Proapoptotic Program Research Articles

Abstract 2129: Aplidin triggers the activation of molecular components of the UPR as part of its pro-apoptotic program in tumor cells.

Abstract Aplidin (APL), a cyclic depsipeptide originally isolated from the marine tunicate Aplidium albicans and currently under phase II/III clinical trials for cancer therapy, induces oxidative stress, activation of Rac1 and phosphorylation of JNK1, which together trigger a rapid apoptotic program in tumor cells. A previously generated APL-resistant HeLa cell line (APL-R) allowed us to find out differences in protein expression with the parental HeLa cells. Among the proteins differentially expressed, several endoplasmic reticulum (ER) stress-related proteins were observed. We found decreased basal levels of BiP in HeLa APL-R, while the levels of Ero1a and phospho-eIF2a were slightly increased when compared to parental HeLa cells, indicative of a higher basal ER stress. In this work, we investigated if APL was inducing a bona fide ER stress in HeLa cells and whether this process was essential in the mechanism of action of the compound. Similarly to that observed with thapsigargin and tunicamycin, two well known ER stress inducing agents, APL triggered the activation of several key molecular components of a classical ER stress induced unfolded protein response (UPR), including the phosphorylation of eIF2a and JNK1, the proteolitic processing of ATF6 and the alternative splicing of XBP1, but not others, such as the accumulation of noxa or the proteolitic activation of caspase 4. Interestingly, although the eIF2a phosphorylation induced by APL in HeLa wt cells correlated with a strong inhibition of protein synthesis, it did not elicit an increased expression of CHOP, a transcription factor involved in the launch of UPR mediated apoptosis. Actually, it was observed a clear reduction of CHOP protein levels after the treatment of HeLa cells with APL, most probably due to its rapid degradation by the proteasome machinery. In summary, as a part of the proapoptotic program triggered by APL in HeLa cells, we observed the induction of ER stress and the activation of several endpoint executioners of the UPR apoptotic branch. Citation Format: Alejandro Losada, Juan F. Martínez-Leal, Alberto Bejarano, Carmen Cuevas, Luis F. Garcia-Fernández, Carlos M. Galmarini. Aplidin triggers the activation of molecular components of the UPR as part of its pro-apoptotic program in tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2129. doi:10.1158/1538-7445.AM2013-2129

Toxicological effects of cigarette smoke on Ana-1 macrophages in vitro

Cigarette smoke exposure is associated with increased risk of different disorders. Immunological dysfunction especially in macrophages is one of important reasons in the initiation, progression and exacerbation of smoke-related pulmonary illnesses. However, it is still obscure how cigarette smoke impacts the vitality and functions of macrophages. In the present study, we examined the effects of cigarette smoke extract (CSE) on mouse Ana-1 macrophages and tried to elucidate the involved mechanism. The results showed CSE induced cell apoptosis accompanied by increased releasing of lactate dehydrogenase (LDH), mitochondrial injury and oxidative stress. It also inhibited anti-apoptosis protein Bcl-2 expression and promoted pro-apoptosis protein Bax and Bad expressions. Moreover, low-dose CSE increased nuclear NF-κB levels of macrophages; on the contrary, high-dose CSE or long-time treatment decreased it. These observations were in correspondence with changes of intracellular ROS level and antioxidant enzymes' activity. Furthermore, pretreatment with 10μM of NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) for 1h significantly enhanced macrophage apoptosis. Taken together, these data implied that mitochondrial dysfunction and oxidative stress played important roles in the injury of Ana-1 cells caused by CSE, which was related to NF-κB pathway; an anti-apoptotic program played a dominant role at low doses/short-term exposure to CSE, whereas a pro-apoptotic program was initiated at high doses/long-term exposure.

