Factors influencing timely diagnosis in neurolymphomatosis.

BACKGROUND: Neurolymphomatosis refers to infiltration of the peripheral nervous system (PNS) by non-Hodgkin lymphoma (NHL). Diagnostic intervals in neurolymphomatosis and factors delaying diagnosis have not been evaluated. We therefore aimed to analyze diagnostic intervals in a large cohort. METHODS: The quality control database at Yale Cancer Center, Section of Neuro-Oncology, was searched for neurolymphomatosis cases diagnosed between 2001 and 2021. Univariate analyses were performed to identify parameters influencing diagnostic intervals. RESULTS: We identified 22 neurolymphomatosis cases including 7 with primary and 15 with secondary disease, which occurred a median (range: 4-144) of 16 months after initial NHL diagnosis. Patients typically presented with painful polyneuropathy (73%), that was asymmetrical and rapidly progressive. Diagnosis was based on PNS biopsy (50%) or integration of neuroimaging findings (50%) with NHL history and diagnostic cerebrospinal fluid examinations. Median interval from symptom onset to diagnosis was 3 months (range: 1-12). Secondary neurolymphomatosis compared to primary disease (median 2 vs. 6 months, p = 0.02), and cases with rapidly-progressive asymmetrical neuropathy as opposed to other presentations (median 2 vs. 6 months; p < 0.001) were diagnosed earlier. Upfront conventional CT compared to other modalities (median 2 vs. 5 months p = 0.04) and nerve root localization as opposed to other disease sites (median 1.5 vs. 4 months; p = 0.04) delayed diagnosis. CONCLUSIONS: NL type and localization, neuropathy course and distribution, and imaging modality selected for initial evaluation influence diagnostic intervals in neurolymphomatosis. Knowledge of this rare entity is critical for early suspicion, and diagnosis.

Systemic deuteration of SCID mice using the water-isotopologue deuterium oxide (D2 O) inhibits tumor growth in an orthotopic bioluminescent model of human pancreatic ductal adenocarcinoma.

Since its initial discovery as a natural isotopologue of dihydrogen oxide (1 H2 O), extensive research has focused on the biophysical, biochemical, and pharmacological effects of deuterated water (2 H2 O [D2 O, also referred to as "heavy water"]). Using a panel of cultured human pancreatic ductal adenocarcinoma (PDAC) cells we have profiled (i) D2 O-induced phenotypic antiproliferative and apoptogenic effects, (ii) redox- and proteotoxicity-directed stress response gene expression, and (iii) phosphoprotein-signaling related to endoplasmic reticulum (ER) and MAP-kinase stress response pathways. Differential array analysis revealed early modulation of stress response gene expression in both BxPC-3 and PANC-1 PDAC cells elicited by D2 O (90%; ≤6 h; upregulated: HMOX1, NOS2, CYP2E1, CRYAB, DDIT3, NFKBIA, PTGS1, SOD2, PTGS2; downregulated: RUNX1, MYC, HSPA8, HSPA1A) confirmed by independent RT-qPCR analysis. Immunoblot-analysis revealed rapid (≤6 h) onset of D2 O-induced MAP-kinase signaling (p-JNK, p-p38) together with ER stress response upregulation (p-eIF2α, ATF4, XBP1s, DDIT3/CHOP). Next, we tested the chemotherapeutic efficacy of D2 O-based drinking water supplementation in an orthotopic PDAC model employing firefly luciferase-expressing BxPC-3-FLuc cells in SCID mice. First, feasibility and time course of systemic deuteration (30% D2 O in drinking water; 21 days) were established using time-resolved whole-body proton magnetic resonance imaging and isotope-ratio mass spectrometry-based plasma (D/H)-analysis. D2 O-supplementation suppressed tumor growth by almost 80% with downregulated expression of PCNA, MYC, RUNX1, and HSP70 while increasing tumor levels of DDIT3/CHOP, HO-1, and p-eIF2α. Taken together, these data demonstrate for the first time that pharmacological induction of systemic deuteration significantly reduces orthotopic tumor burden in a murine PDAC xenograft model.

Mefloquine induces ER stress and apoptosis in BRAFi-resistant A375-BRAFV600E /NRASQ61K malignant melanoma cells targeting intracranial tumors in a bioluminescent murine model.

