Differential role of circulating endothelial progenitor cells in cirrhotic patients with or without hepatocellular carcinoma.

BACKGROUND AND AIMS: Circulating endothelial progenitor cells have a negative prognostic impact in patients with hepatocellular carcinoma, but may play a different role in portal hypertension according to preclinical data. Here, we address this issue for the first time in cirrhotic patients+/-hepatocellular carcinoma. METHODS: Portal hypertension in cirrhotic and hepatocellular carcinoma patients was determined by hepatic venous pressure gradient. Blood cells staining positive for CD34/KDR/AC133 using flow cytometry were characterised as endothelial progenitor cells. Vascular endothelial growth factor levels were determined by ELISA. RESULTS: Endothelial progenitor cells levels in peripheral blood were elevated in cirrhotic (n=23) (mean: 0.12+/-0.06% S.D.) and in hepatocellular carcinoma patients (n=24) (0.14+/-0.09% S.D.) relative to healthy controls (H-group, n=15) (0.06+/-0.04% S.D.) (P=0.056 and P=0.02, respectively). There were higher vascular endothelial growth factor levels in hepatocellular carcinoma patients compared to cirrhotics (P=0.047) and HC (P=0.037). Notably, hepatic venous pressure gradient was positively correlated with vascular endothelial growth factor (r=0.5, P=0.046) but negatively with endothelial progenitor cells levels (r=-0.51, P=0.02) in cirrhotics, but not hepatocellular carcinoma patients. CONCLUSION: Circulating endothelial progenitor cells are increased in patients with portal hypertension+/-hepatocellular carcinoma. The negative correlation of endothelial progenitor cells with hepatic venous pressure gradient suggests a protective role of endothelial progenitor cells in liver cirrhosis whilst vascular endothelial growth factor is associated with high hepatic venous pressure gradient. In contrast, increased endothelial progenitor cells in hepatocellular carcinoma rather reflect tumour specific endothelial progenitor cells mobilisation.

FDG uptake is a surrogate marker for defining the optimal biological dose of the mTOR inhibitor everolimus in vivo.

This study aimed to test whether [(18)F]fluoro-D-glucose (FDG) uptake of tumours measured by positron emission tomography (PET) can be used as surrogate marker to define the optimal biological dose (OBD) of mTOR inhibitors in vivo. Everolimus at 0.05, 0.5, 5 and 15 mg kg(-1) per day was administered to gastric cancer xenograft-bearing mice for 23 days and FDG uptake of tumours was measured using PET from day 1 to day 8. To provide standard comparators for FDG uptake, tumour volume, S6 protein phosphorylation, Ki-67 staining and everolimus blood levels were evaluated. Everolimus blood levels increased in a dose-dependent manner but antitumour activity of everolimus reached a plateau at doses >or=5 mg kg(-1) per day (tumour volume treated vs control (T/C): 51% for 5 mg kg(-1) per day and 57% for 15 mg kg(-1) per day). Correspondingly, doses >or=5 mg kg(-1) per day led to a significant reduction in FDG uptake of tumours. Dose escalation above 5 mg kg(-1) per day did not reduce FDG uptake any further (FDG uptake T/C: 49% for 5 mg kg(-1) per day and 52% for 15 mg kg(-1) per day). Differences in S6 protein phosphorylation and Ki-67 index reflected tumour volume and changes in FDG uptake but did not reach statistical significance. In conclusion, FDG uptake might serve as a surrogate marker for dose finding studies for mTOR inhibitors in (pre)clinical trials.

Characterization of NVX-207, a novel betulinic acid-derived anti-cancer compound.

