Corrigendum: Different pharmacokinetics of tramadol, O-demethyltramadol and N-demethyltramadol in postoperative surgical patients from those observed in medical patients.

[This corrects the article DOI: 10.3389/fphar.2021.656748.].

Role of Oxidative Stress in Vascular Low-Grade Inflammation Initiation Due to Acute Salt Loading in Young Healthy Individuals.

This study aimed to investigate the effect of 7-day high-salt (HS) and the specific role of oxidative stress on vascular low-grade inflammation initiation in young salt-resistant healthy individuals. 30 young healthy individuals adhered to a 7-day low-salt (LS) diet (3.5 g salt/day), followed by a 7-day high-salt (HS) diet (~14.7 g salt/day) protocol. Pro- and anti-inflammatory cytokines, frequencies of peripheral blood Th17 and Treg cells, Th17/Treg ratio, enzymes SGK1, and p38/MAP kinase, as well as biomarkers of endothelial activation and oxidative stress, were measured before and after the 7-day HS diet protocol. Short-term HS diet significantly increased serum level of pro-inflammatory cytokines INF-γ, TNF-α, IL-9, and IL-17A levels, but also of anti-inflammatory cytokines IL-10 and TGF-ß1. Relative amount of total SGK1 significantly increased, following the 7-day HS diet. Increased oxidative stress level, following HS diet, was negatively associated with the frequency of Treg cells. The increase in relative amount of total SGK1 in peripheral mononuclear cells following 7-day HS diet suggests lymphocyte (re)activation, in response to HS intake, resulting in enhanced production of pro-inflammatory (IL-17, INF-γ), but also anti-inflammatory cytokines (IL-10 and TGF-ß1). Increased oxidative stress, due to HS loading, alters immune regulatory mechanisms, presumably via effects on Treg cells.

Consumption of Hen Eggs Enriched with n-3 Polyunsaturated Fatty Acids, Selenium, Vitamin E and Lutein Incites Anti-Inflammatory Conditions in Young, Healthy Participants - A Randomized Study.

BACKGROUND: Dietary supplementation with compounds that possess antioxidant and anti-inflammatory properties (n-3 polyunsaturated fatty acids (PUFAs), selenium, vitamin E, lutein), has been shown to positively correlate with improvements in chronic conditions, although understanding of these combined effects in healthy humans is limited. The study aimed to evaluate the effects of enriched eggs consumption on oxidative status and inflammatory conditions in healthy volunteers. We hypothesized that a three-week diet containing enriched eggs can alter the immune response of healthy adults towards anti-inflammatory conditions. METHODS: 34 participants consumed 3 hard-boiled hen eggs per day (21 days): Control group-regular hen eggs (n-3 PUFAs = 438 mg, selenium = 0.054 mg, lutein = 0.330 mg and vitamin E = 1.785 mg) (N = 14); 4Nutri group-hen eggs enriched with 4 nutrients (n-3 PUFAs = 1026 mg, selenium = 0.06 mg, lutein = 1.85 mg and vitamin E = 3.29 mg) (N = 20). Samples were taken before and after the protocol. Serum concentrations of lipid mediators and cytokines were measured with enzyme-linked immunosorbent assay (ELISA) and antibody-based, magnetic bead reagent kits on the Luminex platform, respectively. Serum oxidative stress and antioxidant capacity were measured using standardized methods, while gene expression in peripheral blood mononuclear cells (PBMCs) was measured via real-time PCR. RESULTS: Decreased serum levels of pro-inflammatory interleukin 17A (IL-17A) and an increased neuronal nitric oxide synthase (nNOS) expression in the 4Nutri group, together with alteration of metabolites produced via cyclooxygenase (COX) pathways in the Control group, suggest a shift towards anti-inflammatory conditions in participants who consumed enriched hen eggs. CONCLUSIONS: Present results suggest that the combined action of n-3 PUFAs and antioxidants may have a protective role in resting, non-inflammatory conditions. CLINICAL TRIAL REGISTRATION: NCT04564690.

ANALGESIC EFFECT OF TRAMADOL IS NOT ALTERED BY POSTOPERATIVE SYSTEMIC INFLAMMATION AFTER MAJOR ABDOMINAL SURGERY.