Molecularly targeted therapeutics have revolutionized the treatment of BRAFV600E -driven malignant melanoma, but the rapid development of resistance to BRAF kinase inhibitors (BRAFi) presents a significant obstacle. The use of clinical antimalarials for the investigational treatment of malignant melanoma has shown only moderate promise, attributed mostly to inhibition of lysosomal-autophagic adaptations of cancer cells, but identification of specific antimalarials displaying single-agent antimelanoma activity has remained elusive. Here, we have screened a focused library of clinically used artemisinin-combination therapeutic (ACT) antimalarials for the apoptotic elimination of cultured malignant melanoma cell lines, also examining feasibility of overcoming BRAFi-resistance comparing isogenic melanoma cells that differ only by NRAS mutational status (BRAFi-sensitive A375-BRAFV600E /NRASQ61 vs. BRAFi-resistant A375-BRAFV600E /NRASQ61K ). Among ACT antimalarials tested, mefloquine (MQ) was the only apoptogenic agent causing melanoma cell death at low micromolar concentrations. Comparative gene expression-array analysis (A375-BRAFV600E /NRASQ61 vs. A375-BRAFV600E /NRASQ61K ) revealed that MQ is a dual inducer of endoplasmic reticulum (ER) and redox stress responses that precede MQ-induced loss of viability. ER-trackerTM DPX fluorescence imaging and electron microscopy indicated ER swelling, accompanied by rapid induction of ER stress signaling (phospho-eIF2α, XBP-1s, ATF4). Fluo-4 AM-fluorescence indicated the occurrence of cytosolic calcium overload observable within seconds of MQ exposure. In a bioluminescent murine model employing intracranial injection of A375-Luc2 (BRAFV600E /NRASQ61K ) cells, an oral MQ regimen efficiently antagonized brain tumor growth. Taken together, these data suggest that the clinical antimalarial MQ may be a valid candidate for drug repurposing aiming at chemotherapeutic elimination of malignant melanoma cells, even if metastasized to the brain and BRAFi-resistant.

TLR4 in skin cancer: From molecular mechanisms to clinical interventions.

The health and economic burden imposed by skin cancer is substantial, creating an urgent need for the development of improved molecular strategies for its prevention and treatment. Cutaneous exposure to solar ultraviolet (UV) radiation is a causative factor in skin carcinogenesis, and TLR4-dependent inflammatory dysregulation is an emerging key mechanism underlying detrimental effects of acute and chronic UV exposure. Direct and indirect TLR4 activation, upstream of inflammatory signaling, is elicited by a variety of stimuli, including pathogen-associated molecular patterns (such as lipopolysaccharide) and damage-associated molecular patterns (such as HMGB1) that are formed upon exposure to environmental stressors, such as solar UV. TLR4 involvement has now been implicated in major types of skin malignancies, including nonmelanoma skin cancer, melanoma and Merkel cell carcinoma. Targeted molecular interventions that positively or negatively modulate TLR4 signaling have shown promise in translational, preclinical, and clinical investigations that may benefit skin cancer patients in the near future.

Haloperoxidases as catalysts in organic synthesis.

Haloperoxidases are very active catalysts for the in situ generation of electrophilic halide species for oxidative halogenation reactions. In the synthetic community, these catalysts, however, are not widely used. The aim of this mini-review is to critically summarise the current state-of-the-art of haloperoxidase catalysis for organic synthesis. We hope that the excellent catalytic performance of these catalysts will trigger more chemists to consider them in their synthesis planning.

A Photoenzymatic NADH Regeneration System.

A photoenzymatic NADH regeneration system was established. The combination of deazariboflavin as a photocatalyst with putidaredoxin reductase enabled the selective reduction of NAD+ into the enzyme-active 1,4-NADH to promote an alcohol dehydrogenase catalysed stereospecific reduction reaction. The catalytic turnover of all the reaction components was demonstrated. Factors influencing the efficiency of the overall system were identified.

Sun sensitivity and sun protective behaviors during sun exposure among indoor office workers in the American Midwest.

