(c) Representative dot plots of cell death analysis and (d) quantification by flow cytometry with annexin-V and propidium iodide (PI) staining in Molt-4 cells after I-CRP and etoposide (ETO) treatments, using Z-DEVD (caspase-3 inhibitor), Z-IETD (caspase-8 inhibitor), Z-LEHD (caspase-9 inhibitor), and QVD (pan-caspase inhibitor). 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 Impurity C of Alfacalcidol 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 ACC-1 cancer cells. To confirm, we evaluated whether the selectivity is Impurity C of Alfacalcidol due to cell lineage or characteristics of cancer cells, testing cytotoxicity in T-acute lymphoblastic leukemia cells and their cell death mechanism. Here, Impurity C of Alfacalcidol 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 Impurity C of Alfacalcidol through proapoptotic and antiapoptotic protein modulation (Bax and Bcl-2) and ROS production, nuclear alterations including DNA damage (assays, it showed an antitumor effect [16, 17]. Several studies reveal its immunomodulatory properties in human and mouse monocytes and macrophages [18, 19] and their cytotoxic effect in different cancer cell lines [20, 21]. In the breast cancer cell line MCF-7, I-CRP inhibits cell growth, suppresses DNA-binding activity of AP-1, decreases c-Jun protein expression, and modulates the mRNA expression of cell death proteins such as NF< 0.005. The data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). The results given in this study represent the mean of at least three independent experiments done in triplicate (mean??SD). 3. Results 3.1. IMMUNEPOTENT-CRP Decreases Selective Cell Viability in Leukemic Cells We assessed whether I-CRP induces selective cytotoxicity in leukemic cells. For this, we analyzed cell viability in the T-acute lymphoblastic leukemia (T-ALL) cell lines Molt-4 and CEM and in the healthy counterpart peripheral blood mononuclear cells (PBMC) and T-lymphocytes (Figure 1). In Figure 1, we show histograms of cell viability analysis in Molt-4 (Figure 1(a)), CEM (Figure 1(b)), PBMC (Figure 1(c)), T-lymphocytes in total PBMC (CD3+) (Figure 1(d)), and in isolated T-lymphocytes (Figure 1(e)) at different concentrations of I-CRP (0.4, 0.6, 0.8, and 1.0?U/mL) at 24 and 48 hours of treatment. In Figure 1(f), we observed that I-CRP decreases cell viability in a time- and concentration-dependent manner in T-ALL cell lines; however, we observed that cell viability of the healthy counterpart was not affected, including T-lymphocytes (CD3+). These results showed that I-CRP decreases selectively the viability in malignant cells only. Open in a separate window Figure 1 Cell viability of T-ALL cell lines and healthy counterpart after Impurity C of Alfacalcidol I-CRP treatment. Representative histograms of cell viability analysis by flow cytometry using calcein-AM staining in (a) Molt-4, (b) CEM, (c) PBMC, (d) CD3+ cells in PBMC, and (e) isolated CD3+ treated with different concentrations (0.4, 0.6, 0.8, and 1.0?U/mL) of I-CRP for 24 and 48 hours. (f) Quantification of cell viability. The results are presented as mean??standard deviation of three different experiments. 3.2. IMMUNEPOTENT-CRP Induces Selective Cell Death in Leukemic Cell Lines To confirm that the loss of cell viability is due to the cytotoxic effect of I-CRP and not due to a metabolic effect, we used a cell death assay analyzing phosphatidylserine (PS) exposure (annexin-V) and membrane permeabilization (propidium iodide, PI) at different concentrations of I-CRP (0.4, 0.6, 0.8, and 1.0?U/mL), after 24 and 48 hours of treatment (Figure 2) in T-ALL cells and the healthy counterpart. As shown in Figures 2(a).