Inhibition of VHL by VH298 Accelerates Pexophagy by Activation of HIF-1α in HeLa Cells.

Autophagy is a pivotal biological process responsible for maintaining the homeostasis of intracellular organelles. Yet the molecular intricacies of peroxisomal autophagy (pexophagy) remain largely elusive. From a ubiquitin-related chemical library for screening, we identified several inhibitors of the Von Hippel-Lindau (VHL) E3 ligase, including VH298, thereby serving as potent inducers of pexophagy. In this study, we observed that VH298 stimulates peroxisomal degradation by ATG5 dependently and escalates the ubiquitination of the peroxisomal membrane protein ABCD3. Interestingly, the ablation of NBR1 is similar to the curtailed peroxisomal degradation in VH298-treated cells. We also found that the pexophagy induced by VH298 is impeded upon the suppression of gene expression by the translation inhibitor cycloheximide. Beyond VHL inhibition, we discovered that roxadustat, a direct inhibitor of HIF-α prolyl hydroxylase, is also a potent inducer of pexophagy. Furthermore, we found that VH298-mediated pexophagy is blocked by silencing HIF-1α. In conclusion, our findings suggest that VH298 promotes pexophagy by modulating VHL-mediated HIF-α transcriptional activity.

Enhanced primary ciliogenesis via mitochondrial oxidative stress activates AKT to prevent neurotoxicity in HSPA9/mortalin-depleted SH-SY5Y cells.

The primary cilium, an antenna-like structure on the cell surface, acts as a mechanical and chemical sensory organelle. Primary cilia play critical roles in sensing the extracellular environment to coordinate various developmental and homeostatic signaling pathways. Here, we showed that the depletion of heat shock protein family A member 9 (HSPA9)/mortalin stimulates primary ciliogenesis in SH-SY5Y cells. The downregulation of HSPA9 enhances mitochondrial stress by increasing mitochondrial fragmentation and mitochondrial reactive oxygen species (mtROS) generation. Notably, the inhibition of either mtROS production or mitochondrial fission significantly suppressed the increase in primary ciliogenesis in HSPA9-depleted cells. In addition, enhanced primary ciliogenesis contributed to cell survival by activating AKT in SH-SY5Y cells. The abrogation of ciliogenesis through the depletion of IFT88 potentiated neurotoxicity in HSPA9-knockdown cells. Furthermore, both caspase-3 activation and cell death were increased by MK-2206, an AKT inhibitor, in HSPA9-depleted cells. Taken together, our results suggest that enhanced primary ciliogenesis plays an important role in preventing neurotoxicity caused by the loss of HSPA9 in SH-SY5Y cells.

PEX13 prevents pexophagy by regulating ubiquitinated PEX5 and peroxisomal ROS.

Peroxisomes are rapidly degraded during amino acid and oxygen deprivation by a type of selective autophagy called pexophagy. However, how damaged peroxisomes are detected and removed from the cell is poorly understood. Recent studies suggest that the peroxisomal matrix protein import machinery may serve double duty as a quality control machinery, where they are directly involved in activating pexophagy. Here, we explored whether any matrix import factors are required to prevent pexophagy, such that their loss designates peroxisomes for degradation. Using gene editing and quantitative fluorescence microscopy on culture cells and a zebrafish model system, we found that PEX13, a component of the peroxisomal matrix import system, is required to prevent the degradation of otherwise healthy peroxisomes. The loss of PEX13 caused an accumulation of ubiquitinated PEX5 on peroxisomes and an increase in peroxisome-dependent reactive oxygen species that coalesce to induce pexophagy. We also found that PEX13 protein level is downregulated to aid in the induction of pexophagy during amino acid starvation. Together, our study points to PEX13 as a novel pexophagy regulator that is modulated to maintain peroxisome homeostasis.Abbreviations: AAA ATPases: ATPases associated with diverse cellular activities; ABCD3: ATP binding cassette subfamily D member; 3ACOX1: acyl-CoA oxidase; 1ACTA1: actin alpha 1, skeletal muscle; ACTB: actin beta; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG12: autophagy related 12; ATG16L1: autophagy related 16 like 1; CAT: catalase; CQ: chloroquine; Dpf: days post fertilization: FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; H2O2: hydrogen peroxide; HA - human influenza hemagglutinin; HBSS: Hanks' Balanced Salt Solution; HCQ; hydroxychloroquine; KANL: lysine alanine asparagine leucine; KO: knockout; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; MTORC2: mechanistic target of rapamycin kinase complex 2; MYC: MYC proto-oncogene, bHLH transcription factor; MZ: maternal and zygotic; NAC: N-acetyl cysteine; NBR1 - NBR1 autophagy cargo receptor; PBD: peroxisome biogenesis disorder; PBS: phosphate-buffered saline; PEX: peroxisomal biogenesis factor; PTS1: peroxisome targeting sequence 1; RFP: red fluorescent protein; ROS: reactive oxygen speciess; iRNA: short interfering RNA; SKL: serine lysine leucine; SLC25A17/PMP34: solute carrier family 25 member 17; Ub: ubiquitin; USP30: ubiquitin specific peptidase 30.

