Genotoxic activity of 1-chloromethylpyrene in stomach epithelium in vivo: insensitivity of the stomach scintillation UDS assay

An acknowledged weakness of current testing programmes for genotoxichazard has been the potential insensitivity of the establishedmouse bone marrow micronucleus test and rat liver unscheduledDNA synthesis (UDS) assays to direct-acting or short-lived mutagens,which may be consumed at the site of initial contact. In suchcases, in vivo test systems sampling tissues such as the skinor the stomach would provide valuable data. To test these principlesa stomach UDS assay was evaluated using the potent locally activemutagen 1-chloromethylpyrene (1-CMP). Contrary to expectations,no UDS response was observed 16 h following 1-CMP dosage byoral gavage. To confirm the integrity of the 1-CMP used forthe stomach UDS assay, a sample of the stored chemical was re-evaluatedin vitro and shown to be still strongly positive in the Amesassay and to have alkylating activity at least 15 min afterincubation at stomach acid pH. No UDS response was observedwhen test dose levels were reduced or when earlier samplingtimes were used. Other genotoxic endpoints were examined instomach. ³²P-Postlabelling analysis revealed high levels ofadduct formation in gastric DNA. An assay utilizing electrophoresisof DNA (the comet assay) showed the occurrence of DNA damagefollowing dosing with 1-CMP in vivo. These positive resultsconfirmed that 1-CMP should be regarded as a potential in vivogenotoxin. The failure to detect a UDS response to 1-CMP instomach was investigated; a strong UDS response was observedin an in vitro hepatocyte UDS assay of 1-CMP indicating thatthe rat was capable of repairing 1-CMP-derived DNA adducts.Pretreatment of rats with hydroxyurea depressed the level ofincorporation of thymidine into DNA both in negative and positive[methyl-N-nitrosoguanidine (MNNG)] controls. The results ofthese studies indicated that the protease digestion method employeddid not selectively or efficiently sample those cells with anyUDS response to 1-CMP or MNNG, and the activity seen for thelatter was most likely due to the presence of S phase cellswithin the digests. As a result of the finding that UDS responseswere not demonstrated for the potent direct-acting mutagens1-CMP and MNNG, the protease digestion/scintillation methodfor stomach UDS does not appear to have general value in a screeningprogramme for locally active genotoxic agents.     

Co-targeting bromodomain and extra-terminal proteins and MCL1 induces synergistic cell death in melanoma

The treatment of melanoma has been markedly improved by the introduction of targeted therapies and checkpoint blockade immunotherapy. Unfortunately, resistance to these therapies remains a limitation. Novel anticancer therapeutics targeting the MCL1 anti-apoptotic protein have shown impressive responses in haematological cancers but are yet to be evaluated in melanoma. To assess the sensitivity of melanoma to new MCL1 inhibitors, we measured the response of 51 melanoma cell lines to the novel MCL1 inhibitor, S63845. Additionally, we assessed combination of this drug with inhibitors of the bromodomain and extra-terminal (BET) protein family of epigenetic readers, which we postulated would assist MCL1 inhibition by downregulating anti-apoptotic targets regulated by NF-kB such as BCLXL, BCL2A1 and XIAP, and by upregulating pro-apoptotic proteins including BIM and NOXA. Only 14% of melanoma cell lines showed sensitivity to S63845, however, combination of S63845 and I-BET151 induced highly synergistic apoptotic cell death in all melanoma lines tested and in an in vivo xenograft model. Cell death was dependent on caspases and BAX/BAK. Although the combination of drugs increased the BH3-only protein, BIM, and downregulated anti-apoptotic proteins such as BCL2A1, the importance of these proteins in inducing cell death varied between cell lines. ABT-199 or ABT-263 inhibitors against BCL2 or BCL2 and BCLXL, respectively, induced further cell death when combined with S63845 and I-BET151. The combination of MCL1 and BET inhibition appears to be a promising therapeutic approach for metastatic melanoma, and presents opportunities to add further BCL2 family inhibitors to overcome treatment resistance.     

4-烷硫基-4-脱氧-4′-去甲表鬼臼毒素的合成和抗肿瘤活性

对4’-去甲表鬼臼毒素的C₄位进行化学修饰,合成和筛选了10个4-烷硫基-4-脱氧-4’-去甲表鬼臼毒素衍生物以进一步研究C₄位不同的原子和取代基与活性之间的关系及寻找结构简单、活性更强的抗肿瘤新药。4’-去甲表鬼臼毒素与硫醇在三氟化硼·乙醚或三氟乙酸存在下生成相应的硫醚,也可用硫醇与4β-溴-4-脱氧-4’-去甲表鬼臼毒素反应生成相应的硫醚。在体外筛选中,化合物10和12抑制L1210白血病细胞的活性与依托泊甙相当或更强,化合物9,10,12和15抑制KB细胞的活性与依托泊甙相当或更强。     

