Molecular therapy for peritoneal dissemination of xenotransplanted human MKN-45 gastric cancer cells with adenovirus mediated Bax gene transfer

BACKGROUND: Gene therapy is an innovative therapeutic approach for cancer. An adenoviral vector expressing the tumour suppressor p53 gene (Ad/p53) is currently under clinical evaluation for various cancers. We recently developed a binary adenoviral vector system that can express the strong proapoptotic gene Bax (Ad/PGK-GV16+Ad/GT-Bax: Ad/Bax). AIMS: To evaluate the potential of Bax gene therapy for gastric cancer, we assessed its antitumour effect in comparison with that of p53. METHODS: The human gastric cancer cell lines MKN-1, MKN-7, MKN-28, and MKN-45 were treated with Ad/Bax or Ad/p53, and cell viability, transgene expression, and caspase activation were assessed in vitro. To compare the antitumour effects of Ad/Bax and Ad/p53 treatment in vivo, subcutaneous tumours and peritoneal dissemination of MKN-45 cells were generated in nude mice. Each mouse underwent intratumoral or intraperitoneal administration of viruses and the growth of implanted tumours was observed after treatment. RESULTS: Treatment with Ad/Bax and Ad/p53 resulted in marked Bax and p53 protein expression and effective apoptosis induction in MKN-1, MKN-7, and MKN-28 cells in vitro. In contrast, MKN-45 cells showed resistance to Ad/p53 and only treatment with Ad/Bax resulted in activation of caspase 3 expression and massive apoptosis. Ad/Bax treatment was more effective in suppressing both subcutaneous and peritoneally disseminated MKN-45 tumours compared with Ad/p53 treatment. CONCLUSION: Ad/Bax treatment significantly inhibited the growth of even p53 resistant gastric cancer in vitro and in vivo. Therefore, adenovirus mediated Bax gene transfer may be useful in gene therapy for gastric cancers.     

Survivin基因沉默对结直肠癌PTTG mRNA的影响

目的探讨重组载体介导的Survivin小干扰RNA(siRNA)对人结肠癌细胞株Lovo中PTTG mRNA的影响。方法前期成功构建的Survivin特异性siRNA真核表达载体转染结肠癌Lovo细胞后,采用实时荧光定量PCR检测PTTG mRNA的表达水平。结果 Survivin基因沉默后24～72 h,与正常对照组相比,Survivin mRNA表达明显降低(P<0.05);基因沉默24和48 h后,pGenesil-2-Survivin 1组和pGenesil-2-Survivin 2组PT-TG mRNA表达显著低于正常对照组(P<0.05)。结论 Survivin基因沉默后结直肠癌细胞PTTG mRNA表达显著下调,提示Survivin和PTTG可能相互促进共同参与结直肠癌的发生发展。     

Lentivirus vectors construction of SiRNA targeting interference GPC3 gene and its biological effects on liver cancer cell lines Huh-7

Objective:To build GPC3 gene short hairpin interference RNA(shRNA)slow virus veclor.observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liver cancer cell growth,and provide theoretical basis for genc therapy of liver cancer.Methods:Hepatocellular carcinoma cell line Huh-7 wsa transfected by a RNA interference technique.GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitative PCR.Targeted GPC3 gene seqnences of small interfering RNA(siRNA)PGC-shRNA-GPC3 were restructured.Stable expression cell linse of siRNA were screened and established with the heplp of liposomes(lipofectamine~(TM2000))as carrier transfcetion of human liver cell lines.In order to validate siRNA interference efficiency.GPC3 siRNA mRNA expression was detected after transfection by using RT-PCR and Western blot.The absorbance value of the cells of blank group,untransfection group and transfection group,the cell cycle and cell apoptosis were calculated,and effects of GPC3 gene nn Huh-7 cell proliferation and apoptosis were observed.Results:In the liver cancer cell lines Huh-7 GPC3 gene showed high expression.PGC-shRNA-GPC3 recombinant plasmid was constructde successfully via sequencing validation.Stable recombinant plasmid transfected into liver cancer cell linse Huh-7can obviously inhibit GPC3 mRNA expression level.Conclusions:The targeted GPC3 siRNA can effectively inhibit the expression of GPC3.     

