Bernd Kronenberger

Tumor regression by combination antisense therapy against Plk1 and Bcl-2

Increased expression of the cell proliferation-associated polo-like kinase 1 (PLK1) and apoptosis-associated BCL-2 genes has been observed in different human malignancies. Inhibition of cell proliferation and reactivation of apoptosis are basic principles in anticancer therapy. The efficiency of this approach is often limited by insuf-ficient targeting and delivery of anticancer drugs into the tumors. Phosphorothioate antisense oligodeoxynucleotides (ODNs) directed against PLK1 and BCL-2 were administered systemically via the tail vein into nude mice bearing A549, MDA-MB-435, and Detroit562 xenografts. To enhance tumor-specific uptake and to reduce systemic toxicity of antisense ODNs membrane electroporation transfer was applied in vivo. Northern and Western blot analyses were used to assess PLK1 and BCL-2 expression. Tumor mass was assessed after resection of tumors. All three cell lines and corresponding xenografts expressed high levels of PLK1 and were sensitive towards antisense PLK1 treatment. Antisense BCL-2 therapy was effective in tumors expressing high levels of BCL-2, but not in A549 cells and corresponding xenografts, which express low levels of BCL-2. Administration of antisense ODNs in a dose of 5u2009mg/kg, twice weekly during four weeks supported by the membrane electroporation transfer, eradicated 60–100% of the xenografted tumors. Antitumor effect in BCL-2 overexpressing MDA-MB-435 cells was synergistic for BCL-2 and PLK1 combination therapy. This study provides evidence that combined systemic administration of antisense ODNs against proliferation and pro- survival associated targets and in vivo electroporation of tumors represents a promising antitumor therapeutic approach.

Differential Stability of Cell-Free Circulating microRNAs: Implications for Their Utilization as Biomarkers

Background MicroRNAs circulating in the blood, stabilized by complexation with proteins and/or additionally by encapsulation in lipid vesicles, are currently being evaluated as biomarkers. The consequences of their differential association with lipids/vesicles for their stability and use as biomarkers are largely unexplored and are subject of the present study. Methods The levels of a set of selected microRNAs were determined by quantitative reverse-transcription PCR after extraction from sera or vesicle- and non-vesicle fractions prepared from sera. The stability of these microRNAs after incubation with RNase A or RNase inhibitor, an inhibitor of RNase A family enzymes was studied. Results The levels of microRNA-1 and microRNA-122, but not those of microRNA-16, microRNA-21 and microRNA-142-3p, declined significantly during a 5-h incubation of the sera. RNase inhibitor prevented the loss of microRNAs in serum as well as the degradation of microRNA-122, a microRNA not expressed in blood cells, in whole blood. Stabilization of microRNA-122 was also achieved by hemolysis. Prolonged incubation of the sera led to enrichment of vesicle-associated relative to non-vesicle-associated microRNAs. Vesicle-associated microRNAs were more resistant to RNase A treatment than the respective microRNAs not associated with vesicles. Conclusions Serum microRNAs showed differential stability upon prolonged incubation. RNase inhibitor might be useful to robustly preserve the pattern of cell-free circulating microRNAs. In the case of microRNAs not expressed in blood cells this can also be achieved by hemolysis. Vesicle-associated microRNAs appeared to be more stable than those not associated with vesicles, which might be useful to disclose additional biomarker properties of miRNAs.

