In vivo monitoring of the anti-angiogenic therapeutic effect of the pan-deacetylase inhibitor panobinostat by small animal PET in a mouse model of gastrointestinal cancers.

INTRODUCTION: Deacetylase inhibitors have recently been established as a novel therapeutic approach to solid and hematologic cancers and have also been demonstrated to possess anti-angiogenic properties. Although these compounds show a good efficacy in vitro and in vivo, no data on monitoring and predicting treatment response are currently available. We therefore investigated the effect of the pan-deacetylase inhibitor panobinostat (LBH589) on gastrointestinal cancer models and the suitability of 2-[(18)F]FGlc-RGD as a specific agent for imaging integrin αvß3 expression during tumor angiogenesis using small animal positron emission tomography (PET). METHODS: The effect of panobinostat on cell viability in vitro was assessed with a label-free impedance based real-time analysis. Nude mice bearing HT29 and HepG2 tumors were treated with daily i.p. injections of 10mg/kg panobinostat for 4 weeks. During this time, tumor size was determined with a calliper and mice were repeatedly subjected to PET imaging. Tumor tissues were analyzed immunohistochemically with a focus on proliferation and endothelial cell markers (Ki-67, Meca-32) and by Western blot applying specific markers of apoptosis. RESULTS: In vitro, panobinostat inhibited the proliferation of HepG2 and HT29 cells. Contrary to the situation in HepG2 tumors in vivo, where panobinostat treatment is known to reduce proliferation and vascularization resulting in a decreased tumor growth, HT29 tumors did not show any effect on these parameters. We demonstrated by Western blotting, that panobinostat induced apoptosis in HT29 tumors in vivo. Longitudinal PET imaging studies in HepG2 tumor-bearing mice using 2-[(18)F]FGlc-RGD demonstrated that the standard uptake value (SUVmax) in HepG2 tumors was significantly decreased by 39% at day 7 after treatment. The comparative PET study using HT29 tumor-bearing animals did not reveal any response of the tumors to panobinostat treatment. CONCLUSIONS: Small-animal PET imaging using 2-[(18)F]FGlc-RGD was successfully applied to the non-invasive monitoring of the HepG2-tumor response to panobinostat in nude mice early after begin of treatment. Thus, PET imaging of angiogenesis using 2-[(18)F]FGlc-RGD could be a valuable tool to monitor panobinostat therapy in further preclinical studies. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: When successfully translated to the clinical surrounding, PET imaging of angiogenesis could therefore facilitate therapy planning and monitoring of therapy success with panobinostat in hepatocellular carcinoma.

Defective autophagosome formation in p53-null colorectal cancer reinforces crocin-induced apoptosis.

Crocin, a bioactive molecule of saffron, inhibited proliferation of both HCT116 wild-type and HCT116 p53(-/-) cell lines at a concentration of 10 mM. Flow cytometric analysis of cell cycle distribution revealed that there was an accumulation of HCT116 wild-type cells in G1 (55.9%, 56.1%) compared to the control (30.4%) after 24 and 48 h of crocin treatment, respectively. However, crocin induced only mild G2 arrest in HCT116 p53(-/-) after 24 h. Crocin induced inefficient autophagy in HCT116 p53(-/-) cells, where crocin induced the formation of LC3-II, which was combined with a decrease in the protein levels of Beclin 1 and Atg7 and no clear p62 degradation. Autophagosome formation was not detected in HCT116 p53(-/-) after crocin treatment predicting a nonfunctional autophagosome formation. There was a significant increase of p62 after treating the cells with Bafilomycin A1 (Baf) and crocin compared to crocin exposure alone. Annexin V staining showed that Baf-pretreatment enhanced the induction of apoptosis in HCT116 wild-type cells. Baf-exposed HCT116 p53(-/-) cells did not, however, show any enhancement of apoptosis induction despite an increase in the DNA damage-sensor accumulation, γH2AX indicating that crocin induced an autophagy-independent classical programmed cell death.

DAPK-HSF1 interaction as a positive-feedback mechanism stimulating TNF-induced apoptosis in colorectal cancer cells.