IMP1 Consensus Recognition Elements Identify Transcripts Affecting Tumor Cell Survival After Ionizing Radiation

An expeditious mechanism for altering gene expression in response to ionizing radiation (IR) is regulation of mRNAs already transcribed. Post-transcriptional regulation may therefore enable radioresistance, characterized by failure to induce pro-apoptotic gene expression programs after IR. As principal modulators of mRNA stability and translational capacity, RNA-binding proteins (RBPs) may post-transcriptionally coordinate the apoptotic response to IR. Overexpression of RBPs including members of the insulin-like growth factor II messenger RBP family (IMP) has been associated with radioresistance.

TNF-α-mediated NF-κB survival signaling impairment by cisplatin enhances JNK activation allowing synergistic apoptosis of renal proximal tubular cells

Cisplatin-induced nephrotoxicity is an important limiting factor for cisplatin use. Tumor necrosis factor-α (TNF-α) is known to contribute to cisplatin-induced nephrotoxicity by inducing an inflammatory process aggravating the primary injury, thereby resulting in acute kidney injury (AKI). The present study investigates the pathways synergistically activated by cisplatin and TNF-α responsible for TNF-α-enhanced cisplatin-induced renal cell injury. To do so, immortalized renal proximal tubular epithelial cells (IM-PTECs) were co-treated with TNF-α and cisplatin. Under these conditions, cisplatin induced dose-dependent apoptosis in IM-PTECs, which was significantly enhanced by TNF-α. Transcriptomic analysis revealed that cisplatin inhibited the typical TNF-α response and cisplatin/TNF-α treatment up-regulated cell death pathways while it down-regulated survival pathways compared to cisplatin alone. In concordance, the gene expression levels of kidney injury markers combined with activation of specific inflammatory mediators were enhanced by cisplatin/TNF-α treatment, resembling the in vivo cisplatin-induced nephrotoxicity response. Furthermore, combined cisplatin/TNF-α treatment inhibited NF-κB nuclear translocation and NF-κB-mediated gene transcription leading to enhanced and prolonged JNK and c-Jun phosphorylation. JNK sustained activation further inhibited NF-κB signaling via a feedback loop mechanism. This led to an alteration in the transcription of the NF-κB-induced anti-apoptotic genes c-IAP2, Bcl-XL, Bruce and Bcl2 and pro-apoptotic genes Bfk and Xaf1 and consequently to sensitization of the IM-PTECs toward cisplatin/TNF-α-induced toxicity. In conclusion, our findings support a model whereby renal cells exposed to both cisplatin and TNF-α switch into a more pro-apoptotic and inflammatory program by altering their NF-κB/JNK/c-Jun balance.

Double-edged sword: a p53 regulator mediates both harmful and beneficial effects in experimental acute kidney injury

Acute kidney injury triggers activation of innate immune responses and of proapoptotic programs such as the p53 pathway. Mulay et al. examine the effects of blocking murine double minute-2 (mdm2), a negative regulator of p53, using a novel chemotherapeutic agent, nutlin-3a, in mouse ischemia-reperfusion injury. Their results indicate that mdm2 promotes renal regeneration by limiting p53-mediated apoptosis but also enhances early inflammation by facilitating DNA binding of nuclear factor-κB independently of p53.

Role of the cellular prion protein in the neuron adaptation strategy to copper deficiency.

Copper transporter 1 (CTR1), cellular prion protein (PrP(C)), natural resistance-associated macrophage protein 2 (NRAMP2) and ATP7A proteins control the cell absorption and efflux of copper (Cu) ions in nervous tissues upon physiological conditions. Little is known about their regulation under reduced Cu availability, a condition underlying the onset of diffused neurodegenerative disorders. In this study, rat neuron-like cells were exposed to Cu starvation for 48 h. The activation of Caspase-3 enzymes and the impairment of Cu,Zn superoxide dismutase (Cu,Zn SOD) activity depicted the initiation of a pro-apoptotic program, preliminary to the appearance of the morphological signs of apoptosis. The transcriptional response related to Cu transport proteins has been investigated. Notably, PrP(C) transcript and protein levels were consistently elevated upon Cu deficiency. The CTR1 protein amount was stable, despite a two-fold increase in the transcript amount, meaning the activation of post-translational regulatory mechanisms. NRAMP2 and ATP7A expressions were unvaried. The up-regulated PrP(C) has been demonstrated to enhance the cell Cu uptake ability by about 50% with respect to the basal transport, and so sustain the Cu delivery to the Cu,Zn SOD cuproenzymes. Conclusively, the study suggests a pivotal role for PrP(C) in the cell adaptation to Cu limitation through a direct activity of ion uptake. In this view, the PrP(C) accumulation observed in several cancer cell lines could be interpreted as a molecular marker of cell Cu deficiency and a potential target of therapeutic interventions against disorders caused by metal imbalances.

