Selective alkylation of beta(II)-tubulin and thioredoxin-1 by structurally related subsets of aryl chloroethylureas leading to either anti-microtubules or redox modulating agents

Aryl chloroethylureas (CEUs) are potent anti-neoplastic agents alkylating specific intracellular proteins such as beta(II)-tubulin. Recently we have identified a new subset of CEU derived from compound 36 that alkylates thioredoxin isoform 1 (Trx-1), inhibits the nuclear translocation of Trx-1, and favors the accumulation of cells in G(0)/G(1) phase. We have evaluated the effects of various substituents and their position on the aromatic ring of a series of derivatives of 36 on (i) the anti-proliferative activity, (ii) the cell cycle progression, (iii) the nuclear translocation of Trx-1, and (iv) their covalent binding to beta-tubulin. The same experiments were performed on representative CEU derivatives where the 2-chloroethyl amino moiety is replaced by either an ethyl, a 2-aminooxazolinyl or a 2-chloroacetyl group. On one hand, our results suggest that CEUs substituted on the phenyl ring at position 3 or 4 by cycloalkyl and substituted cycloalkyl or cycloalkoxy groups inhibit the nuclear translocation of Trx-1 and arrest the cell cycle progression in G(0)/G(1). On the other hand, CEUs substituted by a fused aromatic ring, an aliphatic chain, or a fused aliphatic ring are alkylating beta(II)-tubulin but not Trx-1. Beside the expected inactivity of the ethylurea derivatives, none of the modification to the electrophilic moiety led to cross-selectivity of the drugs toward beta-tubulin but increased the anti-proliferative activity and resulted in mitigated effects on Trx-1 translocation.     

Radiosensitization of glioma cells by TP53-induced glycolysis and apoptosis regulator knockdown is dependent on thioredoxin-1 nuclear translocation

TP53-induced glycolysis and apoptosis regulator (TIGAR) knockdown is proven to radiosensitize glioma cells, but the mechanisms are not fully understood. Thioredoxin-1 (TRX1) is a redox-sensitive oxidoreductase, which plays critical roles in DNA damage signal transduction via nuclear translocation in irradiated cells. Because the TRX1-dependent DNA damage signaling pathway relies on NADPH to maintain the reduced state of TRX1, and TIGAR functions to increase NADPH generation under oxidative stress, in this study, the role of TRX1 in TIGAR abrogation-induced radiosensitization was investigated. It was demonstrated that ionizing radiation (IR)-induced nuclear translocation of TRX1 was significantly inhibited by TIGAR interference and reversed by wild-type (WT)-TRX1 overexpression. In addition, WT-TRX1 overexpression could accelerate the process of DNA damage repair postponed by TIGAR knockdown in irradiated glioma cells. The reduction process of IR-oxidized TRX1 was also delayed by TIGAR knockdown but restored by WT-TRX1 overexpression. Therefore, we conclude that TIGAR knockdown-induced radiosensitization of glioma cells may be dependent on the inhibition of TRX1 nuclear translocation.     

DNMT1 as a molecular target in a multimodality-resistant phenotype in tumor cells

We have previously shown that hydrogen peroxide-resistant permanent (OC-14) cells are resistant to the cytotoxicity of several exogenous oxidative and anticancer agents including H(2)O(2), etoposide, and cisplatin; and we refer to this process as an oxidative multimodality-resistant phenotype (MMRP). Furthermore, OC-14 cells contain increased activator protein 1 activity, and inhibition of activator protein 1 reversed the MMRP. In this study, we show that permanent Rat-1 cell lines genetically altered to overexpress c-Fos also displayed a similar MMRP to H(2)O(2), etoposide, and cisplatin as OC-14 cells. Gene expression analysis of the OC-14 cells and c-Fos-overexpressing cells showed increased DNMT1 expression. Where OC-14 and c-Fos-overexpressing cells were exposed to 5-aza-2'-deoxycytidine, which inhibits DNMT activity, a significant but incomplete reversal of the MMRP was observed. Thus, it seems logical to suggest that DNMT1 might be at least one target in the MMRP. Rat-1 cells genetically altered to overexpress DNMT1 were also shown to be resistant to the cytotoxicity of H(2)O(2), etoposide, and cisplatin. Finally, somatic HCT116 knockout cells that do not express either DNMT1 (DNMT1(-/-)) or DNMT3B (DNMT3B(-/-)) were shown to be more sensitive to the cytotoxicity of H(2)O(2), etoposide, and cisplatin compared with control HCT116 cells. This work is the first example of a role for the epigenome in tumor cell resistance to the cytotoxicity of exogenous oxidative (H(2)O(2)) or systemic (etoposide and cisplatin) agents and highlights a potential role for DNMT1 as a potential molecular target in cancer therapy.     

