T11TS/SLFA-3 Differentially Regulate the Population of Microglia and Brain Infiltrating Lymphocytes to Reduce Glioma by Modulating Intrinsic Bcl-2 Expression rather than p53

Anirban      Ghosh; Joydeep      Mukherjee; Malabika      Bhattacharjee; Pallab      Sarkar; Sagar      Acharya; Swapna      Chaudhuri

Abstract

T11TS/SLFA-3, the glycoprotein isolated from sheep erythrocyte membrane, acts as an antineoplastic agent causing apoptotic elimination of glioma cells through cell mediated immune response. Therefore, elucidation of the proper balance in proliferation and apoptosis of neuroimmune components viz. microglia and brain infiltrating lymphocytes with the neoplastic glial cells was the fundamental issue to establish the efficacy of T11TS as a therapeutic agent in glioma. To decipher its effectivity on proliferation rate of glioma cells, expression of GFAP and cell cycle phase distribution was analyzed with propidium iodide (PI) staining. The apoptotic regulation of interacting immuno-competent microglia, lymphocytes entering into the brain and target glioma cells were elucidated by cytoplasmic DNA fragmentation assay and phosphatidylserine (PS) externalization along with intrinsic p53 and Bcl-2 modulation of the cells. With the reduction of cellular proliferation rate, sharp increase of apoptosis in consecutive doses of T11TS showed the regression of glioma, where an increase of cytosolic p53 and a decrease of Bcl-2 with doses in these neoplastic cells facilitate the process. Resident microglia, the chief immunomodulator of brain, was found to show a low and steady level of proliferation and apoptosis, furnishing it as a stable pool of cells capable of controlling immune reaction in brain compartment. However, microglia showed higher basal level of p53 compared to the other cells in study and being modulated with T11TS dose, it was found to possess a steady level of Bcl-2 that aided to maintain low rate of apoptosis. Brain infiltrating lymphocytes showed increased apoptosis in tumorigenic condition and initial treatment phase mostly due to immuno-suppressive milieu and deprivation of microglial restimulation. But the second dose of T11TS showed reduced apoptosis, enhanced activation of lymphocytes and final dose acting as a regulatory dose, which reduce the infiltrating lymphocytes by apoptotic elimination. Wide fluctuation of cytosolic p53 was observed in these lymphocytes, but anti-apoptotic Bcl-2 was found to modulate apoptosis in the cells. Thus, T11TS was found to differentially regulate the population of immune effector cells against glioma to exert effective effector function in eradication of neoplastic cells where Bcl-2 constitutively suppresses pro-apoptotic function of p53. Regulation of the direction of these balances of cellular life and death in favor of glioma killing and also maintaining homeostasis in brain tissue after reaction established T11TS as an effective therapeutic probe against glioma.

Keywords: T11TS, microglia, brain infiltrating lymphocytes (BIL), glioma, apoptosis, p53, Bcl-2

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