JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
Biological functions of casein kinase 1 isoforms and putative roles in tumorigenesis.
Mol. Cancer
PUBLISHED: 05-16-2014
Show Abstract
Hide Abstract
Isoforms of the casein kinase 1 (CK1) family have been shown to phosphorylate key regulatory molecules involved in cell cycle, transcription and translation, the structure of the cytoskeleton, cell-cell adhesion and receptor-coupled signal transduction. They regulate key signaling pathways known to be critically involved in tumor progression. Recent results point to an altered expression or activity of different CK1 isoforms in tumor cells. This review summarizes the expression and biological function of CK1 family members in normal and malignant cells and the evidence obtained so far about their role in tumorigenesis.
Related JoVE Video
Quantitative Proteomics of the Human Skin Secretome reveal a Reduction of Immune Defense Mediators in Ectodermal Dysplasia Patients.
J. Invest. Dermatol.
PUBLISHED: 04-10-2014
Show Abstract
Hide Abstract
In healthy human skin host defense molecules such as antimicrobial peptides (AMPs) contribute to skin immune homeostasis. In patients with the congenital disease ectodermal dysplasia (ED) skin integrity is disturbed and as a result patients have recurrent skin infections. The disease is characterized by developmental abnormalities of ectodermal derivatives and absent or reduced sweating. We hypothesized that ED patients have a reduced skin immune defense due to the reduced ability to sweat. Therefore, we performed a label-free quantitative proteome analysis of wash solution of human skin from ED patients or healthy individuals. A clear cut difference between both cohorts could be observed in cellular processes related to immunity and host defense. In line with the extensive underrepresentation of proteins of the immune system, dermcidin, a sweat derived AMP, was reduced in its abundance in the skin secretome of ED patients. In contrast, proteins involved in metabolic/catabolic- and biosynthetic processes were enriched in the skin secretome of ED patients. In summary, our proteome profiling provides insights into the actual situation of healthy versus diseased skin. The systematic reduction of immune system and defense related proteins may contribute to the high susceptibility of ED patients to skin infections and altered skin colonization.Journal of Investigative Dermatology accepted article preview online, 27 October 2014. doi:10.1038/jid.2014.462.
Related JoVE Video
Y-box binding protein 1--a prognostic marker and target in tumour therapy.
Eur. J. Cell Biol.
PUBLISHED: 01-28-2014
Show Abstract
Hide Abstract
Y-box binding protein 1 (YB-1) is a multifunctional protein involved in various cellular processes including both transcriptional and translational regulation of target gene expression. Significantly increased YB-1 levels have been reported in a number of human malignancies and shown to be associated with poor prognosis and disease recurrence. Indeed, YB-1 can act as a versatile oncoprotein playing an important role in tumour cell proliferation and progression. Consequently, YB-1 not only proves to be a good prognostic tumour marker, but also may be a promising emerging molecular target for the development of new therapeutical strategies. In this review, we discuss both the role of YB-1 in cancer and specifically in malignant melanoma as well as possible translations into the clinics derived thereof.
Related JoVE Video
Processing of laminin ? chains generates peptides involved in wound healing and host defense.
J Innate Immun
PUBLISHED: 01-18-2014
Show Abstract
Hide Abstract
Laminins play a fundamental role in basement membrane architecture and function in human skin. The C-terminal laminin G domain-like (LG) modules of laminin ? chains are modified by proteolysis to generate LG1-3 and secreted LG4-5 tandem modules. In this study, we provide evidence that skin-derived cells process and secrete biologically active peptides from the LG4-5 module of the laminin ?3, ?4 and ?5 chain in vitro and in vivo. We show enhanced expression and processing of the LG4-5 module of laminin ?3 in keratinocytes after infection and in chronic wounds in which the level of expression and further processing of the LG4-5 module correlated with the speed of wound healing. Furthermore, bacterial or host-derived proteases promote processing of laminin ?3 LG4-5. On a functional level, we show that LG4-5-derived peptides play a role in wound healing. Moreover, we demonstrate that LG4-derived peptides from the ?3, ?4 and ?5 chains have broad antimicrobial activity and possess strong chemotactic activity to mononuclear cells. Thus, the data strongly suggest a novel multifunctional role for laminin LG4-5-derived peptides in human skin and its involvement in physiological processes and pathological conditions such as inflammation, chronic wounds and skin infection.