Death-associated protein kinase (DAPK) is a serine-threonine kinase with tumor suppressor function. Previously, we demonstrated that tumor necrosis factor (TNF) induced DAPK-mediated apoptosis in colorectal cancer. However, the protein-protein interaction network associated with TNF-DAPK signaling still remains unclear. We identified HSF1 as a new DAPK phosphorylation target in response to low concentrations of TNF and verified a physical interaction between DAPK and HSF1 both in vitro and in vivo. We show that HSF1 binds to the DAPK promoter. Transient overexpression of HSF1 protein led to an increase in DAPK mRNA level and consequently to an increase in the amount of apoptosis. By contrast, treatment with a DAPK-specific inhibitor as well as DAPK knockdown abolished the phosphorylation of HSF1 at Ser230 (pHSF1(Ser230)). Furthermore, translational studies demonstrated a positive correlation between DAPK and pHSF1(Ser230) protein expression in human colorectal carcinoma tissues. Taken together, our data define a novel link between DAPK and HSF1 and highlight a positive-feedback loop in DAPK regulation under mild inflammatory stress conditions in colorectal tumors. For the first time, we show that under TNF the pro-survival HSF1 protein can be redirected to a pro-apoptotic program.

Identification of DAPK as a scaffold protein for the LIMK/cofilin complex in TNF-induced apoptosis.

The role of cytoskeleton-associated proteins during TNF-induced apoptosis is not fully understood. A potential candidate kinase that might connect TNF signaling to actin reorganization is the death-associated protein kinase (DAPK). To identify new DAPK interaction partners in TNF-induced apoptosis, we performed a peptide array screen. We show that TNF-treatment enhanced the phosphorylation of LIMK at threonine508 and its downstream target cofilin at serine3 (p-cofilin(Ser3)). Modulation of DAPK activity and expression by DAPK inhibitor treatment, siRNA knockdown, and overexpression affected the phosphorylation of both proteins. We propose a 3D structural model where DAPK functions as a scaffold for the LIMK/cofilin complex and triggers a closer interaction of both proteins under TNF stimulation. Upon TNF a striking redistribution of LIMK, DAPK, and cofilin to the perinuclear compartment was observed. The pro-apoptotic DAPK/LIMK/cofilin multiprotein complex was abrogated in detached cells, indicating that its signaling was no longer needed if cells committed to apoptosis. P-cofilin(Ser3) was strongly accumulated in cells with condensed chromatin, pronounced membrane blebs and Annexin V up-regulation. From studying different cofilin(Ser3) mutants we suggest that p-cofilin(Ser3) is an indicator of TNF-induced apoptosis. Collectively, our findings identify a novel molecular cytoskeleton-associated mechanism in TNF-induced DAPK-dependent apoptosis.

Death-associated protein kinase controls STAT3 activity in intestinal epithelial cells.

The TNF-IL-6-STAT3 pathway plays a crucial role in promoting ulcerative colitis-associated carcinoma (UCC). To date, the negative regulation of STAT3 is poorly understood. Interestingly, intestinal epithelial cells of UCC in comparison to ulcerative colitis show high expression levels of anti-inflammatory death-associated protein kinase (DAPK) and low levels of pSTAT3. Accordingly, epithelial DAPK expression was enhanced in STAT3(IEC-KO) mice. To unravel a possible regulatory mechanism, we used an in vitro TNF-treated intestinal epithelial cell model. We identified a new function of DAPK in suppressing TNF-induced STAT3 activation as DAPK siRNA knockdown and treatment with a DAPK inhibitor potentiated STAT3 activation, IL-6 mRNA expression, and secretion. DAPK attenuated STAT3 activity directly by physical interaction shown in three-dimensional structural modeling. This model suggests that DAPK-induced conformational changes in the STAT3 dimer masked its nuclear localization signal. Alternatively, pharmacological inactivation of STAT3 led to an increase in DAPK mRNA and protein levels. Chromatin immunoprecipitation showed that STAT3 restricted DAPK expression by promoter binding, thereby reinforcing its own activation by inducing IL-6. This novel negative regulation principle might balance TNF-induced inflammation and seems to play an important role in the inflammation-associated transformation process as confirmed in an AOM+DSS colon carcinogenesis mouse model. DAPK as a negative regulator of STAT3 emerges as therapeutic option in the treatment of ulcerative colitis and UCC.