Cycloalkyl-substituted aryl chloroethylureas inhibiting cell cycle progression in G0/G1 phase and thioredoxin-1 nuclear translocation

1-(2-Chloroethyl)-3-(4-cyclohexylphenyl)urea (cHCEU) has been shown to abrogate the presence of thioredoxin-1 into the nucleus through its selective covalent alkylation. In the present letter we have evaluated the structure-activity relationships of the substituents at positions 3 and 4 of the phenyl ring of cHCEU derivatives on cell cycle progression and thioredoxin-1 nuclear translocation. Active CEU derivatives exhibited GI(50) ranging from 1.9 to 49muM on breast carcinoma MCF-7, skin melanoma M21, and colon carcinoma HT-29 cells. On one hand, compounds 1, 2, 9c, 10c, 13, and 14 arrested the cell cycle in G(2)/M phase while CEUs 3, 4, 5c, 6c, 11c, and 12c blocked the cell division in G(0)/G(1) phase. On the other hand, CEUs 2-4, 5c, 7c, 8c, 11c, and 12c abrogated the translocation of thioredoxin-1 while the other CEU derivatives were inactive in that respect. Our results suggest that CEU substituted on the phenyl ring at position 3 or 4 by lower cycloalkyl or cycloalkoxy groups arrest cell progression in G(0)/G(1) phase through mechanism of action different from their antimicrotubule counterparts, presumably via thioredoxin-1 alkylation and modulation of its activity. The mechanism of action of these new molecules is still undetermined. However, the significant accumulation of cells in G(0)/G(1) phase suggests that these molecules may act similarly to known chemopreventive agents against cancers. In addition, the inhibition of Trx-1 nuclear localization also suggests the abrogation of an important chemoresistance mechanism towards a variety of chemotherapeutic agents.     

In silico identification of poly(ADP-ribose)polymerase-1 inhibitors and their chemosensitizing effects against cisplatin-resistant human gastric cancer cells

Poly(ADP-ribose)polymerase-1 (PARP-1) enzyme is involved in the repair of DNA damages made by certain anticancer agents. It is suggested that PARP-1 inhibitors potentiate the cytotoxic effects and circumvent the resistance of DNA-modifying anticancer agents such as cisplatin. In this study, we conducted virtual screening of Korea Chemical Bank database targeting PARP-1 and identified several potent PARP-1 inhibitors with submicromolar IC₅₀ values (77–79 nM). We then examined the chemosensitization of cisplatin by pre-treatment of PARP-1 inhibitors in cisplatin-resistant human gastric cancer cells. Our results show that PARP-1 inhibitors suppress the formation of poly(ADP-ribose) and enhance the cytotoxicity of cisplatin.     

A novel small compound that promotes nuclear translocation of YB-1 ameliorates experimental hepatic fibrosis in mice

Transforming growth factor-β (TGF-β) is considered to be a major factor contributing to liver fibrosis. We have previously shown that nuclear translocation of YB-1 antagonizes the TGF-β/Smad3 signaling in regulating collagen gene expression. More recently, we have demonstrated that the novel small compound HSc025 promotes nuclear translocation of YB-1, resulting in the improvement of skin and pulmonary fibrosis. Here, we presented evidence as to the mechanism by which HSc025 stimulates nuclear translocation of YB-1 and the pharmacological effects of HSc025 on a murine model of hepatic fibrosis. A proteomics approach and binding assays using HSc025-immobilized resin showed that HSc025 binds to the amino acid sequence within the C-tail region of YB-1. In addition, immunoprecipitation experiments and glutathione S-transferase pulldown assays identified poly(A)-binding protein (PABP) as one of the cytoplasmic anchor proteins of YB-1. HSc025 directly binds to YB-1 and interrupts its interaction with PABP, resulting in accelerated nuclear translocation of YB-1. Transfection of cells with PABP siRNA promoted nuclear translocation of YB-1 and subsequently inhibited basal and TGF-β-stimulated collagen gene expression. Moreover, HSc025 significantly suppressed collagen gene expression in cultured activated hepatic stellate cells. Oral administration of HSc025 to mice with carbon tetrachloride-induced hepatic fibrosis improved liver injury as well as the degree of hepatic fibrosis. Altogether, the results provide a novel insight into therapy for organ fibrosis using YB-1 modulators.     