Gene silencing by lentivirus-mediated delivery of siRNA in human CD34+ cells

To derive an efficient system for gene silencing in human hematopoietic stem cells (HSCs) we modified a lentiviral vector for small interfering RNA (siRNA) delivery. For this purpose, an H1 promoter-driven siRNA expression cassette was introduced into a lentiviral vector, and the p53 mRNA was chosen as a target for siRNA-mediated gene silencing. Using the recombinant lentivirus we infected human cord blood-derived CD34+ cells and obtained a transfection efficiency of up to 50%, as determined by expression of enhanced green fluorescent protein (EGFP). In EGFP-positive long-term culture-initiating cell (LTC-IC)- and colony-forming unit cell (CFU-C)-derived cells, we observed a reduction of p53 mRNA of up to 95%. Importantly, this reduction remained stable during several weeks of cell culture. Furthermore, p53 gene silencing resulted in decreased p21 mRNA levels and reduced the sensitivity of CD34+ cells toward the cytotoxic drug etoposide. Thus, lentiviral delivery of siRNA can allow for efficient and stable gene silencing in human HSCs and will be very valuable for further gene function studies.     

Lentivirus-delivered ZEB-1 small interfering RNA inhibits lung adenocarcinoma cell growth in vitro and in vivo

Purpose

To explore the relationship between the expression of ZEB1 gene and the proliferation ability of lung adenocarcinoma cells.

Methods

Immunohistochemistry, Western blot and Real-time PCR were used to detect the expression of ZEB1 gene in lung adenocarcinoma tissue and cell lines compared with adjacent noncancerous region and the human lung fibroblast cell HLF cells. The lentivirus RNA interference technique was used to knock down the expression of ZEB1 in lung adenocarcinoma A549 and H1299 cell lines. Cell cycle and cell apoptosis were measured by FCM assay. In vivo, four groups of 4-week-old nude mice were subcutaneously injected with the stably transfected (ZEB-si, scr-si) cells at a single site to investigate the effect of ZEB1-siRNA in the nude mice tumor growth. In situ apoptosis was detection by TUNEL assay.

Results

ZEB-1 was highly expressed in lung adenocarcinoma tissue and cell lines compared with adjacent noncancerous region and the human lung fibroblast cell HLF cells. ZEB1-siRNA could decrease lung adenocarcinoma cell proliferation by delaying S-phase entry and induce cell apoptosis, which led to the inhibition of the tumorigenicity of A549 and H1299 cell lines. Further investigation showed that injecting the ZEB1-siRNA cells into the nude mice could significantly decrease the tumor growth.

Conclusion

Knockdown of ZEB-1 expression by lentivirus-delivered siRNA may provide a novel therapeutic target for the treatment of lung cancer.     

Pituitary tumor-transforming gene: physiology and implications for tumorigenesis

Pituitary tumor-transforming gene-1 (PTTG1) is overexpressed in a variety of endocrine-related tumors, especially pituitary, thyroid, breast, ovarian, and uterine tumors, as well as nonendocrine-related cancers involving the central nervous, pulmonary, and gastrointestinal systems. Forced PTTG1 expression induces cell transformation in vitro and tumor formation in nude mice. In some tumors, high PTTG1 levels correlate with invasiveness, and PTTG1 has been identified as a key signature gene associated with tumor metastasis. Increasing evidence supports a multifunctional role of PTTG1 in cell physiology and tumorigenesis. Physiological PTTG1 properties include securin activity, DNA damage/repair regulation and involvement in organ development and metabolism. Tumorigenic mechanisms for PTTG1 action involve cell transformation and aneuploidy, apoptosis, and tumorigenic microenvironment feedback. This paper reviews recent advances in our understanding of PTTG1 structure and regulation and addresses known mechanisms of PTTG1 action. Recent knowledge gained from PTTG1-null mouse models and transgenic animals and their potential application to subcellular therapeutic targeting PTTG1 are discussed.     

