Anthony Mansour

Daucus carota pentane-based fractions arrest the cell cycle and increase apoptosis in MDA-MB-231 breast cancer cells

BackgroundDaucus carota L.ssp.carota (wild carrot), an herb used in folk medicine worldwide, was recently demonstrated to exhibit anticancer activity. In this study we examined the anticancer effect of Daucus carota oil extract (DCOE) fractions on the human breast adenocarcinoma cell lines MDA-MB-231 and MCF-7 and clarified the mechanism of action.Methods and resultsUsing the WST assay, the pentane fraction (F1) and 1:1 pentane:diethyl ether fraction (F2) were shown to possess the highest cytotoxicity against both cell lines. Flow cytometric analysis revealed that both fractions induced the accumulation of cells in the sub-G1 phase, increase in apoptotic cell death and chromatin condensation. The increase in apoptosis in response to treatment was also apparent in the increase in BAX and the decrease in Bcl-2 levels as well as the proteolytic cleavage of both caspase-3 and PARP as revealed by Western blot. Furthermore, treatment of MDA-MB-231 cells with either fraction significantly reduced the level of phosphorylated Erk but did not show any effect on phosphorylated Akt. The combined treatment with a potent PI3K inhibitor (wortmannin) and F1 or F2 fraction had a synergistic inhibitory effect on cell survival which shows that these two drugs work on different pathways.ConclusionsThese results suggest that the pentane-based fractions of DCOE possess potential anti-cancer activity that is mainly mediated through the Erk pathway.

Daucus carota Pentane-Based Fractions Suppress Proliferation and Induce Apoptosis in Human Colon Adenocarcinoma HT-29 Cells by Inhibiting the MAPK and PI3K Pathways

Daucus carota L. ssp. carota (Apiacea, wild carrot, Queen Annes lace) has been used in folk medicine throughout the world and recently was shown to possess anticancer and antioxidant activities. This study aims to determine the anticancer activity of the pentane fraction (F1) and the 1:1 pentane:diethyl ether fraction (F2) of the Daucus Carota oil extract (DCOE) against human colon adenocarcinoma cell lines (HT-29 and Caco-2). Treatment of cells with various concentrations of F1 or F2 fractions produced a dose-dependent inhibition of cell proliferation. Flow cytometric analysis indicated that both fractions induced sub-G1 phase accumulation and increased apoptotic cell death. Western blot revealed the activation of caspase-3, PARP cleavage, and a considerable increase in Bax and p53 levels, and a decrease in Bcl-2 level. Treatment of HT-29 cells with either fraction markedly decreased the levels of both phosphorylated Erk and Akt. Furthermore, the combined treatment of F1 or F2 with wortmannin showed no added inhibition of cell survival suggesting an effect of F1 or F2 through the phosphatidyl inositol 3-kinase (PI3K) pathway. This study proposes that DCOE fractions (F1 and F2) inhibit cell proliferation by inducing cell cycle arrest and apoptosis in HT-29 cells through the suppression of mitogen-activated protein kinase (MAPK)/Erk and PI3K/Akt pathways.

Endocrine and Bone Complications in β-Thalassemia Intermedia: Current Understanding and Treatment

Thalassemia intermedia (TI), also known as nontransfusion dependent thalassemia (NTDT), is a type of thalassemia where affected patients do not require lifelong regular transfusions for survival but may require occasional or even frequent transfusions in certain clinical settings and for defined periods of time. NTDT encompasses three distinct clinical forms: β-thalassemia intermedia (β-TI), Hb E/β-thalassemia, and α-thalassemia intermedia (Hb H disease). Over the past decade, our understanding of the molecular features, pathophysiology, and complications of NTDT particularly β-TI has increased tremendously but data on optimal treatment of disease and its various complications are still lacking. In this paper, we shall review a group of commonly encountered complications in β-TI, mainly endocrine and bone complications.

