27 research outputs found

    CYLD deletion triggers nuclear factor-κB-signaling and increases cell death resistance in murine hepatocytes

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    TRAIL-induced apoptosis of hepatocellular carcinoma cells isaugmented by targeted therapies

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    AIM: To analyze the effect of chemotherapeutic drugs and specific kinase inhibitors, in combination with the death receptor ligand tumor necrosis factor-related apoptosis inducing ligand (TRAIL), on overcoming TRAIL resistance in hepatocellular carcinoma (HCC) and to study the efficacy of agonistic TRAIL antibodies, as well as the commitment of antiapoptotic BCL-2 proteins, in TRAIL-induced apoptosis. METHODS: Surface expression of TRAIL receptors (TRAIL-R1-4) and expression levels of the antiapoptotic BCL-2 proteins MCL-1 and BCL-x(L) were analyzed by flow cytometry and Western blotting, respectively. Knock-down of MCL-1 and BCL-x(L) was performed by transfecting specific small interfering RNAs. HCC cells were treated with kinase inhibitors and chemotherapeutic drugs. Apoptosis induction and cell viability were analyzed via flow cytometry and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS: TRAIL-R1 and -R2 were profoundly expressed on the HCC cell lines Huh7 and Hep-G2. However, treatment of Huh7 and Hep-G2 with TRAIL and agonistic antibodies only induced minor apoptosis rates. Apoptosis resistance towards TRAIL could be considerably reduced by adding the chemotherapeutic drugs 5-fluorouracil and doxorubicin as well as the kinase inhibitors LY294002 [inhibition of phosphoinositol-3-kinase (PI3K)], AG1478 (epidermal growth factor receptor kinase), PD98059 (MEK1), rapamycin (mammalian target of rapamycin) and the multi-kinase inhibitor Sorafenib. Furthermore, the antiapoptotic BCL-2 proteins MCL-1 and BCL-x(L) play a major role in TRAIL resistance: knock-down by RNA interference increased TRAIL-induced apoptosis of HCC cells. Additionally, knock-down of MCL-1 and BCL-x(L) led to a significant sensitization of HCC cells towards inhibition of both c-Jun N-terminal kinase and PI3K. CONCLUSION: Our data identify the blockage of survival kinases, combination with chemotherapeutic drugs and targeting of antiapoptotic BCL-2 proteins as promising ways to overcome TRAIL resistance in HCC

    Beyond cell death - antiapoptotic Bcl-2 proteins regulate migration and invasion of colorectal cancer cells in vitro.

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    Migration and invasion of malignant cells are prerequisites for cancer progression and metastasis. The Bcl-2 family of proteins consists of about 25 members and has been extensively studied in the context of apoptosis. Despite the fact that small molecules targeting Bcl-2 proteins have already entered clinical trials, very few studies investigated a role of antiapoptotic Bcl-2 proteins beside cell death in the context of metastasis. The aim of this study was to dissect a potential role of the antiapoptotic Bcl-2 proteins Mcl-1, Bcl-2 and Bcl-xL on migration and invasion of colorectal cancer cells independent of their cell death control function. We used migration and invasion assays as well as three dimensional cell cultures to analyze colorectal cancer cell lines (HT29 and SW480) after siRNA mediated knockdown or overexpression of Mcl-1, Bcl-2 or Bcl-xL. We observed neither spontaneous cell death induction nor impaired proliferation of cells lacking Mcl-1, Bcl-2 or Bcl-xL. In contrast, knockdown of Mcl-1 led to increased proliferation. Strikingly, we demonstrate a profound impairment of both, migration and invasion, of colorectal cancer cells after Mcl-1, Bcl-2 or Bcl-xL knockdown. This phenotype was completely revised in cells overexpressing Mcl-1, Bcl-2 or Bcl-xL. The most pronounced effect among the investigated proteins was observed for Bcl-2. The data presented indicate a pivotal role of Mcl-1, Bcl-2 and Bcl-xL for migration and invasion of colorectal cancer cells independent of their known antiapoptotic effects. Thus, our study illustrates novel antitumoral mechanisms of Bcl-2 protein targeting