Tramadol is a commonly used analgesic in intensive care units (ICUs) for acute postoperative pain. Conversion of tramadol into active metabolites may be impaired in inflammatory states. Catechol-O-methyltransferase may influence pain. The aim of the study was to examine differences in the analgesic effect of tramadol between ICU patients with and without signs of systemic inflammation. Forty-three patients were admitted to ICU after a major abdominal surgery. The patients received a dose of 100 mg of tramadol intravenously every 6 hours during the first 24 hours after surgical procedure. Pain scores were measured by the Numeric Rating Scale before and 30 minutes after tramadol administration in awake patients. Systemic inflammation was considered when at least two of the following postoperative parameters were present in the first 24 hours of ICU admission: fever or hypothermia, tachycardia, pCO2 <4.3 kPa, white blood cells >12000/mm3 or <4000/mm3, or preoperative value of C-reactive protein (CRP) >50 mg/L or/and procalcitonin (PCT) >0.5 mg/L. Catechol-O-methyltransferase was analyzed postoperatively. Fifteen (34.8%) patients met the criteria for systemic inflammation. Tramadol was proven to be an effective analgesic for the treatment of postoperative pain regardless of the presence of systemic inflammation (p<0.05). Lower perception of pain before tramadol application was observed in patients with systemic inflammation, but the difference was not significant. A negative correlation was observed between the preoperative values of CRP and PCT and the analgesic effect of tramadol assessed at the second measurement point (r=-0.358, p=0.03, and r=-0.364, p=0.02, respectively). Catechol-O-methyltransferase variants were not in correlation with pain and opioid consumption. Based on our findings, tramadol is effective in lowering pain scores after major abdominal surgery irrespective of the presence of systemic inflammation.

Different Pharmacokinetics of Tramadol, O-Demethyltramadol and N-Demethyltramadol in Postoperative Surgical Patients From Those Observed in Medical Patients.

Background: Most studies examining tramadol metabolism have been carried out in non-surgical patients and with oral tramadol. The aim of this study was 1) to measure concentrations of tramadol, O-demethyltramadol (ODT), and N-demethyltramadol (NDT) in the surgical patients admitted to the intensive care unit (ICU) within the first 24 postoperative hours after intravenous application of tramadol, and 2) to examine the effect of systemic inflammation on tramadol metabolism and postoperative pain. Methods: A prospective observational study was carried out in the surgical ICU in the tertiary hospital. In the group of 47 subsequent patients undergoing major abdominal surgery, pre-operative blood samples were taken for CYP2D6 polymorphism analysis. Systemic inflammation was assessed based on laboratory and clinical indicators. All patients received 100 mg of tramadol intravenously every 6 h during the first postoperative day. Postoperative pain was assessed before and 30 min after tramadol injections. Tramadol, ODT, and NDT concentrations were determined by high-performance liquid chromatography. Results: CYP2D6 analysis revealed 2 poor (PM), 22 intermediate (IM), 22 extensive (EM), and 1 ultrafast metabolizer. After a dose of 100 mg of tramadol, t1/2 of 4.8 (3.2-7.6) h was observed. There were no differences in tramadol concentration among metabolic phenotypes. The area under the concentration-time curve at the first dose interval (AUC1-6) of tramadol was 1,200 (917.9-1944.4) µg ×h ×L-1. NDT concentrations in UM were below the limit of quantification until the second dose of tramadol was administrated, while PM had higher NDT concentrations compared to EM and IM. ODT concentrations were higher in EM, compared to IM and PM. ODT AUC1-6 was 229.6 (137.7-326.2) µg ×h ×L-1 and 95.5 (49.1-204.3) µg ×h ×L-1 in EM and IM, respectively (p = 0.004). Preoperative cholinesterase activity (ChE) of ≤4244 U L-1 was a cut-off value for a prediction of systemic inflammation in an early postoperative period. NDT AUC1-6 were significantly higher in patients with low ChE compared with normal ChE patients (p = 0.006). Pain measurements have confirmed that sufficient pain control was achieved in all patients after the second tramadol dose, except in the PM. Conclusions: CYP2D6 polymorphism is a major factor in O-demethylation, while systemic inflammation accompanied by low ChE has an important role in the N-demethylation of tramadol in postoperative patients. Concentrations of tramadol, ODT, and NDT are lower in surgical patients than previously reported in non-surgical patients. Clinical Trial Registration: ClinicalTrials.gov, NCT04004481.