Related JoVE Video
Melanoma inhibitory activity promotes melanoma development through activation of YBX1.
Pigment Cell Melanoma Res
PUBLISHED: 03-01-2013
Show Abstract
Hide Abstract
Melanoma inhibitory activity (MIA), a small soluble secreted protein, is functionally important for progression of malignant melanoma. We recently revealed that p54(nrb) acts as a mediator of MIA action. In this study, we characterize the transcriptional regulation of p54(nrb) by MIA to explain MIAs molecular action. We identified one highly conserved region in the p54(nrb) promoter that is necessary and sufficient for MIA-dependent activation. Functional promoter analysis identified the transcription factor YBX1 as the mediator of MIA activation of p54(nrb) transcription. We screened the genome for further potential MIA-regulated genes carrying the element in their promoter regions. Integrating our sequence data with expression data from human melanomas identified a list of 23 potential MIA-YBX1 targets in melanomas. In summary, we present for the first time effects of MIA on transcriptional regulation. Uncovering new potential downstream effectors working via activation of YBX1 supports the important role of MIA in melanoma.
Related JoVE Video
Vemurafenib potently induces endoplasmic reticulum stress-mediated apoptosis in BRAFV600E melanoma cells.
Sci Signal
PUBLISHED: 01-31-2013
Show Abstract
Hide Abstract
The V600E mutation in the kinase BRAF is frequently detected in melanomas and results in constitutive activation of BRAF, which then promotes cell proliferation by the mitogen-activated protein kinase signaling pathway. Although the BRAFV600E kinase inhibitor vemurafenib has remarkable antitumor activity in patients with BRAFV600E-mutated melanoma, its effects are limited by the onset of drug resistance. We found that exposure of melanoma cell lines with the BRAFV600E mutation to vemurafenib decreased the abundance of antiapoptotic proteins and induced intrinsic mitochondrial apoptosis. Vemurafenib-treated melanoma cells showed increased cytosolic concentration of calcium, a potential trigger for endoplasmic reticulum (ER) stress, which can lead to apoptosis. Consistent with an ER stress-induced response, vemurafenib decreased the abundance of the ER chaperone protein glucose-regulated protein 78, increased the abundance of the spliced isoform of the transcription factor X-box binding protein 1 (XBP1) (which transcriptionally activates genes involved in ER stress responses), increased the phosphorylation of the translation initiation factor eIF2? (which would be expected to inhibit protein synthesis), and induced the expression of ER stress-related genes. Knockdown of the ER stress response protein activating transcription factor 4 (ATF4) significantly reduced vemurafenib-induced apoptosis. Moreover, the ER stress inducer thapsigargin prevented invasive growth of tumors formed from vemurafenib-sensitive melanoma cells in vivo. In melanoma cells with low sensitivity or resistance to vemurafenib, combination treatment with thapsigargin augmented or induced apoptosis. Thus, thapsigargin or other inducers of ER stress may be useful in combination therapies to overcome vemurafenib resistance.
Related JoVE Video
Staphylococcus aureus skin colonization is promoted by barrier disruption and leads to local inflammation.
Exp. Dermatol.
PUBLISHED: 01-03-2013
Show Abstract
Hide Abstract
Experimental mouse models of bacterial skin infections that have been described show that pathogenic microorganisms can readily invade the epidermis and dermis to produce localized infections. We used an epicutaneous mouse skin infection model to determine how the level of barrier disruption by tape-stripping correlates with persistence of Staphylococcus aureus skin colonization, concomitant induction of cutaneous inflammation and infection. Furthermore, we investigated how murine skin responds to S. aureus colonization in a physiologic setting by analysing proinflammatory cytokines and antimicrobial peptides in mouse skin. We show that previous cutaneous damage allows skin inflammation to develop and favours S. aureus persistence leading to cutaneous colonization, suggesting an interdependence of cutaneous bacteria and skin. Our study suggests that skin barrier defects favour S. aureus skin colonization, which is associated with profound cutaneous inflammation.
