Benzo[ a ]pyrene-7,8-dione is More Mutagenic than Anti -BPDE on p53 and is Dependent on the Generation of Reactive Oxygen Species

Polycyclic aromatic hydrocarbons (PAHs) may cause lung cancer via metabolic activation. p53 is one of the most commonly mutated tumor suppressor genes in this disease. To determine whether PAH o -quinones cause change-in-function mutations in p53 , we employed a yeast reporter system. Treatment of p53 cDNA with ( - )- anti -7,8-dihydroxy-9 f ,10 f -epoxy-7,8,9,10-tetrahydro-benzo[ a ]pyrene, ( anti -BPDE, an ultimate carcinogen) or submicromolar concentrations of BP-7,8-dione under redox-cycling conditions (NADPH and CuCl 2 ) also caused p53 mutagenesis in a dose-dependent manner. It was found that BP-7,8-dione was 80 times more mutagenic than anti -BPDE under these conditions. p53 mutagenesis by BPQ was attenuated by ROS scavengers and completely abrogated by a combination of superoxide dismutase and catalase, indicating that both superoxide anion and hydroxyl radicals were the responsible mutagens. DNA sequencing demonstrated that over 46% of BPQ-induced mutations were G:C to T:A transversions. Together these data suggest that PAH o -quinones generate an endogenous mutagen (ROS) which leads to p53 inactivation.     

The Aldo-Keto Reductases and Polycyclic Aromatic Hydrocarbon Activation

The aldo-keto reductases (AKRs), are monomeric 37 kDa oxidoreductases that catalyze the NADP + -dependent oxidation of PAH trans -dihydrodiol proximate carcinogens to their reactive and redox-active o -quinones. Five human isoforms have been cloned and expressed as purified recombinant enzymes (AKR1C1-AKR1C4 and AKR1A1). Of these the general metabolic enzyme aldehyde reductase (AKR1A1) displays the highest utilization ratio for the oxidation of ( m ) R,R -benzo[ a ]pyrene-7,8-diol (BP-7,8-diol) to benzo[ a ]pyrene-7,8-dione (BP-7,8-dione). AKR1A1 is coexpressed with CYP1A1 and epoxide hydrolase suggesting that it is exposed to its trans -dihydrodiol substrates in situ. PAH o -quinones produced by AKRs are highly electrophilic yielding bimolecular rate constants for the addition of GSH of at least 1.3 2 10 3 M m 1 min m 1 . By reacting with DNA, PAH o -quinones form both stable and depurinating adducts in vitro. By entering into futile redox-cycles the PAH o -quinones can amplify the production of reactive oxygen species and increase 7,8-dihydro-8-oxo-2-deoxyguanosine (8-oxo-dGuo) formation. The spectrum of DNA-adducts and generation of prooxidants (reactive oxygen) may explain how PAHs can act as complete carcinogens.     

Phosphonamidates as thermally latent initiators in the polymerization of epoxides

Summary Phosphonamidates, O,O-di-tert-butyl 1-piperidinyl phosphonamidate (BP-1) and O-tert-butyl di-1-piperidinyl phosphonamidate (BP-2), were synthesized by the reaction of phosphorus oxychloride and piperidine in the presence of triethylamine, followed by the reaction with tert-butyl alcohol in the presence of sodium hydride. The polymerization of GPE was carried out at 110–190 °C for 12 h with BP-1 and BP-2 as thermally latent initiators (3 mol %). The polymerization with O-tert-butyl P-phenyl 1-piperidinyl phosphonamidate (BP) previously reported was also examined for comparison. No polymerization of GPE took place below 110 °C, whereas it proceeded rapidly above the temperature. The activity order was BP-2 > BP > BP-1. Epikote 828 was cured with BP (5 mol %) at 190 °C to afford the solvent-insoluble gelled epoxy resin quantitatively. A mixture of GPE and phosphonamidate BP (3 mol %) did not react at 50 °C for 4 months. Received: 26 February 2001/Revised version: 12 April 2001/Accepted: 13 April 2001     

Design,Synthesis, Structure-Activity Relationship and Docking Studies of Novel Functionalized Arylvinyl-1,2,4-Trioxanes as Potent Antiplasmodial as well as Anticancer Agents

