Relationship between cytotoxicity,drug accumulation,DNA damage and repair of human ovarian cancer cells treated with doxorubicin: modulation by the tiapamil analog RO11-2933

Summary The effect ofN-(3,4-dimethoxyphenyl)N-methyl-2-(naphthyl)-m-dithiane-2-propylamine hydrochloride (RO11-2933), an analog of the calcium channel blocker tiapamil, on doxorubicin (DOX)-induced cytotoxicity and DNA damage in human ovarian cancer cells sensitive and resistant to DOX was investigated. A2780-DX2, A2780-DX3, and A2780-DX6 cell sublines were characterized by 7-, 26-, and 48-fold resistance after 2 h DOX exposure and 30-, 50-, and 500-fold resistance after 72 h DOX exposure, respectively. Increased drug efflux resulting in a lower intracellular drug accumulation, decreased DOX-induced DNA single-strand breaks (DNA SSBs), and rapid DNA repair correlated with the degree of resistance. In addition, DNA SSBs were rapidly repaired within 8 h in A2780-DX3 cells, whereas no significant repair of DNA SSBs was observed in sensitive cells. In comparison with verapamil, RO11-2933 was found to reverse DOX resistance at lower and nontoxic concentrations (2 M as compared with 10 M verapamil). This reversion was complete in cells with a low degree of resistance (A2780-DX1 and A2780-DX2) but partial in highly resistant cells (A2780-DX3 and A2780-DX6), and continuous exposure to RO11-2933 was essential for optimal reversal of drug resistance. Interestingly, RO11-2933 was found to inhibit the repair of DNA SSBs induced by DOX but not those induced by X-ray. These results suggest that the potentiation of DNA SSBs and the specific inhibition of DNA repair by RO11-2933 in multidrug-resistant cells could be of particular value in overcoming MDR in the clinic.Abbreviations RO11-2933 N-(3,4-dimethoxyphenethyl)-N-methyl-2-(2-naphthyl)-m-dithiane-2-propylamine hydrochloride - DOX doxorubicin-HCl - SSBs single-strand breaks - MDR multidrug resistance This work was supported in part by CA 18420 and CA 21071     

Lack of microvessels in well-differentiated regions of human head and neck squamous cell carcinoma A253 associated with functional magnetic resonance imaging detectable hypoxia, limited drug delivery, and resistance to irinotecan therapy.

PURPOSE: Combination chemotherapy with irinotecan (CPT-11; 50 mg/kg/week x 4 intravenously), followed 24 hour later by 5-fluorouracil (50 mg/kg/week x 4 intravenously), results in 10 and 100% cure rates of animals bearing human head and neck squamous cell carcinoma xenografts A253 and FaDu, respectively. A253 consists of 30% well-differentiated and avascular and 70% poorly differentiated regions with low microvessel density (10/x400), whereas FaDu is uniformly poorly differentiated with higher microvessel density (19/x400). Studies were carried out for determining the role of well-differentiated and avascular regions in drug resistance in A253 and detection of such regions with noninvasive functional magnetic resonance (fMR) imaging. EXPERIMENTAL DESIGN: Tumors were harvested for histopathologic evaluation and immunohistochemistry (CD31, CD34; differentiation marker: involucrin; hypoxia markers: carbonic anhydrase IX, pimonidazole; vascular endothelial factor (VEGF) and Ki67) immediately after fMR imaging following the 3rd dose of chemotherapy. High-performance liquid chromatography determination of intratumoral drug concentration of 7-ethyl-10-hydroxyl-camptothecin and autoradiography with (14)C-labeled CPT-11 was done 2 hours after CPT-11 administration. RESULTS: Although A253 xenografts showed three times higher concentration of 7-ethyl-10-hydroxyl-camptothecin, FaDu was more responsive to therapy. After therapy, A253 tumor consisted mostly (approximately 80%) of well-differentiated regions (positive for involucrin) lacking microvessels with a hypoxic rim (positive for carbonic anhydrase IX and pimonidazole) containing few proliferating (Ki67 positive) poorly differentiated cells. Autoradiography revealed that well-differentiated A253 tumor regions showed 5-fold lower (14)C-labeled CPT-11 concentrations compared with poorly differentiated areas (P < 0.001). Blood oxygen level dependant fMR imaging was able to noninvasively distinguish the hypoxic and well-vascularized regions within the tumors. CONCLUSION: Avascular-differentiated regions in squamous cell carcinoma offer sanctuary to some hypoxic but viable tumor cells (carbonic anhydrase IX and Ki67 positive) that escape therapy because of limited drug delivery. This study provides direct evidence that because of a specific histologic structure, avascular, well-differentiated hypoxic regions in tumors exhibit low drug uptake and represent a unique form of drug resistance.     