Related JoVE Video
YBX1 Is a Modulator of MIA/CD-RAP-Dependent Chondrogenesis.
PLoS ONE
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
MIA/CD-RAP is a small, secreted protein involved in cartilage differentiation and melanoma progression. We recently revealed that p54(nrb) acts as a mediator of MIA/CD-RAP action to promote chondrogenesis and the progression of malignant melanoma. As the molecular mechanism of MIA/CD-RAP action in cartilage has not been defined in detail until now, we aimed to understand the regulation of p54(nrb) transcription in chondrogenesis. We concentrated on the previously described MIA/CD-RAP-dependent regulatory region in the p54(nrb) promoter and characterized the transcriptional regulation of p54(nrb) by MIA/CD-RAP in cartilage. A series of truncated p54(nrb) promoter constructs and mutagenesis analysis revealed that the transcription factor YBX1, which has not been investigated in chondrogenesis thus far, is the mediator of MIA/CD-RAP dependent activation of p54(nrb) transcription. A systematic analysis of genes carrying this binding site in their promoter region revealed further potential MIA/CD-RAP-regulated genes that have been implicated in cartilage differentiation. In summary, we described the effects of MIA/CD-RAP on transcriptional regulation in chondrocytes. Understanding the regulation of p54(nrb) via YBX1 contributes to the understanding of chondrogenesis. Uncovering new downstream effectors that function via the activation of YBX1 supports the important role of MIA/CD-RAP in these processes.
Related JoVE Video
ETS-1/RhoC signaling regulates the transcription factor c-Jun in melanoma.
Int. J. Cancer
PUBLISHED: 06-17-2011
Show Abstract
Hide Abstract
Recently, we discovered that the loss of E-cadherin induces c-Jun protein expression, which is a member of the AP-1 transcription factor family and a key player in the processes of cell proliferation and tumor development and also found in elevated levels in melanomas. Notably, the mRNA level of c-Jun was not affected, suggesting that c-Jun is regulated at post-transcriptional level. Here, we present data that suggest that the dynamic cytoskeletal network, linked to E-cadherin, is involved in the regulation of the c-Jun protein and transcriptional activity. In a signaling cascade, the loss of E-cadherin activates the transcriptional regulator ETS-1 and consequently leads to the induction of RhoC expression that stabilizes c-Jun in melanoma. The link between RhoC and c-Jun seems to be indirect via the cytoskeleton. We conclude that the loss of E-cadherin mediated cell-adhesion induces c-Jun protein expression in a multistep process, offering several possibilities for therapeutic intervention.
Related JoVE Video
The antimicrobial skin barrier in patients with atopic dermatitis.
Curr. Probl. Dermatol.
PUBLISHED: 05-12-2011
Show Abstract
Hide Abstract
Keratinocytes represent the major cell population in the epithelial skin barrier and actively participate in innate immune responses by recognizing pathogenic microorganisms, followed by a fine-tuned production of cytokines, chemokines and antimicrobial peptides or proteins (AMPs). Patients with atopic dermatitis (AD) suffer from a defective permeability barrier which favors pathogen infection indicating that the permeability and antimicrobial barrier functions are interdependent. Several early studies showed that the inducible AMPs LL-37, HBD-2 and HBD-3 are expressed at lower levels in atopic skin compared to psoriatic skin. However, recent data indicate that AMP induction is not compromised in AD patients and that several AMPs are expressed at significantly higher amounts in AD compared to healthy skin. AD patients have an increased susceptibility to Staphylococcus aureus skin infection suggesting that AMP levels expressed by keratinocytes of AD patients might not be sufficient to combat pathogenic skin infection or that AMP function is disturbed. Increasing AMP expression in AD skin and repairing the skin barrier defect might have a therapeutic effect in AD patients enabling the skin to mount an enhanced response to pathogens.
Related JoVE Video
Impact of oncogenic K-RAS on YB-1 phosphorylation induced by ionizing radiation.
Breast Cancer Res.
