Purpose
Previous studies have shown that the novel microtubule poison, JG-03-14, which binds to the colchicine binding site of tubulin, has the capacity to kill breast tumor cells primarily through ...the promotion of autophagy. The current work was designed to determine whether autophagy was, in fact, the primary mode of action as well as susceptibility to JG-03-14 in two additional tumor cell models, the B16/F10 murine melanoma cell line and the HCT-116 human colon cancer cell line.
Methods
Drug cytotoxicity was monitored based on viable cell number and clonogenic survival. Apoptosis was assessed by DAPI staining, the TUNEL assay and/or FACS analysis. Autophagy was monitored based on staining with acridine orange, redistribution and punctuation of RFP-LC3 and electron microscopy as well as p62 degradation. Senescence was evaluated based on β-galactosidase staining and alterations in cell morphology. Drug effects were also evaluated in a murine model of B16/F10 cells that localizes to the lungs while peripheral neuropathy was assessed by three complementary behavioral assays.
Results
Both HCT-116 colon cancer cells and B16/F10 melanoma cells were sensitive to JG-03-14 in that the drug demonstrated tumor cell killing. However, there was minimal induction of apoptosis. In contrast, there was clear evidence for autophagy and autophagic flux while the residual surviving cells appeared to be in a state of irreversible senescence. Inhibition of drug-induced autophagy in either the melanoma cells or the colon carcinoma cells was only slightly protective as the cells instead died by apoptosis. JG-03-14 reduced the size of tumor nodules in mice lungs; furthermore, the drug did not promote peripheral neuropathy.
Conclusions
Taken together with evidence for its actions as a vascular disrupting agent, these observations support the potential utility of JG-03-14 to effectively treat malignancies that might be resistant to conventional chemotherapy through evasion of apoptosis.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Abstract Ischemia/reperfusion (I/R) unleashes cellular events that threaten organ survival. I/R affects endoplasmic reticulum (ER) integrity and initiates the unfolded protein response (UPR). The ...adaptive arm of the UPR attenuates ER stress by increasing expression of chaperones promoting proper protein folding. However, failure to resolve ER stress leads to apoptotis. We recently showed that prolyl hydroxylase inhibition (PHI) attenuated post-ischemic cardiac injury. We hypothesized that PHI attenuated myocardial I/R injury through modulation of the UPR. We show for the first time that PHI activates all three regulatory arms of the UPR in murine microvascular endothelial cells and in mouse hearts. Cardiac I/R activated expression of pro-apoptotic CHOP (2.8 fold, n = 3, p < 0.01). PHI significantly decreased CHOP expression (50%, n = 3, p < 0.05) in post-ischemic hearts. PHI also induced activating transcription factor 4 (3.5 fold, n = 3, p < 0.001), glucose-regulated protein 78 (6 fold, n = 3, p < 0.001) and ER degradation-enhancing α-mannosidase-like protein (2.8 fold, n = 3, p < 0.001) expression in reperfusing hearts. Thus PHI resulted in significant reduction of apoptosis in post-ischemic myocardium. Our studies suggest that PHI induces protective ER stress proteins and attenuates post-ischemic myocardial damage by decreasing the pro-apoptotic components of the UPR.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Gestational trophoblastic diseases, like the complete hydatidiform mole (CHM), are a group of human interrelated neoplasms whose etiology and progression is poorly understood at the molecular level. ...We have previously reported the cloning and expression of a new tumor necrosis factor receptor (TNF-R) related transcript, named CHMS-1 that encodes a potential death domain. Here we show that ectopic expression of the putative CHMS-1 death domain specifically induced apoptosis in a dose-dependent manner, in trophoblastic (JEG-3) and non-trophoblastic (COS-7) cells. We also investigated the expression of apoptosis-related molecules such as Bcl-2 and p53 and demonstrated that Bcl-2 is repressed in CHM while p53 is overexpressed in CHM compared with persistent gestational trophoblastic tumors. Altogether, these data indicate that the CHMS-1 death domain is able to trigger apoptosis, thus suggesting that this new entity might be an important inducer of molar regression mechanisms in women.
