Structural basis for the sequence-dependent effects of platinum–DNA adducts

The differences in efficacy and molecular mechanisms of platinum based anti-cancer drugs cisplatin (CP) and oxaliplatin (OX) have been hypothesized to be in part due to the differential binding affinity of cellular and damage recognition proteins to CP and OX adducts formed on adjacent guanines in genomic DNA. HMGB1a in particular exhibits higher binding affinity to CP-GG adducts, and the extent of discrimination between CP- and OX-GG adducts is dependent on the bases flanking the adducts. However, the structural basis for this differential binding is not known. Here, we show that the conformational dynamics of CP- and OX-GG adducts are distinct and depend on the sequence context of the adduct. Molecular dynamics simulations of the Pt-GG adducts in the TGGA sequence context revealed that even though the major conformations of CP- and OX-GG adducts were similar, the minor conformations were distinct. Using the pattern of hydrogen bond formation between the Pt–ammines and the adjacent DNA bases, we identified the major and minor conformations sampled by Pt–DNA. We found that the minor conformations sampled exclusively by the CP-GG adduct exhibit structural properties that favor binding by HMGB1a, which may explain its higher binding affinity to CP-GG adducts, while these conformations are not sampled by OX-GG adducts because of the constraints imposed by its cyclohexane ring, which may explain the negligible binding affinity of HMGB1a for OX-GG adducts in the TGGA sequence context. Based on these results, we postulate that the constraints imposed by the cyclohexane ring of OX affect the DNA conformations explored by OX-GG adduct compared to those of CP-GG adduct, which may influence the binding affinities of HMG-domain proteins for Pt-GG adducts, and that these conformations are further influenced by the DNA sequence context of the Pt-GG adduct

Walking of antitumor bifunctional trinuclear PtII complex on double-helical DNA

The trinuclear BBR3464 ([{trans-PtCl(NH3)2}2µ-(trans-Pt(NH3)2(H2N(CH2)6NH2)2)]4+) belongs to the polynuclear class of platinum-based anticancer agents. DNA adducts of this complex differ significantly in structure and type from those of clinically used mononuclear platinum complexes, especially, long-range (Pt, Pt) intrastrand and interstrand cross-links are formed in both 5′–5′ and 3′–3′ orientations. We show employing short oligonucleotide duplexes containing single, site-specific cross-links of BBR3464 and gel electrophoresis that in contrast to major DNA adducts of clinically used platinum complexes, under physiological conditions the coordination bonds between platinum and N7 of G residues involved in the cross-links of BBR3464 can be cleaved. This cleavage may lead to the linkage isomerization reactions between this metallodrug and double-helical DNA. Differential scanning calorimetry of duplexes containing single, site-specific cross-links of BBR3464 reveals that one of the driving forces that leads to the lability of DNA cross-links of this metallodrug is a difference between the thermodynamic destabilization induced by the cross-link and by the adduct into which it could isomerize. The rearrangements may proceed in the way that cross-links originally formed in one strand of DNA can spontaneously translocate from one DNA strand to its complementary counterpart, which may evoke walking of the platinum complex on DNA molecule

Cisplatin and Doxorubicin Induce Distinct Mechanisms of Ovarian Follicle Loss; Imatinib Provides Selective Protection Only against Cisplatin

Chemotherapy treatment in premenopausal women has been linked to ovarian follicle loss and premature ovarian failure; the exact mechanism by which this occurs is uncertain. Here, two commonly used chemotherapeutic agents (cisplatin and doxorubicin) were added to a mouse ovary culture system, to compare the sequence of events that leads to germ cell loss. The ability of imatinib mesylate to protect the ovary against cisplatin or doxorubicin-induced ovarian damage was also examined.Newborn mouse ovaries were cultured for a total of six days, exposed to a chemotherapeutic agent on the second day: this allowed for the examination of the earliest stages of follicle development. Cleaved PARP and TUNEL were used to assess apoptosis following drug treatment. Imatinib was added to cultures with cisplatin and doxorubicin to determine any protective effect.Histological analysis of ovaries treated with cisplatin showed oocyte-specific damage; in comparison doxorubicin preferentially caused damage to the granulosa cells. Cleaved PARP expression significantly increased for cisplatin (16 fold, p<0.001) and doxorubicin (3 fold, p<0.01). TUNEL staining gave little evidence of primordial follicle damage with either drug. Imatinib had a significant protective effect against cisplatin-induced follicle damage (p<0.01) but not against doxorubicin treatment.Cisplatin and doxorubicin both induced ovarian damage, but in a markedly different pattern, with imatinib protecting the ovary against damage by cisplatin but not doxorubicin. Any treatment designed to block the effects of chemotherapeutic agents on the ovary may need to be specific to the drug(s) the patient is exposed to

