STI571 reduces TRAIL-induced apoptosis in colon cancer cells: c-Abl activation by the death receptor leads to stress kinase-dependent cell death

Background

In an effort to achieve better cancer therapies, we elucidated the combination cancer therapy of STI571 (an inhibitor of Bcr-Abl and clinically used for chronic myelogenous leukemia) and TNF-related apoptosis-inducing ligand (TRAIL, a developing antitumor agent) in leukemia, colon, and prostate cancer cells.

Methods

Colon cancer (HCT116, SW480), prostate cancer (PC3, LNCaP) and leukemia (K562) cells were treated with STI571 and TRAIL. Cell viability was determined by MTT assay and sub-G1 appearance. Protein expression and kinase phosphorylation were determined by Western blotting. c-Abl and p73 activities were inhibited by target-specific small interfering (si)RNA. In vitro kinase assay of c-Abl was conducted using CRK as a substrate.

Results

We found that STI571 exerts opposite effects on the antitumor activity of TRAIL. It enhanced cytotoxicity in TRAIL-treated K562 leukemia cells and reduced TRAIL-induced apoptosis in HCT116 and SW480 colon cancer cells, while having no effect on PC3 and LNCaP cells. In colon and prostate cancer cells, TRAIL caused c-Abl cleavage to the active form via a caspase pathway. Interestingly, JNK and p38 MAPK inhibitors effectively blocked TRAIL-induced toxicity in the colon, but not in prostate cancer cells. Next, we found that STI571 could attenuate TRAIL-induced c-Abl, JNK and p38 activation in HCT116 cells. In addition, siRNA targeting knockdown of c-Abl and p73 also reduced TRAIL-induced cytotoxicity, rendering HCT116 cells less responsive to stress kinase activation, and masking the cytoprotective effect of STI571.

Conclusions

All together we demonstrate a novel mediator role of p73 in activating the stress kinases p38 and JNK in the classical apoptotic pathway of TRAIL. TRAIL via caspase-dependent action can sequentially activate c-Abl, p73, and stress kinases, which contribute to apoptosis in colon cancer cells. Through the inhibition of c-Abl-mediated apoptotic p73 signaling, STI571 reduces the antitumor activity of TRAIL in colon cancer cells. Our results raise additional concerns when developing combination cancer therapy with TRAIL and STI571 in the future.     

Exclusion of c-Abl from the nucleus restrains the p73 tumor suppression function

The p73alpha protein is a functional homolog of the p53 tumor suppressor. Although the TP53 gene is frequently mutated in human cancers, the TP73 gene is rarely inactivated. We have found that p73alpha is highly expressed in a significant fraction of anaplastic thyroid cancer, whereas it is not detectable in normal thyroid epithelial cells or in papillary and follicular thyroid cancer cells. Interestingly, the tumor suppression function of p73alpha is actively restrained in anaplastic thyroid cancer cells. We have also found that c-Abl tyrosine kinase, an activator of p73, is excluded from the nucleus of p73alpha-positive thyroid cancer cells; whereas c-Abl undergoes nuclear-cytoplasmic shuttling in normal thyroid and p73-negative thyroid cancer cells. We constructed an AblNuk-FK506-binding protein (FKBP) fusion protein to enforce the nuclear accumulation of an inducible Abl kinase. Activation of this nuclear AblNuk-FKBP by dimerization with AP20187 in anaplastic thyroid cancer cells increased the levels of p73alpha and p21Cip1 and caused p73-dependent apoptosis. These results suggest subcellular segregation of c-Abl from p73 to be a strategy for disrupting the tumor suppression function of p73alpha.     