A novel series of synthetic functionalized arylvinyl-1,2,4-trioxanes ( 8 a – p ) has been prepared and assessed for their in vitro antiplasmodial activity against the chloroquine-resistant Pf INDO strain of Plasmodium falciparum by using a SYBR green-I fluorescence assay. Compounds 8 g (IC₅₀=0.051 μM; SI=589.41) and 8 m (IC₅₀=0.059 μM; SI=55.93) showed 11-fold and >9-fold more potent antiplasmodial activity, respectively, as compared to chloroquine (IC₅₀=0.546 μM; SI=36.63). Different in silico docking studies performed on many target proteins revealed that the most active arylvinyl-1,2,4-trioxanes ( 8 g and 8 m ) showed dihydrofolate reductase (DHFR) binding affinities on a par with those of chloroquine and artesunate. The in vitro cytotoxic potentials of 8 a – p were also evaluated against human lung (A549) and liver (HepG2) cancer cell lines along with immortalized normal lung (BEAS-2B) and liver (LO2) cell lines. Following screening, five derivatives viz. 8 a , 8 h , 8 l , 8 m and 8 o (IC₅₀=1.65–31.7 μM; SI=1.08–10.96) were found to show potent cytotoxic activity against (A549) lung cancer cell lines, with selectivity superior to that of the reference compounds artemisinin (IC₅₀=100 μM), chloroquine (IC₅₀=100 μM) and artesunic acid (IC₅₀=9.85 μM; SI=0.76). In fact, the most active 4-naphthyl-substituted analogue 8 l (IC₅₀=1.65 μM; SI >10) exhibited >60 times more cytotoxicity than the standard reference, artemisinin, against A549 lung cancer cell lines. In silico docking studies of the most active anticancer compounds, 8 l and 8 m , against EGFR were found to validate the wet lab results. In summary, a new series of functionalized aryl-vinyl-1,2,4-trioxanes ( 8 a – p ) has been shown to display dual potency as promising antiplasmodial and anticancer agents.     

Cytoplasmic p53β Isoforms Are Associated with Worse Disease-Free Survival in Breast Cancer

TP53 mutations are associated with tumour progression, resistance to therapy and poor prognosis. However, in breast cancer, TP53′s overall mutation frequency is lower than expected (~25%), suggesting that other mechanisms may be responsible for the disruption of this critical tumour suppressor. p53 isoforms are known to enhance or disrupt p53 pathway activity in cell- and context-specific manners. Our previous study revealed that p53 isoform mRNA expression correlates with clinicopathological features and survival in breast cancer and may account for the dysregulation of the p53 pathway in the absence of TP53 mutations. Hence, in this study, the protein expression of p53 isoforms, transactivation domain p53 (TAp53), p53β, Δ40p53, Δ133p53 and Δ160p53 was analysed using immunohistochemistry in a cohort of invasive ductal carcinomas (n = 108). p53 isoforms presented distinct cellular localisation, with some isoforms being expressed in tumour cells and others in infiltrating immune cells. Moreover, high levels of p53β, most likely to be N-terminally truncated β variants, were significantly associated with worse disease-free survival, especially in tumours with wild-type TP53. To the best of our knowledge, this is the first study that analysed the endogenous protein levels of p53 isoforms in a breast cancer cohort. Our findings suggest that p53β may be a useful prognostic marker.     

Mitochondrial Malfunctioning,Proteasome Arrest and Apoptosis in Cancer Cells by Focused Intracellular Generation of Oxygen Radicals

Photofrin/photodynamic therapy (PDT) at sub-lethal doses induced a transient stall in proteasome activity in surviving A549 (p53^+/+) and H1299 (p53^−/−) cells as indicated by the time-dependent decline/recovery of chymotrypsin-like activity. Indeed, within 3 h of incubation, Photofrin invaded the cytoplasm and localized preferentially within the mitochondria. Its light activation determined a decrease in mitochondrial membrane potential and a reversible arrest in proteasomal activity. A similar result is obtained by treating cells with Antimycin and Rotenone, indicating, as a common denominator of this effect, the ATP decrease. Both inhibitors, however, were more toxic to cells as the recovery of proteasomal activity was incomplete. We evaluated whether combining PDT (which is a treatment for killing tumor cells, per se, and inducing proteasome arrest in the surviving ones) with Bortezomib doses capable of sustaining the stall would protract the arrest with sufficient time to induce apoptosis in remaining cells. The evaluation of the mitochondrial membrane depolarization, residual proteasome and mitochondrial enzymatic activities, colony-forming capabilities, and changes in protein expression profiles in A549 and H1299 cells under a combined therapeutic regimen gave results consistent with our hypothesis.     