PUBLISHED: 03-10-2011
Show Abstract
Hide Abstract
Expression of Y-box binding protein-1 (YB-1) is associated with tumor progression and drug resistance. Phosphorylation of YB-1 at serine residue 102 (S102) in response to growth factors is required for its transcriptional activity and is thought to be regulated by cytoplasmic signaling phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways. These pathways can be activated by growth factors and by exposure to ionizing radiation (IR). So far, however, no studies have been conducted on IR-induced YB-1 phosphorylation.
Related JoVE Video
?-Catenin signaling increases during melanoma progression and promotes tumor cell survival and chemoresistance.
PLoS ONE
PUBLISHED: 02-28-2011
Show Abstract
Hide Abstract
Beta-catenin plays an important role in embryogenesis and carcinogenesis by controlling either cadherin-mediated cell adhesion or transcriptional activation of target gene expression. In many types of cancers nuclear translocation of beta-catenin has been observed. Our data indicate that during melanoma progression an increased dependency on the transcriptional function of beta-catenin takes place. Blockade of beta-catenin in metastatic melanoma cell lines efficiently induces apoptosis, inhibits proliferation, migration and invasion in monolayer and 3-dimensional skin reconstructs and decreases chemoresistance. In addition, subcutaneous melanoma growth in SCID mice was almost completely inhibited by an inducible beta-catenin knockdown. In contrast, the survival of benign melanocytes and primary melanoma cell lines was less affected by beta-catenin depletion. However, enhanced expression of beta-catenin in primary melanoma cell lines increased invasive capacity in vitro and tumor growth in the SCID mouse model. These data suggest that beta-catenin is an essential survival factor for metastatic melanoma cells, whereas it is dispensable for the survival of benign melanocytes and primary, non-invasive melanoma cells. Furthermore, beta-catenin increases tumorigenicity of primary melanoma cell lines. The differential requirements for beta-catenin signaling in aggressive melanoma versus benign melanocytic cells make beta-catenin a possible new target in melanoma therapy.
Related JoVE Video
Skin commensals amplify the innate immune response to pathogens by activation of distinct signaling pathways.
J. Invest. Dermatol.
PUBLISHED: 11-04-2010
Show Abstract
Hide Abstract
Little is known about the impact of different microbial signals on skin barrier organ function and the interdependency between resident microflora and pathogenic microorganisms. This study shows that commensal and pathogenic staphylococci differ in their ability to induce expression of antimicrobial peptides/proteins (AMPs) and activate different signaling pathways in human primary keratinocytes. Whereas secreted factors of skin commensals induce expression of the AMPs HBD-3 and RNase7 in primary human keratinocytes via Toll-like receptor (TLR)-2, EGFR, and NF-?B activation, those of pathogenic staphylococci activate the mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT signaling pathways and suppress NF-?B activation. Interestingly, commensal bacteria are able to amplify the innate immune response of human keratinocytes to pathogens by increased induction of AMP expression and abrogation of NF-?B suppression, suggesting that the two activation pathways can act in a synergistic way. These data indicate that commensal and pathogenic microorganisms evolved specific mechanisms to modulate innate immunity of the skin.
Related JoVE Video
The farnesyl transferase inhibitor lonafarnib inhibits mTOR signaling and enforces sorafenib-induced apoptosis in melanoma cells.
