Dynamic transcriptome analysis reveals signatures of paradoxical effect of vemurafenib on human dermal fibroblasts

Abstract Background Vemurafenib (PLX4032) is one of the most frequently used treatments for late-stage melanoma patients with the BRAFV600E mutation; however, acquired resistance to the drug poses as a major challenge. It remains to be determined whether off-target effects of vemurafenib on normal stroma components could reshape the tumor microenvironment in a way that contributes to cancer progression and drug resistance. Methods By using temporally-resolved RNA- and ATAC-seq, we studied the early molecular changes induced by vemurafenib in human dermal fibroblast (HDF), a main stromal component in melanoma and other tumors with high prevalence of BRAFV600 mutations. Results Transcriptomics analyses revealed a stepwise up-regulation of proliferation signatures, together with a down-regulation of autophagy and proteolytic processes. The gene expression changes in HDF strongly correlated in an inverse way with those in BRAFV600E mutant malignant melanoma (MaMel) cell lines, consistent with the observation of a paradoxical effect of vemurafenib, leading to hyperphosphorylation of MEK1/2 and ERK1/2. The transcriptional changes in HDF were not strongly determined by alterations in chromatin accessibility; rather, an already permissive chromatin landscape seemed to facilitate the early accessibility to MAPK/ERK-regulated transcription factor binding sites. Combinatorial treatment with the MEK inhibitor trametinib did not preclude the paradoxical activation of MAPK/ERK signaling in HDF. When administered together, vemurafenib partially compensated for the reduction of cell viability and proliferation induced by trametinib. These paradoxical changes were restrained by using the third generation BRAF inhibitor PLX8394, a so-called paradox breaker compound. However, the advantageous effects on HDF during combination therapies were also lost. Conclusions Vemurafenib induces paradoxical changes in HDF, enabled by a permissive chromatin landscape. These changes might provide an advantage during combination therapies, by compensating for the toxicity induced in stromal cells by less specific MAPK/ERK inhibitors. Our results highlight the relevance of evaluating the effects of the drugs on non-transformed stromal components, carefully considering the implications of their administration either as mono- or combination therapies.

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Abstract 189: HuR, RNA Stabilizing Protein, Regulation of Pluripotency and Differentiation in iPSCs

Circulation Research ◽

10.1161/res.121.suppl_1.189 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Sahana Suresh Babu ◽

Johnson Rajasingh ◽

Wing Tak Wong ◽

Prasanna Krishnamurthy

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Dermal Fibroblast ◽

Critical Role ◽

Human Dermal Fibroblast ◽

Dermal Fibroblasts ◽

General Observation ◽

Transcript Stability ◽

Differentiated Cells ◽

Induced Pluripotent

Background: The Hu family of RNA-binding proteins, HuR (also known as ELAVL1 or human embryonic lethal abnormal vision-like protein), binds to the 3’-untranslated region of mRNAs and regulates transcript stability and translation. Global deletion of HuR is embryonically lethal in mice and plays a critical role in progenitor cell survival and biology. Induced-pluripotent stem cells (iPSC) have distinct transcriptional machinery for the maintenance of pluripotency and achievement of differentiation. However, the exact role of HuR in pluripotency or differentiation of iPSC to cardiomyocytes (iCM) remains unclear. Methods: HuR knockdown in human dermal fibroblast-derived iPSCs was achieved by CRISPR/Cas9 or lentiviral shRNA transduction and subsequently differentiated into cardiomyocytes (iCM). Then, the expression of HuR, pluripotency and cardiomyocyte markers were evaluated on days 0, 1, 3, 6, 8 and 17 following the initiation of differentiation. Results: At basal level, HuR expression was higher in the iPSCs compared to dermal fibroblasts. Upon differentiation of iPSCs into iCM, HuR mRNA expression gradually reduced with significantly lower levels on day 17. As expected, pluripotency markers gradually reduced upon differentiation with significantly lower levels from day 6 onwards. We observed a corresponding increase in ISL1, MESP1 (mesoderm/cardiac progenitor markers) from day 3 through day 8 with a steep fall from day 8 to day 17. This was associated with Myosin light chain-2V and GATA4 expression increases from day 8 through day 17. Interestingly, knockdown of HuR resulted in clumps of colonies with differentiated cells and a corresponding increase in cardiac-troponin positive cells. However, as a general observation, HuR knockdown reduced beating intensity compared to wild type cells. Conclusions: Based on these data, we could speculate that HuR might be necessary for maintenance of pluripotency and loss of which renders cells to differentiate in culture. HuR knockdown yields higher number of c-troponin positive cells but its effect on functional maturity of iCM needs to be further evaluated.