Irritantcy Potential and Sub Acute Dermal Toxicity Study of Pistacia Lentiscus Fatty Oil as a Topical Traditional Remedy

The current study was undertaken to assess safety of Pistacia lentiscus fruits fatty oil (PLFO) as a topical traditional remedy. A primary skin and eye irritation tests were conducted with New Zealand white rabbits to determine the potential for PLFO to produce irritation from a single application. In addition, a sub acute dermal toxicity study was performed on 18 NZW rabbits to evaluate possible adverse effect following application of PLFO for 28 days. Based on the results of the current study, PLFO is classified as slightly irritating to the skin and the eye of rabbits (Primary Irritation Index (P.I.I.) = 1.037; Ocular Irritation Index (O.I.I.) = 5.33 at 1 h). In the sub-acute toxicity test, PLFO produced neither mortality nor significant differences in the body and organ weights between control group and treated rabbits. However, a reversible irritant contact dermatitis was observed in the treated areas from the end of the second week of application until the end of experiment. This local phenomenon was accompanied by a significant skin thickening (P≤0.01) since the 12^th day (ANOVA, F = 11, 07143, P = 0, 00765) which is confirmed with an inflammatory granuloma in histological study. Haematological analysis and blood chemistry values of the 2 groups showed no significant differences in any of the parameters examined. In summary, PLFO is minimally irritating to the eye and skin after a single exposure, but it may cause irritant contact dermatitis and a reversible thickening of skin after prolonged use.     

MR imaging features of focal liver lesions in Wilson disease

Hepatic involvement in Wilson disease (WD) manifests as a diffuse chronic disease in the majority of patients. However, in a subset of patients focal liver lesions may develop, presenting with a wide range of imaging features. The majority of focal liver lesions in patients with WD are benign nodules, but there are reports that have described malignant liver tumors or dysplastic nodules in these patients. Because of the possibility of malignant transformation of liver nodules, major concerns have been raised with respect to the management and follow-up of patients with WD in whom focal liver lesions have been identified. The assessment of liver involvement in patients with WD is generally performed with ultrasonography. However, ultrasonography conveys limited specificity so that magnetic resonance (MR) imaging is often performed to improve lesion characterization. This review was performed to illustrate the spectrum of MR imaging features of focal liver lesions that develop in patients with WD. It is assumed that familiarity with the MR imaging presentation of focal liver lesions in WD may help clarify the actual nature of hepatic nodules in patients with this condition.     

Intensive chemotherapy with mitoxantrone administered as a single injection in patients with high-risk acute myeloid leukemia: results of the EMA 2000 trial

Following a dose-escalation study performed to assess the maximally tolerated dose of high-dose mitoxantrone in a single injection combined with chemotherapy, a phase II trial (EMA 2000 regimen) was performed in patients with refractory or relapsed acute myelogenous leukemia (AML) between October 2000 and December 2003. Sixty-two patients entered the study and received mitoxantrone 45 mg/m² on day 1 in combination with cytarabine and etoposide. Overall, 39 patients (63%) achieved complete remission (CR). Four patients died during remission induction, and 19 patients had resistant disease. Median time to granulocyte and platelet recovery was 34 and 39 days, respectively. The predominant non-hematologic toxicity was infection, with 53% severe infections. Thirty-three of the 39 remitters received subsequent treatment consisting of maintenance chemotherapy courses in 17 patients, allogeneic stem cell transplantation (SCT) in 7 patients, and autologous SCT in 9 patients. The median overall survival of the entire cohort was 8.1 months, with 18% at 2.5 years. EMA chemotherapy using a single injection of mitoxantrone is effective in the treatment of high-risk AML. CR proportion was significantly higher in patients with a first CR duration 6 months when compared with those from a control trial using standard-dose mitoxantrone (90 vs 70%, p=0.03).     

Slit design for efficient and accurate MTF measurement at megavoltage x-ray energies

Empirical determination of the modulation transfer function (MTF) for analog and digital mega-voltage x-ray imagers is a challenging task. The most common method used to determine MTF at megavoltage x-ray energies employs a long, narrow slit formed by two parallel, metal blocks in order to form a "slit beam." In this work, a detailed overview of some of the important considerations of slit design is presented. Based on these considerations, a novel, compact slit, using 19 cm thick tungsten blocks, was designed. The prototype slit was configured to attach to the accessory slot of the gantry of a linear accelerator, which greatly simplified the measurement process. Measurements were performed to determine the presampling MTF at 6 MV for an indirect detection active matrix flat panel imager prototype previously developed for megavoltage imaging applications. In addition, the effects of two important slit design parameters, material type and thickness, on the accuracy of MTF determination were investigated via a Monte Carlo-based theoretical study. Empirically determined MTFs obtained from the prototype slit closely match those from an earlier, less compact slit design based on 40 cm thick steel blocks. The results of the Monte Carlo-based theoretical studies indicate that the prototype slit achieves close-to-ideal performance in terms of accurately determining the MTF by virtue of practically 100% beam attenuation in regions other than the slit gap. Furthermore, the theoretical results suggest that it may be possible to achieve even further reductions in slit thickness without compromising measurement accuracy.     

