Hypermetabolic state is associated with circadian rhythm disruption in mouse and human cancer cells.

Crosstalk between metabolism and circadian rhythms is a fundamental building block of multicellular life, and disruption of this reciprocal communication could be relevant to disease. Here, we investigated whether maintenance of circadian rhythms depends on specific metabolic pathways, particularly in the context of cancer. We found that in adult mouse fibroblasts, ATP levels were a major contributor to signal from a clock gene luciferase reporter, although not necessarily to the strength of circadian cycling. In contrast, we identified significant metabolic control of circadian function across a series of pancreatic adenocarcinoma cell lines. Metabolic profiling of congenic tumor cell clones revealed substantial diversity among these lines that we used to identify clones to generate circadian reporter lines. We observed diverse circadian profiles among these lines that varied with their metabolic phenotype: The most hypometabolic line [exhibiting low levels of oxidative phosphorylation (OxPhos) and glycolysis] had the strongest rhythms, while the most hypermetabolic line had the weakest rhythms. Pharmacological enhancement of OxPhos decreased the amplitude of circadian oscillation in a subset of tumor cell lines. Strikingly, inhibition of OxPhos enhanced circadian rhythms only in the tumor cell line in which glycolysis was also low, thereby establishing a hypometabolic state. We further analyzed metabolic and circadian phenotypes across a panel of human patient-derived melanoma cell lines and observed a significant negative association between metabolic activity and circadian cycling strength. Together, these findings suggest that metabolic heterogeneity in cancer directly contributes to circadian function and that high levels of glycolysis or OxPhos independently disrupt circadian rhythms in these cells.

Senescent glia link mitochondrial dysfunction and lipid accumulation.

Senescence is a cellular state linked to ageing and age-onset disease across many mammalian species1,2. Acutely, senescent cells promote wound healing3,4 and prevent tumour formation5; but they are also pro-inflammatory, thus chronically exacerbate tissue decline. Whereas senescent cells are active targets for anti-ageing therapy6-11, why these cells form in vivo, how they affect tissue ageing and the effect of their elimination remain unclear12,13. Here we identify naturally occurring senescent glia in ageing Drosophila brains and decipher their origin and influence. Using Activator protein 1 (AP1) activity to screen for senescence14,15, we determine that senescent glia can appear in response to neuronal mitochondrial dysfunction. In turn, senescent glia promote lipid accumulation in non-senescent glia; similar effects are seen in senescent human fibroblasts in culture. Targeting AP1 activity in senescent glia mitigates senescence biomarkers, extends fly lifespan and health span, and prevents lipid accumulation. However, these benefits come at the cost of increased oxidative damage in the brain, and neuronal mitochondrial function remains poor. Altogether, our results map the trajectory of naturally occurring senescent glia in vivo and indicate that these cells link key ageing phenomena: mitochondrial dysfunction and lipid accumulation.

Hypermetabolic state is associated with circadian rhythm disruption in mouse and human cancer cells.

Crosstalk between cellular metabolism and circadian rhythms is a fundamental building block of multicellular life, and disruption of this reciprocal communication could be relevant to degenerative disease, including cancer. Here, we investigated whether maintenance of circadian rhythms depends upon specific metabolic pathways, particularly in the context of cancer. We found that in adult mouse fibroblasts, ATP levels were a major contributor to overall levels of a clock gene luciferase reporter, although not necessarily to the strength of circadian cycling. In contrast, we identified significant metabolic control of circadian function in an in vitro mouse model of pancreatic adenocarcinoma. Metabolic profiling of a library of congenic tumor cell clones revealed significant differences in levels of lactate, pyruvate, ATP, and other crucial metabolites that we used to identify candidate clones with which to generate circadian reporter lines. Despite the shared genetic background of the clones, we observed diverse circadian profiles among these lines that varied with their metabolic phenotype: the most hypometabolic line had the strongest circadian rhythms while the most hypermetabolic line had the weakest rhythms. Treatment of these tumor cell lines with bezafibrate, a peroxisome proliferator-activated receptor (PPAR) agonist shown to increase OxPhos, decreased the amplitude of circadian oscillation in a subset of tumor cell lines. Strikingly, treatment with the Complex I antagonist rotenone enhanced circadian rhythms only in the tumor cell line in which glycolysis was also low, thereby establishing a hypometabolic state. We further analyzed metabolic and circadian phenotypes across a panel of human patient-derived melanoma cell lines and observed a significant negative association between metabolic activity and circadian cycling strength. Together, these findings suggest that metabolic heterogeneity in cancer directly contributes to circadian function, and that high levels of glycolysis or OxPhos independently disrupt circadian rhythms in these cells.

