1,Targeting Transcription Of Survivin Promoter In The Liver Cancer Stem Cells 2,TACE In Combination With HIFU For Unresectable Hepatocellular Carcinoma: A Meta-Analysis

Primary liver cancer is a malignant tumor of high malignancy and poor prognosis, known as the "the King of cancer ". Hepatocellular carcinoma (HCC) is the fourth leading cause of death from cancer in the world, with an increasing incidence in the United States and the China. Globally, HCC accounts for approximately 5.4% of all cancers and causes 610,000 deaths per year. Most patients with HCCs are diagnosed either at an intermediate to advanced stage or at the point with poor liver function, and few meaningful therapeutic options are available. With a poor prognosis and limited systemic treatment options, approximately 80% of patients die within a year of diagnosis. Overall efficacy of HCC is not satisfactory, partly due to the tumor cells to radiotherapy and chemotherapy resistance.The theory of Cancer stem cells (CSCs) provides a new way for the anti-tumor therapy. The majority of tumor cell populations have differentiated and matured, which is no proliferation ability. Conventional radiotherapy and chemotherapy is to kill off this part of the cells, so CSCs of proliferation and tumor formation ability survived. This part of CSCs becomes the root of cancer recurrence and treatment resistance. Therefore, exploring the role of CSCs in the occurrence and development of tumor, developing targeting treatment of CSCs, and killing or inhibiting tumor stem cells through different means, are expected to become the new field of study. It is reported that the presence of functional liver cell lines of liver cancer stem cells (LCSCs). Effective way to remove stem cells is very necessary for liver cancer to get a good anti-liver cancer. Human CD133 gene,which is expressed in a variety of cancer stem cells, is a new marker of cancer stem cells (including LCSCs).Molecular targeted cancer therapy (MTCT) is the "personalized" or "individualized" approaches toward cancer which targets the particular molecular or genetic changes. CSCs represent a distinct subpopulation of cancer cells of integral importance. May molecular targeted therapy choose as a treatment for CSCs? Some research explored that the differences of gene expression in CSCs and non-tumor cell population. The regulation of gene expression should be an ideal means of treating cancer.Adenovirus, a wide range of carriers in cancer gene therapy, can enhance the foreign gene transduction and transcriptional targeting and greatly improve the therapeutic effect. However, to date, there have been two limitations to the clinical application of these agents, i.e. poor viral infectivity and tumor specificity. To bypass these problems, a new generation of vectors is being developed, which selectively target tumor cells and which allow for increased viral replication. This can be achieved by use of tumor-specific promoters (TSP). Therefore, to some extent, the evaluation of transcription activity of TSP in tumor is not only of great value in targeted gene therapy, but also is a significant therapeutic end point in human clinical trails.Survivin, the smallest member of the inhibitor of apoptosis protein (IAP) family, is overexpressed in transformed cell lines and in all the most common human cancers and undetectable in terminally differentiated adult tissues. kuang ea al. reveals that the survivin promoter possesses a high transcriptional activity in all three established HCC cell lines and may serve as a useful tool for transcriptional targeting gene therapy of HCCs.Jin XD et al. found that low-LET X-rays induced dose-dependent increases in survivin expression. Nandi S showed that the survivin gene was up-regulated to f10-fold in U118MG, indicating a strong radiation-inducible promoter. This study is to reveal the activity of the survivin gene promoter in liver cancer stem cells and evaluate the possible application of this promoter as a radioinducible promoter in tumor gene therapy.ObjectiveTo isolate and characterization of CD133 positive cells from of liver stem cells by MACS sorting and investigate its biological characteristics. To Construct Survivin promoter-mediated recombinant adenovirus vector, determine the promoter activity in liver stem cells, evaluate the adenovirus with respect to radioinducible properties, test the capacity of our novel adenovirus to effectively target CD133~+ liver cancer stem cells and provide experimental basis for targeted therapy.Methods1. Isolation and characterization of CD133~+ LCSCs from the HepG2 liver cancer by MACS sorting. The percentage of CD133~+ LCSCs was evalating by flow cytometry.2. To observe the growth of the isolated CD133~+ LCSCs in serum-free medium and in medium containing 10% FBS.3. To examine the impact of radiation on HepG2, we evaluated the percentage of CD133~+ LCSCs before and after radiation exposure by FACS.4. To determine whether CD133~+ cells are more tumorigenic than their CD133- counterparts in vitro and vivo, we compared their proliferative, clonal ability and tumorigenicity using proliferation, colony formation assay and tumor development experiments, respectively.5. To examine the specific expression of possible candidate for promoter-inducible elements, we evaluated the level of Survivin mRNA expression after different dose radiation exposure by RT-PCR.6. The genomic fragment encoding the Survivin promoter was isolated cloned into the KpnI/HindIII sites of the luciferase reporter plasmid pGL3-Basic. We examined the luciferase activity in the CD133~+ cells.7. We examined the promoter activity in the CD133~+ HepG2 cells in response to low-dose radiation. 