Shikonin targets cytosolic thioredoxin reductase to induce ROS-mediated apoptosis in human promyelocytic leukemia HL-60 cells

Shikonin, a major active component of the Chinese herbal plant Lithospermum erythrorhizon, has been applied for centuries in traditional Chinese medicine. Although shikonin demonstrates potent anticancer efficacy in numerous types of human cancer cells, the cellular targets of shikonin have not been fully defined. We report here that shikonin may interact with the cytosolic thioredoxin reductase (TrxR1), an important selenocysteine (Sec)-containing antioxidant enzyme with a C-terminal -Gly-Cys-Sec-Gly active site, to induce reactive oxygen species (ROS)-mediated apoptosis in human promyelocytic leukemia HL-60 cells. Shikonin primarily targets the Sec residue in TrxR1 to inhibit its physiological function, but further shifts the enzyme to an NADPH oxidase to generate superoxide anions, which leads to accumulation of ROS and collapse of the intracellular redox balance. Importantly, overexpression of functional TrxR1 attenuates the cytotoxicity of shikonin, whereas knockdown of TrxR1 sensitizes cells to shikonin treatment. Targeting TrxR1 with shikonin thus discloses a previously unrecognized mechanism underlying the biological activity of shikonin and provides an in-depth insight into the action of shikonin in the treatment of cancer.     

Ifosfamide induces acute renal failure via inhibition of the thioredoxin reductase activity

The present study investigated the impact of ifosfamide (IFO) on renal thioredoxin reductase (TrxR) activity. In mice treated with IFO for 6 h, TrxR activity significantly decreased in a dose-dependent manner. Subsequently, acute renal failure (ARF) occurred dose-dependently. Like IFO, the well-established TrxR-specific inhibitor auranofin suppresfssed renal TrxR activity and generated ARF too. TrxR was inactivated by IFO preferentially over other antioxidant parameters at 6 h; however, it recovered nearly to normal levels within 12 h. When auranofin was administered at 6 h after IFO treatment, the recovery at 12 h was sharply attenuated. Consequently, ARF was pronouncedly exacerbated. IFO within its maximum tolerated dose did not considerably deplete renal glutathione. However, escalating IFO dose strikingly attacked both the thioredoxin and the glutathione systems, resulting in lethality, which implies that glutathione depletion sensitizes IFO-induced nephrotoxicity and cosuppression of both systems causes more severe toxicological consequences than suppressing the thioredoxin system alone. Indeed, combining IFO with buthionine sulfoximine, an inhibitor of glutathione synthesis, induced much more severe ARF than IFO alone did. Taken together, inhibition of renal TrxR activity can be considered as a pivotal mechanism of IFO-induced ARF, and individuals with lower levels of renal glutathione are at high risk of incurring ARF after IFO treatment.     

The effects of hexavalent chromium on thioredoxin reductase and peroxiredoxins in human bronchial epithelial cells

Inhalational exposure to hexavalent chromium (Cr(VI)) compounds (e.g., chromates) is of concern in many Cr-related industries and their surrounding environments. The bronchial epithelium is directly exposed to inhaled Cr(VI). Cr(VI) species gain easy access inside cells, where they are reduced to reactive Cr species, which may also contribute to the generation of reactive oxygen species. The thioredoxin (Trx) system promotes cell survival and has a major role in maintaining intracellular thiol redox balance. Previous studies with normal human bronchial epithelial cells (BEAS-2B) demonstrated that chromates cause dose- and time-dependent oxidation of Trx1 and Trx2. The Trx’s keep many intracellular proteins reduced, including the peroxiredoxins (Prx’s). Prx1 (cytosolic) and Prx3 (mitochondrial) were oxidized by Cr(VI) treatments that oxidized all, or nearly all, of the respective Trx’s. Prx oxidation is therefore probably the result of a lack of reducing equivalents from Trx. Trx reductases (TrxR’s) keep the Trx’s largely in the reduced state. Cr(VI) caused pronounced inhibition of TrxR, but the levels of TrxR protein remained unchanged. The inhibition of TrxR was not reversed by removal of residual Cr(VI) or by NADPH, the endogenous electron donor for TrxR. In contrast, the oxidation of Trx1, Trx2, and Prx3 was reversible by disulfide reductants. Prolonged inhibition of TrxR in Cr(VI)-treated cells might contribute to the sustained oxidation of Trx’s and Prx’s. Reduced Trx binds to an N-terminal domain of apoptosis signaling kinase (ASK1), keeping ASK1 inactive. Cr(VI) treatments that significantly oxidized Trx1 resulted in pronounced dissociation of Trx1 from ASK1. Overall, the effects of Cr(VI) on the redox state and function of the Trx’s, Prx’s, and TrxR in the bronchial epithelium could have important implications for redox-sensitive cell signaling and tolerance of oxidant insults.     

