Low levels of Caspase-3 predict favourable response to 5FU-based chemotherapy in advanced colorectal cancer: Caspase-3 inhibition as a therapeutic approach.

Colorectal cancer (CRC) is one of the most common cancers in the Western world. 5-Fluorouracil (5FU)-based chemotherapy (CT) remains the mainstay treatment of CRC in the advanced setting, and activates executioner caspases in target cells. Executioner caspases are key proteins involved in cell disassembly during apoptosis. Activation of executioner caspases also has a role in tissue regeneration and repopulation by stimulating signal transduction and cell proliferation in neighbouring, non-apoptotic cells as reported recently. Tissue microarrays (TMAs) consisting of tumour tissue from 93 stage II and III colon cancer patients were analysed by immunohistochemistry. Surprisingly, patients with low levels of active Caspase-3 had an increased disease-free survival time. This was particularly pronounced in patients who received 5FU-based adjuvant CT. In line with this observation, lower serum levels of active Caspase-3 were found in patients with metastasised CRC who revealed stable disease or tumour regression compared with those with disease progression. The role of Caspase-3 in treatment responses was explored further in primary human tumour explant cultures from fresh patient tumour tissue. Exposure of explant cultures to 5FU-based CT increased the percentage of cells positive for active Caspase-3 and Terminal Deoxynucleotidyl Transferase dUTP Nick end Labelling (TUNEL), but also the expression of regeneration and proliferation markers β-Catenin and Ki-67, as well as cyclooxygenase-2 (COX-2). Of note, selective inhibition of Caspase-3 with Ac-DNLD-CHO, a selective, reversible inhibitor of Caspase-3, significantly reduced the expression of proliferation markers as well as COX-2. Inhibition of COX-2 with aspirin or celecoxib did not affect Caspase-3 levels but also reduced Ki-67 and β-Catenin levels, suggesting that Caspase-3 acted via COX-2 to stimulate cell proliferation and tissue regeneration. This indicates that low levels of active Caspase-3 may represent a new predictor of CT responsiveness, and inhibition of Caspase-3, or antagonising downstream effectors of Caspase-3 paracrine signalling, such as COX-2 may improve patient outcomes following CT in advanced CRC

Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1

The wogonin-containing herb Scutellaria baicalensis has successfully been used for curing various diseases in traditional Chinese medicine. Wogonin has been shown to induce apoptosis in different cancer cells and to suppress growth of human cancer xenografts in vivo. However, its direct targets remain unknown. In this study, we demonstrate for the first time that wogonin and structurally related natural flavones, for example, apigenin, chrysin and luteolin, are inhibitors of cyclin-dependent kinase 9 (CDK9) and block phosphorylation of the carboxy-terminal domain of RNA polymerase II at Ser2. This effect leads to reduced RNA synthesis and subsequently rapid downregulation of the short-lived anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) resulting in apoptosis induction in cancer cells. We show that genetic inhibition of Mcl-1 or CDK9 expression by siRNA is sufficient to mimic flavone-induced apoptosis. Pull-down and in silico docking studies demonstrate that wogonin directly binds to CDK9, presumably to the ATP-binding pocket. In contrast, wogonin does not inhibit CDK2, CDK4 and CDK6 at doses that inhibit CDK9 activity. Furthermore, we show that wogonin preferentially inhibits CDK9 in malignant compared with normal lymphocytes. Thus, our study reveals a new mechanism of anti-cancer action of natural flavones and supports CDK9 as a therapeutic target in oncology

Suppression of Mcl-1 via RNA interference sensitizes human hepatocellular carcinoma cells towards apoptosis induction