CT7 (MAGE-C1) Promotes Survival and Interacts with STAT1 and PIASy in Multiple Myeloma Cells

Abstract Abstract 1807 The type I Melanoma Antigen GEnes MAGE-A3 and CT7 (MAGE-C1) are detected in more than 75% of primary multiple myeloma specimens, and their expression is correlated with proliferation and progression of disease. We previously showed that MAGE-A3 inhibits apoptosis in human myeloma cell lines (HMCL) and primary cells in part through ubiquitinylation of the prototypical tumor suppressor p53, which targets it for proteasomal degradation and inhibits its pro-apoptotic transcriptional program (Nardiello et al, Clin Cancer Res, 2011; 17:4309). However, silencing of MAGE-A3 in HMCL and primary cells that lacked functional p53, either through deletions or mutations, also resulted in apoptosis. Silencing of CT7 alone did not affect survival of HMCL that lacked p53, but it did increase their sensitivity to chemotherapy-induced apoptosis. Many type I MAGE proteins, including MAGE-A3, bind to the RING domain protein TRIM28/Kap1 through their highly conserved MAGE Homology Domain (MHD) to form E3 ubiquitin ligase complexes, but it was unknown if CT7 also associated with TRIM28. These results lead to the hypothesis that CT7 is a survival factor for myeloma cells that can act through p53-independent mechanisms. To investigate the biochemical activity of CT7 and identify non-p53 pathways regulated by type I MAGE in myeloma cells, we analyzed protein-protein interactions with CT7 by two methods. We immunoprecipitated (IP'ed) CT7 from lysates of HMCL followed by sequencing of co-IP'ed proteins by mass spectroscopy and we performed yeast two-hybrid screening with a bait construct containing the MHD of CT7. CT7 was reciprocally co-IP'ed from HMCL lysates with TRIM28, demonstrating that it, too, associated with this RING domain protein. These methods also revealed two novel interactions with CT7. CT7 reciprocally co-IP'ed with STAT1, a transcription factor that plays a critical role in receptor-mediated signaling for cytokines such as interferon α/β. CT7 also interacted with Protein Inhibitor of Activated STAT y (PIASy), a modified RING domain protein that negatively regulates STAT proteins through its Small Ubiquitin-like MOdifier (SUMO) ligase activity, which sequesters target proteins out of the nucleus and into the cytoplasm. Surprisingly, phosphorylated STAT1 (pSTAT1) was detected in lysates from unstimulated HMCL, indicating a tonic level of activation, but most or all of the pSTAT1 was in the cytoplasm, suggesting that its transcriptional activity was being blocked through exclusion from the nucleus. Phospho-STAT1 also appeared to preferentially associate with the CT7/TRIM28 complex. Phospho-STAT1 did not appear to be ubiquitinylated in these HMCL, but SUMO2/3 modification of pSTAT1 was detected. These results suggest that CT7 negatively regulates pSTAT1 activity in HMCL by sequestering the transcription factor out of the nucleus through SUMO modification. This may be a direct result of CT7-mediated SUMOylation in partnership with PIASy, or indirectly due to CT7/TRIM28-mediated ubiquitinylation events that result in activation of a SUMO ligase such as PIASy. These findings suggest the possibility that complexes containing type I MAGE proteins may also have SUMO ligase activity and identify STAT1 as a novel non-p53 biochemical pathway regulated by these genes. Disclosures: No relevant conflicts of interest to declare.