J. Invest. Dermatol.
PUBLISHED: 10-14-2010
Show Abstract
Hide Abstract
Farnesyl transferase inhibitors (FTIs) inhibit the farnesylation of proteins, including RAS and RHEB (Ras homolog enriched in brain). RAS signals to the RAF-MEK-ERK (MAPK) and PI3K-AKT-mTOR (AKT) signaling pathways, which have a major role in melanoma progression. RHEB positively regulates mammalian target of rapamycin (mTOR). We investigated the effects of the FTI lonafarnib alone and in combination with MAPK (mitogen-activated protein kinase) or AKT (acutely transforming retrovirus AKT8 in rodent T-cell lymphoma) pathway inhibitors on proliferation, survival, and invasive tumor growth of melanoma cells. Lonafarnib alone did not sufficiently inhibit melanoma cell growth. Combinations of lonafarnib with AKT pathway inhibitors did not significantly increase melanoma cell growth inhibition. In contrast, combinations of lonafarnib with MAPK pathway inhibitors yielded additional growth-inhibiting effects. In particular, the combination of the FTI lonafarnib with the pan-RAF inhibitor sorafenib synergistically inhibited melanoma cell growth, significantly enhanced sorafenib-induced apoptosis, and completely suppressed invasive tumor growth in monolayer and organotypic cultures, respectively. Apoptosis induction was associated with upregulation of the endoplasmic reticulum stress-related transcription factors p8 and CHOP (CAAT/enhancer binding protein (C/EBP) homologous protein), and downregulation of the antiapoptotic Bcl-2 (B-cell lymphoma-2) family protein Mcl-1(myeloid cell leukemia 1). Lonafarnib did not affect MAPK and AKT but did affect mTOR signaling. Together, these findings suggest that the FTI lonafarnib inhibits mTOR signaling and enforces sorafenib-induced apoptosis in melanoma cells and may therefore represent an effective alternative for melanoma treatment.
Related JoVE Video
Suppression of casein kinase 1alpha in melanoma cells induces a switch in beta-catenin signaling to promote metastasis.
Cancer Res.
PUBLISHED: 08-10-2010
Show Abstract
Hide Abstract
Casein kinase 1 alpha (CK1alpha) is a multifunctional Ser/Thr kinase that phosphorylates several substrates. Among those is beta-catenin, an important player in cell adhesion and Wnt signaling. Phosphorylation of beta-catenin by CK1alpha at Ser45 is the priming reaction for the proteasomal degradation of beta-catenin. Interestingly, aside from this role in beta-catenin degradation, very little is known about the expression and functional role of CK1alpha in tumor cells. Here, we show that CK1alpha expression in different tumor types is either strongly suppressed or completely lost during tumor progression and that CK1alpha is a key factor determining beta-catenin stability and transcriptional activity in tumor cells. CK1alpha reexpression in metastatic melanoma cells reduces growth in vitro and metastasis formation in vivo, and induces cell cycle arrest and apoptosis, whereas suppression of CK1alpha in primary melanoma cells induces invasive tumor growth. Inactivation of CK1alpha promotes tumor progression by regulating a switch in beta-catenin-mediated signaling. These results show that melanoma cells developed an efficient new mechanism to activate the beta-catenin signaling pathway and define CK1alpha as a novel tumor suppressor.
Related JoVE Video
Severity of Staphylococcus aureus infection of the skin is associated with inducibility of human beta-defensin 3 but not human beta-defensin 2.
Infect. Immun.
PUBLISHED: 04-19-2010
Show Abstract
Hide Abstract
Gram-positive bacteria are the predominant cause of skin infections. Antimicrobial peptides (AMPs) are believed to be of major importance in skins innate defense against these pathogens. This study aimed at providing clinical evidence for the contribution of AMP inducibility to determining the severity of Gram-positive skin infection. Using real-time PCR, we determined the induction of human beta-defensin 2 (HBD-2), HBD-3, and RNase 7 by comparing healthy and lesional mRNA levels in 32 patients with Gram-positive skin infection. We then examined whether AMP induction differed by disease severity, as measured by number of recurrences and need for surgical drainage in patients with Staphylococcus aureus-positive lesions. We found that HBD-2 and -3, but not RNase 7, mRNA expression was highly induced by Gram-positive bacterial infection in otherwise healthy skin. Less induction of HBD-3, but not HBD-2, was associated with more-severe S. aureus skin infection: HBD-3 mRNA levels were 11.4 times lower in patients with more than 6 recurrences (P = 0.01) and 8.8 times lower in patients reporting surgical drainage (P = 0.01) than in the respective baseline groups. This suggests that inducibility of HBD-3 influences the severity of Gram-positive skin infection in vivo. The physiological function of HBD-2 induction in this context remains unclear.