Inhibition of thioredoxin reductase by mansonone F analogues: Implications for anticancer activity

Mammalian thioredoxin reductase (TrxR), a ubiquitous selenocysteine containing oxidoreductase, catalyzes the NADPH-dependent reduction of oxidized thioredoxin (Trx). TrxR has been suggested as a potential target for anticancer drugs development for its overexpression in human tumors and its diverse functions in intracellular redox control, cell growth and apoptosis. Mansonone F (MF) compounds have been shown to exhibit antiproliferative effects, but their complex mechanisms are unknown. In the present study, we have investigated the effects of some synthesized MF analogues on TrxR and HeLa cells. The studies of the mode of inhibition and the interactions of IG3, one of the most potent MF analogues, with TrxR showed MF compounds could be partly reduced by the C-terminal selenolthiol active site, and possibly by the N-terminal dithiol motif and/or FAD domain of TrxR simultaneously, accompanied by redox cycling with the generation of superoxide anion radicals. In addition, MF analogues exhibited the potential to inhibit the growth of HeLa cells and reduce TrxR activity in cell lysates. The cell cycle was arrested in G2/M phase and apoptosis was induced in a dose-dependent manner. Furthermore, our results showed that IG3-treated HeLa cells induced the change of intracellular ROS. Taken together, the reported results here suggest that inhibition of TrxR by MF analogues provides a possible complex mechanism for explaining the anticancer activity of MF compounds.     

2-Hydroxycurcuminoid induces apoptosis of human tumor cells through the reactive oxygen species-mitochondria pathway

2-Hydroxycinnamaldehyde (HCA) and curcumin have been reported to have antitumor effects against various human tumor cells in vitro and in vivo by generation of ROS. Aldehyde-free HCA analogs were synthesized based on the structure of curcumin, which we have called 2-hydroxycurcuminoids. The hydroxyl group of curcuminoids enhances the ability to generate ROS. 2-Hydroxycurcuminoid (HCC-7) strongly inhibited the growth of SW620 colon tumor cells with a GI₅₀ value of 7 μM, while the parent compounds, HCA and curcumin, displayed GI₅₀ values of 12 and 30 μM, respectively. HCC-7 was found to induce apoptosis through the reactive oxygen species-mitochondria pathway and cell cycle arrest at G2/M phase.     

Oxidative stress and hepatic Nox proteins in chronic hepatitis C and hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common liver cancer and a leading cause of cancer-related mortality in the world. Hepatitis C virus (HCV) is a major etiologic agent of HCC. A majority of HCV infections lead to chronic infection that can progress to cirrhosis and, eventually, HCC and liver failure. A common pathogenic feature present in HCV infection, and other conditions leading to HCC, is oxidative stress. HCV directly increases superoxide and H₂O₂ formation in hepatocytes by elevating Nox protein expression and sensitizing mitochondria to reactive oxygen species generation while decreasing glutathione. Nitric oxide synthesis and hepatic iron are also elevated. Furthermore, activation of phagocytic NADPH oxidase (Nox) 2 of host immune cells is likely to exacerbate oxidative stress in HCV-infected patients. Key mechanisms of HCC include genome instability, epigenetic regulation, inflammation with chronic tissue injury and sustained cell proliferation, and modulation of cell growth and death. Oxidative stress, or Nox proteins, plays various roles in these mechanisms. Nox proteins also function in hepatic fibrosis, which commonly precedes HCC, and Nox4 elevation by HCV is mediated by transforming growth factor β. This review summarizes mechanisms of oncogenesis by HCV, highlighting the roles of oxidative stress and hepatic Nox enzymes in HCC.     