RNA-binding Protein Insulin-like Growth Factor mRNA-binding Protein 3 (IMP-3) Promotes Cell Survival via Insulin-like Growth Factor II Signaling after Ionizing Radiation

Ionizing radiation (IR) induces proapoptotic gene expression programs that inhibit cell survival. These programs often involve RNA-binding proteins that associate with their mRNA targets to elicit changes in mRNA stability and/or translation. The RNA-binding protein IMP-3 is an oncofetal protein overexpressed in many human malignancies. IMP-3 abundance correlates with tumor aggressiveness and poor prognosis. As such, IMP-3 is proving to be a highly significant biomarker in surgical pathology. Among its many mRNA targets, IMP-3 binds to and promotes translation of insulin-like growth factor II (IGFII) mRNA. Our earlier studies showed that reducing IMP-3 abundance with siRNAs reduced proliferation of human K562 chronic myeloid leukemia cells because of reduced IGF-II biosynthesis. However, the role of IMP-3 in apoptosis is unknown. Here, we have used IR-induced apoptosis of K562 cells as a model to explore a role for IMP-3 in cell survival. Knockdown of IMP-3 with siRNA increased susceptibility of cells to IR-induced apoptosis and led to reduced IGF-II production. Gene reporter assays revealed that IMP-3 acts through the 5' UTR of IGFII mRNA during apoptosis to promote translation. Finally, culture of IR-treated cells with recombinant IGF-II partially reversed the effects of IMP-3 knockdown on IR-induced apoptosis. Together, these results indicate that IMP-3 acts in part through the IGF-II pathway to promote cell survival in response to IR. Thus, IMP-3 might serve as a new drug target to increase sensitivity of CML cells or other cancers to IR therapy.

Abstract 3850: CDKN2a deficiency exposes a requirement for RhoA in maintenance of non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related death world-wide. Tumorigenesis is a multistep process that involves several genetic aberrations. For example, K-RAS (onc-K-RAS), the most commonly mutated oncogene in human cancer, is expressed in about 25% of NSCLC. In addition, NSCLC often carry mutations or epigenetic modifications that affect the expression of the CDKN2a locus, which comprises the p16INK4a and the p14ARF (p19ARF in mice) tumor suppressor genes, which in turn regulate the retinoblastoma and the p53 tumor suppressor pathways. NSCLC tumorigenesis is faithfully recapitulated in transgenic mice where onc-K-Ras expression invariably induces adenomas and, after a long latency, adenocarcinomas. To characterize the consequence of concomitant onc-K-RAS expression and CDKN2a inactivation, we generated p16CDK4/p19ARF null mice expressing onc-K-Ras in the respiratory epithelium. We determined that Cdkn2a deficiency promotes rapid progression of lung adenomas to adenocarcinomas, which display aggressive features. We determined the status of activation of oncogenic signaling networks downstream of onc-K-Ras with phosphospecific antibodies and by expression profiling. We discovered that NSCLC that arises in CDKN2A deficient mice display dysregulation of RhoA, a small GTPase implicated in the regulation of the cytoskeleton, cell adhesion, and motility of cancer cells in culture. These activities are pertinent to tumorigenesis, however it is not known whether RhoA plays a role in the maintenance of established tumors. Surprisingly, we determined that RhoA is required for the survival of NSCLC derived lines expressing onc-K-RAS and deficient for either INK4a or the p53 tumor suppressor both when cultured in vitro and as xenografts in vivo. Cell death was due to a proapoptotic program and was accompanied by disruption of the cytoskeleton. Moreover, by performing loss and gain of function experiments we identified focal adhesion kinase (FAK) protein as the principal downstream target of RhoA responsible for this effect. Experiments in vivo with specific inhibitors are ongoing and will be presented at the meeting. Our findings reveal a previously unappreciated context dependent requirement of the RhoGTPase to maintain viability of established NSCLC. We propose that RhoA and its signaling network are novel therapeutic targets in NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3850. doi:10.1158/1538-7445.AM2011-3850