Related JoVE Video
Interaction of C-type lectin-like receptors NKp65 and KACL facilitates dedicated immune recognition of human keratinocytes.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 03-01-2010
Show Abstract
Hide Abstract
Many well-known immune-related C-type lectin-like receptors (CTLRs) such as NKG2D, CD69, and the Ly49 receptors are encoded in the natural killer gene complex (NKC). Recently, we characterized the orphan NKC gene CLEC2A encoding for KACL, a further member of the human CLEC2 family of CTLRs. In contrast to the other CLEC2 family members AICL, CD69, and LLT1, KACL expression is mostly restricted to skin. Here we show that KACL is a non-disulfide-linked homodimeric surface receptor and stimulates cytotoxicity by human NK92MI cells. We identified the corresponding activating receptor on NK92MI cells that is encoded adjacently to the CLEC2A locus and binds KACL with high affinity. This CTLR, termed NKp65, stimulates NK cytotoxicity and release of proinflammatory cytokines upon engagement of cell-bound KACL. NKp65, a distant relative of the human activating NK receptor NKp80, possesses an amino-terminal hemITAM that is required for NKp65-mediated cytotoxicity. Finally, we show that KACL expression is mainly restricted to keratinocytes. Freshly isolated keratinocytes express KACL and are capable of stimulating NKp65-expressing cells in a KACL-dependent manner. Thus, we report a unique NKC-encoded receptor-ligand system that may fulfill a dedicated function in the immunobiology of human skin.
Related JoVE Video
Peptides derived from the human laminin alpha4 and alpha5 chains exhibit antimicrobial activity.
Peptides
PUBLISHED: 02-04-2010
Show Abstract
Hide Abstract
Laminins are a family of heterotrimeric extracellular matrix glycoproteins in the basement membrane of different tissues and are composed of alpha, beta, and gamma chains. In mammals, five different alpha chains, three beta chains, and three gamma chains have been identified that assemble into 15 different laminins. Each alpha-chain possesses a C-terminal globular domain which can be subdivided into the five subdomains LG1-LG5. LG1-LG3 modules are connected to LG4-LG5 by a linker domain which is known to be sensitive to proteolytic processing. Here, we show that peptides derived from the human laminin alpha4 and alpha5 chain, exhibit a dose-dependent antimicrobial activity against gram-positive and gram-negative bacteria. Furthermore, we show that these peptides permeabilize the bacterial membrane and are able to bind to bacterial DNA. Interestingly, the ability to kill the microorganisms correlated with their ability to bind to heparin. These data suggest that extracellular matrix components are able to protect the respective tissues from invading pathogens and are part of the host defense response.
Related JoVE Video
Constitutive expression of the antimicrobial peptide RNase 7 is associated with Staphylococcus aureus infection of the skin.
J. Infect. Dis.
PUBLISHED: 11-19-2009
Show Abstract
Hide Abstract
Staphylococcus aureus infections of the skin are a public health problem of growing importance. Antimicrobial peptides in human skin are believed to play an important role in innate defense against intruding pathogens. This study aimed to clarify whether their baseline expression influences the propensity of healthy individuals to develop S. aureus-positive skin infections.
Related JoVE Video
Staphylococcus aureus mutant screen reveals interaction of the human antimicrobial peptide dermcidin with membrane phospholipids.
Antimicrob. Agents Chemother.
PUBLISHED: 07-13-2009
Show Abstract
Hide Abstract
Antimicrobial peptides (AMPs) form an important part of the innate host defense. In contrast to most AMPs, human dermcidin has an anionic net charge. To investigate whether bacteria have developed specific mechanisms of resistance to dermcidin, we screened for mutants of the leading human pathogen, Staphylococcus aureus, with altered resistance to dermcidin. To that end, we constructed a plasmid for use in mariner-based transposon mutagenesis and developed a high-throughput cell viability screening method based on luminescence. In a large screen, we did not find mutants with strongly increased susceptibility to dermcidin, indicating that S. aureus has no specific mechanism of resistance to this AMP. Furthermore, we detected a mutation in a gene of unknown function that resulted in significantly increased resistance to dermcidin. The mutant strain had an altered membrane phospholipid pattern and showed decreased binding of dermcidin to the bacterial surface, indicating that dermcidin interacts with membrane phospholipids. The mode of this interaction was direct, as shown by assays of dermcidin binding to phospholipid preparations, and specific, as the resistance to other AMPs was not affected. Our findings indicate that dermcidin has an exceptional value for the human innate host defense and lend support to the idea that it evolved to evade bacterial resistance mechanisms targeted at the cationic character of most AMPs. Moreover, they suggest that the antimicrobial activity of dermcidin is dependent on the interaction with the bacterial membrane and might thus assist with the determination of the yet unknown mode of action of this important human AMP.
