Tumor Stem Cell Researches On Colorectal Cell Line SW620 AND Adenovirus-mediated TRAIL As Targeting Gene Therapy For Primary Colorectal Cancer

Part 1:CD133 is regarded as one marker of tumor initiating cells in many tumors including colorectal cancer. O'Brien and Ricci et al. have proved in primary colorectal tumors that there being colorectal tumor stem cells (initiating cells) which are marked by CD 133 antigen, and the tumor initiating cells are responsible for tumor proliferation, recurrence, metastasis and drug resistance. Using a genetic knockin lacZ reporter mouse model, Shmelkov et al. challenged this increasingly influential viewpoint and drew two important conclusions oppugning the former opinions. First, CD133 is widely distributed throughout the full range of tumor epithelial cells in colon other than limited in a few cells. Second, CD 133 negative cells of colon tumors are also tumorigenic and are more inclined to metastasize. Based on the two distinct opinions, we assume that the expression of CD133 is different among the tumor cells and the quantitative but not qualitative analyses of CD 133 abundance are necessary to determine the relationship between CD133 expression and tumor stem cell characteristics. To verify this hypothesis, colorectal cancer cell line SW620 was cultured and sorted into CD133Hi, CD133Mid and CD133Low subgroups using magnetic microbeads to compare their xenograft biological characteristics. The results showed that the CD133Hi subgroup of SW620 is more close to the tumor initiating cells in biological characteristics than CD133Mid and CD133low subgroups, but the CD133low subgroup still keep the ability of tumorigenicity. It supported that tumor initiating cells are more correlated to the abundance of CD 133. Part 2:Tumour necrosis factor-related apoptosis-inducing ligand or Apo2 ligand (TRAIL/Apo2L) is a member of the tumour necrosis factor (TNF) superfamily of cytokines that induces apoptosis upon binding to its death domain-containing transmembrane receptors, death receptors 4 and 5 (DR4, DR5), and then activate caspase-8 by death domain to induce apoptosis in tumor cells. In the meantime, TRAIL preferentially induces apoptosis in cancer cells while exhibiting little or no toxicity in normal cells. Many researches demonstrated that TRAIL delivered by adenovirus can induce apoptosis and suppress growth in a wide range of tumor cell lines in in vivo and in vitro circumstances. But recent findings report TRAIL resistance in some cell lines requiring combined treatments with sensitizing agents as standard chemotherapeutics can enhance tumor cells sensitivity to TRAIL. To assess whether combination of targeted gene agent TRAIL with chemo agent (5-Fu and TAXOL) can enhance the sensitivity of tumor cells to TRAIL as well as decrease TRAIL resistance, we detect the treatment of recombinant adenoviral vector expressing the TRAIL protein(Ad/TRAIL) in combination with chemotherapeutic drug 5-Fu or TAXOL to primary colorectal cancer tumors ex vivo an in vivo, and the Synergetic effects in nude mice tumor xenograft model. By detecting the expression of TRAIL, DR4, caspase-3, caspase-8 and tumor apoptosis rate, we can realize the possible anti-cancer mechanism of TRAIL. The immunohistochemical results show high expression of TRAIL protein and high expression of caspase-3 in the end pathway of TRAIL induced apoptosis in subgroups containing Ad/TRAIL. It reflects that caspase related apoptosis pathway is activated by TRAIL which led to increase of apoptosis product. The TUNUL staining show us that tumor cells infected with Ad/TRAIL collapse or fall in apoptosis by direct infection or by bystander effect. The in vivo experiments indicate that tumor grows slowly in treatment groups than in control groups and the tumor-bearing mice in treatment groups suvive longer than in control groups. The combination of Ad/TRAIL and chemotherapeutics will significantly increased survival rate compared to the control mice or mice receiving Ad-/TRAIL alone. All the results abovementioned support the anti-tumor characters of Ad/TRAIL agent. No obvious side effects were found showing mild damage to normal cells of the body. Our findings demonstrate that Ad/TRAIL gene products activated by promoter hTERT exert effective therapeutic and toxic effects to primary colorectal tumor cells and will exhibit robust tumoricidal activity against human primary tumors in clinical practice with minimal toxic side effects.