HLA-A, -B, -C, -DRB1, -DQA1, and -DQB1 allele and haplotype frequencies defined by next generation sequencing in a population of East Croatia blood donors.

Next-generation sequencing (NGS) is increasingly used in transplantation settings, but also as a method of choice for in-depth analysis of population-specific HLA genetic architecture and its linkage to various diseases. With respect to complex ethnic admixture characteristic for East Croatian population, we aimed to investigate class-I (HLA-A, -B, -C) and class-II (HLA-DRB1, -DQA1, -DQB1) HLA diversity at the highest, 4-field resolution level in 120 healthy, unrelated, blood donor volunteers. Genomic DNA was extracted and HLA genotypes of class I and DQA1 genes were defined in full-length, -DQB1 from intron 1 to 3' UTR, and -DRB1 from intron 1 to intron 4 (Illumina MiSeq platform, Omixon Twin algorithms, IMGT/HLA release 3.30.0_5). Linkage disequilibrium statistics, Hardy-Weinberg departures, and haplotype frequencies were inferred by exact tests and iterative Expectation-Maximization algorithm using PyPop 0.7.0 and Arlequin v3.5.2.2 software. Our data provide first description of 4-field allele and haplotype frequencies in Croatian population, revealing 192 class-I and class-II alleles and extended haplotypic combinations not apparent from the existing 2-field HLA reports from Croatia. This established reference database complements current knowledge of HLA diversity and should prove useful in future population studies, transplantation settings, and disease-associated HLA screening.

Comparative analysis of abdominal fluid cytokine levels in ovarian hyperstimulation syndrome (OHSS).

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is a rare, yet severe, iatrogenic complication of ovulation induction therapy during assisted reproductive procedures. Our group previously detected atypical cells in the ascitic fluid of OHSS patients, although no malignancy developed during follow up. Here, the aim was to perform a comparative analysis of the cytokines present in the abdominal fluid of patients affected by OHSS versus patients with advanced ovarian cancer, a benign adnexal mass, or ovarian endometriosis. METHODS: This prospective, non-randomized study was conducted at the Clinical Center of the University of Pecs Department of Obstetrics and Gynecology/Reproductive Center between October 2016 and March 2018. Abdominal fluid samples were obtained from 76 patients and subjected to Luminex analysis. The samples were collected from patients with OHSS (OHSS; n = 16), advanced ovarian cancer (OC; n = 22), a benign adnexal mass (BAM; n = 21), or ovarian endometriosis (EM; n = 17). Data were subjected to the non-parametric Kruskal-Wallis test and Spearman's rank correlation coefficient to identify statistical differences between the four study groups. RESULTS: Leukocytosis and hemoconcentration were detected in the peripheral blood of OHSS patients. Abdominal fluid analysis further revealed significantly higher levels of interleukin (IL)-6, IL-8, IL-10, and transforming growth factor (TGF)-ß in both the OHSS and OC groups compared to the BAM and EM groups. The highest concentration of vascular endothelial growth factor (VEGF) was detected in the OC group, while a significantly lower level was detected in the OHSS group. Moreover, VEGF levels in OC and OHSS groups were significantly elevated compared to the levels in the BAM and EM groups. CONCLUSIONS: Vasoactive and hematogenic cytokines were present at higher levels in both the OHSS and OC abdominal fluid samples compared to the fluid samples obtained from the peritoneal cavity of the BAM patients. It is possible that these cytokines play an important role in the formation of ascites.

Association of IL-1ß and IL-10 Polymorphisms with Prostate Cancer Risk and Grade of Disease in Eastern Croatian Population.

Single nucleotide polymorphisms (SNPs) in the promotor regions of cytokine genes included in angiogenesis may influence prostate cancer (PCa) development via regulation of the pathways of tumor angiogenesis. The aim of the present study was to investigate the association of IL-1 female +3954 (rs1143634) and IL-10-1082 (rs1800896) polymorphisms with PCa risk and aggressiveness in eastern Croatian patients. One hundred twenty PCa patients and 120 benign prostatic hyperplasia (BPH) controls were genotyped using real-time PCR (LightCycler Instrument, Roche Diagnostics) and the melting curve analysis method. There was no significant difference in the frequency of genotypes for the two polymorphisms between PCa patients and controls (Χ2 = 0.857, p = 0.355 for IL-female 1; Χ2 = 0.026, p = 0.872 for IL-10). Carriers of the IL-10-1082A>G variant were found to be associated with the Gleason score (GS) > 7 (AA versus GA+GG, OR = 3.47, 95% CI 1.11-10.88, p = 0.033). There was no significant difference in the frequency of genotypes for the two polymorphisms and the presence of metastatic disease in PCa patients. These results suggest that tested SNPs associated with differential production of IL-1 female and IL-10 are not risk factors for PCa and do not correlate with the presence of distant metastasis in eastern Croatians. We found that IL-10-1082 GA+/or GG carriers have a higher risk of developing PCa with GS > 7 in eastern Croatians.