Change in Pulse Wave Velocity and Short-Term Development of Cardiovascular Events in the Hemodialysis Population

The association between single measurements of carotid‐femoral pulse wave velocity (cfPWV) and cardiovascular (CV) events is driven by late events beyond 12 months of follow‐up. This prospective study compares single measurements of cfPWV vs the 2‐year delta cfPWV and the association with short‐term development of CV events in hemodialysis patients. cfPWV was performed at t=0 and t=1 two years later, and patients were followed‐up for development of CV events through 12 months (n=66). In Cox regression models adjusted for CV risk factors, history of CV events and delta cfPWV remained associated with the development of CV events (hazard ratio for prior CV events=8.9, P=.03; hazard ratio for delta cfPWV=1.14; P=.002). When delta cfPWV was substituted for single cfPWV measurement, none of the single measures were associated with new CV events. The change in cfPWV, but not single measurements of cfPWV, was associated with the development of CV events through 12 months.

Antioxidant and hepatoprotective activities of the oil fractions from wild carrot (Daucus carota ssp. carota)

Abstract Context: Wild carrot, Daucus carota L. ssp. carota (Apiacae), is widely distributed throughout the world and has various uses in traditional medicine in Lebanon. Objective: The present study aimed to fractionate and analyze the chemical composition of the Daucus carota oil extract (DCOE) fractions and to evaluate their antioxidant and hepatoprotective properties in vitro and in vivo. Materials and methods: DCOE was chromatographed on silica gel column to produce four fractions: pentane (F1), 50:50 pentane:diethyl ether (F2), diethyl ether (F3), and 93:7 chloroform: methanol (F4). Qualitative and quantitative analyses of oil fractions were performed by GC-MS and HPLC techniques. The in vitro antioxidant properties were assessed using DPPH, FIC, and ferric-reducing antioxidant power (FRAP) assays. The hepatoprotective property was determined by examining the levels of serum markers (alanine transaminase (ALT) and aspartate transaminase (AST)) and hepatic antioxidant (superoxide dismutase (SOD), catalase (CAT), and glutathione-S-transferase (GST)) enzymes in CCl4-intoxicated mice pretreated with intraperitoenal 50, 100, or 200 mg/kg b.w. of the oil fractions for 5 d. Results: GCMS analysis of F2 revealed the presence of 2-himachalen-6-ol (61.4%) which is reported for the first time in Daucus carota species. F3 and F4 were rich in phenolics and flavonoids and demonstrated significant DPPH activity (IC50 = 0.29 and 0.38 mg/ml, respectively) and high FRAP values (225.11 and 437.59 µmol FeSO4/g, respectively). The sesquiterpene-rich fraction F1 had the highest FIC ability (IC50 = 0.28 mg/ml). Pretreatment with F1 and F4 reversed the CCl4-induced decrease in SOD, CAT, and GST levels and reduced significantly hepatic damage. Discussion and conclusion: The current results suggested that wild carrot oil fractions exhibited a unique chemical composition and possessed significant antioxidant activities as well as hepatoprotective effects against CCl4-induced hepatotoxicity.

A novel xylene-free deparaffinization method for the extraction of proteins from human derived formalin-fixed paraffin embedded (FFPE) archival tissue blocks.

Graphical abstract

Efficient and Cost-Effective Alternative Treatment for Recurrent Urinary Tract Infections and Interstitial Cystitis in Women: A Two-Case Report

Urinary tract infections (UTIs) are among the most common bacterial infections affecting women. UTIs are primarily caused by Escherichia coli, which increases the likelihood of a recurrent infection. We encountered two cases of recurrent UTIs (rUTIs) with a positive E. coli culture, not improving with antibiotics due to the development of antibiotic resistance. An alternative therapeutic regimen based on parsley and garlic, L-arginine, probiotics, and cranberry tablets has been given. This regimen showed a significant health improvement and symptoms relief without recurrence for more than 12 months. In conclusion, the case supports the concept of using alternative medicine in treating rUTI and as a prophylaxis or in patients who had developed antibiotic resistance.

