The redox antimalarial dihydroartemisinin targets human metastatic melanoma cells but not primary melanocytes with induction of NOXA-dependent apoptosis.

Recent research suggests that altered redox control of melanoma cell survival, proliferation, and invasiveness represents a chemical vulnerability that can be targeted by pharmacological modulation of cellular oxidative stress. The endoperoxide artemisinin and semisynthetic artemisinin-derivatives including dihydroartemisinin (DHA) constitute a major class of antimalarials that kill plasmodium parasites through induction of iron-dependent oxidative stress. Here, we demonstrate that DHA may serve as a redox chemotherapeutic that selectively induces melanoma cell apoptosis without compromising viability of primary human melanocytes. Cultured human metastatic melanoma cells (A375, G361, LOX) were sensitive to DHA-induced apoptosis with upregulation of cellular oxidative stress, phosphatidylserine externalization, and activational cleavage of procaspase 3. Expression array analysis revealed DHA-induced upregulation of oxidative and genotoxic stress response genes (GADD45A, GADD153, CDKN1A, PMAIP1, HMOX1, EGR1) in A375 cells. DHA exposure caused early upregulation of the BH3-only protein NOXA, a proapototic member of the Bcl2 family encoded by PMAIP1, and genetic antagonism (siRNA targeting PMAIP1) rescued melanoma cells from apoptosis indicating a causative role of NOXA-upregulation in DHA-induced melanoma cell death. Comet analysis revealed early DHA-induction of genotoxic stress accompanied by p53 activational phosphorylation (Ser 15). In primary human epidermal melanocytes, viability was not compromised by DHA, and oxidative stress, comet tail moment, and PMAIP1 (NOXA) expression remained unaltered. Taken together, these data demonstrate that metastatic melanoma cells display a specific vulnerability to DHA-induced NOXA-dependent apoptosis and suggest feasibility of future anti-melanoma intervention using artemisinin-derived clinical redox antimalarials.

DCPIP (2,6-dichlorophenolindophenol) as a genotype-directed redox chemotherapeutic targeting NQO1*2 breast carcinoma.

Accumulative experimental evidence suggests feasibility of chemotherapeutic intervention targeting human cancer cells by pharmacological modulation of cellular oxidative stress. Current efforts aim at personalization of redox chemotherapy through identification of predictive tumour genotypes and redox biomarkers. Based on earlier research demonstrating that anti-melanoma activity of the pro-oxidant 2,6-dichlorophenolindophenol (DCPIP) is antagonized by cellular NAD(P)H:quinone oxidoreductase (NQO1) expression, this study tested DCPIP as a genotype-directed redox chemotherapeutic targeting homozygous NQO1*2 breast carcinoma, a common missense genotype [rs1800566 polymorphism; NP_000894.1:p.Pro187Ser] encoding a functionally impaired NQO1 protein. In a panel of cultured breast carcinoma cell lines and NQO1-transfectants with differential NQO1 expression levels, homozygous NQO1*2 MDA-MB231 cells were hypersensitive to DCPIP-induced caspase-independent cell death that occurred after early onset of oxidative stress with glutathione depletion and loss of genomic integrity. Array analysis revealed upregulated expression of oxidative (GSTM3, HMOX1, EGR1), heat shock (HSPA6, HSPA1A, CRYAB) and genotoxic stress response (GADD45A, CDKN1A) genes confirmed by immunoblot detection of HO-1, Hsp70, Hsp70B', p21 and phospho-p53 (Ser15). In a murine xenograft model of human homozygous NQO1*2-breast carcinoma, systemic administration of DCPIP displayed significant anti-tumour activity, suggesting feasibility of redox chemotherapeutic intervention targeting the NQO1*2 genotype.

GLO1 overexpression in human malignant melanoma.