c-erbB-2和p53基因产物在实验性大肠癌裸鼠移植瘤和转移瘤中的表达

目的探讨c-erbB-2和p53基因产物在实验性人大肠癌裸鼠移植瘤和转移瘤中的表达及其在肝转移中的作用。方法用具有不同生物学物特性的人大肠癌细胞株HT29(低分化腺癌)和Lovo细胞株(未分化癌)进行BALB/C裸鼠脾内接种,分别制成人大肠癌肝转移模型。应用快速免疫组化法分别检测裸鼠移植瘤和转移瘤中c-erbB-2和p53基因产物的表达情况。结果 p53蛋白在两种细胞株接种的移植瘤及转移瘤中的表达虽有一定的变化,但无统计学意义。c-erbB-2基因产物在Lovo细胞株接种后的移植瘤及转移瘤中的阳性表达率间有显著的统计学差异(p<0.05)。结论 p53基因突变可能发生在癌转移之前,而c-erbB-2基因产物变化可能与癌转移有关。     

靶向甲硫氨酸腺苷转移酶2A基因小干扰RNA抑制肝癌细胞生长的研究

目的研究靶向甲硫氨酸腺苷转移酶(MAT)2A基因的小干扰RNA(siRNA)对肝癌细胞生长和细胞凋亡的影响。方法以MAT 2A为目的基因,以产生siRNA质粒载体pSilence-2.1-U6为表达模板,细胞内转录合成4条siRNA;并构建携带荧光素酶报告基因的重组质粒载体plucA-MAT 2A。脂质体转染法将重组质粒载体plucA-MAT 2A与产生siRNA的质粒pSilence-2.1-U6共转染293 T细胞,定量检测荧光素酶活性,初步筛选出抑制荧光素酶表达的有效siRNA,然后将有效的siRNA转染Bel-7402肝癌细胞,半定量逆转录聚合酶链反应检测MAT 2A mRNA表达,并检测转染后肝癌细胞MAT的活性,进一步采用四甲基偶氮唑盐法观察siRNA对肝癌细胞生长的抑制率,用流式细胞仪检测siRNA对肝癌细胞凋亡的影响。结果所合成的4条siRNA中有2条抑制荧光素酶表达,抑制效率分别为81%和89%,并特异性抑制肝癌细胞MAT 2A表达,降低了肝癌细胞中MAT活性,抑制肝癌细胞的生长,诱导肝癌细胞凋亡。结论靶向MAT 2A基因的siRNA抑制肝癌细胞生长,诱导肝癌细胞凋亡;MAT 2A是肝癌基因治疗的一个很有希望的靶位点。     

Induction of cell proliferation arrest and apoptosis in hepatoma cells through adenoviral-mediated transfer of p53 gene

BACKGROUND/AIM: Loss of p53 function is common in hepatocellular carcinoma and is associated with an extremely poor prognosis. The aim of the study was to evaluate the biologic effect of adenoviral-mediated gene transfer of wild-type p53 gene in four hepatoma cell lines with different p53 genetic makeup. METHODS: Recombinant adenovirus expressing wild-type p53 was used. Recombinant adenoviruses with either an empty expression cassette or expressing beta-galactosidase gene served as controls. RESULTS: High-level expression of wild-type p53 was achieved with adenoviral-mediated gene transfer. The expressed p53 protein showed nuclear localization and its expression was associated with an induction of p21 and bax expression. Expression of the p53 gene was associated with inhibition of tumor cell proliferation and induction of apoptosis. Expression of p53 was also associated with an upregulation of CD95 (Apo-1/Fas) gene expression, which may predispose the tumor cells to undergo apoptosis induced by the Fas Ligand/Fas cytolytic pathway. An additional anti-tumor effect, in terms of allowing the replication-defective adenovirus to replicate, was observed in hepatoma cells with homozygous deletion of p53 genes and to a lesser extent, hepatoma cells with mutated p53 genes. CONCLUSIONS: These data showed that adenoviral-mediated gene transfer is effective in delivering p53 gene to tumor cells, and the multiple pathways involved in their antitumor activities.     