FoxO1 and SIRT1 Regulate β-Cell Responses to Nitric Oxide

For many cell types, including pancreatic β-cells, nitric oxide is a mediator of cell death; paradoxically, nitric oxide can also activate pathways that promote the repair of cellular damage. In this report, a role for FoxO1-dependent transcriptional activation and its regulation by SIRT1 in determining the cellular response to nitric oxide is provided. In response to nitric oxide, FoxO1 translocates from the cytoplasm to the nucleus and stimulates the expression of the DNA repair gene GADD45α, resulting in FoxO1-dependent DNA repair. FoxO1-dependent gene expression appears to be regulated by the NAD(+)-dependent deacetylase SIRT1. In response to SIRT1 inhibitors, the FoxO1-dependent protective actions of nitric oxide (GADD45α expression and DNA repair) are attenuated, and FoxO1 activates a proapoptotic program that includes PUMA (p53-up-regulated mediator of apoptosis) mRNA accumulation and caspase-3 cleavage. These findings support primary roles for FoxO1 and SIRT1 in regulating the cellular responses of β-cells to nitric oxide.

A microRNA-dependent program controls p53-independent survival and chemosensitivity in human and murine squamous cell carcinoma

The p53 tumor suppressor, a central mediator of chemosensitivity in normal cells, is functionally inactivated in many human cancers. Therefore, a central challenge in human cancer therapy is the identification of pathways that control tumor cell survival and chemosensitivity in the absence of functional p53. The p53-related transcription factors p63 and p73 exhibit distinct functions—p73 mediates chemosensitivity while p63 promotes proliferation and cell survival—and are both overexpressed in squamous cell carcinomas (SCCs). However, how p63 and p73 interact functionally and govern the balance between prosurvival and proapoptotic programs in SCC remains elusive. Here, we identify a microRNA-dependent mechanism of p63/p73 crosstalk that regulates p53-independent survival of both human and murine SCC. We first discovered that a subset of p63-regulated microRNAs target p73 for inhibition. One of these, miR-193a-5p, expression of which was repressed by p63, was activated by proapoptotic p73 isoforms in both normal cells and tumor cells in vivo. Chemotherapy caused p63/p73-dependent induction of this microRNA, thereby limiting chemosensitivity due to microRNA-mediated feedback inhibition of p73. Importantly, inhibiting miR-193a interrupted this feedback and thereby suppressed tumor cell viability and induced dramatic chemosensitivity both in vitro and in vivo. Thus, we have identified a direct, microRNA-dependent regulatory circuit mediating inducible chemoresistance, whose inhibition may provide a new therapeutic opportunity in p53-deficient tumors.

ATF4 and the integrated stress response are induced by ethanol and cytochrome P450 2E1 in human hepatocytes

Molecular mechanisms underlying alcoholic liver disease (ALD) are still not fully understood. Activating transcription factor-4 (ATF4) is the master coordinator of the integrated stress response (ISR), an adaptive pathway triggered by multiple stressors. which can promote cell death and induce metabolic dysregulation if the stress is intense or prolonged. The aim of this study was to assess the effect of alcohol on the ISR signaling pathway in human liver cells and to define the role of cytochrome P450 2E1 (CYP2E1) in this response. Primary cultured human hepatocytes and human HepG2 cells over-expressing CYP2E1 by adenoviral infection were exposed to ethanol (25-100mM) for 8-48h. Ethanol treatment of both liver cells up-regulated ATF4 as well as the pro-survival and the pro-apoptotic transcriptional program of the ISR. Indeed, in CYP2E1-expressing HepG2 cells exposed to ethanol, the expression of ISR target genes (HMOX-1, GCLC, AsnS, IGFBP-1, GADD34,CHOP, ATF3, CHAC1) was induced. Up-regulation of ATF4 and the ISR transcriptional program was decreased by addition of the anti-oxidant glutathione. Several mechanisms mediated ATF4 protein induction, including, at early times, the phosphorylation of eIF2α which controls ATF4 translation, and, at later times, increased mRNA level and increased stability of the protein. A decrease in cell survival was also observed. This study demonstrates that both CYP2E1 and ethanol induce ATF4 and the integrated stress response, a pathway which coordinates signals from multiple stresses, as well as established risk factors for ALD, and can display detrimental cellular effects upon prolonged activation.