Evidence for cancer-associated expression of NADPH oxidase 1 (Nox1)-based oxidase system in the human stomach

Helicobacter pylori infection has been suggested to stimulate expression of the NADPH oxidase 1 (Nox1)-based oxidase system in guinea pig gastric epithelium, whereas Nox1 mRNA expression has not yet been documented in the human stomach. PCR of human stomach cDNA libraries showed that Nox1 and Nox organizer 1 (NOXO1) messages were absent from normal stomachs, while they were specifically coexpressed in intestinal- and diffuse-type adenocarcinomas including signet-ring cell carcinoma. Immunohistochemistry showed that Nox1 and NOXO1 proteins were absent from chronic atrophic gastritis (15 cases), adenomas (4 cases), or surrounding tissues of adenocarcinomas (45 cases). In contrast, Nox1 and its partner proteins were expressed in intestinal-type adenocarcinomas (19/21 cases), diffuse-type adenocarcinomas (15/15 cases), and signet-ring cell carcinomas (9/9 cases). Confocal microscopy revealed that Nox1, NOXO1, Nox activator 1, and p22^phox were predominantly associated with Golgi apparatus in these cancer cells, while diffuse-type adenocarcinomas also contained cancer cells having Nox1 and its partner proteins in their nuclei. Nox1-expressing cancer cells exhibited both gastric and intestinal phenotypes, as assessed by expression of mucin core polypeptides. Thus, the Nox1-base oxidase may be a potential marker of neoplastic transformation and play an important role in oxygen radical- and inflammation-dependent carcinogenesis in the human stomach.     

Methylselenol,a selenium metabolite,modulates p53 pathway and inhibits the growth of colon cancer xenografts in Balb/c mice

It is has been hypothesized that methylselenol is a critical selenium metabolite for anticancer activity in vivo. In this study, we used a protein array which contained 112 different antibodies known to be involved in the p53 pathway to investigate the molecular targets of methylselenol in human HCT116 colon cancer cells. The array analysis indicated that methylselenol exposure changed the expression of 11 protein targets related to the regulation of cell cycle and apoptosis. Subsequently, we confirmed these proteins with the Western blotting approach, and found that methylselenol increased the expression of GADD 153 and p21 but reduced the level of c-Myc, E2F1 and Phos p38 MAP kinase. Similar to our previous report on human HCT116 colon cancer cells, methylselenol also inhibited cell growth and led to an increase in G1 and G2 fractions with a concomitant drop in S-phase in mouse colon cancer MC26 cells. When the MC26 cells were transplanted to their immune-competent Balb/c mice, methylselenol-treated MC26 cells had significantly less tumor growth potential than that of untreated MC26 cells. Taken together, our data suggest that methylselenol modulates the expression of key genes related to cell cycle and apoptosis and inhibits colon cancer cell proliferation and tumor growth.     

c-Abl mediates angiotensin II-induced apoptosis in podocytes

Angiotensin II (Ang II) has been reported to cause podocyte apoptosis in rats both in vivo and in vitro studies. However, the underlying mechanisms are poorly understood. In the present study, we investigated the role of the nonreceptor tyrosine kinase c-Abl in Ang II-induced podocyte apoptosis. Male Sprague–Dawley rats in groups of 12 were administered either Ang II (400 kg/kg/min) or Ang II + STI-571 (50 mg/kg/day) by osmotic minipumps. In addition, 12 rats-receiving normal saline served as the control. Glomeruli c-Abl expression was carried out by real time PCR, Western blotting and immunolabeled, and occurrence of apoptosis was carried out by TUNEL staining and transmission electron microscopic analysis. In vitro studies, conditionally immortalized mouse podocytes were treated with Ang II (10^?9–10^?6 M) in the presence or absence of either c-Abl inhibitor, Src-I1, specific c-Abl siRNA, or c-Abl plasmid alone. Quantification of podocyte c-Abl expression and c-Abl phosphorylation at Y245 and Y412 was carried out by real time PCR, Western blotting and immunofluorescence imaging. The nuclear c-Abl and p53 were quantified by co-immunoprecipitation and Western blotting studies. Podocyte apoptosis was analysed by flow cytometry and Hoechst-33342 staining. c-Abl expression was demonstrated in rat kidney podocytes in vivo and cultured mouse podocytes in vitro. Ang II-receiving rats displayed enhanced podocyte c-Abl expression. And Ang II significantly stimulated c-Abl expression in cultured podocytes. Furthermore Ang II upregulated podocyte c-Abl phosphorylation at Y245 and Y412. Ang II also induced an increase of nuclear p53 protein and nuclear c-Abl-p53 complexes in podocytes and podocyte apoptosis. Down-regulation of c-Abl expression by c-Abl inhibitor (Src-I1) as well as specific siRNA inhibited Ang II-induced podocyte apoptosis; conversely, podoctyes transfected with c-Abl plasmid displayed enhanced apoptosis. These findings indicate that c-Abl may mediates Ang II-induced podocyte apoptosis, and inhibition of c-Abl expression can protect podocytes from Ang II-induced injury.     