BACKGROUND: Development of betulinic acid derivatives for clinical use has been hampered by adverse pharmacological and physico-chemical characteristics of this class of compounds. We here present a novel semi-synthetic betulinic acid-derived drug candidate well suited for further clinical development. MATERIALS AND METHODS: In vitro activity and mode of action of NVX-207 were determined using normal as well as cancer cell lines. Gene expression profiling was performed with Affymetrix U133 microarrays. NVX-207 binding partners were identified using a heterobifunctional chemical crosslinker system. Potential binding proteins were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. Clinical studies were conducted in canine cancer patients suffering from spontaneously arising pre-treated tumours. RESULTS: NVX-207 showed anti-tumour activity (mean IC(50) = 3.5 microM) against various human and canine cell lines. NVX-207-induced apoptosis was associated with activation of the intrinsic apoptotic pathway via cleavage of caspases -9, -3, -7 and of poly (ADP-ribose) polymerase (PARP). Global gene expression profiling demonstrated regulation of genes associated with lipid metabolism, most notably an upregulation of genes coding for insulin-induced gene 1 (Insig-1), low-density lipoprotein receptor (LDL-R) and of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA). NVX-207 bound to apolipoprotein A-I, a major regulator of lipid metabolism and cholesterol transport. A phase I/II study in dogs suffering from naturally occurring cancer receiving local treatment of NVX-207 (10 mg mL(-1)) showed excellent clinical responses including a complete remission in so far 5/5 treated animals. CONCLUSIONS: NVX-207 is well tolerated and has significant anti-cancer activity in vitro and in vivo in dogs with treatment-resistant malignancies.

Suppression of mTOR complex 2-dependent AKT phosphorylation in melanoma cells by combined treatment with rapamycin and LY294002.

BACKGROUND: Inhibition of mTOR complex 1 (mTORC1) with rapamycin leads to phosphorylation of AKT in some cancer cells, with unknown biological consequences. The role of this phosphorylation in melanoma is unknown, although preliminary clinical data indicate poor activity of rapalogues in melanoma. OBJECTIVES: We aimed at elucidating the role of AKT phosphorylation after mTORC1 inhibition in melanoma cells. METHODS: Western blotting, apoptosis assays, cell cycle analyses and viability assays were performed to analyse the effects of rapamycin and LY294002 treatment on melanoma cells. For suppression of mTOR complex 2 (mTORC2) an siRNA directed against rictor was used. RESULTS: Rapamycin showed limited effects on cell viability but resulted in strong and lasting AKT phosphorylation in melanoma cells. Combined PI3K/mTOR inhibition with LY294002 had pronounced effects on viability but also led to increased AKT phosphorylation after prolonged treatment. In contrast, combination of rapamycin plus LY294002 suppressed AKT phosphorylation. Suppression of AKT phosphorylation did not correlate with decreases in cell viability. Inhibition of mTORC2 led to reduced levels of phosphorylated AKT. CONCLUSIONS: mTORC1 inhibition with rapamycin and with LY294002 can lead to AKT phosphorylation in melanoma cells via mTORC2. Combination of rapamycin and LY294002 suppresses AKT phosphorylation but without significant effect on treatment efficacy.

A clinical trial on absorption and N-acetylation of oral and rectal mesalazine.

BACKGROUND: Mesalazine (5-ASA) is a standard treatment for ulcerative colitis. Extent of absorption and N-acetylation determine systemic exposure to 5-ASA, and are thereby relevant for the safety of the treatment. The aim of the study was to compare absorption and N-acetylation of 5-ASA following rectal or oral drug administration. Healthy subjects were compared to patients with ulcerative colitis to evaluate the impact of chronic inflammation of colorectal mucosa on disposition of 5-ASA. MATERIALS AND METHODS: First, 12 healthy adults were randomized to receive 2 g of 5-ASA by each of four different formulations: oral delayed release granules, 30 mL enema, 60 mL rectal foam, and 120 mL rectal foam. Second, 12 patients with active ulcerative colitis received 60 mL rectal foam. Pharmacokinetic analysis was performed by determination of 5-ASA and its acetylated, pharmacologically inactive metabolite (Ac-5-ASA) in plasma and urine. RESULTS: First, systemic exposure to 5-ASA was markedly lower after rectal drug administration as compared to oral dosing (P < 0.001; e.g. median relative bioavailability of 60 mL rectal foam: 36%). Second, N-acetylation of rectal 5-ASA was lower in patients than in healthy subjects [area under the curve (AUC) ratio Ac-5-ASA/5-ASA: 1.6 +/- 0.5 vs. 2.3 +/- 0.4, mean +/- SD, P < 0.01]. High peak plasma concentrations of 5-ASA were correlated with high microscopic disease activity (r = 0.67, P < 0.05). CONCLUSIONS: Rectal delivery of 5-ASA results in low systemic drug exposure with potentially reduced toxicity in comparison with oral drug administration. Chronic inflammation of colorectal mucosa might be a relevant source of variability in pharmacokinetics of 5-ASA.