Interactions between the multikinase inhibitor sorafenib and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were examined in malignant hematopoietic cells. Pretreatment (24 h) of ...U937 leukemia cells with 7.5 mu mol/L sorafenib dramatically increased apoptosis induced by sublethal concentrations of TRAIL/Apo2L (75 ng/mL). Similar interactions were observed in Raji, Jurkat, Karpas, K562, U266 cells, primary acute myelogenous leukemia blasts, but not in normal CD34 super(+) bone marrow cells. Sorafenib/TRAIL-induced cell death was accompanied by mitochondrial injury and release of cytochrome c, Smac, and AIF into the cytosol and caspase-9, caspase-3, caspase-7, and caspase-8 activation. Sorafenib pretreatment down-regulated Bcl-xL and abrogated Mcl-1 expression, whereas addition of TRAIL sharply increased Bid activation, conformational change of Bak (ccBak) and Bax (ccBax), and Bax translocation. Ectopic Mcl-1 expression significantly attenuated sorafenib/TRAIL-mediated lethality and dramatically reduced ccBak while minimally affecting levels of ccBax. Similarly, inhibition of the receptor-mediated apoptotic cascade with a caspase-8 dominant-negative mutant significantly blocked sorafenib/TRAIL-induced lethality but not Mcl-1 down-regulation or Bak/Bax conformational change, indicating that TRAIL-mediated receptor pathway activation is required for maximal lethality. Sorafenib/TRAIL did not increase expression of DR4/DR5, or recruitment of procaspase-8 or FADD to the death-inducing signaling complex (DISC), but strikingly increased DISC-associated procaspase-8 activation. Sorafenib also down-regulated cFLIP sub(L), most likely through a translational mechanism, in association with diminished eIF4E phosphorylation, whereas ectopic expression of cFLIP sub(L) significantly reduced sorafenib/TRAIL lethality. Together, these results suggest that in human leukemia cells, sorafenib potentiates TRAIL-induced lethality by down-regulating Mcl-1 and cFLIP sub(L), events that cooperate to engage the intrinsic and extrinsic apoptotic cascades, culminating in pronounced mitochondrial injury and apoptosis. Cancer Res 2007; 67(19):9490-500
Abstract
The ability of HDAC inhibitors (HDACIs) to induce sustained activation of NF-κB (p65/RelA) has been attributed to reversible RelA acetylation (Chen et al., Science 293:1653-7, 2001), a ...phenomenon that diminishes HDACI antineoplastic activity (Dai et al., Mol Cell Biol. 25:5429-44, 2005). However, the process by which HDACIs initially activate NF-κB has yet to be elucidated. To address this question, mechanisms underlying HDAC inhibitor (HDACI)-mediated NF-κB activation were investigated in human myeloid leukemia cells. Exposure of U937 and other leukemia cells to the pan-HDACI LBH-589 induced reactive oxygen species (ROS) and p65/RelA activation, the NF-κB-dependent induction of Mn-SOD2 mRNA and protein accompanied by ROS elimination, and sequential induction of XRCC1 (single strand) followed by γ-H2A. X (double strand) DNA breaks. LBH-589 lethality was significantly attenuated by siRNA knockdown of the chromatin-linked DNA damage protein histone H1.2. U937 IκBα super-repressor cells lacking serine 32 and 36 IκBα phosphorylation sites displayed diminished HDACI-mediated NF-κB activation/Mn-SOD2 induction, accompanied by enhanced ROS accumulation, DNA damage, and apoptosis. In contrast, TRAF2 siRNA knockdown blocked TNFα- but not HDACI-mediated NF-κB activation and lethality. The Mn-SOD2 mimetic TBAP prevented HDACI-induced NF-κB activation and nuclear localization, while dramatically attenuating DNA damage and apoptosis. Notably, LBH-589 exposure activated ATM (on serine 1981) and increased its association with NEMO, events comprising a recently described “inside-out” nuclear NF-κB activation pathway (Wu et al., Science 311:1110-1, 2006). Notably, siRNA NEMO or ATM knockdown blocked HDACI-mediated NF-κB nuclear translocation/activation, diminished MnSOD2 induction, and potentiated oxidative injury, DNA damage and cell death. Similarly, SUMOylation-site mutant NEMO (K277A and/or K309A) cells exposed to LBH-589 displayed diminished association of ATM with NEMO, reduced NEMO and p65/RelA nuclear localization/activation, and MnSOD2 down-regulation. These events were accompanied by increased ROS production, γ-H2A. X formation, and enhanced lethality. Collectively, these findings indicate that in human leukemia cells, HDACIs activate the cytoprotective NF-κB pathway through an ATM/NEMO-dependent process involving the induction of ROS and DNA damage. They also raise the possibility that blocking NF-κB activation via the atypical DNA damage-related ATM/NEMO nuclear pathway has the potential to enhance HDACI antileukemic activity.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3652.
