The Synergistic Effect Of RhoB And Taxol On Gastric Cancer

Gastric cancer is one of the most common malignancies throughout the world, especially in East Asia. The increasing approaches have not improved the effect against gastric cancer. Taxol has been used to treat a wide variety of cancers since its approval by the FDA in 1992. Although treatment with taxol against gastric cancer has led to the improvement in the duration and quality of life for some cancer patients, drug resistance is a major obstacle to improve the overall response and survival of patients. It was documented that RhoB might involved in the taxol induced apoptosis, but treatment with taxol is hampered because of the low expression of RhoB in many malignancies, including gastric cancer. Thus, we proposed that study of the synergistic effects of RhoB and taxol against gastric cancer may enhance the effect of taxol treatment and raise the value of its potential use in cancer therapy.[Objectives](1) Construction of the inducible expression vector of RhoB and set up the gastric cancer sub cell lines of inducible RhoB expression. (2) Study of the effect of RhoB on the malignant biological behaviors of gastric cancer cells. (3) Exploring the synergistic effect of RhoB and taxol on gastric cancer cells.[Methods](1) The RhoB cDNA was expanded form pcEFL-GST-RhoB plasmid byPCR, then the RhoB cDNA fragment was inserted into pGene/V5-His plasmidflagment. The pSwitch and pGene/V5-His-RhoB plasmids were transfectedinto AGS cells with Lipofectamine 2000. (2) The expression of RhoB proteinwas detected with western blot after the transfected cells were induced for 24hours by mifepristone. (3) MTT assay was performed to evaluate the potentialeffects of RhoB on AGS cell growth. (4) Hoechest/PI staining and Annexin Vflow cytometric experiments were performed to cell apoptosis. (5) Flowcytometry was done to study the effects of RhoB on cell cycle. (6) To furtherinvestigate whether RhoB was involved in chemotherapeutic sensitivities inAGS cells, the sensitivities to taxol, MMC, ADR, VCR and CPPD wereinvestigated by MTT assay. (7) The inhibiting effects of combining RhoB andtaxol on AGS cell growth were tested by MTT assay. (8) To study thecombining effects with RhoB and taxol on cell apoptosis, Hoechest/PIstaining and Annexin V flow cytometric experiments were performed. (9)Flow cytometry was taken to study the effects of different concentration oftaxol on cell cycle. (10) The synergistic effect of RhoB and taxol on cell cyclewas study by Flow cytometry.[Results](1) The inducible expression vector of RhoB was constructed and identified by digestion with BamH I and EcoR I and DNA sequencing. (2) The stable clones, that RhoB protein can be induced by 1 X 10 M mifepristone, were screened out after the transfaction of RhoB inducible expression vector. (3) As the increasing of RhoB expression induced by mifepristone, the proliferation of the transfected cells was significantly inhibited. (4) The upregulation of RhoB did not contribute to the change of...