Expression of thioredoxin in bleomycin-injured airway epithelium: possible role of protection against bleomycin induced epithelial injury

Bleomycin (BLM) is an anticancer drug, administration of which leads to severe lung injury, in which the generation of intracellular reactive oxygen species (ROS) is thought to participate in that. Thioredoxin (TRX) has been found to function as a powerful antioxidant by reducing ROS, and thus protecting against ROS-mediated cytotoxicity. However, a protective role of TRX in BLM-induced lung injury has not been determined. In the present study, we therefore attempted to clarify this issue. Human TRX-transfected L929 murine fibrosarcoma cells were more resistant to BLM-induced cytotoxicity than the parental and the control transfected cells, indicating that TRX plays the protective role in BLM-induced cytotoxicity. Next, we examined TRX expression in the lung of in vivo model of BLM-induced lung injury and BLM-stimulated bronchial epithelial cells in vitro to clarify the role of TRX in BLM-induced lung injury. In the lungs of BLM-treated mice, the expression of TRX was strongly induced in bronchial epithelial cells. TRX expression was also up-regulated at both the mRNA and protein levels in cultured BEC with the treatment with BLM. However, the expression of other major antioxidants, such as Cu/Zn-SOD, Mn-SOD, catalase and glutathione peroxidase, was not affected by BLM. These observations suggest that the cellular reduction and oxidation (redox) state modified by TRX is involved in the BLM resistancy and the induction of TRX in bronchial epithelial cells might play a protective role in BLM-induced lung injury.     

Thioredoxin-dependent redox regulation of p53-mediated p21 activation

Thioredoxin (TRX) is a dithiol-reducing enzyme that is induced by various oxidative stresses. TRX regulates the activity of DNA-binding proteins, including Jun/Fos and nuclear factor-kappaB. TRX also interacts with an intranuclear reducing molecule redox factor 1 (Ref-1), which enhances the activity of Jun/Fos. Here, we have investigated the role of TRX in the regulation of p53 activity. Electrophoretic mobility shift assay showed that TRX augmented the DNA binding activity of p53 and also further potentiated Ref-1-enhanced p53 activity. Luciferase assay revealed that transfection of TRX enhanced p53-dependent expression of p21 and further intensified Ref-1-mediated p53 activation. Furthermore, Western blot analysis revealed that p53-dependent induction of p21 protein was also facilitated by transfection with TRX. Overexpression of transdominant negative mutant TRX (mTRX) suppressed the effects of TRX or Ref-1, showing a functional interaction between TRX and Ref-1. cis-Diamminedichloroplatinum (II) (CDDP) induced p53 activation and p21 transactivation. The p53-dependent p21 transactivation induced by CDDP was inhibited by mTRX overexpression, suggesting that TRX-dependent redox regulation is physiologically involved in p53 regulation. CDDP also stimulated translocation of TRX from the cytosol into the nucleus. Hence, TRX-dependent redox regulation of p53 activity indicates coupling of the oxidative stress response and p53-dependent repair mechanism.     

N-Phenyl-N'-(2-chloroethyl)ureas (CEU) as potential antineoplastic agents. Part 2: role of omega-hydroxyl group in the covalent binding to beta-tubulin

Tubulin is the target of many anticancer drugs, including N-phenyl-N'-(2-chloroethyl)urea (CEU). Unlike most anti-beta-tubulin agents, CEUs are protein monoalkylating agents binding through their N'-(2-chloroethyl)urea moiety to an amino acid nearby the colchicine-binding site on beta-tubulin isoform-2. Following the previously synthesized and attractive N-(3-omega-hydroxyalkylphenyl)-N'-(2-chloroethyl)urea that exhibited growth inhibitory activity at the nanomolar level, we investigated the importance of lower alkyl and alkoxy groups to evaluate the effect of hydroxylated group and chain length on both cell growth inhibition and the mechanism of action of CEU. Here, we describe the preparation of two new series of CEU and show that the most potent CEU derivatives beside the omega-hydroxylated 1f were 2f and 3e, respectively. We have confirmed that the pentyl substituted CEUs 1f, 2f, and 3e are still covalently binding to beta-tubulin and still arrest cell division in G(2)/M phase.     

Cell death by reactive oxygen species generated from water-soluble cationic metalloporphyrins as superoxide dismutase mimics.