Ligand-free mitochondria-localized mutant AR-induced cytotoxicity in spinal bulbar muscular atrophy.

Spinal bulbar muscular atrophy (SBMA), the first identified CAG-repeat expansion disorder, is an X-linked neuromuscular disorder involving CAG-repeat-expansion mutations in the androgen receptor (AR) gene. We utilized CRISPR-Cas9 gene editing to engineer novel isogenic human induced pluripotent stem cell (hiPSC) models, consisting of isogenic AR knockout, control and disease lines expressing mutant AR with distinct repeat lengths, as well as control and disease lines expressing FLAG-tagged wild-type and mutant AR, respectively. Adapting a small-molecule cocktail-directed approach, we differentiate the isogenic hiPSC models into motor neuron-like cells with a highly enriched population to uncover cell-type-specific mechanisms underlying SBMA and to distinguish gain- from loss-of-function properties of mutant AR in disease motor neurons. We demonstrate that ligand-free mutant AR causes drastic mitochondrial dysfunction in neurites of differentiated disease motor neurons due to gain-of-function mechanisms and such cytotoxicity can be amplified upon ligand (androgens) treatment. We further show that aberrant interaction between ligand-free, mitochondria-localized mutant AR and F-ATP synthase is associated with compromised mitochondrial respiration and multiple other mitochondrial impairments. These findings counter the established notion that androgens are requisite for mutant AR-induced cytotoxicity in SBMA, reveal a compelling mechanistic link between ligand-free mutant AR, F-ATP synthase and mitochondrial dysfunction, and provide innovative insights into motor neuron-specific therapeutic interventions for SBMA.

Time-of-day specificity of anticancer drugs may be mediated by circadian regulation of the cell cycle.

Circadian rhythms are an integral part of physiology, underscoring their relevance for the treatment of disease. We conducted cell-based high-throughput screening to investigate time-of-day influences on the activity of known antitumor agents and found that many compounds exhibit daily rhythms of cytotoxicity concomitant with previously reported oscillations of target genes. Rhythmic action of HSP90 inhibitors was mediated by specific isoforms of HSP90, genetic perturbation of which affected the cell cycle. Furthermore, clock mutants affected the cell cycle in parallel with abrogating rhythms of cytotoxicity, and pharmacological inhibition of the cell cycle also eliminated rhythmic drug effects. An HSP90 inhibitor reduced growth rate of a mouse melanoma in a time-of-day-specific manner, but efficacy was impaired in clock-deficient tumors. These results provide a powerful rationale for appropriate daily timing of anticancer drugs and suggest circadian regulation of the cell cycle within the tumor as an underlying mechanism.

RNU (Foxn1 RNU-Nude) Rats Demonstrate an Improved Ability to Regenerate Muscle in a Volumetric Muscle Injury Compared to Sprague Dawley Rats.

Products developed for skeletal muscle regeneration frequently incorporate allogeneic and xenogeneic materials to elicit a regenerative response to heal skeletal muscle wounds. To avoid graft rejection in preclinical studies, immunodeficient rodents are used. Whether the immunodeficiency alters the host response to the material in skeletal muscle has not been studied. In this study, we hypothesized that an allogeneic acellular skeletal muscle grafts implanted in an immunodeficient rat (RNU, Foxn1-deficient) would exhibit better new muscle fiber formation compared to grafts implanted in immunocompetent Sprague Dawley (SD) rats. Decellularized SD skeletal muscle matrix (DMM) was implanted in the gastrocnemius (N = 8 rats/group). 56 days after surgery, animal gait was examined and animals were euthanized. Muscle force was assessed and fiber number as well as immune cell infiltrate was measured by histomorphometry and immunohistochemistry. Animal gait and percent recovery of muscle force were unchanged in both groups, but newly regenerated muscle fibers increased in RNU rats. Macrophage staining for CD68 was higher in RNU rats than in SD rats. These data show differences in muscle regeneration between animal models using the same biomaterial treatment, but these differences could not be ascribed to the immune response. Overall, our data provide awareness that more studies are needed to understand how host responses to biomaterials differ based on the animal model used.

