Endostatin and physical exercise in young female and male athletes and controls.

It was suggested that endostatin, an angiogenic mediator, is influenced by physical exercise. We performed bicycle stress testing in 88 healthy non-smoking female and male individuals, divided into athlete and non-athlete groups. Serum endostatin and norepinephrine were measured at rest, after reaching maximum workload and after 20 min of recovery. At baseline, both female and male controls showed significant lower levels compared to female and male athletes (89.39±15.32 resp. 93.39±15.00 ng/ml; p<0.001 vs. 128.81±20.84 resp. 147.52±27.72; p<0.001). An increase in endostatin levels in both groups and sexes was associated with bicycle stress testing (p for all groups<0.001). The extent of endostatin increase was comparable in both groups and sexes and varied between 23-27%. Significance was obscured when the performance was entered as covariate. Acutely induced physical strain leads to an increase in endostatin levels in athletes and controls of both sexes, the extent of increase depending on the extent of workload. An athletic lifestyle with >3 h of endurance training/week seems to lead to higher long-term endostatin levels which might play a role in the connection between sports and cardiovascular prevention.

Multifactorial anticancer effects of digalloyl-resveratrol encompass apoptosis, cell-cycle arrest, and inhibition of lymphendothelial gap formation in vitro.

BACKGROUND: Digalloyl-resveratrol (di-GA) is a synthetic compound aimed to combine the biological effects of the plant polyhydroxy phenols gallic acid and resveratrol, which are both radical scavengers and cyclooxygenase inhibitors exhibiting anticancer activity. Their broad spectrum of activities may probably be due to adjacent free hydroxyl groups. METHODS: Protein activation and expression were analysed by western blotting, deoxyribonucleoside triphosphate levels by HPLC, ribonucleotide reductase activity by (14)C-cytidine incorporation into nascent DNA and cell-cycle distribution by FACS. Apoptosis was measured by Hoechst 33258/propidium iodide double staining of nuclear chromatin and the formation of gaps into the lymphendothelial barrier in a three-dimensional co-culture model consisting of MCF-7 tumour cell spheroids and human lymphendothelial monolayers. RESULTS: In HL-60 leukaemia cells, di-GA activated caspase 3 and dose-dependently induced apoptosis. It further inhibited cell-cycle progression in the G1 phase by four different mechanisms: rapid downregulation of cyclin D1, induction of Chk2 with simultaneous downregulation of Cdc25A, induction of the Cdk-inhibitor p21(Cip/Waf) and inhibition of ribonucleotide reductase activity resulting in reduced dCTP and dTTP levels. Furthermore, di-GA inhibited the generation of lymphendothelial gaps by cancer cell spheroid-secreted lipoxygenase metabolites. Lymphendothelial gaps, adjacent to tumour bulks, can be considered as gates facilitating metastatic spread. CONCLUSION: These data show that di-GA exhibits three distinct anticancer activities: induction of apoptosis, cell-cycle arrest and disruption of cancer cell-induced lymphendothelial disintegration.

[Quality management in medical laboratories]. / Qualitätsmanagement im medizinischen Labor.

During the 20th century understanding for quality has changed and international and national requirements for quality have been published. Therefore also medical branches started to establish quality management systems. Quality assurance has always been important for medical laboratories. Certification according to the standard ISO 9001 and accreditation according to the standard ISO 17025 have been the proof of fulfilling quality requirements. The relatively new standard ISO 15189 is the first standard for medical laboratories. This standard includes technical and management requirements for the medical laboratory. The main focus is the proof of competence within the personnel. As this standard is accepted throughout the European Union an increase in accreditations of medical laboratories is predictable.

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.

A novel PCR-based point-of-care method facilitates rapid, efficient, and sensitive diagnosis of influenza virus infection.