8. Construction of Survivin promoter-mediated recombinant adenovirus vector with luciferase reporter pAd-sur-luc by recombinant DNA technology. We examined the luciferase activity in the CD133~+ cells to see whether our pAd-sur-luc could preferentially target CD133~+ cells.9. We examined the promoter activity in the CD133~+ cells in response to pAd-sur-luc in conjunction with radiation.Results1. The percentage of CD133~+ LCSCs was 1.54±0.26% and 89.58±0.72% before and after sorting, respectively(p < 0.05). MACS technology is an ideal method for purify and separate CD133~+ cells from the HepG2 cell line, with high purity and no influence of cell activity.2. The isolated CD133~+ LCSCs generate suspension spheres which have certain stem properties in serum-free medium, and increased as days passed by. But the differentiation were induced in medium containing 10% FBS in 24h. So CD133~+ cell subpopulation has stronger ability to differentiate in vitro.3. The percentage of CD133~+ LCSCs was 3.91±0.21% and 1.24±0.36% after 2Gy XRT or not, respectively(p<0.05), suggesting that radiation induce the increase of CD133~+ cells.4. CD133~+ cells isolated from the HepG2 cell line possess higher proliferative and clonogenic potential in vitro and higher tumorigenicity ability in vivo than CD133- cells. 5. The mRNA levels of Survivin were up-regulated on exposure to radiation, especially in 2Gy radiation, indicating that survivin promoter is a strong low-dose radiation-inducible promoter.6. The recombinant pGL3B-SUR-PRO was successfully generated and sequenced. The constructed vector was transfected into CD133~+ HepG2 cells and the result showed that pGL3B-SUR-PRO has activity in CD133~+ HepG2 cells.7. pGL3B-SUR-PRO showed an increase activity in response to low-dose radiation.8. The recombinant pAd-Sur-luc was successfully constructed with improved two-step homologous recombination protocol, which drives the expression of a reporter luciferase gene and sequenced. The constructed vector was transfected into CD133~+ HepG2 cells and the result showed that the CD133~+ enriched cells are targeted by pAd-Sur-luc in vitro.9. Low-dose radiation enhances survivin-mediated virotherapy against liver cancer stem cells.ConclusionsMACS technology is an ideal method for purify and separate CD133~+ cells from the HepG2 cell line, with high purity and has no influence of cell activity. CD133~+ cell subpopulation has stronger ability to differentiate, higher proliferative and clonogenic potential in vitro and higher tumorigenicity ability in vivo. Low-dose radiation enhances survivin-mediated virotherapy against liver cancer stem cells. Objective: To evaluate the therapeutic effects of Transcatheter arterial chemoembolization (TACE) plus High Intensity Focused Ultrasound (HIFU) for unresectable hepatocellular carcinoma (UHCC).Methods: We performed a systematic review and meta-analysis of Chinese literature by searching the Chinese Biomedical Database, Wei-Pu database and Wan-Fang database and Chinese scientific Journals database (up to June 2010). Randomized controlled trials (RCTs) and non-randomized studies comparing TACE plus HIFU with TACE alone in treating UHCC were identified using a pre-defined search strategy. Outcomes such as tumor response and survival between the two operations were compared by using the methods provided by the Cochrane Handbook for Systematic Reviews of Interventions. According to the test of heterogeneity, a fixed-effect model or a random effect model was used and the odds ratio (OR) was the principal measure of effects.Results: Nine controlled trials with a total of 736 patients compared the effect of TACE plus HIFU with TACE alone in treating UHCC. TACE plus HIFU Significantly improved the 0.5 year survival (OR=5.95; 95% CI 3.43-1033; P=0.000), 1 year survival (OR=3.25; 95% CI 2.31-4.59; P=0.000), 2 year survival (OR=3.34; 95% CI 1.93-5.76; P=0.000), 3 year survival (OR=2.97; 95% CI 1.77-4.98; P=0.000) and tumor response [CR+PR (OR=2.43; 95% CI 1.55–3.82; p=0.000); CR+PR+SD (OR=2.75; 95% CI 1.83–4.13; p=0.000)] of patients. The findings were stable in sensitivity analyses and were not affected by publication bias.Conclusions: Transcatheter arterial chemoembolization (TACE) plus High Intensity Focused Ultrasound (HIFU) was more therapeutically beneficial. However, considering the strength of the evidence, additional randomized controlled trials are needed before combined therapy can be recommended routinely.