Abstract
PIG7 localizes to lysosomal membrane in leukemia cells. Our previous work has shown that transduction of pig7 into a series of leukemia cell lines did not result in either apoptosis or differentiation of most tested cell lines. Interestingly, it did significantly sensitize these cell lines to chemotherapeutic drugs. Here, we further investigated the mechanism underlying pig7-induced improved sensitivity of acute leukemia cells to chemotherapy. Our results demonstrated that the sensitization effect driven by exogenous pig7 was more effective in drug-resistant leukemia cell lines which had lower endogenous pig7 expression. Overexpression of pig7 did not directly activate the caspase apoptotic pathway, but decreased the lysosomal stability. The expression of pig7 resulted in lysosomal membrane permeabilization (LMP) and lysosomal protease (e.g. cathepsin B, D, L) release. Moreover, we also observed increased reactive oxygen species (ROS) and decreased mitochondrial membrane potential (ΔΨm) induced by pig7. Some autophagy markers such as LC3I/II, ATG5 and Beclin-1, and necroptosis maker MLKL were also stimulated. However, intrinsic antagonism such as serine/cysteine protease inhibitors Spi2A and Cystatin C prevented downstream effectors from triggering leukemia cells, which were only on the “verge of apoptosis”. When combined with chemotherapy, LMP increased and more proteases were released. Once this process was beyond the limit of intrinsic antagonism, it induced programmed cell death cooperatively via caspase-independent and caspase-dependent pathways.
Highlights
Pig7 (P53 induced gene 7) was initially believed to be one of the key genes involved in P53-induced apoptosis, serving as a bridge between P53 expression and apoptosis [1]
In order to explore the mechanism behind this effect, we selected two ordinary leukemia cell lines (K562 and HL60), two chemotherapy resistant cell lines (K562/ADM, HL60/ADM), and primary bone marrow mononuclear cells from five acute myeloid leukemia patients
Among the four cell lines, the IC50 values of VP16 and ADM at 48 h for K562/ADM cells, which had the lowest expression of endogenous pig7, were reduced from 407.3 μg/ml and 4.01 μg/ml for the Plent6.3 group to 79.6 μg/ml and 0.28 μg/ml for the Pig7 groups, respectively
Summary
Pig (P53 induced gene 7) was initially believed to be one of the key genes involved in P53-induced apoptosis, serving as a bridge between P53 expression and apoptosis [1]. It was later found that Pig had two completely different transcripts: LITAF (LPS-Induced TNF Alpha Factor) and SIMPLE (Small Integral Membrane Protein of Lysosome/late Endosome). LITAF acts as a transcriptional promoter in LPS-induced TNF alpha expression [2, 3], and SIMPLE functions as an intrinsic protein in the early endosomal/lysosomal membrane. SIMPLE expression in leukemia cells was found to be lower than in healthy cells When these leukemia cells were treated with chemotherapeutic drugs (e.g. PB, ATRA, and VP16), we found a significant increase (22.5–27.8 times) in endogenous expression of pig during cellular apoptosis. These results indicated www.impactjournals.com/oncotarget that pig may be closely related to cellular apoptosis. When combined with chemotherapies such as using VP16 and daunorubicin, we found that there was a significant enhancement in the drug sensitivity of the tested leukemia cells [6, 7]
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