DNA repair and cholesterol-mediated drug efflux induce dose-dependent chemoresistance in nutrient-deprived neuroblastoma cells.

Neuroblastoma is a solid, heterogeneous pediatric tumor. Chemotherapy is widely used to treat neuroblastoma. However, dose-dependent responses and chemoresistance mechanisms of neuroblastoma cells to anticancer drugs remain challenging. Here, we investigated the dose-dependent effects of topotecan on human neuroblastoma cells (SK-N-SH, SH-SY5Y, and SK-N-BE) under various nutrient supply conditions. Serum-starved human neuroblastoma cells showed reduced toxicity. Their survival rate increased upon treatment with a high concentration (1 µM) of topotecan. Quantitative profiling of global and phosphoproteome identified 12,959 proteins and 48,812 phosphosites, respectively, from SK-N-SH cells. Network analysis revealed that topotecan upregulated DNA repair and cholesterol-mediated topotecan efflux, resulting in topotecan resistance. Results of DNA damage assay, cell cycle, and quantitative analyses of membrane cholesterol supported the validity of these resistance factors and their applicability to all neuroblastoma cells. Our results provide a model for high dose-dependent chemoresistance in neuroblastoma cells that could enable a patient-dependent chemotherapy screening strategy.

Probing drug delivery and mechanisms of action in 3D spheroid cells by quantitative analysis.

Human tumor cells in a 3-dimensional (3D) spheroid can reflect the characteristics of solid tumors by forming cell-cell interactions and microenvironments. This makes 3D cell culture useful for preclinical stability and drug efficacy tests. In this study, the drug delivery and action mechanisms in SK-N-SH neuroblastoma cells cultured in 3D spheroids were quantitatively compared to those cultured in 2D monolayers using confocal microscopy imaging and inductively coupled plasma-mass spectrometry. In the 3D spheroids, cisplatin only accessed the surface, accumulating in the cells on the spheroid exterior. As a result, an increased cellular amount of cisplatin was required to obtain similar cytotoxicity in the 3D spheroid cells to that in 2D monolayers. The mechanisms of reduction of drug efficacy by dimethyl sulfoxide (DMSO) in the 3D spheroid cells compared to those in the 2D monolayer cells were further investigated. DMSO reduced the drug cytotoxicity by forming stable DMSO-substituted compounds that inhibited the cellular uptake of cisplatin and DNA-Pt adduct formation. The quantitative analysis used in this study is promising for understanding drug delivery and drug action mechanisms in cells in various microenvironments.

JNK suppresses melanogenesis by interfering with CREB-regulated transcription coactivator 3-dependent MITF expression.

Melanogenesis is a critical self-defense mechanism against ultraviolet radiation (UVR)-induced skin damage and carcinogenesis; however, dysregulation of melanin production and distribution causes skin-disfiguring pigmentary disorders. Melanogenesis is initiated by UVR-induced cAMP generation and ensuing activation of transcription factor CREB, which induces expression of the master melanogenic regulator MITF. Recent studies have demonstrated that recruitment of CRTCs to the CREB transcription complex is also required for UVR-stimulated melanogenesis. Therefore, modulation of cAMP-CRTC/CREB-MITF signaling may be a useful therapeutic strategy for UVR-associated skin pigmentary disorders. Methods: We identified the small-molecule Ro31-8220 from CREB/CRTC activity screening and examined its melanogenic activity in cultured mouse and human melanocytes as well as in human skin. Molecular mechanisms were deciphered by immunoblotting, RT-PCR, promoter assays, tyrosinase activity assays, immunofluorescent examination of CRTC3 subcellular localization, and shRNA-based knockdown. Results: Ro31-8220 suppressed basal and cAMP-stimulated melanin production in melanocytes and human melanocyte co-culture as well as UVR-stimulated melanin accumulation in human skin through downregulation of MITF and tyrosinase expression. Mechanistically, down regulation of MITF expression by Ro31-8220 was due to inhibition of transcriptional activity of CREB, which was resulted from phosphorylation-dependent blockade of nuclear translocation of CRTC3 via JNK activation. The selective JNK activator anisomycin also inhibited melanin production through phosphoinhibition of CRTC3, while JNK inhibition enhanced melanogenesis by stimulating CRTC3 dephosphorylation and nuclear migration. Conclusions: Melanogenesis can be enhanced or suppressed via pharmacological modulation of a previously unidentified JNK-CRTC/CREB-MITF signaling axis. As Ro31-8220 potently inhibits UVR-stimulated melanin accumulation in human skin, suggesting that small-molecule JNK-CRTC signaling modulators may provide therapeutic benefit for pigmentation disorders.