OBJECTIVE: The aim of this single-centre study was the comparative analysis of the GeneXpert (Cepheid Inc.) and the LIAT (Roche) system for the rapid polymerase chain reaction (PCR)-based detection of influenza A (IA) and influenza B (IB) viruses. PATIENTS AND METHODS: During the 2017-2018 flu season, 651 prospectively collected samples (throat and nasal swabs) of patients with symptoms of influenza-like illness or acute respiratory infection were tested for the presence of IA and IB viruses using the GeneXpert and LIAT systems. To evaluate the usefulness for near-patient testing, a LIAT system was installed at the Department of Emergency Medicine, and sample testing was performed on site. Reference testing of all samples was performed with the Xpert Flu assay and for 313 samples in addition with the Xpert Xpress Flu/RSV (respiratory syncytial virus) assay at the central laboratory. Analysis of all samples was carried out within 24 hr after collection. RESULTS: Overall, 267 of the 651 samples analysed were positive for influenza viruses in at least one of the three assays investigated (IA, 88; IB, 179). The overall rates of agreement between the LIAT assay and the Xpert Flu assay was 96.0% for the detection of IA and IB viruses. The sensitivity and specificity of the LIAT assay compared to the Xpert Flu assay for the detection of IA was 98.80% (95% confidence interval (CI) 93.47-99.97%) and 99.12% (95% CI, 97.96% to 99.71%) and for the detection of IB 98.76% (95% CI 95.58-99.85%), and 96.33% (95% CI 94.26-97.81%), respectively. The LIAT assay showed a statistically significant higher detection rate of IB virus than the Xpert Flu assay (p <0.01). No significant difference was found between the detection rate of the LIAT assay and the Xpert Xpress Flu/RSV assay. The mean time to the availability of a definite test result was significantly shorter with the on-site LIAT system than the GeneXpert system (mean 59 min saving time; p <0.01). CONCLUSION: The LIAT system represents a robust and highly sensitive point-of-care device for the rapid PCR-based detection of influenza A and influenza B viruses.

Long-term endurance training increases serum cathepsin S levels in healthy female subjects.

BACKGROUND: Circulating cathepsin S (CS) has been associated with a lower risk for breast cancer in a large Swedish cohort. Long-term physical activity has been shown to have beneficial effects on the development of various cancer subtypes, in particular breast and colorectal cancers. The aim of this study was to investigate the effect of long-term endurance sport on CS levels in females. MATERIAL AND METHODS: Thirty-six of 40 subjects completed the study. Subjects were told to increase their activity pensum for 8 months reaching 150 min/week moderate or 75 min/week intense exercise. Ergometries were performed at the beginning and the end of the study to prove/quantify the performance gain. Blood samples were drawn at baseline and every 2 months. Serum CS levels were measured by ELISA. To analyse the change and the progression of CS, Wilcoxon rank sum and Friedman tests were used. RESULTS: The sportive group (performance gain by > 4.9%) showed a significant increase of CS levels from 3.32/2.73/4.09 to 4.00/3.09/5.04 ng/ml (p = 0.008) corresponding to an increase of 20.5%. CONCLUSIONS: We could show a significant increase of circulating CS levels in healthy female subjects induced by long-term physical activity. CS, occurring in the tumour microenvironment, is well-known to promote tumour growth, e.g. by ameliorating angiogenesis. However, the role of circulating CS in cancer growth is not clear. As physical activity is known as preventive intervention, in particular concerning breast and colorectal cancers, and long-term physical activity leads to an increase of CS levels in female subjects, circulating CS might even be involved in this protective effect. TRIAL REGISTRATION: Clinical trial registration: NCT02097199.

Von Willebrand factor indicates bacterial translocation, inflammation, and procoagulant imbalance and predicts complications independently of portal hypertension severity.