c-Abl Is an Upstream Regulator of Acid Sphingomyelinase in Apoptosis Induced by Inhibition of Integrins αvβ3 and αvβ5

Inhibition of integrins αvβ3/αvβ5 by the cyclic function-blocking peptide, RGDfV (Arg-Gly-Asp-Phe-Val) can induce apoptosis in both normal cells and tumor cells. We show that RGDfV induced apoptosis in ECV-304 carcinoma cells, increased activity and mRNA expression of acid sphingomyelinase (ASM), and increased ceramides C(16), C(18∶0), C(24∶0) and C(24∶1) while decreasing the corresponding sphingomyelins. siRNA to ASM decreased RGDfV-induced apoptosis as measured by TUNEL, PARP cleavage, mitochondrial depolarization, and caspase-3 and caspase-8 activities, as well as by annexinV in a 3D collagen model. These findings indicate a causal role for ASM in RGDfV-induced apoptosis in ECV-304. We have shown that c-Abl, a non-receptor tyrosine kinase, also mediates RGDfV-induced apoptosis. However, c-Abl, has not been previously linked to ASM in any system. Here we show that STI-571 (imatinib, inhibitor of c-Abl) inhibited RGDfV-induced ASM activity. Furthermore, STI-571 and c-Abl-siRNA both inhibited RGDfV-induced increase in ASM mRNA, but ASM-siRNA did not affect c-Abl phosphorylation or expression, supporting that c-Abl regulates the RGDfV-induced increase in ASM expression. These studies implicate ASM as a mediator of apoptosis induced by inhibition of integrins αvβ3/αvβ5, and for the first time place c-Abl as an upstream regulator of ASM expression and activity.     

Role of c-Abl kinase in DNA mismatch repair-dependent G2 cell cycle checkpoint arrest responses

Current published data suggest that DNA mismatch repair (MMR) triggers prolonged G(2) cell cycle checkpoint arrest after alkylation damage from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by activating ATR (ataxia telangiectasia-Rad3-related kinase). However, analyses of isogenic MMR-proficient and MMR-deficient human RKO colon cancer cells revealed that although ATR/Chk1 signaling controlled G(2) arrest in MMR-deficient cells, ATR/Chk1 activation was not involved in MMR-dependent G(2) arrest. Instead, we discovered that disrupting c-Abl activity using STI571 (Gleevec, a c-Abl inhibitor) or stable c-Abl knockdown abolished MMR-dependent p73alpha stabilization, induction of GADD45alpha protein expression, and G(2) arrest. In addition, inhibition of c-Abl also increased the survival of MNNG-exposed MMR-proficient cells to a level comparable with MMR-deficient cells. Furthermore, knocking down GADD45alpha (but not p73alpha) protein levels affected MMR-dependent G(2) arrest responses. Thus, MMR-dependent G(2) arrest responses triggered by MNNG are dependent on a human MLH1/c-Abl/GADD45alpha signaling pathway and activity. Furthermore, our data suggest that caution should be taken with therapies targeting c-Abl kinase because increased survival of mutator phenotypes may be an unwanted consequence.     