Association study of cytochrome P450 1A1*2A polymorphism with prostate cancer risk and aggressiveness in Croatians.

Cytochrome P450 1A1 (CYP1A1) is an enzyme participating in the bioactivation of various endogenous and environmental reactive compounds that can bind to DNA and thus induce cancerogenesis. Gene encoding the enzyme is expressed in the prostate tissue and is polymorphic. CYP1A1*2A gene polymorphism is associated with elevated enzyme activity and/or inducibility which can lead to accumulation of genotoxic compounds and consequently to cancerogenesis. We examined the association of this polymorphism with prostate cancer (PCa) risk and aggressiveness. The case-control study consisted of 120 PCa patients and 120 benign prostatic hyperplasia (BPH) controls, in Croatian population. Regarding aggressiveness, PCa patients were grouped according to the Gleason score (GS), tumor stage (T) and existence of distant metastasis (M). The polymorphism was analyzed using real-time polymerase chain reaction (PCR). We did not observe association of mutated allele with PCa risk, neither with PCa aggressiveness. Furthermore, frequency of polymorphic genotype was slightly higher in BPH group (16.6% vs. 14.2%, respectively) and also in less aggressive form of PCa (20.4% vs. 9.6% for GS < 7; 15.6% vs. 9.1% for T < 3; 16.7% vs. 10.0% for no distant metastasis). Comparing our findings with other published results, we can assume that the ethnicity influence the genotype distribution and thus may affect the etiology of PCa, even possibly in the way to cause an opposite effect among different ethnic groups. Given the small number of participants, results should be validated on the larger sample size.

Interleukin-6 polymorphism and prostate cancer risk in population of Eastern Croatia.

Recent studies suggest that chronic inflammation is crucial in the development and progression of prostate cancer (CaP). Interleukin-6 (IL-6) is a proinflammatory cytokine that plays an important role in intraprostatic inflammation and thus carcinogenesis. The -174G > C polymorphism of IL-6 gene has been associated with high IL-6 producer phenotype and an increased risk for CaP. The aim of this study was to evaluate the association between the mentioned IL-6 polymorphism and CaP risk, as well as to compare the genotype frequency between the different tumour grades of CaP, in population of Eastern Croatia. We analyzed the IL-6 polymorphism in 120 CaP patients and 120 controls with benign prostatic hyperplasia (BPH). CaP patients and BPH controls did not statistically differ in studied IL-6 polymorphism. Furthermore, high IL-6 producer genotypes (GG or GC) were more frequent in controls than in CaP group (86.7% vs 80.8%, respectively, p = 0.147). Also, no statistically significant difference in IL-6 high and low producer genotype frequency was noticed between well, moderately and poorly differentiated tumours. Our results, taken together with other studies on the subject, suggest that IL-6 - 174 single nucleotide polymorphism (SNP) distribution may differ between various ethnic groups and that a single cytokine gene polymorphism has probably just a minor effect on CaP susceptibility. Further studies should be performed to clarify the link between SNPs of different cytokines and the risk for CaP.

Lack of association between TNF-alpha promoter polymorphism and prostate carcinoma susceptibility in eastern Croatian population.

