Application of “macromolecular crowding” in vitro to investigate the naphthoquinones shikonin, naphthazarin and related analogues for the treatment of dermal scars

Fan, Chen; Lim, Lay Keng Priscilla; Loh, See Qi; Lim, Kimberley Ying Ying; Upton, Zee; Leavesley, David I.

doi:10.1016/j.cbi.2019.108747

Cited by 21 publications

(16 citation statements)

References 71 publications

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“…However, their change in behavior when cultured on plastic surfaces complicates in vitro studies 13 . Several reports have proposed stimulating primary fibroblasts in a crowded macromolecular environment to help overcome some of these difficulties 15 – 18 , 24 . In this model, we combined the macromolecular crowding with dermal fibroblasts and translational biomarkers.…”

Section: Discussionmentioning

confidence: 99%

“…This phenotype change has traditionally been overcome by using 3D cultures 14 . Recently, macromolecular crowding has been examined as a means to imitate the crowded in vivo environment surrounding the fibroblasts 15 – 17 . Macromolecular crowding involves the use of a soluble macromolecule to occupy space and thereby exclude volume 18 .…”

Section: Introductionmentioning

confidence: 99%

“…Macromolecular crowding involves the use of a soluble macromolecule to occupy space and thereby exclude volume 18 . This affects the concentration and time course of the appearance of soluble components such as proteins and proteases 15 .…”

mentioning

confidence: 99%

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Dermal fibroblasts have different extracellular matrix profiles induced by TGF-β, PDGF and IL-6 in a model for skin fibrosis

Juhl

Bondesen

Hawkins

et al. 2020

Sci Rep

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Different stimulants might induce different extracellular matrix profiles. It is essential to gain an understanding and quantification of these changes to allow for focused anti-fibrotic drug development. This study investigated the expression of extracellular matrix by dermal fibroblast mimicking fibrotic skin diseases as SSc using clinically validated biomarkers. Primary healthy human dermal fibroblasts were grown in media containing FICOLL. The cells were stimulated with PDGF-AB, TGF-β1, or IL-6. Anti-fibrotic compounds (iALK-5, Nintedanib) were added together with growth factors. Biomarkers of collagen formation and degradation together with fibronectin were evaluated by ELISAs in the collected supernatant. Immunohistochemical staining was performed to visualize fibroblasts and proteins, while selected gene expression levels were examined through qPCR. TGF-β and PDGF, and to a lesser extent IL-6, increased the metabolic activity of the fibroblasts. TGF-β primarily increased type I collagen and fibronectin protein and gene expression together with αSMA. PDGF stimulation resulted in increased type III and VI collagen formation and gene expression. IL-6 decreased fibronectin levels. iALK5 could inhibit TGF-β induced fibrosis while nintedanib could halt fibrosis induced by TGF-β or PDGF. Tocilizumab could not inhibit fibrosis induced in this model. The extent and nature of fibrosis are dependent on the stimulant. The model has potential as a pre-clinical model as the fibroblasts fibrotic phenotype could be reversed by an ALK5 inhibitor and Nintedanib.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Dermal fibroblasts have different extracellular matrix profiles induced by TGF-β, PDGF and IL-6 in a model for skin fibrosis

Juhl

Bondesen

Hawkins

et al. 2020

Sci Rep

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“…In accordance with previous publications, several other organspecific fibrotic models induced by MMC supplementation have been developed (Chen et al, 2009;Graupp et al, 2015;Graupp et al, 2018;Fan et al, 2019;Good et al, 2019;Fan et al, 2020;Rønnow et al, 2020;De Pieri et al, 2021). It is worth noting that cocktails of pro-inflammatory cytokines have been used for more accurate recapitulation of fibrosis in vitro (Chawla and Ghosh, 2018) and such approach should be studied further in the future in combination with MMC.…”

Section: Discussionmentioning

confidence: 61%

Adapting the Scar-in-a-Jar to Skin Fibrosis and Screening Traditional and Contemporary Anti-Fibrotic Therapies

Coentro

May

Prince

et al. 2021

Front. Bioeng. Biotechnol.