Non-invasive diagnostic tests for Helicobacter pylori infection

BACKGROUND: Helicobacter pylori (H pylori) infection has been implicated in a number of malignancies and non-malignant conditions including peptic ulcers, non-ulcer dyspepsia, recurrent peptic ulcer bleeding, unexplained iron deficiency anaemia, idiopathic thrombocytopaenia purpura, and colorectal adenomas. The confirmatory diagnosis of H pylori is by endoscopic biopsy, followed by histopathological examination using haemotoxylin and eosin (H & E) stain or special stains such as Giemsa stain and Warthin-Starry stain. Special stains are more accurate than H & E stain. There is significant uncertainty about the diagnostic accuracy of non-invasive tests for diagnosis of H pylori. OBJECTIVES: To compare the diagnostic accuracy of urea breath test, serology, and stool antigen test, used alone or in combination, for diagnosis of H pylori infection in symptomatic and asymptomatic people, so that eradication therapy for H pylori can be started. SEARCH METHODS: We searched MEDLINE, Embase, the Science Citation Index and the National Institute for Health Research Health Technology Assessment Database on 4 March 2016. We screened references in the included studies to identify additional studies. We also conducted citation searches of relevant studies, most recently on 4 December 2016. We did not restrict studies by language or publication status, or whether data were collected prospectively or retrospectively. SELECTION CRITERIA: We included diagnostic accuracy studies that evaluated at least one of the index tests (urea breath test using isotopes such as13C or14C, serology and stool antigen test) against the reference standard (histopathological examination using H & E stain, special stains or immunohistochemical stain) in people suspected of having H pylori infection. DATA COLLECTION AND ANALYSIS: Two review authors independently screened the references to identify relevant studies and independently extracted data. We assessed the methodological quality of studies using the QUADAS-2 tool. We performed meta-analysis by using the hierarchical summary receiver operating characteristic (HSROC) model to estimate and compare SROC curves. Where appropriate, we used bivariate or univariate logistic regression models to estimate summary sensitivities and specificities. MAIN RESULTS: We included 101 studies involving 11,003 participants, of which 5839 participants (53.1%) had H pylori infection. The prevalence of H pylori infection in the studies ranged from 15.2% to 94.7%, with a median prevalence of 53.7% (interquartile range 42.0% to 66.5%). Most of the studies (57%) included participants with dyspepsia and 53 studies excluded participants who recently had proton pump inhibitors or antibiotics.There was at least an unclear risk of bias or unclear applicability concern for each study.Of the 101 studies, 15 compared the accuracy of two index tests and two studies compared the accuracy of three index tests. Thirty-four studies (4242 participants) evaluated serology; 29 studies (2988 participants) evaluated stool antigen test; 34 studies (3139 participants) evaluated urea breath test-13C; 21 studies (1810 participants) evaluated urea breath test-14C; and two studies (127 participants) evaluated urea breath test but did not report the isotope used. The thresholds used to define test positivity and the staining techniques used for histopathological examination (reference standard) varied between studies. Due to sparse data for each threshold reported, it was not possible to identify the best threshold for each test.Using data from 99 studies in an indirect test comparison, there was statistical evidence of a difference in diagnostic accuracy between urea breath test-13C, urea breath test-14C, serology and stool antigen test (P = 0.024). The diagnostic odds ratios for urea breath test-13C, urea breath test-14C, serology, and stool antigen test were 153 (95% confidence interval (CI) 73.7 to 316), 105 (95% CI 74.0 to 150), 47.4 (95% CI 25.5 to 88.1) and 45.1 (95% CI 24.2 to 84.1). The sensitivity (95% CI) estimated at a fixed specificity of 0.90 (median from studies across the four tests), was 0.94 (95% CI 0.89 to 0.97) for urea breath test-13C, 0.92 (95% CI 0.89 to 0.94) for urea breath test-14C, 0.84 (95% CI 0.74 to 0.91) for serology, and 0.83 (95% CI 0.73 to 0.90) for stool antigen test. This implies that on average, given a specificity of 0.90 and prevalence of 53.7% (median specificity and prevalence in the studies), out of 1000 people tested for H pylori infection, there will be 46 false positives (people without H pylori infection who will be diagnosed as having H pylori infection). In this hypothetical cohort, urea breath test-13C, urea breath test-14C, serology, and stool antigen test will give 30 (95% CI 15 to 58), 42 (95% CI 30 to 58), 86 (95% CI 50 to 140), and 89 (95% CI 52 to 146) false negatives respectively (people with H pylori infection for whom the diagnosis of H pylori will be missed).Direct comparisons were based on few head-to-head studies. The ratios of diagnostic odds ratios (DORs) were 0.68 (95% CI 0.12 to 3.70; P = 0.56) for urea breath test-13C versus serology (seven studies), and 0.88 (95% CI 0.14 to 5.56; P = 0.84) for urea breath test-13C versus stool antigen test (seven studies). The 95% CIs of these estimates overlap with those of the ratios of DORs from the indirect comparison. Data were limited or unavailable for meta-analysis of other direct comparisons. AUTHORS' CONCLUSIONS: In people without a history of gastrectomy and those who have not recently had antibiotics or proton ,pump inhibitors, urea breath tests had high diagnostic accuracy while serology and stool antigen tests were less accurate for diagnosis of Helicobacter pylori infection.This is based on an indirect test comparison (with potential for bias due to confounding), as evidence from direct comparisons was limited or unavailable. The thresholds used for these tests were highly variable and we were unable to identify specific thresholds that might be useful in clinical practice.We need further comparative studies of high methodological quality to obtain more reliable evidence of relative accuracy between the tests. Such studies should be conducted prospectively in a representative spectrum of participants and clearly reported to ensure low risk of bias. Most importantly, studies should prespecify and clearly report thresholds used, and should avoid inappropriate exclusions