Expression of the target receptor CD33 in CD34+/CD38-/CD123+ AML stem cells.

BACKGROUND: CD33 (Siglec-3) is becoming increasingly important as a target of antibody-mediated therapy in acute myeloid leukaemia (AML). In normal myelopoiesis, expression of CD33 is restricted to advanced stages of differentiation, whereas primitive stem cells do not express CD33. In the present study, we asked whether leukaemic stem cells in patients with AML express CD33. MATERIALS AND METHODS: A multicolour-staining technique was applied in 11 patients with AML, and leukaemic progenitors defined as CD34(+)/CD38(-)/CD123(+) cells. AML stem cells were purified by cell sorting and were examined for expression of CD33 mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: In all patients in whom the majority of myeloblasts expressed CD33 (n = 8), AML progenitors reacted with the CD33 antibody P67.6. Repopulation experiments utilizing irradiated NOD/SCID mice confirmed that AML stem cells in these patients reside within the CD33(+) subpopulation of the leukaemic clone. Moreover, highly purified AML stem cells (> 98% purity) from patients with CD33(+) AML were found to express CD33 mRNA in RT-PCR analyses. CD33 was neither detectable on CD34(+)/CD38(-) cells in normal bone marrow nor on leukaemic stem cells in patients with CD33-negative AML. CONCLUSIONS: Leukaemic stem cells in patients with CD33(+) AML express CD33. This observation is in favour of novel treatment concepts employing CD33-targeting antibodies in AML.

Antisense molecules for targeted cancer therapy.

The efficacy of traditional anti-cancer agents is hampered by toxicity to normal tissues, due to the lack of specificity for malignant cells. Recent advances in our understanding of molecular genetics and tumor biology have led to the identification of signaling pathways and their regulators implicated in tumorigenesis and malignant progression. Consequently, novel biological agents were designed which specifically target key regulators of cell survival and proliferation activated in malignant cells and thus are superior to unspecific cytotoxic agents. Antisense molecules comprising conventional single-stranded antisense oligonucleotides (ASO) and small interfering RNA (siRNA) inhibit gene expression on the transcript level. Thus, they specifically target the genetic basis of cancer and are particularly useful for inhibiting the expression of oncogenes the protein products of which are inaccessible to small molecules or inhibitory antibodies. Despite the somewhat disappointing results of recent antisense oncology trials, the identification of new cancer targets and ongoing progress in ASO and siRNA technology together with improvements in tumor targeted delivery have raised new hopes that this fascinating intervention concept will eventually translate into enhanced clinical efficacy.

Bcl-x(L) antisense oligonucleotides radiosensitise colon cancer cells.