BACKGROUND/PURPOSE: Sun sensitivity, a confounder between sun exposure and sun protection, is often overlooked. We examined how sun exposure and sun protection behaviors among indoor workers varied by sun sensitivity. METHODS: Sun exposure and sun protection diaries over a 45-day period from Midwestern United States indoor workers were examined. We categorized sun sensitivity (fair and non-fair complexion) using tanning inability and sunburn tendency. Total exposure (sunrise to sunset) and peak exposure (10 am and 4 pm) lasting at least 60 minutes were examined. Percentages of time using sun protection were reported. We determined associations between fair complexion, mean sun exposure, and mean sun protection times with logistic regression. RESULTS: Fair individuals spent less time in the sun than non-fair individuals, but a greater proportion of time using sun protection behaviors, including sunscreen with SPF 30+ (odds ratio (OR) = 1.36; 95% confidence interval (95% CI) = 0.98, 1.87)), or wearing long-sleeved shirts (OR = 2.89; 95% CI = 1.24, 6.73). CONCLUSION: Fair individuals spent less time in the sun and practiced more sun protective behaviors than non-fair individuals. This complex association between sun sensitivity, sun protection and sun exposure has not consistently been addressed in studies of skin cancer.

The quinone methide aurin is a heat shock response inducer that causes proteotoxic stress and Noxa-dependent apoptosis in malignant melanoma cells.

Pharmacological induction of proteotoxic stress is rapidly emerging as a promising strategy for cancer cell-directed chemotherapeutic intervention. Here, we describe the identification of a novel drug-like heat shock response inducer for the therapeutic induction of proteotoxic stress targeting malignant human melanoma cells. Screening a focused library of compounds containing redox-directed electrophilic pharmacophores employing the Stress & Toxicity PathwayFinder(TM) PCR Array technology as a discovery tool, a drug-like triphenylmethane-derivative (aurin; 4-[bis(p-hydroxyphenyl)methylene]-2,5-cyclohexadien-1-one) was identified as an experimental cell stress modulator that causes (i) heat shock factor transcriptional activation, (ii) up-regulation of heat shock response gene expression (HSPA6, HSPA1A, DNAJB4, HMOX1), (iii) early unfolded protein response signaling (phospho-PERK, phospho-eIF2α, CHOP (CCAAT/enhancer-binding protein homologous protein)), (iv) proteasome impairment with increased protein-ubiquitination, and (v) oxidative stress with glutathione depletion. Fluorescence polarization-based experiments revealed that aurin displays activity as a geldanamycin-competitive Hsp90α-antagonist, a finding further substantiated by molecular docking and ATPase inhibition analysis. Aurin exposure caused caspase-dependent cell death in a panel of human malignant melanoma cells (A375, G361, LOX-IMVI) but not in non-malignant human skin cells (Hs27 fibroblasts, HaCaT keratinocytes, primary melanocytes) undergoing the aurin-induced heat shock response without impairment of viability. Aurin-induced melanoma cell apoptosis depends on Noxa up-regulation as confirmed by siRNA rescue experiments demonstrating that siPMAIP1-based target down-regulation suppresses aurin-induced cell death. Taken together, our data suggest feasibility of apoptotic elimination of malignant melanoma cells using the quinone methide-derived heat shock response inducer aurin.

Nrf2 modulates contractile and metabolic properties of skeletal muscle in streptozotocin-induced diabetic atrophy.

The role of Nrf2 in disease prevention and treatment is well documented; however the specific role of Nrf2 in skeletal muscle is not well described. The current study investigated whether Nrf2 plays a protective role in an STZ-induced model of skeletal muscle atrophy. Modulation of Nrf2 through siRNA resulted in a more robust differentiation of C2C12s, whereas increasing Nrf2 with sulforaphane treatment inhibited differentiation. Diabetic muscle atrophy was not dramatically influenced by Nrf2 genotype, since no differences were observed in total atrophy (all fiber types combined) between WT+STZ and KO+STZ animals. Nrf2-KO animals however illustrated alterations in muscle size of Fast, Type II myosin expressing fibers. KO+STZ animals show significant alterations in myosin isoform expression in the GAST. Similarly, KO controls mimic both WT+STZ and KO+STZ muscle alterations in mitochondrial subunit expression. PGC-1α, a well-established player in mitochondrial biogenesis and myosin isoform expression, was decreased in KO control, WT+STZ and KO+STZ SOL muscle. Similarly, PGC-1α protein levels are correlated with Nrf2 levels in C2C12s after modulation by Nrf2 siRNA or sulforaphane treatment. We provide experimental evidence indicating Nrf2 plays a role in myocyte differentiation and governs molecular alterations in contractile and metabolic properties in an STZ-induced model of muscle atrophy.

