Gintonin-Enriched Panax ginseng Extract Fraction Sensitizes Renal Carcinoma Cells to TRAIL-Induced Apoptosis through DR4/5 Upregulation

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising chemotherapeutic agent because of its selective apoptotic action on cancer cells. However, resistance to TRAIL-induced apoptosis remains a challenge in many cancers. The gintonin-enriched Panax ginseng extract fraction (GEF) has diverse pharmacological benefits. We explored the combined efficacy of GEF and TRAIL in inducing apoptosis in human renal cell carcinoma (RCC) cells. The effect of GEF treatment on the viability, clonogenic potential, wound healing, and TRAIL-induced apoptotic signaling of RCC cells was studied in vitro. Our investigation revealed that GEF pre-treatment sensitized RCC cells to TRAIL-induced apoptosis, as evidenced by DNA fragmentation and cell proliferation, colony formation, and migration inhibition. This sensitization was linked to the upregulation of death receptors 4 and 5 and alterations in apoptotic protein expression, notably, the decreased expression of the Mu-2-related death-inducing gene, a novel anti-apoptotic protein. Our findings underscore the necessity of caspase activation for GEF/TRAIL-induced apoptosis using the pan-caspase inhibitor Z-VAD-FMK. This study demonstrates that GEF sensitizes human RCC cells to TRAIL-induced apoptosis by upregulating DR4/5 and modulating apoptotic protein expression. These findings suggest a promising strategy for overcoming TRAIL resistance in cancer therapy and highlight the potential of GEF as a valuable adjunct to TRAIL-based treatments.

Keywords: apoptosis; gintonin-enriched fraction; human renal cell carcinoma; mu-2-related death-inducing gene; tumor necrosis factor-related apoptosis-inducing ligand.

Figure 1

Gintonin-enriched Panax ginseng extract fraction (GEF) enhances the sensitivity of human renal cell carcinoma (RCC) cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). (A,B) 786-O and A498 cells were pre-treated with varying GEF concentrations or DMSO for 12 h, followed by TRAIL treatment with or without GEF for another 12 h. The cell viability was assessed using the WST-1 assay. (C) Morphological changes occurred in the cells after 12 h of pre-treatment with GEF followed by TRAIL treatment (40× magnification). The data are represented as the mean ± standard deviation (SD) of three independent experiments, with statistical significance denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the untreated control group.

Figure 2

The combination of gintonin-enriched Panax ginseng extract fraction (GEF) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) suppresses the clonogenic formation and migration of human renal cell carcinoma (RCC) cells. (A) 786-O and A498 cells were pre-treated with and without GEF for 12 h, followed by TRAIL treatment with and without GEF for an additional 12 h before reseeding. The colony formation results are shown on the left, with the percentage of stained colonies displayed in the graph on the right. (B,C) The cells were scratched and pre-treated similarly before wound size measurement. The data are represented as the mean ± standard deviation (SD) of three independent experiments, with statistical significance denoted as * p < 0.05, and *** p < 0.001 compared to the untreated control group.

Figure 3

The combination of GEF and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis and DNA fragmentation in human renal cell carcinoma (RCC) cells. (A) 786-O and A498 cells were pre-treated with and without GEF for 12 h, followed by TRAIL treatment with and without GEF for another 12 h. Flow cytometry analysis was conducted to assess cell apoptosis. (B,C) The cells underwent similar pre-treatment before terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining to observe DNA fragmentation. The proportion of TUNEL-positive cells was quantified. The data are represented as the mean ± standard deviation (SD) of three independent experiments, with statistical significance denoted as * p < 0.05, and *** p < 0.001 compared to the untreated control group.

Figure 4

Gintonin-enriched Panax ginseng extract fraction (GEF) elevated death receptors 4 and 5 (DR4/5) expression in human renal cell carcinoma (RCC) cells. (A) 786-O and A498 cells underwent 12 h pre-treatment with GEF, followed by 12 h treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). RT-qPCR was used to analyze the DR4 and DR5 mRNA levels. (B) Cells received pre-treatment with GEF for 12 h before exposure to various TRAIL concentrations for another 12 h. Western blot analysis quantified the DR4 and DR5 protein levels relative to that in the controls. (C) After a 24 h treatment with GEF or DMSO, flow cytometry assessed the cell surface expression of DR4 and DR5. The data are represented as the mean ± standard deviation (SD) of three independent experiments, with statistical significance denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001; n.s., non-significant compared to the untreated control group.

Figure 5

The combination of gintonin-enriched Panax ginseng extract fraction (GEF) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) regulated the expression of apoptotic proteins. (A) 786-O and A498 cells underwent a 12 h pre-treatment with specified GEF concentrations, followed by a 12 h treatment with specified TRAIL concentrations. Western blot analysis assessed the apoptotic protein expression levels relative to the controls. (B,C) Cells received 12 h of pre-treatment with specified GEF concentrations before exposure to specified TRAIL concentrations for an additional 12 h. Immunoprecipitation using death receptors 4 (DR4) and DR5 Abs was followed by Western blot analysis of caspase-8 and Fas-associated death domain (FADD). The fold change of caspase-8 and FADD was normalized to DR4 or DR5 and to the untreated control group (=1.00).

Figure 6

Gintonin-enriched Panax ginseng extract fraction (GEF)/ tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis relies on the caspase activity in human renal cell carcinoma (RCC) cells. (A) 786-O and A498 cells were pre-treated with 20 µM Z-VAD-FMK for 1 h, followed by 12 h treatment with and without GEF. Subsequently, the cells received a 12 h treatment with and without TRAIL. Cell apoptosis was quantified using flow cytometry. (B) 786-O and A498 cells underwent a 1 h pre-treatment with 20 µM Z-VAD-FMK, followed by a 12 h treatment with specified GEF concentrations. A subsequent 12 h treatment with specified TRAIL concentrations was performed, and the cell viability was assessed using the WST-1 assay. The data are represented as the mean ± standard deviation (SD) of three independent experiments, with statistical significance denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the untreated control group.

Figure 7

Schematic representation illustrating the induction of apoptosis by the combined action of gintonin-enriched Panax ginseng extract fraction (GEF) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Abbreviations: Bax, Bcl-2–associated X protein; Bcl-2, B-cell lymphoma-2; Bid, BH3 interacting-domain death agonist; DISC, death-inducing signaling complex; DR4/5, death receptors 4 and 5; FADD, Fas-associated death domain; LPA, lysophosphatidic acid; MuD, Mu-2-related death-inducing gene; tBid, truncated Bid.