Mutant p53 Mediates Sensitivity to Cancer Treatment Agents in Oesophageal Adenocarcinoma Associated with MicroRNA and SLC7A11 Expression

TP53 gene mutations occur in 70% of oesophageal adenocarcinomas (OACs). Given the central role of p53 in controlling cellular response to therapy we investigated the role of mutant (mut-) p53 and SLC7A11 in a CRISPR-mediated JH-EsoAd1 TP53 knockout model. Response to 2 Gy irradiation, cisplatin, 5-FU, 4-hydroxytamoxifen, and endoxifen was assessed, followed by a TaqMan OpenArray qPCR screening for differences in miRNA expression. Knockout of mut-p53 resulted in increased chemo- and radioresistance (2 Gy survival fraction: 38% vs. 56%, p < 0.0001) and in altered miRNA expression levels. Target mRNA pathways analyses indicated several potential mechanisms of treatment resistance. SLC7A11 knockdown restored radiosensitivity (2 Gy SF: 46% vs. 73%; p = 0.0239), possibly via enhanced sensitivity to oxidative stress. Pathway analysis of the mRNA targets of differentially expressed miRNAs indicated potential involvement in several pathways associated with apoptosis, ribosomes, and p53 signaling pathways. The data suggest that mut-p53 in JH-EsoAd1, despite being classified as non-functional, has some function related to radio- and chemoresistance. The results also highlight the important role of SLC7A11 in cancer metabolism and redox balance and the influence of p53 on these processes. Inhibition of the SLC7A11-glutathione axis may represent a promising approach to overcome resistance associated with mut-p53.     

Evaluation of a Novel Oncolytic Adenovirus Silencing SYVN1

Oncolytic adenoviruses are promising new anticancer agents. To realize their full anticancer potential, they are being engineered to express therapeutic payloads. Tumor suppressor p53 function contributes to oncolytic adenovirus activity. Many cancer cells carry an intact TP53 gene but express p53 inhibitors that compromise p53 function. Therefore, we hypothesized that oncolytic adenoviruses could be made more effective by suppressing p53 inhibitors in selected cancer cells. To investigate this concept, we attenuated the expression of the established p53 inhibitor synoviolin (SYVN1) in A549 lung cancer cells by RNA interference. Silencing SYVN1 inhibited p53 degradation, thereby increasing p53 activity, and promoted adenovirus-induced A549 cell death. Based on these observations, we constructed a new oncolytic adenovirus that expresses a short hairpin RNA against SYVN1. This virus killed A549 cells more effectively in vitro and inhibited A549 xenograft tumor growth in vivo. Surprisingly, increased susceptibility to adenovirus-mediated cell killing by SYVN1 silencing was also observed in A549 TP53 knockout cells. Hence, while the mechanism of SYVN1-mediated inhibition of adenovirus replication is not fully understood, our results clearly show that RNA interference technology can be exploited to design more potent oncolytic adenoviruses.     

Polycondensation of squaric acid with N-alkylcarbazoles

Summary 1,2-Dihydroxycyclobutene-3,4-dione (squaric acid) was polycondensed with N-ethyl- and N-(1-butyl)carbazole in polyphosphoric acid to give polymers having 36–43% of 1,2-oriented squarate units. The polymers are insoluble in organic solvents or sulfuric acid. Condensation of 1,2-dichlorocyclobutene-3,4-dione (squaryl dichloride) with N-1-butyl-carbazole in nitrobenzene gave 100% of 1,2-oriented squarate units. This polymer is soluble in most organic solvents, and the molecular weight (¯Mw) is 1900 (DP=6). The electrical conductivity was low, <10^–9 (ohm cm)^–1, and did not increase on treatment with iodine.     