Carnitine Protects against MPP+-Induced Neurotoxicity and Inflammation by Promoting Primary Ciliogenesis in SH-SY5Y Cells.

Primary cilia help to maintain cellular homeostasis by sensing conditions in the extracellular environment, including growth factors, nutrients, and hormones that are involved in various signaling pathways. Recently, we have shown that enhanced primary ciliogenesis in dopamine neurons promotes neuronal survival in a Parkinson's disease model. Moreover, we performed fecal metabolite screening in order to identify several candidates for improving primary ciliogenesis, including L-carnitine and acetyl-L-carnitine. However, the role of carnitine in primary ciliogenesis has remained unclear. In addition, the relationship between primary cilia and neurodegenerative diseases has remained unclear. In this study, we have evaluated the effects of carnitine on primary ciliogenesis in 1-methyl-4-phenylpyridinium ion (MPP+)-treated cells. We found that both L-carnitine and acetyl-L-carnitine promoted primary ciliogenesis in SH-SY5Y cells. In addition, the enhancement of ciliogenesis by carnitine suppressed MPP+-induced mitochondrial reactive oxygen species overproduction and mitochondrial fragmentation in SH-SY5Y cells. Moreover, carnitine inhibited the production of pro-inflammatory cytokines in MPP+-treated SH-SY5Y cells. Taken together, our findings suggest that enhanced ciliogenesis regulates MPP+-induced neurotoxicity and inflammation.

Post-Translational Modifications of ATG4B in the Regulation of Autophagy.

Autophagy plays a key role in eliminating and recycling cellular components in response to stress, including starvation. Dysregulation of autophagy is observed in various diseases, including neurodegenerative diseases, cancer, and diabetes. Autophagy is tightly regulated by autophagy-related (ATG) proteins. Autophagy-related 4 (ATG4) is the sole cysteine protease, and four homologs (ATG4A-D) have been identified in mammals. These proteins have two domains: catalytic and short fingers. ATG4 facilitates autophagy by promoting autophagosome maturation through reversible lipidation and delipidation of seven autophagy-related 8 (ATG8) homologs, including microtubule-associated protein 1-light chain 3 (LC3) and GABA type A receptor-associated protein (GABARAP). Each ATG4 homolog shows a preference for a specific ATG8 homolog. Post-translational modifications of ATG4, including phosphorylation/dephosphorylation, O-GlcNAcylation, oxidation, S-nitrosylation, ubiquitination, and proteolytic cleavage, regulate its activity and ATG8 processing, thus modulating its autophagic activity. We reviewed recent advances in our understanding of the effect of post-translational modification on the regulation, activity, and function of ATG4, the main protease that controls autophagy.

Nalfurafine Hydrochloride, a κ-Opioid Receptor Agonist, Induces Melanophagy via PKA Inhibition in B16F1 Cells.

