Biofabrication has proved to be a versatile and valuable tool for tissue engineering and cancer research in order to mimic different tumor microenvironments. In the present study, four different cell lines, including two melanoma cell lines, Mel Im and Mel Wei, and two breast cancer cell lines, MDA-MB-231 and MCF-7, were tested in combination with four hydrogels. The hydrogels were a composite ink consisting of alginate, hyaluronic acid and gelatine (Alg/HA/Gel), pre-crosslinked oxidized alginate with gelatine (ADA-GEL), alginate with methyl cellulose (Meth-Alg) and acrylated hyaluronic acid (HAA). Rheological analysis and shear stress calculations, printability assays and dynamic mechanical analysis were performed on all the hydrogels. The cell lines were then mixed into the hydrogels, printed and examined over a period of 14 days. The focus was on cell survival in the gel, metabolic activity and cell cycle analysis using FUCCI reporters. The results showed that all hydrogels were well printable and Meth-Alg was the softest gel. The cell lines survived the printing process in all inks, but there were significant differences in the metabolic activity and the predominant cell cycle stage. In Alg/HA/Gel, the cells grew in spheroid colonies. ADA-GEL proved to be a good bioink for all cell lines, which enabled proliferation, migration and supported the metabolic activity of the cells, while Meth-Alg offered pores for spreading and proliferating cells. However, it was shown that HAA resulted in the lowest cell number and metabolic activity for all cell lines due to its high polymer content, leading in senescence. Our data demonstrated that depending on the hypothesis, all inks are valuable approaches for breast cancer and melanoma models.
MIA (melanoma inhibitory activity) has been previously isolated from the tissue culture supernatant of melanoma cell lines as an autoregulatory activity, inhibiting thymidine incorporation. However, subsequent analyses of melanocytic tumours in vivo have correlated enhanced MIA expression with progression of melanocytic tumours, conflicting with the idea that MIA acts as a tumour suppressor. To investigate the role of MIA in vivo, we have therefore generated a panel of stably transfected B16 cell clones secreting different amounts of MIA. The capacity of these cell clones to form lung metastases in syngeneic C57Bl6 mice was strictly correlated to the level of MIA secretion, but the clones did not differ with respect to their proliferation in vitro. In summary, we suggest that MIA plays a causal role in promoting the metastasis of malignant melanomas, involving inhibition of tumour cell attachment to extracellular matrix molecules within their local milieu.
Cold atmospheric plasma (CAP) has been gaining increasing interest as a new approach for the treatment of skin diseases or wounds. Although this approach has demonstrated promising antibacterial activity, its exact mechanism of action remains unclear. This study explored in vitro and in vivo whether CAP influences gene expression and molecular mechanisms in keratinocytes. Our results revealed that a 2 min CAP treatment using the MicroPlaSter s in analogy to the performed clinical studies for wound treatment induces expression of IL-8, TGF-s1, and TGF-s2. In vitro and in vivo assays indicated that keratinocyte proliferation, migration, and apoptotic mechanisms were not affected by the CAP treatment under the applied conditions. Further, we observed that antimicrobial peptides of the s-defensin family are upregulated after CAP treatment. In summary, our results suggest that a 2 min application of CAP induces gene expression of key regulators important for inflammation and wound healing without causing proliferation, migration or cell death in keratinocytes. The induction of s-defensins in keratinocytes describes an absolutely new plasma strategy. Activation of antimicrobial peptides supports the well-known antibacterial effect of CAP treatment, whereas the mechanism of s-defensin activation by CAP is not investigated so far.
Zusammenfassung
Weltweit ist in den letzten 30 Jahren in der hellhautigen
Bevolkerung eine Zunahme des malignen
Melanoms der Haut zu beobachten. So
betragt beispielsweise die geschatzte Inzidenz
im Grosraum Munchen derzeit 14 bis 16 pro
100 000 Einwohner. Wahrend die fruhzeitig diagnostizierten
Melanome meist durch chirurgische
Exzision geheilt werden konnen, ist die
Prognose des metastasierenden malignen Melanoms
bis heute in der Regel infaust. Da immer
noch etwa 20 Prozent der Melanompatienten
trotz aller Fruherkennungsprogramme an der
Progression ihrer Erkrankung sterben, ist das Interesse
an Laboruntersuchungen und bildgebenden
Verfahren zur Untersuchung asymptomatischer
Patienten mit Metastasen sehr gros.