We investigated the effect on cell death of reactive oxygen species induced by water-soluble cationic metalloporphyrins with superoxide dismutase (SOD) activity. The SOD activity of 5,10,15,20-tetrakis(4-N-methylpyridyl)]porphine (MPy(4)P) containing Fe, Mn or Cu was measured using a cytochrome c assay by the xanthine/xanthine oxidase system and stopped-flow kinetic analysis. Cell viability of four cell lines treated with metalloporphyrins, mitomycin c (MMC), or cisplatin was estimated by a trypan blue exclusion assay. FeMPy(4)P with a high SOD activity showed a significant cytotoxicity compared with MMC and cisplatin, while CuMPy(4)P without SOD activity exhibited no cytotoxicity. However, MnMPy(4)P showing an SOD activity as high as that of FeMPy(4)P did not indicate cytotoxicity. These findings suggest that FeMPy(4)P as SOD mimic converts intracellular O2(*-) to H(2)O(2) and that it rapidly reacts with H(2)O(2) to form *OH, causing DNA damage and inducing cell death. On the other hand, MnMPy(4)P did not participate in the Fenton reaction, so that DNA damage in the cells treated with MnMPy(4)P was not observed. In addition, the cytotoxicity by the metalloporphyrin was inversely correlated with the SOD activity of the cells and the selective damage at cellular and DNA levels was confirmed. We believe that for an anticancer drug with antioxidant ability O(2)(*-) is useful as a target molecule to induce selective cell death between cancer and normal cells and that metalloporphyrins showing SOD activity and Fenton-like reaction are a new class of anticancer agents.     

Inhibition of ERβ induces resistance to cisplatin by enhancing Rad51-mediated DNA repair in human medulloblastoma cell lines

Cisplatin is one of the most widely used and effective anticancer drugs against solid tumors including cerebellar tumor of the childhood, Medulloblastoma. However, cancer cells often develop resistance to cisplatin, which limits therapeutic effectiveness of this otherwise effective genotoxic drug. In this study, we demonstrate that human medulloblastoma cell lines develop acute resistance to cisplatin in the presence of estrogen receptor (ER) antagonist, ICI182,780. This unexpected finding involves a switch from the G2/M to G1 checkpoint accompanied by decrease in ATM/Chk2 and increase in ATR/Chk1 phosphorylation. We have previously reported that ERβ, which is highly expressed in medulloblastomas, translocates insulin receptor substrate 1 (IRS-1) to the nucleus, and that nuclear IRS-1 binds to Rad51 and attenuates homologous recombination directed DNA repair (HRR). Here, we demonstrate that in the presence of ICI182,780, cisplatin-treated medulloblastoma cells show recruitment of Rad51 to the sites of damaged DNA and increase in HRR activity. This enhanced DNA repair during the S phase preserved also clonogenic potential of medulloblastoma cells treated with cisplatin. In conclusion, inhibition of ERβ considered as a supplemental anticancer therapy, has been found to interfere with cisplatin-induced cytotoxicity in human medulloblastoma cell lines.     

Inhibition of heme oxygenase-1 enhances the chemosensitivity of laryngeal squamous cell cancer Hep-2 cells to cisplatin

It has been previously reported that cisplatin is a well-known anticancer drug being used against a wide range of malignancies including head and neck, ovarian and non-small cell lung carcinoma, and demonstrated its anticancer activity by reacting with DNA or changing cell structure, immune response, reactive oxygen species level (ROS). In this research we proved that cisplatin induced cell injuries and heme oxygenase-1 (HO-1) expression in laryngeal squamous cell cancer Hep-2 cells through ROS generation. The induction of HO-1 clearly protected Hep-2 cells from cisplatin-induced cell death and ROS reaction, and the inhibitor of HO-1 enhanced the cell death and ROS generation induced by cisplatin. Furthermore, the HO-1 expression induced by cisplatin was strongly inhibited by the knockdown of nuclear factor-erythroid-2-related factor-2 (Nrf-2), and the oxidative damages induced by cisplatin were significantly enhanced. Therefore, it may be concluded that the inhibition of HO-1 or the knockdown of Nrf-2 significantly enhanced cisplatin’s anticancer effects on Hep-2 cells. In clinic, with the overexpression of HO-1 in laryngeal squamous cancer tissues, the combination of cisplatin with the inhibitor of HO-1 or Nrf-2 siRNA may act as a new method to the treatment of laryngeal squamous cancer.