Warner B. Bair

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.

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.

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

SummaryRecent 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.

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.

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

Abstract 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.