Allicin Induces Gastric Cancer Cells SGC-7901 Apoptosis And Its Mechanism

BackgroundGastric cancer is the second leading cause of cancer-related deaths in the world, with an estimated-760,000 cases of gastric cancer are diagnosed annually worldwide,. Gastric cancer is often asymptomatic, with only nonspecific symptoms in its early stages. Thus, by the time symptoms occur, the cancer has generally metastasized to other regions of the body. The cause of gastric cancer remains unknown, however it is more common in 55 years or older men. In addition, a high salt diet and low vitamin intake increase the risk of gastric cancer. Pre-existing conditions, such as pernicious anemia, atrophic gastritis, and intestinal polyps also increase the risk and additional risk factors include hereditary nonpolyposis colon cancer syndrome, Li-Fraumeni syndrome, a family history of gastrointestinal cancer, and blood type A. A diet high in raw fruits and vegetables, citrus fruits, and fiber may lower the risk for gastric cancer. Surgery is still the first line of treatment for gastric cancer, with chemotherapy showing limited effectiveness. The prognosis of gastric cancer remains poor; nevertheless, if detected early, long-term survival of gastric cancer is very favorable. Therefore, novel approaches for early detection, prevention, and treatment strategy are needed. Our research objective was to develop novel agents for the prevention and treatment of this deadly disease.Recently, it has become new ways to extract the active ingredients from the traditional Chinese medicine for cancer prevention and treatment.Garlic is widely used for its pungent flavor as a seasoning and in China, garlic has been consumed and used to treat various ailments for over three thousand years. More recently, epidemiological studies have shown that dietary intake of allium-containing food, such as garlic, decreased the risk of developing various types of malignancies, although the underlying molecular mechanisms remain to be defined. Allicin, diallyl thiosulfinate, is the main biologically active compound derived from garlic. It was first isolated and studied in the laboratory by Chester J. Cavallito in 1944 where it was found to exhibit antibacterial and anti-fungal properties. It is formed by the enzyme alliinase on alliin (S-allyl-L-cysteine sulfoxide), with alliin is chiral and only occurs as a racemic enzyme. This racemic form can also be generated by the oxidation of diallyl disulfide. Alliinase and alliin are enclosed in different compartments within the garlic cloves, with the intact garlic cloves not containing allicin. When the garlic clove is crushed, alliin and alliinase interact to form allicin, pyruvic acid and ammonia.About the anti-tumor mechanism of allicin, allicin blocks the synthesis of carcinogens, inhibit activation of carcinogens and shows anti-mutagenic, anti-distortion effect.In a previous study by our group, allicin was demonstrated to induce mitotic arrest of gastric cancer cells, and one of its possible mechanisms is due to enhancing microtubule depolymerization by elevating the expression of cyclin Bl. In the current study, we further investigated the effects of allicin on gastric cancer cells and evaluated its antitumor mechanism. However, the allicin-induced apoptosis in gastric cancer cells and its mechanism remains to be further explored.Cisplatin is the most widely used for chemotherapy drugs. However, clinical effect of cisplatin is dose-dependent, clinical side effects is serious when application of high-dose, which limits of cisplatin high-dose in the clinical application, Therefore, the efficacy of cisplatin is still need to be improved. In order to improve the clinical efficacy, the conventional way of chemotherapy is to combinate drugs. it is needs further study whethe allicin combined with cisplatin increase the rate of cancer cell apoptosis.In our present study, gastric cancer line SGC-7901 cells as a model, to study the allicin induced gastric cancer SGC-7901 cells apoptosis and its mechanism, and to study the synergistic effect of allicin combined with cisplatin inducing gastric cancer cell apoptosis.Materials and methods1. The MTT assay was used to detect cell viability.2. To observe the morphological changes of SGC-7901 cells by inverted microscope.3. To observe the morphological changes of SGC-7901 cells by transmission electron microscopy.4. To observe the morphological changes of SGC-7901 cells by Rh123 and propidium iodide staining.5. Gastric cancer cells undergoing apoptosis were further determined using an Annexin V/FITC apoptosis kit.6. Analysis of alterations in mitochondrial membrane potential by the fluorescent dye rhodamine 123 Staining.7. Immunocytochemistry and western blot was used to detect gene expression.8. Q-RT-PCR was used to detect gene expression.9. The data were summarized from at least three independent experiments. The results were expressed as the mean±standard deviation and evaluated using one-or two-way analysis of variance (ANOVA) followed by the Student's t-test. Statistical significance was defined as P<0.05.Results1. We found that allicin reduced cell viability in a dose-and time-dependent manner, partly through induction of apoptosis in gastric cancer cells. Allicin, after 24h