BACKGROUND: Hepatocelluar carcinoma (HCC) is one of the most common cancers worldwide and a major cause of cancer-related mortality. HCC is highly resistant to currently available chemotherapeutic drugs. Defects in apoptosis signaling contribute to this resistance. Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 protein family which interferes with mitochondrial activation. In a previous study we have shown that Mcl-1 is highly expressed in tissues of human HCC. In this study, we manipulated expression of the Mcl-1 protein in HCC cells by RNA interference and analyzed its impact on apoptosis sensitivity of HCC cells in vitro. METHODS: RNA interference was performed by transfecting siRNA to specifically knock down Mcl-1 expression in HCC cells. Mcl-1 expression was measured by quantitative real-time PCR and Western blot. Induction of apoptosis and caspase activity after treatment with chemotherapeutic drugs and different targeted therapies were measured by flow cytometry and fluorometric analysis, respectively. RESULTS: Here we demonstrate that Mcl-1 expressing HCC cell lines show low sensitivity towards treatment with a panel of chemotherapeutic drugs. However, treatment with the anthracycline derivative epirubicin resulted in comparatively high apoptosis rates in HCC cells. Inhibition of the kinase PI3K significantly increased apoptosis induction by chemotherapy. RNA interference efficiently downregulated Mcl-1 expression in HCC cells. Mcl-1 downregulation sensitized HCC cells to different chemotherapeutic agents. Sensitization was accompanied by profound activation of caspase-3 and -9. In addition, Mcl-1 downregulation also increased apoptosis rates after treatment with PI3K inhibitors and, to a lower extent, after treatment with mTOR, Raf I and VEGF/PDGF kinase inhibitors. TRAIL-induced apoptosis did not markedly respond to Mcl-1 knockdown. Additionally, knockdown of Mcl-1 efficiently enhanced apoptosis sensitivity towards combined treatment modalities: Mcl-1 knockdown significantly augmented apoptosis sensitivity of HCC cells towards chemotherapy combined with PI3K inhibition. CONCLUSION: Our data suggest that specific downregulation of Mcl-1 by RNA interference is a promising approach to sensitize HCC cells towards chemotherapy and molecularly targeted therapies

Development of Mouse Hepatocyte Lines Permissive for Hepatitis C Virus (HCV)

The lack of a suitable small animal model for the analysis of hepatitis C virus (HCV) infection has hampered elucidation of the HCV life cycle and the development of both protective and therapeutic strategies against HCV infection. Human and mouse harbor a comparable system for antiviral type I interferon (IFN) induction and amplification, which regulates viral infection and replication. Using hepatocytes from knockout (ko) mice, we determined the critical step of the IFN-inducing/amplification pathways regulating HCV replication in mouse. The results infer that interferon-beta promoter stimulator (IPS-1) or interferon A receptor (IFNAR) were a crucial barrier to HCV replication in mouse hepatocytes. Although both IFNARko and IPS-1ko hepatocytes showed a reduced induction of type I interferons in response to viral infection, only IPS-1-/- cells circumvented cell death from HCV cytopathic effect and significantly improved J6JFH1 replication, suggesting IPS-1 to be a key player regulating HCV replication in mouse hepatocytes. We then established mouse hepatocyte lines lacking IPS-1 or IFNAR through immortalization with SV40T antigen. Expression of human (h)CD81 on these hepatocyte lines rendered both lines HCVcc-permissive. We also found that the chimeric J6JFH1 construct, having the structure region from J6 isolate enhanced HCV replication in mouse hepatocytes rather than the full length original JFH1 construct, a new finding that suggests the possible role of the HCV structural region in HCV replication. This is the first report on the entry and replication of HCV infectious particles in mouse hepatocytes. These mouse hepatocyte lines will facilitate establishing a mouse HCV infection model with multifarious applications

Synergistic induction of cell death in liver tumor cells by TRAIL and chemotherapeutic drugs via the BH3-only proteins Bim and Bid

Although death receptors and chemotherapeutic drugs activate distinct apoptosis signaling cascades, crosstalk between the extrinsic and intrinsic apoptosis pathway has been recognized as an important amplification mechanism. Best known in this regard is the amplification of the Fas (CD95) signal in hepatocytes via caspase 8-mediated cleavage of Bid and activation of the mitochondrial apoptosis pathway. Recent evidence, however, indicates that activation of other BH3-only proteins may also be critical for the crosstalk between death receptors and mitochondrial triggers. In this study, we show that TNF-related apoptosis-inducing ligand (TRAIL) and chemotherapeutic drugs synergistically induce apoptosis in various transformed and untransformed liver-derived cell lines, as well as in primary human hepatocytes. Both, preincubation with TRAIL as well as chemotherapeutic drugs could sensitize cells for apoptosis induction by the other respective trigger. TRAIL induced a strong and long lasting activation of Jun kinase, and activation of the BH3-only protein Bim. Consequently, synergistic induction of apoptosis by TRAIL and chemotherapeutic drugs was dependent on Jun kinase activity, and expression of Bim and Bid. These findings confirm a previously defined role of TRAIL and Bim in the regulation of hepatocyte apoptosis, and demonstrate that the TRAIL–Jun kinase–Bim axis is a major and important apoptosis amplification pathway in primary hepatocytes and liver tumor cells