DAPK plays an important role in panobinostat-induced autophagy and commits cells to apoptosis under autophagy deficient conditions.

The histone deacetylase inhibitor (HDACi) LBH589 has been verified as an effective anticancer agent. The identification and characterization of new targets for LBH589 action would further enhance our understanding of the molecular mechanisms involved in HDACi therapy. The role of the tumor suppressor death-associated protein kinase (DAPK) in LBH589-induced cytotoxicity has not been investigated to date. Stable DAPK knockdown (shRNA) and DAPK overexpressing (DAPK+++) cell lines were generated from HCT116 wildtype colon cancer cells. LBH589 inhibited cell proliferation, reduced the long-term survival, and up-regulated and activated DAPK in colorectal cancer cells. Moreover, LBH589 significantly suppressed the growth of colon tumor xenografts and in accordance with the in vitro studies, increased DAPK levels were detected immunohistochemically. LBH589 induced a DAPK-dependent autophagy as assessed by punctuate accumulation of LC3-II, the formation of acidic vesicular organelles, and degradation of p62 protein. LBH589-induced autophagy seems to be predominantly caused by DAPK protein interactions than by its kinase activity. Caspase inhibitor zVAD increased autophagosome formation, decreased the cleavage of caspase 3 and PARP but didn't rescue the cells from LBH589-induced cell death in crystal violet staining suggesting both caspase-dependent as well as caspase-independent apoptosis pathways. Pre-treatment with the autophagy inhibitor Bafilomycin A1 caused caspase 3-mediated apoptosis in a DAPK-dependent manner. Altogether our data suggest that DAPK induces autophagy in response to HDACi-treatment. In autophagy deficient cells, DAPK plays an essential role in committing cells to HDACi-induced apoptosis.

IL-33 regulates TNF-α dependent effects in synovial fibroblasts.

The recently described IL-33 acts as a pro-inflammatory cytokine, inducing the expression of multiple responses in the target cells. Although a nuclear localization of IL-33 has been described, its exact functional relevance is presently unknown. The present study was conducted to analyze the effects of IL-33 on the TNF-α induced synthesis of the pro-inflammatory mediators IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) and the pro-destructive molecules matrix metalloproteinase-1 (MMP-1), MMP-3, and TIMP-1 of rheumatoid arthritis synovial fibroblast (RA-SFs) using RNA overexpression and silencing. TNF-α significantly induced IL-33 mRNA expression and protein synthesis in RA-SFs. TNF-α-induced IL-33 protein expression was mediated via p38 signaling. Immunohistochemistry for IL-33 clearly showed that nuclear translocation of IL-33 was induced in TNF-α stimulated RA-SFs. IL-33 overexpression enhanced TNF-α-induced pro-inflammatory and pro-destructive functions in RA-SFs. IL-33 silencing significantly downregulated TNF-α-induced pro-inflammatory functions, whereas TNF-α-induced pro-destructive functions were less influenced by IL-33 silencing. This study identifies IL-33 as a critical regulator/enhancer of TNF-α-induced functions in RA-SFs, pointing to a central role of this cytokine in the perpetuation of pro-inflammatory and pro-destructive processes in rheumatoid arthritis (RA) and other inflammatory and degenerative diseases.

Apoptosis signalling activated by TNF in the lower gastrointestinal tract--review.

The tumor necrosis factor (TNF) gene is an immediate early gene, rapidly transcribed in a variety of cell types following exposure to a broad range of pathogens and signals of inflammation and stress. Regulation of TNF gene expression at the transcriptional level is cell type- and stimulus-specific, involving epigenetic mechanisms or miRNAs. A better knowledge of the molecular mechanisms that control TNF gene regulation and TNF signalling will provide deeper understanding of the initiation and development of apoptotic and inflammatory processes triggered by TNF cytokine in the gut. The described efforts to embed TNF in clinical treatment regiments reflect its attractive effectiveness in killing tumor cells. Whether the described strategies will achieve the success of incorporating TNF in lower gastrointestinal tract therapy for inflammatory diseases and cancer remains to be determined.

