From old alkylating agents to new minor groove binders.

Alkylating agents represent the oldest class of anticancer agents with the approval of mechloretamine by the FDA in 1949. Even though their clinical use is far beyond the use of new targeted therapies, they still occupy a major place in the treatment of specific malignancies, sometimes representing the unique option for the treatment of refractory tumors. Here, we are reviewing the major classes of alkylating agents, with a particular focus on the latest generations of compounds that specifically target the minor groove of the DNA. These naturally occurring derivatives have a unique mechanism of action that explains the recent regain of interest in developing new classes of alkylating agents that could be used in combination with other anticancer drugs to enhance tumor response in the clinic.

The polyphenolic ellagitannin vescalagin acts as a preferential catalytic inhibitor of the α isoform of human DNA topoisomerase II.

Polyphenolic ellagitannins are natural compounds that are often associated with the therapeutic activity of plant extracts used in traditional medicine. They display cancer-preventing activity in animal models by a mechanism that remains unclear. Potential targets have been proposed, including DNA topoisomerases II (Top2). Top2α and Top2ß, the two isoforms of the human Top2, play a crucial role in the regulation of replication, transcription, and chromosome segregation. They are the target of anticancer agents used in the clinic such as anthracyclines (e.g., doxorubicin) or the epipodophyllotoxin etoposide. It was recently shown that the antitumor activity of etoposide was due primarily to the inhibition of Top2α, whereas inhibition of Top2ß was responsible for the development of secondary malignancies, pointing to the need for more selective Top2α inhibitors. Here, we show that the polyphenolic ellagitannin vescalagin preferentially inhibits the decatenation activity of Top2α in vitro, by a redox-independent mechanism. In CEM cells, we also show that transient small interfering RNA-mediated down-regulation of Top2α but not of Top2ß conferred a resistance to vescalagin, indicating that the α isoform is a preferential target. We further confirmed that Top2α inhibition was due to a catalytic inhibition of the enzyme because it did not induce DNA double-strand breaks in CEM-treated cells but prevented the formation of Top2α- rather than Top2ß-DNA covalent complexes induced by etoposide. To our knowledge, vescalagin is the first example of a catalytic inhibitor for which cytotoxicity is due, at least in part, to the preferential inhibition of Top2α.

S100-B protein as a screening tool for the early assessment of minor head injury.

STUDY OBJECTIVE: A computed tomography (CT) scan has high sensitivity in detecting intracranial injury in patients with minor head injury but is costly, exposes patients to high radiation doses, and reveals clinically relevant lesions in less than 10% of cases. We evaluate S100-B protein measurement as a screening tool in a large population of patients with minor head injury. METHODS: We conducted a prospective observational study in the emergency department of a teaching hospital (Bordeaux, France). Patients with minor head injury (2,128) were consecutively included from December 2007 to February 2009. CT scans and plasma S100-B levels were compared for 1,560 patients. The main outcome was to evaluate the diagnostic value of the S100-B test, focusing on the negative predictive value and the negative likelihood ratio. RESULTS: CT scan revealed intracranial lesions in 111 (7%) participants, and their median S100-B protein plasma level was 0.46 µg/L (interquartile range [IQR] 0.27 to 0.72) versus 0.22 µg/L (IQR 0.14 to 0.36) in the other 1,449 patients. With a cutoff of 0.12 µg/L, traumatic brain injuries on CT were identified with a sensitivity of 99.1% (95% confidence interval [CI] 95.0% to 100%), a specificity of 19.7% (95% CI 17.7% to 21.9%), a negative predictive value of 99.7% (95% CI 98.1% to 100%), a positive likelihood ratio of 1.24 (95% CI 1.20 to 1.28), and a negative likelihood ratio of 0.04 (95% CI 0.006 to 0.32). CONCLUSION: Measurement of plasma S100-B on admission of patients with minor head injury is a promising screening tool that may be of help to support the clinician's decision not to perform CT imaging in certain cases of low-risk head injury.

Protein arginine (N)-methyl transferase 7 (PRMT7) as a potential target for the sensitization of tumor cells to camptothecins.