Selective autophagy controls cellular homeostasis by degrading unnecessary or damaged cellular components. Melanosomes are specialized organelles that regulate the biogenesis, storage, and transport of melanin in melanocytes. However, the mechanisms underlying melanosomal autophagy, known as the melanophagy pathway, are poorly understood. To better understand the mechanism of melanophagy, we screened an endocrine-hormone chemical library and identified nalfurafine hydrochlorides, a κ-opioid receptor agonist, as a potent inducer of melanophagy. Treatment with nalfurafine hydrochloride increased autophagy and reduced melanin content in alpha-melanocyte-stimulating hormone (α-MSH)-treated cells. Furthermore, inhibition of autophagy blocked melanosomal degradation and reversed the nalfurafine hydrochloride-induced decrease in melanin content in α-MSH-treated cells. Consistently, treatment with other κ-opioid receptor agonists, such as MCOPPB or mianserin, inhibited excessive melanin production but induced autophagy in B16F1 cells. Furthermore, nalfurafine hydrochloride inhibited protein kinase A (PKA) activation, which was notably restored by forskolin, a PKA activator. Additionally, forskolin treatment further suppressed melanosomal degradation as well as the anti-pigmentation activity of nalfurafine hydrochloride in α-MSH-treated cells. Collectively, our data suggest that stimulation of κ-opioid receptors induces melanophagy by inhibiting PKA activation in α-MSH-treated B16F1 cells.

BRCA1-BARD1 regulates transcription through modulating topoisomerase IIß.

RNA polymerase II (Pol II)-dependent transcription in stimulus-inducible genes requires topoisomerase IIß (TOP2B)-mediated DNA strand break and the activation of DNA damage response signalling in humans. Here, we report a novel function of the breast cancer 1 (BRCA1)-BRCA1-associated ring domain 1 (BARD1) complex in this process. We found that BRCA1 is phosphorylated at S1524 by the kinases ataxia-telangiectasia mutated and ATR during gene activation, and that this event is important for productive transcription. Our biochemical and genomic analyses showed that the BRCA1-BARD1 complex interacts with TOP2B in the EGR1 transcription start site and in a large number of protein-coding genes. Intriguingly, the BRCA1-BARD1 complex ubiquitinates TOP2B, which stabilizes TOP2B binding to DNA while BRCA1 phosphorylation at S1524 controls the TOP2B ubiquitination by the complex. Together, these findings suggest the novel function of the BRCA1-BARD1 complex in the regulation of TOP2B and Pol II-mediated gene expression.

Primary Ciliogenesis by 2-Isopropylmalic Acid Prevents PM2.5-Induced Inflammatory Response and MMP-1 Activation in Human Dermal Fibroblasts and a 3-D-Skin Model.

Particulate matters (PMs) increase oxidative stress and inflammatory response in different tissues. PMs disrupt the formation of primary cilia in various skin cells, including keratinocytes and melanocytes. In this study, we found that 2-isopropylmalic acid (2-IPMA) promoted primary ciliogenesis and restored the PM2.5-induced dysgenesis of primary cilia in dermal fibroblasts. Moreover, 2-IPMA inhibited the generation of excessive reactive oxygen species and the activation of stress kinase in PM2.5-treated dermal fibroblasts. Further, 2-IPMA inhibited the production of pro-inflammatory cytokines, including IL-6 and TNF-α, which were upregulated by PM2.5. However, the inhibition of primary ciliogenesis by IFT88 depletion reversed the downregulated cytokines by 2-IPMA. Moreover, we found that PM2.5 treatment increased the MMP-1 expression in dermal fibroblasts and a human 3-D-skin model. The reduced MMP-1 expression by 2-IPMA was further reversed by IFT88 depletion in PM2.5-treated dermal fibroblasts. These findings suggest that 2-IPMA ameliorates PM2.5-induced inflammation by promoting primary ciliogenesis in dermal fibroblasts.

Triamterene induces autophagic degradation of lysosome by exacerbating lysosomal integrity.