Advanced colon cancer is a malignancy with poor response to various treatment modalities including ionising radiation (IR) and chemotherapy. Both IR and chemotherapeutic agents have been shown to act by inducing apoptosis, a type of cell death antagonised by the Bcl-x(L) gene product. Since approximately 60% of human colon cancers express Bcl-x(L), it was the aim of this study to explore the potential of Bcl-x(L) antisense oligonucleotides as a novel radiosensitisation strategy. Caco-2 colon cancer cells were treated with Bcl-x(L) antisense oligonucleotides in combination with IR or cisplatin, and Bcl-x(L) protein expression, apoptosis, cell viability and clonogenic survival were examined. Bcl-x(L) antisense oligonucleotide specifically reduced the Bcl-x(L) protein level by almost 50% in Caco-2 cells. The decreased threshold for the induction of apoptosis resulted in a 300% increase of apoptosis after IR or cisplatin treatment and led to a 60% reduction of cell proliferation beyond response rates achieved with IR. These data suggest that Bcl-x(L) is an important factor contributing to the treatment resistance of human colon cancer. Specific reduction of Bcl-x(L) protein levels by antisense oligonucleotides qualifies as a promising therapeutic strategy for colon cancer that may help overcome resistance and improve clinical outcome in this malignancy.

Compact, broad-bandwidth fiber laser for sub-2-microm axial resolution optical coherence tomography in the 1300-nm wavelength region.

A novel, compact, user friendly fiber laser with a broad emission bandwidth (MenloSystems, lambdac = 1375 nm, deltalambda = 470 nm, Pout = 4 mW) was used to achieve unprecedented sub-2-microm axial resolution optical coherence tomography (OCT) in nontransparent biological tissue in the 1300-nm wavelength region. Fresh human skin and arterial biopsies were imaged ex vivo with approximately 1.4-microm axial and approximately 3-microm lateral resolution and 95-dB sensitivity, demonstrating the great potential for clinical OCT applications of this stable, low-cost, and turn-on-key fiber laser.

Bcl-2 antisense oligonucleotides chemosensitize human gastric cancer in a SCID mouse xenotransplantation model.

We used Bcl-2 antisense oligonucleotides (G3139) to chemosensitize human gastric cancer by downregulation of Bcl-2 expression in vivo. Oligonucleotides and cisplatin were administered systemically in a human gastric cancer SCID mouse model, and Bcl-2 expression, apoptosis, tumor size, and survival were assessed. Used alone, G3139 treatment led to downregulation of Bcl-2 and moderate tumor reduction compared to saline control. G3139 combined with cisplatin treatment markedly enhanced the antitumor effect of cisplatin (70% tumor size reduction vs. cisplatin alone), associated with increased apoptosis measured in tumor biopsy specimens. Combined treatment with G3139 and cisplatin prolonged survival of the tumor-bearing SCID mice by more than 50% without adding significant drug-related toxicity. Treatment with Bcl-2 antisense oligonucleotides is thus a promising novel approach to enhance antitumor activity of cisplatin or other drugs used in gastric cancer therapy and warrants further evaluation in clinical trials.

Differential expression levels of Par-4 in melanoma.

The pro-apoptotic prostate apoptosis response-4 gene product Par-4 sensitizes prostate cells to the induction of programmed cell death. In this study we examined Par-4 expression in human melanoma cell lines and melanoma metastases. The heterogeneous expression detected prompted us to investigate the biological relevance of Par-4 in a human melanoma xenotransplantation model. Overexpression of Par-4 by transfection decreased tumour development in xenotransplanted A375-C6 melanoma cells in SCID mice and correlated to an increase in tumour cell apoptosis. These data suggest that high expression of the pro-apoptotic protein Par-4 could qualify as a prognostic marker in human melanoma.

Chemosensitisation of malignant melanoma by BCL2 antisense therapy.