Reconnaissance par des protéines de l'ADN modifié par des complexes du platine antitumoraux

In our work we studied the recognition of DNA-platinum cross-links by different cellular proteins. We have shown that cisplatin-GG adducts within double stranded DNA are weak but specific recognition targets for the HMG domain of LEF-1. In the second part of the thesis we analyzed duplexes containing site-specific adducts of the platinum compound BBR3464 and cisplatin by DNase I footprinting. It was shown that the platinum adducts which induce different conformational alterations in DNA are differently recognized by DNase I. In the third part we demonstrated that cisplatin is considerably more efficient in DNA-protein cross-linking than transplatin and DNA-protein cross-links were shown to be more persistent that intrastrand CLs. In the last part of the thesis, we examined the stability of 1,2-GG-intrastrand CLs within the GGG sequence. We have shown that these CLs were unstable and evolved with half-life times of tens of days at 30C.Dans notre travail, la reconnaissance des adduits ADN-platine par différentes protéines a été étudiée. Nous avons montré que des adduits ADN-platine présentent un site de reconnaissance spécifique mais de faible affinité pour le domaine HMG de LEF-1. Dans une autre étude nous avons utilisé l empreinte à la DNase I pour analyser des doubles brins d ADN contenant un adduit spécifique du complexe de platine BBR3464 et du cisplatine. Nous avons montré que les différents adduits du platine sont reconnus différemment par la DNase I. Concernant les complexes covalents ADN-platine-protéine nous avons montré que le cisplatine est plus efficace dans la formation d un complexe ADN-platine-protéine que le transplatine et ces complexes sont moins bien réparés que les complexes ADN-platine. Nous avons également examiné la stabilité de l adduit 1,2- GG- intrabrin du cisplatine sur les séquences contenant GGG. Nous avons montré que ces adduits s isomérisent lentement en adduits 2,3- GG- intrabrin.PARIS5-BU Saints-Pères (751062109) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Mass spectrometry evidence for cisplatin as a protein cross-linking reagent

Cisplatin is a potent anti-cancer drug, which functions by cross-linking adjacent DNA guanine residues. However within one day of injection, 65~98% of the platinum in the blood plasma is protein-bound. It is generally accepted that cisplatin binds to methionine and histidine residues, but what is often underappreciated is that platinum from cisplatin has a 2+ charge and can form up to four bonds. Thus, it has the potential to function as a cross-linker. In this report, the cross-linking ability of cisplatin is demonstrated by Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) with the use of standard peptides, the 16.8 kDa protein calmodulin (CaM), but was unsuccessful for the 64 kDa protein hemoglobin. The high resolution and mass accuracy of FTICR MS along with the high degree of fragmentation of large peptides afforded by collisionally activated dissociation (CAD) and electron capture dissociation (ECD) are shown to be a valuable means of characterizing cross-linking sites. Cisplatin is different from current cross-linking reagents by targeting new functional groups, thioethers, and imidazoles groups, which provides complementarity with existing cross-linkers. In addition, platinum(II) inherently has two positive charges which enhance the detection of cross-linked products. Higher charge states not only promote the detection of cross-linking products with less purification, but result in more comprehensive MS/MS fragmentation and can assist the assignment of modification sites. Moreover, the unique isotopic pattern of platinum flags cross-linking products and modification sites by mass spectrometry

Carvacrol induces cytotoxicity in human cervical cancer cells but causes cisplatin resistance: Involvement of MEK-ERK activation

Carvacrol has been shown to possess anticancer activity but the mechanism is unknown, as well as the possibility of interaction with anticancer drugs. The aim of this study was to investigate the role of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling in carvacrol-induced human cervical cancer HeLa cell cytotoxicity. In addition, we studied sensitization of HeLa cells to cisplatin (CP) by carvacrol. Both carvacrol and CP showed dose-dependent cytotoxicity against HeLa cells and activated ERK1/2. The MEK inhibitor PD325901 suppressed ERK expression and further increased cytotoxicity of carvacrol but increased viability of CP-treated cells by modulating apoptosis. The MEK inhibitor also increased microtubule-associated protein 1A/1B- light chain 3 (LC3B) expression in CP treatment. Co-treatment with CP and carvacrol resulted in increased viability of the cancer cells compared to CP treatment, which was associated with the suppression of apoptosis. MEK inhibition decreased the cell viability, without changes in apoptosis. Concomitantly, carvacrol increased CP-induced expression of LC3B, which was enhanced by MEK inhibition. The results of the current study suggest the opposite role of ERK1/2 in carvacrol and CP- induced HeLa cell cytotoxicity. Interestingly, carvacrol induced CP resistance in HeLa cells through ERK1/2-independent suppression of apoptosis and ERK1/2-dependent modulation of autophagy