Phenethyl isothiocyanate induces DNA damage-associated G2/M arrest and subsequent apoptosis in oral cancer cells with varying p53 mutations

Phenethyl isothiocyanate (PEITC) is a naturally occurring cruciferous vegetable-derived compound that inhibits cell growth and induces apoptosis in oral cancer cells. However, the exact mechanism of PEITC action has not been fully elucidated. This study investigated the molecular mechanism and anticancer potential of PEITC in oral squamous cell carcinoma (OSCC) cells with various p53 statuses. PEITC inhibited the growth of OC2, SCC4, and SCC25 cells (functional p53 mutants) in a dose-dependent manner with low toxicity to normal cells. Treatment with PEITC induced reactive oxygen species production, nitric oxide generation, and GSH depletion and triggered DNA damage response as evidenced by flow cytometry, 8-OHdG formation, and comet assay. Furthermore, the subsequent activation of ATM, Chk2, and p53 as well as the increased expression of downstream proteins p21 and Bax resulted in a G2/M phase arrest by inhibiting Cdc25C, Cdc2, and cyclin B1. The PEITC-induced apoptotic cell death, following a diminished mitochondrial transmembrane potential, reduced the expression of Bcl-2 and Mcl-1, released mitochondrial cytochrome c, and activated caspase 3 and PARP cleavage. The p53 inhibitor pifithrin-α and the antioxidants N-acetylcysteine and glutathione (GSH) protected the cells from PEITC-mediated apoptosis. However, mito-TEMPO, catalase, apocynin, and L-NAME did not prevent PEITC-induced cell death, suggesting that PEITC induced G2/M phase arrest and apoptosis in oral cancer cells via a GSH redox stress and oxidative DNA damage-induced ATM–Chk2–p53-related pathway. These results provide new insights into the critical roles of both GSH redox stress and p53 in the regulation of PEITC-induced G2/M cell cycle arrest and apoptosis in OSCCs.     

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

Thioredoxin systems, composed of thioredoxin reductase (TrxR), thioredoxin (Trx) and NADPH, play important roles in maintaining cellular redox homeostasis and redox signaling. Recently the cytosolic Trx1 system has been shown to be a cellular target of arsenic containing compounds. To elucidate the relationship of the structure of arsenic compounds with their ability of inhibiting TrxR1 and Trx1, and cytotoxicity, we have investigated the reaction of Trx1 system with seven arsenic trithiolates: As(Cys)₃, As(GS)₃, As(Penicillamine)₃, As(Mercaptoethanesulfonate)₃, As(Mercaptopurine)₃, As(2-mercaptopyridine)₃ and As(2-mercaptopyridine N-oxide)₃. The cytotoxicity of these arsenicals was consistent with their ability to inhibit TrxR1 in vitro and in cells. Unlike other arsenicals, As(Mercaptopurine)₃ which did not show inhibitory effects on TrxR1 had very weak cytotoxicity, indicating that TrxR1 is a reliable drug target for arsenicals. Moreover, the two aromatic compounds As(2-mercaptopyridine)₃ and As(2-mercaptopyridine N-oxide)₃ showed stronger cytotoxicity than the others. As(2-mercaptopyridine)₃ which selectively oxidized two structural cysteines (Cys62 and Cys69) in Trx1 showed mild improvement in cytotoxicity. As(2-mercaptopyridine N-oxide)₃ oxidized all the Cys residues in Trx1, exhibiting the strongest cytotoxicity. Oxidation of Trx1 by As(2-mercaptopyridine)₃ and As(2-mercaptopyridine N-oxide)₃ affected electron transfer from NADPH and TrxR1 to peroxiredoxin 1 (Prx1), which could result in the reactive oxygen species elevation and trigger cell death process. These results suggest that oxidation of structural cysteine residues in Trx1 by aromatic group in TrxR1-targeting drugs may sensitize tumor cells to cell death, providing a novel approach to regulate cellular redox signaling and also a basis for rational design of new anticancer agents.     

Upregulation of aldose reductase by homocysteine in type II alveolar epithelial cells

Homocysteine (Hcy) has recently been recognized as an integral component of several disorders. However, the association between hyperhomocysteinemia (HHcy) and pulmonary disease is not well understood. The combination of two-dimensional electrophoresis and tandem mass spectrometry detected and identified proteins that are differentially expressed in human type II alveolar epithelial cells (A549 cells) treated by Hcy. We found that aldose reductase (AR) showed more abundant expression in the cells. Further, Hcy (100-500microM) could induce a time- and dose-dependent upregulation of AR protein levels. Immunohistochemical staining of cross-sections from HHcy mice lungs also revealed increased expression of AR protein. Intracellular levels of reactive oxygen species (ROS) were remarkably elevated in A549 cells treated with Hcy. Pretreatment of A549 cells with catalase and SOD significantly suppressed the Hcy-induced AR expression, which suggests the involvement of ROS in this process. The major signaling pathway mediating the upregulation of AR was demonstrated to be the Ras/Raf/ERK1/2 pathway. In addition, Hcy might reduce surfactant protein B (SP-B) expression in the cells, which could be significantly attenuated by Alrestatin, an AR inhibitor, indicating a damaging role of Hcy-induced AR elevation in the lung. These results show a novel and unanticipated link between HHcy and AR upregulation that may be a risk factor in pulmonary disease of patients with HHcy.     