Omi／HtrA2对人肝癌细胞系凋亡的调控作用研究

目的探讨丝氨酸蛋白激酶Omi／HtrA2在肝癌细胞中在促凋亡基因p53与抑凋亡基因XIAP之间调控机制的研究,并探讨其今后用于靶向治疗的可能性。方法用Western blotting的方法半定量测定4种不同的肝细胞株（L02、HepG2、Hep3B、Huh7）中p53、XIAP、Omi／HtrA2蛋白的表达量,以及使用Omi／HtrA2特异性抑制剂ucf-101后各自的表达量。结果在p53表达不同的细胞株中XIAP蛋白和Omi／HtrA2蛋白的表达也是不同的,p53可上调Omi／HtrA2蛋白表达,下调XIAP蛋白表达。使用ucf-101后,肝癌细胞中XIAP蛋白表达明显上调。结论Omi／HtrA2是p53与XIAP之间凋亡调控作用的信号因子之一,参与细胞凋亡的调控,有可能作为肿瘤细胞凋亡的治疗靶点。     

TIP30 inhibits growth of HCC cell lines and inhibits HCC xenografts in mice in combination with 5-FU

Hepatocellular carcinoma (HCC) is an aggressive cancer with a poor prognosis. The specific cellular gene alterations responsible for hepatocarcinogenesis are not well known. Previous works showed that loss of TIP30, also called CC3, a putative tumor suppressor, increased the incidence of hepatocellular carcinoma in mice, and some clinical samples of human HCC tissues had aberrant expression of TIP30. Here, we report that the introduction of TIP30 by an adenovirus vector into HCC cell lines that had decreased expressions of TIP30 inhibited cell proliferation, decreased anchorage-dependent growth, suppressed invasion through the extracellular matrix, and inhibited tumorigenesis in nude mice. Moreover, exogenous expression of Tip30 sensitized HCC cells to cytotoxic drugs and to apoptosis induced by tumor necrosis factor-related ligands in vitro. Ectopic expression of TIP30 in HCC cells enhanced p53 expression and decreased Bcl-2/Bcl-xL expression. Treatment of nude mice bearing subcutaneously established HCC tumors with a combination of an adenovirus expressing TIP30 and the cytotoxic drug 5-fluorouracil completely suppressed tumor growth and prolonged survival. In conclusion, TIP30 may play an important role in the suppression of hepatocarcinogenesis by acting as a tumor suppressor. Overexpression of TIP30 might be a promising candidate as a treatment for HCC that would increase sensitivity to chemotherapeutic drugs.     

An intronless homolog of human proto-oncogene hPTTG is expressed in pituitary tumors: evidence for hPTTG family

A novel proto-oncogene, PTTG (Pituitary Tumor Transforming Gene), was isolated in our laboratory by virtue of its increased expression in rat pituitary tumor cell lines. Cells which overexpress human or rat PTTG form tumors in athymic mice. hPTTG is highly expressed in cancer cell lines, pituitary adenomas and in normal testis, suggesting that hPTTG protein has different tissue-specific interactions in normal cells and in cancer. Alternatively, different hPTTG gene family members may be functional in normal development and in tumorigenesis. While mapping the chromosomal location of hPTTG to 5q33, we discovered a second gene, hPTTG2, which is intronless and maps to chromosome 4p12. Using gene-specific oligonucleotide hybridization in a PCR-ELISA assay, we determined that hPTTG2 is expressed in both normal and tumorous pituitary. However, high levels of hPTTG mRNA in cancer cell lines are due to increased expression of hPTTG1. Thus, this family of proto-oncogenes appears to differentially participate in tumor-specific pathogenesis.     