The maintenance of lysosomal integrity is essential for lysosome function and cell fate. Damaged lysosomes are degraded by lysosomal autophagy, lysophagy. The mechanism underlying lysophagy remains largely unknown; this study aimed to contribute to the understanding of this topic. A cell-based screening system was used to identify novel lysophagy modulators. Triamterene (6-phenylpteridine-2,4,7-triamine) was identified as one of the most potent lysophagy inducers from the screening process. We found that triamterene causes lysosomal rupture without affecting other cellular organelles and increases autophagy flux in HepG2 cells. Damaged lysosomes in triamterene-treated cells were removed by autophagy-mediated pathway, which was inhibited by depletion of the autophagy regulator, ATG5 or SQSTM1. In addition, treatment of triamterene decreased the integrity of lysosome and cell viability, which were rescued by removing the triamterene treatment in HepG2 cells. Hence, our data suggest that triamterene is a novel lysophagy inducer through the disruption of lysosomal integrity.

Tetraarsenic oxide affects non-coding RNA transcriptome through deregulating polycomb complexes in MCF7 cells.

Non-coding RNAs (ncRNAs) play important and diverse roles in mammalian cell biology and pathology. Although the functions of an increasing number of ncRNAs have been identified, the mechanisms underlying ncRNA gene expression remain elusive and are incompletely understood. Here, we investigated ncRNA gene expression in Michigan cancer foundation 7 (MCF7), a malignant breast cancer cell line, on treatment of tetraarsenic oxide (TAO), a potential anti-cancer drug. Our genomic analyses found that TAO up- or down-regulated ncRNA genes genome-wide. A subset of identified ncRNAs with critical biological and clinical functions were validated by real-time quantitative polymerase chain reaction. Intriguingly, these TAO-regulated genes included CDKN2B-AS, HOXA11-AS, SHH, and DUSP5 that are known to interact with or be targeted by polycomb repressive complexes (PRCs). In addition, the PRC subunits were enriched in these TAO-regulated ncRNA genes and TAO treatment deregulated the expression of PRC subunits. Strikingly, TAO decreased the cellular and gene-specific levels of EZH2 expression and H3K27me3. In particular, TAO reduced EZH2 and H3K27me3 and increased transcription at MALAT1 gene. Inhibiting the catalytic activity of EZH2 using GSK343 increased representative TAO-inducible ncRNA genes. Together, our findings suggest that the expression of a subset of ncRNA genes is regulated by PRC2 and that TAO could be a potent epigenetic regulator through PRCs to modulate the ncRNA gene expression in MCF7 cells.

Melasolv induces melanosome autophagy to inhibit pigmentation in B16F1 cells.

The melanosome is a specialized membrane-bound organelle that is involved in melanin synthesis, storage, and transportation. In contrast to melanosome biogenesis, the processes underlying melanosome degradation remain largely unknown. Autophagy is a process that promotes degradation of intracellular components' cooperative process between autophagosomes and lysosomes, and its role for process of melanosome degradation remains unclear. Here, we assessed the regulation of autophagy and its contributions to depigmentation associated with Melasolv (3,4,5-trimethoxycinnamate thymol ester). B16F1 cells-treated with Melasolv suppressed the α-MSH-stimulated increase of melanin content and resulted in the activation of autophagy. However, introduction of bafilomycin A1 strongly suppressed melanosome degradation in Melasolv-treated cells. Furthermore, inhibition of autophagy by ATG5 resulted in significant suppression of Melasolv-mediated depigmentation in α-MSH-treated cells. Taken together, our results suggest that treatment with Melasolv inhibits skin pigmentation by promoting melanosome degradation via autophagy activation.

Ursolic acid inhibits pigmentation by increasing melanosomal autophagy in B16F1 cells.

