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  • br Fig IFN and TRAIL expression after


    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).
    IFN-β and TRAIL because of stresses such as serum depletion, which resulted in necrotic cell death in H460 Sphingosine-1-phosphate and suppressed tumor growth in a murine model. Interestingly, type I IFN mRNA, particularly IFN-β, was expressed after the second wash step of ASCs prior to transplantation, while TRAIL was not expressed after any wash. When the ASCs were washed with PBS and HBSS supplemented with 10% FBS, IFN-β was not expressed. These results suggest that IFN-β ex-pression may be regulated by serum depletion in high-density cell culture or during the washing step. IFN-β may act in an autocrine fashion to induce TRAIL expression, but only during cell culture. In macrophages, serum deprivation was found to induce apoptosis through the autocrine secretion of type I IFNs [38]. Type I/II IFNs are well-known stimulators that can induce TRAIL expression in various cells [30,33,34,39–41]. In our system, IFN-β was expressed in ASCs cultured at high density (40K) or in serum-free conditions as well as during the washing step prior to ASC transplantation. However, TRAIL mRNA and protein expression was observed only in ASCs cultured at high density (40K) or in serum-free media. These results suggest that the autocrine effects of IFN-β combined with sufficient cellular energy sources (e.g., ATP) and/or building blocks (e.g., glucose, amino acids) are required for TRAIL expression. However, since the washing solution lacked ATP and the building blocks for IFN-β protein synthesis, it was hypothesized that TRAIL would not be expressed even if IFN-β mRNA was expressed.
    IFN-β and TRAIL induced apoptotic cell death in H460 cells. However, when H460 cells were indirectly co-cultured with 5K-ASCs, IFN-β induced necrotic cell death in H460 cells. These dying cells, which were still attached to the bottom of the culture plate, were al-most identical to the necrotic H460 cells observed at the beginning of co-culture with 40K-ASCs. Rapidly growing tumors may easily undergo hypoxia and nutrient (e.g., glucose) deficiency due to a lack of blood supply; these metabolic stresses have been reported to mainly cause 
    necrosis during carcinogenesis [42,43]. Indeed, treatment of H460 cells with TRAIL in the absence of both serum and glucose induced necrotic cell death, as indicated by cell morphology. Taken together, we con-cluded that serum deficiency causes ASCs to express IFN-β, leading to increased TRAIL expression. TRAIL then induces necroptosis in lung cancer H460 cells in an environment lacking serum and glucose.
    Nevertheless, although 40K-ASCs induced the death of H460 cells very efficiently in vitro, the tumor weight reduction in our murine xe-nograft model was statistically significant, but not dramatic. These re-sults suggest that ASCs may act very differently depending on the mi-croenvironment. In fact, when 40K-ASCs expressing IFN-β and TRAIL were re-seeded at a low density (5K), IFN-β and TRAIL mRNA was ex-pressed during sub-culturing, but not TRAIL protein. Thus, we hy-pothesized that TRAIL protein expression is regulated by the nutritional status (i.e., serum and glucose) of the microenvironment. In addition to tumor growth inhibition, TRAIL plays an important role in regulating immune reactions [44–46]. Therefore, ASCs expressing TRAIL may be useful for treatment of cancer as well as various inflammatory diseases. In the future, the regulation of TRAIL expression by the micro-environment is expected to enhance the therapeutic effects of stem cell-based treatments for inflammatory disease and cancer.