BACKGROUND: Elevated plasma von Willebrand factor antigen (vWF) has been shown to indicate the presence of clinically significant portal hypertension, and thus, predicts the development of clinical events in patients with cirrhosis. AIM: To investigate the impact of bacterial translocation and inflammation on vWF, as well as the association between vWF and procoagulant imbalance. Moreover, we assessed whether vWF predicts complications of cirrhosis, independent of the severity of portal hypertension. METHODS: Our study population comprised 225 patients with hepatic venous pressure gradient (HVPG) ≥ 10 mm Hg without active bacterial infections or hepatocellular carcinoma. RESULTS: vWF correlated with markers of bacterial translocation (lipopolysaccharide-binding protein [LBP; ρ = 0.201; P = 0.021]), inflammation (interleukin 6 [IL-6; ρ = 0.426; P < 0.001] and C-reactive protein [CRP; ρ = 0.249; P < 0.001]), and procoagulant imbalance (factor VIII/protein C ratio; ρ = 0.507; P < 0.001). Importantly, the associations between vWF and these parameters were independent of HVPG. Moreover, vWF (per 10%) independently predicted variceal bleeding (hazard ratio [HR]: 1.08 [95% confidence interval (95% CI): 1.01-1.16]; P = 0.023), requirement of paracentesis (HR: 1.05 [95% CI: 1.01-1.1]; P = 0.023) and bacterial infections (HR: 1.04 [95% CI: 1-1.09]; P = 0.04) including spontaneous bacterial peritonitis (HR: 1.09 [95% CI: 0.999-1.18]; P = 0.053) on a trend-wise level. After backward elimination, vWF (HR: 1.05 [95% CI: 1.02-1.08]; P = 0.003) and CRP (per 10 mg/L; HR: 1.53 [95% CI: 1.14-2.05]; P = 0.005) remained in the final model for transplant-free mortality. Finally, the independent prognostic value of vWF/CRP groups for mortality was confirmed by competing risk analysis. CONCLUSION: Our results demonstrate that vWF is not only a marker of portal hypertension but also independently linked to bacterial translocation, inflammation and procoagulant imbalance, which might explain its HVPG-independent association with most clinical events. Prognostic groups based on vWF/CRP efficiently discriminate between patients with a poor 5-year survival and patients with a favourable prognosis.

Antitumor effects of 3,3',4,4',5,5'-hexahydroxystilbene in HL-60 human promyelocytic leukemia cells.

Resveratrol (3,4',5-trihydroxystilbene, RV) exerts remarkable cytostatic and cytotoxic effects against a multitude of human cancer cell lines. Since the introduction of additional hydroxyl groups was supposed to increase the biological activity of RV, we have synthesized a number of polyhydroxylated stilbene analogues as potential antitumor agents. In this study, the activity of 3,3',4,4',5,5'-hexahydroxystilbene (M8) was investigated in HL-60 human promyelocytic leukemia cells. Employing a growth inhibition assay, incubation with M8 and RV resulted in IC50 values of 6.25 and 12 microM, respectively. Using a specific Hoechst/propidium iodide double staining method, we found that M8 was able to induce apoptosis in concentrations significantly lower than those of RV. In addition, M8 arrested cells in the S phase and totally depleted cells in the G2-M phase of the cell cycle (143% and 0% of control after treatment with 12.5 microM M8, respectively). We therefore believe that this promising agent deserves further preclinical and in vivo testing.

Resveratrol, an ingredient of wine, acts synergistically with Ara-C and tiazofurin in HL-60 human promyelocytic leukemia cells.

Resveratrol (RV), a naturally occurring stilbene derivative, is a potent free radical scavenger causing a number of biochemical and antineoplastic effects. It was shown to induce differentiation and apoptosis in leukemia cells and was also identified as an inhibitor of ribonucleotide reductase (RR), a key enzyme of DNA synthesis. In this study, we report about the biochemical effects of RV in HL-60 human promyelocytic leukemia cells. RV effectively inhibited in situ RR activity. Furthermore, incubation of HL-60 cells with RV significantly decreased intracellular dCTP, dTTP, dATP and dGTP concentrations. In growth inhibition and clonogenic assays, RV acted synergistically with both Ara-C and tiazofurin in HL-60 cells. We conclude that RV could become a viable candidate as one compound in the combination chemotherapy of leukemia and therefore deserves further in vitro and in vivo testing.

Targeted multiple biomarker approach in predicting cardiovascular events in patients with diabetes.