Viral kinetics during antiviral therapy in patients with chronic hepatitis C and persistently normal ALT levels

The aim of the present study was to compare viral kinetics between patients with chronic hepatitis C and persistently normal alanine aminotransferase (ALT) levels and those with elevated ALT levels. Kinetic parameters were derived from nonlinear, least square fitting of serum hepatitis C virus RNA quantifications collected from patients with chronic hepatitis C and persistently normal (n = 20) and elevated (n = 19) ALT levels before and during treatment with 180 μg pegylated interferon α‐2a once weekly plus daily ribavirin. Patients with chronic hepatitis C and persistently normal ALT levels showed a trend to lower pretreatment infected cell loss (δ) (P = .13) but no differences in efficacy of blocking virus production (ϵ) and infected cell loss during treatment (mδ) compared with patients with elevated ALT levels. Differences were significant for ϵ (P = .02) and δ (P = .04) when applying updated “healthy” levels for ALT (0.75 times and 0.63 times upper limit of normal for male and female patients, respectively). A significant reduction of the kinetic parameters ϵ, δ, and mδ was observed in patients with elevated γ‐glutamyltranspeptidase (GGT) levels compared with patients with normal GGT levels (P = .02, P = .005, and P = .02, respectively). In conclusion, viral kinetics are similar in patients with chronic hepatitis C and persistently normal ALT levels and those with elevated ALT levels. However, in patients with elevated GGT levels, a major association with reduced efficacy of blocking virus production and lower infected cell loss was observed. These data show that virological response in patients with chronic hepatitis C is less associated with baseline ALT than with GGT levels. (HEPATOLOGY 2004;40:1442–1449.)

High levels of the soluble programmed death-ligand (sPD-L1) identify hepatocellular carcinoma patients with a poor prognosis

AIMnImmunotherapy in cancer is a recent and very promising approach, namely the inhibition of the PD/programmed death-ligand 1 (PD-L1) axis. Here we aimed to investigate the prognostic value of a soluble form of PD-L1 in hepatocellular carcinoma (HCC) patients.nnnMETHODSnHCC patients were prospectively recruited and soluble programmed death-ligandxa01xa0(sPD-L1) levels were determined. sPD-L1 levels were compared to stages of cirrhosisxa0and HCC. The association of the sPD-L1 levels and overall survival (OS) was assessed.nnnRESULTSnTwo hundred fifteen patients with HCC were prospectively included. The median serum sPD-L1 concentration in patients with HCC was 0.5xa0ng/ml (range 0.03-6.04). Soluble PD-L1 levels positively correlated with the stage of cirrhosis and with stages of HCC. Furthermore, sPD-L1 correlated positively with a marker of macrophage activation (sCD163) and inflammation (C-reactive protein). The cut-off for high-level sPD-L1 (>0.8xa0ng/ml) was defined by sPD-L1 levels determined in a healthy control cohort. Patients with high serum sPD-L1 concentrations had an increased mortality risk (hazard ratio 3.340, 95 % confidence interval 1.609-6.934, P<0.001), while very low PD-L1 levels seem to come along with better prognosis. High sPD-L1 levels were associated with mortality independently from cirrhosis stage, alpha-fetoprotein and sCD163 levels in a multivariate Cox regression model.nnnCONCLUSIONSnWe conclude that a high sPD-L1 level is a possible prognostic indicator for a poor outcome in HCC patients. The predictive value of sPD-L1 levels for a successful anti-PD1/PD-L1 therapy should be investigated in the future.

Improving Drug Penetrability with iRGD Leverages the Therapeutic Response to Sorafenib and Doxorubicin in Hepatocellular Carcinoma.

iRGD is a derivative of the integrin-binding peptide RGD, which selectively increases the penetrability of tumor tissue to various coadministered substances in several preclinical models. In this study, we investigated the ability of iRGD to improve the delivery of sorafenib and doxorubicin therapy in hepatocellular carcinoma (HCC) using established mouse models of the disease. A contrast-enhanced MRI method was developed in parallel to assess the in vivo effects of iRGD in this setting. We found that iRGD improved the delivery of marker substances to the tumors of HCC-bearing mice about three-fold without a parallel increase in normal tissues. Control peptides lacking the critical CendR motif had no effect. Similarly, iRGD also selectively increased the signal intensity from tumors in Gd-DTPA-enhanced MRI. In terms of antitumor efficacy, iRGD coadministration significantly augmented the individual inhibitory effects of sorafenib and doxorubicin without increasing systemic toxicity. Overall, our results offered a preclinical proof of concept for the use of iRGD coadministration as a strategy to widen the therapeutic window for HCC chemotherapy, as monitored by Gd-DTPA-enhanced MRI as a noninvasive, clinically applicable method to identify iRGD-reactive tumors.