A single nuclear polymorphisms (SNPs) in the promoter region of the tumor necrosis factor-alpha (TNF-alpha) gene are involved in regulation of expression levels of TNF-alpha and therefore are associated with oncogenesis of several cancers. Aim of our study was to investigate the effect of G--->A polymorphism at -308 position in the promoter region of the TNF-alpha gene on prostate cancer (CalphaP) susceptibility in a subset of patients from Eastern Croatia. Study population consisted of 240 patients (120 with CalphaP, 120 controls). They were genotyped for TNF-alpha G-308A polymorphism using real-time PCR (LightCycler Instrument, Roche Diagnostics) and melting curve analysis method. X(2) test was used to compare distribution of TNF-alpha polymorphism genotypes between patients and control group. Relative risk was estimated by the odds ratio (OR). There was no significant statistical difference (X(2)=0.000, DF=1, p=1, OR=1, 95%CI=0.5537-1.8059) between patients and control group. Besides, data of CalphaP patients were stratified according to pathohistological diagnosis (PHD) by Gleason score and groups were compared according to TNF-alpha genotypes. Also, all patients and CalphaP patients were grouped according to prostate volume (V) into three groups: V<50 mL, V50-100 mL, V>100 mL. These groups were also compared according to TNF-alpha genotypes. There were no significant statistical differences between any of groups. Our findings suggest that TNF-alpha -308 SNP is not associated with CalphaP in Eastern Croatia population.

Permanent positive charge strongly influences DNA/RNA binding and antiproliferative activity of urea-phenanthridinium conjugates.

An introduction of the permanent positive charge by methylation of heterocyclic nitrogen on a series of previously studied bis-urea phenanthridine derivatives substantially changed their interactions with DNA and RNA as well as biological activity. At variance to non-methylated analogues, novel methylated derivatives interacted with DNA/RNA not only at pH 5 but also at pH 7, and some compounds switched the DNA binding mode from the minor groove binding (non-methylated derivatives) to the intercalation (novel, methylated derivatives). Moreover, selective ds-RNA over ds-DNA thermal stabilization of previously observed non-methylated derivatives was reversed for novel, methylated derivatives. The variation of a linker length connecting two urea-phenanthridinium conjugates regulated their binding modes toward double stranded polynucleotides. All novel compounds were able to distinguish between polynucleotides of A-T(U) and G-C basepair composition by a specific fluorescence change. Moreover, the introduction of the permanent positive charge on the phenanthridinium moiety resulted in significantly higher biological potency in respect to non-methylated analogues.

Minor structural differences of monomethine cyanine derivatives yield strong variation in their interactions with DNA, RNA as well as on their in vitro antiproliferative activity.

Comparison of binding properties of a series of monomethine cyanine derivatives to ds-DNA and ds-RNA revealed significant impact of the properties of substituent attached to the longer axis of aromatic core. Namely, it seems that only compounds 7, 8 characterised by length of longer axis not exceeding the length of longer axis of basepairs could intercalate into ds-DNA and ds-RNA, while the increased substituent length and additional possibility of hydrogen bonds formation directed binding of 1-6 into ds-DNA minor groove. Consequent ds-RNA over ds-DNA selectivity of 7 and 8 is the most appealing and rather rare property among small molecules. The interactions of 1-8 with ss-RNA were strongly dependent on both, structure of compound and base composition of RNA. The cytotoxicity screening of compounds 1-8 by MTT test revealed considerable antiproliferative activity against solid tumours and especially toward haematological malignancies (IC(50)=0.001-6.6 microM), whereby normal human aortic endothelial cells (HAEC) were significantly less affected (IC(50)=1-200 microM). The cells of chronic myeloid leukaemia in blast crisis (K562) were especially sensitive to all tested compounds (IC(50)=0.001-0.6 microM), while normal lymphocytes were more resistant (IC(50)=0.01-1 microM). Results of uptake and intracellular distribution of compounds 1 and 2 in the living cells showed that they do not bind primarily to nuclear DNA but their fluorescence is scattered through the whole cells. A detailed mechanism of antitumor activity of tested molecules remains to be investigated.

Biological properties of 4-methyl-2,7-diamino-5,10-diphenyl-4,9-diazapyrenium hydrogensulfate (ADAP).

