| Part A:Expression and clinical significance of periostin in colon cancerPurpose:Colon cancer is a common malignancy in the digestive system. Early detection and appropriate management are critical for improving the survival of colon cancer patients. Currently, there are many non-invasive methods for colon cancer screening and serum carcinoembryonic antigen (CEA) is one of the most commonly used biomarkers. However, the sensitivity and specificity of serum CEA are relatively low. Periostin is a matricellular protein and highly expresses in many tissues, especially tumor tissues. The aim of this part is to analyze the expression and clinical significance of periostin in colon cancer.Methods:Twelve healthy controls,6males and6females and aged48-75years, were enrolled. We also enrolled20random colon cancer patients who were admitted between April2011and April2012. The patient group included13males and7females, with a median age of62years old (38-81years old). Pathological diagnosis was performed on all patients. Data on preoperative CEA levels were available. None of the patients received preoperative chemotherapy or radiotherapy. Eleven patients (55%) presented lymph node involvement,6liver metastasis, and1lung metastasis. Well-, moderately-, and poorly-defferentiated carcinomas were observed in5,7, and8patients, respectively. TNM I Ⅱ, Ⅲ and Ⅳ were found in5,4,4, and7cases, respectively. Semi-quantitative RT-PCR and Western blot analysis were done to examine the mRNA and protein expression of periostin in colon cancer and adjacent colon tissues. Serum periostin levels in colon cancer patients and healthy controls were measured by ELISA. The correlations between serum periostin and clinicopathological parameters were analyzed.Results:RT-PCR analysis revealed that the mRNA abundance of periostin was significantly higher in colon cancer tissues (1.35±0.61) than that in adjacent colon tissues (0.33±0.21)(P<0.05). Western blot analysis showed that the periostin protein level was consistently increased in colon cancer tissues compared to adjacent colon tissues (P<0.05). ELISA demonstrated that colon cancer patients had significantly higher concentrations of serum periostin than that in healthy controls (55.04±11.50ng/mL vs.23.31±6.72ng/mL, P<0.05). Serum periostin levels were significantly associated with lymph node metastasis, distant metastasis, and TNM stage. However, there was no significant association between serum periostin and age, gender, tumor size, or serum CEA.Conclusions:Periostin is upregulated in colon cancer. Serum periostin is significantly correlated with lymph node metastasis, distant metastasis, and advanced tumor stage. Part B:Biological roles of periostin in colon cancer metastasisPurpose:Distant metastasis is a major cause of cancer-related deaths. Epithelial-mesenchymal transition (EMT) is a key event contributing to cancer cell invasion. The aim of this part is to determine the effects of periostin overexpression on cell proliferation, colony formation, adhesion, invasion, and EMT in colon cancer cells.Methods:Human full-length periostin cDNA was amplified by PCR and cloned into pcDNA3.1(+) expression vector. The resultant periostin-expressing plasmid and empty vector was individually transfected into HT-29and SW480cells and G418was used to select stable clones. Cell viability was assessed by MTT assay, transformation activity by colony formation assay, adhesive capacity by cell adhesion assay, and invasiveness by transwell invasion assay. Changes in cell morphology were examined by a contrast-phase microscope. The EMT biomarkers, E-cadherin and vimentin, were measured by Western blot analysis.Results:Compared to untransfected and empty vector-transfected colon cancer cells, periostin-transfected cells had an8-10-fold elevation in the periostin expression. Overexpression of periostin did not affect the cell proliferation, compared to untransfected and empty vector-transfected cells (P>0.05). The colony formation rate was78±7%and67±13%in periostin-expressing SW480and HT-29cells, respectively, and80±14%and64±11%in empty vector-transfected SW480and HT-29cells, respectively (P>0.05). Cell adhesion assay revealed that periostin overexpression significantly enhanced the adhesion of colon cancer cells to collagen I (P<0.05relative to empty vector-transfected cells). The results of the transwell assay showed that the invaded cell number was greater in the periostin overexpression group than that in the empty vector group (98±9cells/field vs.51±7cells/field; P<0.05). Enforced expression of periostin resulted in EMT in colon cancer cells, as proved by decreased E-cadherin and increased vimentin.Conclusions:Periostin overexpression significantly promotes the cell adhesion and invasion as well as EMT in colon cancer cells. However, cell proliferation and transformation activity of colon cancer cells are not affected by periostin overexpression. Part C:Periostin induces chemoresistance in colon cancer cellsPurpose:Chemoresistance is a major obstacle for successful anticancer therapy. Periostin has been linked with decreased apoptosis and resistance to chemotherapy in cancer cells. In this study, we sought to explore the association of periostin and chemoresistance in colon cancer cells.Methods:We examined periostin at mRNA and protein expression changes in SW480and HT-29colon cancer cells exposed to anticancer drugs, oxaliplatin or5-fluorouracil alone, for48h. Small interfering RNA technology was employed to specifically silence periostin expression. A survivin-expressing plasmid was constructed and was co-transfected with periostin siRNA into SW480and HT-29cells,and after incubation for24h, the cells were treated with oxaliplatin and5-fluorouracil alone for another48h. Annexin-V/propidium iodide staining was performed to measure the effects of periostin on drug-induced apoptosis. Western blot analysis was done to examine the protein expression of cleaved caspase-3, poly(ADP-ribose) polymerase (PARP) and survivin. To investigate the involvement of the PBK/Akt pathway in periostin-mediated upregulation of survivin, SW480and HT-29cells were exposed to20μM LY294002(a specific PI3K inhibitor) for1h before transfection with periostin-expressing plasmids or empty vector.Results:Elevated expression of periostin, at both mRNA and protein levels, was observed in colon cancer cells exposed to drugs. Silencing of periostin significantly (P<0.01) enhanced drug-induced apoptosis in colon cancer cells, accompanyed with accumulation of cleaved caspase-3and PARP. Mechanistic studies revealed that periostin silencing remarkably (P<0.01) suppressed the protein expression of survivin, an antiapoptotic protein. Restoration of survivin inhibited chemotherapeutic drug-induced apoptosis in periostin-depleted SW480and HT-29cells. Moreover, periostin overexpression increased the expression of survivin and the phosphorylation of Akt, which was significantly (P<0.05) attenuated by the pretreatment with LY294002, an inhibitor of phosphatidylinositol3-kinase (PI3K). Conclusions:Our results demonstrate that the upregulation of periostin confers chemoresistance in colon cancer cells through PI3K/Akt signaling-mediated induction of survivin expression. |
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