Abstract 2992: Transcriptional silencing of amplicons by histone deacetylase inhibition

Abstract Inhibition of cancer cell growth by histone deacetylase (HDAC) inhibitors has been observed in a number of malignancies. Multiple mechanisms have been proposed to explain the cancer-specific cytotoxic effect of HDAC inhibition, including transcriptional activation of pro-apoptotic and differentiation programs that become epigenetically silenced during tumorigenesis. Here, we report a direct transcriptional repression mechanism of histone deacetylase inhibition that selectively targets genomic amplicons, providing genetic and epigenetic alterations as a basis for the cancer selective cytotoxicity of these drugs. Our study implicates distinct epigenetic structures at the oncogenic amplicons that might be targeted for cancer drug development. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 2992.

TGF-β/Smad-1 signaling activates regenerative pro-survival mechanisms, whereas TGF-β/Smad-2/3 leads to pro-apoptotic programs in primary mouse hepatocytes

Phosphorylation of the signaling molecules Smad2/3 is considered as classical TGF-β signal transduction pathway. However, in an increasing number of cell types, including endothelial cells, hepatocytes and hepatic stellate cells, TGF-β phosphorylates Smad1, originally dedicated as BMP responsive branch. Since no discrimination of these parallel pathways was done up to now in liver cells, aim of the present study was to identify TGF-β/Smad1 target genes in cultured hepatocytes. Using microarray technology, TGF-β regulated early-response genes were identified either under control conditions or after adenoviral overexpression of Smad6, a specific inhibitor of Smad1 phosphorylation, which does not affect Smad2/3. Additionally TGF-β regulated genes were identified upon overexpression of Smad7, which blocks phosphorylation of all three Smads. With this experimental setup TGF-β/Smad1 targets could be distinguished from Smad2/3 and non-Smad target genes. TGF-β induced 53 genes within 1h by greater or equal to 1.6-fold and down-regulated 10 genes. Those genes which were mainly regulated via the Smad1 pathway included proteins involved in proliferation, differentiation, EMT (e.g. Id1, -2, -3, PDGF-A and –B, Snai1) as well as inhibitory proteins like Smad6 itself, Cish or the TGF-β pseudo-receptor Bambi. One of the strongest induced TGF-β/Smad1 target genes was the iron regulatory hormone hepcidin and the strongest down-regulated target gene was the cytochrome P450 1a1 (Cyp1a1). In summary, our data show that (i) TGF-β regulates a huge number of target genes within only 1 hour, (ii) the most strongly regulated genes (Id1, hepcidin, Id3, Cyp1a1) belong to the group of Smad1 targets and (iii) pro-apoptotic genes like Jun-B, Fos-B and Tieg1 are Smad2/3 regulated, implying that the alternative TGF-β/Smad1 route relates more to cell survival and regeneration. These findings explain to some extent the frequently observed opposing effects of TGF-β, e.g. on proliferation or apoptosis.

AATF mediates an antiapoptotic effect of the unfolded protein response through transcriptional regulation of AKT1

Endoplasmic reticulum (ER) stress-mediated cell death plays an important role in the pathogenesis of chronic diseases including diabetes and neurodegeneration. Although pro-apoptotic programs activated by ER stress have been extensively studied, identification and characterization of anti-apoptotic programs that counteract ER stress is currently incomplete. Through the gene expression profiling of β-cells lacking WFS1, a causative gene for Wolfram syndrome, we have discovered a novel anti-apoptotic gene of the unfolded protein response (UPR), apoptosis antagonizing transcription factor (AATF). Here we study the regulation of AATF, identify its target genes, and determine the basis for its anti-apoptotic activities in response to ER stress. We show that AATF is induced by ER stress through the PERK-eIF2α pathway and transcriptionally activates the Akt1 gene through Stat3, which sustains Akt1 activation and promotes cell survival. Ectopic expression of AATF or a constitutively active form of AKT1 confers on cells resistance to ER stress-mediated cell death, whereas RNAi-mediated knockdown of AATF or AKT1 renders cells sensitive to ER stress. We also discovered positive crosstalk between the AATF and WFS1 signaling pathways. Thus, WFS1-deficiency or AATF-deficiency mediates a self-perpetuating cycle of cell death. Our results reveal a novel anti-apoptotic program relevant to treatment for diseases caused by ER stress-mediated cell death.