Glyoxalase I [lactoylglutathione lyase (EC 4.4.1.5) encoded by GLO1] is a ubiquitous cellular defense enzyme involved in the detoxification of methylglyoxal, a cytotoxic byproduct of glycolysis. Accumulative evidence suggests an important role of GLO1 expression in protection against methylglyoxal-dependent protein adduction and cellular damage associated with diabetes, cancer, and chronological aging. On the basis of the hypothesis that GLO1 upregulation may play a functional role in glycolytic adaptations of cancer cells, we examined GLO1 expression status in human melanoma tissue. Quantitative reverse transcription polymerase chain reaction analysis of a cDNA tissue array containing 40 human melanoma tissues (stages III and IV) and 13 healthy controls revealed pronounced upregulation of GLO1 expression at the mRNA level. Immunohistochemical analysis of a melanoma tissue microarray confirmed upregulation of glyoxalase I protein levels in malignant melanoma tissue versus healthy human skin. Consistent with an essential role of GLO1 in melanoma cell defense against methylglyoxal cytotoxicity, siRNA interference targeting GLO1-expression (siGLO1) sensitized A375 and G361 human metastatic melanoma cells towards the antiproliferative, apoptogenic, and oxidative stress-inducing activity of exogenous methylglyoxal. Protein adduction by methylglyoxal was increased in siGLO1-transfected cells as revealed by immunodetection using a monoclonal antibody directed against the major methylglyoxal-derived epitope argpyrimidine that detected a single band of methylglyoxal-adducted protein in human LOX, G361, and A375 total cell lysates. Using two-dimensional proteomics followed by mass spectrometry the methylglyoxal-adducted protein was identified as heat shock protein 27 (Hsp27; HSPB1). Taken together, our data suggest a function of GLO1 in the regulation of detoxification and target adduction by the glycolytic byproduct methylglyoxal in malignant melanoma.

Antimelanoma activity of the redox dye DCPIP (2,6-dichlorophenolindophenol) is antagonized by NQO1.

Altered redox homeostasis involved in the control of cancer cell survival and proliferative signaling represents a chemical vulnerability that can be targeted by prooxidant redox intervention. Here, we demonstrate that the redox dye 2,6-dichlorophenolindophenol (DCPIP) may serve as a prooxidant chemotherapeutic targeting human melanoma cells in vitro and in vivo. DCPIP-apoptogenicity observed in the human melanoma cell lines A375 and G361 was inversely correlated with NAD(P)H:quinone oxidoreductase (NQO1) expression levels. In A375 cells displaying low NQO1 activity, DCPIP induced apoptosis with procaspase-3 and PARP cleavage, whereas G361 cells expressing high levels of enzymatically active NQO1 were resistant to DCPIP-cytotoxicity. Genetic (siRNA) or pharmacological (dicoumarol) antagonism of NQO1 strongly sensitized G361 cells to DCPIP apoptogenic activity. DCPIP-cytotoxicity was associated with the induction of oxidative stress and rapid depletion of glutathione in A375 and NQO1-modulated G361 cells. Expression array analysis revealed a DCPIP-induced stress response in A375 cells with massive upregulation of genes encoding Hsp70B' (HSPA6), Hsp70 (HSPA1A), heme oxygenase-1 (HMOX1), and early growth response protein 1 (EGR1) further confirmed by immunodetection. Systemic administration of DCPIP displayed significant antimelanoma activity in the A375 murine xenograft model. These findings suggest feasibility of targeting tumors that display low NQO1 enzymatic activity using DCPIP.

The experimental chemotherapeutic N6-furfuryladenosine (kinetin-riboside) induces rapid ATP depletion, genotoxic stress, and CDKN1A(p21) upregulation in human cancer cell lines.

Cytokinins and cytokinin nucleosides are purine derivatives with potential anticancer activity. N(6)-furfuryladenosine (FAdo, kinetin-riboside) displays anti-proliferative and apoptogenic activity against various human cancer cell lines, and FAdo has recently been shown to suppress tumor growth in murine xenograft models of human leukemia and melanoma. In this study, FAdo-induced genotoxicity, stress response gene expression, and cellular ATP depletion were examined as early molecular consequences of FAdo exposure in MiaPaCa-2 pancreas carcinoma, A375 melanoma, and other human cancer cell lines. FAdo, but not adenosine or N(6)-furfuryladenine (FA), displayed potent anti-proliferative activity that was also observed in human primary fibroblasts and keratinocytes. Remarkably, massive ATP depletion and induction of genotoxic stress as assessed by the alkaline comet assay occurred within 60-180min of exposure to low micromolar concentrations of FAdo. This was followed by rapid upregulation of CDKN1A and other DNA damage/stress response genes (HMOX1, DDIT3, and GADD45A) as revealed by expression array and Western analysis. Pharmacological and siRNA-based genetic inhibition of adenosine kinase (ADK) suppressed FAdo cytotoxicity and also prevented ATP depletion and p21 upregulation suggesting the importance of bioconversion of FAdo into the nucleotide form required for drug action. Taken together our data suggest that early induction of genotoxicity and energy crisis are important causative factors involved in FAdo cytotoxicity.

The cinnamon-derived Michael acceptor cinnamic aldehyde impairs melanoma cell proliferation, invasiveness, and tumor growth.