Heparin regulates colon cancer cell growth through p38 mitogen-activated protein kinase signalling

Objectives:  Heparin acts as an extracellular stimulus capable of activating major cell signalling pathways. Thus, we examined the putative mechanisms utilized by heparin to stimulate HT29, SW1116 and HCT116 colon cancer cell growth.
Materials and methods:  Possible participation of the mitogen-activated protein kinase (MAPK) cascade on heparin-induced HT29, SW1116 and HCT116 colon cancer cell growth was evaluated using specific MAPK cascade inhibitors, Western blot analysis, real-time quantitative PCR and FACS apoptosis analysis.
Results:  Treatment with a highly specific p38 kinase inhibitor, SB203580, significantly (50–70%) inhibited heparin-induced colon cancer cell growth, demonstrating that p38 MAPK signalling is involved in their heparin-induced proliferative response. This was shown to be correlated with increased (up to 3-fold) phosphorylation of 181/182 threonine/tyrosine residues on p38 MAP kinase. Furthermore, heparin inhibited cyclin-dependent kinase inhibitor p21 ^{WAF1 / CIP1} and p53 tumour suppressor gene and protein expression up to 2-fold or 1.8-fold, respectively, and stimulated cyclin D1 expression up to 1.8-fold, in these cell lines through a p38-mediated mechanism. On the other hand, treatment with heparin did not appear to affect HT29, SW1116 and HCT116 cell levels of apoptosis.
Conclusions:  This study demonstrates that an extracellular glycosaminoglycan, heparin, finely modulates expression of genes crucial to cell cycle regulation through specific activation of p38 MAP kinase to stimulate colon cancer cell growth.     

p47phox participates in activation of RelA in endothelial cells

Activation of endothelial cell NF-kappaB by interleukin (IL)-1 constitutes an event critical to the progression of the innate immune response. In this context, oxidants have been associated with NF-kappaB activation, although the molecular source and mechanism of targeting have remained obscure. We found that RelA, essential for NF-kappaB activation by IL-1, was associated with the NADPH oxidase adapter protein p47(phox) in yeast two-hybrid, coprecipitation, and in vitro binding studies. RelA and p47-GFP also colocalized in endothelial cells in focal submembranous dorsoventral protrusions. Overexpression of p47(phox) synergized with IL-1beta in the activation of an artificial kappaB-luciferase reporter and specifically augmented IL-1beta-induced RelA transactivation activity. p47(phox) overexpression also greatly increased IL-1beta-stimulated RelA phosphorylation, whereas it had no effect on I-kappaB degradation or on RelA nuclear translocation or kappaB binding. The tandem SH3 domains of p47(phox) were found to associate with a proline-rich mid-region of RelA (RelA-PR) located between the Rel homology and transactivation domains. The RelA-PR peptide blocked interaction of p47(phox) and RelA, and ectopic expression of RelA-PR abrogated IL-1beta-induced transactivation of the NF-kappaB-dependent E-selectin promoter. Further, suppression of NADPH oxidase function through the inhibitor diphenylene iodonium, the superoxide dismutase mimetic Mn(III) tetrakis(4-benzoic acid)porphyrin (MnTBAP), or expression of a dominant interfering mutant of a separate NADPH oxidase subunit (p67(V204A)) decreased IL-1beta-induced E-selectin promoter activation, suggesting that p47(phox) facilitates NF-kappaB activation through linkage with the NADPH oxidase. IL-1beta rapidly increased tyrosine phosphorylation of IL-1 type I receptor-associated proteins, suggesting that oxidants may operate through inactivation of local protein-tyrosine phosphatases in the proximal IL-1beta signaling pathway leading to RelA activation.     

Abelson interactor protein-1 positively regulates breast cancer cell proliferation, migration, and invasion

Abelson interactor protein-1 (ABI-1) is an adaptor protein involved in actin reorganization and lamellipodia formation. It forms a macromolecular complex containing Hspc300/WASP family verprolin-homologous proteins 2/ABI-1/nucleosome assembly protein 1/PIR121 or Abl/ABI-1/WASP family verprolin-homologous proteins 2 in response to Rho family-dependent stimuli. Due to its role in cell mobility, we hypothesized that ABI-1 has a role in invasion and metastasis. In the present study, we found that weakly invasive breast cancer cell lines (MCF-7, T47D, MDA-MB-468, SKBR3, and CAMA1) express lower levels of ABI-1 compared with highly invasive breast cancer cell lines (MDA-MB-231, MDA-MB-157, BT549, and Hs578T), which exhibit high ABI-1 levels. Using RNA interference, ABI-1 was stably down-regulated in MDA-MB-231, which resulted in decreased cell proliferation and anchorage-dependent colony formation and abrogation of lamellipodia formation on fibronectin. Down-regulation of ABI-1 decreased invasiveness and migration ability and decreased adhesion on collagen IV and actin polymerization in MDA-MB-231 cells. Additionally, compared with control parental cells, ABI-1 small interfering RNA-transfected cells showed decreased levels of phospho-PDK1, phospho-Raf, phospho-AKT, total AKT, and AKT1. These data suggest that ABI-1 plays an important role in the spread of breast cancer and that this role may be mediated via the phosphatidylinositol 3-kinase pathway.     

