Cancer Letters br sTRAIL and mbTRAIL expression
Cancer Letters 440–441 (2019) 202–210
sTRAIL and mbTRAIL expression in 5K-ASCs and 40K-ASCs after 5 days of culture. The mean fluorescence intensity of mbTRAIL increased by approximately 5-fold in 40K-ASCs on day 5 compared to that in 5K-ASCs on day 1. The mean fluorescence intensity of DR5 slightly in-creased in 5K-ASCs at day 5 and 40K-ASCs at both day 1 and 5, but there was no significant diﬀerence between the three groups (Fig. 1F). Furthermore, sTRAIL was detected in 20K-ASCs and 40K-ASCs in a cell dose-dependent manner (Fig. 1G). Taken together, these results in-dicate that when ASCs were cultured at a density > 20K, IFN-β was expressed, which, in turn, induced TRAIL expression.
Next, we investigated whether 40K-ASCs expressing IFN-β and TRAIL would induce cell death in the TRAIL-sensitive H460 lung cancer cells. H460 Ku-0059436 were incubated with concentrated- or normal CM re-covered from 40K-ASCs cultured for 1–5 days (40K-ASC-CM) or in-directly co-cultured with 40K-ASCs for the same time period using a Transwell system. H460∗ cells were directly co-cultured with 40K-ASCs. When the H460 cells were treated with concentrated- or normal 40K-ASC-CM, their viability was lowest in the 5-day 40K-ASC-CM group, but the viability in the concentrated CM group was also lower similar to that in the 5 ng/ml rTRAIL group (Fig. 2A). When the H460 cells were co-cultured with 40K-ASCs, the cellular contents, indicative of necrosis, leaked out of the cells, leading to the presence of floating cytosolic particles in the culture medium (Fig. 2B). To determine whether this leakage of cellular contents was related to the necrotic death of H460 cells co-cultured with 40K-ASCs, annexin-V-PE/7-AAD staining was performed. H460 cells co-cultured with 40K-ASCs were annexin-V-negative/7-AAD-positive, suggesting necrotic cell death (Fig. 2C). In addition, when H460∗ cells were cultured alone, the G0/G1 population was high at day 2, but dead-sub-G0 populations were rarely observed (Fig. 2D). However, approximately 30% of H460∗ cells directly co-cultured with 40K-ASCs showed sub-G0 cell death from day 1, and more than 60% sub-G0 cell death was observed on day 2 (Fig. 2D). These results suggest that 40K-ASCs could induce necrotic cell death in H460 cells.
3.3. IFN-β and TRAIL stimulate necrotic cell death in H460 cells
Since 40K-ASCs expressed both IFN-β and TRAIL and induced ne-crotic cell death in H460 cells, we next determined whether both IFN-β and TRAIL were required to induce necrotic cell death. Compared to control H460 cells, H460 cells cultured with either IFN-β or TRAIL for 3 days showed substantially increased apoptotic cell death, confirmed by morphological microscopy and annexin-V/7-AAD staining (Fig. 3A) and the cleavage of Cas-3 and Cas-8 (Fig. 3B). IFN-β and TRAIL induced the apoptotic body, early apoptotic population (annexin-V-positive/7-AAD-negative, 17.3 ± 2.4% for IFN-β and 25.0 ± 3.2% for TRAIL), and the expression of cleaved-cas-3 and -8 in H460 cells (Fig. 3A and B). However, when H460 cells were indirectly co-cultured with 5K-ASCs, IFN-β induced necrotic cell death in H460 cells. The dying cells, which were still attached to the bottom of the culture plate, were identical to the necrotic H460 cells observed at the beginning of the co-culture with 40K-ASCs, and the late apoptotic (annexin-V-positive/7-AAD-positive, 19.9 ± 0.8%) and necrotic population (annexin-V-negative/7-AAD-positive, 24.5 ± 2.7%) was observed, but not early apoptotic popula-tion (annexin-V-positive/7-AAD-negative) (Fig. 3A). Additionally, both Cas-3 and cleaved Cas-3 levels were reduced compared to those in IFN-β-treated H460 cells, and cleaved Cas-8 was not observed in H460 cells co-cultured with 5K-ASCs under IFN-β treatment (Fig. 3B). Incubation with 40K-ASC-CM induced cell death in H460 cells, but their viability was recovered by neutralization with anti-IFN-β and anti-TRAIL anti-bodies. Moreover, cell viability was recovered synergistically at low concentrations of anti-IFN-β (0.05 μg/ml) and anti-TRAIL (0.1 μg/ml) antibody treatment (Fig. 3C). As previously reported, glucose deficiency