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Novel quinazolinone MJ-29 triggers endoplasmic reticulum stress and intrinsic apoptosis in murine leukemia WEHI-3 cells and inhibits leukemic mice.

ABSTRACT: The present study was to explore the biological responses of the newly compound, MJ-29 in murine myelomonocytic leukemia WEHI-3 cells in vitro and in vivo fates. We focused on the in vitro effects of MJ-29 on ER stress and mitochondria-dependent apoptotic death in WEHI-3 cells, and to hypothesize that MJ-29 might fully impair the orthotopic leukemic mice. Our results indicated that a concentration-dependent decrease of cell viability was shown in MJ-29-treated cells. DNA content was examined utilizing flow cytometry, whereas apoptotic populations were determined using annexin V/PI, DAPI staining and TUNEL assay. Increasing vital factors of mitochondrial dysfunction by MJ-29 were further investigated. Thus, MJ-29-provaked apoptosis of WEHI-3 cells is mediated through the intrinsic pathway. Importantly, intracellular Ca(2+) release and ER stress-associated signaling also contributed to MJ-29-triggered cell apoptosis. We found that MJ-29 stimulated the protein levels of calpain 1, CHOP and p-eIF2? pathways in WEHI-3 cells. In in vivo experiments, intraperitoneal administration of MJ-29 significantly improved the total survival rate, enhanced body weight and attenuated enlarged spleen and liver tissues in leukemic mice. The infiltration of immature myeloblastic cells into splenic red pulp was reduced in MJ-29-treated leukemic mice. Moreover, MJ-29 increased the differentiations of T and B cells but decreased that of macrophages and monocytes. Additionally, MJ-29-stimulated immune responses might be involved in anti-leukemic activity in vivo. Based on these observations, MJ-29 suppresses WEHI-3 cells in vitro and in vivo, and it is proposed that this potent and selective agent could be a new chemotherapeutic candidate for anti-leukemia in the future.

SUBMITTER: Lu CC

PROVIDER: S-EPMC3360742 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Novel quinazolinone MJ-29 triggers endoplasmic reticulum stress and intrinsic apoptosis in murine leukemia WEHI-3 cells and inhibits leukemic mice.

Lu Chi-Cheng CC Yang Jai-Sing JS Chiang Jo-Hua JH Hour Mann-Jen MJ Lin Kuei-Li KL Lin Jen-Jyh JJ Huang Wen-Wen WW Tsuzuki Minoru M Lee Tsung-Han TH Chung Jing-Gung JG

PloS one 20120525 5

The present study was to explore the biological responses of the newly compound, MJ-29 in murine myelomonocytic leukemia WEHI-3 cells in vitro and in vivo fates. We focused on the in vitro effects of MJ-29 on ER stress and mitochondria-dependent apoptotic death in WEHI-3 cells, and to hypothesize that MJ-29 might fully impair the orthotopic leukemic mice. Our results indicated that a concentration-dependent decrease of cell viability was shown in MJ-29-treated cells. DNA content was examined uti ...[more]

PMID: 22662126

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Project description:BackgroundEndoplasmic reticulum (ER) calcium storage and release play important roles in B lymphocyte maturation, survival, antigen-dependent cell activation and immunoglobulin synthesis. Calcium is accumulated in the endoplasmic reticulum (ER) by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes). Because lymphocyte function is critically dependent on SERCA activity, it is important to understand qualitative and quantitative changes of SERCA protein expression that occur during B lymphoid differentiation and leukemogenesis.MethodsIn this work we investigated the modulation of SERCA expression during the pharmacologically induced differentiation of leukemic precursor B lymphoblast cell lines that carry the E2A-PBX1 fusion oncoprotein. Changes of SERCA levels during differentiation were determined and compared to those of established early B lymphoid differentiation markers. SERCA expression of the cells was compared to that of mature B cell lines as well, and the effect of the direct inhibition of SERCA-dependent calcium transport on the differentiation process was investigated.ResultsWe show that E2A-PBX1+ leukemia cells simultaneously express SERCA2 and SERCA3-type calcium pumps; however, their SERCA3 expression is markedly inferior to that of mature B cells. Activation of protein kinase C enzymes by phorbol ester leads to phenotypic differentiation of the cells, and this is accompanied by the induction of SERCA3 expression. Direct pharmacological inhibition of SERCA-dependent calcium transport during phorbol ester treatment interferes with the differentiation process.ConclusionThese data show that the calcium pump composition of the ER is concurrent with increased SERCA3 expression during the differentiation of precursor B acute lymphoblastic leukemia cells, that a cross-talk exists between SERCA function and the control of differentiation, and that SERCA3 may constitute an interesting new marker for the study of early B cell phenotype.