treatment with 15μg/ml allicin, after 24h treatment with 120μg/ml allicin, reduced the viable cells by 95.3±2.61%, and by 78.3±2.63%; The same concentration of allicin reduced the viable cells deeply along with the allicin treatment longer time.Allicin, after 24h treatment with 30μg/ml allicin, after 48h treatment with 30μg/ml allicin, after 72h treatment with 30μg/ml allicin, reduced the viable cells by 93.89±3.01%, by 70.1±4.5%and by 60.1±2.5%.2. The morphological changes of SGC-7901 cells by inverted microscope:cell volume were smaller, cells were round, which showed dead cells increased significantly.3. We performed transmission electron microscope detection of morphology changes following treatment of the SGC-7901 cells with 30 g/ml allicin for 48 h and found that the allicin-treated cancer cells had reduced size, increased chromatin condensation, cytoplasm density, cytoplasm vacillation, and marinated nuclear chromatin; cell nucleus was broken, suggesting that the cells underwent apoptosis. However, control cells showed normal nuclei and cytoplasm and microvillies on the cell surface.4. We chemically stained the gastric cancer cells with Rh123 and propidium iodide and found that after treatment with 30μg/ml allicin for 48h, SGC-7901 cells showed chromatin condensation and fragmentation of the cells, nucleus with PI staining, and enhanced fluorescence, indicating that cell lose viability, whereas the control cells not manifested the character.5. Annexin V/FITC assay showed the rate of apoptotic cells, the control group was 2.48±0.5%, the group of treatment with 30μg/ml allicin for 48h was increased to 15.14±1.5%, Comparing with the control group, the difference was significant(P<0.05).6. The test of mitochondrial membrane potential demonstrated that control cells showed strong staining for Rh123, indicating a negative membrane potential of the mitochondria and the accumulation of the lipophilic dye, whereas allicin-treated cells were depolarized and the cell population was shifted to the left, indicating that allicin induced significant disruption of the mitochondrial potential membrane potential (ΔΨm), respectively 90.81±3.48%and 47.75±5.41%.7. Immunohistochemistry and western blot showed that Caspase 3, Bax, Cytochrome c, Fas and Caspase 8 protein expression of allicin treatmeat group 发"increased, compared with control group.8. Real-time PCR showed that Caspase 3 mRNA,Bax mRNA,Cytochrome c mRNA and Fas mRNA of allicin treatmeat group amplificated, compared with control group.9. Allicin(30μg/ml) combined with cisplatin(2μg/ml) reduced the viable cells by 81.12±3.58%,42.33±3.86%and 22.12±4.12%, after 24h treatment, after 48h treatment, after 72h treatment by MTT test.The result showed that allicin combined with cisplatin played a synergistic effect on reducing cell viability, the difference was significant compared with the control group, the allicin group and cisplatin group.10. We chemically stained the gastric cancer cells with Rh123 and propidium iodide and found that after treatment with allicin(30μg/ml) combined with cisplatin(2μg/ml) for 48h, SGC-7901 cells showed chromatin condensation and fragmentation of the cells, nucleus with PI staining, and enhanced fluorescence, indicating that cell lose viability, whereas the control cells not manifested the character.11. Annexin V/FITC assay showed the rate of apoptotic cells, the group of allicin(30μg/ml) combined with cisplatin(2μg/ml) for 48h, was 38.3±0.5%, the control group was 2.48±0.5%, the group of treatment with 30μg/ml allicin for 48h was increased to 15.14±1.5%, the group of treatment with 2μg/ml cisplatin for 48h was 30.96±0.5%, The result showed that allicin combined with cisplatin play a synergistic effect on inducing apoptosis, the difference was significant compared with the control group, the allicin group and cisplatin group.12. The test of mitochondrial membrane potential demonstrated mitochondrial membrane potential was 55.26±4.88%in the group of allicin(30μg/ml) combined with cisplatin(2μg/ml) for 48h, the control group was 90.81±3.48%.13. Immunohistochemistry and western blot showed that Caspase 3, Bax, Cytochrome c, Fas and Caspase 8 protein expression increased in allicin(30μg/ml) combined with cisplatin(2μg/ml) for 48h, compared with control group. Conclusion1. Allicin induced gastric cancer cells to apoptosis.2. Allicin promoted the expression of Bax, Cytochrome c, reduced mitochondrial membrane potential, and activated of Caspase 3, which showed that mitochondrial apoptosis pathway was involved in gastric cancer cells apoptosis.3. Allicin promoted the expression of Fas and Caspase 8 expression, induced gastric cancer cells apoptosis.which showed death receptor pathway was involved in gastric cancer cells apoptosis.4. The result showed that allicin combined with cisplatin play a synergistic effect on inducing apoptosis in gastric cancer cells.5. Mitochondrial pathway and death receptor pathway were both involved during the process of allicin combined with cisplatin inducing gastric cancer cells to apoptosis. The result showed that allicin combined with cisplatin played a synergistic effect on inducing gastric cancer cells to apoptosis, which mechanism was that mitochondrial pathway and death receptor pathway were both involved.