Downregulation of ornithine decarboxylase by pcDNA-ODCr inhibits gastric cancer cell growth in vitro

Ornithine decarboxylase (ODC), the first rate-limiting enzyme of polyamine biosynthesis, was found to be associated with cell growth, proliferation and transformation. ODC gene expression in gastric cancer was increased and its level was positively correlated with the degree of malignity of gastric mucosa and development of gastric lesions. In order to evaluate the therapeutic effects of antisense RNA of ODC on gastric cancer, an antisense RNA of ODC expressing plasmid pcDNA-ODCr which delivered a 120 bp fragment complementary to the initiation codon of ODC gene was constructed and transfected to gastric cancer cells SGC7901 and MGC803. Expression of ODC in gastric cancer cells was determined by western blot. Cell proliferation was assessed by MTS assay. Cell cycle was analyzed by flow cytometry and Matrigel assay was performed to assess the ability of gastric cancer cell invasiveness. The results showed that the ODC gene expression in gastric cancer cells transfected with the pcDNA-ODCr was downregulated efficiently. Tumor cell proliferation was suppressed significantly, and cell cycle was arrested at G1 phase. Gastric cancer cells had reduced invasiveness after gene transfer. Our study suggested that antisense RNA of ODC expressing plasmid pcDNA-ODCr had antitumor activity by inhibiting the expression of ODC, and downregulation of ODC expression using a gene therapy approach might be a novel therapeutic strategy for gastric cancer.     

Molecular consequences of silencing mutant K-ras in pancreatic cancer cells: justification for K-ras-directed therapy

Mutation of the K-ras gene is an early event in the development of pancreatic adenocarcinoma and, therefore, RNA interference (RNAi) directed toward mutant K-ras could represent a novel therapy. In this study, we examine the phenotypic and molecular consequences of exposure of pancreatic tumor cells to mutant-specific K-ras small interfering RNA. Specific reduction of activated K-ras via RNAi in Panc-1 and MiaPaca-2 cells resulted in cellular changes consistent with a reduced capacity to form malignant tumors. These changes occur through distinct mechanisms but likely reflect an addiction of each cell line to oncogene stimulation. Both cell lines show reduced proliferation after K-ras RNAi, but only MiaPaca-2 cells showed increased apoptosis. Both cell lines showed reduced migration after K-ras knockdown, but changes in integrin levels were not consistent between the cell lines. Both cell lines showed alteration of the level of GLUT-1, a metabolism-associated gene that is downstream of c-myc, with Panc-1 cells demonstrating decreased GLUT-1 levels, whereas MiaPaca-2 cells showed increased levels of expression after K-ras knockdown. Furthermore, after K-ras RNAi, there was a reduction in angiogenic potential of both Panc-1 and MiaPaca-2 cells. Panc-1 cells increased the level of expression of thrombospondin-1, an endogenous inhibitor of angiogenesis, whereas MiaPaca-2 cells decreased the production of vascular endothelial growth factor, a primary stimulant of angiogenesis in pancreatic tumors. We have found that silencing mutant K-ras through RNAi results in alteration of tumor cell behavior in vitro and suggests that targeting mutant K-ras specifically might be effective against pancreatic cancer in vivo.     

Interferon-α and interleukin-12 gene therapy of cancer: interferon-α induces tumor-specific immune responses while interleukin-12 stimulates non-specific killing

Cytokine gene therapy is applied in clinical studies of tumors, and IFN-alpha and IL-12 are widely used for cancer immunotherapy. Using a poorly immunogenic murine colorectal cancer cell line, MC38, we compared antitumor effects of IFN-alpha and IL-12. Transduced MC38 cell lines expressing IFN-alpha or IL-12 (MC38-IFNalpha or MC38-IL12, respectively) were established using retroviral vectors. Transduction of IFN-alpha or IL-12 gene to MC38 cells significantly reduced tumorigenicity in immunocompetent mice. When tumor-free mice initially injected with MC38-IFNalpha or MC38-IL12 cells were reinjected contralaterally with wild-type MC38 cells (MC38-WT) after 35 days, 7 of 12 or 2 of 12 mice rejected MC38-WT cells, respectively. In therapy-model mice with established tumor derived from MC38-WT cells, inoculation of gene-transduced cells significantly suppressed growth of the tumor in MC38-IFNalpha-inoculated groups, but not in the IL-12-inoculated group. Immunohistologic and flow cytometric analyses showed marked infiltration of CD8(+) cells in wild-type tumors of mice inoculated with IFN-alpha-expressing cells. Leukocyte-depletion experiments implicated CD8(+) T cells in tumor rejection induced by IFN-alpha-transduction; both CD8(+) T cells and natural killer cells were implicated in the more modest antitumor effect from IL-12 expression. To investigate induction of tumor-specific immune responses, we stimulated splenocytes from tumor-free mice twice in vitro with genetically modified MC38 cells. In vitro stimulations with MC38-IFNalpha cells induced definite MC38-specific lysis, but not stimulations with MC38-IL-12 cells. Injecting combination of MC38-IFNalpha and MC38-IL-12 cells caused an additive antitumor effect in the therapy model. These data suggested that IFN-alpha induces cytotoxic T lymphocytes and elicits long-lasting tumor-specific immunity, whereas IL-12 seems to stimulate non-specific killing. With additional refinements, combined IFN-alpha and IL-12 gene therapy might warrant clinical trials.     