Mechanisms underlying histone deacetylase inhibitor (HDACI)-mediated NF-IordmB activation were investigated in human leukemia cells. Exposure of U937 and other leukemia cells to LBH-589 induced ...reactive oxygen species (ROS) followed by single strand (XRCC1) and double strand (g-H2AX) DNA breaks. Notably, LBH-589 lethality was markedly attenuated by small interfering RNA (siRNA) knockdown of the DNA damage-linked histone, H1.2. LBH-589 triggered p65/RelA activation, NF-IordmB-dependent induction of Mn-SOD2, and ROS elimination. Interference with LBH-589-mediated NF-IordmB activation (e.g. in IIordmBa super-repressor transfected cells) diminished HDACI-mediated Mn-SOD2 induction and increased ROS accumulation, DNA damage, and apoptosis. The Mn-SOD2 mimetic TBAP (manganese(III)-tetrakis 4-benzoic acid porphyrin) prevented HDACI-induced ROS and NF-IordmB activation while dramatically attenuating DNA damage and cell death. In contrast, TRAF2 siRNA knockdown, targeting receptor-mediated NF-IordmB activation, blocked TNFa- but not HDACI-mediated NF-IordmB activation and lethality. Consistent with ROS-mediated DNA damage, LBH-589 exposure activated ATM (on serine 1981) and increased its association with NEMO. Significantly, siRNA NEMO or ATM knockdown blocked HDACI-mediated NF-IordmB activation, resulting in diminished MnSOD2 induction and enhanced oxidative DNA damage and cell death. In accord with the recently described DNA damage/ATM/NEMO pathway, SUMOylation site mutant NEMO (K277A or K309A) cells exposed to LBH-589 displayed diminished ATM/NEMO association, NEMO and p65/RelA nuclear localization/activation, and MnSOD2 up-regulation. These events were accompanied by increased ROS production, g-H2AX formation, and cell death. Together, these findings indicate that in human leukemia cells, HDACIs activate the cytoprotective NF-IordmB pathway through an ATM/NEMO/SUMOylation-dependent process involving the induction of ROS and DNA damage and suggest that blocking NF-IordmB activation via the atypical ATM/NEMO nuclear pathway can enhance HDACI antileukemic activity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The role of ROS production on DNA damage and potentiation of fludarabine (F) lethality by the HDAC inhibitor LAQ-824 was investigated in human leukemia cells. Pre-exposure (24 h) of U937, HL-60, ...Jurkat, or K562 cells to LAQ-824 (40nM) followed by F (0.4µM) dramatically potentiated apoptosis (≥75%). LAQ-824 triggered an early reactive oxygen species (ROS) peak (30′- 3 h), which declined by 6 h, following LAQ-824 induced Mn-SOD2. LAQ-824/F lethality was significantly diminished by either ROS scavengers NAC (N-acetylcysteine) or Mn-TBAP (manganeseIII-tetrakis 4-benzoic acid porphyrin) or ectopic Mn-SOD2 expression and conversely, increased by Mn-SOD2 antisense knockdown. During this interval, LAQ-824 induced early (4-8 h) increases in γ-H2AX which persisted (48 h) secondary to LAQ-824-mediated inhibition of DNA repair (e.g., down-regulation of Ku86 and Rad50, increased Ku70 acetylation, diminished Ku70 and Ku86 DNA binding activity, and downregulated DNA repair genes
BRCA1, CHEK1, and RAD51
). Addition of fludarabine further potentiated DNA damage which was incompatible with cell survival, and triggered multiple pro-apoptotic signals including activation of nuclear caspase 2 and release of histone H1.2 into the cytoplasm. The latter event induced activation of Bak and culminated in pronounced mitochondrial injury and apoptosis. These findings provide a mechanistic basis for understanding the role of early HDACI-induced ROS generation and modulation of DNA repair processes in potentiation of nucleoside analog-mediated DNA damage and lethality in leukemia. Moreover, they demonstrate for the first time the link between HDAC inhibitor-mediated ROS generation and the recently reported DNA damage observed in cells exposed to these agents.
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