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Wound Healing Potential of Spirulina Protein on CCD-986sk Cells

Marine Drugs ◽

10.3390/md17020130 ◽

2019 ◽

Vol 17 (2) ◽

pp. 130

Author(s):

Ping Liu ◽

Jeong-Wook Choi ◽

Min-Kyeong Lee ◽

Youn-Hee Choi ◽

Taek-Jeong Nam

Keyword(s):

Wound Healing ◽

Dermal Fibroblast ◽

Fibroblast Cell ◽

Underlying Mechanism ◽

Human Dermal Fibroblast ◽

Dermal Fibroblasts ◽

Cyclin Dependent Kinase ◽

Pi3k Inhibitor Ly294002 ◽

And Migration ◽

Proliferation And Migration

Wound healing is a dynamic and complex process. The proliferation and migration of dermal fibroblasts are crucial for wound healing. Recent studies have indicated that the extracts from Spirulina platensis have a positive potential for wound healing. However, its underlying mechanism is not fully understood. Our previous study showed that spirulina crude protein (SPCP) promoted the viability of human dermal fibroblast cell line (CCD-986sk cells). In this study, we further investigated the wound healing effect and corresponding mechanisms of SPCP on CCD-986sk cells. Bromodeoxyuridine (BrdU) assay showed that SPCP promoted the proliferation of CCD-986sk cells. The wound healing assay showed that SPCP promoted the migration of CCD-986sk cells. Furthermore, cell cycle analysis demonstrated that SPCP promoted CCD-986sk cells to enter S and G2/M phases from G0/G1 phase. Western blot results showed that SPCP significantly upregulated the expression of cyclin D1, cyclin E, cyclin-dependent kinase 2 (Cdk2), cyclin-dependent kinase 4 (Cdk4), and cyclin-dependent kinase 6 (Cdk6), as well as inhibited the expression of CDK inhibitors p21 and p27 in CCD-986sk cells. In the meanwhile, SPCP promoted the phosphorylation and activation of phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt). However, the phosphorylation of Akt was significantly blocked by PI3K inhibitor (LY294002), which in turn reduced the SPCP-induced proliferation and migration of CCD-986sk cells. Therefore, the results presenting in this study suggested that SPCP can promote the proliferation and migration of CCD-986sk cells; the PI3K/Akt signaling pathway play a positive and important role in these processes.

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Dermal Olfactory Receptor OR51B5 Is Essential for Survival and Collagen Synthesis in Human Dermal Fibroblast (Hs68 Cells)

International Journal of Molecular Sciences ◽

10.3390/ijms22179273 ◽

2021 ◽

Vol 22 (17) ◽

pp. 9273

Author(s):

Bomin Son ◽

Wesuk Kang ◽

Soyoon Park ◽

Dabin Choi ◽

Taesun Park

Keyword(s):

Cell Survival ◽

Collagen Synthesis ◽

Dermal Fibroblast ◽

Olfactory Receptors ◽

Camp Response Element Binding ◽

Human Dermal Fibroblast ◽

Skin Aging ◽

Dermal Fibroblasts ◽

Nasal Tissue ◽

Extracellular Matrix Components

Skin dermis comprises extracellular matrix components, mainly collagen fibers. A decrease in collagen synthesis caused by several factors, including ultraviolet (UV) irradiation and stress, eventually causes extrinsic skin aging. Olfactory receptors (ORs) were initially considered to be specifically expressed in nasal tissue, but several ORs have been reported to be present in other tissues, and their biological roles have recently received increasing attention. In this study, we aimed to characterize the role of ORs in cell survival and collagen synthesis in dermal fibroblasts. We confirmed that UVB irradiation and dexamethasone exposure significantly decreased cell survival and collagen synthesis in Hs68 dermal fibroblasts. Moreover, we demonstrated that the mRNA expression of 10 ORs detectable in Hs68 cells was significantly downregulated in aged conditions compared with that in normal conditions. Thereafter, by individual knockdown of the 10 candidate ORs, we identified that only OR51B5 knockdown leads to a reduction of cell survival and collagen synthesis. OR51B5 knockdown decreased cAMP levels and dampened the downstream protein kinase A/cAMP-response element binding protein pathway, downregulating the survival- and collagen synthesis-related genes in the dermal fibroblasts. Therefore, OR51B5 may be an interesting candidate that plays a role in cell survival and collagen synthesis.