Plant extracts of the family Lauraceae: a potential resource for chemopreventive agents that activate the nuclear factor-erythroid 2-related factor 2/antioxidant response element pathway.

Cells and tissues counteract insults from exogenous or endogenous carcinogens through the expression of genes encoding antioxidants and phase II detoxifying enzymes regulated by antioxidant response element promoter regions. Nuclear factor-erythroid 2-related factor 2 plays a key role in regulating the antioxidant response elements-target gene expression. Hence, the Nrf2/ARE pathway represents a vital cellular defense mechanism against damage caused by oxidative stress and xenobiotics, and is recognized as a potential molecular target for discovering chemopreventive agents. Using a stable antioxidant response element luciferase reporter cell line derived from human breast cancer MDA-MB-231 cells combined with a 96-well high-throughput screening system, we have identified a series of plant extracts from the family Lauraceae that harbor Nrf2-inducing effects. These extracts, including Litsea garrettii (ZK-08), Cinnamomum chartophyllum (ZK-02), C. mollifolium (ZK-04), C. camphora var. linaloolifera (ZK-05), and C. burmannii (ZK-10), promoted nuclear translocation of Nrf2, enhanced protein expression of Nrf2 and its target genes, and augmented intracellular glutathione levels. Cytoprotective activity of these extracts against two electrophilic toxicants, sodium arsenite and H2O2, was investigated. Treatment of human bronchial epithelial cells with extracts of ZK-02, ZK-05, and ZK-10 significantly improved cell survival in response to sodium arsenite and H2O2, while ZK-08 showed a protective effect against only H2O2. Importantly, their protective effects against insults from both sodium arsenite and H2O2 were Nrf2-dependent. Therefore, our data provide evidence that the selected plants from the family Lauraceae are potential sources for chemopreventive agents targeting the Nrf2/ARE pathway.

Exposure to chlorinated drinking water alters the murine fecal microbiota.

An abundant body of scientific studies and regulatory guidelines substantiates antimicrobial efficacy of freshwater chlorination ensuring drinking water safety in large populations worldwide. In contrast to the purposeful use of chlorination ensuring antimicrobial safety of drinking water, only a limited body of research has addressed the molecular impact of chlorinated drinking water exposure on the gut microbiota. Here, for the first time, we have examined the differential effects of drinking water regimens stratified by chlorination agent [inorganic (HOCl) versus chloramine (TCIC)] on the C57BL/6J murine fecal microbiota. To this end, we exposed C57BL/6J mice to chlorinated drinking water regimens followed by fecal bacterial microbiota analysis at the end of the three-week feeding period employing 16S rRNA sequencing. α-diversity was strongly reduced when comparing chlorinated versus control drinking water groups and community dissimilarities (ß-diversity) were significant between groups even when comparing HOCl and TCIC. We detected significant differences in fecal bacterial composition as a function of drinking water chlorination observable at the phylum and genus levels. Differential abundance analysis of select amplicon sequence variants (ASVs) revealed changes as a function of chlorination exposure [up: Lactobacillus ASV1; Akkermansia muciniphila ASV7; Clostridium ss1 ASV10; down: Ileibacterium valens ASV5; Desulfovibrio ASV11; Lachnospiraceae UCG-006 ASV15]. Given the established complexity of murine and human gastrointestinal microbiota and their role in health and disease, the translational relevance of the chlorination-induced changes documented by us for the first time in the fecal murine microbiota remains to be explored.

Inhibition of UV-Induced Stress Signaling and Inflammatory Responses in SKH-1 Mouse Skin by Topical Small-Molecule PD-L1 Blockade.