PRMT7 belongs to the protein arginine methyl-transferases family. We show that downregulation of PRMT7alpha and beta isoforms in DC-3F hamster cells was associated with increased sensitivity to the Top1 inhibitor camptothecin (CPT). This effect was not due to a change in Top1 contents or catalytic activity, or to a difference in the reversal of DNA breaks. Overexpression of PRMT7alpha and beta in DC-3F cells had no effect on CPT sensitivity, whereas it conferred a resistance to DC-3F/9-OH-E cells for which both isoforms are reduced by two- to three-fold as compared to DC-3F parental cells. Finally, downregulation of the human PRMT7 could also sensitize HeLa cells to CPT, suggesting that it could be used as a target to potentiate CPT derivatives.

Synthesis and antiproliferative activity of aryl- and heteroaryl-hydrazones derived from xanthone carbaldehydes.

In order to explore the antiproliferative effect associated with the xanthone framework, several arylhydrazonomethyl derivatives were synthesized from various isomeric 1,3-dihydroxyxanthone carbaldehydes. Variation in the position of the aldehydic function led to three sets of compounds, bearing the hydrazonomethyl chain at positions 5, 6 or 7 on the xanthone nucleus, respectively. The antiproliferative effect of the compounds was evaluated in vitro using the MTT colorimetric method against two human cancer cell lines (MCF-7, breast adenocarcinoma, and KB 3.1, squamous cell oral carcinoma) for two time periods (24 h and 72 h). Among the series, four compounds exhibited interesting growth inhibitory effects against both the cell lines, with IC(50) values in the micromolar concentration range. When compared with doxorubicin, the xanthone derivatives showed moderate cytotoxic effects. Surprisingly, unlike doxorubicin, these compounds displayed no significant time-dependent change in the concentration causing 50% inhibitory effect in proliferation. This unusual cytotoxicity profile led to the hypothesis that these molecules could be endowed with a mechanism of action distinct to that of doxorubicin.

Inhibition of topoisomerase I cleavage activity by thiol-reactive compounds: importance of vicinal cysteines 504 and 505.

DNA topoisomerase I (Top1) is a nuclear enzyme that plays a crucial role in the removal of DNA supercoiling associated with replication and transcription. It is also the target of the anticancer agent, camptothecin (CPT). Top1 contains eight cysteines, including two vicinal residues (504 and 505), which are highly conserved across species. In this study, we show that thiol-reactive compounds such as N-ethylmaleimide and phenylarsine oxide can impair Top1 catalytic activity. We demonstrate that in contrast to CPT, which inhibits Top1-catalyzed religation, thiolation of Top1 inhibited the DNA cleavage step of the reaction. This inhibition was more pronounced when Top1 was preincubated with the thiol-reactive compound and could be reversed in the presence of dithiothreitol. We also established that phenylarsine oxide-mediated inhibition of Top1 cleavage involved the two vicinal cysteines 504 and 505, as this effect was suppressed when cysteines were mutated to alanines. Interestingly, mutation of Cys-505 also altered Top1 sensitivity to CPT, even in the context of the double Cys-504 to Cys-505 mutant, which relaxed supercoiled DNA with a comparable efficiency to that of wild-type Top1. This indicates that cysteine 505, which is located in the lower Lip domain of human Top1, is critical for optimal poisoning of the enzyme by CPT and its analogs. Altogether, our results suggest that conserved vicinal cysteines 504 and 505 of human Top1 play a critical role in enzyme catalytic activity and are the target of thiol-reactive compounds, which may be developed as efficient Top1 catalytic inhibitors.

The chemistry of wine polyphenolic C-glycosidic ellagitannins targeting human topoisomerase II.

Polyphenolic nonahydroxyterphenoyl-containing C-glycosidic oak ellagitannins are found in wine as a result of the aging of this beverage in oak-made barrels. Once in the slightly acidic wine (pH approximately 3-4), some of these complex natural products such as (-)-vescalagin (1), but not its C-1 epimer (-)-castalagin (2), can capture grape-derived nucleophilic entities such as ethanol, the flavanols catechin (10a) and epicatechin (10b), the anthocyanin oenin (13b), and the thiolic glutathione (16) to furnish condensation products with retention of configuration at the C-1 locus. A computer-aided rationale of this high diastereoselectivity is given. These condensation products can contribute to the modulation of organoleptic properties of the wine, as evidenced by the 23 nm bathochromic shift color absorbance observed with the novel oenin-based anthocyano-ellagitannin (15b). Hydrolysis of 1 under solvolytic conditions furnished another novel compound that we refer to as vescalene (21), in addition to the known (-)-vescalin (18). Of pharmacological importance is the fact that most of these found-in-wine water-soluble ellagitannin derivatives are much more potent than etoposide (VP-16) at inhibiting top2-mediated DNA decatenation in vitro (top2=topoisomerase II)). The known (-)-vescalin (18) and the novel vescalene (21) fully inhibited top2 at 10 microM concentration!