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Skin fibrosis still constitutes an unmet clinical need. Although pharmacological strategies are at the forefront of scientific and technological research and innovation, their clinical translation is hindered by the poor predictive capacity of the currently available in vitro fibrosis models. Indeed, customarily utilised in vitro scarring models are conducted in a low extracellular matrix milieu, which constitutes an oxymoron for the in-hand pathophysiology. Herein, we coupled macromolecular crowding (enhances and accelerates extracellular matrix deposition) with transforming growth factor β1 (TGFβ1; induces trans-differentiation of fibroblasts to myofibroblasts) in human dermal fibroblast cultures to develop a skin fibrosis in vitro model and to screen a range of anti-fibrotic families (corticosteroids, inhibitors of histone deacetylases, inhibitors of collagen crosslinking, inhibitors of TGFβ1 and pleiotropic inhibitors of fibrotic activation). Data obtained demonstrated that macromolecular crowding combined with TGFβ1 significantly enhanced collagen deposition and myofibroblast transformation. Among the anti-fibrotic compounds assessed, trichostatin A (inhibitors of histone deacetylases); serelaxin and pirfenidone (pleiotropic inhibitors of fibrotic activation); and soluble TGFβ receptor trap (inhibitor of TGFβ signalling) resulted in the highest decrease of collagen type I deposition (even higher than triamcinolone acetonide, the gold standard in clinical practice). This study further advocates the potential of macromolecular crowding in the development of in vitro pathophysiology models.

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“…Our analysis of 8 SHI analogs identified that, in common with SHI, DO-SHI and β,β-SHI also possess viability-inhibiting ability against HSF. 14 Our review of the literature found that the effects of DO-SHI and β,β-SHI on melanoma related cells are largely unknown. Rajasekar et al 15 reported that the extract from the roots of L. erythrorhizon (LEH) induces apoptosis in various melanoma cell lines and the potential active components are SHI, DO-SHI, β-hydroxyisovalerylshikonin, acetylshikonin, and isobutyrylshikonin 15 ; however, LEH is a mixture of compounds, the identity of which and mechanisms of each component are still unknown.…”

mentioning

confidence: 99%

Investigating the Effects of Shikonin, Deoxyshikonin, and (β,β-Dimethylacryl)Shikonin on Melanoma Cell Lines

Upton

Leavesley

et al. 2020

Natural Product Communications

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Melanoma is the most lethal form of various skin cancers and contributes to more than 79% of all skin cancer deaths. Although there are numerous therapies available for melanoma, the high rate of recurrence in melanoma post-therapy remains a challenging issue for both patients and clinicians. Apoptosis is one of the foundations for cancer treatment as deficient apoptosis is one of the most essential reasons for the formation of tumour tissues. Shikonin (SHI), an active component extracted from Lithospermum erythrorhizon, has been broadly demonstrated to possess antitumorigenic property due to its apoptosis-inducing ability in various cancer cell lines. The analogs of SHI, such as deoxyshikonin (DO-SHI) and (β,β-dimethylacryl)shikonin (β,β-SHI), have also been found to possess similar bioactivities. The apoptosis-inducing ability of SHI and its analogs enable them to be potential anticancer therapies. In this study reported herein, we investigated the effects of SHI, DO-SHI, and β,β-SHI on both human (A375) and mouse (B16-F0 and B16-F10) melanoma cell lines. Cell viability was measured using Alamar blue assay, while cell migration was detected using scratch assay. Cell apoptosis was captured using terminal deoxynucleotidyl dUTP nick end labeling and fluorescence activated cell sorting. Signaling pathway activation was detected using Western blotting. Our results revealed that SHI, DO-SHI, and β,β-SHI reduce cell viability, inhibit cell migration, and induce apoptosis in melanoma cell lines. These 3 molecules-induced apoptosis in A375 is regulated via mitogen-activated protein kinase/caspase 3 signaling pathway. In particular, DO-SHI and β,β-SHI induce higher apoptosis rate in A375 and B16-F0 compared to SHI. The data from this study demonstrate that DO-SHI and β,β-SHI offer potential new reagents for managing melanoma.

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Application of “macromolecular crowding” in vitro to investigate the naphthoquinones shikonin, naphthazarin and related analogues for the treatment of dermal scars

Cited by 21 publications

References 71 publications

Dermal fibroblasts have different extracellular matrix profiles induced by TGF-β, PDGF and IL-6 in a model for skin fibrosis

Dermal fibroblasts have different extracellular matrix profiles induced by TGF-β, PDGF and IL-6 in a model for skin fibrosis

Adapting the Scar-in-a-Jar to Skin Fibrosis and Screening Traditional and Contemporary Anti-Fibrotic Therapies

Investigating the Effects of Shikonin, Deoxyshikonin, and (β,β-Dimethylacryl)Shikonin on Melanoma Cell Lines

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