Project description:Novel quinazolinone compounds have been studied in the field of drug discovery for a long time. Among their broad range of pharmacological effects, certain compounds effectively inhibit cancer cell proliferation. MJ?33 is a quinazolinone derivative with proposed anticancer activities that was synthesized in our laboratory. The present study aimed to evaluate the anticancer activity of MJ?33 in fluorouracil (5FU)?resistant colorectal cancer cells (HT?29/5FUR) and to investigate the underlying molecular mechanisms. The cell viability assay results indicated that HT?29/5FUR cell viability was inhibited by MJ?33 treatment in a concentration?dependent manner compared with the control group. The cellular morphological alterations observed following MJ?33 treatment indicated the occurrence of apoptosis and autophagy, as well as inhibition of cell proliferation in a time?dependent manner compared with the control group. The acridine orange, LysoTracker Red and LC3?green fluorescent protein staining results indicated that MJ?33 treatment significantly induced autophagy compared with the control group. The DAPI/TUNEL dual staining results demonstrated increased nuclear fragmentation and condensation following MJ?33 treatment compared with the control group. The Annexin V apoptosis assay and image cytometry analysis results demonstrated a significant increase in apoptotic cells following MJ?33 treatment compared with the control group. The western blotting results demonstrated markedly decreased Bcl?2, phosphorylated (p)?BAD, pro?caspase?9 and pro?caspase?3 expression levels, and notably increased cytochrome c and apoptotic peptidase activating factor 1 expression levels following MJ?33 treatment compared with the control group. Moreover, the expression levels of autophagy?related proteins, including autophagy related (ATG)?5, ATG?7, ATG?12, ATG?16, p62 and LC3?II, were increased following MJ?33 treatment compared with the control group. Furthermore, MJ?33?treated HT?29/5FUR cells displayed decreased expression levels of p?AKT and p?mTOR compared with control cells. The results suggested that MJ?33?induced apoptosis was mediated by AKT signaling, and subsequently modulated via the mitochondria?dependent signaling pathway. Therefore, the results suggested that suppression of AKT/mTOR activity triggered autophagy in the HT?29/5FUR cell line. In summary, the results indicated that MJ?33 inhibited HT?29/5FUR cell viability, and induced apoptosis and autophagy via the AKT/mTOR signaling pathway. The present study may provide novel insight into the anticancer effects and mechanisms underlying MJ?33 in 5FU?resistant colorectal cancer cells.

Project description:BACKGROUND: Zearalenone (ZEA) is a phytoestrogen from Fusarium species. The aims of the study was to identify mode of human leukemic cell death induced by ZEA and the mechanisms involved. METHODS: Cell cytotoxicity of ZEA on human leukemic HL-60, U937 and peripheral blood mononuclear cells (PBMCs) was performed by using 3-(4,5-dimethyl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Reactive oxygen species production, cell cycle analysis and mitochondrial transmembrane potential reduction was determined by employing 2',7'-dichlorofluorescein diacetate, propidium iodide and 3,3'-dihexyloxacarbocyanine iodide and flow cytometry, respectively. Caspase-3 and -8 activities were detected by using fluorogenic Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC) and Ile-Glu-Thr-Asp-7-amino-4-methylcoumarin (IETD-AMC) substrates, respectively. Protein expression of cytochrome c, Bax, Bcl-2 and Bcl-xL was performed by Western blot. The expression of proteins was assessed by two-dimensional polyacrylamide gel-electrophoresis (PAGE) coupled with LC-MS2 analysis and real-time reverse transcription polymerase chain reaction (RT-PCR) approach. RESULTS: ZEA was cytotoxic to U937 > HL-60 > PBMCs and caused subdiploid peaks and G1 arrest in both cell lines. Apoptosis of human leukemic HL-60 and U937 cell apoptosis induced by ZEA was via an activation of mitochondrial release of cytochrome c through mitochondrial transmembrane potential reduction, activation of caspase-3 and -8, production of reactive oxygen species and induction of endoplasmic reticulum stress. Bax was up regulated in a time-dependent manner and there was down regulation of Bcl-xL expression. Two-dimensional PAGE coupled with LC-MS2 analysis showed that ZEA treatment of HL-60 cells produced differences in the levels of 22 membrane proteins such as apoptosis inducing factor and the ER stress proteins including endoplasmic reticulum protein 29 (ERp29), 78 kDa glucose-regulated protein, heat shock protein 90 and calreticulin, whereas only ERp29 mRNA transcript increased. CONCLUSION: ZEA induced human leukemic cell apoptosis via endoplasmic stress and mitochondrial pathway.

Dataset Information

Novel quinazolinone MJ-29 triggers endoplasmic reticulum stress and intrinsic apoptosis in murine leukemia WEHI-3 cells and inhibits leukemic mice.

Publications

Novel quinazolinone MJ-29 triggers endoplasmic reticulum stress and intrinsic apoptosis in murine leukemia WEHI-3 cells and inhibits leukemic mice.

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