Fig 2: EV domain of EVDRL enhances the apoptosis-inducing efficacy of DRL.(A and B) Cell viability of BLBC-BM lines after 72-hour treatment (A) and WB of cleavage of caspases and PARP in BLBC-BM lines after 8-hour treatment (B) with control media (Ctrl), EV, DRL, EV + DRL, and EVDRL (n = 3, technical replicates). (C) Confocal images of unstimulated BMET02 cells stably expressing EGFR-YFP and transiently transfected with DR5-CFP. (D) Real-time FRET (sensitized emission) imaging in NIH-3T3 cells coexpressing DR5-CFP and EGFR-YFP. Ratio images depicting the bottom focal plane of the cell show FRET before (left image) and 30 min after treatment EVDRL (right image). (E) Schematic of EGFR inhibitors used for blocking experiments of EVDRL. (F and G) Cell viability (F) and WB showing cleavage of caspases and PARP (G) of BLBC-BM lines after 24-hour treatments with DRL and EVDRL after pretreatment with various concentrations of cetuximab (n = 3, technical replicates). (H) Coimmunoprecipitation (Co-IP) and WB analyses showing EGFR-EVDRL-DR4/5 complex formation in the presence of EVDRL and the attenuation of the complex by cetuximab in BMET02 (n = 2, technical replicates). (I) Cell viability of BLBC-BM lines treated by EVDRL for 24 hours with or without pretreatment by 1 µM erlotinib (n = 3, technical replicates). ns, not significant. (J) Flow cytometry showing reduction of cell surface expression of DR4/5 in BMET02 lines with CRISPR-Cas9 knockout (KO) of DR4, DR5, and DR4/5. (K) Cell viability of BMET02-DR4/5 KO lines after 72-hour treatment with DRL and EVDRL (n = 3, technical replicates). (L) Left: Co-IP and WB analyses showing levels of DRL bound to DR4/5 in BLBC-BM lines after 8-hour treatment with separated EV plus DRL (S) or combined EVDRL (C). Right: Quantification of levels of DRL bound to DR4/5 (n = 3). (M) Schematic showing functional difference between DRL (left) and EVDRL (right).

Fig 3: HDAC Inhibitors cause upregulation of death receptor expression and sensitize HepG2 cells to TRAIL NPs(A and B) HepG2 death receptor (DR4 and DR5) expression by (A) western blot and (B) flow cytometry after 24-h vorinostat treatment. (C–E) HepG2 cell viability 48 h after transfection with either mTRAIL or sTRAIL NPs and treated with varying doses of (C) vorinostat, (D) sodium butyrate, and (E) MS-275. Data represented as mean ± SEM of n = 3 replicate wells. Significant differences between sTRAIL and mTRAIL-treated cells are determined by two-way ANOVA and Sidak’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001.

Fig 4: BLBC is sensitive to EVDRL, and EV domain of EVDRL enhances apoptosis-inducing effect of DRL depending on tumor cell surface EGFR expression.(A) Representative microphotograph of low and high (the insets) magnifications of H&E and immunohistochemistry of EGFR and DR4/5 in patient samples of TNBC- BM. Scale bars, 100 µm (main images) and 10 µm (insets). (B) Schematic showing the construction of anti-EGFR VHH-DRL (EVDRL) and anti-EGFR scFv-DRL (ESDRL) proteins. (C) Cell viability of BLBC-BM lines after 72-hour treatment with control media (Ctrl), ESDRL, or EVDRL. (n = 3, technical replicates). (D) WB showing cleavage of caspases and poly(ADP-ribose) polymerase (PARP) in BLBC-BM lines after 8-hour treatment with Ctrl, ESDRL, or EVDRL (n = 3, technical replicates). (Loading control–adjusted ratios are provided under blots; only cleaved part was quantified). (E) Cell viability of 18 BC cell lines after 72-hour treatment with different concentrations of Ctrl, EV, DRL, or EVDRL (n = 3, technical replicates). (F) Correlation between cell surface DR4/5 expression and growth inhibition effect of DRL at the time point of 24 hours. (G) Correlation between cell surface EGFR expression and growth inhibition ratio between DRL and EVDRL at the time point of 24 hours. (H) Correlation between cell surface DR5 and EGFR expression and the growth inhibition efficacy of EVDRL. (I) WB showing phosphorylation of EGFR and its downstream elements in BLBC-BM lines with EGF treatment after pretreatment with various concentrations of EVDRL (n = 3, technical replicates). (J) WB showing cleavage of caspases and PARP in BLBC-BM lines after 8-, 16-, and 24-hour treatment with Ctrl or EVDRL (n = 3, technical replicates). (K) Caspase-Glo 3/7 assay of BLBC-BM lines after 8-hour treatment with Ctrl or EVDRL (n = 3, technical replicates).

Fig 5: Overexpression of IAP members, TRAIL receptors DR4, DR5 and BCL-2 members in colorectal adenocarcinoma cell lines: DLD-1 (lane 2), HCT116 (lane 3), SW620 (lane 4), HT29 (lane 5), RKO (lane 6) and Colo-205 (lane 7), compared to Caco2, an intermediate colorectal adenoma cell line (lane 1). a: Relative RNA and protein levels of cIAP-1, cIAP-2, XIAP, SURVIVIN, DR4 and DR5 genes were evaluated by Real Time PCR and W.B analysis respectively in colon adenocarcinoma cell lines. The analysis was performed in triplicates and the ± SD is shown. Columns indicate relative RNA levels normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Proteins are quantified against a-Tubulin. b: Protein levels of other (anti)apoptotic factors in colon adenocarcinoma cell lines by W.B. Data are representative for three independent experiments