OBJECTIVE: 4-Methyl-2,7-diamino-5,10-diphenyl-4,9-diazapyrenium hydrogensulfate (ADAP) is a potential antitumor compound because of its DNA and RNA intercalating ability. In this study, cellular uptake, intracellular distribution as well as mechanism of action, antitumor activity in vitro and toxicity in vivo of ADAP were investigated. METHODS: Based on the fluorescence properties of ADAP, its entry and distribution into live cells were analyzed by fluorescence microscopy. The in vitro antiproliferative activity was determined using MTT test. For screening of topoisomerase II-targeted effects of ADAP, the cell-free assay and immunoband depletion assay were used. Expression of the genes c-mos, c-N-ras, c-Ki-ras, c-H-ras, p53 and caspase 3 in Caco-2 cells treated with ADAP was examined by RT-PCR. Toxicity in vivo was determined using C3HHf/Bu Zgr/Hr mice treated by single or multiple doses of ADAP at a concentration of 25 mg/kg. RESULTS: ADAP in microM concentrations entered into MIAPaCa-2 cell's cytoplasm in 5 min and into nuclei in 60 min after administration. Intracellular distribution of ADAP depended on the period of treatment time. ADAP (0.1-100 microM) strongly inhibited the growth of both mouse (FsaR, SCCVII) and human tumor cells (HeLa, Caco-2, HT-29, MIAPaCa-2, HBL, HEp-2, SW620, MCF-7) compared to its weak cytotoxicity on controls and normal cells (WI38). Results of both topoisomerase II assays showed that ADAP is not a topoisomerase II poison. Expression of investigated genes was dependent on the incubation time, except for p53 and c-H-ras. Morphological changes in tissues and organs of mice were not observed. Results of patohistological analysis have been confirmed by hematological and clinical-chemical analysis of blood of treated and non-treated animals. CONCLUSION: ADAP is a strongly bioactive compound with antitumor potential in vitro. The antitumor potential in vivo remains to be identified.

Tumor-cell-targeted methionine-enkephalin analogues containing unnatural amino acids: design, synthesis, and in vitro antitumor activity.

A series of new peptides (8-25) containing different unnatural amino acids of the adamantane type (1-6), was synthesized. Possible cytotoxic activity on human cervical adenocarcinoma (HeLa), larynx carcinoma (HEp-2), colon carcinomas (HT-29, Caco-2), poorly differentiated cells from lymph node metastasis of colon carcinoma (SW-620), mammary gland adenocarcinoma (MCF-7), and melanoma (HBL) cells were tested by the MTT assay. The results were compared with the effect of methionine-enkephalin (Tyr-Gly-Gly-Phe-Met, or opioid growth factor, OGF), and its shorter N-terminal fragments. Peptide analogues containing C(alpha alpha)-dialkylated glycine (Aaa1, 1) or C(alpha)-alkylated glycine (Aaa2, 2) amino acid residues showed antitumor activity against melanoma, larynx carcinoma, colon carcinomas, and colon metastasis cell lines in vitro. The pentapeptide Tyr-(R,S)-Aaa2-Gly-Phe-Met (18) was the most effective analogue especially against the most antitumor drug-resistant cell lines HEp-2 and SW-620. Apoptosis as a mode of cell death was confirmed in these tumor cells after exposure to pentapeptide 18.

The influence of cell growth media on the stability and antitumour activity of methionine enkephalin.

Studies with cultured tumour cell lines are widely used in vitro to evaluate peptide-induced cytotoxicity as well as molecular and biochemical interactions. The objectives of this study were to investigate the influence of the cell culture medium on peptide metabolic stability and in vitro antitumour activity. The degradation kinetics of the model peptide methionine enkephalin (Met-E, Tyr-Gly-Gly-Phe-Met), demonstrated recently to play an important role in the rate of proliferation of tumour cells in vitro and in vivo, were investigated in cell culture systems containing different amounts of fetal bovine serum (FBS). The influence of enzyme inhibitors (bestatin, captopril, thiorphan) on the Met-E degradation was also investigated. The results obtained in the Dulbecco's modified Eagle medium containing 10% FBS indicated a rapid degradation of Met-E (t(1/2) = 2.8 h). Preincubation of the medium with a mixture of peptidase inhibitors reduced the hydrolysis of Met-E, as shown by the increased half-life to 10 h. The in vitro activity of Met-E against poorly differentiated cells from lymph node metastasis of colon carcinoma (SW620) and human larynx carcinoma (HEp-2) cells was determined. Tumour cells were grown for 3 weeks prior to the experiment in a medium supplemented with 10%, 5% or 2% FBS. Statistically significant to mild or no suppression of cell proliferation was observed in all cultures. In both cell lines, a significant suppression of cell growth by a combination of peptidase inhibitors and Met-E, compared with cells exposed to the peptide alone and cells grown in the absence of Met-E, was observed. This study indicated that caution must be exercised in interpreting the antiproliferative effects of peptide compounds in conventional drug-response assays.