| BACKGROUND:Gastric cancer remains one of the most common malignancies in the world. Invasion, metastasis and recurrence are the major causes of gastric cancer patients'death and the key factors affecting clinical treatment and prognosis. Cancer cell require sufficient energy supplement for its rapidly growth. Warburg identified a particular metabolic pathway in carcinomas, characterized by the anaerobic degeneration of glucose ever in the normal supply oxygen(known as "Warburg effect". Transketolase (TK) is a rate-limiting enzyme in the non-oxidative branch of pentose phosphate pathway(PPP) and play an important role in degradation of glucose in cancer cell. Additonally, the main functions of PPP are to allow glucose conversion to ribose for mucleic acid synthesis. Recently, a novel transketolase-like enzyme (TKTL1) was found to be expressed at high levels in several malignancies and was correlated with the tumor metastasis and poor prognosis. Increased expression of TKTL1can promoted tumor growth and enhanced the metastatic potential. However, the underlying mechanisms for TKTL1promote tumor invasion are still unclear. p53(wild type) gene, one of the anti-oncogenes, can response to DNA damage, inducing DNA repair genes, and initiates apoptosis if DNA strand breaks fail to repair. Mutation of p53gene is correlated with carcinogenesis, invasion and metastasis. Recently, p53has been considered of playing a key role in the regulation of PPP. However, the correlation between TKTL1and p53expressions in gastric cancer is still unclear and need further investigation. OBJECTIVE:To investigate the character and mechanisms of TKTL1in gastric cancerMETHODS:1. The expression of TKTL1and p53in101specimens of gastric cancer tissues and25specimens of normal gastric mucosa tissues was detected by immunohistochemistry (IHC). The relationship between their expression and clinicopathological features, prognosis was statistically analyzed.2. The specific TKTL1small interference RNA (siRNA) plasmid was constructed and transfected into gastric cancer SGC-7901cells using lipofectamine2000. Three groups of cells were used in the in vitro studies, including blank control group (untransfected cells), negative control group (cells transfected with the non-silencing siRNA) and TKTL1siRNA group (cells transfected with the TKTL1siRNA). The expression of TKTL1mRNA was detected by real-time reverse transcription-polymerase chain reaction (RT-PCR). The expression of TKTL1protein was measured by western blot. Cell proliferation was assessed using methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The cell cycle phase distribution was determined by flow cytometry. The expression of wild type p53was also detected by real-time RT-PCR and western blot.3. Suspensions of SGC-7901tumor cells were implanted in the subcutaneous of nude mice. The animals were randomly divided into three groups (n=6) that received the following treatment:blank control group (0.9%sodium chloride intratumorally), negative control group (non-silencing siRNA plasmid+liposome intratumorally) and the TKTL1siRNA group (TKTL1siRNA plasmid+liposome intratumorally). The volume of each tumor was measured once a week. Three weeks after the treatment of therapy, the mice were sacrificed and the weight of the tumor was recorded. The expression of TKTL1protein in tumor xenograft was detected by IHC.RESULTS:1. The expression of TKTL1and mutant p53in gastric cancer tissues was significantly higher than that in normal gastric mucosa tissues (P<0.05). Overexpression of TKTL1and p53was statistically significantly associated with the size of tumor, depth of invasion, TNM stage and numbers of lymph node metastasis (P<0.05), moreover a significant correlation was observed between mutant p53expression and histologic type (P=0.017). Next, the expression of TKTL1and p53was significantly positive correlation in gastric cancer tissues (r=0.476, P<0.01). In univariate analysis, elevated TKTL1expression was associated with5-year survival rate (P=0.001). Moreover, the impact of p53expression on5-year survival rate was statistically significant (P<0.001). In Cox multivariate analysis, expression of TKTL1and p53immunostaining was all found to be independent prognostic factors (P<0.05).2. TKTL1siRNA plasmid was constructed successfully and transfected into the SGC-7901cells efficiently. Compared to negative control group, the mRNA and protein level of TKTL1in TKTL1siRNA group were reduced by69.2%and63.4%(P<0.01), respectively. The growth of cells was significantly decreased in TKTL1siRNA group compare to blank control group or negative control group (P<0.01). Compared to blank control group or negative control group, specific TKTL1siRNA significantly increased the number of cells in the G0/G1phase (P<0.01), significantly decreased the number of cells of in the S phase (P<0.05) and G2/M phase (P<0.05), and significantly decreased the proliferation index (P<0.01). In the meantime, the expression of wild type p53mRNA and protein level in TKTL1siRNA group were increased significantly compared to blank control group or negative control group (P<0.01).3. The model of gastric cancer SGC-7901cell in subcutaneous of nude mice was established. Both the volume and the weight of the tumors in TKTL1siRNA group were significantly decreased compared to blank control group (P<0.01) or negative control group (P<0.01). The rate of tumor inhibition in the TKTL1siRNA treated xenograft tumors volume was approximately47.3%. The expression of TKTL1protein was higher in the blank control group or negative control group compared to the TKTL1siRNA group.CONCLUSION:1. Overexpression of TKTL1and mutant p53is closely related with progression and metastasis of gastric cancer, and might be regarded as factors of poor prognosis in gastric cancer.2. TKTL1siRNA led to the efficient and specific inhibition of TKTL1expression in gastric cancer SGC-7901cells. Silencing of TKTL1expression inhibits the proliferation, and expression of wild type p53in SGC-7901cells was increased.3. Intratumoral injection with TKTL1siRNA plasmid inhibits the growth of SGC-7901tumor xenografts and the expression of TKTL1protein.
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