Safety and pharmacokinetics of recombinant human hepatocyte growth factor (rh-HGF) in patients with fulminant hepatitis: a phase I/II clinical trial, following preclinical studies to ensure safety

<p>Abstract</p> <p>Background</p> <p>Hepatocyte growth factor (HGF) stimulates hepatocyte proliferation, and also acts as an anti-apoptotic factor. Therefore, HGF is a potential therapeutic agent for treatment of fatal liver diseases. We performed a translational medicine protocol with recombinant human HGF (rh-HGF), including a phase I/II study of patients with fulminant hepatitis (FH) or late-onset hepatic failure (LOHF), in order to examine the safety, pharmacokinetics, and clinical efficacy of this molecule.</p> <p>Methods</p> <p>Potential adverse effects identified through preclinical safety tests with rh-HGF include a decrease in blood pressure (BP) and an increase in urinary excretion of albumin. Therefore, we further investigated the effect of rh-HGF on circulatory status and renal toxicity in preclinical animal studies. In a clinical trial, 20 patients with FH or LOHF were evaluated for participation in this clinical trial, and four patients were enrolled. Subjects received rh-HGF (0.6 mg/m²/day) intravenously for 12 to 14 days.</p> <p>Results</p> <p>We established an infusion method to avoid rapid BP reduction in miniature swine, and confirmed reversibility of renal toxicity in rats. Although administration of rh-HGF moderately decreased BP in the participating subjects, this BP reduction did not require cessation of rh-HGF or any vasopressor therapy; BP returned to resting levels after the completion of rh-HGF infusion. Repeated doses of rh-HGF did not induce renal toxicity, and severe adverse events were not observed. Two patients survived, however, there was no evidence that rh-HGF was effective for the treatment of FH or LOHF.</p> <p>Conclusions</p> <p>Intravenous rh-HGF at a dose of 0.6 mg/m²was well tolerated in patients with FH or LOHF; therefore, it is desirable to conduct further investigations to determine the efficacy of rh-HGF at an increased dose.</p

Epidermal growth factor regulates Mcl-1 expression through the MAPK-Elk-1 signalling pathway contributing to cell survival in breast cancer

Myeloid cell leukaemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 family that is elevated in a variety of tumour types including breast cancer. In breast tumours, increased Mcl-1 expression correlates with high tumour grade and poor patient survival. We have previously demonstrated that Her-2 levels correspond to increased Mcl-1 expression in breast tumours. Epidermal growth factor (EGF) receptor signalling is frequently deregulated in breast cancer and leads to increased proliferation and survival. Herein, we determined the critical downstream signals responsible for the EGF mediated increase of Mcl-1 and their role in cell survival. We found that both Mcl-1 mRNA and protein levels are rapidly induced upon stimulation with EGF. Promoter analysis revealed that an Elk-1 transcription factor-binding site is critical for EGF activation of the Mcl-1 promoter. Furthermore, we found that knockdown of Elk-1or inhibition of the Erk signalling pathway was sufficient to block EGF upregulation of Mcl-1 and EGF mediated cell survival. Using chromatin immunoprecipitation and biotin labelled probes of the Mcl-1 promoter, we found that Elk-1 and serum response factor are bound to the promoter after EGF stimulation. To determine whether Mcl-1 confers a survival advantage, we found that knockdown of Mcl-1 expression increased apoptosis whereas overexpression of Mcl-1 inhibited drug induced cell death. In human breast tumours, we found a correlation between phosphorylated Elk-1 and Mcl-1 protein levels. These results indicate that the EGF induced activation of Elk-1 is an important mediator of Mcl-1 expression and cell survival and therefore a potential therapeutic target in breast cancer