Melanosomes are specialized membrane-bound organelles that are involved in melanin synthesis. Unlike melanosome biogenesis, the melanosome degradation pathway is poorly understood. Among the cellular processes, autophagy controls degradation of intracellular components by cooperating with lysosomes. In this study, we showed that ursolic acid inhibits skin pigmentation by promoting melanosomal autophagy, or melanophagy, in melanocytes. We found that B16F1 cells treated with ursolic acid suppressed alpha-melanocyte stimulating hormone (α-MSH) stimulated increase in melanin content and activated autophagy. In addition, we found that treatment with ursolic acid promotes melanosomal degradation, and bafilomycin A1 inhibition of autophagosome-lysosome fusion blocked the removal of melanosomes in α-MSH-stimulated B16F1 cells. Furthermore, depletion of the autophagy-related gene 5 (ATG5) resulted in significant suppression of ursolic acid-mediated anti-pigmentation activity and autophagy in α-MSH-treated B16F1 cells. Taken together, our results suggest that ursolic acid inhibits skin pigmentation by increasing melanosomal degradation in melanocytes.

Primary cilia mediate mitochondrial stress responses to promote dopamine neuron survival in a Parkinson's disease model.

A primary cilium is an antenna-like structure on the cell surface that plays a crucial role in sensory perception and signal transduction. Mitochondria, the 'powerhouse' of the cell, control cell survival, and death. The cellular ability to remove dysfunctional mitochondria through mitophagy is important for cell survival. We show here that mitochondrial stress, caused by respiratory complex inhibitors and excessive fission, robustly stimulates ciliogenesis in different types of cells including neuronal cells. Mitochondrial stress-induced ciliogenesis is mediated by mitochondrial reactive oxygen species generation, subsequent activation of AMP-activated protein kinase and autophagy. Conversely, abrogation of ciliogenesis compromises mitochondrial stress-induced autophagy, leading to enhanced cell death. In mice, treatment with mitochondrial toxin, MPTP elicits ciliary elongation and autophagy in the substantia nigra dopamine neurons. Blockade of cilia formation in these neurons attenuates MPTP-induced autophagy but facilitates dopamine neuronal loss and motor disability. Our findings demonstrate the important role of primary cilia in cellular pro-survival responses during mitochondrial stress.

Loss of RNA binding protein, human antigen R enhances mitochondrial elongation by regulating Drp1 expression in SH-SY5Y cells.

Mitochondria are essential for providing the energy necessary for neuronal function. Dysregulation of mitochondrial dynamics has been linked with the pathogenesis of many neurodegenerative diseases. Dynamin related protein 1 (Drp1) participates in fission activity in the mitochondria, and post-translational modifications to Drp1 modulate complex mitochondrial dynamics. However, the regulation of Drp1 at the post-transcriptional level remains poorly understood. In this study, we found that the RNA-binding protein Hu antigen R (HuR) post-transcriptionally regulates Drp1 expression. HuR interacts with Drp1 mRNA at its 3' untranslated region. Depletion of HuR reduces Drp1 expression, which leads to mitochondrial elongation in SH-SY5Y neuroblastoma cells. In contrast, ectopic expression of HuR enhances Drp1 expression, which promotes mitochondrial fragmentation in response to treatment with the mitochondrial complex 1 inhibitor MPP+. In addition, depletion of HuR suppressed the generation of mitochondrial ROS and cytotoxicity in MPP+ treated cells. Taken together, these findings suggest that HuR controls mitochondrial morphology via regulation of Drp1.

P-TEFb Regulates Transcriptional Activation in Non-coding RNA Genes.