OBJECTIVE: We hypothesised that biomarkers representing different pathophysiological pathways of atherosclerosis namely growth differentiation factor 15 (GDF-15), N-terminal pro B-type natriuretic peptide (NT-proBNP) and high-sensitive troponin T (hs-TnT) could enhance cardiovascular risk prediction in patients with type 2 diabetes mellitus. METHODS: This is a prospective study in 746 patients with type 2 diabetes mellitus, who were followed up for 60 months. The primary endpoint was defined as unplanned hospitalisation for cardiovascular disease or death. The prognostic performance of the biomarkers of interest (GDF-15 in comparison with NT-proBNP and hs-TnT) was evaluated in univariate as well as in stepwise Cox regression models. HRs are presented per standard unit increase. RESULTS: The primary endpoint was registered in 171 patients (22.9%). In univariate Cox regression models, GDF-15 as well as hs-TnT provided significant prognostic information. Even after adjusting for established cardiovascular risk factors, GDF-15, hs-TnT and NT-proBNP remained strong independent predictors of the endpoint (logGDF-15: HR 1.37, p<0.01, CI 1.12 to 1.68; loghs-TnT: HR 1.43, p<0.01, CI 1.13 to 1.1.82; logNT-proBNP: HR 1.45, p<0.01, CI 1.26 to 1.66). The number of elevated markers showed a strong complementarity to predict future long-term risk. Adding hs-TnT and GDF-15 to a zero model already including NT-proBNP led to a net reclassification improvement (NRI) of 33.6% (CI 16.0% to 50.8%, NRI for patients with event: 11.1% CI -4.7% to 26.6%, for patients without event: 22.5% CI 13.6% to 30.5%). CONCLUSIONS: GDF-15 and hs-TnT are strong independent cardiovascular biomarkers augmenting the predictive value of NT-proBNP in patients with diabetes.

Growth promotion by homocysteine but not by homocysteic acid: a role for excessive growth in homocystinuria or proliferation in hyperhomocysteinemia?

Excessive growth of long bones in patients with homocystinuria is still unexplained and previous work incriminating homocysteic acid could not be confirmed by others. In vitro studies from our laboratory showed that homocysteine stimulated growth in a clonogenic assay. This observation made us study plasma cyclin dependent kinase (CDK), homocyst(e)ine and homocysteic acid in 10 patients with homocystinuria and 20 controls. In addition, homocysteine and homocysteic acid were tested in a clonogenic assay to correlate the growth promoting activity with CDK. Plasma CDK (protein) correlated strongly with homocysteine (r=0.84) but not with homocysteic acid. Supernatants of the clonogenic assay samples showed up to three times higher CDK levels in the presence of homocyst(e)ine but not homocysteic acid. In vitro data and the strong correlation between homocysteine and CDK suggest a role for homocysteine stimulating CDK, the starter of mitosis, with subsequent stimulation of growth.

Trimidox, an inhibitor of ribonucleotide reductase, induces apoptosis and activates caspases in HL-60 promyelocytic leukemia cells.

Ribonucleotide reductase (RR) is the rate-limiting enzyme for the de novo synthesis of deoxyribonucleotides. Its activity is significantly increased in tumor cells related to the proliferation rate. Therefore, the enzyme is considered to be an excellent target for cancer chemotherapy. In the present study, we investigated whether the antineoplastic effects of trimidox (3,4, 5-trihydroxybenzamidoxime), a novel inhibitor of RR, were due to induction of apoptosis.HL-60 cells were incubated with various concentrations of trimidox. Consequently, cell morphology, DNA condensation, annexin binding, DNA fragmentation, and signature type cleavage of poly(ADP-ribose)polymerase and gelsolin were determined. We also tested the involvement of CD95 and CD95 ligand in apoptosis induction. Furthermore, we examined the c-myc expression of HL-60 cells after incubation with trimidox in order to elucidate a possible association between c-myc expression and induction of apoptosis in the case of trimidox. Trimidox incubation caused a time-dependent increase of c-myc RNA expression and this was accompanied by the induction of apoptosis. Apoptosis was triggered independently of CD95 by the activation of caspases and PARP cleavage. We conclude that trimidox is able to induce programmed cell death. The induction of apoptosis was demonstrated by various biochemical and morphological methods and seems to be associated with the induction of c-myc. Apoptosis was induced by the activation of caspases and without change of the CD95 and CD95 ligand expression.

Activation of caspases and induction of apoptosis by novel ribonucleotide reductase inhibitors amidox and didox.