Combining BET and HDAC inhibitors synergistically induces apoptosis of melanoma and suppresses AKT and YAP signaling

Histone acetylation marks have an important role in controlling gene expression and are removed by histone deacetylases (HDACs). These marks are read by bromodomain and extra-terminal (BET) proteins and novel inhibitiors of these proteins are currently in clinical development. Inhibitors of HDAC and BET proteins have individually been shown to cause apoptosis and reduce growth of melanoma cells. Here we show that combining the HDAC inhibitor LBH589 and BET inhibitor I-BET151 synergistically induce apoptosis of melanoma cells but not of melanocytes. Induction of apoptosis proceeded through the mitochondrial pathway, was caspase dependent and involved upregulation of the BH3 pro-apoptotic protein BIM. Analysis of signal pathways in melanoma cell lines resistant to BRAF inhibitors revealed that treatment with the combination strongly downregulated anti-apoptotic proteins and proteins in the AKT and Hippo/YAP signaling pathways. Xenograft studies showed that the combination of inhibitors was more effective than single drug treatment and confirmed upregulation of BIM and downregulation of XIAP as seen in vitro. These results support the combination of these two classes of epigenetic regulators in treatment of melanoma including those resistant to BRAF inhibitors.     

Antitumour prodrug development using cytochrome P450 (CYP) mediated activation

An ideal cancer chemotherapeutic prodrug is completely inactive until metabolized by a tumour-specific enzyme, or by an enzyme that is only metabolically competent towards the prodrug under physiological conditions unique to the tumour. Human cancers, including colon, breast, lung, liver, kidney and prostate, are known to express cytochrome P450 (CYP) isoforms including 3A and 1A subfamily members. This raises the possibility that tumour CYP isoforms could be a focus for tumour-specific prodrug activation. Several approaches are reviewed, including identification of prodrugs activated by tumour-specific polymorphic CYPs, use of CYP-gene directed enzyme prodrug therapy and CYPs acting as reductases in hypoxic tumour regions. The last approach is best exemplified by AQ4N, a chemotherapeutic prodrug that is bioreductively activated by CYP3A. This study shows that freshly isolated murine T50/80 mammary carcinoma and RIF-1 fibrosarcoma 4-electron reduces AQ4N to its cytotoxic metabolite, AQ4 (T50/80 Km = 26.7 microM, Vmax = 0.43 microM/mg protein/min; RIF-1 Km = 33.5 microM, Vmax = 0.42 microM/mg protein/min) via AQM, a mono-N-oxide intermediate (T50/80 Km = 37.5 microM; Vmax = 1.4 microM/mg protein/min; RIF-1 Km = 37.5 microM; Vmax = 1.2 microM/mg protein/ min). The prodrug conversion was dependent on NADPH and inhibited by air or carbon monoxide. Cyp3A mRNA and protein were both present in T50/80 carcinoma grown in vivo (RIF-1 not measured). Exposure of isolated tumour cells to anoxia (2 h) immediately after tumour excision increased cyp3A protein 2-3-fold over a 12 h period, after which time the cyp protein levels returned to the level found under aerobic conditions. Conversely, cyp3A mRNA expression showed an initial 3-fold decrease under both oxic and anoxic conditions; this returned to near basal levels after 8-24 h. These results suggest that cyp3A protein is stabilized in the absence of air, despite a decrease in cyp3A mRNA. Such a 'stabilization factor' may decrease cyp3A protein turnover without affecting the translation efficiency of cyp3A mRNA. Confirmation of the CYP activation of AQ4N bioreduction was shown with human lymphoblastoid cell microsomes transfected with CYP3A4, but not those transfected with CYP2B6 or cytochrome P450 reductase. AQ4N is also reduced to AQ4 in NADPH-fortified human renal cell carcinoma (Km = 4 microM, Vmax = 3.5 pmol/mg protein/min) and normal kidney (Km = 4 microM, Vmax = 4.0 pmol/mg protein/min), both previously shown to express CYP3A. Germane to the clinical potential of AQ4N is that although both normal and tumour cells are capable of reducing AQ4N to its cytotoxic species, the process requires low oxygen conditions. Hence, AQ4N metabolism should be restricted to hypoxic tumour cells. The isoform selectivity of AQ4N reduction, in addition to its air sensitivity, indicates that AQ4N haem coordination and subsequent oxygen atom transfer from the active-site-bound AQ4N is the likely mechanism of N-oxide reduction. The apparent increase in CYP3A expression under hypoxia makes this a particularly interesting application of CYPs for tumour-specific prodrug activation.     