G1/S cell cycle regulators mediate effects of circadian dysregulation on tumor growth and provide targets for timed anticancer treatment.

Circadian disruption has multiple pathological consequences, but the underlying mechanisms are largely unknown. To address such mechanisms, we subjected transformed cultured cells to chronic circadian desynchrony (CCD), mimicking a chronic jet-lag scheme, and assayed a range of cellular functions. The results indicated a specific circadian clock-dependent increase in cell proliferation. Transcriptome analysis revealed up-regulation of G1/S phase transition genes (myelocytomatosis oncogene cellular homolog [Myc], cyclin D1/3, chromatin licensing and DNA replication factor 1 [Cdt1]), concomitant with increased phosphorylation of the retinoblastoma (RB) protein by cyclin-dependent kinase (CDK) 4/6 and increased G1-S progression. Phospho-RB (Ser807/811) was found to oscillate in a circadian fashion and exhibit phase-shifted rhythms in circadian desynchronized cells. Consistent with circadian regulation, a CDK4/6 inhibitor approved for cancer treatment reduced growth of cultured cells and mouse tumors in a time-of-day-specific manner. Our study identifies a mechanism that underlies effects of circadian disruption on tumor growth and underscores the use of treatment timed to endogenous circadian rhythms.

Targeting Biophysical Cues: a Niche Approach to Study, Diagnose, and Treat Cancer.

Probing the biophysical properties of the tumor niche offers a new perspective in cancer mechanobiology, and supports the development of next-generation diagnostics and therapeutics for cancer, in particular for metastasis.

Mechanoresponsive stem cells to target cancer metastases through biophysical cues.

Despite decades of effort, little progress has been made to improve the treatment of cancer metastases. To leverage the central role of the mechanoenvironment in cancer metastasis, we present a mechanoresponsive cell system (MRCS) to selectively identify and treat cancer metastases by targeting the specific biophysical cues in the tumor niche in vivo. Our MRCS uses mechanosensitive promoter-driven mesenchymal stem cell (MSC)-based vectors, which selectively home to and target cancer metastases in response to specific mechanical cues to deliver therapeutics to effectively kill cancer cells, as demonstrated in a metastatic breast cancer mouse model. Our data suggest a strong correlation between collagen cross-linking and increased tissue stiffness at the metastatic sites, where our MRCS is specifically activated by the specific cancer-associated mechano-cues. MRCS has markedly reduced deleterious effects compared to MSCs constitutively expressing therapeutics. MRCS indicates that biophysical cues, specifically matrix stiffness, are appealing targets for cancer treatment due to their long persistence in the body (measured in years), making them refractory to the development of resistance to treatment. Our MRCS can serve as a platform for future diagnostics and therapies targeting aberrant tissue stiffness in conditions such as cancer and fibrotic diseases, and it should help to elucidate mechanobiology and reveal what cells "feel" in the microenvironment in vivo.

Mesenchymal stem cells engineered to express selectin ligands and IL-10 exert enhanced therapeutic efficacy in murine experimental autoimmune encephalomyelitis.

Systemic administration of mesenchymal stem cells (MSCs) affords the potential to ameliorate the symptoms of Multiple Sclerosis (MS) in both preclinical and clinical studies. However, the efficacy of MSC-based therapy for MS likely depends on the number of cells that home to inflamed tissues and on the controlled production of paracrine and immunomodulatory factors. Previously, we reported that engineered MSCs expressing P-selectin glycoprotein ligand-1 (PSGL-1) and Sialyl-Lewis(x) (SLeX) via mRNA transfection facilitated the targeted delivery of anti-inflammatory cytokine interleukin-10 (IL-10) to inflamed ear. Here, we evaluated whether targeted delivery of MSCs with triple PSGL1/SLeX/IL-10 engineering improves therapeutic outcomes in mouse experimental autoimmune encephalomyelitis (EAE), a murine model for human MS. We found PSGL-1/SLeX mRNA transfection significantly enhanced MSC homing to the inflamed spinal cord. This is consistent with results from in vitro flow chamber assays in which PSGL-1/SleX mRNA transfection significantly increased the percentage of rolling and adherent cells on activated brain microvascular endothelial cells, which mimic the inflamed endothelium of blood brain/spinal cord barrier in EAE. In addition, IL-10-transfected MSCs show significant inhibitory activity on the proliferation of CD4(+) T lymphocytes from EAE mice. In vivo treatment with MSCs engineered with PSGL-1/SLeX/IL-10 in EAE mice exhibited a superior therapeutic function over native (unmodified) MSCs, evidenced by significantly improved myelination and decreased lymphocytes infiltration into the white matter of the spinal cord. Our strategy of targeted delivery of performance-enhanced MSCs could potentially be utilized to increase the effectiveness of MSC-based therapy for MS and other central nervous system (CNS) disorders.