OBJECTIVE: Amidox and didox are two polyhydroxy-substituted benzohydroxamic acid derivatives that belong to a new class of ribonucleotide reductase (RR) inhibitors. RR is the rate-limiting enzyme for de novo deoxyribonucleotide synthesis, and its activity is significantly increased in tumor cells in proportion to the proliferation rate. Therefore, RR is a target for antitumor therapy. MATERIALS AND METHODS: HL-60 and K562 leukemia cells were treated with increasing doses of amidox and didox. Thereafter, the mode of cytotoxic drug action was determined by Hoechst 33258/propidium iodide (HO/PI) double staining, annexin binding, DNA fragmentation, and caspase activation. This was correlated to the decrease in dNTP levels. Staining with HO/PI and binding of fluorescein isothiocyanate-conjugated annexin V to externalized phosphatidylserine were used to quantify apoptosis. RESULTS: Low doses of amidox or didox resulted in an increase of apoptotic HL-60 cells within 48 hours. Higher doses (50 microM amidox or 250 microM didox) led to rapid induction of apoptosis, which could be detected as early as 4 hours after treatment. After 48 hours with these concentrations, almost 100% of the HL-60 cells died by apoptosis without an increase in necrosis. K562 cells were found to be resistant to amidox but not to didox. In HL-60 cells, upstream caspase 8 is processed in response to didox, whereas caspases 8 and 9 are processed upon amidox treatment. Didox-induced apoptosis, but not amidox-induced apoptosis, can be correlated with the decrease in dNTP levels. The results suggests that amidox induces several apoptosis mechanisms in HL-60 cells. In contrast, only caspase 9 is activated by didox in K562 cells, and because amidox hardly induces apoptosis in this cell line, no caspase cleavage is observed. CONCLUSIONS: Didox triggers distinct apoptosis pathways in HL-60 and K562 cells.

Cyclosporine metabolism in patients after kidney, bone marrow, heart-lung, and liver transplantation in the early and late posttransplant periods.

Cyclosporine is used in the prevention of allograft rejection. Owing to its narrow therapeutic index, regular monitoring of the whole blood levels of cyclosporine is required. We observed that immunoassays measured significantly higher cyclosporine levels than did high-performance liquid chromatography (HPLC) over time after transplantation. As cyclosporine metabolites cross-react even with immunoassays, this observation might be due to alterations of the cyclosporine metabolism. We analyzed cyclosporine metabolite concentrations in the early and in the late posttransplantation periods in 127 patients after kidney, bone marrow, heart-lung, and liver transplantation by HPLC and determined whole blood levels of cyclosporine by 4 immunoassays (enzyme-multiplied immunoassay [EMIT], cloned enzyme donor immunoassay [CEDIA], AxSYM [Abbott Laboratories, Chicago, IL], and TDx [Abbott Laboratories]). Despite reduced dose, we found significantly higher cyclosporine concentrations measured by the EMIT, AxSYM, and TDx assays in various patient groups. These results are due to the increased metabolite/cyclosporine ratio in the late posttransplantation period. In particular, the metabolites AM1 and AM19 increased significantly over time in bone marrow transplant recipients. Therefore, cyclosporine levels measured by immunoassays should be interpreted with caution.

Association of CD38 antigen expression with other prognostic parameters in early stages of chronic lymphocytic leukemia.

The expression of the surface molecule CD38 on B cell chronic lymphocytic leukemia (B-CLL) cells has recently been described as a prognostic marker for patient survival. We have analyzed CD19/CD38 expression in 81 patients with predominantly early stages of B-CLL (69 Binet A, seven Binet B, five Binet C). Sixty-two patients (77%) had less than 30% CD38+/CD19+ cells, while 19 (23%) had > or = 30%. There was a significant association between Binet stages (A vs. B+C, p < 0.0001), Rai stages (0-II vs. III+IV, p < 0.001) and CD38 expression, confirming the published cut-off level of 30%. A particularly strong association between CD38 expression was found with soluble CD23 (sCD23) levels of > or = 2000 U/ml (p < 0.0001) and beta2-microglobulin (beta2 MG) serum levels of > or = 3 mg/l (p < 0.0001) indicating that CD38 is a marker of tumor mass as well as disease progression. A borderline association was found with lymphocyte doubling time (LDT) < 12 months (p = 0.05) due to low patient numbers, while there was no association with age, sex or immunoglobulin deficiency. Discordant results were obtained in a number of patients: 10 of 69 patients (14%) with Binet A had a CD38 > or = 30% while three of seven patients with Binet B had a CD38 < 30%. In these two subgroups CD38 and other prognostic factors gave discrepant results. Due to the early stage and short median observation time (12 months. range 1-24 months), calculations concerning patient survival were not performed. However, our data show a strong association between CD38 and other known prognostic factors. The results also suggest that this factor is not always reliable in Binet A patients.