Variable regulation of sensitivity to retinoic acid-induced differentiation in wild-type and retinoic acid-resistant HL-60 cells

The initial cell association and metabolic conversion of retinoic acid (RA) by HL-60 cells in serum-free, transferrin/insulin-supplemented, RPMI 1640 medium was greater than or equal to 10-fold greater than in RPMI 1640 medium containing 10% fetal bovine serum (FBS). This was paralleled under the serum-free conditions by 10-fold greater sensitivity to RA-induced differentiation, which was partially reversed by the addition of purified bovine serum albumin to the same concentration present in 10% FBS. In serum-free HL-1 medium, HL-60 cell sensitivity to RA-induced differentiation was approximately 250-fold less than in serum-free RPMI 1640 medium but, in this comparison, there was little difference in RA cell association or metabolism. A greater than 200-fold RA-resistant HL-60 subline had RA cell-association and metabolism rates similar to those of wild-type cells under all culture conditions. No significant qualitative differences in the high performance liquid chromatography elution patterns of polar metabolites were observed under any circumstances. These results indicate that inherent cellular properties, not associated with gross differences in RA uptake or metabolism, primarily determined the relative sensitivity or insensitivity of HL-60 cells to RA-induced differentiation but that RA responsiveness was markedly regulated by extracellular factors, one of which, serum albumin, appeared to act by decreasing the initial cell association and metabolism of RA, whereas other, as yet unidentified exogenous factors, may have acted independently of these functions.     

Acadesine (AICA-riboside) improves postischemic cardiac recovery.

To test if acadesine (5-aminoimidazole-4-carboxamide riboside), a purine precursor, has cardioprotective effects, 16 dogs were placed on total cardiopulmonary bypass and subjected to global myocardial ischemia. Hemodynamic recovery was compared between a control (n = 8) group receiving standard cardioplegia and an acadesine (n = 8) group pretreated with intravenous acadesine (2.5 mg.kg-1.min-1 for 5 minutes, then 0.5 mg.kg-1.min-1) before ischemia, during ischemia, and until 10 minutes after removal of the aortic cross-clamp. Additionally, in the acadesine group the cardioplegia also contained 20 mumol/L acadesine. While the dogs were on cardiopulmonary bypass, global warm myocardial ischemia was induced by aortic cross-clamping for 5 minutes under normothermic conditions to simulate an angioplasty accident. Five minutes after aortic cross-clamping, hypothermic cardioplegia (30 mL/kg) was administered. The left anterior descending coronary artery was occluded before the first infusion of cardioplegia to simulate poor cardioplegia delivery that can occur during an emergency coronary artery bypass procedure after an angioplasty accident. The left anterior descending artery occlusion was released, and additional cardioplegia (15 mL/kg) infusions were made every 30 minutes thereafter during 120 minutes of cardioplegic ischemia. Thirty minutes after reperfusion, all animals in both groups were weaned from bypass and recovery data were obtained to compare with baseline preischemic values. There were no significant differences in heart rate, left atrial pressure, or systemic vascular resistance between groups after weaning from bypass. Peak developed pressure recovered to 79% +/- 19% (mean +/- standard deviation) of baseline in the acadesine group compared with 56% +/- 22% in the control group (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Spinal fusion for back pain: a clinical and radiological review.

Eighty-one patients who had spinal fusions performed for back pain over a 7-year period were reviewed; 74% were satisfied with the outcome of their surgery, mainly because of the degree of pain relief obtained. Based on lateral radiographs of the fusion area in flexion and extension, there was a 34% pseudarthrosis rate in first-time fusions. However, there was no clear relationship between the integrity of fusion and clinical success, indicating that many factors other than bony fusion influence the eventual outcome of the operation.     

A dose escalation study of carboplatin and ifosfamide in advanced ovarian cancer

Summary A dose escalation study of carboplatin (CBDCA) and ifosfamide was carried out in 35 patients with advanced ovarian carcinoma to determine the toxicity and therapeutic effect of this combination. In all, 13 patients had recurrent ovarian carcinoma, 11 had abdominal carcinomatosis of probable ovarian origin and 9 had newly diagnosed stage III/IV ovarian cancer. Myelosuppression was the major dose-limiting toxicity. At a dose of 400 mg/m² CBDCA plus 5,000 mg/m² ifosfamide, 61% of courses were associated with grade 3 leukopenia and 27%, with grade 3 thrombocytopenia. The lowest leukocyte and platelet counts occurred at a median of 14 days after treatment and cytopenia persisted for a median of 8 days. Myelotoxicity was cumulative with successive courses at this dose level, whereas at a dose of 400 mg/m² CBDCA plus 4,000 mg/m² ifosfamide it was possible to deliver the planned six courses of treatment. No other untoward toxicities were observed. A clinical response was achieved in 16/33 patients (49%), with 10 complete remissions (CRs), of which 3 were pathologically confirmed at laparotomy. No significant dose-response relationship was demonstrated in this heterogeneous group of patients. The predicted median duration of response is 12 months. CBDCA plus ifosfamide is an active combination therapy for ovarian cancer that merits further comparison with CBDCA alone. The recommended doses for six courses are 400 mg/m² CBDCA plus 4,000 mg/m² ifosfamide.