    Bcl-xL and Myeloid cell leukaemia-1 contribute to apoptosis resistance of colorectal cancer cells

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    AIM: To explore the role of Bcl-xL and Myeloid cell leukaemia (Mcl)-1 for the apoptosis resistance of colorectal carcinoma (CRC) cells towards current treatment modalities

    Pan-Bcl-2 Inhibitor Obatoclax Delays Cell Cycle Progression and Blocks Migration of Colorectal Cancer Cells

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    <div><p>Despite the fact that new treatment regimes have improved overall survival of patients challenged by colorectal cancer (CRC), prognosis in the metastatic situation is still restricted. The Bcl-2 family of proteins has been identified as promising anti cancer drug target. Even though small molecules targeting Bcl-2 proteins are in clinical trials, little is known regarding their effects on CRC. The aim of this study was to preclinically investigate the value of ABT-737 and Obatoclax as anticancer drugs for CRC treatment. The effects of the BH3-mimetics ABT-737 and Obatoclax on CRC cells were assessed using viability and apoptosis assays. Wound healing migration and boyden chamber invasion assays were applied. 3-dimensional cell cultures were used for long term assessment of invasion and proliferation. Clinically relevant concentrations of pan-Bcl-2 inhibitor Obatoclax did not induce cell death. In contrast, the BH3-mimetic ABT-737 induced apoptosis in a dose dependent manner. Obatoclax caused a cell line specific slowdown of CRC cell growth. Furthermore, Obatoclax, but not ABT-737, recovered E-Cadherin expression and led to impaired migration and invasion of CRC cells. The proliferative capacity and invasiveness of CRC cells was strikingly inhibited by low dose Obatoclax in long term 3-dimensional cell cultures. Obatoclax, but not ABT-737, caused a G1-phase arrest accompanied by a downregulation of Cyclin D1 and upregulation of p27 and p21. Overexpression of Mcl-1, Bcl-x<sub>L</sub> or Bcl-2 reversed the inhibitory effect of Obatoclax on migration but failed to restore the proliferative capacity of Obatoclax-treated CRC cells. The data presented indicate broad and multifaceted antitumor effects of the pan-Bcl-2 inhibitor Obatoclax on CRC cells. In contrast to ABT-737, Obatoclax inhibited migration, invasion and proliferation in sublethal doses. In summary, this study recommends pan-Bcl-2 inhibition as a promising approach for clinical trials in CRC.</p></div

    Western blot analysis for E-Cadherin and antiapoptotic Bcl-2 proteins in CRC cells treated with Obatoclax.

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    <p>(<b>A</b>) Western blotting for E-Cadherin in four CRC cell lines after 24 h treatment with Obatoclax in escalating doses (left). Corresponding densitometric analysis relative to untreated controls and adjusted to Tubulin as loading control. (<b>B</b>) Western blotting for Mcl-1, Bcl-2 and Bcl-x<sub>L</sub> in HT29 cells (left) and SW480 cells (right) after 24 h treatment with Obatoclax. Tubulin served as a loading control. Western blots are representative for at least three blots from independent experiments.</p

    Migration of CRC cells treated with Obatoclax for 7 days in 3D scaffolds.

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    <p>(<b>A</b>) Representative pictures of HT29 cells in scaffolds after 7 days treatment with Obatoclax (Hematoxylin and Eosin staining. Scale bar applies for both pictures) (<b>B</b>) Corresponding analysis of invasion depth in scaffolds. Assays were performed in triplicates. Bars represent mean ± SD. Assays are representative of at least three independent experiments. ***p<0.001. Oba  =  Obatoclax.</p

    Invasion of SW480 cells treated with Obatoclax for 72 h in Matrigel coated Boyden chambers.

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    <p>SW480 cells were seeded into the upper chamber of a transwell. 48 h after seeding, nuclei on the lower surface were visualized by Hoechst staining. (<b>A</b>) Representative pictures of lower insert surface after Hoechst staining (scale bar indicate magnification for all panels). (<b>B</b>) Five fields of view per insert were counted. N = 5 per group. Values are expressed as mean ± SD. Assays are representative of at least three independent experiments. ***p<0.001. Oba  =  Obatoclax, ctrl  =  control.</p
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