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Effect of MMP/TIMP Balancing of Cynoglossus semilaevis Shell Extracts on Skin Protection

Fishes ◽

10.3390/fishes6030034 ◽

2021 ◽

Vol 6 (3) ◽

pp. 34

Author(s):

Soo-Cheol Choi ◽

In-Ah Lee

Keyword(s):

Skin Diseases ◽

Dermal Fibroblast ◽

Functional Materials ◽

Human Dermal Fibroblast ◽

Cynoglossus Semilaevis ◽

Dermal Fibroblasts ◽

Skin Tissue ◽

Dependent Manner ◽

Functional Material ◽

Concentration Dependent Manner

Cynoglossus semilaevis shell is a by-product of the Cynoglossus semilaevis, a species of fish mainly distributed along the west coast of Korea. As its skin is very tough and difficult to process, it is not useful as food. For this reason, most of it is discarded except for a small amount that is used as feed, which results in environmental pollution. Considering this, there is a need for research on the development of functional materials using Cynoglossus semilaevis shell. This study focused on the mechanism of in vitro expression function of Cynoglossus semilaevis shell extract (CSE) for skin tissue in human dermal fibroblasts that induced or did not induce wrinkles by UV-B irradiation and aims to use it as a functional material for human skin beauty or wrinkle improvement through extraction and purification. According to the ELISA results using human dermal fibroblast cells, CSE reduced MMP-1 and elastase activity by up to 21.89% and 12.04%, respectively, in a concentration-dependent manner, and increased PIP synthesis by up to 62.24% in a concentration-dependent manner. The RT-PCR test results using mRNA showed the MMP-1, 2, and 3 expression levels were suppressed in the CSE-treated group compared to the UVB-induced group and caused a concentration-dependent increase in TIMP-1 in the CSE-treat group. These results suggest that CSE can maintain and improve skin tissue conditions through MMP/TIMP balancing in human dermal fibroblast cell lines and indicate its potential as a functional material for improving skin diseases and suppressing photo-aging.

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Attachment and Proliferation of Human Dermal Fibroblasts onto ECM-Immobilized PLGA Films

Key Engineering Materials ◽

10.4028/www.scientific.net/kem.288-289.291 ◽

2005 ◽

Vol 288-289 ◽

pp. 291-294

Author(s):

Hyun Joo Son ◽

Dong Wook Han ◽

H.H. Kim ◽

Hee Joong Kim ◽

In Seop Lee ◽

...

Keyword(s):

Cell Attachment ◽

Dermal Fibroblast ◽

Supporting Cell ◽

Human Dermal Fibroblast ◽

Dermal Fibroblasts ◽

Water Soluble ◽

Seeding Density ◽

Tetrazolium Salt ◽

Cell Seeding ◽

Plga Film

In this study, human dermal fibroblast behaviors onto non-porous PLGA (75:25) films immobilized with 1, 10 and 100 µg/ml collagen (CN) or fibronectin (FN) were investigated according to different cell-seeding densities (1,000, 10,000 and 100,000 cells/ml). Cell attachment and proliferation were assessed using water soluble tetrazolium salt. The results indicated that 1 µg/ml of FN-immobilized PLGA film demonstrated significantly (p < 0.05) superior cellular attachment to the intact PLGA film after 4 hr of incubation. Moreover, the number of attached cells was shown to be directly proportional to that of initially seeded cells. After 48 hr, the cells showed significantly (p < 0.05) higher proliferation onto 1 or 10 µg/ml of FN-immobilized PLGA films than onto other PLGA films, regardless of the initial cell-seeding density. In terms of CN-immobilization, cell proliferation was appreciably increased but it was relatively lower than FN-immobilization. These results suggested that ECM-immobilization can enhance the cell affinity of hydrophobic scaffolds and be used to potential applications for tissue engineering by supporting cell growth.