Inhibition of sodium butyrate-induced apoptosis in recombinant Chinese hamster ovary cells by constitutively expressing antisense RNA of caspase-3

Sodium butyrate (NaBu) can enhance the expression of genes controlled by some of the mammalian promoters, but it can also inhibit cell growth and induce cellular apoptosis. Thus, the beneficial effect of using a higher concentration of NaBu on a foreign protein expression is compromised by its cytotoxic effect on cell growth. To overcome this cytotoxic effect of NaBu, the expression vector of antisense RNA of caspase-3 was constructed and transfected to recombinant Chinese hamster ovary (rCHO) cells producing a humanized antibody. Using this antisense RNA strategy, rCHO cells (B3) producing a low level of caspase-3 proenzyme were established. When batch cultures of both B3 cells and control cells transfected with antisense RNA-deficient plasmid were performed in the absence of NaBu, both cells showed similar profiles of cell growth and antibody production. Compared with control cell culture, under the condition of 5 mM NaBu addition at the exponential growth phase, expression of antisense RNA of caspase-3 significantly suppressed the NaBu-induced apoptosis of B3 cells and extended culture longevity by >2 days if the culture was terminated at cell viability of 50%. However, compared with control cell culture, the final antibody concentration of B3 cell culture was not increased in the presence of NaBu, which may be due to the loss of cellular metabolic capability resulted from the depolarization of mitochondrial membrane. Taken together, this study suggests that, although expression of antisense RNA of caspase-3 does not improve antibody productivity of rCHO cells, it can suppress NaBu-induced apoptotic cell death of rCHO cells and thereby may reduce problems associated with cellular disintegration.     

低氧提高肿瘤细胞反义VEGF165基因表达

为了探讨反义VEGF1 65基因对食管癌的抑制作用 ,并初步探讨利用肿瘤低氧微环境改善基因治疗的效果 ,采用PCR技术和DNA重组技术构建了含低氧反应元件的真核表达载体 ,并用此载体构建了含荧光素酶报告基因和反义VEGF1 65基因的重组载体。用脂质体将重组载体导入食管癌细胞 ,体外用化学发光光度计测定低氧对报告基因表达的调节和ELISA法间接测定低氧对反义VEGF基因表达的调节作用。体内利用裸鼠皮下移植实验研究低氧对反义VEGF1 65基因抑瘤作用的影响。体外实验表明 ,用带低氧反应元件的重组真核表达载体转染食管癌细胞 ,在低氧培养下可以使报告基因的表达提高 3 780 % ,并可以显著提高反义VEGF1 65基因的表达 ,体内用带低氧反应元件的载体将反义VEGF1 65基因导入食管癌细胞中 ,其抑瘤效果显著优于不含该元件的载体 ,抑瘤率分别为 71 .7%和 5 6 .1 %。反义VEGF1 65基因能显著抑制食管癌的生长 ;利用肿瘤低氧可以实现治疗基因的自主调节 ,改善基因治疗的效果     

Inhibition of glucose transporter gene expression by antisense nucleic acids in HL-60 leukemia cells.

Glucose is the basic source of energy for mammalian cells. The energy-independent transport of glucose down its concentration gradient is mediated by the facilitative glucose transporter family (GLUT). It has long been recognised that glucose transporter genes are overexpressed in many human cancer cells, to help provide extra energy for the rapid growth of cancer cells. In the present study, antisense oligonucleotides and plasmid-derived antisense RNA against GLUT-1 gene were synthesized and transfected into human leukemia HL-60 cells to investigate the effect of these antisense nucleic acids on tumour growth. Our results show that antisense nucleic acids inhibited the proliferation of HL-60 cells by 50-60% and the mRNA expression of GLUT-1 gene was suppressed as detected by Northern hybridization.     