Sickle cell nephropathy: an update on pathophysiology, diagnosis, and treatment

Sickle cell nephropathy is a major complication of sickle cell disease. It manifests in different forms, including glomerulopathy, proteinuria, hematuria, and tubular defects, and frequently results in end-stage renal disease (ESRD). Different pathophysiologic mechanisms have been proposed to explain the development of nephropathy in SCD, where hemolysis and vascular occlusion are the main contributors in the manifestations of this disease. Markers of renal injury, such as proteinuria and tubular dysfunction, have been associated with outcomes among patients with sickle cell nephropathy and provide means for early detection of nephropathy and screening prior to progression to renal failure. In small-sized clinical trials, hydroxyurea has demonstrated to be effective in slowing the progression to ESRD. Dialysis and renal transplantation represent the last resort for patients with sickle cell nephropathy. Nevertheless, despite the availability of diagnostic and therapeutic strategies, sickle cell nephropathy remains a challenging and under-recognized complication for patients with sickle cell disease.

Transfusion Therapy in Children With Sickle Cell Disease.

Hydroxyurea, blood transfusions, and hematopoietic stem cell transplantation represent the 3 disease-modifying therapies in children with sickle cell disease (SCD). Blood transfusions play an increasingly important role in both prevention and management of SCD complications in this age group. This review will focus on the indications of blood transfusion in children with SCD and modalities of its administration. It will also highlight the complications of this life-saving therapy and ways of optimizing transfusion to minimize its associated risks.

Abstract LB-288: Optimized xylene-free protein extraction method from formalin-fixed paraffin-embedded tissue sections for western blot analysis

Background: Formalin fixed paraffin embedded (FFPE) tissues remains the standard method for fixation and storage of tissues for clinical pathology use. Protein extraction from these tissues remains challenging due to the reduced quality and amount of extracted proteins. Despite multiple successful attempts, isolation of proteins from FFPE tissue sections necessitates routine use of xylene, a highly toxic organic solvent. We previously showed that proteins can be efficiently extracted with a novel technique that utilizes hot distilled water as a substitute for xylene with a quality adequate for western blot analysis. However, its major drawback is the need to use an entire or major part of the paraffin-embedded tissue block. To address these issues we developed a new xylene-free method for protein extraction from FFPE tissue sections of around 8 μm thickness. Methods: A total of 44 different types of FFPE tissues sections of 8 μm thickness were obtained from various archived FFPE specimens which include: 17 colorectal cancer (5-6 years), 7 breast cancer (3 years), 3 thyroid cancer (2 years), 4 ovarian cancer (2 years), 8 uterine cancer (1 year), and 5 prostate cancer (1 year). Deparaffinization was conducted by gentle treatment of each section with hot distilled water (≈90°C) for less than 10 seconds. Deparaffinized tissues were then placed in a cell lysis buffer and Laemli buffer and incubated at 100°C for 5-10 minutes. The extracted proteins were quantified using NanoDrop Spectrophotometer and evaluated using western blot analysis for the presence of AKT and beta-actin. Results: Using this method, a significant amount of proteins was successfully isolated with an average amount of 2.670 μg/μl ± 0.623 or 1068 μg/8 μm tissue section. Compared to the standard protein extraction method using xylene the amount of proteins extracted in this experiment is at least four times greater. Protein extracts were of good quality and efficiently analyzed by western blot experiment. Moreover, the isolated proteins showed a similar migration pattern compared to the positive control protein on SDS-PAGE but with better bands’ intensity and clarity. Protein kinase B (PKB/AKT) was successfully identified in all specimens, and beta-actin protein was resolved with an efficiency higher than 80%. Hence, this technique has enabled us to efficiently extract and detect selected proteins from archived samples for up to 6 years. Conclusion: We developed an efficient, safe, cost-effective, and rapid method to isolate proteins from FFPE tissues sections and adequate for western blot analysis. Hence, utilizing this technique can further aid in the identification of tissue protein biomarkers of various diseases, monitor cancer progression, and improve the diagnosis. It remains to be determined if this method of protein extraction form FFPE is adequate for use in proteomic analyses. Citation Format: Anthony Mansour, Noha Bejjani, Carole Dagher, Rajaa Chatila, Wissam H. Faour. Optimized xylene-free protein extraction method from formalin-fixed paraffin-embedded tissue sections for western blot analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-288. doi:10.1158/1538-7445.AM2015-LB-288