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[Clinical and economic evaluation of the introduction of the combinazion trametinib + dabrafenib in the management of advanced melanoma in the Italian market]

Farmeconomia Health economics and therapeutic pathways ◽

10.7175/fe.v17i3s.1279 ◽

2016 ◽

Vol 17 (3S) ◽

pp. 1-32

Author(s):

Lorenzo Pradelli ◽

Paolo Ascierto

Keyword(s):

Impact Analysis ◽

Acquired Resistance ◽

Braf Inhibitor ◽

Mek Inhibitor ◽

Advanced Melanoma ◽

Rapid Response ◽

Antitumor Activities ◽

Development Of Resistance ◽

Skin Cancers ◽

Italian Market

Melanoma is the most aggressive type of all skin cancers. In Italy the incidence is increasing both in men and in women with 13,800 new cases expected in 2016. The advanced melanoma therapy has changed in recent years with the use of immunotherapy and targeted therapies. In particular, treatment with BRAF inhibitors in patients with advanced BRAF V600 mutated melanoma has shown high rates of rapid response and survival. Due to development of acquired resistance with disease progression the rapid response observed with BRAF inhibitor therapy is not long lasting. Combining a BRAF inhibitor with a MEK inhibitor may help to delay the development of resistance and to enhance the antitumor activities with a further increase in the response and survival rate. Trametinib, an inhibitor of MEK kinases, and dabrafenib, an inhibitor of BRAF kinase, have authorizations as monotherapies and in combination with each other for treating adults with unresectable or metastatic melanoma with BRAF V600 mutation. Purpose of this report is to describe the combination in terms of clinical efficacy, safety, and economic impact. In particular, a cost-effectiveness analysis and a budget impact analysis were performed in order to evaluate the combination versus monotherapy and the financial sustainability of trametinib + dabrafenib on the Italian market.[In Italian]

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Bovine vitreous gel can reactivate replicative senescence of human dermal fibroblast

Indonesian Journal of Biotechnology ◽

10.22146/ijbiotech.28228 ◽

2018 ◽

Vol 23 (1) ◽

pp. 48

Author(s):

Maria Vianny Sansan ◽

Sunardi Radiono ◽

Muhamad Eko Irawanto ◽

Yohanes Widodo Wirohadidjojo

Keyword(s):

Hyaluronic Acid ◽

Replicative Senescence ◽

Dermal Fibroblast ◽

Human Dermal Fibroblast ◽

Dermal Fibroblasts ◽

Influential Factor ◽

Proliferation Index ◽

Potential Candidate ◽

Chronic Ulcers ◽

Tgf Β1

The most influential factor in the poor healing of chronic ulcers is replicative senescence of fibroblasts that are unresponsive to TGF-β1 stimulation. Cellular replicative senescence can be induced by cultivating normal human dermal fibroblasts (HDFs) in a serum-starved medium. In addition, increasing microenviroment mechanical forces by hyaluronic acid can ameliorate the TGF-β1 signaling of these senescent cells. One of natural resources of hyaluronic acid is bovine vitreous gel. In order to evaluate the effect of bovine-vitreous gel on replicative senescence of fibroblasts, we used various levels of bovine vitreous gel diluted in Dulbecco’s modified Eagle’s medium to stimulate cellular activities of serum-starved HDFs. Those cellular activities were compared to the control media, standardized hyaluronic acid, and to normal HDFs. Our results show that replicative senescence of HDFs treated with 50% bovine vitreous gel exhibited a significantly higher proliferation index, migration rate, and collagen deposition than those cultured in control media, and they displayed an equal level of cellular activity with the HDFs exposed only to standardized hyaluronic acid. We concluded that bovine vitreous gel can be used to stimulate replicative senescence of HDFs and therefore a potential candidate material to stimulate healing of chronic ulcers.