Fur das maligne Melanom gab es bisher keinen,
auserhalb von experimentellen und klinischen
Studien stehenden Tumormarker. Dies hat sich
seit einiger Zeit geandert. Es stehen nun kommerziell
erhaltliche Tumormarker wie S100β
und das Protein „melanoma inhibitory activity“
(MIA) zur Verfugung. Auf diese sowie auf die
Wertigkeit experimenteller Prognosefaktoren
wird im Folgenden eingegangen.
Summary
Tumour markers in malignant melanoma
The incidence of malignant melanoma of the
skin has increased in Caucasian populations
over the last 30 years. The estimated incidence
in the Munich area is currently 14 to 16 cases
per 100,000 inhabitants. While early treatment
by surgical excision is usually curative, the prognosis
of the metastatic malignant melanoma
remains poor. As around 20 per cent of melanoma
patients die of advanced disease despite
early detection programs, laboratory investigations
and imaging procedures have an important
role to play in asymptomatic patients
with metastases. Until recently, tumor markers
for malignant melanoma have been purely experimental.
This has recently changed, with the
advent of some commercially available tumor
markers such as S100β and MIA („melanoma inhibitory
activity“). This review discusses these
tumor markers along with experimental prognostic
markers.
MIA/CD-RAP is a small, soluble protein secreted from malignant melanoma cells and from chondrocytes. Recent evidence has identified MIA/CD-RAP as the prototype of a small family of extracellular proteins adopting an SH3 domain-like fold. It is thought that interaction between MIA/CD-RAP and specific epitopes in extracellular matrix proteins regulates the attachment of tumor cells and chondrocytes. In order to study the consequences of MIA/CD-RAP deficiency in vivo, we generated mice with a targeted gene disruption. The complete absence of MIA/CD-RAP mRNA and protein expression was demonstrated by reverse transcriptase, Western blot analysis, and enzyme-linked immunosorbent assay measurements of whole-embryo extracts. MIA−/− mice were viable and developed normally, and histological examination of the organs by means of light microscopy revealed no major abnormalities. In contrast, electron microscopic studies of cartilage composition revealed subtle defects in collagen fiber density, diameter, and arrangement, as well as changes in the number and morphology of chondrocytic microvilli. Taken together, our data indicate that MIA/CD-RAP is essentially required for formation of the highly ordered ultrastructural fiber architecture in cartilage and may have a role in regulating chondrocyte matrix interactions.
Regain methionine is essential for cellular metabolism and methylthioadenosine phosphorylase (MTAP), the rate-limiting enzyme in the methionine and adenine salvage pathway, catalyzes the phosphorylation of 5'-deoxy-5'-(methylthio)adenosine (MTA), which is a by-product of polyamine synthesis. The polyamine metabolic pathway has been implicated in the pathogenesis of chronic liver disease, and previous studies have shown that pharmacological doses of MTA (500µM and higher) exhibited anti-fibrotic effects in murine models of hepatic injury.
Small molecules like aliphatic polycations, named polyamines, were proven to be essential for hepatic growth and regeneration. Additionally, different stages of liver regeneration are regulated by a variety of factors such as the liver growth associated protein ALR, augmenter of liver regeneration. Also, increased ALR levels were detected for various types of acute liver disease, and in addition, we could find increased expression of ALR in livers from patients with cirrhosis, hepatocellular and cholangiocellular carcinoma [1]. On the other hand polyamines are known to contribute to carcinogenesis and tumour progression by increased levels due to over-expression of ornithine decarboxylase (ODC) [2], the rate limiting key enzyme of polyamine metabolism and further, to be correlated with the degree of malignancy in hepatocellular carcinomas [3]. Therefore, using primary human hepatocytes in vitro we investigated the effect of ALR on the biosynthesis of intracellular polyamines. We demonstrated by HPLC analysis that recombinant ALR enhanced intracellular hepatic levels of putrescine, spermidine and spermine within 9–12h after treatment. The activation of polyamine biosynthesis was dose dependent with putrescine showing the strongest increases. Additionally, ALR treatment induced mRNA expression of ornithine decarboxylase and S-adenosylmethionin decarboxylase, both key enzymes of polyamine biosynthesis. Further, ALR induced c-myc mRNA expression, a regulator of ODC expression and therefore we assume that ALR exerts its augmenting effects on liver regeneration as well as a putative role of ALR in hepatocellular carcinoma through stimulation of signalling pathways leading an activated polyamine metabolism regulated by increased c-myc as well as ODC and AdoMetDC mRNA expression.
