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Abstract
Immunotherapies strengthen the immune system to fight multiple diseases such as infections, immunodeficiencies, and autoimmune diseases, and recently, they are being used as an adjuvant in cancer treatment. IMMUNEPOTENT-CRP (I-CRP) is an immunotherapy made of bovine dialyzable leukocyte extract (bDLE) that has chemoprotective and immunomodulatory effects in different cellular populations of the immune system and antitumor activity in different cancer cell lines. Our recent results suggest that the antineoplastic effect of I-CRP is due to the characteristics of cancer cells. To confirm, we evaluated whether the selectivity is due to cell lineage or characteristics of cancer cells, testing cytotoxicity in T-acute lymphoblastic leukemia cells and their cell death mechanism. Here, we assessed the effect of I-CRP on cell viability and cell death. To determine the mechanism of cell death, we tested cell cycle, mitochondrial and nuclear alterations, and caspases and reactive oxygen species (ROS) and their role in cell death mechanism. Our results show that I-CRP does not affect cell viability in noncancer cells and induces selective cytotoxicity in a dose-dependent manner in leukemic cell lines. I-CRP also induces mitochondrial damage through proapoptotic and antiapoptotic protein modulation (Bax and Bcl-2) and ROS production, nuclear alterations including DNA damage (γ-H2Ax), overexpression of p53, cell cycle arrest, and DNA degradation. I-CRP induced ROS-dependent apoptosis in leukemic cells. Overall, here, we show that I-CRP cytotoxicity is selective to leukemic cells, inducing ROS-dependent apoptosis. This research opens the door to further exploration of their role in the immune system and the cell death mechanism that could potentially work in conjunction with other therapies including hematological malignances.
Highlights
Regulated cell death (RCD) is a mechanism by which the cell activates its own machinery for self-destruction; it involves structured signaling cascades and molecularly defined effector mechanisms and is important for the maintenance of tissues [1,2,3,4]
We tested phosphorylation of H2Ax (c-H2Ax) in Molt-4 cells; we found that the percentage of c-H2Ax increases from 3% to 32% in treated cells (Figure 4(a)), indicating that I-CRP induces DNA damage. en, we analyzed p53 expression and found it increased from 18% to 38% in treated cells (Figure 4(b)). e western blot analysis (Figure 4(c)) confirmed overexpression of p53 in treated cells with I-CRP. ese results suggest that I-CRP has a role in cell cycle arrest and cell death
We assessed loss of mitochondrial membrane potential (LMMP) during reactive oxygen species (ROS) inhibition; in the analysis, we present that N-acetyl cysteine (NAC) avoids LMMP induced by I-CRP (Figure 7(b)). e c-H2AX analysis shows that DNA damage (c-H2AX) diminishes when we block ROS production (Figure 7(c)). e caspase-3 activation analysis showed that the activation of caspase-3 induced by
Summary
Regulated cell death (RCD) is a mechanism by which the cell activates its own machinery for self-destruction; it involves structured signaling cascades and molecularly defined effector mechanisms and is important for the maintenance of tissues [1,2,3,4]. Apoptosis is the most widely described RCD mechanism; it is characterized by cell shrinkage (pyknosis), membrane blebbing, apoptotic body formation, DNA fragmentation (karyorrhexis), and chromatin condensation [5,6,7]. T-cell acute lymphoblastic leukemia (T-ALL) is a type of cancer derived from the bone marrow that affects T-lymphocytes and is the most common cause of cancer in children worldwide [8, 9]. Malignant progression is promoted by the myeloprotection provided by the bone marrow and evasion of the host’s immune system, evidencing the resistance capacity to treatments of the leukemic cells [8, 10]
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