Many non-coding RNAs (ncRNAs) serve as regulatory molecules in various physiological pathways, including gene expression in mammalian cells. Distinct from protein-coding RNA expression, ncRNA expression is regulated solely by transcription and RNA processing/stability. It is thus important to understand transcriptional regulation in ncRNA genes but is yet to be known completely. Previously, we identified that a subset of mammalian ncRNA genes is transcriptionally regulated by RNA polymerase II (Pol II) promoter-proximal pausing and in a tissue-specific manner. In this study, human ncRNA genes that are expressed in the early G1 phase, termed immediate early ncRNA genes, were monitored to assess the function of positive transcription elongation factor b (P-TEFb), a master Pol II pausing regulator for protein-coding genes, in ncRNA transcription. Our findings indicate that the expression of many ncRNA genes is induced in the G0-G1 transition and regulated by P-TEFb. Interestingly, a biphasic characteristic of P-TEFb-dependent transcription of serum responsive ncRNA genes was observed: Pol II carboxyl-terminal domain phosphorylated at serine 2 (S2) was largely increased in the transcription start site (TSS, -300 to +300) whereas overall, it was decreased in the gene body (GB, > +350) upon chemical inhibition of P-TEFb. In addition, the three representative, immediate early ncRNAs, whose expression is dependent on P-TEFb, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), nuclear enriched abundant transcript 1 (NEAT1), and X-inactive specific transcript (XIST), were further analyzed for determining P-TEFb association. Taken together, our data suggest that transcriptional activation of many human ncRNAs utilizes the pausing and releasing of Pol II, and that the regulatory mechanism of transcriptional elongation in these genes requires the function of P-TEFb. Furthermore, we propose that ncRNA and mRNA transcription are regulated by similar mechanisms while P-TEFb inhibition unexpectedly increases S2 Pol II phosphorylation in the TSSs in many ncRNA genes. One Sentence Summary: P-TEFb regulates Pol II phosphorylation for transcriptional activation in many stimulus-inducible ncRNA genes.

Fine particulate matter (PM2.5) inhibits ciliogenesis by increasing SPRR3 expression via c-Jun activation in RPE cells and skin keratinocytes.

Exposure to fine particulate matter (PM) with diameter <2.5 µm (PM2.5) causes epithelium injury and endothelial dysfunction. Primary cilia are sensory organelles that transmit extracellular signals into intracellular biochemical responses and have roles in physiology. To date, there have been no studies investigating whether PM2.5 affects primary cilia in skin. We addressed this in the present study using normal human epidermal keratinocytes (NHEKs) and retinal pigment epithelium (RPE) cells. We found that formation of primary cilium is increased in differentiated NHEKs. However, treatment with PM2.5 blocked increased ciliogenesis in NHEKs and RPE cells. Furthermore, PM2.5 transcriptionally upregulated small proline rich protein 3 (SPRR3) expression by activating c-Jun, and ectopic expression of SPRR3 inhibits suppressed the ciliogenesis. Accordingly, treatment with c-Jun activator (anisomycin) induced SPRR3 expression, whereas the inhibitor (SP600125) recovered the ciliated cells and cilium length in PM2.5-treated cells. Moreover, c-Jun inhibitor suppressed upregulation of SPRR3 in PM2.5-treated cells. Taken together, our finding suggested that PM2.5 inhibits ciliogenesis by increasing SPRR3 expression via c-Jun activation in RPE cells and keratinocytes.

Down-regulated TMED10 in Alzheimer disease induces autophagy via ATG4B activation.

Several studies have shown that dysfunction of macroautophagy/autophagy is associated with many human diseases, including neurodegenerative disease and cancer. To explore the molecular mechanisms of autophagy, we performed a cell-based functional screening with SH-SY5Y cells stably expressing GFP-LC3, using an siRNA library and identified TMED10 (transmembrane p24 trafficking protein 10), previously known as the γ-secretase-modulating protein, as a novel regulator of autophagy. Further investigations revealed that depletion of TMED10 induced the activation of autophagy. Interestingly, protein-protein interaction assays showed that TMED10 directly binds to ATG4B (autophagy related gene 4B cysteine peptidase), and the interaction is diminished under autophagy activation conditions such as rapamycin treatment and serum deprivation. In addition, inhibition of TMED10 significantly enhanced the proteolytic activity of ATG4B for LC3 cleavage. Importantly, the expression of TMED10 in AD (Alzheimer disease) patients was considerably decreased, and downregulation of TMED10 increased amyloid-ß (Aß) production. Treatment with Aß increased ATG4B proteolytic activity as well as dissociation of TMED10 and ATG4B. Taken together, our results suggest that the AD-associated protein TMED10 negatively regulates autophagy by inhibiting ATG4B activity.Abbreviations: Aß: amyloid-ß; AD: Alzheimer disease; ATG: autophagy related; BECN1: beclin 1; BiFC: bimolecular fluorescence complementation; CD: cytosolic domain; GFP: green fluorescent protein; GLUC: Gaussia luciferase; IP: immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LD: luminal domain; PD: Parkinson disease; ROS: reactive oxygen species; siRNA: small interfering RNA; SNP: single-nucleotide polymorphisms; TD: transmembrane domain; TMED10: transmembrane p24 trafficking protein 10; VC: C terminus of Venus fluorescent protein; VN: N terminus of Venus fluorescent protein.

