Muktheshwar Gandesiri

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.

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.

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.

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.

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

ABSTRACT 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 (pHSF1Ser230). Furthermore, translational studies demonstrated a positive correlation between DAPK and pHSF1Ser230 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.

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.

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 αvβ3 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.

Abstract A21: Autophagy: A potential target for colorectal cancer therapeutics.

Colorectal cancer is among the leading causes of death worldwide. Here, we investigate the molecular mechanism of crocin-induced apoptosis in colorectal cancer cells. Crocin was shown here to inhibit proliferation of both HCT wildtype and HCT p53-/- cell lines at a concentration of 10 mM. Flow cytometric analysis of cell cycle distribution revealed that there was an accumulation of HCT wildtype cells in G1 compared to the control after 24 and 48 hours of crocin treatment, respectively. However, crocin induced only mild G2 arrest in HCT p53-/- after 24 hours. Crocin induced apoptosis in both cell lines in a time dependent manner. Nevertheless, p53 deficient cells were not able to repair the damage induced by crocin. Crocin induced inefficient autophagy in HCT p53-/- cells where crocin induced the formation of LC3-II which was combined by a decrease in the protein levels of beclin 1 and Atg7 and no clear p62 degradation. Autophagosome formation was not detected in HCT 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, indicating an effective autophagic flux. Annexin V staining showed that Baf-pretreatment enhanced the induction of apoptosis in HCT wildtype cells. Baf-exposed-HCT p53-/- cells did not 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. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A21. Citation Format: Amr Amin, Khuloud Bajbouj, Adrian Koch, M. Gandesiri, Regine Schneider-Stock. Autophagy: A potential target for colorectal cancer therapeutics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A21.

Abstract 4673A: DAPK-mediated phosphorylation of HSF1 enhances apoptosis level upon TNF in colorectal carcinoma cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Objectives: Tumor necrosis factor ≤ (TNF) is an inflammatory cytokine, which is released upon different stimuli, including irradiation. Recently it has been shown, that the Death-associated protein kinase (DAPK) mediates TNF-induced apoptosis in colon cancer cells [1]. Here, we aimed to identify new DAPK binding partners and to characterize the functional role of novel protein interaction complexes during TNF-induced apoptosis in colon cancer cells. Methods/Results: HCT116 colorectal cancer cells were cultured for 6 to 48 hours in either normal or TNF-conditioned medium. For phosphopeptide microarray (PPM) whole cell lysates were incubated on peptide platforms with radioactive-labeled P33. Apoptosis was detected by Annexin V staining and caspase 3 cleavage in Western Blotting. PPM analysis revealed heat shock transcription factor 1 (HSF1) as a new potential substrate of DAPK phosphorylation under TNF-stimulation. DAPK Co-IP, co-immunofluorescence (Co-IF), and mass spectrometry showed that DAPK interacts with HSF1 after TNF-treatment. Phosphorylation on serine residues of HSF1 is necessary for HSF1 nuclear translocation and the initiation of transcription of its target genes. Among them, Ser 230 is part of a consensus phosphorylation motif for DAPK. Maximal level of pHSF1Ser230 was observed at the time point where massive apoptosis was induced. Co-IF microscopy confirmed enrichment of pHSF1Ser230 in the nucleus already after 24 hours of TNF-stimulation. EMSA and Chromatin-IP revealed that pHSF1Ser230 binds to the heat shock response element in the DAPK promoter region and enhances its transcriptional activity. Exogenous over-expression of HSF1 protein led to a significant increase in mRNA DAPK levels and consequently to an enforcement of apoptosis. As expected, DAPK knockdown cells did not show any variation in pHSF1Ser230 level, supporting again that DAPK is an important mediator of the TNF-driven signaling pathway in colorectal cancer cells. The significance of the DAPK/pHSF1Ser230 interaction for response prediction was evaluated by immunohistochemical staining on tissue microarrays of colorectal cancer before and after radiotherapy. Conclusion: Our data show a novel functional interaction between HSF1 and DAPK under TNF-stress and highlight a positive feedback mechanism in DAPK-regulation. These results help to understand cell death pathways in response to radiotherapy. [1].Bajbouj K.et al., Am J Pathol. (2009) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4673A. doi:1538-7445.AM2012-4673A

Abstract 4779: TNF-induced Cofilin 1 phosphorylation by LIMK in colorectal tumor cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Objectives: Recently, we have shown that the pro-inflammatory cytokine TNF induces apoptosis in HCT116 colorectal tumor cells. Among the first apoptotic signs is a drastic change in cell morphology through modulation of the cytoskeleton. In order to identify and to characterize phosphorylation events and kinase targets associated with early cytoskeletal reorganization after TNF-treatment, we performed a peptide array screen. Methods: HCT116 tumor cells were cultured for 6h to 72 hours in either normal or TNF- conditioned medium, and radioactive labelled lysates were hybridized to a PepChip kinase full slide array for screening the phosphorylation status of over 400 proteins at over 1400 potential phosphorylation sites. For verification, cell lysates were collected and co-immunoprecipitated. The precipitated proteins were analyzed by western blotting. We verified the subcellular localization of proteins by co-immunofluorescence. Results: Pepscan array and western blotting revealed that cofilin 1 is markedly phosphorylated on Serine 3 (pCofSer3) after 24 and 48 hours of TNF-treatment. A potential up-stream kinase of cofilin is LIMK1, a serine/threonine protein kinase known to be involved in actin cytoskeleton reorganization through phosphorylation and inactivation of cofilin 1. Interaction between LIMK/Cofilin1/pCofSer3 was verified by co-immunoprecipitation and co-immunofluorescence. F-actin, cofilin1 and LIMK colocalized in membrane ruffles and lamellipodia, consistent with a cofilin-dependent actin cytoskeletal remodelling after TNF treatment. The effects were reversed by ROCK 1 inhibition. Conclusion: Our data show that TNF-treatment in HCT116 tumor cells induces the formation of a LIMK/Cofilin1/pCofSer3 protein complex that is localized in membrane ruffles and lamellipodia, suggesting that TNF-induced cytoskeletal alterations early during apoptosis are regulated through cofilin signalling. 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 4779. doi:10.1158/1538-7445.AM2011-4779