The effect of adenovirus expressing wild-type p53 on 5-fluorouracil chemosensitivity is related to p53 status in pancreatic cancer cell lines

AIM: There are conflicting data about p53 function on cellular sensitivity to the cytotoxic action of 5-fluorouracil (5-FU). Therefore the objective of this study was to determine the combined effects of adenovirus-mediated wild-type (wt) p53 gene transfer and 5-FU chemotherapy on pancreatic cancer cells with different p53 gene status. METHODS: Human pancreatic cancer cell lines Capan-1(p53mut), Capan-2(p53wt), FAMPAC(p53mut), PANC1(p53mut), and rat pancreatic cancer cell lines AS(p53wt) and DSL6A(p53null) were used for in vitro studies. Following infection with different ratios of Ad-p53-particles (MOI) in combination with 5-FU, proliferation of tumor cells and apoptosis were quantified by cell proliferation assay (WST-1) and FACS (PI-staining). In addition, DSL6A syngeneic pancreatic tumor cells were inoculated subcutaneously in to Lewis rats for in vivo studies. Tumor size, apoptosis (TUNEL) and survival were determined. RESULTS: Ad-p53 gene transfer combined with 5-FU significantly inhibited tumor cell proliferation and substantially enhanced apoptosis in all four cell lines with an alteration in the p53 gene compared to those two cell lines containing wt-p53. In vivo experiments showed the most effective tumor regression in animals treated with Ad-p53 plus 5-FU. Both in vitro and in vivo analyses revealed that a sublethal dose of Ad-p53 augmented the apoptotic response induced by 5-FU. CONCLUSION: Our results suggest that Ad-p53 may synergistically enhance 5-FU-chemosensitivity most strikingly in pancreatic cancer cells lacking p53 function. These findings illustrate that the anticancer efficacy of this combination treatment is dependent on the p53 gene status of the target tumor cells.     

Synergistic anticancer effect of exogenous wild-type p53 gene combined with 5-FU in human colon cancer resistant to 5-FU in vivo

AIM To investigate the anticancer effect of a recombinant adenovirus-mediated p53(r Ad-p53) combined with 5-fluorouracil(5-FU) in human colon cancer resistant to 5-FU in vivo and the mechanism of r Ad-p53 in reversal of 5-FU resistance.METHODS nude mice bearing human colon cancer SW480/5-FU(5-FU resistant) were randomly assigned to four groups(n = 25 each): control group, 5-FU group, r Ad-p53 group, and r Ad-p53 + 5-FU group. At 24 h, 48 h, 72 h, 120 h and 168 h after treatment, 5 mice were randomly selected from each group and sacrificed using an overdose of anesthetics. The tumors were removed and the protein expressions of p53, protein kinase C(PKC), permeability-glycoprotein(P-gp) and multidrug resistance-associated protein 1(MRP1)(Western blot) and apoptosis(TUNEL) were determined.RESULTS The area ratios of tumor cell apoptosis were larger in the r Ad/p53 + 5-FU group than that in the control, 5-FU and r Ad/p53 groups(P 0.05), and were larger in the r Ad/p53 group than that of the control group(P 0.05) and the 5-FU group at more than 48 h(P 0.05). The p53 expression was higher in the r Ad/p53 and the r Ad/p53 + 5-FU groups than that of the control and 5-FU groups(P 0.05), and were higher in the r Ad/p53 + 5-FU group than that of the r Ad/p53 group(P 0.05). Overexpression of PKC, P-gp and MRP1 was observed in the 5-FU and control groups. In the r Ad/p53 + 5-FU group, the expression of P-gp and MRP1 was lower that of the control and 5-FU groups(P 0.05), and the expression of PKC was lower than that of the control, 5-FU and r Ad/p53 groups at more than 48 h(P 0.05). In the r Ad/p53 group, the expression of P-gp and MRP1 was lower that of the control and 5-FU groups at more than 48 h(P 0.05), and the expression of PKC was lower than that of the control and 5-FU groups at more than 120 h(P 0.05).CONCLUSION5-FU combined with r Ad-p53 has a synergistic anticancer effect in SW480/5-FU(5-FU resistance), which contributes to reversal of 5-FU resistance.     

