| Background and aims:Gastric cancer is one of the most prevalent digestive carcinomas, ranking the first place in mortality of patients suffered with malignancies. Therefore, gastric cancer have already affected the health of people worldwide. Taken together, it is important to study the epidemiology and molecular biology as well as pathogenesis of gastric cancer. To date, the median 5-year survival rate of patients after surgery is approximately 95%, however, the total efficacy of surgery in patients in advanced stage is unsatisfactory, as about 50% patients died within 5 years. As most of the patients with gastric cancer are definitely diagnozed when the disease progresses into advanced stage, it is difficult to completely cure by surgery. Therefore, more and more doctors have payed attention to chemotherapy. However, its severe side effects and chemo-resistance have significantly limited its wide use, although chemotherapy can benefit patients in advanced gastric cancer. Together, the top priority is to investige the mechanism of chemo-resistance.One of the most studied doxorubicin (DOX), a member of theanthracycline group, is one of the most widely used anti-cancer drugs. It was originally isolated from Streptomyces peucetius in the 1960s. One of its anti-cancer mechanisms is its ability of interfering with topoisomeraseⅡ(topoⅡ). When topoisomeraseⅡ(topoⅡ) unwinds and cleaves DNA, DOX can directly bind to the intermediate cleavable products and inhibits the re-combination of the cleaved duplex, thus resulting in a doublestrand break (DSB)PTEN gene is a cancer suppressor gene which was concurrently discovered and nominated by 3 independent research groups in 1997; it was also referred to as MMAC1 or TEP1. PTEN gene is located in the chromosome 10q23. The whole length of PTEN gene is approximately 200kb. It consists of 9 exons and 8 introns and encodes a protein of 403 amino acids, with a molecular weight of 53 kDa. The N-terminal of the molecule consists of a catalytic domain which can hold a larger phosphoinositide substrate, whereas the C-terminal is composed of a C2 domain that adjusts the membrane location of PTEN protein, as well as a PDZ domain that controls the interaction between PTEN and other proteins and 2 PEST sequences, which are responsible for the stability and biological activity of the protein. It has been reported that loss of PTEN expression has been found in a wide range of human cancers, including human brain, breast, prostate, endometrial and ovarian cancers. Recently, researches have demonstrated that PTEN are capable of enhancing the sensitivity of cancer cells to certain anticancer agents. It has been found that gene therapy with PTEN is an effective way on reversing chemoresistance of drug-resistant cancer cells and PTEN has become a new potential target in sensitizing cancer cells to chemotherapy.Via several signal transduction pathways, PTEN is involved in multiple biological actions, including cell proliferation, cell apopotosis, cell traffic, as well as cell metastasis and selfrenewel of stem cell. Of note, one of the most important functions of PTEN is its ability to control the activation of PI3K pathway. After activaction of PI3K, PIP2 can be phosphorylated into PIP3, an important second messeger. PIP3 can activate the transduction of phosphoinositide/dependent kinase. In addition, PIP3 can bind specifically to PH domain located in N-terminal of AKT protein, forming a dipolymer. Then phosphorelation of AKT is strengthened. The highly-conserved phosphotase domain of PTEN protein consists of two specific allysineresidues, which can specifically negatively regulate PIP3, resulting in inactivation of the PI3K/Akt pathway. Inctivated PKB/Akt pathway is involved in cell survival and cell cycling.The aims of this research are to identify:1) The expression of PTEN of gastric cancer cell lines in mRNA and protein levels after administration of chemotherapeutic drugs and the possible mechanism; 2) Whether the stable transfection of PTEN may enhance the chemotherapeutic sensitivity of gastric cancer cells in vitro; 3) Whether selective knockdown the expression of PTEN may suppress the chemotherapeutic sensitivity of gastric cancer cells in vitro or not; 4) Whether PTEN may strengthen the chemotherapeutic sensitivity of gastric cancer cells in vivo by using animal models.Methods:1) Human gastric adenocarcinoma cell line BGC-823 was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics