| Objective:To explore the inhibitive effect of phenethyl isothiocyanate(PEITC) on the migration, invasion as well as chemotherapeutic sensitivity of human gastric cancer cell line MGC-803. In addition, it also aims to clarify the mechanism by which PEITC influences TGF-β1R to regulate EMT and alter gastric cancer stem cell phenotype.Methods:1. Growth inhibition of PEITC on gastric cancer cell line MGC-803 was detected by the CCK-8 assay, after which the most appropriate concentration was determined.2. Cell ability of adhesion induced by TGF-β1 were studied by cell adhesion assay which was compared with the siRNA group. Meanwhile, the effect of PEITC on cell invasion and migration was detected by Tranwell assay.3. Immunofluorescence and Western blot were used to detect the changes of the protein relative to metastasis and EMT resulted from PEITC intervention.4. The affect of PEITC on the chemotherapeutic sensitivity of gastric cancer cell line MGC-803 was explored by use of CCK-8 assay, Hoechst33258 nuclear staining and Flow cytometric analysis as well.5. The apoptosis related protein was researched by Western blot assay.6. Flow cytometric analysis was applied in order to study the influence of PEITC on stem cell phenotype of gastric cancer cell line MGC-803.7. Flow cytometric was used to isolate different subtypes of gastric cancer cell line MGC-803, subsequent to which, colony formation assay and xenograft tumor-formation assay were both conducted to verified the characteristics of gastric cancer stem cells with different subtypes.Results:1. After the treatment with 20μM PEITC for 24h, the inhibitive rate of gastric cancer cell line MGC-803 could reach to 48.6%.2. Compared with the control and the TGF-β1 group, the adhesion, migration and invasion ability of human gastric cell line MGC-803 were obviously decreased by treating with PEITC (24μM) for 24 hours.3. PEITC could also downregulate the E-cad/N-cad conversion, reduce the expression of TGF-β1 and upregulate the expression of Vimentin, Twist, MMP-9 and MMP-14.4. 10μM of PEITC could improve the chemotherapeutic sensitivity of human gastric cancer cell line MGC-803 to DDP, which could be decreased by TGF-β1 to make gastric cancer cells less sensitive to chemotherapy.5. Hoechst33258 assay discovered that combined application of PEITC and DDP could enhance the capability of DDP to damage the nuclear structure so as to induce apoptosis of human gastric cancer, which was similar to the activity of siRNA.6. Flow cytometric analysis showed that PEITC was able to raise the early apoptosis rate of DDP from 14.2% to 40.0%, while the result of siRNA was 46.2%.7. Western blot assay confirmed that PEITC could promote the shear of caspase 3 and excitation of c-caspase 3, improve the chemotherapeutic sensitivity of human gastric cancer cell line MGC-803 to DDP, which was similar to siRNA while contrary to TGF-β1. Combined with the part one experiments, we might consider it related to the prohibitive effect of PEITC on EMT induced by TGF-β1.8. PEITC could downregulate gastric cancer stem cell biomarkers including CD133, CD44, CD71 and upregulate NESTIN, which was similar to siRNA while contrary to TGF-β1. Since the regular changes of CD24 were not detected, we chose CD 133, CD44 and NESTIN as biomarkers to conduct isolation.9. Xenograft tumor-formation assay demonstrated that CD133+CD44+NESTIN-cells has much stronger cancer stem cell characteristics.Conclusion:PEITC could inhibit the proliferation, adhesion, migration and invasion ability of human GC cell line MGC-803, the mechanism of which could be explained that PEITC could regulate the EMT and active MMPs through adjusting the TGF-β1 receptor. Small dose of PEITC could improve the chemotherapeutic sensitivity of human gastric cancer cell line MGC-803 to DDP, which could be relative to the regulation of EMT as well as CSCs phenotype. PEITC could suitably change the CSCs biomarkers of gastric cancer cell line MGC-803, it could downregulate gastric cancer stem cell biomarkers including CD 133, CD44, CD71 and upregulate NESTIN, decrease the percentage of stem cells in GC cells, which might be connected to its effect on EMT via regulating the TGF-β1 receptor. |
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