Disruption of the mitochondrial thioredoxin system as a cell death mechanism of cationic triphenylmethanes

Abstract

Alteration of mitochondrial structure and function is a hallmark of cancer cells different from normal cells and thus targeting mitochondria emerges as an novel approach for cancer therapy. Mitochondrial thioredoxin2 (Trx2) system is critical for cell viability, but the role in cancer biology is not well understood. Recently some cationic triphenylmethanes such as brilliant green (BG) and gentian violet showed antitumor and antiangiogenic activity by unknown mechanisms. Here we demonstrate that BG killed cells at nanomolar concentrations and targeted mitochondrial Trx2, which was oxidized and degraded. HeLa cells were more sensitive to BG than fibroblasts. In HeLa cells, Trx2 down-regulation by siRNA resulted in increased sensitivity to BG, whereas for fibroblasts, the same treatments had no effect. BG was visualized to accumulate in mitochondria and cause a rapid and dramatic decrease of mitochondrial Trx2 protein. With a redox Western blot method, we found that treatment with BG caused oxidation of both Trx1 and Trx2, followed by release of cytochrome c and apoptosis inducing factor from mitochondria into cytosol. Moreover, this treatment resulted in an elevation of mRNA level of Lon protease, a protein quality control enzyme in mitochondrial matrix, suggesting that the oxidized Trx2 may be degraded by Lon protease.

Oxidation of structural cysteine residues in thioredoxin 1 by aromatic arsenicals enhances cancer cell cytotoxicity caused by the inhibition of thioredoxin reductase 1

“…A moderately elevated ROS level in cancer cells increased metabolic activity via oncogenes can promote tumor growth and survival by stimulating a series of enzymes, such as mitogen-activated protein kinase (MAPK) and extracellular signalregulated kinase (ERK) and cyclin D1 [17]. However, a heavily elevated level of ROS can induce cell death through multiple pathways, such as activation of ASK1, translocation of cytochrome c from mitochondria to cytosol and induction of p53 expression, or oxidation of nucleotides in nucleotide pool [12,26,30,31]. Many studies suggest the cellular redox pathways can be a promising target in anticancer therapy [17,29].…”

“…After incubating with primary and corresponding secondary antibodies, target proteins were detected with chemiluminescence Reagent Plus (Perkin Elmer Life Sciences, Shelton, CT, USA). The mRNA levels were measured by Quantitative real-time PCR analysis as previously described [26].…”

“…Detection of redox state of pure Trx proteins were performed according to the method described previously [19,26]. Briefly, Trxs were pre-reduced with incubation of 30 mM dithiothreitol (DTT) at 37°C for 30 min.…”

“…To determine the exact thiols in Trx1 to react As6, we analyzed the effects of As6 on the redox state of Trx1, Trx2 and a Trx1 C62S/ C73S double mutant by a redox Western blot electrophoresis method [19,26]. Unlike Trx1, which has three extra structural cysteine residues besides two in the active site, Trx2 only has two Cys residues in its classic active site -Cys-Gly-Pro-Cys- [11,12].…”

Potential suppressive effects of gentian violet on human breast cancer MDA-MB-231 cells in vitro: Comparison with gemcitabine

“…Furthermore, recent studies have suggested the angiogenic and anticancer properties of GV, and this chemical is currently experiencing renewed interest in medical applications (11,12). Our recent study demonstrated that GV inhibits nuclear factor-κB (NF-κB) activity, and that this agent can potently enhance osteoblast differentiation and mineralization, but suppress the differentiation to osteoclasts (13).…”