Related JoVE Video
Inhibition of PI3K-AKT-mTOR signaling sensitizes melanoma cells to cisplatin and temozolomide.
J. Invest. Dermatol.
PUBLISHED: 06-23-2009
Show Abstract
Hide Abstract
In melanoma, the PI3K-AKT-mTOR (AKT) and RAF-MEK-ERK (MAPK) signaling pathways are constitutively activated and appear to play a role in chemoresistance. Herein, we investigated the effects of pharmacological AKT and MAPK pathway inhibitors on chemosensitivity of melanoma cells to cisplatin and temozolomide. Chemosensitivity was tested by examining effects on growth, cell cycle, survival, expression of antiapoptotic proteins, and invasive tumor growth of melanoma cells in monolayer and organotypic culture, respectively. MAPK pathway inhibitors did not significantly increase chemosensitivity. AKT pathway inhibitors consistently enhanced chemosensitivity yielding an absolute increase of cell growth inhibition up to 60% (P<0.05, combination therapy vs monotherapy with inhibitors or chemotherapeutics). Cotreatment of melanoma cells with AKT pathway inhibitors and chemotherapeutics led to a 2- to 3-fold increase of apoptosis (P<0.05, combination therapy vs monotherapy) and completely suppressed invasive tumor growth in organotypic culture. These effects were associated with suppression of the antiapoptotic Bcl-2 family protein Mcl-1. These data suggest that inhibition of the PI3K-AKT-mTOR pathway potently increases sensitivity of melanoma cells to chemotherapy.
Related JoVE Video
Dermcidin-derived peptides show a different mode of action than the cathelicidin LL-37 against Staphylococcus aureus.
Antimicrob. Agents Chemother.
PUBLISHED: 04-13-2009
Show Abstract
Hide Abstract
Dermcidin (DCD) is an antimicrobial peptide which is constitutively expressed in eccrine sweat glands. By postsecretory proteolytic processing in sweat, the DCD protein gives rise to anionic and cationic DCD peptides with a broad spectrum of antimicrobial activity. Many antimicrobial peptides induce membrane permeabilization as part of their killing mechanism, which is accompanied by a loss of the bacterial membrane potential. In this study we show that there is a time-dependent bactericidal activity of anionic and cationic DCD-derived peptides which is followed by bacterial membrane depolarization. However, DCD-derived peptides do not induce pore formation in the membranes of gram-negative and gram-positive bacteria. This is in contrast to the mode of action of the cathelicidin LL-37. Interestingly, LL-37 as well as DCD-derived peptides inhibit bacterial macromolecular synthesis, especially RNA and protein synthesis, without binding to microbial DNA or RNA. Binding studies with components of the cell envelope of gram-positive and gram-negative bacteria and with model membranes indicated that DCD-derived peptides bind to the bacterial envelope but show only a weak binding to lipopolysaccharide (LPS) from gram-negative bacteria or to peptidoglycan, lipoteichoic acid, and wall teichoic acid, isolated from Staphylococcus aureus. In contrast, LL-37 binds strongly in a dose-dependent fashion to these components. Altogether, these data indicate that the mode of action of DCD-derived peptides is different from that of the cathelicidin LL-37 and that components of the bacterial cell envelope play a role in the antimicrobial activity of DCD.
Related JoVE Video
miR-137 inhibits the invasion of melanoma cells through downregulation of multiple oncogenic target genes.
