Background: Recently, several genome-wide association studies (GWAS) have independently found numerous loci at which common single-nucleotide polymorphisms (SNPs) modestly influence the risk of developing colorectal cancer. The aim of this study was to test 11 loci, reported to be associated with an increased or decreased risk of colorectal cancer: 8q23.3 (rs16892766), 8q24.21 (rs6983267), 9p24 (rs719725), 10p14 (rs10795668), 11q23.1 (rs3802842), 14q22.2 (rs4444235), 15q13.3 (rs4779584), 16q22.1 (rs9929218), 18q21.1 (rs4939827), 19q13.1 (rs10411210) and 20p12.3 (rs961253), in a Swedish-based cohort. Methods: The cohort was composed of 1786 cases and 1749 controls that were genotyped and analysed statistically. Genotype–phenotype analysis, for all 11 SNPs and sex, age of onset, family history of CRC and tumour location, was performed. Results: Of eleven loci, 5 showed statistically significant odds ratios similar to previously published findings: 8q23.3, 8q24.21, 10p14, 15q13.3 and 18q21.1. The remaining loci 11q23.1, 16q22.1, 19q13.1 and 20p12.3 showed weak trends but somehow similar to what was previously published. The loci 9p24 and 14q22.2 could not be confirmed. We show a higher number of risk alleles in affected individuals compared to controls. Four statistically significant genotype–phenotype associations were found; the G allele of rs6983267 was associated to older age, the G allele of rs1075668 was associated with a younger age and sporadic cases, and the T allele of rs10411210 was associated with younger age. Conclusions: Our study, using a Swedish population, supports most genetic variants published in GWAS. More studies are needed to validate the genotype–phenotype correlations.

The role of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) and the ADP-ribosylation inhibitor 3-aminobenzamide (3-ABA) in the cytotoxicity induced by the novel antitumoral cyanoguanidine CHS 828 was investigated in the human lymphoma cell line U-937 GTB. Exposing cells to CHS 828 and 3-ABA in combination resulted in a 100-fold higher IC(50) compared to exposure to CHS 828 alone. CHS 828 did not activate PARP, measured as PARP-activity and formation of poly(ADP-ribose). The ATP-levels and levels of extracellular acidification rate of cells exposed to CHS 828 in combination with 3-ABA were maintained for a longer period than for cells exposed to CHS 828 alone. To characterize the mode of cell death, caspase-3 activity and gross morphology were assessed. 3-ABA increased and delayed the caspase-3 activity in cells exposed to CHS 828. Cells exposed to high concentrations of CHS 828 showed a necrotic morphology, while high concentrations of CHS 828 in combination with 3-ABA switched the mode of cell death, generating an apoptotic morphology. The results indicate that the cytotoxicity and morphology induced by CHS 828 is not due to PARP activation but can be modulated by the ADP-ribosylation inhibitor 3-ABA.

1. The present study was aimed at elucidating the apoptosis inhibitory properties of the cyanoguanidine CHS 828. CHS 828 exhibits impressive cytotoxic activity in vitro and in vivo. Apoptosis is not its main mode of cytotoxic effect, and we have previously proposed a dual mechanism, where CHS 828 inhibits its own cell death pathways. 2. Etoposide on the other hand, is a well-established anticancer agent with documented effect in a number of malignancies, induces apoptosis through extensively studied caspase dependent pathways. 3. Here we studied the combined effect of the two drugs in the human lymphoma cell line U-937 GTB. Cytotoxicity was evaluated as total viability measured by the fluorometric microculture cytotoxicity assay (FMCA). Caspase activity was assessed by colorimetric detection of specific cleavage products for caspases 3, 8 and 9, respectively. Morphology was evaluated in May-Grünwald/Giemsa stained preparations. Interaction analysis based on FMCA results of simple combination exposure revealed impressive synergistic effect on cell kill. 4. Detailed investigations of the kinetics involved showed that short pre-exposure (0-12 h) to CHS 828 enhanced caspase activation by etoposide, while longer pre-exposure (18-48 h) inhibited both caspase activation and apoptotic morphology otherwise induced by etoposide. The present results support the theory that CHS 828 block specific cell death pathways. 5. The synergistic results are promising for future combination trials in animals, however, different dosing schedules should be considered, in order to investigate whether the above findings translate into the in vivo setting.