Therapeutic Potential of Rottlerin for Skin Hyperpigmentary Disorders by Inhibiting the Transcriptional Activity of CREB-Regulated Transcription Coactivators.

Exposure to UVR stimulates the cAMP signaling pathway, which leads to melanin deposits in skin tissues. Although melanogenesis can be beneficial by protecting skin from UVR-induced damage, excessive or uneven deposits of melanin can cause various skin hyperpigmentation disorders. Because cAMP-responsive element binding protein (CREB) and CREB-regulated transcription coactivators (CRTC) play a major role in conveying cAMP signals that induce transcription of microphthalmia-associated transcription factor and melanin production, we screened for a CREB or CRTC inhibitor and identified rottlerin (Rot) as a potent inhibitor of its transcriptional activity. Rot suppressed melanin production in both basal and cAMP-stimulated cultured melanocytes by downregulating melanogenic gene expression. In addition, topical application of Rot on the tails of mice decreased melanin accumulation. Mechanistically, we showed that Rot decreased the mitochondrial membrane potential, which then activated AMPK, leading to the phosphorylation and nuclear exclusion of CRTC3 and suppressing the expression of CREB target genes, including MITF. Our study demonstrates that Rot is an active antimelanogenic agent and suggests that screening for an inhibitor of CREB or CRTC transcriptional activity is a promising strategy by which to discover better drugs to treat skin hyperpigmentary disorders.

NEDD4L limits cAMP signaling through ubiquitination of CREB-regulated transcription coactivator 3.

The transcription factor cAMP-responsive element-binding protein (CREB) is involved in a variety of physiologic processes. Although its activity appears to be largely correlated with its phosphorylation status, cAMP-mediated dephosphorylation and the subsequent nuclear migration of the CREB-regulated transcription factors (CRTCs) are required to stimulate CREB transcriptional activity. Among the 3 identified mammalian homologs of CRTCs, CRTC3 has been shown to be expressed predominantly in adipose tissues in response to catecholamine signals that regulate lipid metabolism. Here, we show that prolonged cAMP signaling down-regulates CRTC3 in a proteasome-dependent manner and that neural precursor cell-expressed developmentally down-regulated gene 4-like (NEDD4L), a specific ubiquitin ligase for CRTC3, is responsible for this process. By recognizing the PY motif of CRTC3, NEDD4L interacts with CRTC3 and promotes its polyubiquitination. Interaction between NEDD4L and CRTC3 is further boosted by cAMP signaling, and this enhanced interaction appears to be dependent on the cAMP-mediated phosphorylation of NEDD4L at the Ser448 site. Furthermore, we show that food withdrawal stimulates NEDD4L phosphorylation in mice, which then show a decrease of adipose tissue CRTC3 protein levels. Together, these results suggest that NEDD4L plays a key role in the feedback regulation of cAMP signaling by limiting CRTC3 protein levels.-Kim, Y.-H., Yoo, H., Hong, A.-R., Kwon, M., Kang, S.-W., Kim, K., Song, Y. NEDD4L limits cAMP signaling through ubiquitination of CREB-regulated transcription coactivator 3.

Novel regulation of melanogenesis by adiponectin via the AMPK/CRTC pathway.

Several studies observed that adiponectin, an important adipokine that improves glucose metabolism by regulating AMP-activated protein kinase (AMPK) signaling, is dermatologically beneficial. In our recent microarray data, we found that adiponectin expression was lower in lesional skin than in non-lesional skin of melasma patients. Given that AMPK is a key adiponectin signaling mediator, we investigated the role of adiponectin and AICAR, a cell-permeable AMPK activator, in melanogenesis. We herein showed that adiponectin and AICAR downregulated MITF, tyrosinase, TRP-1, and DCT expression and reduced melanin content in normal human and mouse melanocytes. The depigmenting effect of adiponectin was mediated via AMPK activation, which induced the inhibitory phosphorylation of CREB-regulated transcription co-activators (CRTCs) and subsequent suppression of the novel CRTC/CREB pathway in melanocytes. These findings suggest that adiponectin and its analogs are useful as a clinical strategy for treating hyperpigmentation disorders.

Distinct Fragmentation Pathways of Anticancer Drugs Induced by Charge-Carrying Cations in the Gas Phase.