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Ultrastructural and metabolic effects of products of activated mononuclear leucocytes on cultured human dermal fibroblasts

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100113299 ◽

1984 ◽

Vol 42 ◽

pp. 682-683

Author(s):

I. Stachura ◽

Matias Pardo ◽

Jennifer Worrall ◽

Theresa L. Whiteside

Keyword(s):

Plasma Membranes ◽

Dermal Fibroblast ◽

Human Dermal Fibroblast ◽

Dermal Fibroblasts ◽

Metabolic Effects ◽

Normal Human Skin ◽

Normal Human ◽

Mononuclear Leucocytes ◽

Distinctive Phenotype

Products of antigen- or mitogen-activated mononuclear leucocytes (ML) are known to modulate fibroblast proliferation and collagen production in vitro. In tissue, ML accumulate at sites of inflammation and are probably involved in the process of fibrosis. We have established that supernatants (SN) of concanavalin A-activated ML increase synthesis of glycosaminoglycan (GAG) in human dermal fibroblast (DF) cultures). When explants of normal human skin were cultured in CMRL 1066 medium supplemented with 15% pooled human serum and containing MLSN the outgrowing DF acquired a distinctive phenotype. In comparison to control DF, the cells treated with MLSN exhibited a marked increase in the number of intracytoplasmic organelles especially dilated cisternae of RER filled with electrondense material, abundant lysosomes, prominent Golgi apparatus and bundles of microfilaments often extending beyond the cell boundaries. Cell surfaces were shaggy and floccular material accumulated in patches along the plasma membranes.

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A large-conductance calcium-regulated K+ channel in human dermal fibroblast mitochondria

Biochemical Journal ◽

10.1042/bcj20160732 ◽

2016 ◽

Vol 473 (23) ◽

pp. 4457-4471 ◽

Cited By ~ 10

Author(s):

Anna Kicinska ◽

Bartlomiej Augustynek ◽

Bogusz Kulawiak ◽

Wieslawa Jarmuszkiewicz ◽

Adam Szewczyk ◽

...

Keyword(s):

Membrane Potential ◽

Potassium Channel ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Dermal Fibroblast ◽

Human Dermal Fibroblast ◽

Dermal Fibroblasts ◽

K Channel ◽

Inner Mitochondrial Membrane ◽

Bkca Channels

Potassium channels have been found in the inner mitochondrial membrane of various cells. These channels regulate the mitochondrial membrane potential, respiration and production of reactive oxygen species. In the present study, we identified the activity of a mitochondrial large-conductance Ca2+-regulated potassium channel (mitoBKCa channel) in mitoplasts isolated from a primary human dermal fibroblast cell line. A potassium selective current was recorded with a mean conductance of 280 ± 2 pS in a symmetrical 150 mM KCl solution. The mitoBKCa channel was activated by the Ca2+ and by potassium channel opener NS1619. The channel activity was irreversibly inhibited by paxilline, a selective inhibitor of the BKCa channels. In isolated fibroblast mitochondria NS1619 depolarized the mitochondrial membrane potential, stimulated nonphosphorylating respiration and decreased superoxide formation. Additionally, the α- and β-subunits (predominantly the β3-form) of the BKCa channels were identified in fibroblast mitochondria. Our findings indicate, for the first time, the presence of a large-conductance Ca2+-regulated potassium channel in the inner mitochondrial membrane of human dermal fibroblasts.

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Effects Of (1→3), (1→6)-β-D-Glucan Behavior in Human Dermal Fibroblast Cells under Serum Starvation

Key Engineering Materials ◽

10.4028/www.scientific.net/kem.342-343.401 ◽

2007 ◽

Vol 342-343 ◽

pp. 401-404 ◽

Cited By ~ 1

Author(s):

Yeon I Woo ◽

Hyun Joo Son ◽

Hye Ryeon Lim ◽

Mi Hee Lee ◽

Hyun Sook Baek ◽

...

Keyword(s):

Wound Healing ◽

Dermal Fibroblast ◽

Human Dermal Fibroblast ◽

Positive Influence ◽

Dermal Fibroblasts ◽

Serum Starvation ◽

Healing Agent ◽

And Migration ◽

Free Serum

Glucans have been reported to stimulate immunity and to promote wound healing. Adult human dermal fibroblast (aHDF) cultured in serum free (serum-starvation). Proliferation of aHDF was measured at various concentrations of β-glucan by MTT assay, and migration was observed for 36h on microscope. The result of fibroblast bioassay, β-glucan had positive influence. In this study, the direct effects of β-glucan on proliferation and migration of human dermal fibroblasts were examined in vitro. That means β-D-glucan has the effect to enhance proliferation and aHDF migration speed, and has the potential as a wound healing agent.

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