Background: CHS 828 is a novel cyanoguanidine with cytotoxic properties which was recently shown to induce an early increase in extracellular acidification. This could hypothetically be exploited for combination with drugs interfering with, or being dependent on, pH for their effect. Methods: The isobole method and the additive model were used to evaluate the combinations CHS 828-amiloride and CHS 828-mitomycin C (MMC) in the lymphoma cell line U-937 GTB and in primary cultures of tumour cells from patients. Results: Amiloride, which blocks the Na+/H+ antiport, shifted the CHS 828 dose-response curve to the left in a synergistic manner according to the additive interaction model. MMC is a bioreductive drug with enhanced cytotoxicity at acidic pH. A lowering of pH induced by CHS 828 would theoretically create a favourable environment for bioreduction of MMC. The interaction between these drugs was mainly classified as additive by both methods, but was synergistic at the highest effect level tested. In addition, there were sub-additive to synergistic interactions between CHS 838 and MMC in 76% of the haematological samples tested. Conclusions: Circumstantial evidence indicated that the mechanisms for the interactions could be pH independent. Thus, the interaction between CHS 828 and amiloride was synergistic while the interaction between CHS 828 and MMC in tumour cells was at least additive.

N-(6-(4-chlorophenoxy)hexyl)-N'-cyano-N''-4-pyridylguanidine, CHS 828, is a new anti-neoplastic agent with promising anti-tumor activity both in vitro and in vivo. To characterize the metabolic events over time, the lymphoma cell line U-937 GTB was exposed to CHS 828 and the structurally related mitochondrial inhibitor meta-iodobenzylguanidine (MIBG). There was an instant stimulation of the extracellular acidification rate in response to CHS 828 as measured by the Cytosensor microphysiometer. MIBG shared this metabolic feature. During the first 24 hours of CHS 828 exposure, the consumption of glucose was not significantly affected, but was thereafter shut off. CHS 828 exposure induced a slight increase in lactate production, with 114% of control after 8 hours and 126% for MIBG. Only limited cytotoxicity from CHS 828 exposure was observed after 24 hours and ATP levels remained at 65% of control. Thereafter, the ATP level decreased rapidly and subsequently cell death appeared. Thus, the CHS 828-induced increase in metabolic activity was associated with increased lactate production, probably from increased glycolytic activity. However, CHS 828 also appears to induce a late inhibition of glucolysis leading to energy depletion and subsequent cell death.

The pharmacology and clinical application of three guanidino-containing compounds are reviewed in this commentary with special focus on a new member of this group of drugs. CHS 828 [N-(6-(4-chlorophenoxy)hexyl)-N'-cyano-N" -4-pyridylguanidine]. m-Iodobenzylguanidine (MIBG) and methylglyoxal bis(guanylhydrazone) (MGBG) have been extensively studied, preclinically as well as clinically. and have established use as anticancer agents. MIBG has structural similarities to the neurotransmitter, norepinephrine, and MGBG is a structural analog of the natural polyamine spermidine. CHS 828 is a pyridyl cyanoguanidine newly recognized as having cytotoxic effects when screening antihypertensive compounds. Apart from having the guanidino groups in common, there are many differences between these drugs in both structure and their mechanisms of action. However, they all inhibit mitochondrial function, a seemingly unique feature among chemotherapeutic drugs. In vitro in various cell lines and primary cultures of patient tumor cells and in vivo in various rumor models, CHS 828 has cytotoxic properties unlike any of the standard cytotoxic drugs with which it has been compared. Among these are non-cross-resistance to standard drugs and pronounced activity in tumor models acknowledged to be highly drug-resistant. Similar to MIBG, CHS 828 induces an early increase in extracellular acidification, due to stimulation of the glycolytic flux. Furthermore, ATP levels decrease, and the syntheses of DNA and protein are shut off after approximately 30 hr of exposure, indicating active cell death. CHS 828 is now in early clinical trials, the results of which are eagerly awaited. (C) 2001 Elsevier Science Inc. All rights reserved.

Combination of CHS 828 and etoposide in vitro - from cytotoxic synergy to complete inhibition of apoptosis