in a humidified 5%CO2 atmosphere at 37℃; 2) Gastric cancer cell line BGC-823 cells were treated with doxorubicin (DOX). Cell proliferation and apoptosis were evaluated by MTT and Annexin V-FITC-PI double staining. RT-PCR and Western blot were used to detect the level of PTEN mRNA and protein expressed in BGC-823 treated by doxorubicin, respectively; 3) BGC-823 cells were trasfected using Notchl siRNA, followed by administration of doxorubicin. Then the expression level of PTEN and Notchl was detected; 4) pEAK8-PTEN plasmid tranfected BGC-823 cell line cells, followed by administration of doxorubicin. The apoptosis and survival rate as well as the expression level of both PTEN and p-AKT were detected.; 5) BGC-823 cells were trasfected using PTEN siRNA, followed by administration of doxorubicin. Then proliferation and apoptosis of these cells as well as the expression level of PTEN were detected.6) pEAK8-PTEN-BGCC-823 and pEAK8-BGCC-823cells were planted on the nude mice. Then the effects of doxorubicin were compared by determining the tumor size, and calculating the apoptotic index in tumor tissues by TUNEL assay.Results:1) After treatment with doxorubicin, the survival rates of 3h,6h and 12h in gastric cancer cells were (84.93±2.05)%, (75.35±2.11)% and (44.94±2.62) %, respectivley. It was a time-dependent manner. There was also a significant difference when compared with control [(98.24±0.60)%] (P<0.05). After 12 hours’ exposoure to doxorubicin, the apoptosis rate of cells was (31.37±3.58)%, with significant difference compared with control[(2.59±0.92)%] (P< 0.05).2) The expression of PTEN and Notchl in both mRNA and protein levels was significantly increased in a time-dependent manner after administration of doxorubicin. There was a significant difference (P< 0.05).3)The expression of Notchl in gastric cancer cells was decreased after transfected with Notchl siRNA. After administration of doxorubicin, there was a significant increase in both Notchl and PTEN.4) After stable transfection with pEAK8-PTEN plasmid, the expression of PTEN mRNA and protein in pEAK8-BGC-823 and BGC-823 cells was minimal, without significant difference (P>0.05). However, it is much higher in pEAK8-PTEN-BGC-823 than that in pEAK8-BGC-823 and BGC-823 (P<0.05).5) After 12h of administration of 0.3 umol/L doxorubicin, the survival rates of pEAK8-PTEN-BGC-823, BGC823 and pEAK8-B GC-823 were (22.45±1.05)%, (40.57±1.21)% and (37.61±0.22)%, respectively. The suppressive ability of doxorubicin on pEAK8-PTEN-BGC-823 is significantly stronger than BGC823 and pEAK8-BGC-823, (P<0.05). There was no significant difference between pEAK8-BGC-823 and BGC823 (P>0.05); flow data showed that after 12 hours’exposure to doxorubicin (0.3umol/L), the appoptosis rate of pEAK8- PTEN-BGC-823 approached nearly (45.2±4.5)%, with significant difference (P<0.05) in comparison with pEAK8-BGC-823 (28.9±3.2)% and BGC823 (27.8±2.6)%.6) After stable transfection with PTEN, the expression of PTEN is significantly increased. The expression of p-AKT was decreased compared with vector plasmid, (P< 0.05). The expression of PTEN in cells treated with doxorubicin was obviously increased and much higher than cells treated with the vector, while p-AKT is converse.7) The survival rate of cells transfected with PTEN siRNA was (76.52±3.24) %, whereas the survival rate of the control was (43.52±4.69)%. There was significant difference (P< 0.05). After treatment with doxorubicin, the apoptosis rate of cells transfected with PTEN siRNA was (10.35±1.04)%, whereas the control was (31.37±3.58)%. There was significant difference (P< 0.05) 8) In vivo experiment showed that stable transfection of PTEN plasmid strengthened the anti-tumor effect in gastric cancer xneografts and increased the apoptosis rate of tumor cells.Conclusions:The chemotherapeutic drugs doxorubicin can suppress the growth and induce apoptosis of BGC-823 obviously. And the level of PTEN mRNAand protein was increased. The reason that the level of PTEN increased after treated doxorubicin in gastric cancer may be due to Notchl activation. Stable transfection of gastric cancer cell line BGC-823 with PTEN can strengthen its sensitivity to chemotherapy. PTEN siRNA transfected BGC-823 can decrease the chemosensitivity. Therefore, it will pave a new way to the combination of gene therapy and chemotherapy to treat cancer. |
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