Effect of 2-Methoxyestradiol, Buthionine Sulfoximine and Hydrogen Peroxide on the Viability of Renal Carcinoma Cell Lines (ACHN and ACVB)

Abstract

Reactive oxygen species (ROS) are constantly generated in cells by several biological metabolic processes. A moderate level of intracellular ROS is thought to be important as a stimulant of cellular proliferation. However, the overproduction of ROS can result in detrimental cellular damage, including lipid peroxidation, DNA damage, protein oxidation and enzyme inactivation, which can ultimately lead to cell death.1,2) Aerobic cells contain several antioxidant enzymes, such as superoxide dismutase (SOD), catalase and various peroxidases, to maintain appropriate intracellular ROS levels and to prevent against oxidative damage. Generally, the cellular SOD activity appeared to be a little lower in malignant tissues than in normal tissues.3) 2-Methoxyestradiol (2-ME), an endogenous metabolite of 17b-estradiol, is present in human blood and urine. 2-ME is produced by 2-hydroxylation and subsequent O-methylation 4) and is known to have a low binding activity to the estrogen receptor.5) 2-ME has been shown to have potent anti-proliferative effects on various types of human cancer cells, including leukaemia, multiple myeloma, prostate cancer, gastric carcinoma and hepatoma.6-10) 2-ME has been reported to induce an accumulation of O 2 · Ϫ by inhibiting SOD, and to make G 2 /M-phase arrest accompanied by the inhibition of tubulin polymerization. [6][7][8][9][10] This compound is currently in clinical trial in the United States.8) Buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, has been used in clinical trials of cancer treatment. 11,12) However, the combination of the effect of 2-ME and the modification of ROS generation and the metabolic system have not been investigated in renal carcinoma cell lines.Renal carcinoma is one of the drug resistant malignancies in humans. ACHN is a cell line of human renal cell carcinoma, and ACVB is a vinblastine-resistant cell line of ACHN. ACHN has been used to study cytotoxic effects of several anticancer agents. For example, synergistic cytotoxicity by the combination of anti-Fas monoclonal antibody with adriamycin in ACHN had been reported. 13) In the present study, we examined the effect of combination with 2-ME, BSO and hydrogen peroxide (H 2 O 2 ) on the viability of human renal carcinoma cell lines (ACHN and ACVB). We have also analyzed the relationship between cell viability and intracellular ROS levels. MATERIALS AND METHODSChemicals 2-ME, estradiol, dihydroethidium (HEt), 2Ј,7Ј-dichlorofluoresin diacetate (DCFH-DA) and buthionine sulfoximine were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). RPMI-1640 was obtained from Invitrogen (Rockvill, MD, U.S.A.). Vinblastine, H 2 O 2 and 5Ј,5Ј-dithiobis 2-nitrobenzoic acid (DTNB) were obtained from Wako Pure Chemicals (Osaka). A WST-1 cell counting kit was obtained from Dojindo (Kumamoto). Fetal bovine serum (FBS) was obtained from ICN (Aurora, OH, U.S.A.).Cell Line and Cell Culture Human renal carcinoma cell line (ACHN) and its vinblastine resistant cell line (ACVB) were received from Prof. Hirai of Kobe ...

Nuclear Receptor 4A1 (NR4A1) as a Drug Target for Renal Cell Adenocarcinoma

“…The functions of TXNDC5 and IDH1 have not been characterized in RCC and it is also possible that NR4A1 regulates other similar genes and this is currently being investigated. Since previous studies show that other ROS-inducing antineoplastic agents inhibit growth and survival of RCC [ 42 , 43 ], the induction of ROS by C-DIM/NR4A1 antagonists contributed to the anticancer activity of these compounds.…”

Predictive model for recurrence of renal cell carcinoma by comparing pre‐ and postoperative urinary metabolite concentrations

“…So far, metabolites identified in this study have been reported in these three cell lines. [27][28][29] In 786-O, metabolites of glycolysis and the TCA cycle were identified. 27 Damaraju et al 28 reported that nucleoside metabolites with antitumor effects, such as glycine, increased in Caki-1.…”

“…27 Damaraju et al 28 reported that nucleoside metabolites with antitumor effects, such as glycine, increased in Caki-1. Additionally, Hirai et al 29 showed that the anticancer effect was related to an increase in the intracellular contents of glutaminolysis in ACHN. As cancer bioavailability was associated with intracellular concentrations of metabolites from these results, we can assume that evaluating intracellular metabolites would be helpful to understand the relationship between recurrence and urinary metabolites.…”