Reactive oxygen species mediate thymoquinone-induced apoptosis and activate ERK and JNK signaling.

Thymoquinone (TQ), a component of black seed essential oil, is known to induce apoptotic cell death and oxidative stress, however, the direct involvement of oxidants in TQ-induced cell death has not been established yet. Here, we show that TQ inhibited the proliferation of a panel of human colon cancer cells (Caco-2, HCT-116, LoVo, DLD-1 and HT-29), without exhibiting cytotoxicity to normal human intestinal FHs74Int cells. Further investigation in DLD-1 revealed that apoptotic cell death is the mechanism for TQ-induced growth inhibition as confirmed by flow cytometry, M30 cytodeath and caspase-3/7 activation. Apoptosis was induced via the generation of reactive oxygen species (ROS) as evidenced by the abrogation of TQ apoptotic effect in cells preincubated with the strong antioxidant N-acetyl cysteine (NAC). TQ increased the phosphorylation states of the mitogen-activated protein kinases (MAPK) JNK and ERK, but not of p38. Their activation was completely abolished in the presence of NAC. Using PD98059 and SP600125, specific ERK and JNK inhibitors, the two kinases were found to possess pro-survival activities in TQ-induced cell death. These data present evidence linking the pro-oxidant effects of TQ with its apoptotic effects in colon cancer and prove a protective role of MAPK.

Predominant activation of MAP kinases and pro-destructive/pro-inflammatory features by TNF alpha in early-passage synovial fibroblasts via TNF receptor-1: failure of p38 inhibition to suppress matrix metalloproteinase-1 in rheumatoid arthritis.

OBJECTIVE: To examine the relative importance of tumour necrosis factor-receptor 1 (TNF-R1) and TNF-R2 and their signalling pathways for pro-inflammatory and pro-destructive features of early-passage synovial fibroblasts (SFB) from rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Cells were stimulated with tumour necrosis factor (TNF)alpha or agonistic anti-TNF-R1/TNF-R2 monoclonal antibodies. Phosphorylation of p38, ERK and JNK kinases was assessed by western blot; proliferation by bromodesoxyuridine incorporation; interleukin (IL)6, IL8, prostaglandin E(2) (PGE(2)) and matrix metalloproteinase (MMP)-1 secretion by ELISA; and MMP-3 secretion by western blot. Functional assays were performed with or without inhibition of p38 (SB203580), ERK (U0126) or JNK (SP600125). RESULTS: In RA- and OA-SFB, TNFalpha-induced phosphorylation of p38, ERK or JNK was exclusively mediated by TNF-R1. Reduction of proliferation and induction of IL6, IL8 and MMP-1 were solely mediated by TNF-R1, whereas PGE(2) and MMP-3 secretion was mediated by both TNF-Rs. In general, inhibition of ERK or JNK did not significantly alter the TNFalpha influence on these effector molecules. In contrast, inhibition of p38 reversed TNFalpha effects on proliferation and IL6/PGE(2) secretion (but not on IL8 and MMP-3 secretion). The above effects were comparable in RA- and OA-SFB, except that TNFalpha-induced MMP-1 secretion was reversed by p38 inhibition only in OA-SFB. CONCLUSION: In early-passage RA/OA-SFB, activation of MAPK cascades and pro-inflammatory/pro-destructive features by TNFalpha is predominantly mediated by TNF-R1 and, for proliferation and IL6/PGE(2) secretion, exclusively regulated by p38. Strikingly, RA-SFB are insensitive to p38 inhibition of MMP-1 secretion. This indicates a resistance of RA-SFB to the inhibition of pro-destructive functions and suggests underlying structural/functional alterations of the p38 pathway, which may contribute to the pathogenesis or therapeutic sensitivity of RA, or both.