breast cancer (BC). Methods. Ficoll-Hypaque gradient centrifugation. Results. The data obtained indicate that some mechanisms of LSM generation/utilization in stimulated crude HPBL were significantly altered in BC compared to norm. Particularly, the reliable generation of arachidonyl-1,2-diacylglycerol (1,2-DAG) at the initial step (5 s) of cell stimulation observed in norm was depressed in BC and reached the value below the basal level in unstimulated cells. It is important that the disturbances in 1,2-DAG formation in HPBL obtained from patients with BC were identical with those observed earlier in other forms of cancer. Conclusions. We conclude that the regularities revealed are common characteristics for all the types of malignancy studied and can be used as additional testing parameters for cancer definition and individual correction of the chemotherapy programs for disease treatment.

Introduction.The establishment of many tumors can occur as a consequence of innate and/or adaptive immune systems escape.Among many factors that are involved in tumor immune escape, the regulatory T cells (T reg ) in particular appear to play an important role.It was reported that compared with normal individuals the patients with several types of cancer have an increased prevalence of T reg cells that coexpress CD4, CD25 and Treg marker FOXP3 in the peripheral blood [1,2], tumor microenvironment and tumor draining lymph nodes.The increase in T reg cells suppressing autoimmunity may also inhibit the immune response against cancer, as evidenced by improved tumor rejection and survival of the tumor-bearing mice that have undergone T reg depletion.However, little has been done to explore the possible relations between the T reg prevalence in the peripheral blood and changes in the lymphocyte signaling machineries.Consequently, it was interesting to investigate the regularities of diverse lipid second messenger (LSM) molecules formation at the early (seconds) time points of peripheral blood lymphocytes costimulation by anti-CD3/CD28 in norm and breast cancer (BC).
Isolation and stimulation of HPBL.HPBL were isolated from the heparinized whole blood by standard Ficoll-Hypaque gradient centrifugation described by Innes et al.Preliminary labeling of HPBL by [ 14 C]AA TLC was performed as described earlier [3].Anti-CD3 and anti-CD28 antibodies (2.5 mg) were added to 1 ml of [ 14 C]AA-labeled HPBL suspension (10 8 cells/ml) and incubated for 30 min at 4 o C.After the addition of equal volume of Eagle's medium the excess of unbound anti-bodies was removed by centrifugation (650 g, 15 min).After this, resuspended (10 6 cells/ml) HPBL were stimulated by 10 mg/ml IgG at 37 o C in a final volume of 0.5 ml.Reactions were terminated after 5, 10, 30, or 60 s by adding 2 ml of cold chloroform-methanol (1:2, v/v).The cells incubated in the absence of antibodies were used as negative controls.
Lipid analysis.Lipids were extracted according to Bligh and Dyer standard method and fractionated by TLC as described earlier [3].Lipid fractions were visualized on TLC plates in iodine vapor and identified by comparison with chromatographically pure lipid standards.The distribution of radioactivity in spots was detected by radioscanning (Thin-layer Scanner II LB 2723, Germany).The spots corresponding to different lipid fractions were scrapped into scintillation vials and the amount of radioactivity was estimated using liquid scintillation counter («Roche-Bioelectronique Kontron», France).
Results and discussion.In order to determine the kinetics and nature of LSMs production in activated T cells, we used [ 14 C]AA-prelabeled HPBL, and analyzed the changes in the level of AA-labeled 1,2-diacylglycerol (1,2-DAG), lysophosphatidylcholine (LPC), free AA, monoacylglycerole (MAG) and triacylglycerol (TAG) at the different (5, 10, 30 and 60 s) time points of T cell costimulation by crosslinked anti-CD3/CD28 mAbs.
According to the data obtained at both early (5 s) and relatively sustained (60 s) stages of HPBL stimulation, there are significant differences in the levels of studied LSM molecules in BC compared to norm.It is important that at the initial step (5 s) of cell stimulation the 1,2-DAG level decreased and reached the value below the basal level in unstimulated cells, confirming early depression of phosphoinositide (PI)-cycle as a result of PI-specific phospholipase C inhibition.Notably, the inhibition of 1,2-DAG quick formation was identical with those, observed in ovarian cancer (OC) and three different types of leukemia [3] studied earlier.
At the relatively sustained stage (60 s) following the anti-CD3/CD28 costimulation of HPBL obtained from BC patients, the reliable increase in MAG and TAG levels was observed, correlating with the reduced free AA accumulation.This indicates the possible changes in metabolic pathways realizing fractional interconversions of these lipids.Importantly, the alterations in diverse LSMs generation/utilization dynamic processes in the stimulated crude HPBL, obtained from different patients with BC, display similar regularities but have certain individual characteristics for each patient.The same fact was observed also in OC as well as in two acute forms of leukemia.So, some of disturbances in different LSMs quick formation processes observed in BC are common characteristics for all the types of cancer studied by us.
We conclude that some of disturbances revealed in malignancy can be used as additional testing parameters for cancer definition and individual correction of the chemotherapy programs for disease treatment (Tadevosyan et al., Patents, AM2256, G01N 33/49, 2008; AM 2211, GO1N 33/53, 2009).