Anti-melanogenic activity of schaftoside in Rhizoma Arisaematis by increasing autophagy in B16F1 cells.

Skin pigmentation involves multiple processes, including melanin synthesis, transport, and melanosome release. Melanin content determines skin color and protects against UV radiation-induced damage. Autophagy is a cooperative process between autophagosomes and lysosomes that degrades cellular components and organelles. In the present study, B16F1 cells were treated with Rhizoma Arisaematis extract (RA) and assessed for pigmentation and autophagy regulation. RA treatment suppressed the α-MSH-stimulated increase of melanogenesis and down-regulated the expression of tyrosinase and TRP1 proteins in B16F1 cells. In addition, autophagy was activated in RA-treated cells. Inhibition of autophagy reduced the anti-melanogenic activity of RA in α-MSH-treated B16F1 cells. We identified schaftoside as an effector molecule by LC-MS analysis of RA. Consistently, treatment of schaftoside showed anti-melanogenic effect and induced autophagy activation in B16F1 cells. Inhibition of autophagy by 3 MA treatment reduced the anti-melanogenic effect of the schaftoside and recovered expression level of melanogenesis regulators in α-MSH-treated B16F1 cells. Taken together, our results suggest that schaftoside from RA inhibits skin pigmentation through modulation of autophagy.

Up-regulation of UVRAG by HDAC1 Inhibition Attenuates 5FU-induced Cell Death in HCT116 Colorectal Cancer Cells.

The ultraviolent irradiation resistance-associated gene (UVRAG), a component of the Beclin 1/autophagy-related 6 complex, regulates the autophagy initiation step and functions in the DNA-damage response. UVRAG is frequently mutated in various cancer types, and mutations of UVRAG increase sensitivity to chemotherapy by impairing DNA-damage repair. In this study, we addressed the epigenetic regulation of UVRAG in colorectal cancer cells. UVRAG expression was increased in cells treated with histone deacetylase (HDAC) inhibitors, such as valproic acid and suberoylanilide hydroxamic acid. Down-regulation of HDAC1 enhanced UVRAG expression in colorectal cancer cells. In addition, both chemical and genetic inhibition of HDAC1 reduced the activation of caspase-3 and cytotoxicity in 5-fluorouracil (5FU)-treated cancer cells. In contrast, UVRAG overexpression inhibited caspase activation and cell death in 5FU-treated cells. Taken together, our findings suggest that up-regulation of UVRAG by HDAC1 inhibition potentiates DNA-damage-mediated cell death in colorectal cancer cells.

Inhibition of never in mitosis A (NIMA)-related kinase-4 reduces survivin expression and sensitizes cancer cells to TRAIL-induced cell death.

The tumor necrosis factor-related apoptosis inducing ligand (TRAIL) preferentially induces apoptosis in cancer cells. However, many tumors are resistant to TRAIL-induced apoptosis, and resistance mechanisms are not fully understood. To identify novel regulatory molecules of TRAIL resistance, we screened a siRNA library targeting the human kinome, and NEK4 (NIMA-related kinase-4) was identified. Knockdown of NEK4 sensitized TRAIL-resistant cancer cells and in vivo xenografts to cell death. In contrast, over expression of NEK4 suppressed TRAIL-induced cell death in TRAIL-sensitive cancer cells. In addition, loss of NEK4 resulted in decrease of the anti-apoptotic protein survivin, but an increase in apoptotic cell death. Interestingly, NEK4 was highly upregulated in tumor tissues derived from patients with lung cancer and colon cancer. These results suggest that inhibition of NEK4 sensitizes cancer cells to TRAIL-induced apoptosis by regulation of survivin expression.