J. Invest. Dermatol.
Show Abstract
Hide Abstract
MicroRNAs are small noncoding RNAs that regulate gene expression and have important roles in various types of cancer. Previously, miR-137 was reported to act as a tumor suppressor in different cancers, including malignant melanoma. In this study, we show that low miR-137 expression is correlated with poor survival in stage IV melanoma patients. We identified and validated two genes (c-Met and YB1) as direct targets of miR-137 and confirmed two previously known targets, namely enhancer of zeste homolog 2 (EZH2) and microphthalmia-associated transcription factor (MITF). Functional studies showed that miR-137 suppressed melanoma cell invasion through the downregulation of multiple target genes. The decreased invasion caused by miR-137 overexpression could be phenocopied by small interfering RNA knockdown of EZH2, c-Met, or Y box-binding protein 1 (YB1). Furthermore, miR-137 inhibited melanoma cell migration and proliferation. Finally, miR-137 induced apoptosis in melanoma cell lines and decreased BCL2 levels. In summary, our study confirms that miR-137 acts as a tumor suppressor in malignant melanoma and reveals that miR-137 regulates multiple targets including c-Met, YB1, EZH2, and MITF.
Related JoVE Video
The multiple facets of dermcidin in cell survival and host defense.
J Innate Immun
Show Abstract
Hide Abstract
Eccrine sweat glands, which are distributed over the whole bodies of primates and humans, have long been regarded mainly to have a function in thermoregulation. However, the discovery of dermcidin-derived antimicrobial peptides in eccrine sweat demonstrated that sweat actively participates in the constitutive innate immune defense of human skin against infection. In the meantime, a number of studies proved the importance of dermcidin in skin host defense. Several reports also state that peptides processed from the dermcidin precursor protein exhibit a range of other biological functions in neuronal and cancer cells. This review summarizes the evidence gathered until now concerning the expression of dermcidin and the functional relevance of dermcidin-derived peptides.
Related JoVE Video
MAPK and PI3K/AKT mediated YB-1 activation promotes melanoma cell proliferation which is counteracted by an autoregulatory loop.
Exp. Dermatol.
Show Abstract
Hide Abstract
Y-box binding protein 1 (YB-1) is an oncogenic transcription and translation factor and is overexpressed in several types of cancer. Our previous data showed that YB-1 is upregulated and translocated to the nucleus during melanoma progression and that YB-1 is an important transcription factor regulating proliferation, survival, migration, invasion and chemosensitivity of melanoma cells. It has been suggested that YB-1 is activated and translocated to the nucleus after S102-phosphorylation in the DNA binding domain. In this study, we show that activation of YB-1 by S102-phosphorylation and nuclear translocation is increased during melanoma progression using a human tissue microarray with 100 melanocytic lesions. Furthermore, we analysed the mechanisms governing the expression and activity of YB-1 in melanoma cells. We show that the PI3K/AKT and p53 signalling, growth factors and chemotherapeutic agents increase YB-1 promoter activity. This, however, resulted in no or only modest increase in YB-1 protein expression. We show that the MAPK and PI3K/AKT signalling pathways, both activated in melanoma cells, as well as p53 overexpression increase YB-1 S102-phosphorylation, whereas NF?B signalling inhibits phosphorylation. Overexpression of YB-1 in melanoma cells inhibits translation efficiency and by this proliferation and survival of melanoma cells indicating that there is an autoregulatory loop restricting YB-1 protein expression. These data suggest that there is a tightly regulated feedback mechanism regulating YB-1 expression and activation, necessary for proper cell cycle progression of melanoma cells.
Related JoVE Video
Structure-activity analysis of the dermcidin-derived peptide DCD-1L, an anionic antimicrobial peptide present in human sweat.
J. Biol. Chem.
Show Abstract
Hide Abstract
Dermcidin encodes the anionic amphiphilic peptide DCD-1L, which displays a broad spectrum of antimicrobial activity under conditions resembling those in human sweat. Here, we have investigated its mode of antimicrobial activity. We found that DCD-1L interacts preferentially with negatively charged bacterial phospholipids with a helix axis that is aligned flat on a lipid bilayer surface. Upon interaction with lipid bilayers DCD-1L forms oligomeric complexes that are stabilized by Zn(2+). DCD-1L is able to form ion channels in the bacterial membrane, and we propose that Zn(2+)-induced self-assembly of DCD-1L upon interaction with bacterial lipid bilayers is a prerequisite for ion channel formation. These data allow us for the first time to propose a molecular model for the antimicrobial mechanism of a naturally processed human anionic peptide that is active under the harsh conditions present in human sweat.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.