N-(6-(4-chlorophenoxy)hexyl)-N'-cyano-N"-4-pyridylguanidine (CHS 828) is a new guanidino-containing compound with antitumoral activity both in vitro and in vivo. Its activity profile differs from those of standard cytotoxic drugs but the mechanism of action is not yet fully understood. CHS 828 is presently in early phase I and II clinical trials. In the present study, the pharmacodynamic effects at the cellular level of CHS 828 was compared to another compound containing two guanidino groups, methylglyoxal-bis(guanylhydrazone) (MGBG). MGBG is known to inhibit the synthesis of polyamines, which are important in, e.g., proliferation and macromolecular synthesis. The concentration-response relationship of CHS 828 closely resembled that of MGBG and the drugs were similar with respect to inhibition of DNA and protein synthesis. On the other hand, CHS 828 induced a significant increase in cellular metabolism while MGBG did not. The cytotoxic effect of MGBG was reversed by the addition of exogenous polyamines, while that of CHS 828 was unaffected. Unlike MGBG, there was also no effect of CHS 828 on the levels of decarboxylating enzymes in the polyamine biosynthesis. In conclusion, CHS 828 does not appear to share any major mechanisms of action with the polyamine synthesis inhibitor MGBG. Further studies will be required to define the exact mechanism of action of CHS 828.

Antitumour properties of the cyanoguanidine CHS 828 and analogues were discovered in 1997. CHS 828 is presently in clinical phase I/II trials. This thesis encompasses in vitro studies of the kinetics and mode of cell death induced in the human cell line U-937 GTB, by CHS 828 and the standard antitumour drug etoposide.Etoposide induces apoptosis in U-937 GTB within 4 h. The cells exhibited apoptotic morphology, including condensed and fragmented nuclei and formation of apoptotic bodies, activation of caspase 3 and 8, and DNA fragmentation, visualised by TdT-mediated dUTP nick end-labelling (TUNEL).CHS 828 induced few and weak signs of apoptosis. Metabolic activity was the only parameter affected during the first 24 h of exposure. After ~30 h, proliferation (DNA synthesis) and protein synthesis ceased, and viability started to decrease towards 10% at 72 h. Morphology and ultrastructure of dying/dead cells showed predominant necrosis. The decrease in viability was postponed by protein synthesis inhibition or maintenance of ATP levels by 3-aminobenzamide. In addition, 3-aminobenzamide switched morphology towards apoptosis. Continuous co-exposure to CHS 828 and etoposide resulted in impressive cell kill synergy in U-937 GTB cells at effect levels of 30-70%. Pre-exposure to CHS 828 for 18 h or more, on the other hand, resulted in diminished cell kill and inability to activate the apoptotic machinery upon etoposide stimulation, evaluated by morphology and caspase activity.In summary, CHS 828 induced cell death is predominantly non-apoptotic, does not involve caspases and can be postponed by maintained protein synthesis and ATP levels.

CHS 828, a newly recognized pyridyl cyanoguanidine, has shown promising antitumor activity both in vitro and in vivo and is presently in early phase I clinical trial in collaboration with EORTC. In this study, the effects of CHS 828 and a series of analogues on extracellular acidification and cytotoxicity were compared with those of m-iodobenzylguanidine (MIBG) in human tumor cells. The extracellular acidification rate was measured using the Cytosensor microphysiometer, and determination of cytotoxicity and proliferation was [14C] performed by the fluorometric microculture cytotoxicity assay (FMCA) and measurement of [14C]thymidine and leucine uptake. CHS 828 significantly increased the acidification rate during the first 15–24 hr in a concentration-dependent manner. This effect was abolished by removal of glucose from the medium, substituted with 10 mM of pyruvate, indicating stimulated glycolysis as the source of the increased acidification rate. However, CHS 828 induced cytotoxicity at concentrations well below those that affected the rate of acidification; when a series of closely related pyridylguanidine analogues were tested and compared, no apparent relationship between cytotoxicity and acidification could be discerned. Furthermore, comparable increases in the acidification rate were evident in one subline with high-grade resistance to the cytotoxic actions of CHS 828. The results indicate that CHS 828 may share the inhibitory actions of MIBG on mitochondrial respiration with a subsequent increase in glycolysis and acidification rate. However, this mechanism of action appears neither necessary nor sufficient to fully explain the cytotoxic actions of CHS 828 in human tumor cells, actions which remain to be mechanistically clarified.

