| ObjectiveInvestigate the expression and correlations of Beclinl, mTOR, ULK1 and LC3, and explore their correlations with the clinic characteristics of human colorectal carcinoma. Examine the expression of multidrug resistance proteins LRP, GST-π and TOPO II, and discuss the correlations between autophagy and MDR and their impacts on prognosis of human colorectal carcinoma.Methods1. Formalin-fixed and paraffin-embedded CRC specimens and the adjacent noncancerous tissues from 202 patients were obtained from the archives of the Department of Pathology, Binzhou Medical University Hospital, China. The study group consisted of 202 colorectal cancer patients who were admitted to our hospital between January 2004 and July 2008. They underwent curative resection at the Department of Gastroenterological Surgery, Binzhou Medical University Hospital, China. None of the patients had undergone either radiotherapy or chemotherapy prior to sample collection. The tumors were classified according to the criteria defined by the WHO International Histological Classification of Tumors. The follow-up period was defined as the interval from the date of operation to the date of death or the last follow-up. All patients were observed until July 2012, ranged from 10 to 89 months (median,65.5 months).2.Immunohistochemistry was employed to detect the expression of Beclin1, mTOR, ULK1, LC3, and MDR proteins in colorectal carcinoma. Their correlations with clinic characteristics and impacts on colorectal carcinoma were studied. Real-time PCR and Western blot methods were used to detect the expression of Beclinl, mTOR, ULK1 and LC3, and the results were compared with the immunohistochemistry results.Results1.The expression of Beclinl, mTOR, ULK1 and LC3 in fresh CRC tissues was 90%,52.5%,92.5% and 87.5% and was higher than that in adjacent noncancerous tissues (P≤0.05). Real-time PCR and Western blot results indicated that the expression of Beclinl, mTOR, ULK1 and LC3 in colorectal cancer tissues were significantly higher than those in the adjacent noncancerous tissues (P<0.01).2. The expression of LRP, GST-π and TOPO II was 94.55%,71.78% and 97.03% in CRC, respectively. The expression of Beclin1 was found to have a positive correlation with LRP and GST-π (r=0.140, P=0.047 and r=0.160, P=0.023), and no correlation was found between Beclinl and TOPO II. LC3 was positively associated with LRP and GST-π (r=0.162, P=0.021; r=0.142, P=0.044), and no correlation was found between LC3 and TOPO Ⅱ. There were no significant correlations between the expression of mTOR and ULK1 and MDR proteins in CRC (P>0.05).3.Immunohistochemistry results showed that the expression of Beclinl, mTOR, ULK1 and LC3 in cancer tissues were 90.74%,45.06%,93.07% and 85.80% and were higher than that in noncancerous tissues (P≤0.05).The expression of mTOR was related to cell differentiation (P≤0.05); the expression of LC3 was related to cell differentiation and lymph node metastasis.No significant correlations were found between Beclinl, ULK1 and clinicopathologic parameters. Beclinl was positively correlated with ULK1 and LC3 in CRC (r=0.623, P≤0.01 and r=0.552, P<0.01).mTOR was negatively correlated with ULK1 and LC3 in CRC (r=-0.507, P<0.01; r=-0.200, P≤0.01). No significant correlation was found between Beclinl and mTOR (P>0.05).4. The expression of Beclinl, mTOR, ULK1 and LC3 was significantly increased in CRC compared with normal colorectal mucosa tissues. High expression of mTOR was associated with lymphnode metastasis (P≤0.01). High expression of LC3 was associated with cell differentiation and lymph node metastasis of CRC (P≤0.01).No significant associations were found between Beclinl, ULK1 and clinicopathological parameters of CRC (P>0.05).The median survival time of 202 cases patients were 60.5 months, Kaplan-Meier analysis revealed that Beclinl, mTOR, ULK1, LC3, GST-π and lymph node metastasis were prognostic parameters. The five-year survival rate of patients with Beclinl-positive expression was as high as 61.2%, whereas that with Beclinl-negative expression was 35.8%(P≤0.01).The five-year survival rate of patients with mTOR-negative expression was as high as 52.1%, whereas that with mTOR-positive expression was 78.7%(P≤0.01).The five-year survival rate of patients with ULK1-positive expression was as high as 63.5%, whereas that with ULK1-negative expression was 33.2%(P≤0.01). The five-year survival rate among patients with LC3-positive expression was 64.3%, compared to 32.1% for patients with LC3-negative expression (P≤0.01). The five-year survival rate of CRC patients with high expression of GST-π was 43.1%, whereas that with GST-π negative expression was 58.6%. Cox proportional hazard model showed that mTOR, LC3, GST-π and lymphnode metastasis were independent prognosis factors of patients with colorectal carcinoma.Kaplan-Meier analysis showed that CRC patients with positive expression of Beclinl,ULK1, LC3 and negative expression of mTOR, GST-π and no lymph node metastasis have longer survival time after radical surgery. Cox regression analysis showed that mTOR, LC3,GST-π and lymph node metastasis were independent prognosis factors of CRC.Conclusion1.The expression of autophagy related genes were increased in CRC,which maybe contributed to the tumorigenesis and progression of CRC.2. High expression of mTOR was associated with lymph node metastasis, and high expression of LC3 was associated with cell differentiation and lymph node metastasis of CRC.3.Beclinl plays a role in the promotion of autophagic activity and mTOR is involved in the inhibition of autophagy, they may co-regulate the autophagic activity of CRC via different cell signal pathways.4. The increasing autophagic activity was significantly associated with MDR and prognosis of patients with CRC. |
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