RGO-SMEDDS inhibits HCC proliferation by inducing apoptosis and G2 phase arrest.
Functional assays were performed to assess the anti-tumor effects of RGO-SMEDDS. First, we examined the effects of 0, 0.15, 0.30, 0.60, and 1.20 µM RGO-SMEDDS on the proliferation of HCC and LO2 cells by CCK8 assay (Fig. 1A). RGO-SMEDDS inhibited the proliferation of liver cancer cells in a dose-dependent manner and did no harm to normal cells within the prescribed concentration range. In order to determine an appropriate administration concentration, we assessed the semi-lethal dose of RGO-SMEDDS for Huh7 and HepG2 cells by IC50 assay (Fig. 1B). The RGO-SMEDDS IC50s for Huh7 and HepG2 were 1.2 µM and 2.0 µM, respectively. And this dose was used as the treatment concentration for subsequent experiments in vitro. Clone formation was used to assess the effects of RGO-SMEDDS on cell viability. RGO-SMEDDS significantly inhibited the viability of Huh7 and HepG2 cells (Fig. 1C). As judged by FCM, RGO-SMEDDS increased the percentage of cells in the G2/M phase compared to control (Fig. 1D). Furthermore, RGO-SMEDDS induced a higher apoptosis rate as judged by FCM with V-FITC and PI staining (Fig. 1E). Overall, RGO-SMEDDS effectively produced anti-tumor effects by inhibiting cell proliferation, reducing cell viability, inducing cell cycle arrest, and promoting cell apoptosis.
RGO-SMEDDS suppresses xenograft tumor growth in vivo.
The above experiments demonstrated the anti-tumor effects of RGO-SMEDDS in vitro. To assess the effects of RGO-SMEDDS on HCC in vivo, Subcutaneous xenograft models were constructed in nude mice and C57B/L mice. As shown in (Fig. 2A-F), tumor size of RGO-SMEDDS-treated mice was significantly reduced compared to control mice, indicating significant inhibition of liver cancer growth. Furthermore, more obvious nuclear fragmentation and proliferation inhibition was found in RGO-treated group via HE and IHC staining assay (Fig. 2G).
RGO- SMEDDS inhibits HCC invasion, migration, and angiogenesis.
The effects of RGO-SMEDDS on metastasis were assessed. By migration assay, RGO-SMEDDS reduced cell migration (Fig. 3A) and by invasion assay, RGO-SMEDDS significantly weakened the ability of HCC cells to invade barriers (Fig. 3B). The effect of RGO-SMEDDS on angiogenesis was measured by tubule formation assay (Fig. 3C). HUEVC cells were cultured in conditioned medium of HepG2 and Huh7 cells treated or not with RGO-SMEDDS for 48 h. RGO-SMEDDS significantly inhibited angiogenesis induced by HepG2 and Huh7 cells. In addition, we performed qRT-PCR and WB analysis to investigate the mechanism of RGO on angiogenesis (Fig. 3D-E). Compared to control, the expression of VEGF and VEGFR in RGO-SMEDDS-treated HCC cells and tissues was significantly down-regulated. Similar results were found by IHC, where RGO-SMEDDS significantly down-regulated VEGF and VEGFR levels in tumor tissues (Fig. 3F). Further, the number of blood vessels in the treated group was much less than in control. Taken together, these results demonstrated RGO-SMEDDS can effectively inhibit the migration, invasion, and angiogenesis of HCC cells.
RGO-SMEDDS inhibits the production of immunosuppressive cytokine and M2 macrophages.