Increased concentrations of the amino acid homocysteine—hyperhomocysteinemia—are correlated with atherosclerotic and thrombotic diseases (1)(2). High concentrations can be lowered by diet and dietary supplements with vitamins B12, folate, and pyridoxine. Homocysteine is produced in the metabolism of the essential amino acid methionine and is converted by cystathionine β-synthase to cystathionine and by methionine synthase back to methionine. These enzymatic reactions are dependent on sufficient concentrations of the vitamins B12, folate, and pyridoxine. Knowledge of biological, postprandial, and orthostatic variations are important in judging significant changes in results and error sources in blood sampling conditions (3), and several studies on the biological variation of plasma homocysteine have been published (4)(5)(6). Fasting blood samples traditionally have been recommended for plasma homocysteine measurement because postprandial changes produce a modest decrease in the first hours and an increase after 8 h (7)(8). Orthostatic changes can also be important in the monitoring of homocysteine in patients with atherosclerotic and thrombotic diseases. Because most homocysteine is bound to albumin, the decrease with supine posture is expected to be 5–10%. In this study we examined the day-to-day, postprandial, and orthostatic variations of plasma total homocysteine. Blood samples were obtained from 19 healthy hospital employees (11 women and 8 men) ages 19–60 years (median age, 44 years). None of these individuals was or became pregnant or had medical diseases. The intake of oral contraceptives, intermittent asthma and allergy medicines, nonsteroidal antiinflammatory drugs, acetaminophen, and multivitamins was allowed during the study. Multivitamins were taken on a regular basis by four persons. Blood hemoglobin, erythrocyte folate, and serum cobalamin concentrations in all subjects were within the reference intervals. Participants provided fasting blood samples after arrival for work (0800 to 1000) and nonfasting samples after lunch (1215 …

Microphysiometry is a non-invasive, physiological method where measurement of metabolic activity can be made on living human tumor cells. Indirect measurement of the extracellular acidification is measured over a pH-sensitive silicon membrane. In this study microphysiometry was employed for the study of cytotoxic agents used in therapy of cancer. Standard cytotoxic drugs with different postulated mechanisms of action were investigated using cell lines as well as primary cultures of patient tumor cells. Each investigated cytotoxic drug induced a characteristic pattern of metabolic activity. From these patterns, key features, like stimulation and inhibition of acidification, the time point when the response curves of the drugs fall below the control curve, and the maximum inhibition of acidification at 20 h, could be quantified. Most of the investigated drugs showed some initial stimulation of acidification rate during the experiments. For drugs producing a reduced metabolic rate at 20 h a concentration-response relationship was observed. The drug effects measured at 20 h were irreversible and correlated reasonably well with parallel measurements of membrane integrity using a standard cytotoxicity test. The results demonstrate the feasibility of 'on-line' measurements of metabolic activity using this approach and also revealed an unexpected variety of drug response profiles.

Microphysiometry is a non-invasive, physiological method where measurement of metabolic activity can be made on living human tumor cells. Indirect measurement of the extracellular acidification is measured over a pH-sensitive silicon membrane. In this study microphysiometry was employed for the study of cytotoxic agents used in therapy of cancer. Standard cytotoxic drugs with different postulated mechanisms of action were investigated using cell lines as well as primary cultures of patient tumor cells. Each investigated cytotoxic drug induced a characteristic pattern of metabolic activity. From these patterns, key features, like stimulation and inhibition of acidification, the time point when the response curves of the drugs fall below the control curve, and the maximum inhibition of acidification at 20 h, could be quantified. Most of the investigated drugs showed some initial stimulation of acidification rate during the experiments. For drugs producing a reduced metabolic rate at 20 h a concentration-response relationship was observed. The drug effects measured at 20 h were irreversible and correlated reasonably well with parallel measurements of membrane integrity using a standard cytotoxicity test. The results demonstrate the feasibility of 'on-line' measurements of metabolic activity using this approach and also revealed an unexpected variety of drug response profiles.

Patients with homozygous homocystinuria are at greatly increased risk for development of atherosclerosis and thrombosis (1). Elevated plasma levels of homocysteine (HCY) are caused by reduced enzymatic catabolism or reduced enzymatic remethylation of HCY, due to either hereditary enzyme defects or to nutrional deficiencies of vitamins functioning as cofactors. However, several recent studies have suggested that persons with mildly elevated plasma levels of HCY also are at increased risk for coronary heart disease. (2-4).There are some indications that dietary n-3 polyunsaturated fatty acids (PUFAs) may offer protection against coronary heart disease (5-6). Several mechanisms may be involved, including beneficial effects of n-3 PUFAs on plasma lipids, platelet and leukocyte reactivity, blood pressure and vasoreactivity (7). Interestingly, Olszewski el al. recently found HCY-levels to be lowered 36% in 15 type IIa or IIb hyperlipemic men by n-3 PUFA supplementation.A possible beneficial effect of n-3 PUFA on the incidence of coronary heart disease was initially suggested from studies in Greenland Inuits by our group (8). We therefore investigated plasma levels of homocystein in a group of traditionally living Greenland Inuits with a diet consisting mainly of marine food and with a very high content of n-3 PUFAs. (C) 1997 Elsevier Science Ltd.