Modulation of 5-fluorouracil resistance in human colon tumor cell lines by azidothymidine.

5-fluorouracil (5-FU), an inhibitor of thymidylate synthase (EC 2.1.1.45), is clinically used in the treatment of several solid tumors, including colorectal, head and neck, gastric, and pancreatic cancer. The drug effectively inhibits deoxynucleoside triphosphate de novo synthesis. However, this inhibition can be circumvented by increased thymidine kinase (EC 2.7.1.21) activity. In the present study we examined the effects of 5-FU combined with azidothymidine (AZT), a competitive inhibitor of thymidine kinase in human colon tumor cells in vitro, including three 5-FU resistant cell lines. The cells were simultaneously incubated with various concentrations of 5-FU (0.015 to 150 microM) and AZT (20 to 300 microM) for 6 days. 5-FU alone yielded an IC50 of 18 microM in the parental CCL 227 cell line and IC50s of 470 and 1100 microM in the 5-FU resistant cell lines as determined by a MTT chemosensitivity assay. Addition of 100 microM AZT alone, a drug concentration that can be achieved in patients, had no effect on the growth of the cell lines examined. However, when added simultaneously with 5-FU, the IC50s of 5-FU synergistically decreased to 10 microM in the sensitive and to 360 or 760 microM in the resistant cell lines, respectively. Our results demonstrate that the combination of 5-FU with AZT synergistically inhibited the growth of 5-FU resistant cells, suggesting the use of 5-FU in combination with AZT for the treatment of 5-FU sensitive as well as resistant human colon tumors.

Sensitizing human colon carcinoma HT-29 cells to cisplatin by cyclopentenylcytosine, in vitro and in vivo.

Cyclopentenylcytosine (CPEC) is cytotoxic to HT-29 cells in vitro and in vivo. Treatment with CPEC resulted in sensitizing HT-29 cells to cisplatin (CDDP), as evidenced by synergistic cytotoxicity. CPEC exhibits potent cytotoxicity to HT-29 cells in vitro, 2 and 24 h exposure providing an LC50 of 2.4 and 0.46 microM, respectively. Exposure of HT-29 cells to CDDP for 2 h resulted in an LC50 of 26 microM. Treatment of HT-29 cells with 1.0 or 1.25 microM CPEC and then incubating with CDDP showed synergistic cytotoxicity. Lesser synergy at very high concentrations of CPEC was demonstrated when HT-29 cells were first exposed to CDDP and then incubated with CPEC. Combination index calculations showed synergistic cytotoxicity in HT-29 cells when CPEC was combined with CDDP. Synergistic antitumor activity was demonstrable in vivo in mice transplanted with HT-29 tumor when treated with a combination of CPEC and CDDP without undue toxicity, since no excessive loss in mouse body weight or overt pathology was observed. CPEC had no influence on the total DNA adduct formation and CDDP did not affect the intracellular levels of CPEC or its metabolites, suggesting that enhanced CDDP cytotoxicity resulted from a step subsequent to excision of platinum-cross-linked DNA. These studies support a new approach for augmenting cytotoxic effect of CPEC with CDDP in treating human colon carcinoma.

Iron binding capacity of didox (3,4-dihydroxybenzohydroxamic acid) and amidox (3,4-dihydroxybenzamidoxime) new inhibitors of the enzyme ribonucleotide reductase.