Protein kinase A mediates cAMP-induced tyrosine phosphorylation of the epidermal growth factor receptor

An increase in the intracellular cAMP concentration induces tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) followed by activation of extracellular signal-regulated kinases 1/2 (ERK1/2). In this report we demonstrate that these effects of cAMP are mediated via activation of protein kinase A (PKA). Chemical inhibition of PKA suppressed forskolin-induced EGFR tyrosine phosphorylation and ERK1/2 activation in PC12 cells. Furthermore, forskolin failed to induce significant tyrosine phosphorylation of the EGFR and ERK1/2 activation in PKA-defective PC12 cells. Forskolin-induced EGFR tyrosine phosphorylation was also observed in A431 cells and in membranes isolated from these cells. Phosphoamino acid analysis indicated that the recombinant catalytic subunit of PKA elicited phosphorylation of the EGFR on both tyrosine and serine but not threonine residues in A431 membranes. Together, our data indicate that activation of PKA mediates the effects of cAMP on the EGFR and ERK1/2. While PKA may directly phosphorylate the EGFR on serine residues, PKA-induced tyrosine phosphorylation of the EGFR occurs by an indirect mechanism.

Serum sphingolipidomic analyses reveal an upregulation of C16- ceramide and sphingosine-1-phosphate in hepatocellular carcinoma

We have recently shown that major alterations of serum sphingolipid metabolites in chronic liver disease associate significantly with the stage of liver fibrosis in corresponding patients. In the current study we assessed via mass spectrometry serum concentrations of sphingolipid metabolites in a series of 122 patients with hepatocellular carcinoma (HCC) compared to an age- and sex-matched series of 127 patients with cirrhosis. We observed a highly significant upregulation of long and very long chain ceramides (C16-C24) in the serum of patients with HCC as compared to patients with cirrhosis (P < 0.001). Accordingly, dihydro-ceramides, synthetic precursors of ceramides and notably sphingosine, sphingosine-1-phosphate (S1P) and sphinganine-1-phosphate (SA1P) were upregulated in patients with HCC (P < 0.001). Especially the diagnostic accuracy of C16-ceramide and S1P, assessed by receiver operating curve (ROC) analysis, showed a higher area under the curve (AUC) value as compared to alpha fetoprotein (AFP) (0.999 and 0.985 versus 0.823, P < 0.001 respectively). In conclusion, serum levels of sphingolipid metabolites show a significant upregulation in patients with HCC as compared to patients with cirrhosis. Particularly C16-ceramide and S1P may serve as novel diagnostic markers for the identification of HCC in patients with liver diseases. Our data justify further investigations on the role of sphingolipids in HCC.

BAX and caspase-5 frameshift mutations and spontaneous apoptosis in colorectal cancer with microsatellite instability

Background and aimsHereditary nonpolyposis colorectal cancer (HNPCC) and a subset of sporadic colorectal cancers are characterized by microsatellite instability (MSI) and inactivating frameshift mutations of target genes. Inactivation of BAX, caspase-5 (cas-5), and other genes coding for pro-apoptotic proteins might contribute to tumor progression by enhancing escape from apoptosis. The aim of this study was to further characterize the role of BAX and cas-5 inactivation for spontaneous apoptosis.MethodsTwenty-five colorectal cancers with MSI were analyzed for frameshift mutations in the BAX (G)8 and cas-5 (A)10 tract by fluorescence PCR, cloning, and sequencing. The rate of spontaneous apoptosis was examined by in situ DNA nick end-labeling. The results were compared with 25 stage-matched microsatellite stable (MSS) colorectal cancers.ResultsIn colorectal cancer with MSI frameshift mutations in BAX and cas-5 were present in 16 of 25 (64%) and in 12 of 25 (48%) tumors, respectively, whereas neither mutant BAX nor cas-5 alleles were detected in all stage-matched sporadic MSS colorectal cancer. Tumors with MSI showed a higher apoptotic rate than MSS tumors (2.5±1.0 vs. 2.1±0.7; p <0.05), whereas the presence of BAX or cas-5 frameshift mutations had only minor influence on this finding (2.4±1.1% and 2.5±0.9%, respectively).ConclusionMismatch-repair deficiency itself is associated with increased spontaneous apoptosis, not further accelerated by either inactivating BAX or cas-5 frameshift mutations.