인간 A549 폐암세포에서 비스테로이드성 항염증제와 genistein의 복합처리에 의한 NAG-1 의존적 세포사멸 증진 효과

A number of studies have demonstrated that the regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) can reduce the risks of colorectal, oesophageal and lung cancers. NSAIDs have been shown to exert their anti-cancer effects through inducing apoptosis in cancer cells. The susceptibility of tumor cells to anti-tumor drug-induced apoptosis appears to depend on the balance between pro-apoptotic and anti-apoptotic programs such as nuclear factor kB (NF-kB), phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) and MEK1/2-ERK1/2 pathways. We examined the effects of pro-survival PI3K and ERK1/2 signal pathways on cell cycle arrest and apoptosis in response to NSAIDs including sulindac sulfide and NS398. We show that simultaneous inhibition of the Akt/PKB and ERK1/2 signal cascades could synergistically enhance the potential pro-apoptotic activities of sulindac sulfide and NS398. Similar enhancement was observed in cells treated with sulindac sulfide or NS398 and 100 μM genistein, an inhibitor of receptor tyrosine kinases (RTKs) that are upstream of PI3K and MEK1/2 signaling. We further demonstrate that NAG-1 is induced and plays a critical role(s) in apoptosis by NSAIDs-based combined treatment. In sum, our results show that combinatorial treatment of sulindac sulfide or NS398 and genistein results in a highly synergistic induction of apoptotic cell death to increase the chemopreventive effects of the NSAIDs, sulindac sulfide and NS398.

FOXO3a elicits a pro-apoptotic transcription program and cellular response to human lung carcinogen nicotine-derived nitrosaminoketone (NNK)

Long-term carcinogen exposure exerts continuous pressure on key mechanisms that repair or eliminate carcinogen-damaged cells giving rise to selective failures that contribute to lung cancer. FOXO3a is a transcription factor that elicits a protective response to diverse cellular stresses. Although implicated as a tumor suppressor, its role in sporadic cancer is uncertain. We recently observed that FOXO3a gene inactivation occurs frequently in carcinogen-induced lung adenocarcinoma (LAC). This suggests that FOXO3a may play a role in LAC suppression by eliciting a protective response to carcinogenic stress. Here we investigated this possibility by examining the role of FOXO3a in the cellular response to nicotine-derived nitrosaminoketone (NNK), a lung carcinogen implicated as a cause of human LAC. We show that restoration of FOXO3a in FOXO3a-deficient LAC cells increases sensitivity to apoptosis caused by a DNA-damaging intermediate of NNK. Prior to this cellular outcome, FOXO3a is functionally activated and mediates a large-scale transcription program in response to this damage involving a significant modulation of 440 genes. Genes most significantly represented in this program are those with roles in cell growth and proliferation>protein synthesis>gene expression>cell death>cell cycle. The results of this study show that FOXO3a directs an anti-carcinogenic transcription program that culminates in the elimination of carcinogen-damaged cells. This suggests that FOXO3a is a potential suppressor of carcinogenic damage in LAC.