The immune checkpoint ligand PD-L1 has emerged as a molecular target for skin cancer therapy and might also hold promise for preventive intervention targeting solar UV light-induced skin damage. In this study, we have explored the role of PD-L1 in acute keratinocytic photodamage testing the effects of small-molecule pharmacological inhibition. Epidermal PD-L1 upregulation in response to chronic photodamage was established using immunohistochemical and proteomic analyses of a human skin cohort, consistent with earlier observations that PD-L1 is upregulated in cutaneous squamous cell carcinoma. Topical application of the small-molecule PD-L1 inhibitor BMS-202 significantly attenuated UV-induced activator protein-1 transcriptional activity in SKH-1 bioluminescent reporter mouse skin, also confirmed in human HaCaT reporter keratinocytes. RT-qPCR analysis revealed that BMS-202 antagonized UV induction of inflammatory gene expression. Likewise, UV-induced cleavage of procaspase-3, a hallmark of acute skin photodamage, was attenuated by topical BMS-202. NanoString nCounter transcriptomic analysis confirmed downregulation of cutaneous innate immunity- and inflammation-related responses, together with upregulation of immune response pathway gene expression. Further mechanistic analysis confirmed that BMS-202 antagonizes UV-induced PD-L1 expression both at the mRNA and protein levels in SKH-1 epidermis. These data suggest that topical pharmacological PD-L1 antagonism using BMS-202 shows promise for skin protection against photodamage.

Vitamin B6: beyond coenzyme functions.

Endogenous reactive intermediates such as photoexcited states of tissue chromophores, reactive oxygen species (ROS), reactive carbonyl species (RCS), and transition metal ions are mediators of tissue damage involved in initiation and progression of human pathologies including tumorigenesis, atherosclerosis, diabetes, and neurodegenerative disease. A large body of evidence now suggests that B6 vitamers antagonize the harmful activity of endogenous reactive intermediates fulfilling a very different role than that established as a cofactor for numerous enzymes. In this chapter, the structural basis of vitamin B6 activity as a potent antioxidant, metal chelator, carbonyl scavenger, and photosensitizer is presented and the physiological relevance is discussed.

Phenotypic identification of the redox dye methylene blue as an antagonist of heat shock response gene expression in metastatic melanoma cells.

Repurposing approved and abandoned non-oncological drugs is an alternative developmental strategy for the identification of anticancer therapeutics that has recently attracted considerable attention. Due to the essential role of the cellular heat shock response in cytoprotection through the maintenance of proteostasis and suppression of apoptosis, small molecule heat shock response antagonists can be harnessed for targeted induction of cytotoxic effects in cancer cells. Guided by gene expression array analysis and a phenotypic screen interrogating a collection of 3,7-diamino-phenothiazinium derivatives, we have identified the redox-drug methylene blue (MB), used clinically for the infusional treatment of methemoglobinemia, as a negative modulator of heat shock response gene expression in human metastatic melanoma cells. MB-treatment blocked thermal (43 °C) and pharmacological (celastrol, geldanamycin) induction of heat shock response gene expression, suppressing Hsp70 (HSPA1A) and Hsp27 (HSPB1) upregulation at the mRNA and protein level. MB sensitized melanoma cells to the apoptogenic activity of geldanamycin, an Hsp90 antagonist known to induce the counter-regulatory upregulation of Hsp70 expression underlying cancer cell resistance to geldanamycin chemotherapy. Similarly, MB-cotreatment sensitized melanoma cells to other chemotherapeutics (etoposide, doxorubicin). Taken together, these data suggest feasibility of repurposing the non-oncological redox drug MB as a therapeutic heat shock response antagonist for cancer cell chemosensitization.

The Glycolysis-derived α-Dicarbonyl Metabolite Methylglyoxal is a UVA-photosensitizer Causing the Photooxidative Elimination of HaCaT Keratinocytes with Induction of Oxidative and Proteotoxic Stress Response Gene Expression†.

