Synergistic anticancer effects of everolimus (RAD001) and Rhein on gastric cancer cells via phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway

Abstract

Everolimus (RAD001) is a mTOR inhibitor and is widely used for the treatment of gastric cancer (GC). Evidence suggests that Rhein has anticancer effect on GC. But the synergistic effect and mechanism of RAD001 and Rhein combination on GC is not clear. The current study aims to clarify the combination of RAD001 and Rhein in GC treatment. We found Rhein dose-dependently repressed MGC-803 cell viability (50% inhibition concentration (IC50) value = 94.26 μM). Rhein (80 μM) significantly suppressed GC cell proliferation and invasion. RAD001 dose-dependently repressed MGC-803 cells viability (IC50 value = 45.41 nM). The combination of Rhein and RAD001 repressed MGC-803 cells viability, invasion, and proliferation compared to the administration of Rhein or RAD001 alone. Protein levels of epithelial-mesenchymal transition (EMT)-related molecules E-cadherin, N-cadherin and Vimentin expressions were significantly affected by the combination of Rhein and RAD001. The combination of Rhein and RAD001 significantly facilitated cell apoptosis and up-regulated expressions of cell apoptosis and cycle-related protein p53, cyclin-dependent kinase 4 (CDK4) and cyclin D1 compared to the administration of Rhein or RAD001 alone. Moreover, the combination of Rhein and RAD001 repressed the expressions of phosphorylation-phosphoinositide-3-kinase (p-PI3K), p-protein kinase B (p-AKT) and p-mammalian target of rapamycin (p-mTOR). Finally, the combination of RAD001 and Rhein significantly decreased tumor weight and volume, suppressed the expressions of p-PI3K, p-Akt and p-mTOR, and repressed cell proliferation marker Ki-67 expression, which exerted synergistic cancer prevention in GC in vivo. Overall, the combination of Rhein and RAD001 exert synergistic cancer prevention in GC via PI3K/Akt/mTOR pathway.

Keywords: Gastric cancer; Pi3k/Akt/mTOR pathway; RAD001; Rhein.

Figure 1.

Rhein suppressed MGC-803 cell proliferation and invasion. A. structure of Rhein. B. MGC-803 cells were treated with 0, 10, 20, 40, 60, 80, 160 μM Rhein. after 48 hours, IC50 value for Rhein dose response was calculated by MTT assay. C. MGC-803 cells were treated with 0, 80 μM Rhein, MGC-803 cell invasion was detected using transwell assay. D. EdU-staining was applied to detect MGC-803 cell proliferation. EdU-positive cells of MGC-803 cells after 0, 80 μM Rhein treatment for 48 h (×100). EdU was in red. DAPI was in blue. *p < 0.05.

Figure 2.

Synergistic inhibition impacts of Rhein and RAD001 on MGC-803 cell proliferation and invasion. A. structure of RAD001. B. MGC-803 cells were treated with 0, 2.5, 5, 10, 20, 40, 80, 160 nM RAD001. after 48 hours, IC50 value for RAD001 dose response was calculated using MTT. C. Rhein (80 μM) and RAD001 (40 nM) were used for MGC-803 cells, then cell viability was measured using MTT. D. Rhein (80 μM) and RAD001 (40 nM) were used for MGC-803 cells, then MGC-803 cell invasion was detected using transwell. E. EdU-staining was applied to detect MGC-803 cell proliferation. EdU-positive cells of MGC-803 cells after 80 μM Rhein or 40 nM RAD001 treatment for 48 h (×100). F. Light microscope images of MGC-803 cells were observed. G. protein levels of EMT-related molecules E-cadherin, N-cadherin, vimentin were measured using Western blotting. *p < 0.05.

Figure 3.

Synergistic promotion impacts of Rhein and RAD001 on MGC-803 cell apoptosis. Rhein (80 μM) and RAD001 (40 nM) were used to treat MGC-803 cells. A. Rhein and RAD001 combination in MGC-803 cell apoptosis was detected using flow cytometery. B. Rhein and RAD001 combination in MGC-803 cell apoptosis was detected by TUNEL assay. C. The effect of Rhein and RAD001 combination on p53, CDK4 and Cyclin D1expressions was measured using Western blotting. *p < 0.05.

Figure 4.

Synergistic inhibition impacts of Rhein and RAD001 on PI3K/AKT/mTOR pathway. A. KEGG pathway enrichment analysis associated with the target polygonum multiflorum. B. The effect of Rhein and RAD001 combination on PI3K/AKT/mTOR pathway proteins expressions was measured using Western blotting. *p < 0.05.

Figure 5.

Synergistic inhibition effects of Rhein and RAD001 on MGC-803 cell proliferation in vivo. A-C. Mice received Rhein (60 mg/kg) and RAD001 (5 mg/kg) by oral administration. Tumor weight and tumor volume were detected at each group. D. The expressions of PI3K/AKT/mTOR pathway proteins were detected using Western blotting. E. Immunohistochemical (IHC) staining of tumor cell proliferation marker Ki-67 expression. *p < 0.05.