The Complete Loss of p53 Expression Uniquely Predicts Worse Prognosis in Colorectal Cancer

p53 immunohistochemistry is considered an accurate surrogate marker reflecting the underlying TP53 mutation status and has utility in tumor diagnostics. In the present study, 269 primary CRCs were immunohistochemically evaluated for p53 expression to assess its utility in diagnostic pathology and prognostication. p53 expression was wild-type in 59 cases (23%), overexpressed in 143 cases (55%), completely lost in 50 cases (19%), and cytoplasmic in 10 cases (4%). p53 immunoreactivity was associated with tumor size (p = 0.0056), mucus production (p = 0.0015), and mismatch repair (MMR) system status (p < 0.0001). Furthermore, among CRCs with wild-type p53 expression, a significantly higher number of cases had decreased CDX2 than those with p53 overexpression (p = 0.012) or complete p53 loss (p = 0.043). In contrast, among CRCs with p53 overexpression, there were significantly fewer ALCAM-positive cases than p53 wild-type cases (p = 0.0045). However, no significant association was detected between p53 immunoreactivity and the “stem-like” immunophenotype defined by CDX2 downregulation and ALCAM-positivity. Multivariate Cox hazards regression analysis identified tubular-forming histology (hazard ratio [HR] = 0.17, p < 0.0001), younger age (HR = 0.52, p = 0.021), and female sex (HR = 0.55, p = 0.046) as potential favorable factors. The analysis also revealed complete p53 loss (HR = 2.16, p = 0.0087), incomplete resection (HR = 2.65, p = 0.0068), and peritoneal metastasis (HR = 5.32, p < 0.0001) as potential independent risk factors for patients with CRC. The sub-cohort survival analyses classified according to chemotherapy after surgery revealed that CRC patients with wild-type p53 expression tended to have better survival than those with overexpression or complete loss after chemotherapy. Thus, immunohistochemistry for p53 could be used for the prognostication and chemotherapy target selection of patients with CRC.     

Comparative study of unsaturated halo and nonhalo polyesters and inhibition of double-base rocket propellants

Unsaturated halo and nonhalo polyesters, based on tetrachlorophthalic anhydride, tetrabromophthalic anhydride, and phthalic anhydride with diethylene glycol and maleic anhydride, have been synthesized, using a “2-step” polyesterification process. These were mixed with 35% styrene monomer and 0.02% hydroquinone. The structural aspects of halo and nonhalo polyesters have been studied by IR and NMR. Further, these polyesters have been characterized for various properties. A comparison of various properties suggests that tensile strength, bond strength, heat resistance, flame retardance, gel time, and oxygen index follow the order as BP-35 > CP-35 > NHP-35, whereas the order for other properties, such as exotherm peak temperature, thermal conductivity, % elongation, and nitroglycerine absorption, is BP-35 < CP-35 < NHP-35. Variation in the properties of cured halo and nonhalo polyesters has been discussed in terms of structural considerations. The nature of P-t profile of inhibited double-base (DB) rocket propellants on static evaluation suggests that CP-35 is a potential inhibitor for DB propellants. © 1993 John Wiley & Sons, Inc.     

Differences in the Relative Abundance of ProBDNF and Mature BDNF in A549 and H1299 Human Lung Cancer Cell Media

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, has been linked to several human malignancies and shown to promote tumorigenesis. The purpose of this study was to explore the relative abundance of pro-brain-derived neurotrophic factor (proBDNF) and mature BDNF (mBDNF) in A549 (p53 wild-type) and H1299 (p53-null) lung cancer cell media. Higher levels of proBDNF were detected in the media of A549 cells than in H1299 cell media. Using inhibitors, we found that the levels of proBDNF and mBDNF in the media are likely regulated by PI3K, AKT, and NFκB. However, the largest change in these levels resulted from MMP2/9 inhibition. Blocking p53 function in A549 cells resulted in increased mBDNF and decreased proBDNF, suggesting a role for p53 in regulating these levels. The ratio of proBDNF/mBDNF was not affected by MMP2 knockdown but increased in the media of both cell lines upon knockdown of MMP9. Downregulation of either MMP2 or MMP9 by siRNA showed that MMP9 siRNA treatment of either A549 or H1299 cells resulted in decreased cell viability and increased apoptosis, an effect diminished upon the same treatment with proBDNF immunodepleted media, suggesting that MMP9 regulates the cytotoxic effects induced by proBDNF in lung cancer cells.     

