br and TRAIL mRNA was expressed
and TRAIL mRNA was expressed during sub-culturing, but TRAIL pro-tein expression was not observed (Fig. 5E and F). These results suggest that serum-free high-density culture of stem A 61603 may induce IFN-β and TRAIL expression.
Although nude mice co-injected with H460 tumor cells and 40K-ASCs showed reduced tumor weight in our xenograft tumor model, 5K-ASCs also significantly reduced tumor weight (Fig. 4). We therefore investigated whether inducing IFN-β and TRAIL expression before transplanting 5K-ASCs into experimental animals would increase their anti-tumor activity. First, we examined whether IFN-β and TRAIL could be expressed in 5K-ASCs after four washes with PBS and HBSS—a routine procedure to completely remove serum components before transplantation. 5K-ASCs cultured for 5 days were treated with trypsin, washed three times with PBS and once with HBSS, resuspended in HBSS, and incubated at room temperature for the indicated times. IFN-β mRNA expression was observed in the washed 5K-ASCs; it gradually increased until 4 h and decreased thereafter (Fig. 6A). However, TRAIL mRNA and protein expression was not detected (Fig. 6A and B). Next, we investigated whether IFN-β and TRAIL expression could be detected in 5K-ASCs at any of the five washing steps, and whether the expression of these cytokines would be altered when ASCs were washed with PBS
or HBSS supplemented with serum. Interestingly, IFN-β mRNA expres-sion was observed only in ASCs after serum-free washing (Fig. 6C–F). Moreover, when 5K-ASCs were washed with serum-free PBS, IFN-β mRNA expression was observed after the second wash (Fig. 6C). Taken together, these results indicate that when ASCs were exposed to stresses such as serum starvation at a high cell density, IFN-β was expressed, leading to the induction of TRAIL expression in normal media. How-ever, during washing with PBS or HBSS, TRAIL was not expressed even if IFN-β mRNA was expressed. It is possible that when ASCs are cultured at a high concentration, the serum concentration gradually decreases, causing cellular stress that may lead to the expression of type I IFNs and TRAIL.
Despite our increasing knowledge of the potential role of MSCs in medical treatments, the reported roles in promotion or suppression of tumor growth conflict. Until now, the promotion of tumor growth was thought to be regulated by various trophic factors secreted by MSCs. However, only a few reports have analyzed the factors playing crucial roles in tumor growth suppression. In this study, 40K-ASCs expressed
Fig. 3. Eﬀects of IFN-β and TRAIL on cell death in H460 cells. A) Morphological changes in H460 cells with 5K-ASCs treated with IFN-β or TRAIL for 3 days (100 × ) and necrotic cell death in H460 cells by 5 ng/ml TRAIL for 1 day in serum- and glucose-deficient medium. The morphology of H460 cells was observed using a light microscope (100 × ) and cell death mode was analyzed by flow cytometer after annexin-V-PE/7-AAD staining. B) Immunoblot of caspase (Cas)-3 and Cas-8 in H460 cells with 5K-ASCs treated with IFN-β or TRAIL for 3 days. C) Viability of H460 cells treated with 40K-ASC-CM after the neutralization of IFN-β and/or TRAIL. H460 cells were cultured with 40K-ASC-CM and treated with anti-IFN-β and/or anti-TRAIL neutralizing antibodies for 24 h. Cell viability was assessed by an MTT assay. Error bars represent the mean ± SD of triplicate wells. Data were obtained from one of three independent experiments. *P ≤ 0.05 and **P ≤ 0.01.
Fig. 4. Anti-proliferative eﬀect of ASCs in H460 xenograft tumor models. A) Changes in tumor volume 3 weeks after ASC injection. For 3 weeks after co-injection of H460 and ASCs cultured at 5K or 40K, the tumor volume was measured weekly using Vernier calipers. B) Morphology of xenograft mice and tumor mass. At 3 weeks after injection of ASCs, mice were sacrificed by cervical dislocation (Control, n = 3; 5K, n = 5; 40K, n = 6), and the tumor mass was separated and photographed. Representative images from three mice are shown. C) Tumor weight at 3 weeks after injection with ASCs. Tumor weight was measured using an electronic balance. Error bars represent the mean ± SD of experimental mice. **P ≤ 0.01.
Fig. 5. IFN-β and TRAIL expression after serum starvation in 40K-ASCs or sub-culture of 5-day-cultured 40K-ASCs into 5K. A, C) mRNA expression of IFN-β and TRAIL in 5K- and 40K-ASCs after serum starvation. 5K- and 40K-ASCs were cultured in serum-free medium for the indicated time points, and then mRNA expression was evaluated by RT-PCR. B, D) TRAIL expression in 5K- and 40K-ASCs cultured with serum-free media for the indicated time points. TRAIL expression was detected by immunoblotting. E–F) IFN-β and TRAIL expression in 5K-ASCs seeded from 5-day-cultured 40K-ASCs. 40K-ASCs were cultured for 5 days and sub-cultured at 5K for the indicated time points. Total RNA and proteins were isolated from the ASCs, and the expression of IFN-β and TRAIL was analyzed by RT-PCR (C) and im-munoblotting (D).