With the growth of the pharmaceutical industry, structural elucidation of drugs and derivatives using tandem mass spectrometry (MS2) has become essential for drug development and pharmacokinetics studies because of its high sensitivity and low sample requirement. Thus, research seeking to understand fundamental relationships between fragmentation patterns and precursor ion structures in the gas phase has gained attention. In this study, we investigate the fragmentation of the widely used anticancer drugs, doxorubicin (DOX), vinblastine (VBL), and vinorelbine (VRL), complexed by a singly charged proton or alkali metal ion (Li+, Na+, K+) in the gas phase. The drug-cation complexes exhibit distinct fragmentation patterns in tandem mass spectra as a function of cation size. The trends in fragmentation patterns are explicable in terms of structures derived from ion mobility mass spectrometry (IM-MS) and theoretical calculations. Graphical Abstract ᅟ.

Structural Characterization of Anticancer Drug Paclitaxel and Its Metabolites Using Ion Mobility Mass Spectrometry and Tandem Mass Spectrometry.

Paclitaxel (PTX) is a popular anticancer drug used in the treatment of various types of cancers. PTX is metabolized in the human liver by cytochrome P450 to two structural isomers, 3'-p-hydroxypaclitaxel (3p-OHP) and 6α-hydroxypaclitaxel (6α-OHP). Analyzing PTX and its two metabolites, 3p-OHP and 6α-OHP, is crucial for understanding general pharmacokinetics, drug activity, and drug resistance. In this study, electrospray ionization ion mobility mass spectrometry (ESI-IM-MS) and collision induced dissociation (CID) are utilized for the identification and characterization of PTX and its metabolites. Ion mobility distributions of 3p-OHP and 6α-OHP indicate that hydroxylation of PTX at different sites yields distinct gas phase structures. Addition of monovalent alkali metal and silver metal cations enhances the distinct dissociation patterns of these structural isomers. The differences observed in the CID patterns of metalated PTX and its two metabolites are investigated further by evaluating their gas-phase structures. Density functional theory calculations suggest that the observed structural changes and dissociation pathways are the result of the interactions between the metal cation and the hydroxyl substituents in PTX metabolites.

Molecular cloning, regulation, and functional analysis of two GHS-R genes in zebrafish.

Mammalian ghrelin is derived from stomach and regulates growth hormone release and appetite by modulating GHS-R (Growth hormone secretagogue receptor) activity. Zebrafish has been developed as a forward genetic screening model system and previous screening identified a number of genes involved in multiple signaling pathways. In this system, ghrelin has been identified and its function and regulation have been shown to be highly conserved to that of mammals. Here, we identified three isoforms of zGHS-R1 and one of zGHS-R2 (zGHS-R2a), and characterized their expression, regulation and function. Three isoforms of zGHS-R1, which we named zGHS-R1a, zGHS-R1b, and zGHS-R1c, are generated by alternative splicing. The expression of zGHS-R1 is highly enriched in brain, intestine tissue, and skin tissues. Compared to zGHS-R1, the expression pattern of zGHS-R2a is rather evenly distributed. A 15 day fasting elevated expression of zGHS-R1 and zGHS-R2 transcripts in anterior intestine tissues, but not in brain. Whereas zGHS-R1a, zGHS-R1c, and zGHS-R2a appear to be presented on the plasma membrane, the localization of zGHS-R1b seems to be restricted in the intracellular region. Treatment of ghrelin agonist, L692,585 or goldfish ghrelin peptides but not rat ghrelin, elevated intracellular Ca(2+) level and phosphorylation of ERK in HEK-293 cells expressing zGHS-R1a, but not zGHS-R1b, zGHS-R1c, or zGHS-R2a. It appears that besides core ghrelin peptide sequence of GS/TSF additional amino acids are required for the activation of zGHS-R1a, as rat ghrelin induces neither intracellular Ca(2+) mobilization nor ERK phosphrylation. These results suggest that ghrelin system in zebrafish is highly conserved to that of mammals, and thus is an ideal in vivo model for dissecting ghrelin system.

Cu2+-induced intermolecular static excimer formation of pyrenealkylamine.

Synthesis of monopyrenylalkylamine derivative 1 and its fluorescence behavior for Cu2+ in H2O/CH3CN (1:1, v/v) were investigated. Upon Cu2+ binding, 1, bearing a sulfonamide group, exhibited a marked excimer emission at 455 nm along with a weak monomer emission at 375 nm. The excimer emission, driven by formation of an intermolecular pyrenyl static excimer upon Cu2+ binding to the sulfonamide group, is rationalized by experimental and theoretical DFT calculation results.