Hydroxyurea (HU) induces fetal hemoglobin (Hb F) production in patients with sickle cell anemia. The therapeutic dosage of HU used for Hb F induction often elicits myelosuppression, which becomes its major associated complication. We examined the effect of HU on hemoglobin modulation and the role of radical scavengers on these induced changes. In vitro exposure of human blood to various concentrations of HU at predetermined time intervals induced a progressive dose‐dependent oxidation (MetHb formation) of both adult (Hb AA) and sickle (Hb SS) hemoglobins. The oxidative effect of HU on Hb SS was 3 times greater than its effect on Hb AA. Similar but less profound changes were observed in H2O2‐treated samples. Hb F was, however, observed to be relatively resistant to HU‐induced oxidative damage. A substantial protective effect of Hb by α‐tocopherol, ascorbic acid, and D‐mannitol was observed during pretreatment of Hb AA and Hb SS blood samples. Analyses of the hemoglobins and their globin chain components by high‐performance liquid chromatography revealed a considerable protective effect by these free radical scavengers. These results indicate that the HU‐induced damage of hemoglobin and their component globin chains can be reduced by radical scavengers. J. Clin. Lab. Anal. 15:1–7, 2001. © 2001 Wiley‐Liss, Inc.

No abstract

Emerging studies have identified the overexpression of Arginase-1 (ARG-1) in a variety of human malignancies, including cancer of the colon, leading to the promotion of tumor cell growth and invasion. Inactivation of the p53 tumor suppressor protein is known to involve somatic mutation or binding of viral oncoproteins to the p53 protein. However, several types of malignant and premalignant tissues harbor a genetically wild-type, but transcriptionally inactive, form of p53, often localized in the cytoplasm. The transactivation of the inactive p53 gene is thought to lead to its tumor suppressor function. The present report showed that the treatment of cultured human colon cancer cell lines (HT-29 and HCT-116) with 10 or 20 µM chloroquine (CQ), a known anti-rheumatoid/antimalarial drug, inhibited arginase activity with a concomitant nuclear accumulation of the p53 protein. By using specific antibodies it was demonstrated that the decreased in arginase activity correlated with the reduction of arg-1 levels in the cells. Upon treatment of cells with 20 µM CQ, the levels of arginase activity or arg-1 decreased by 43.5 % or 38.3 % in HT-29 cells and 40.9 or 39.4 % in HCT-116 respectively. Further the treatment of cells with 20 µM CQ for 48 h led to an increased level of p53 in the nucleus in comparison with the control samples (p <0.01). The nuclear:cytosol ratios of p53 levels were 5.3 (control) and 83 (20 µM CQ) in HCT-116 (wild type) cells and 2.6 (control) and 40.9 (20 µM CQ) in the HT-29 (mutant type) cells. These results indicate that the CQ-induced inhibition of arg-1 led to the nuclear accumulation of p53 in colon cancer cells. Thus the inhibition of arg-1 by CQ appears to protect the p53 tumor suppressor function.In an effort to expand our understanding of the molecular link between β-catenin levels, increased nuclear accumulation of p53 and CQ-mediated inhibition of arginase activity, HT-29 cells were co-incubated with CQ (0-50 µM) for 48 h and the level of β-catenin were determined by western blot assay. The results of this study indicate that CQ inhibited the β-catenin protein in a dose dependent manner. This decrease in the β-catenin level corresponded to an increase in the nuclear accumulation of p53 and corresponding diminution of intracellular Arg-1. In conclusion, the data presented herein represent a new and important improvement in understanding the pleiotropic effects of CQ on arg-1, with specific reference to its participation in the enhancement of the nuclear accumulation of p53 with the resultant down regulation of β-catenin protein. Thus this observation that CQ may inhibit the initial step of the arginine-polyamine pathway that results in the enhanced nuclear accumulation of p53 and a concomitant down regulation of β-catenin protein may provide a paradigm for targeted therapy in colorectal cancer chemoprevention and other malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1627. doi:1538-7445.AM2012-1627

Colorectal cancer (CRC) is the third-leading malignancy in the United States. This year alone, about 141,210 new cases of colorectal cancer will be diagnosed and more than 49,380 people will die from the disease. Strategies have focused on early screening practices and the prevention of the development of colonic adenomas that serve as precursors of invasive colon cancers. We have recently showed that chloroquine (CQ) an anti-malarial and anti-rheumatoid agent has the capacity to competitively inhibit intracellular arginase-1 (ARG-1) and that this inhibition increases the nuclear localization of the cell cycle regulator p53 protein in colon cancer cell lines. However, the mechanism of CQ-induced inhibition of ARG-1 that results in the up-regulation of p53 is unclear. The goal of the current study was to examine the chemopreventive efficacy of CQ in the development of colonic neoplasia and to further evaluate the possible mechanism(s) of action. In this regard, six weeks old Apc(Min/+) mice were treated daily (except Saturdays and Sundays) with 0-50 mg CQ/kg body wt. via oral gavage for forty days. Post-treatment, mice were sacrificed and jejunum and colon were retrieved and preserved in 10% formalin for observation of any gross pathological changes. The results of this study showed an increased in prevalence of colonic adenomas in untreated mice compared with treated mice (< 0.05). Interestingly, histochemical analysis showed significant numbers of adenomas with high-grade dysplasia (as evidenced by the presence of increased fibrous tissue core) in untreated control mice in comparison with drug-treated mice (<0.05).Since emerging studies have identified the overexpression of ARG-1 in a variety of human malignancies, including cancer of the colon, we then investigated the impact of CQ on ARG-1 levels. To this end, HT-29 cells were co-incubated with CQ (0-50 μM) for 24 h and the levels of ARG-1 were determined by western blot assay. Surprisingly, our result showed that CQ-induced inhibition of ARG-1 level corresponded to the diminution of β-catenin protein via to its phosphorylation. Further immuno- fluorescence analysis showed an increased level of nuclear localization of GFP tagged p53 (GFP-p53) and p21 in transiently transfected HT-29 cells upon CQ treatment. The observation that targeting ARG-1 results in a corresponding decrease in β-catenin level is intriguing, since the Wnt/β-catenin signaling is fundamental to colon carcinogenesis and spontaneous tumorigenesis in Apc(Min/+) mice and patients with familial adenomatous polyposis.In conclusion, our cumulative findings indicate that CQ ameliorates the development of colonic neoplasia and that the inhibition of ARG-1 and β-catenin may contribute to its chemopreventive effects in intestinal tumorigenesis. Citation Format: Efe W. Iyamu, Mohammad S. Niaz, Aramandla Ramesh, Amos Sakwe, Kieosha Williams, Billy R. Ballard, Samuel E. Adunyah. Targeted inhibition of arginase-1 may underline the suppression of the development of colonic neoplasia in a transgenic APC Min mouse model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3710. doi:10.1158/1538-7445.AM2013-3710