Multilayered fibroblasts constructed by accelerated cellular self-assembly and applications for regenerative medicine.

Regenerative medicine research requires animal experiments to evaluate the treatment effects. According to the 3Rs principles, alternative models have been developed and utilized to evaluate the efficacy and safety of new products. Three-dimensional (3D) cell cultures have been recognized for their relevant structures and biological functions akin to native tissues. They can better represent in vivo conditions than two-dimensional (2D) cell cultures. Herein, we present a fast and simple technique for the construction of 3D dermal fibroblasts (3D-DFs) without exogenous scaffolds. The 3D-DFs can be obtained within 3 days by seeding DFs at a level that exceeds their confluent density and culturing in the presence of ascorbic acid. The 3D-DFs had a compact multilayer structure, as revealed from their histology. The collagen content of the resulting 3D-DFs drastically increased compared to in a monolayer. The 3D-DF-derived extracellular matrix can serve for the 3D culturing of other cells. A gap closure assay was performed with the 3D-DFs to represent a 3D-wounded dermal model. Interestingly, the multilayered structure of the 3D-DFs could be regenerated after wounding even when cultured in the absence of ascorbic acid. Moreover, skin grafting of the 3D-DFs was demonstrated in vitro using wounded full-thickness skin models as an alternative to animal experiments. The 3D-DFs will potentially be useful for regenerative medicine or as tissue models for in vitro studies.

Avicequinone B sensitizes anoikis in human lung cancer cells.

BACKGROUND: During metastasis, cancer cells require anokis resistant mechanism to survive until reach the distant secondary tissues. As anoikis sensitization may benefit for cancer therapy, this study demonstrated the potential of avicequinone B, a natural furanonaphthoquinone found in mangrove tree (Avicenniaceae) to sensitize anoikis in human lung cancer cells. METHODS: Anoikis inducing effect was investigated in human lung cancer H460, H292 and H23 cells that were cultured in ultra-low attachment plate with non-cytotoxic concentrations of avicequinone B. Viability of detached cells was evaluated by XTT assay at 0-24 h of incubation time. Soft agar assay was performed to investigate the inhibitory effect of avicequinone B on anchorage-independent growth. The alteration of anoikis regulating molecules including survival and apoptosis proteins were elucidated by western blot analysis. RESULTS: Avicequinone B at 4 µM significantly induced anoikis and inhibited proliferation under detachment condition in various human lung cancer cells. The reduction of anti-apoptotic proteins including anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) and myeloid cell leukemia 1 (Mcl-1) associating with the diminution of integrin/focal adhesion kinase (FAK)/Proto-oncogene tyrosine-protein kinase (Src) signals were detected in avicequinone B-treated cells. CONCLUSIONS: Avicequinone B sensitized anoikis in human lung cancer cells through down-regulation of anti-apoptosis proteins and integrin-mediated survival signaling.

Peptides extracted from edible mushroom: Lentinus squarrosulus induces apoptosis in human lung cancer cells.

CONTEXT: Lentinus squarrosulus Mont. (Polyporaceae) is an interesting source of diverse bioactive compounds. OBJECTIVE: This is the first study of the anticancer activity and underlying mechanism of peptides extracted from Lentinus squarrosuls. MATERIALS AND METHODS: Peptides were isolated from the aqueous extract of L. squarrosulus by employing solid ammonium sulphate precipitation. They were further purified by ion-exchange chromatography on diethylaminoethanol (DEAE)-cellulose and gel filtration chromatography on Sephadex G25. Anticancer activity was investigated in human lung cancer H460, H292 and H23 cells cultured with 0-40 µg/mL of peptide extracts for 24 h. Cell viability and mode of cell death were evaluated by MTT and nuclear staining assay, respectively. Western blotting was used to investigate the alteration of apoptosis-regulating proteins in lung cancer cells treated with peptide extracts (0-20 µg/mL) for 24 h. RESULTS: The cytotoxicity of partially-purified peptide extracts from L. squarrosulus was indicated with IC50 of ∼26.84 ± 2.84, 2.80 ± 2.14 and 18.84 ± 0.30 µg/mL in lung cancer H460, H292 and H23 cells, respectively. The extracts at 20 µg/mL induced apoptosis through the reduction of anti-apoptotic Bcl-2 protein (∼0.5-fold reduction) and up-regulation of BAX (∼4.5-fold induction), a pro-apoptotic protein. Furthermore, L. squarrosulus peptide extracts (20 µg/mL) also decreased the cellular level of death receptor inhibitor c-FLIP (∼0.6-fold reduction). CONCLUSIONS AND DISCUSSION: This study provides the novel anticancer activity and mechanism of L. squarrosulus peptide extracts, which encourage further investigation and development of the extracts for anticancer use.