The qRT-PCR was used to assess cytokine mRNA expression levels in HCC cells and tumor tissues treated with or without RGO-SMEDDS(Fig. 4A). RGO-SMEDDS significantly reduced the expression levels of IL2, IL4, IL10, IL13, and M-CSF1, which are closely related to M2 macrophage differentiation. Based on these results we speculated that RGO-SMEDDS inhibited M2-polarized macrophage induced by HCC cells. In order to verify this speculation, we added RGO-SMEDDS to the THP1-HCC co-culture system, and investigated the mRNA expression level of M2 macrophage markers in THP1 cells by qRT-PCR (Fig. 4B). The expression levels of IL-10, CD163, and Arg1 were significantly down-regulated in THP1 cells from RGO-SMEDDS added co-cultures system. Moreover, we found RGO-SMEDDS to directly inhibit IL10, CD163, and Arg1 in THP1 cells, which indicated that RGO-SMEDDS could not only prevent the M2-polarized macrophage induced by HCC cells, but also directly inhibit the formation of M2 macrophages. IHC was used to assess the proportion of M2-like macrophages surrounding tumor tissues (Fig. 4C), and the results demonstrated the number of M2 macrophages to be significantly less in RGO-SMEDDS-treated mice compared to control. Therefore, RGO-SMEDDS improved the immunosuppressive microenvironment by inhibiting the M2-type differentiation of macrophages.
EGFR may be a potential target of Oridonin.
In order to explore the anti-tumor mechanism of Oridonin, the 3D structure (obtained from PubChem) was used to predict Oridonin targets by use of Pharmmapper (Fig. 5A). GO enrichment analysis of predicted Oridonin targets were mainly tyrosine kinases, which are closely related to tumor occurrence and development (Fig. 5B, C). AutoDock 1.1.2 software was used to simulate molecular docking between Oridonin and the tyrosine kinase receptor, EGFR (Fig. 5D-F). We found Oridonin to form hydrogen bonds with the amino acid residue, Arg841, and to form hydrophobic interactions with amino acid residue Cys797, Val726, and Leu844. Moreover, Oridonin fit well within the activation cavity of EGFR (docking score − 7.2 kcal/mol). These results suggest Oridonin to be a potential inhibitor of EGFR.
RGO-SMEDDS suppresses HCC stemness via the EGFR/AKT and GSK3α/β signaling pathways.
IHC staining demonstrated EGFR and P-EGFR levels to be significantly lower than control with RGO treatment (Fig. 6A).By WB, EGFR and P-EGFR were detected in HCC cells treated with RGO-SMEDDS and EGF(Fig. 6B). The results showed that RGO-SMEDDS effectively inhibited EGFR phosphorylation induced by EGF. In order to investigate the anti-tumor mechanism of RGO-SMEDDS, we detected downstream molecules of the EGFR signaling by WB (Fig. 6C). With RGO-SMEDDS treatment, the levels of AKT/p70S6K and GSK3α/β were significantly reduced compared to control group, suggesting that RGO-SMEDDS may exert an anti-tumor effect by inhibition of HCC stemness. A sphere formation assay was used to confirm this speculation. Results demonstrated that RGO-SMEDDS treatment significantly reduced the number of spheres compared to control (Fig. 6D, E). In addition, the results of qRT-PCR showed that RGO-SMEDDS down-regulated mRNA expression of stemness marker both in HCC cells and tumor tissues (Fig. 6G). By WB, similar results were obtained (Fig. 6F). These results demonstrated RGO-SMEDDS to attenuate HCC stemness by suppressing the EGFR/AKT and GSK3α/β signaling pathways.
RGO-SMEDDS is a safe and effective anti-tumor drug.
Within 14 days after administration, the mice in the 1000 mg/kg group showed sluggish activity within 2–3 hours after administration and the other group (0, 100, 200, mg/kg) were in good condition without significant weight loss. To assess RGO-SMEDDS toxicity, HE staining was performed on tissue sections of major organs. No histopathological changes were found in the brain, gastric, colon, heart, liver, spleen, lung and kidney tissues, indicating that RGO-SMEDDS has a relatively low degree of toxicity (sFig. 2). Since no half-lethal dose of RGO-SMEDDS was found, we performed an LD50 test with the maximum dose (1,000 mg/kg). The results of HE staining were shown in Fig. 7 and are consistent with previous experiments.