[4] suggested that the capability of internalized CTXs to disrupt lysosome determines their cytotoxicity in human lung cancer A549 cells and leukemia HL60 cells. AMPK. Overexpression of PP2Ac mitigated the CTX3-induced AMPK phosphorylation. CTX3-induced autophagy was via AMPK-mediated suppression of the Akt/mTOR pathway. Removal of Ca2+ or suppression of AMPK phosphorylation inhibited the CTX3-induced cell death. CTX3 was unable to induce autophagy and apoptosis in U937 cells expressing constitutively active Akt. Met-modified CTX3 retained its membrane-perturbing activity, however, it did not induce AMPK activation and death of U937 cells. These results conclusively indicate that CTX3 induces autophagy and apoptosis in U937 cells via the Ca2+/PP2A/AMPK axis, and suggest that the membrane-perturbing activity of CTX3 is not crucial for the cell BRL-15572 death signaling pathway induction. CTX on myoblast cells. AMPK has been demonstrated to be involved in mitochondrial biogenesis [13,14] and lysosomal biogenesis [15]. Some studies have revealed that AMPK acts as a tumor suppressor or oncogene in different malignancy cells [16]. Other studies indicate that AMPK-mediated signaling elicits death in BRL-15572 cancer cells via autophagy and/or apoptosis [17]. Therefore, it is intriguing to explore the role of the AMPK-mediated pathway in the cytotoxicity of CTXs. Prior studies have shown that human myeloid leukemia cells are highly susceptible to the cytotoxicity of CTXs [4,7,18]. Therefore, we investigated whether the AMPK-associated pathway is an important mediator in CTX3-induced death of human leukemia U937 cells. 2. Results Treatment with CTX3 reduced the U937 survival of cells in a concentration- and time-dependent manner with an IC50 value of approximately 150 nM for a 4-h treatment (Physique 1A). Hence, we used this dose of CTX3 to study the mechanism of its cytotoxicity. CTX3 treatment increased the number of annexin V-FITC staining U937 cells (Physique 1B). In line with this, the CTX-treated cells showed degradation of procaspase-3 and PARP (Physique 1C), whereas the caspase inhibitors inhibited the cell death induced by CTX3 (Physique 1D). These results indicated that CTX3 induces apoptosis in U937 cells. Open in a separate window Physique 1 Cobra cardiotoxin (CTX)3 induced apoptotic death of U937 cells. (A) Effect of CTX3 around the viability of U937 cells. Cells were incubated with indicated CTX3 concentrations for 4 h. (Inset) U937 cells were treated BRL-15572 with 150 nM CTX3 for indicated time periods. Cell viability was decided using methlythiazolyldiphenyl-tetrazolium bromide (MTT) assay. Results are expressed as the percentage of cell survival relative to the control. Each value is the mean SD of three impartial experiments with triplicate measurements; (B) Flow cytometry analyses of annexin V-PI double staining CTX3-treated cells. U937 cells were incubated with 150 nM CTX3 for 4 h. Rabbit polyclonal to PAAF1 Around the flow cytometric scatter graphs, the left lower quadrant represents remaining live cells. BRL-15572 The right lower quadrant represents the population of early apoptotic cells. The right upper quadrant represents the accumulation of late apoptotic cells; (C) Western blot analyses of procaspase-3 and poly(ADP-ribose) polymerase (PARP) degradation in CTX3-treated cells. U937 cells were incubated with 150 nM CTX3 for 4 h; (D) Viability of CTX3-treated cells was restored by pretreatment with caspase inhibitors. U937 cells were pretreated with 10 M Z-VAD-FMK (pan-caspase inhibitor) or Z-DEVD-FMK (caspase-3 inhibitor) for 1 h, and then incubated with 150 nM CTX3 for 4 h. Each value is the mean SD of three impartial experiments with triplicate measurements (* < 0.05). To examine whether CTX3-induced apoptosis is related to mitochondrial dysfunction, the mitochondrial membrane potential (m) of CTX3-treated cells was thus measured using TMRM fluorescence. CTX3 caused a marked loss of m in U937 cells, as exhibited by flow cytometry analysis (Physique 2A). Mcl-1, Bcl-2, or Bcl-xL suppression has been shown to induce mitochondrial permeability and m loss [19]. Immunoblotting analyses showed that CTX3 induced downregultion of Mcl-1, Bcl-2, and Bcl-xL expression in U937 cells BRL-15572 (Physique 2B). Moreover, we used 10-N-nonyl acridine orange (NAO), that binds to cardiolipin around the mitochondrial membrane, to measure the mitochondrial mass. Compared to the untreated control.