Exogenous marker-engineered mesenchymal stem cells detect cancer and metastases in a simple blood assay.

INTRODUCTION: Mesenchymal stem cells (MSCs) are adult multipotent stem cells that possess regenerative and immunomodulatory properties. They have been widely investigated as therapeutic agents for a variety of disease conditions, including tissue repair, inflammation, autoimmunity, and organ transplantation. Importantly, systemically infused MSCs selectively home to primary and metastatic tumors, though the molecular mechanisms of tumor tropism of MSCs remain incompletely understood. We have exploited the active and selective MSCs homing to cancer microenvironments to develop a rapid and selective blood test for the presence of cancer. METHODS: We tested the concept of using transplanted MSCs as the basis for a simple cancer blood test. MSCs were engineered to express humanized Gaussia luciferase (hGluc). In a minimally invasive fashion, hGluc secreted by MSCs into circulation as a reporter for cancer presence, was assayed to probe whether MSCs co-localize with and persist in cancerous tissue. RESULTS: In vitro, hGluc secreted by engineered MSCs was detected stably over a period of days in the presence of serum. In vivo imaging showed that MSCs homed to breast cancer lung metastases and persisted longer in tumor-bearing mice than in tumor-free mice (P < 0.05). hGluc activity in blood of tumor-bearing mice was significantly higher than in their tumor-free counterparts (P < 0.05). CONCLUSIONS: Both in vitro and in vivo data show that MSCs expressing hGluc can identify and report small tumors or metastases in a simple blood test format. Our novel and simple stem cell-based blood test can potentially be used to screen, detect, and monitor cancer and metastasis at early stages and during treatment.

Delivery of an engineered HGF fragment in an extracellular matrix-derived hydrogel prevents negative LV remodeling post-myocardial infarction.

Hepatocyte growth factor (HGF) has been shown to have anti-fibrotic, pro-angiogenic, and cardioprotective effects; however, it is highly unstable and expensive to manufacture, hindering its clinical translation. Recently, a HGF fragment (HGF-f), an alternative c-MET agonist, was engineered to possess increased stability and recombinant expression yields. In this study, we assessed the potential of HGF-f, delivered in an extracellular matrix (ECM)-derived hydrogel, as a potential treatment for myocardial infarction (MI). HGF-f protected cardiomyocytes from serum-starvation and induced down-regulation of fibrotic markers in whole cardiac cell isolate compared to the untreated control. The ECM hydrogel prolonged release of HGF-f compared to collagen gels, and in vivo delivery of HGF-f from ECM hydrogels mitigated negative left ventricular (LV) remodeling, improved fractional area change (FAC), and increased arteriole density in a rat myocardial infarction model. These results indicate that HGF-f may be a viable alternative to using recombinant HGF, and that an ECM hydrogel can be employed to increase growth factor retention and efficacy.

Chemokine treatment rescues profound T-lineage progenitor homing defect after bone marrow transplant conditioning in mice.

Development of T cells in the thymus requires continuous importation of T-lineage progenitors from the bone marrow via the circulation. Following bone marrow transplant, recovery of a normal peripheral T-cell pool depends on production of naïve T cells in the thymus; however, delivery of progenitors to the thymus limits T-lineage reconstitution. Here, we examine homing of intravenously delivered progenitors to the thymus following irradiation and bone marrow reconstitution. Surprisingly, following host conditioning by irradiation, we find that homing of lymphoid-primed multipotent progenitors and common lymphoid progenitors to the thymus decreases more than 10-fold relative to unirradiated mice. The reduction in thymic homing in irradiated mice is accompanied by a significant reduction in CCL25, an important chemokine ligand for thymic homing. We show that pretreatment of bone marrow progenitors with CCL25 and CCL21 corrects the defect in thymic homing after irradiation and promotes thymic reconstitution. These data suggest new therapeutic approaches to promote T-cell regeneration.

Trafficking to the thymus.