C3-fullero-tris-methanodicarboxylic acid protects cerebellar granule cells from apoptosis

Buckminsterfullerenols were recently investigated for their protective properties in different models of acute and chronic neurodegeneration. We tested C3-fullero-tris-methanodicarboxylic acid in our in vitro model of apoptotic neuronal death, which consists of shifting the culture K+ concentration from 25 to 5 mM for rat cerebellar granule cells. The impairment of mitochondrial respiratory function as well as chromatin derangement and fragmentation of DNA in apoptotic oligonucleosomes that occur in these conditions were protected by this compound in a concentration-dependent way. To assess whether antioxidant activity could account for the rescue of cerebellar granule cells from apoptosis, we tested the fullerene derivative under FeSO4-induced oxidative stress and found significant protection. Thus, we visualized membrane and cytoplasmic peroxides and reactive oxygen species and found a significant reduction of the species after 24 h in 5 mM K+ with the fullerene derivative. Such evidence suggests that this compound exerts a protective role in cerebellar granule cell apoptosis, likely reducing the oxidative stress.     

Cardiolipin deficiency leads to decreased cardiolipin peroxidation and increased resistance of cells to apoptosis

Cardiolipin (CL), a unique mitochondrial phospholipid synthesized by CL synthase (CLS), plays important, yet not fully understood, roles in mitochondria-dependent apoptosis. We manipulated CL levels in HeLa cells by knocking down CLS using RNA interference and selected a clone of CL-deficient cells with ~ 45% of its normal content. ESI–MS analysis showed that the CL molecular species were the same in CL-deficient and CL-sufficient cells. CL deficiency did not change mitochondrial functions (membrane potential, reactive oxygen species generation, cellular ATP levels) but conferred resistance to apoptosis induced by actinomycin D (ActD), rotenone, or γ-irradiation. During ActD-induced apoptosis, decreased CL peroxidation along with suppressed cytochrome (cyt) c release was observed in CL-deficient cells, whereas Bax translocation to mitochondria remained similar to that in CL-sufficient HeLa cells. The amounts of loosely bound cyt c (releasable under high ionic strength conditions) were the same in CL-deficient and CL-sufficient cells. Given that CL peroxidation during apoptosis is catalyzed by CL/cyt c complexes and CL oxidation products are essential for cyt c release from mitochondria, our results suggest that CL deficiency prevents adequate assembly of productive CL/cyt c complexes and CL peroxidation, resulting in increased resistance to apoptosis.     

Differential regulation of expression of cytosolic and mitochondrial thioredoxin reductase in rat liver and kidney

Adequate supply of selenium (Se) is critical for synthesis of selenoproteins through selenocysteine insertion mechanism. To explore this process we investigated the expression of the cytosolic and mitochondrial isoenzymes of thioredoxin reductase (TrxR1 and TrxR2) in response to altered Se supply. Rats were fed diets containing different quantities of selenium and the levels of TrxR1 and TrxR2 protein and their corresponding mRNAs were determined in liver and kidney. Expression of the two isoenzymes was differentially affected, with TrxR1 being more sensitive to Se depletion than TrxR2 and greater changes in liver than kidney. In order to determine if the selenocysteine incorporation sequence (SECIS) element was critical in this response liver and kidney cell lines (H4 and NRK-52E) were transfected with reporter constructs in which expression of luciferase required read-through at a UGA codon and which contained either the TrxR1 or TrxR2 3'UTR, or a combination of the TrxR1 5' and 3'UTRs. Cell lines expressing constructs with the TrxR1 3'UTR demonstrated no response to restricted Se supply. In comparison the Se-deficient cells expressing constructs with the TrxR2 3'UTR showed considerably less luciferase activity than the Se-adequate cells. No disparity of response to Se supply was observed in the constructs containing the different TrxR1 5'UTR variants. The data show that there is a prioritisation of TrxR2 over TrxR1 during Se deficiency such that TrxR1 expression is more sensitive to Se supply than TrxR2 but this sensitivity of TrxR1 was not fully accounted for by TrxR1 5' or 3'UTR sequences when assessed using luciferase reporter constructs.     