β-Catenin-Specific Inhibitor,iCRT14, Promotes BoHV-1 Infection-Induced DNA Damage in Human A549 Lung Adenocarcinoma Cells by Enhancing Viral Protein Expression

Oncolytic bovine herpesvirus type 1 (BoHV-1) infection induces DNA damage in human lung adenocarcinoma cell line A549. However, the underlying mechanisms are not fully understood. We found that BoHV-1 infection decreased the steady-state protein levels of p53-binding protein 1 (53BP1), which plays a central role in dictating DNA damage repair and maintaining genomic stability. Furthermore, BoHV-1 impaired the formation of 53BP1 foci, suggesting that BoHV-1 inhibits 53BP1-mediated DNA damage repair. Interestingly, BoHV-1 infection redistributed intracellular β-catenin, and iCRT14 (5-[[2,5-Dimethyl-1-(3-pyridinyl)-1H-pyrrol-3-yl]methylene]-3-phenyl-2,4-thiazolidinedione), a β-catenin-specific inhibitor, enhanced certain viral protein expression, such as the envelope glycoproteins gC and gD, and enhanced virus infection-induced DNA damage. Therefore, for the first time, we provide evidence showing that BoHV-1 infection disrupts 53BP1-mediated DNA damage repair and suggest β-catenin as a potential host factor restricting both virus replication and DNA damage in A549 cells.     

Approximations for non-symmetric truncated sequential and repeated significance tests

Let X₁, X₂…X_N be a random sample from a N(μ, l) population. Using the B-value process {B(t); 0 ≤ t ≤ 1} introduced by Lan and Wittes (1988) one ran design a sequential test for H₀ : μ = μ₀ versus H₁ : μ > μ₀ for some functions γ1(t) > γ2(t): if B(t) > γ₁(t) stop and reject H₀ , if B(t) < γ2(t) stop and do not reject H₀ , otherwise take one more observation. When t = 1, stop anyway rejecting H₀ if B(1) > γ1(1). We suggest some approximations which are usually upper bounds for the significance level, power of the tests and lower bounds for the expected stopping time. Simulations show that the approximations get better as N → ∞ and/or μ → ∞. The exact values are difficult to obtain while the suggested approximations are very easy to compute in the case of linear and RST type boundaries and simulation results show that they are quite reasonable in several cases, even for moderate N.     

Ambient PM exposure and DNA methylation in tumor suppressor genes: a cross-sectional study

Exposure to ambient air particles matter (PM) has been associated with increased risk of lung cancer. Aberrant tumor suppressor gene promoter methylation has emerged as a promising biomarker for cancers, including lung cancer. Whether exposure to PM is associated with peripheral blood leukocyte (PBL) DNA methylation in tumor suppressor genes has not been evaluated. In 63 male healthy steel workers with well-characterized exposure to metal-rich particles nearby Brescia, Italy, we evaluated whether exposure to PM and metal components was associated with PBL DNA methylation in 4 tumor suppressor genes (i.e., APC, p16, p53 and RASSF1A). Blood samples were obtained on the 1^st (baseline) and 4^th day (post-exposure) of the same work week and DNA methylation was measured using pyrosequencing. A linear mixed model was used to examine the correlations of the exposure with promoter methylation levels. Mean promoter DNA methylation levels of APC or p16 were significantly higher in post-exposure samples compared to that in baseline samples (p-values = 0.005 for APC, and p-value = 0.006 for p16). By contrast, the mean levels of p53 or RASSF1A promoter methylation was decreased in post-exposure samples compared to that in baseline samples (p-value = 0.015 for p53; and p-value < 0.001 for RASSF1A). In post-exposure samples, APC methylation was positively associated with PM₁₀ (β = 0.27, 95% CI: 0.13-0.40), and PM₁ (β = 0.23, 95% CI: 0.09-0.38). In summary, ambient PM exposure was associated with PBL DNA methylation levels of tumor suppressor genes of APC, p16, p53 and RASSF1A, suggesting that such methylation alterations may reflect processes related to PM-induced lung carcinogenesis.     