Cellular oxidative stress contributes to solar ultraviolet (UV) radiation-induced skin photoaging and photocarcinogenesis. Light-driven electron and energy transfer reactions involving non-DNA chromophores are a major source of reactive oxygen species (ROS) in skin, and the molecular identity of numerous endogenous chromophores acting as UV-photosensitizers has been explored. Methylglyoxal (MG), a glycolytic byproduct bearing a UV-active α-dicarbonyl-chromophore, is generated under metabolic conditions of increased glycolytic flux, associated with posttranslational protein adduction in human tissue. Here, we undertook a photophysical and photochemical characterization of MG substantiating its fluorescence properties (Stokes shift), phosphorescence lifetime, and quantum yield of singlet oxygen (1 O2 ) formation. Strikingly, upon UV-excitation (290 nm), a clear emission (around 490 nm) was observed (phosphorescence-lifetime: 224.2 milliseconds). At micromolar concentrations, MG acts as a UVA-photosensitizer targeting human HaCaT-keratinocytes inducing photooxidative stress and caspase-dependent cell death substantiated by zVADfmk-rescue and Alexa-488 caspase-3 flow cytometry. Transcriptomic analysis indicated that MG (photoexcited by noncytotoxic doses of UVA) elicits expression changes not observable upon isolated MG- or UVA-treatment, with upregulation of the proteotoxic (CRYAB, HSPA6) and oxidative (HMOX1) stress response. Given the metabolic origin of MG and its role in human pathology, future investigations should address the potential involvement of MG-photosensitizer activity in human skin photodamage.

The Drinking Water and Swimming Pool Disinfectant Trichloroisocyanuric Acid Causes Chlorination Stress Enhancing Solar UV-Induced Inflammatory Gene Expression in AP-1 Transgenic SKH-1 Luciferase Reporter Mouse Skin.

Freshwater sanitation and disinfection using a variety of chemical entities as chlorination agents is an essential public health intervention ensuring water safety for populations at a global scale. Recently, we have published our observation that the small molecule oxidant, innate immune factor and chlorination agent HOCl antagonize inflammation and photocarcinogenesis in murine skin exposed topically to environmentally relevant concentrations of HOCl. Chlorinated isocyanuric acid derivatives (including the chloramines trichloroisocyanuric acid [TCIC] and dichloroisocyanuric acid [DCIC]) are used worldwide as alternate chlorination agents serving as HOCl precursor and stabilizer compounds ensuring sustained release in aqueous environments including public water systems, recreational pools and residential hot tubs. Here, for the first time, we have examined the cutaneous TCIC-induced transcriptional stress response (in both an organotypic epidermal model and in AP-1 luciferase reporter SKH-1 mouse skin), also examining molecular consequences of subsequent treatment with solar ultraviolet (UV) radiation. Taken together, our findings indicate that cutaneous delivery of TCIC significantly enhances UV-induced inflammation (as profiled at the gene expression level), suggesting a heretofore unrecognized potential to exacerbate UV-induced functional and structural cutaneous changes. These observations deserve further molecular investigations in the context of TCIC-based freshwater disinfection with health implications for populations worldwide.

D-Penicillamine targets metastatic melanoma cells with induction of the unfolded protein response (UPR) and Noxa (PMAIP1)-dependent mitochondrial apoptosis.

D-Penicillamine (3,3-dimethyl-D-cysteine; DP) is an FDA-approved redox-active D-cysteine-derivative with antioxidant, disulfide-reducing, and metal chelating properties used therapeutically for the control of copper-related pathology in Wilson's disease and reductive cystine-solubilization in cystinuria. Based on the established sensitivity of metastatic melanoma cells to pharmacological modulation of cellular oxidative stress, we tested feasibility of using DP for chemotherapeutic intervention targeting human A375 melanoma cells in vitro and in vivo. DP treatment induced caspase-dependent cell death in cultured human metastatic melanoma cells (A375, G361) without compromising viability of primary epidermal melanocytes, an effect not observed with the thiol-antioxidants N-acetyl-L-cysteine (NAC) and dithiothreitol. Focused gene expression array analysis followed by immunoblot detection revealed that DP rapidly activates the cytotoxic unfolded protein response (UPR; involving phospho-PERK, phospho-eIF2α, Grp78, CHOP, and Hsp70) and the mitochondrial pathway of apoptosis with p53 upregulation and modulation of Bcl-2 family members (involving Noxa, Mcl-1, and Bcl-2). DP (but not NAC) induced oxidative stress with early impairment of glutathione homeostasis and mitochondrial transmembrane potential. SiRNA-based antagonism of PMAIP1 expression blocked DP-induced upregulation of the proapoptotic BH3-only effector Noxa and prevented downregulation of the Noxa-antagonist Mcl-1, rescuing melanoma cells from DP-induced apoptosis. Intraperitoneal administration of DP displayed significant antimelanoma activity in a murine A375 xenograft model. It remains to be seen if melanoma cell-directed induction of UPR and apoptosis using DP or improved DP-derivatives can be harnessed for future chemotherapeutic intervention.