Atresia of the common pulmonary vein

A newborn girl with atresia of the common pulmonary vein, presented immediately after birth with severe cyanosis and acidosis. The diagnosis of totally obstructed total pulmonary venous return was made by cross-sectional echocardiography. Subsequent cardiac catheterization failed to demonstrate the site of pulmonary venous return. Necropsy showed the pulmonary veins to be connected bilaterally to an atretic common pulmonary vein. There was no obvious alternative pathway for pulmonary venous return.     

UHRF1 regulation of the Keap1–Nrf2 pathway in pancreatic cancer contributes to oncogenesis

The cellular defence protein Nrf2 is a mediator of oncogenesis in pancreatic ductal adenocarcinoma (PDAC) and other cancers. However, the control of Nrf2 expression and activity in cancer is not fully understood. We previously reported the absence of Keap1, a pivotal regulator of Nrf2, in ～70% of PDAC cases. Here we describe a novel mechanism whereby the epigenetic regulator UHRF1 suppresses Keap1 protein levels. UHRF1 expression was observed in 20% (5 of 25) of benign pancreatic ducts compared to 86% (114 of 132) of pancreatic tumours, and an inverse relationship between UHRF1 and Keap1 levels in PDAC tumours (n = 124) was apparent (p = 0.002). We also provide evidence that UHRF1‐mediated regulation of the Nrf2 pathway contributes to the aggressive behaviour of PDAC. Depletion of UHRF1 from PDAC cells decreased growth and enhanced apoptosis and cell cycle arrest. UHRF1 depletion also led to reduced levels of Nrf2‐regulated downstream proteins and was accompanied by heightened oxidative stress, in the form of lower glutathione levels and increased reactive oxygen species. Concomitant depletion of Keap1 and UHRF1 restored Nrf2 levels and reversed cell cycle arrest and the increase in reactive oxygen species. Mechanistically, depletion of UHRF1 reduced global and tumour suppressor promoter methylation in pancreatic cancer cell lines, and KEAP1 gene promoter methylation was reduced in one of three cell lines examined. Thus, methylation of the KEAP1 gene promoter may contribute to the suppression of Keap1 protein levels by UHRF1, although our data suggest that additional mechanisms need to be explored. Finally, we demonstrate that K‐Ras drives UHRF1 expression, establishing a novel link between this oncogene and Nrf2‐mediated cellular protection. Since UHRF1 over‐expression occurs in other cancers, its ability to regulate the Keap1–Nrf2 pathway may be critically important to the malignant behaviour of these cancers. © 2015 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Induction of biphasic DNA double strand breaks and activation of multiple repair protein complexes by DNA topoisomerase I drug 7-ethyl-10-hydroxy-camptothecin

Camptothecins demonstrate a broad spectrum of antitumor activity. Although they are known to trap DNA topoisomerase I on DNA, form cleavable complexes, and generate DNA breaks upon collision with DNA or RNA polymerases, the precise mechanisms predictive for antitumor activity remain to be identified. Recent studies using panels of colorectal and breast cancer cell lines indicate that events downstream of cleavable complexes are more relevant. In this study, we chose SN-38, an active metabolite of irinotecan, to characterize DNA double strand breaks and repair mechanisms induced by this type of drugs using a human head and neck squamous cell carcinoma cell line A253. The results showed that 2-h exposure of cells to an IC(50) concentration of SN-38 induces biphasic DNA double-strand break (DSBs): an immediate phase, which was greatly reduced within 8 h, and a lagging phase, culminating 24 h after drug removal. Three DNA double-strand break repair protein complexes were activated: DNA-dependent protein kinase (DNA-PK), NBS1-MRE11-RAD50, and BRCA1. Aphidicolin, a DNA polymerase inhibitor, abolished both phase I DSBs and the activation of repair protein complexes, suggesting that they resulted from the collision between the cleavable complex and DNA polymerase of S-phase cells. This is in contrast to ionizing radiation-induced activation of DNA-PK and NBS1-MRE11-RAD50 complexes that occur predominantly among non-S-phase cells. The trigger for phase II DSBs cannot be abolished by aphidicolin. The data also indicate that DNA fragments in the size of 50 to 200 kilobases were detected in the lagging phase. This suggests that the late DNA DSBs were associated with apoptotic cell death.