BACKGROUND: Chemoresistance of malignant melanoma has been linked to expression of the proto-oncogene BCL2. Antisense oligonucleotides (ASO) targeted against BCL2 mRNA decreased BCL2 protein concentrations, increased tumour-cell apoptosis, and led to tumour responses in a mouse xenotransplantation model when combined with systemic dacarbazine. This phase I-II clinical study investigated the combination of BCL2 ASO (augmerosen, Genasense, G3139) and dacarbazine in patients with advanced malignant melanoma expressing BCL2. METHODS: In a within-patient dose-escalation protocol, 14 patients with advanced malignant melanoma were given augmerosen intravenously or subcutaneously in daily doses of 0.6-6.5 mg/kg plus standard dacarbazine treatment (total doses up to 1000 mg/m2 per cycle). Toxicity was scored by common toxicity criteria. Plasma augmerosen concentrations were assayed by high-performance liquid chromatography. In serial tumour biopsy samples, BCL2 protein concentrations were measured by western blotting and tumour-cell apoptosis was assessed. FINDINGS: The combination regimen was well tolerated, with no dose-limiting toxicity. Haematological abnormalities were mild to moderate. Lymphopenia was common, but no febrile neutropenia occurred. Higher doses of augmerosen were associated with transient fever. Four patients had liver-function abnormalities that resolved within 1 week. Steady-state plasma concentrations of augmerosen were attained within 24 h, and increased with administered dose. By day 5, daily doses of 1.7 mg/kg and higher led to a median 40% decrease in BCL2 protein in melanoma samples compared with baseline, concomitantly with increased tumour-cell apoptosis, which was greatly increased after dacarbazine treatment. Six patients have shown antitumour responses (one complete, two partial, three minor). The estimated median survival of all patients now exceeds 12 months. INTERPRETATION: Systemic administration of augmerosen downregulated the target BCL2 protein in metastatic cancer. Such downregulation of BCL2, combined with standard anticancer therapy, offers a new approach to the treatment of patients with resistant neoplasms.

Erythropoietin receptor expression in human melanoma cells.

Erythropoietin is well known for its role in the control of erythropoiesis, where it acts by binding to its cognate receptor (EpoR) on the surface of erythroid progenitor cells. Here we present the novel finding that the EpoR is also expressed in cells of the melanocytic lineage. It is expressed in transformed cell lines established from normal melanocytes and also in established human melanoma cell lines derived from melanoma metastases, but not in normal primary human melanocytes. The analysis of individual subclones isolated from spontaneously transformed melanocytes revealed that approximately 50% of all the clones examined expressed the EpoR. Further analysis of the individual growth characteristics of EpoR-positive and EpoR-negative clones indicated that, under standard cell culture conditions, expression of the receptor did not affect cell growth. Expression of this receptor is consequently most likely driven by an event that is associated with, but not absolutely required for, the transformed phenotype. While the definite function of this receptor in melanoma cells is still unknown and additional studies are required, our findings support the hypothesis that the EpoR may serve as a progression marker for human melanoma. This observation might be useful in the early diagnosis of melanoma.

A novel Ras antagonist regulates both oncogenic Ras and the tumor suppressor p53 in colon cancer cells.

BACKGROUND: In colon cancer, K-Ras oncogenes, which appear to be linked to chemoresistance and poor prognosis, are activated in more than 50% of cases, whereas the tumor suppressor gene p53 is mutationally altered in about 70% of all cases. The transcription factor p53, which is frequently mutated at codon 273, maintains wild-type configuration and possibly carries out residual functions. Although blocking of activated K-Ras may constitute a rational therapeutic concept for this treatment-resistant malignancy, a strategy influencing both oncogenic Ras and the tumor suppressor p53 may be even more promising. MATERIALS AND METHODS: We evaluated the effects of S-trans, trans-farnesyl-thiosalicylic acid (FTS), a novel Ras antagonist on human SW480 and HT-29 colon cancer cells, which both harbor a p53 His273 mutation but express activated K-Ras and wild-type, but overexpressed, H-Ras, respectively. Besides cell growth and morphology, levels of cellular Ras proteins, regulation of p53 and p21(waf1/cip1) expression were analyzed by immunoblotting. The cell cycle arresting potential of FTS was quantified by flow cytometry. RESULTS: We demonstrate that FTS treatment alters the morphology and blocks the growth of SW480 and HT-29 colon cancer cells by both reducing the total amount of Ras and up-regulating the tumor suppressor p53. Furthermore, FTS caused an upregulation of the cyclin-cyclin-dependent kinase (CDK) inhibitor p21(waf1/cip1) and blocked the cell cycle. p53 antisense oligonucleotides not only reduced the level of p53 proteins but correspondingly also blocked the expression of p21(waf1/cip1) in FTS-treated colon cancer cells. CONCLUSIONS: FTS, a unique compound capable of regulating both oncogenic Ras and the tumor suppressor p53 may prove particularly useful for the therapy of colon cancer and other treatment-resistant malignancies where Ras is altered and p53 is either wild-type or mutated in positions that allow residual p53 functions.