Fecal chymotrypsin and elastase-1 determination on one single stool collected at random: diagnostic value for exocrine pancreatic status.

OBJECTIVE: The secretin-cholecystokinin test is the "gold standard" to evaluate exocrine pancreatic function. But this direct duodenal intubation test is invasive, particularly in children, time-consuming, and expensive. For several years, indirect noninvasive tests of pancreatic insufficiency have been developed, such as fecal chymotrypsin (FChT) and fecal elastase-1 (FEL-1) measurements. Generally, elastase-1 is truly admitted to be the most relevant test of exocrine pancreatic status. However, so far, no consensus for stool collection protocol exists. The aim of our study was to investigate the diagnostic advantage from measuring fecal proteases in stool samples collected for two or three consecutive days in comparison to one single stool sample collected at random. DESIGN: A total of 69 children were divided into group A (stool samples collected for three consecutive days) and group B (stool samples collected for two consecutive days). These two groups included pancreatic-sufficient patients (PS) and severe pancreatic-insufficient patients (PI). One single determination of fecal chymotrypsin activity and of fecal elastase-1 concentration was realized on each stool. RESULTS: The same relatively important intraindividual variability of fecal proteases was observed in group A and B (mean coefficients of variation (CVs) 36% vs. 40.2% for chymotrypsin, 22.2% vs. 26.8% for elastase-1). No significant PS or severe PI diagnostic discordance was observed between 1, 2, or 3 days of stool collections. CONCLUSION: Our study clearly shows that the determination of fecal proteases on one single stool collected at random is sufficient to evaluate pancreatic exocrine status for PS and severe PI.

Mass transfer, clearance and plasma concentration of procalcitonin during continuous venovenous hemofiltration in patients with septic shock and acute oliguric renal failure.

OBJECTIVES: To measure the mass transfer and clearance of procalcitonin (PCT) in patients with septic shock during continuous venovenous hemofiltration (CVVH), and to assess the mechanisms of elimination of PCT. SETTING: The medical department of intensive care. DESIGN: A prospective, observational study. PATIENTS: Thirteen critically ill patients with septic shock and oliguric acute renal failure requiring continuous venovenous postdilution hemofiltration with a high-flux membrane (AN69 or polyamide) and a 'conventional' substitution volume (< 2.5 l/hour). MEASUREMENTS AND MAIN RESULTS: PCT was measured with the Lumitest PCT Brahms(R) in the prefilter and postfilter plasma, in the ultrafiltrate at the beginning of CVVH (T0) and 15 min (T15'), 60 min (T60') and 6 hours (T6h) after setup of CVVH, and in the prefilter every 24 hours during 4 days. Mass transfer was determined and the clearance and the sieving coefficient were calculated according to the mass conservation principle. Plasma and ultrafiltrate clearances, respectively, at T15', T60' and T6h were 37 +/- 8.6 ml/min (not significant) and 1.8 +/- 1.7 ml/min (P < 0.01), 34.7 +/- 4.1 ml/min (not significant) and 2.3 +/- 1.8 ml/min (P < 0.01), and 31.5 +/- 7 ml/min (not significant) and 5 +/- 2.3 ml/min (P < 0.01). The sieving coefficient significantly increased from 0.07 at T15' to 0.19 at T6h, with no difference according to the nature of the membrane. PCT plasma levels were not significantly modified during the course of CCVH. CONCLUSIONS: We conclude that PCT is removed from the plasma of patients with septic shock during CCVH. Most of the mass is eliminated by convective flow, but adsorption also contributes to elimination during the first hours of CVVH. The effect of PCT removal with a conventional CVVH substitution fluid rate (<2.5 l/hour) on PCT plasma concentration seems to be limited, and PCT remains a useful diagnostic marker in these septic patients. The impact of high-volume hemofiltration on the PCT clearance, the mass transfer and the plasma concentration should be evaluated in further studies.

C-reactive protein and procalcitonin as markers of mortality in hemodialysis patients: a 2-year prospective study.