The effect of adenovirus expressing wild-type p53 on 5-fiuorouracil chemosensitivity is related to p53 status in pancreatic cancer cell lines

AIM: There are conflicting data about p53 function on cellular sensitivity to the cytotoxic action of 5-fluorouracil (5-FU).Therefore the objective of this study was to determine the combined effects of adenovirus-mediated wild-type (wt) p53gene transfer and 5-FU chemotherapy on pancreatic cancer cells with different p53 gene status.METHODS: Human pancreatic cancer cell lines Capan-1p53mut,Capan-2p53wt, FAMPACp53mut, PANC1p53mut, and rat pancreatic cancer cell lines ASp53wt and DSL6Ap53null were used for in vitro studies. Following infection with different ratios of Adp53-particles (MOI) in combination with 5-FU, proliferation of tumor cells and apoptosis were quantified by cell proliferation assay (WST-1) and FACS (PI-staining). In addition, DSL6A syngeneic pancreatic tumor cells were inoculated subcutaneously in to Lewis rats for in vivo studies.Tumor size, apoptosis (TUNEL) and survival were determined.RESULTS: Ad-p53 gene transfer combined with 5-FU significantly inhibited tumor cell proliferation and substantially enhanced apoptosis in all four cell lines with an alteration in the p53 gene compared to those two cell lines containing wt-p53. In vivo experiments showed the most effective tumor regression in animals treated with Ad-p53 plus 5-FU. Both in vitro and in vivo analyses revealed that a sublethal dose of Ad-p53 augmented the apoptotic response induced by 5-FU.CONCLUSION: Our results suggest that Ad-p53 may synergistically enhance 5-FU-chemosensitivity most strikingly in pancreatic cancer cells lacking p53 function. These findings illustrate that the anticancer efficacy of this combination treatment is dependent on the p53 gene status of the target tumor cells.     

芹菜素对肝癌细胞生长及基因表达的影响

目的:探讨芹菜素(apigenin)对Huh-7肝癌细胞生长及基因表达的影响.方法:采用四甲基偶氮唑蓝(MTT)比色法、平板克隆形成实验、流式细胞术分别检测芹菜素对Huh-7细胞增殖、克隆形成、周期及凋亡的影响:通过动物模型观察芹菜素对裸鼠人肝癌Huh-7移植瘤肿瘤质量及体积的影响;采用基因芯片技术检测芹菜素作用前后Huh-7细胞全基因组序列,分析其基因表达差异;采用qRT-PCR、Westem blot技术验证基因芯片结果.结果:与对照组相比,不同浓度的芹菜素(5、10、20 mg/L)处理Huh-7细胞后,芹菜素对Huh-7细胞的生长有显著的抑制作用(IC50=10.5 mg/L±0.3 mg/L).细胞周期阻滞于G2/M期、降低G0-G1期细胞的比例、并促进细胞凋亡和抑制移植瘤的生长.全基因芯片发现芹菜素可改变Huh-7细胞中1 764个功能性基因的表达.在这些差异表达的基因中,大多数与核酸结合、转运、接触和酶调节活性、转录调节、细胞骨架结构和黏附、信号转导、代谢、凋亡以及免疫反应等有关.其中最重要的发现是芹菜素显著下调IL-4R和USP18基因表达,qRT-PCR、Westem blot检测结果与芯片结果相符.结论:芹菜素可能通过阻滞细胞周期于G2/M期并诱导细胞凋亡从而抑制体内体外Huh-7细胞的生长,并影响多种基因的表达.