The continuous production of T lymphocytes requires that hematopoietic progenitors developing in the bone marrow migrate to the thymus. Rare progenitors egress from the bone marrow into the circulation, then traffic via the blood to the thymus. It is now evident that thymic settling is tightly regulated by selectin ligands, chemokine receptors, and integrins, among other factors. Identification of these signals has enabled progress in identifying specific populations of hematopoietic progenitors that can settle the thymus. Understanding the nature of progenitor cells and the molecular mechanisms involved in thymic settling may allow for therapeutic manipulation of this process, and improve regeneration of the T lineage in patients with impaired T cell numbers.

Expression of functional P-selectin glycoprotein ligand 1 on hematopoietic progenitors is developmentally regulated.

T cell development requires periodic importation of hematopoietic progenitors into the thymus. The receptor-ligand pair P-selectin and P-selectin glycoprotein ligand 1 (PSGL-1) are critically involved in this process. In this study, we examined the expression of functional PSGL-1 on bone marrow hematopoietic progenitors. We demonstrate that functional PSGL-1 is expressed at low levels on hematopoietic stem cells, but upregulated on the cell surface of progenitors that bear other homing molecules known to be important for thymic settling. We found that progenitors able to home to the thymus expressed high levels of PSGL-1 transcripts compared with hematopoietic stem cells. We further demonstrate that hematopoietic progenitors lacking fucosyltransferase 4 and 7 do not express functional PSGL-1, and do not home efficiently to the thymus. These studies provide insight into the developmentally regulated expression of a critical determinant involved in progenitor homing to the thymus.

The anti-inflammatory TIPE2 is an inhibitor of the oncogenic Ras.

The connection between cancer and inflammation is widely recognized, yet the underlying molecular mechanisms are poorly understood. We report here that TIPE2 provides a molecular bridge from inflammation to cancer by targeting the Ras signaling pathway. TIPE2 binds the Ras-interacting domain of the RalGDS family of proteins, which are essential effectors of activated Ras. This binding prevented Ras from forming an active complex, thereby inhibiting the activation of the downstream signaling molecules Ral and AKT. Consequently, TIPE2 deficiency led to heightened activation of Ral and AKT, resistance to cell death, increased migration, and dysregulation of exocyst complex formation. Conversely, TIPE2 overexpression induced cell death and significantly inhibited Ras-induced tumorigenesis in mice. Importantly, TIPE2 expression was either completely lost or significantly downregulated in human hepatic cancer. Thus, TIPE2 is an inhibitor of both inflammation and cancer, and a potential drug target for inflammatory and neoplastic diseases.

Delivery of progenitors to the thymus limits T-lineage reconstitution after bone marrow transplantation.

T-cell production depends on the recruitment of hematopoietic progenitors into the thymus. T cells are among the last of the hematopoietic lineages to recover after bone marrow transplantation (BMT), but the reasons for this delay are not well understood. Under normal physiologic conditions, thymic settling is selective and either CCR7 or CCR9 is required for progenitor access into the thymus. The mechanisms of early thymic reconstitution after BMT, however, are unknown. Here we report that thymic settling is briefly CCR7/CCR9-independent after BMT but continues to rely on the selectin ligand PSGL-1. The CCR7/CCR9 independence is transient, and by 3 weeks after BMT these receptors are again strictly required. Despite the normalization of thymic settling signals, the rare bone marrow progenitors that can efficiently repopulate the thymus are poorly reconstituted for at least 4 weeks after BMT. Consistent with reduced progenitor input to the thymus, intrathymic progenitor niches remain unsaturated for at least 10 weeks after BMT. Finally, we show that thymic recovery is limited by the number of progenitors entering the thymus after BMT. Hence, T-lineage reconstitution after BMT is limited by progenitor supply to the thymus.

Fusion of EML4 and ALK is associated with development of lung adenocarcinomas lacking EGFR and KRAS mutations and is correlated with ALK expression.