乳腺癌中Abi1和c-Abl及WAVE 2的表达及其相互关系

目的研究乳腺癌组织中Abi1、c-Abl和WAVE2蛋白的表达及其相互关系。方法应用免疫组化SP法检测66例乳腺癌组织和24例正常乳腺组织中Abi1、c-Abl和WAVE2蛋白的表达情况。结果 1.正常乳腺组织与乳腺癌组织相比,Abi1和WAVE2蛋白表达有显著性差异(P0.05),而c-Abl蛋白表达无显著性差异(P0.05),但有定位的改变。2.Abi1强阳性率与乳腺癌的肿瘤大小、组织学分级、淋巴结转移及临床分期呈负相关性(P0.05);与患者年龄无关(P0.05)。c-Abl阳性率及WAVE2强阳性率均与乳腺癌的组织学分级、淋巴结转移及临床分期呈负相关性(P0.05);与患者年龄及肿瘤大小无关(P0.05)。3.Abi1蛋白表达与c-Abl和WAVE2蛋白表达呈正相关(P0.05)。结论乳腺癌中Abi1低表达与预后不良有关。乳腺癌中Abi1蛋白表达的变化可影响c-Abl蛋白的定位和WAVE2蛋白的表达。推测Abi1在Abl/Abi1/WAVE2通路中具有至关重要的地位。     

Protein kinase PKC delta and c-Abl are required for mitochondrial apoptosis induction by genotoxic stress in the absence of p53, p73 and Fas receptor

Doxorubicin, cis-diamminedichloroplatinum (II) and 5-fluorouracil used in chemotherapy induce apoptosis in Hep3B cells in the absence of p53, p73, and functional Fas. Since mediators remain unknown, the requirement of PKC delta (PKCdelta) and c-Abl was investigated. Suppression of c-Abl or PKCdelta expression using SiRNAs impaired PARP cleavage, Gleevec and/or rottlerin inhibited the induction of the subG1 phase and the increase of reactive oxygen species level. Co-precipitations and phosphorylations to mitochondria of c-Abl, PKCdelta and Bcl-X(L/s) were induced. A depolarization of the mitochondrial membrane and activations of caspase-2 and -9 were observed. We propose that, in the absence of p53, p73 and Fas, genotoxic drugs could require both PKCdelta and c-Abl to induce apoptosis through the mitochondrial pathway.     

A positive role for c-Abl in Atm and Atr activation in DNA damage response

DNA damage triggers Atm- and/or Atr-dependent signaling pathways to control cell cycle progression, apoptosis, and DNA repair. However, how Atm and Atr are activated is not fully understood. One of the downstream targets of Atm is non-receptor tyrosine kinase c-Abl, which is phosphorylated and activated by Atm. The current view is that c-Abl relays pro-apoptotic signals from Atm to p73 and p53. Here we show that c-Abl deficiency resulted in a broad spectrum of defects in cell response to genotoxic stress, including activation of Chk1 and Chk2, activation of p53, nuclear foci formation, apoptosis, and DNA repair, suggesting that c-Abl might also act upstream of the DNA damage-activated signaling cascades in addition to its role in p73 and p53 regulation. Indeed, we found that c-Abl is required for proper activation of both Atm and Atr. c-Abl is bound to the chromatin and shows enhanced interaction with Atm and Atr in response to DNA damage. c-Abl can phosphorylate Atr on Y291 and Y310 and this phosphorylation appears to have a positive role in Atr activation under genotoxic stress. These findings suggest that Atm-mediated c-Abl activation in cell response to double-stranded DNA breaks might facilitate the activation of both Atm and Atr to regulate their downstream cellular events.     