High dose of ascorbic acid induces cell death in mesothelioma cells

Malignant mesothelioma is an asbestos-related fatal disease with no effective cure. Recently, high dose of ascorbate in cancer treatment has been reexamined. We studied whether high dose of ascorbic acid induced cell death of four human mesothelioma cell lines. High dose of ascorbic acid induced cell death of all mesothelioma cell lines in a dose-dependent manner. We further clarified the cell killing mechanism that ascorbic acid induced reactive oxygen species and impaired mitochondrial membrane potential. In vivo experiment, intravenous administration of ascorbic acid significantly decreased the growth rate of mesothelioma tumor inoculated in mice. These data suggest that ascorbic acid may have benefits for patients with mesothelioma.     

斑蝥素酸镁对人肝癌细胞SMMC-7721及其裸鼠皮下移植瘤的影响

【目的】探讨斑蝥素酸镁对人肝癌细胞SMMC-7721及其裸鼠皮下移植瘤的影响。【方法】1、采用磺酰罗丹明染色法(SRB法)检测不同浓度斑蝥素酸镁在体外对人肝癌细胞SMMC-7721增殖的抑制作用;2、流式细胞术检测斑蝥素酸镁对人肝癌细胞SMMC-7721细胞周期和细胞凋亡的影响;3、Hoechst33342染色观察斑蝥素酸镁对人肝癌细胞SMMC-7721细胞形态的影响;4、透射电镜观察斑蝥素酸镁作用后人肝癌细胞SMMC-7721超微结构的变化;5、建立人肝癌细胞SMMC-7721裸鼠皮下移植瘤模型,实验组每只裸鼠瘤周注射斑蝥素酸镁6.26×10?5 mmol,对照组给予相同容积的无菌生理盐水瘤周注射,计算抑瘤率;6、原位末端标记染色(TUNEL)法检测人肝癌裸鼠皮下移植瘤组织细胞凋亡情况。【结果】1、斑蝥素酸镁对人肝癌细胞SMMC-7721有比较明显的抑制作用,抑制率随药物浓度的增加而升高,呈剂量效应关系,其半数抑制浓度(IC50)为1.79?mol/L;2、流式细胞检测结果显示:人肝癌细胞SMMC-7721在斑蝥素酸镁的作用下,G0/G1期细胞减少,G2/M期细胞增加,细胞出现G2/M期阻滞;细胞凋亡率随斑蝥素酸镁浓度加大而逐渐增加;3、Hoechst33342染色镜下显示:斑蝥素酸镁作用后人肝癌细胞SMMC-7721出现凋亡细胞形态特征;4、透射电镜观察:斑蝥素酸镁作用后人肝癌细胞SMMC-7721出现细胞核异形、染色质聚集成团、边集,见凋亡小体;5、斑蝥素酸镁组肿瘤体积、重量显著小于生理盐水组(P﹤0.05),抑瘤率为49%;6、TUNEL法提示斑蝥素酸镁组移植瘤组织细胞凋亡率显著高于生理盐水组(χ2=92.609,P﹦0.000)。【结论】斑蝥素酸镁对人肝癌细胞SMMC-7721在体内外均有抑制增殖作用,并可以诱导肿瘤细胞凋亡,其凋亡的发生与细胞分裂期阻滞有关。     

Pyridoxamine protects intestinal epithelium from ionizing radiation-induced apoptosis

Reactive oxygen species (ROS) and reactive carbonyl species (RCS) are the major causes of biological tissue damage during exposure to ionizing radiation (IR). The existing strategies to protect normal tissues from the detrimental effects of IR suffer from several shortcomings including highly toxic side effects, unfavorable administration routes, and low efficacy. These shortcomings emphasize a need for radioprotective treatments that combine effectiveness with safety and ease of use. In this paper, we demonstrate that pyridoxamine, a ROS and RCS scavenger with a very favorable safety profile, can inhibit IR-induced gastrointestinal epithelial apoptosis in cell culture and in an animal model. Pyridoxamine was more effective at protecting from radiation-induced apoptosis than amifostine, a synthetic thiol compound and the only FDA-approved radioprotector. We suggest that pyridoxamine has potential as an effective and safe radioprotective agent.