Ginsenoside Rg3 suppresses vasculogenic mimicry by impairing DVL3-maintained stemness via PAAD cell-derived exosomal miR-204 in pancreatic adenocarcinoma

Vasculogenic mimicry (VM) is an angiogenesis-independent process that potentially contributes to the poor clinical outcome of anti-angiogenesis therapy in multiple malignant cancers, including pancreatic adenocarcinoma (PAAD). Several studies have shown that ginsenoside Rg3, a bioactive component of ginseng, holds considerable potential for cancer treatment. Our previous work has proved that Rg3 can inhibit VM formation in PAAD. However, its underlying mechanism remains unclear. To explore the underlying mechanism by which Rg3 affects VM formation in PAAD. We first investigated the effects of Rg3 on the cellular phenotypes of two PAAD cell lines (SW-1990 and PCI-35), and the expression of EMT- and stemness-related proteins. SW-1990 cells were adopted to construct xenograft models, and the anti-tumor effects of Rg3 in vivo were validated. Subsequently, we isolated the exosomes from the two PAAD cell lines with Rg3 treatment or not, and explored whether Rg3 regulated VM via PAAD cell-derived exosomes. MiRNA sequencing, clinical analysis, and rescue experiments were performed to investigate whether and which miRNA was involved. Subsequently, the target gene of miRNA was predicted using the miRDB website (https://mirdb.org/), and rescue experiments were further conducted to validate those in vitro and in vivo. Rg3 indeed exhibited excellent anti-tumor effects both in vitro and in vivo, with inhibitory effects on EMT and stemness of PAAD cells. More interestingly, Rg3-treated PAAD cell-derived exosomes suppressed the tube-forming ability of HUVEC and PAAD cells, with a decrease in stemness-related protein expression, indicating that Rg3 inhibited both angiogenesis and VM processes. Subsequently, we found that Rg3 induced the up-regulation of miR-204 in PAAD cell-derived exosomes, and miR-204 alone inhibited tube and sphere formation abilities of PAAD cells like exosomes. Specifically, miR-204 down-regulated DVL3 expression, which was involved in regulating cancer cell stemness, and ultimately affected VM. The in vivo experiments further indicated that Rg3-treated SW-1990 cell-derived exosome-inhibited tumor growth, VM formation, and stemness-related protein expression can be abrogated by DVL3 overexpression. Ginsenoside Rg3 increased the PAAD cell-derived exosomal miR-204 levels, which subsequently inhibited its target genes DVL3 expression in the receptor PAAD cells, and the down-regulated DVL3 broke stemness maintenance, ultimately suppressing VM formation of PAAD. Our findings revealed a novel mechanism by which Rg3 exerted its anti-tumor activity in PAAD via inhibiting VM, and provided a promising strategy to make up for the deficiency of anti-angiogenesis therapy in cancer.