Endoplasmic Reticulum Stress and Impairment of Ribosome Biogenesis Mediate the Apoptosis Induced by Ocimum x africanum Essential Oil in a Human Gastric Cancer Cell Line

Abstract

Background and Objectives: Gastric cancer remains a major unmet clinical problem worldwide. Although conventional medical treatments are available, their curative effects are generally unsatisfactory. Consequently, it remains necessary to search natural products for potential alternatives in treating gastric cancer patients. Ocimum x africanum Lour. is a culinary herb that has been used in folk medicine for various diseases, but little is known regarding its anti-cancer activity against gastric cancer cells. In the current study, we focus on the anti-cancer mechanisms of O. x africanum essential oil (OAEO) in the AGS human gastric cancer cell line. Materials and Methods: After OAEO treatment, AGS cell viability was evaluated by MTT assay. Cell migration and apoptotic nuclear morphology were determined by wound-healing assay and DAPI staining, respectively. Gene expression levels of apoptosis-related genes were quantified by qRT-PCR. Differential protein expression was determined with an LC-MS/MS-based proteomics approach to identify the key proteins that may be important in the anti-cancer mechanisms of OAEO on AGS cells. The chemical constituents of OAEO were identified by GC-MS analysis. Results: We found OAEO to exhibit a potent growth-inhibiting effect on AGS cells, with an IC₅₀ value of 42.73 µg/mL. After OAEO treatment for 24 h, AGS cell migration was significantly decreased relative to the untreated control. OAEO-treated AGS cells exhibited common features of apoptotic cell death, including cell shrinkage, membrane blebbing, chromatin condensation, and nuclear fragmentation. Apoptotic cell death was confirmed by qRT-PCR for apoptosis-related genes, revealing that OAEO decreased the expression of anti-apoptotic genes (BCL2 and BCL-xL) and activated pro-apoptotic genes and apoptotic caspase genes (TP53, BAX, CASP9, CASP12, and CASP3). Moreover, expression of CASP8 was not changed after treatment. Proteomic analysis revealed that OAEO may produce a signature effect on protein clusters relating to unfolded protein accumulation, thereby inducing severe ER stress and also impairing ribosome synthesis. STRING analysis revealed seven up-regulated and 11 down-regulated proteins, which were significantly associated with protein folding and ribosome biogenesis, respectively. Using GC-MS analysis, 6-methyl-5-hepten-2-one, citral, neral, and linalool were found to be the major chemical constituents in OAEO. Conclusions: Taken together, these results indicate that OAEO has a potential anti-proliferative effect on AGS cells. Our molecular findings show evidence supporting an important role of ER stress and ribosome biogenesis impairment in mediating the induction of cell death by OAEO through the mitochondrial-apoptotic pathway. This study, therefore, provides fundamental knowledge for future applications using OAEO as an alternative therapy in gastric cancer management.

Keywords: AGS gastric cancer; ER stress; Ocimum x africanum; apoptosis; essential oil; ribosome biogenesis.

Figure 1

Flow diagram of this study.

Figure 2

Inhibition of cell viability by OAEO and 5-FU in AGS cells, as determined by MTT assay. Data are presented as the mean ± standard deviation of three independent experiments. * p < 0.05 vs. control.

Figure 3

Effect of OAEO on AGS cell migration as determined by in vitro wound-healing assay. (A) Representative images of the initial wound area (0 h) and the remaining cell-free area (24 h) in OAEO-treated and vehicle control cultures. (B) Rates of cell migration (%) were quantified by comparing the wound area before and after treatment. Data are presented as mean ± standard deviation from triplicate experiments. * p < 0.05 vs the vehicle control group.

Figure 4

(A) Morphology of vehicle control and OAEO-treated AGS cells at 6, 12, and 24 h, as observed by inverted microscope. (B) Nuclear morphology of vehicle control and OAEO-treated AGS cells at 6, 12, and 24 h, as visualized by DAPI staining. All determinations were performed in triplicate as independent experiments.

Figure 5

Effects of OAEO on apoptosis-related gene expression in AGS-treated cells. Expression levels of TP53, BAX, BCL2, BCL-xL, CASP8, CASP12, CASP9, and CASP3 were determined by qRT–PCR at 6, 12, and 24 h (blue bars: untreated control; red bars: OAEO treatment). Relative gene expression data are presented as mean ± SD. * p < 0.05 vs. vehicle control cells.

Figure 6

Protein–protein interaction networks for up-regulated (A) and down-regulated (B) proteins according to the STRING database.