Ribonucleotide reductase is the rate limiting enzyme of deoxynucleoside triphosphate synthesis and is considered to be an excellent target of cancer chemotherapy. Didox and amidox are newly synthesized compounds, which inhibit this enzyme and have in vitro and in vivo antitumor activity. We have now investigated the capability of didox and amidox to interfere with the iron metabolism. We show by photometric and polarographic methods, that didox and amidox are capable of forming an iron complex. However, their cytotoxic action cannot be completely circumvented by addition of Fe-ammoniumcitrate, indicating that the iron complexing capacity may not be responsible for the mechanism of action of these compounds. When L1210 leukemia cells were incubated with the didox-iron or amidox-iron complex itself, changes of the 50% growth inhibitory capacity of the complex in comparison with didox or amidox alone could be shown. We conclude, that didox and amidox are capable of forming iron complexes, but in contrast to other agents, the anticancer activity cannot be contributed to this effect alone. Future studies will have to elucidate the molecular mechanism of action of these new and promising anticancer agents.

Studies on the antitumor activity and biochemical actions of cyclopentenyl cytosine against human colon carcinoma HT-29 in vitro and in vivo.

Cyclopentenyl cytosine (CPEC) is cytotoxic to several tumor cell lines. CPEC inhibits CTP synthesis resulting in depletion of cytidylate pools. The aim of this study was to examine CPEC's cytotoxic and antitumor activity in vitro and in vivo against human colon carcinoma HT-29, and to relate its action on CTP synthesis. CPEC exhibits potent cytotoxicity in vitro to HT-29 cells with an LC50 (concentration that is lethal to the survival of 50% cell colonies) of 2.4 microM and 0.46 microM following 2 h and 24 h exposure, respectively. Incubation of cells with CPEC for 2 h resulted in a dose-dependent decrease in cytidylate pools. The in vivo antitumor activity of CPEC in athymic mice transplanted subcutaneously (s.c.) with 3 million HT-29 cells was examined. Antitumor activity of CPEC was elucidated in early-staged tumor, wherein CPEC (1.5 mg/kg, QD x 9 or 3 mg/kg, QOD x 9) was administered intraperitoneally (i.p.) 24 h after tumor implantation and it resulted in a significant reduction in tumor weight to 48% of control. The effect of CPEC on established solid tumors in vivo was examined in athymic mice transplanted s.c. 14 days earlier with HT-29 cells and treated i.p. with 1.5 mg/kg CPEC, QD x 5 for 4 courses, with a 10 day-interval between courses. This treatment resulted in a significant reduction in tumor weight (72%) in the treated group. HPLC analysis of HT-29 tumor obtained from mice after treatment with CPEC showed a depletion of the CTP concentration reaching a nadir at 8 h. In conclusion, the present studies demonstrate potent antitumor activity of CPEC against freshly transplanted and established human colon carcinoma which can be corroborated with the drug's biochemical actions.

Enhanced effects of adriamycin by combination with a new ribonucleotide reductase inhibitor, trimidox, in murine leukemia.

Ribonucleotide reductase is the rate limiting enzyme of de novo DNA synthesis; its activity is significantly increased in tumor cells related to the proliferation rate. Therefore the enzyme is considered to be an excellent target for cancer chemotherapy. In the present study we tested the in vitro and in vivo antitumor effects of a drug combination using trimidox (3,4,5-trihydroxybenzamidoxime), a novel inhibitor of ribonucleotide reductase with adriamycin, a widely used anticancer drug. This combination was selected because adriamycin generates free radicals being responsible for cardiotoxic side effects; trimidox has been shown to be a good free radical scavenger. The in vitro cytotoxic effect of the drug combination was examined in L1210 mouse leukemia cells employing a MTT chemosensitivity assay. Incubation of these cells with adriamycin and trimidox together yielded less than additive cytotoxic effects compared to either drug alone. These effects were not caused by the involvement of p-glycoprotein mediated drug efflux. However, when the effect of trimidox and adriamycin in combination was examined in L1210 leukemia bearing mice antitumor effects of adriamycin could be enhanced by the presence of trimidox. Our data indicate, that the in vivo combination of adriamycin together with trimidox might be beneficial for the treatment of malignancies.