Efficacy of amantadine on quality of life in patients with chronic hepatitis C treated with interferon-α and ribavirin : results from a randomized, placebo-controlled, double-blind trial

Aim The aim of this study was to investigate whether amantadine reduces deterioration of quality of life in patients with chronic hepatitis C during and after treatment with interferon-&agr; (IFN-&agr;) and ribavirin. Patients and methods In this randomized, prospective, placebo-controlled, multicenter trial, previously untreated patients with chronic hepatitis C were treated with IFN-&agr; plus ribavirin [17] and randomized for treatment with amantadine (200u2009mg/day, orally, n=136) or placebo (n=131). Quality of life was assessed with the ‘Profile of Mood States’ scale and the ‘Everyday Life’ questionnaire at baseline, treatment week (TW) 8, TW24, TW48, and at follow-up. Results Early during treatment at TW8, quality of life was not different between patients in the control and the amantadine group. At TW24, the control group but not the amantadine group, however, showed significant deterioration of the modalities depression, fatigue, and vigor compared with baseline. Especially, nonresponders in the amantadine group showed significantly lower deterioration of depression, anger, mind function, everyday life, and zest for life than those in the placebo group. After treatment, the beneficial effects of amantadine disappeared. Conclusion The addition of amantadine to IFN-&agr; plus ribavirin combination therapy may reduce deterioration of depression, fatigue, and vigor during treatment but does not affect quality of life after treatment.

Dynamics of CD81 expression on lymphocyte subsets during interferon‐α‐based antiviral treatment of patients with chronic hepatitis C

CD81 is a hepatitis C virus (HCV) coreceptor with important functions in lymphocytes. During treatment, CD81 expression may be changed directly by the antiviral therapy or indirectly by reduction of the HCV serum level. The regulation of CD81 on lymphocyte subtypes has not been investigated so far and may be relevant for the control of viral infection and treatment response. CD81 was analyzed by flow cytometry in CD8(+), CD4(+), CD19(+), and CD56(+) lymphocyte subtypes from 20 patients with chronic hepatitis C before, during, and after antiviral treatment with pegylated interferon‐α (IFN‐α) and ribavirin. A sustained virologic response (SVR) was achieved in 11 patients. Dynamics of CD81 were investigated in correlation with HCV‐RNA dynamics and the outcome of therapy. During treatment, the following typical patterns of CD81 regulation were observed: down‐regulation on CD8(+) T cells (P=0.022) and most significantly, on CD56(+) natural killer cells (P<0.001), transient up‐regulation on CD19(+) B cells (P=0.006), and weak and late down‐regulation on CD4(+) T cells (P=0.028). During treatment, CD81 expression was not associated with the HCV‐RNA serum level on all lymphocyte subtypes. After end of treatment, CD81 increased again in CD8(+) and CD56(+) cells (P=0.001, P=0.002). On CD8(+) T cells post‐treatment, CD81 remained lower in patients who achieved a SVR compared with patients who failed to eliminate HCV after treatment (P=0.033). Lymphocyte subsets show different patterns of CD81 response before and during antiviral treatment, which are associated with administration of IFN‐α and antiviral response.