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.

Autophagic-lysosomal dysregulation downstream of cathepsin B inactivation in human skin fibroblasts exposed to UVA.

Recently, using 2D-DIGE proteomics we have identified cathepsin B as a novel target of UVA in human Hs27 skin fibroblasts. In response to chronic exposure to noncytotoxic doses of UVA (9.9 J cm(-2), twice a week, 3 weeks), photooxidative impairment of cathepsin B enzymatic activity occurred with accumulation of autofluorescent aggregates colocalizing with lysosomes, an effect mimicked by pharmacological antagonism of cathepsin B using the selective inhibitor CA074Me. Here, we have further explored the mechanistic involvement of cathepsin B inactivation in UVA-induced autophagic-lysosomal alterations using autophagy-directed PCR expression array analysis as a discovery tool. Consistent with lysosomal expansion, UVA upregulated cellular protein levels of the lysosomal marker glycoprotein Lamp-1, and increased levels of the lipidated autophagosomal membrane constituent LC3-II were detected. UVA did not alter expression of beclin 1 (BECN1), an essential factor for initiation of autophagy, but upregulation of p62 (sequestosome 1, SQSTM1), a selective autophagy substrate, and α-synuclein (SNCA), an autophagic protein substrate and aggresome component, was observed at the mRNA and protein level. Moreover, UVA downregulated transglutaminase-2 (TGM2), an essential enzyme involved in autophagolysosome maturation. Strikingly, UVA effects on Lamp-1, LC3-II, beclin 1, p62, α-synuclein, and transglutaminase-2 were mimicked by CA074Me treatment. Taken together, our data suggest that UVA-induced autophagic-lysosomal alterations occur as a consequence of impaired autophagic flux downstream of cathepsin B inactivation, a novel molecular mechanism potentially involved in UVA-induced skin photodamage.

Vemurafenib Drives Epithelial-to-Mesenchymal Transition Gene Expression in BRAF Inhibitor‒Resistant BRAFV600E/NRASQ61K Melanoma Enhancing Tumor Growth and Metastasis in a Bioluminescent Murine Model.

BRAF inhibitor (BRAFi) resistance compromises long-term survivorship of patients with malignant melanoma, and mutant NRAS is a major mediator of BRAFi resistance. In this study, employing phenotypic and transcriptomic analysis of isogenic melanoma cells that differ only by NRAS mutational status (BRAFi-sensitive A375-BRAFV600E/NRASQ61 vs. BRAFi-resistant A375-BRAFV600E/NRASQ61K), we show that BRAFi (vemurafenib) treatment selectively targets BRAFV600E/NRASQ61K cells upregulating epithelial-to-mesenchymal transition (EMT) gene expression, paradoxically promoting invasiveness and metastasis in vitro and in vivo. First, NanoString nCounter transcriptomic analysis identified the upregulation of specific gene expression networks (EMT and EMT to metastasis) as a function of NRASQ61K status. Strikingly, BRAFi treatment further exacerbated the upregulation of genes promoting EMT in BRAFV600E/NRASQ61K cells (with opposing downregulation of EMT-driver genes in the BRAFV600E/NRASQ61 genotype) as detected by EMT-focused RT2 Profiler qPCR array analysis. In BRAFV600E/NRASQ61K cells, BRAFi treatment enhanced proliferation and invasiveness, together with activation of phosphorylated protein kinase B (Ser473), with opposing phenotypic effects observable in BRAFV600E/NRASQ61 cells displaying downregulation of phosphorylated protein kinase B and phosphorylated extracellular signal-regulated kinase 1/2. In a SCID mouse bioluminescent melanoma metastasis model, BRAFi treatment enhanced lung tumor burden imposed by BRAFV600E/NRASQ61K cells while blocking BRAFV600E/NRASQ61 metastasis. These preclinical data document the BRAFi-driven enhancement of tumorigenesis and metastasis in BRAFi-resistant human BRAFV600E/NRASQ61K melanoma, a finding with potential clinical implications for patients with NRAS-driven BRAFi-resistant tumors receiving BRAFi treatment.