| [Background and Objectives]Gastric cancer is one of the most common cancers worldwide. Although gastric cancer incidence and mortality rates has fallen over the last30years, but remains a leading cause of cancer-related death worldwide, second only to lung cance. Infiltrative growth and metastasis are two lethal processes of gastric cancer. Despite the possibility of curative surgery, the prognosis of advanced gastric cancer remains very poor.Today, the standard therapies for gastric cancer include surgery, chemotherapy, and herbal therapy. These therapies are often used in combination, and they are the best hope for gastric cancer sufferers. Now, the use of herbal medicines for gastric cancer is widespread because of their safety and few side effects. Rheum palmatum L. is one of the ancient and best-known plant medicines, which has been used in China for thousands of years. Emodin (1,3,8-Tri-hydroxy-6-methylanthraquinine) is an anthraquinone derivative isolated mainly from the root and rhizome of it. Some studies have indicated that emodin has a number of biological properties, including anti-proliferation, anti-inflammatory, anti-pain and antiviral activities. Emodin has also been reported to exhibit an anticancer activity on human lung, liver, and ovarian cancers. However, there is relatively little information in the published literature with regard to the anticancer mechanism of emodin.In this study, we investigated the levels of PRL-3and Bmi-1mRNA expression in gastric cancer tissues and paired adjacent normal gastric tissues, evaluated their clinical significance in gastric cancer. Additionally, we investigated the effects of emodin on SGC-7901cells proliferation, apoptosis, regulation of PRL-3expression and PI3K/AKT pathway, thereby finding a new potential therapeutic target for aggressive gastric cancer.[Methods]MTT assay for cell viability Cell viability was determined by MTT assay. The cells (6×103) were seeded in96-well plates and were incubated at37℃in5%CO2/95%air. After24h, these cells were treated with emodin at different (15,30,45and60μM) concentrations and incubated for24,48and72h. The control group was treated with the equivalent amount of dimethyl sulphoxide (DMSO, the maximum of0.2%of the assay mixture was used). Apoptotic cell determination by AO/EB method After24hours with emodin treatment, SGC-7901cells were collected by using trypsin. After being washed by PBS, the cells were resuspended in PBS. Mixde with proper acridine orange and ethidium bromide solution, apoptosic cells in resuspended solution were calculated with fluoroscope.Apoptotic cell determination by Annexin V/PI staining assay Various concentrations of emodin were added to the wells and incubated for an additional24h (the final concentrations of emodin were15,30,45and60μM). The cells were resuspended in200μl of binding buffer.10ul of Annexin V-FITC and5ul of propidium iodide were added to the resuspended cells. After incubation at room temperature for15min in the dark,300μl of binding buffer was added to the resuspended cells, and then the stained cells were analyzed by flow cytometry.Expression of PRL-3mRNA in gastric cancer tissues and adjacent normal gastric tissues52patients with gastric cancer admitted to Affiliated Hospital to Yangzhou University for surgical treatment were included into this study. SYBR Green Ⅰ Real-time fluorescent quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to study the level of PRL-3mRNA expression in gastric cancer and paired adjacent normal gastric tissues (5cm from carcinoma). The human glyceraldehydes-3-phosphate dehydrogenase(hGAPDH) gene was used for normalization of gene expression levels.Clinical significance of PRL-3and Bmi-1mRNA in gastric cancer by real-time quantitative PCR SYBR Green Ⅰ Real-time fluorescent quantitative reverse transcriptase polymerase chain reaction was used to study the levels of PRL-3and Bmi-1mRNA expression in gastric cancer. The human glyceraldehydes-3-phosphate dehydrogenase (hGAPDH) gene was used for normalization of gene expression levels. The relationship between the levels of PRL-3and Bmi-1mRNA expression and clinicopathologic factors in gastric cancer were analyzed statistically.Clinical significance of PRL-3and Bmi-1in gastric cancer by immunohistochemistry anaysis A total of116gastric adenocarcinoma cases were used. Immunohistochemical staining S-P method was performed on tissue Paraffin section. Correlations between the expression of PRL-3protein, Bmi-1protein, and clinicopathological features of gastric cancers were analyzed.Analysis of PRL-3mRNA alteration after emodin treatment by real-time quantitative PCR After treated with15,30,45and60μM emodin for24h, SGC-7901cells were harvested by trypsinization and RNA was extracted by using TRIZOL(?) reagent. Real-time fluorescence detection was carried out with Applied Biosystems7500Real Time PCR System.Analysis of PRL-3alteration after emodin treatment by western blot analysis After treated with15,30,45and60μM emodin for24h, the cells were lysed in RIPA buffer containing protease inhibitor cocktail. Proteins were electrophoresed on SDS-polyacrylamide gels and transferred to a PVDF membrane. Immunoreactive bands were detected using DAB method.Analysis of Akt, phospho-Thr308-Akt, phospho-Ser473-Akt and Bcl-2family proteins after emodin treatment by western blot analysis After treated with15,30,45and60μM emodin for24h, Proteins were electrophoresed on SDS-polyacrylamide gels and transferred to a PVDF membrane. Primary antibodies were incubated with membranes overnight at4℃. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody, and then immunoreactive bands were detected using DAB method.[Results]1. After treatment with15,30,45and60μM emodin for24,48and72h, emodin decreased the cell viability in concentration and time-dependent manners. Cell viability was inhibited by13.71%at the lower concentration as15μM. Maximum inhibition (80.98%) was observed at60μM.2. Apoptotic changes were analyzed by using AO/EB method. After treated with15,30,45and60uM emodin for24h, apoptotic cells were increased (4.05%into14.79%,27.84,35.15%and43.63%, respectively).3. Early apoptotic changes were identified using AnnexinV/PI staining. After treated with15,30,45and60μM emodin for24h, the early apoptotic cells were increased (3.52%into11.63%,23.58,30.45%and37.25%, respectively).4. PRL-3mRNA can be detected in all specimens. The level of PRL-3mRNA expression in gastric cancer was significantly higher than in paired adjacent normal gastric tissues(P<0.01).5. The level of PRL-3protein expression in gastric cancer was significantly higher than in paired adjacent normal gastric tissues(P<0.01).6. PRL-3and Bmi-1mRNA can be detected in all specimens. The levels of PRL-3and Bmi-1mRNA expression in gastric cancer showed no association with patient’s gender, patient’s age. However, the levels of PRL-3and Bmi-1mRNA expression were correlated with tumor size, TNM staging, lymph node metastasis and the depth of tumor invasion. In addition, PRL-3mRNA expression was correlated with tumor histodifferentiation.7. PRL-3protein expression was irrelated with gender, age, tumor size and tumor histodifferentiation of gastric carcinoma (P>0.05), but was closely related with TNM staging, lymph node metastasis and the depth of tumor invasion. Bmi-1protein expression was irrelated with gender, age and tumor histodifferentiation, but was closely related with TNM staging, lymph node metastasis, the depth of tumor invasion and tumor size.8. Emodin treated SGC-7901cells showed a significant decrease in PRL-3mRNA expression when compared with control (P<0.01). Emodin at the concentration of15μM decreased PRL-3mRNA of SGC-7901cells by32.47%, while at concentration of60μM; the level of PRL-3mRNA was decreased by87.72%.9. After SGC-7901cells were treated with15,30,45and60μM emodin for24h respectively, PRL-3expression was down-regulated in a concentration-dependent (15-60μM) manner. The emodin-mediated changes in the protein levels of PRL-3coincided well with the mRNA levels as evidenced by real-time quantitative PCR results.10. After SGC-7901cells were treated with15,30,45and60μM emodin for24h respectively; the levels of phospho-Thr308-Akt and phospho-Ser473-Akt were reduced in a concentration-dependent manner. However, emodin treatment did not alter the level of total Akt protein expression.11. Exposure of SGC-7901cells with15,30,45and60μM emodin caused in a significant decrease in Bcl-2while increase in Bax protein levels after24h treatment. The ratio of Bax/Bcl-2was significantly increased in a concentration-dependent manner with emodin treatment.[Conclusion]1. Emodin inhibits SGC-7901cell viability in a concentration-and time-dependent manner. Apoptosis rates increased in a concentration-dependent manner after emodin treatment, it suggested that apoptosis induction of emodin involved in its antitumor effect.2. The level of PRL-3mRNA expression in gastric cancer was significantly higher than in paired adjacent normal gastric tissues, it might be a novel molecular marker for aggressive gastric cancer.3. High expressions of PRL-3and Bmi-1are associated with gastric cancer progression, and they might be novel molecular markers for aggressive gastric cancer. The combined detection of two tumor markers can increase diagnosis rate for gastric cancer, which shows some clinical value.4. PRL-3expression level decreased when SCG-7901cells were treated with emodin, which is positively related with apoptotic rate, all these indicated down-regulation of PRL-3is involved in the proliferation inhibition and apoptosis induced by emodin in human gastric carcinoma cell line SGC-7901.5. Emodin-mediated inactivation of AKT could be involved in the anti-proliferative/pro-apoptotic response of SGC-7901cells to emodin. The ratio of Bax/Bcl-2was significantly increased in a concentration-dependent manner with emodin treatment, suggesting that SGC-7901cell apoptosis occurs with the involvement of Bcl-2family proteins. The down-regulation of PI3K/AKT signaling pathway contributes to emodin-induced SGC-7901gastric cancer cell line apoptosis. |
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