Redox dysregulation in cancer cells represents a chemical vulnerability that can be targeted by pro-oxidant redox intervention. Dietary constituents that contain an electrophilic Michael acceptor pharmacophore may therefore display promising chemopreventive and chemotherapeutic anti-cancer activity. Here, we demonstrate that the cinnamon-derived dietary Michael acceptor trans-cinnamic aldehyde (CA) impairs melanoma cell proliferation and tumor growth. Feasibility of therapeutic intervention using high doses of CA (120 mg/kg, po, daily, 10 days) was demonstrated in a human A375 melanoma SCID mouse xenograft model. Low-micromolar concentrations (IC(50)< 10 microM) of CA, but not closely related CA derivatives devoid of Michael acceptor activity, suppressed proliferation of human metastatic melanoma cell lines (A375, G361, LOX) with G1 cell-cycle arrest, elevated intracellular ROS, and impaired invasiveness. Expression array analysis revealed that CA induced an oxidative stress response in A375 cells, up-regulating heme oxygenase 1, sulfiredoxin 1 homolog, thioredoxin reductase 1, and other genes, including the cell-cycle regulator and stress-responsive tumor suppressor gene cyclin-dependent kinase inhibitor 1A, a key mediator of G1-phase arrest. CA, but not Michael-inactive derivatives, inhibited NF-kappaB transcriptional activity and TNFalpha-induced IL-8 production in A375 cells. These findings support a previously unrecognized role of CA as a dietary Michael acceptor with potential anti-cancer activity.

Experimental therapeutics: targeting the redox Achilles heel of cancer.

Reactive oxygen species (ROS) have recently emerged as promising targets for anticancer drug discovery. Constitutively elevated levels of cellular oxidative stress and dependence on mitogenic and anti-apoptotic ROS signaling represent a specific vulnerability of malignant cells that can be selectively targeted by novel pro- and antioxidant redox chemotherapeutics. This review discusses small-molecule anticancer redox drugs currently in various phases of preclinical and clinical development that are characterized by their unique mechanism of action, including small-molecule superoxide dismutase and catalase mimetics, bioreductively activated pro-oxidant redox catalysts, metal-based pro-oxidants, hypoxia-selective free radical precursors, and specific antagonists of the cancer cell antioxidant glutathione or thioredoxin redox systems. Based on ongoing redox biomarker discovery and validation, future redox phenotyping and genotyping may guide the selection of novel redox chemotherapeutics that efficiently target the redox Achilles heel of the individual tumor.

Increased skin carcinogenesis in a keratinocyte directed thioredoxin-1 transgenic mouse.

Thioredoxin-1 is a low molecular weight redox protein that protects cells against oxidant damage. Thioredoxin-1 levels are increased in the epidermal layer of sun-damaged human skin. Thioredoxin-1 levels are also increased in several human primary tumors where its expression is associated with increased tumor cell proliferation, decreased apoptosis and aggressive tumor growth. We have investigated whether increased thioredoxin-1 levels in skin can lead to increased tumor formation using transgenic mice with mouse thioredoxin-1 expressed in keratinocytes under the control of the keratinocyte-14 (K14) promoter. Thioredoxin-1 protein expression was increased 2-fold in the keratinocyte layer of the transgenic mice. The skin was macroscopically and histologically normal but in the two-stage model of carcinogenesis using topical dimethylbenzanthracene (DMBA) as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoting agent, there was a 6-fold increase in the number of papillomas per mouse and a 3-fold increase in papilloma size in the K14 thioredoxin-1 transgenic mice compared with non-transgenic littermates. Thus, increased thioredoxin-1 in keratinocytes acts as an enhancer of carcinogenesis in the DMBA/TPA two-stage model of skin carcinogenesis in mice.

Inhibitory effects of sodium salicylate and acetylsalicylic acid on UVB-induced mouse skin carcinogenesis.

We conducted an in vivo carcinogenesis experiment to determine the efficacy of topical aspirin and sodium salicylate (NAS) in preventing UVB-induced nonmelanoma skin cancer. Hairless SKH-1 mice were randomly divided into eight treatment groups. They were treated topically with either 40 or 10 micromol aspirin or NAS three times weekly before 9 kJ/m(2) UVB irradiation. The experiment was carried out over 25 weeks. Both dose levels of NAS significantly inhibited (P < 0.05) the rate of tumor formation when compared with vehicle control. The 40 micromol dose of aspirin significantly inhibited the rate of tumor formation (P < 0.05), whereas the 10 micromol dose had no inhibitory effect when compared with the vehicle control. To investigate the mechanism of this inhibition, we studied UVB-induced thymine dimer formation in the epidermis of the mouse skin. We found that NAS inhibited UVB-induced thymine dimer formation (P = 0.0001), whereas aspirin did not. Therefore, we conclude that NAS prevents UVB-induced tumor growth and formation through a sunscreen effect; whereas, the moderate inhibition of aspirin may be because of a molecular event, such as the inhibition of various UVB signaling pathways.