Association of the ERK1/2 and p38 kinase pathways with nitric oxide-induced apoptosis and cell cycle arrest in colon cancer cells

To investigate the mechanism by which nitric oxide (NO) induces cell death in colon cancer cells, we compared two types of colon cancer cells with different p53 status: HCT116 (p53 wild-type) cells and SW620 (p53-deficient) cells. We found that S-nitrosoglutathione (GSNO), the NO donor, induced apoptosis in both types of colon cancer cells. However, SW620 cells were much more susceptible than HCT116 cells to apoptotic death by NO. We investigated the role of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 kinase on NO-induced apoptosis in both types of colon cancer cells. GSNO treatment effectively stimulated activation of the ERK1/2 and p38 kinase in both types of cells. In HCT116 cells, pretreatment with PD98059, an inhibitor of ERK1/2, or SB203580, an inhibitor of p38 kinase, had no marked effect on GSNO-induced apoptosis. However, in SW620 cells, SB203580 significantly reduced the NO-induced apoptosis, whereas PD098059 increases NO-induced apoptosis. Furthermore, we found evidence of cell cycle arrest of the G₀/G₁ phase in SW620 cells but not in HCT116 cells. Inhibition of ERK1/2 with PD098059, or of p38 kinase with SB203580, reduced the GSNO-induced cell cycle arrest of the G₀/G₁ phase in SW620 cells. We therefore conclude that NO-induced apoptosis in colon cancer cells is mediated by a p53-independent mechanism and that the pathways of ERK1/2 and p38 kinase are important in NO-induced apoptosis and in the cell cycle arrest of the G₀/G₁ phase.     

曲古抑菌素A对结肠癌细胞株SW480细胞周期影响的机制研究

为了研究组蛋白去乙酰化酶(HDACs)抑制剂曲古抑菌素A(TSA)对结肠癌细胞周期和凋亡的影响,初步探讨TSA作用细胞周期的可能机制,将人结肠癌细胞系SW480经TSA处理后,运用流式细胞术检测细胞周期、凋亡以及细胞周期素的变化,最后采用western-blot对细胞周期相关的基因进行检测.结果表明,TSA处理细胞后,TSA能够延缓细胞周期G1-S进程,阻滞细胞于G1期,并且影响细胞周期素cyclinE、cyclinA聚集,而对凋亡无明显的影响.Western-blot显示,TSA能够上调p21Waf1/Cip1、p27Kip1的表达,下调CDK2、cyclinE以及cycli-nA的表达.以上结果说明在结肠癌细胞中,TSA能够通过上调p21Waf1/Cip1、p27Kip1的表达以及下调CDK2、cy-clinE、cyclinA的表达,从而阻滞细胞周期于G1期,最终影响肿瘤细胞的生长,以上研究为HDAC抑制剂应用于结肠癌治疗提供了理论依据.     

c-Abl is involved in high glucose-induced apoptosis in embryonic E12.5 cortical neural progenitor cells from the mouse brain

Hyperglycemia causes direct apoptosis of neural progenitor cells (NPCs) in diabetic-induced neural tube defects in embryos. However, the underlying mechanisms are poorly understood. The present study is aimed to investigate the specific cellular proteins that may be involved in NPCs apoptosis as well as mechanisms by which the proteins regulate the oxidative stress-induced NPCs apoptosis. Our present results have shown that the expression of c-Abl was up-regulated in NPCs exposed to high glucose in vitro . The increased c-Abl was localized mainly in the nucleus. High glucose also induced an increase in nuclear p53 protein levels and the p53-c-Abl complex in NPCs. Administration of reactive oxygen species scavengers decreased the protein level of c-Abl, p53 and NPCs apoptosis. Inhibition of c-Abl reduced NPCs apoptosis and the nuclear protein level of p53 in response to high glucose. These results demonstrate that c-Abl is involved in the reactive oxygen species-activated apoptotic pathways in NPCs apoptosis. Inhibition of c-Abl may protect NPCs against insults induced by high glucose via the modulation of NPCs apoptotic machinery.