Nuclear Bile Acid Receptor FXR Protects against Intestinal Tumorigenesis

Bile acids have been considered intestinal tumor promoters, and because they are natural ligands for the nuclear receptor FXR, we examined the role of FXR in intestinal tumorigenesis. Using gain- and loss-of-function studies, we found that FXR suppresses intestinal tumorigenesis in vivo. Loss of FXR in the ApcMin/+ and in the chronic colitis mouse models of intestinal tumorigenesis resulted in early mortality and increased tumor progression via promotion of Wnt signaling by infiltrating neutrophils and macrophages and tumor necrosis factor alpha production. Treatment with the bile acid binding resin cholestyramine did not modify the intestinal tumor susceptibility of FXR-/- mice, indicating that loss of FXR and not merely elevated bile acid concentrations increases susceptibility to tumorigenesis. Activation of FXR induced a proapoptotic program in the differentiated normal colonic epithelium as well as transformed colonocytes. Our data suggest that it is unlikely that the tumor-promoting activity of bile acids occurs as a function of their ability to activate FXR. However, FXR activity is relevant to the pathogenesis of intestinal cancer. When FXR is absent in the intestine, there is a promotion of Wnt signaling with expansion of the basal proliferative compartment, and a concomitant reduction in the apical differentiated apoptosis-competent compartment. When FXR is activated in the intestine and in colon cancer cells, there is an induction of apoptosis and removal of genetically altered cells, which may otherwise progress to complete transformation. Thus, from a therapeutic standpoint, strategies aimed at reactivating FXR expression in colon tumors might be useful in treatment of colon cancer.

Roscovitine inhibits thyroid hormone‐induced tail regression of the frog tadpole and reveals a role for cyclin C/Cdk8 in the establishment of the metamorphic gene expression program

The involvement of phosphorylation signaling pathways in postembryonic development of the frog is poorly understood. The thyroid hormone, 3, 5, 3'-triiodothyronine (T(3)), is essential for inducing tadpole metamorphosis and we show that the cyclin-dependent kinase (Cdk) inhibitor, roscovitine, prevents T(3)-dependent regression of cultured tail tips from Rana catesbeiana tadpoles. Using tail tips from precociously induced and naturally metamorphosing tadpoles, our data suggest that protein phosphorylation is important in the establishment of the T(3)-dependent proapoptotic gene expression program. Our evidence indicates that Cdk8 is the most likely candidate for this proapoptotic activity with the expression of its regulatory subunit, cyclin C, identified as a novel T(3)-responsive gene. We suggest that this activity is crucial for the genetic reprogramming required for tail regression and demonstrate that protein phosphorylation is important in T(3)-induced apoptosis in normal cells during development.

Differential Control of the CCAAT/Enhancer-binding Protein β (C/EBPβ) Products Liver-enriched Transcriptional Activating Protein (LAP) and Liver-enriched Transcriptional Inhibitory Protein (LIP) and the Regulation of Gene Expression during the Response to Endoplasmic Reticulum Stress

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers a stress response program that protects cells early in the response and can lead to apoptosis during prolonged stress. The basic leucine zipper transcription factor, CCAAT/enhancer-binding protein beta (C/EBPbeta), is one of the genes with increased expression during ER stress. Translation of the C/EBPbeta mRNA from different initiation codons leads to the synthesis of two transcriptional activators (LAP-1 and -2) and a transcriptional repressor (LIP). The LIP/LAP ratio is a critical factor in C/EBPbeta-mediated gene transcription. It is shown here that the LIP/LAP ratio decreased by 5-fold during the early phase of ER stress and increased by 20-fold during the late phase, mostly because of changes in LIP levels. The early decrease in LIP required degradation via the proteasome pathway and phosphorylation of the translation initiation factor, eIF2alpha. The increased LIP levels during the late phase were due to increased synthesis and increased stability of the protein. It is proposed that regulation of synthesis and degradation rates during ER stress controls the LIP/LAP ratio. The importance of C/EBPbeta in the ER-stress response program was demonstrated using C/EBPbeta-deficient mouse embryonic fibroblasts. It is shown that C/EBPbeta attenuates expression of pro-survival ATF4 target genes in late ER stress and enhances expression of cell death-associated genes downstream of CHOP. The inhibitory effect of LIP on ATF4-induced transcription was demonstrated for the cat-1 amino acid transporter gene. We conclude that regulation of LIP/LAP ratios during ER stress is a novel mechanism for modulating the cellular stress response.