Pharmacokinetics and pharmacodynamics of the novel H1-receptor antagonist emedastine in healthy volunteers.

OBJECTIVE: Emedastine is a novel H1-receptor antagonist with pre-clinically well-documented anti-allergic effects. Here, we set out to study the relationship between emedastine pharmacokinetics and the suppressive effect on histamine-induced wheals and flares, and to compare these effects to placebo and cetirizine. METHODS: Emedastine (4 mg q.d.), emedastine (2 mg b.i.d.), cetirizine (10 mg q.d.) and placebo were administered to healthy volunteers in a double-blind, cross-over fashion. On day 1 and day 5 (steady state) following drug administration, wheals and flares were induced by skin-prick testing with 1 mg ml(-1) or 10 mg ml(-1) histamine. RESULTS: Following the administration of 4 mg emedastine q.d., mean area under the concentration-time curve (AUC)0-24 values of 34.49 +/- 24.07 ng h ml(-1) and 47.05 +/- 36.12 ng h ml(-1) were attained on day 1 and day 5, respectively. Following the administration of emedastine (2 mg b.i.d.) mean AUC0-24 values were 29.75 +/- 19.92 ng h ml(-1) and 46.13 +/- 38.50 ng h ml(-1) on day 1 and day 5, respectively. Histamine-induced wheals and flares were significantly more effectively suppressed by emedastine and cetirizine than placebo. At pharmacokinetic steady-state levels, no significant difference could be found in the potency between cetirizine and emedastine (2 mg b.i.d.). CONCLUSION: Emedastine displays pharmacodynamic properties comparable with cetirizine and therefore qualifies as a safe and alternative compound with H1-receptor antagonist properties. Additional larger studies may be needed to substantiate potential benefits of cetirizine over emedastine after single-dose administration.

Effects of betulinic acid alone and in combination with irradiation in human melanoma cells.

Recently, betulinic acid was identified as a highly selective inhibitor of human melanoma growth and was reported to induce apoptosis in these cells. We have investigated the growth-inhibitory properties of this compound alone and in combination with ionizing radiation in a panel of established human melanoma cell lines as well as in normal human melanocytes. Betulinic acid strongly and consistently suppressed the growth and colony-forming ability of all human melanoma cell lines investigated. In combination with ionizing radiation the effect of betulinic acid on growth inhibition was additive in colony-forming assays. Betulinic acid also induced apoptosis in human melanoma cells as demonstrated by Annexin V binding and by the emergence of cells with apoptotic morphology. The growth-inhibitory action of betulinic acid was more pronounced in human melanoma cell lines than in normal human melanocytes. Notably, despite the induction of apoptosis, analysis of the expression of Bcl-2 family members in betulinic-acid-treated cells revealed that expression of the anti-apoptotic protein Mcl-1 was induced. Furthermore, the antiproliferative action of betulinic acid seemed to be independent of the p53 status. The properties of betulinic acid make it an interesting candidate, not only as a single agent but also in combination with radiotherapy. We conclude that the strictly additive mode of growth inhibition in combination with irradiation suggests that the two treatment modalities may function by inducing different cell death pathways or by affecting different target cell populations.