BACKGROUND: The anaplastic lymphoma kinase (ALK) gene is frequently involved in translocations that lead to gene fusions in a variety of human malignancies, including lymphoma and lung cancer. Fusion partners of ALK include NPM, EML4, TPM3, ATIC, TFG, CARS, and CLTC. Characterization of ALK fusion patterns and their resulting clinicopathological profiles could be of great benefit in better understanding the biology of lung cancer. RESULTS: RACE-coupled PCR sequencing was used to assess ALK fusions in a cohort of 103 non-small cell lung carcinoma (NSCLC) patients. Within this cohort, the EML4-ALK fusion gene was identified in 12 tumors (11.6%). Further analysis revealed that EML4-ALK was present at a frequency of 16.13% (10/62) in patients with adenocarcinomas, 19.23% (10/52) in never-smokers, and 42.80% (9/21) in patients with adenocarcinomas lacking EGFR and KRAS mutations. The EML4-ALK fusion was associated with non-smokers (P = 0.03), younger age of onset (P = 0.03), and adenocarcinomas without EGFR/KRAS mutations (P = 0.04). A trend towards improved survival was observed for patients with the EML4-ALK fusion, although it was not statistically significant (P = 0.20). Concurrent deletion in EGFR exon 19 and fusion of EML4-ALK was identified for the first time in a Chinese female patient with an adenocarcinoma. Analysis of ALK expression revealed that ALK mRNA levels were higher in tumors positive for the EML-ALK fusion than in negative tumors (normalized intensity of 21.99 vs. 0.45, respectively; P = 0.0018). However, expression of EML4 did not differ between the groups. CONCLUSIONS: The EML4-ALK fusion gene was present at a high frequency in Chinese NSCLC patients, particularly in those with adenocarcinomas lacking EGFR/KRAS mutations. The EML4-ALK fusion appears to be tightly associated with ALK mRNA expression levels. RACE-coupled PCR sequencing is a highly sensitive method that could be used clinically for the identification of EML4-ALK-positive patients.

Controllable microfluidic synthesis of multiphase drug-carrying lipospheres for site-targeted therapy.

We report the production of micrometer-sized gas-filled lipospheres using digital (droplet-based) microfluidics technology for chemotherapeutic drug delivery. Advantages of on-chip synthesis include a monodisperse size distribution (polydispersity index (sigma) values of <5%) with consistent stability and uniform drug loading. Photolithography techniques are applied to fabricate novel PDMS-based microfluidic devices that feature a combined dual hydrodynamic flow-focusing region and expanding nozzle geometry with a narrow orifice. Spherical vehicles are formed through flow-focusing by the self-assembly of phospholipids to a lipid layer around the gas core, followed by a shear-induced break off at the orifice. The encapsulation of an extra oil layer between the outer lipid shell and inner bubble gaseous core allows the transport of highly hydrophobic and toxic drugs at high concentrations. Doxorubicin (Dox) entrapment is estimated at 15 mg mL(-1) of particles packed in a single ordered layer. In addition, the attachment of targeting ligands to the lipid shell allows for direct vehicle binding to cancer cells. Preliminary acoustic studies of these monodisperse gas lipospheres reveal a highly uniform echo correlation of greater than 95%. The potential exists for localized drug concentration and release with ultrasound energy.

Cinnamoyl-based Nrf2-activators targeting human skin cell photo-oxidative stress.

Strong experimental evidence suggests the involvement of photo-oxidative stress mediated by reactive oxygen species as a crucial mechanism of solar damage relevant to human skin photoaging and photocarcinogenesis. Based on the established role of antioxidant response element (ARE)-mediated gene expression in cancer chemoprevention, we tested the hypothesis that small molecule Nrf2-activators may serve a photo-chemopreventive role by targeting skin cell photo-oxidative stress. A luciferase-based reporter gene assay was used as a primary screen for the identification of novel agents that modulate the Nrf2-Keap1 signaling pathway. A series of cinnamoyl-based electrophilic Michael acceptors including cinnamic aldehyde and methyl-1-cinnamoyl-5-oxo-2-pyrrolidine-carboxylate was identified as potent Nrf2-activators. Hit confirmation was performed in a secondary screen, based on immunodetection of Nrf2 protein upregulation in human Hs27 skin fibroblasts, HaCaT keratinocytes, and primary skin keratinocytes. Bioefficacy profiling of positive test compounds in skin cells demonstrated compound-induced upregulation of hemeoxygenase I and NAD(P)H-quinone oxidoreductase, two Nrf2 target genes involved in the cellular antioxidant response. Pretreatment with cinnamoyl-based Nrf2-activators suppressed intracellular oxidative stress and protected against photo-oxidative induction of apoptosis in skin cells exposed to high doses of singlet oxygen. Our pilot studies suggest feasibility of developing cinnamoyl-based Nrf2-activators as novel photo-chemopreventive agents targeting skin cell photo-oxidative stress.