TP53 Targeted Deep Sequencing of Cell-Free DNA in Esophageal Squamous Cell Carcinoma Using Low-Quality Serum: Concordance with Tumor Mutation

Circulating cell-free DNA (cfDNA) is emerging as a potential tumor biomarker. CfDNA-based biomarkers may be applicable in tumors without an available non-invasive screening method among at-risk populations. Esophageal squamous cell carcinoma (ESCC) and residents of the Asian cancer belt are examples of those malignancies and populations. Previous epidemiological studies using cfDNA have pointed to the need for high volumes of good quality plasma (i.e., >1 mL plasma with 0 or 1 cycles of freeze-thaw) rather than archival serum, which is often the main available source of cfDNA in retrospective studies. Here, we have investigated the concordance of TP53 mutations in tumor tissue and cfDNA extracted from archival serum left-over from 42 cases and 39 matched controls (age, gender, residence) in a high-risk area of Northern Iran (Golestan). Deep sequencing of TP53 coding regions was complemented with a specialized variant caller (Needlestack). Overall, 23% to 31% of mutations were concordantly detected in tumor and serum cfDNA (based on two false discovery rate thresholds). Concordance was positively correlated with high cfDNA concentration, smoking history (p-value = 0.02) and mutations with a high potential of neoantigen formation (OR; 95%CI = 1.9 (1.11–3.29)), suggesting that tumor DNA release in the bloodstream might reflect the effects of immune and inflammatory context on tumor cell turnover. We identified TP53 mutations in five controls, one of whom was subsequently diagnosed with ESCC. Overall, the results showed that cfDNA mutations can be reliably identified by deep sequencing of archival serum, with a rate of success comparable to plasma. Nonetheless, 70% non-identifiable mutations among cancer patients and 12% mutation detection in controls are the main challenges in applying cfDNA to detect tumor-related variants when blindly targeting whole coding regions of the TP53 gene in ESCC.     

Regioselective Reduction of Quinolines Catalyzed by Rhodium and Iridium Complexes with mono-, di-, and tri-dentated Phosphine Ligands

The systems prepared in situ by addition of the corresponding equivalents of the respective phosphine (mono-, di- and tri-dentated), called M₂Cl₂(COE)₄/n phosphine (M = Rh, Ir; and COE = cyclooctene), are efficient and regioselective precatalysts for the hydrogenation of quinoline, isoquinoline, 5,6- and 7,8-benzoquinoline and acridine. The Rh systems were more active than the corresponding Ir ones, being the systems with 1,1,1-tris(diphenylphosphinomethyl)ethane (triphos) more active than those with 1,2-bis(diphenylphosphino)ethane (dppe), except for the case of acridine, where the inversed tendencies were observed (Ir > Rh and dppe > triphos). The systems with triphenylphosphine showed the lowest activities.     

Gene Mutation Analysis in EGFR Wild Type NSCLC Responsive to Erlotinib: Are There Features to Guide Patient Selection?

Tyrosine kinase inhibitors (TKIs) are very efficacious in non-small-cell lung cancer (NSCLC) patients harboring activating Epidermal Growth Factor Receptor (EGFR) mutations. However, about 10% of EGFR wild type (wt) patients respond to TKI, with unknown molecular mechanisms of sensitivity. We considered a case series of 34 EGFR wt NSCLC patients responsive to erlotinib after at least one line of therapy. Responsive patients were matched with an equal number of non-responsive EGFR wt patients. A panel of 26 genes, for a total of 214 somatic mutations, was analyzed by MassARRAY^® System (Sequenom, San Diego, CA, USA). A 15% KRAS mutation was observed in both groups, with a prevalence of G12C in non-responders (80% vs. 40% in responders). NOTCH1, p53 and EGFR-resistance-related mutations were found more frequently in non-responders, whereas EGFR-sensitizing mutations and alterations in genes involved in proliferation pathways were more frequent in responders. In conclusion, our findings indicate that p53, NOTCH1 and exon 20 EGFR mutations seem to be related to TKI resistance. KRAS mutations do not appear to influence the TKI response, although G12C mutation is more frequent in non-responders. Finally, the use of highly sensitive methodologies could lead to the identification of under-represented EGFR mutations potentially associated with TKI sensitivity.