OBJECTIVE: We have previously shown in a transversal study that PCT combined to CRP is associated to an altered nutritional status in hemodialysis patients. In a 2-year prospective study, we have assessed the relationship between markers of inflammation or nutrition and mortality. DESIGN: Two-year prospective study, in 61 patients dialyzed in our unit (29 M/32 F, age 63 +/- 15 years, on dialysis for 76 +/- 94 months, 12 hrs/wk, on high-flux (HF) membrane for 25 patients and low-flux (LF) for 36 patients, without reuse). Kt/V was 1.53 +/- 0.30. SETTING: Hospital-based dialysis unit. MAIN OUTCOME MEASURE: CRP, PCT, ferritin, albumin, and prealbumin, were measured in 04/99 (T0) and every 6 months thereafter. Interleukin-6 (IL6) and fibrinogen were measured at the start of study. The outcome and the causes of death of patients were noted in 58 patients, 3 patients were lost of follow-up. RESULTS: The mortality (24 deaths) was 42% at 2 years in this hospital based unit. The main causes of mortality were cardiovascular diseases (71%) and infection (17%). Patients were classified according to their CRP (CRP+ if CRP > or = 5 mg/L; n = 40), and PCT values (PCT + if PCT > or = 0.5 ng/mL; n = 25). IL6 level was > or = 10 pg/mL for 95% of the patients. Mortality was higher in the CRP+ group (Kaplan-Meier test P < .01) but not in the PCT or IL6 positive patients. All patients of the CRP+ group at T0 remained CRP+. Only 56% of patients of PCT+ remained positive at 6 months. When patients were grouped according to CRP quartile the difference on survival remained significant (P = .03), patients who were classified in the third and fourth quartile (upper than 9.9 mg/L), exhibited a higher rate of mortality than the lower quartile. The concomitant presence of a high level of PCT and CRP was associated with a worsened nutritional status at T0 but PCT level had no influence on 2-year mortality. CONCLUSION: In this 2-year prospective study in a hospital-based cohort of high-risk hemodialysis patients, elevated CRP, but not raised PCT, was associated with increased mortality. Inflammation remained present throughout a 2-year follow-up in patients with an initial CRP higher than 5 mg/L. An upper value of CRP above 9.9 mg/L is independly predictive of mortality, mainly from cardiovascular causes. The association of high PCT and CRP was no more predictive of mortality than high CRP.

Determinants of the cytotoxicity of irinotecan in two human colorectal tumor cell lines.

PURPOSE: Irinotecan is a drug of the camptothecin family that has proven activity in advanced colon cancer, with about 20% responses in untreated as well as in 5-fluorouracil-resistant tumors. Irinotecan is considered as a prodrug which needs to be activated to SN-38 by carboxylesterases to become able to interact with its target, topoisomerase I. The work reported here intended to identify the determinants of the cytotoxicity of irinotecan in two human colorectal tumor cell lines, LoVo and HT-29, at the level of the target of the drug and at the level of the availability of the active metabolite to the target. RESULTS: The cytotoxicity of irinotecan and SN-38 markedly differed in the two cell lines: irinotecan IC(50) values were 15.8 microM for LoVo cells and 5.17 microM for HT-29 cells; SN-38 IC(50) values were 8.25 n M for LoVo cells and 4.50 n M for HT-29 cells. Topoisomerase I expression (at the mRNA and the protein levels) and catalytic activity were similar in the two cell lines. Irinotecan induced similar amounts of cleavable complexes at its IC(50) in both cell lines. SN-38 induced a concentration-dependent formation of cleavable complexes, which was not significantly different in the two cell lines. Expression of the carboxylesterase CES1 was higher in HT-29 than in LoVo cells. Expression of the carboxylesterase gene CES2 was comparable in the two cell lines and much higher than CES1 gene expression. Carboxylesterase activity was extremely low using p-nitrophenylacetate as a substrate (1.45 and 1.84 pmol/min per mg proteins) and could not even be detected using irinotecan as a substrate. Cell accumulation of irinotecan was markedly different, reaching consistently higher levels in HT-29 cells than in LoVo cells. CONCLUSIONS: Our results indicate that (1) the cytotoxicity of irinotecan was likely due to the drug itself and not to its metabolite SN-38, and (2) that irinotecan uptake was more predictive of its cytotoxicity than topoisomerase I availability and activity in these two cell lines.