Intratumoral interferon-α gene transfer enhances tumor immunity after allogeneic hematopoietic stem cell transplantation

One of the major challenges in the treatment of solid cancers by allogenic hematopoietic stem cell transfer (alloHSCT) is the specific enhancement of antitumor immunity. Interferon (IFN) is a cytokine with pleiotropic biological functions including an immunomoduration, and our preclinical studies have shown that an intratumoral IFN-α gene transfer induced strong local tumor control and systemic tumor-specific immunity. In the present study, we examined whether the IFN-α gene transfer could enhance recognition of tumor-associated antigens by donor T cells and augment the antitumor activity of alloHSCT. First, when a mouse IFN-α adenovirus vector (Ad-mIFN) was injected into subcutaneous xenografts of syngeneic renal and colon cancer cells, tumor growth was significantly suppressed in a dose-dependent manner. A significant tumor cell death and infiltration of immune cells was recognized in the Ad-mIFN-injected tumors, and the dendrtic cells isolated from the tumors showed a strong Th1-oriented response. The antitumor effect of Ad-mIFN was then examined in a murine model of minor histocompatibility antigen-mismatched alloHSCT. The intratumoral IFN-α gene transfer caused significant tumor suppression in the alloHSCT recipients, and this suppression was evident not only in the gene-transduced tumors but also in simultaneously inoculated distant tumors which did not receive the vector injection. A cytotoxicity assay showed specific tumor cell lysis by donor T cells responding to IFN-α. Graft-versus-host disease was not exacerbated serologically or clinically in the mice treated with IFN-α. This combination strategy deserves evaluation in future clinical trials for human solid cancers.     

Silencing of human c-myc oncogene expression by poly-DNP-RNA

Deregulation of c-myc oncogene expression drives the progression of many different types of cancer. Recent experimental data suggest that even brief inhibition of c-myc expression may be sufficient to permanently stop tumor growth and induce regression of tumors. Previous efforts in developing an inhibitor to silence the c-myc gene were hampered by low efficacy and lack of sequence specificity. Here, we report the synthesis of an antisense RNA inhibitor based on a new 21-nt sequence on a poly- DNP-RNA platform that can specifically inhibit cancer cell growth by silencing c-myc gene expression. Both c-myc mRNA and protein levels were significantly decreased in MCF-7 cells following treatment with this antisense DNP-RNA inhibitor. The control compounds with sense or mismatched sequence were inactive. When c-myc transgenic mice were each treated with a single dose of the antisense RNA inhibitor, in vivo silencing of c-myc gene expression was observed for up to 72 hours by real-time RT-PCR. Similar treatment of c-myc transgenic mice with unmodified (native) homologous small interfering RNA (siRNA) had no effect on the mRNA concentration of the c-myc gene. Injection of this short antisense poly-DNP-RNA into mice did not induce the synthesis of DNP-binding immunoglobulins in the host. The observed in vivo gene silencing by this antisense RNA inhibitor suggests its possible use as a therapeutic agent for cancers involving the deregulation of c-myc gene expression.     

An overview of gene therapy in head and neck cancer

Gene therapy is a new treatment modality in which new gene is introduced or existing gene is manipulated to cause cancer cell death or slow the growth of the tumor. In this review, we have discussed the different treatment approaches for cancer gene therapy; gene addition therapy, immunotherapy, gene therapy using oncolytic viruses, antisense ribonucleic acid (RNA) and RNA interference-based gene therapy. Clinical trials to date in head and neck cancer have shown evidence of gene transduction and expression, mediation of apoptosis and clinical response including pathological complete responses. The objective of this article is to provide an overview of the current available gene therapies for head and neck cancer.     

Long non-coding RNA OIP5-AS1 promotes pancreatic cancer cell growth through sponging miR-342-3p via AKT/ERK signaling pathway

Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1), a long non-coding RNA (lncRNA), has been reported to link with the progression of some cancers. However, its biological functions and underlying molecular mechanisms in pancreatic cancer are largely unknown. The aim of this study was to investigate the role of lncRNA OIP5-AS1 in pancreatic cancer. Quantitative real-time PCR analysis revealed that OIP5-AS1 is highly expressed in pancreatic cancer tissues versus adjacent non-tumor tissues. In vitro functional assays showed that downregulation of OIP5-AS1 or overexpression of miR-342-3p inhibited the proliferation, decreased Ki67 expression, and induced cell cycle arrest in pancreatic cancer cells. The expression of cyclinD1, CDK4, and CDK6 was decreased by knockdown of OIP5-AS1. Moreover, we found that OIP5-AS1 acted as a miR-342-3p sponge to suppress its expression and function. Dual-luciferase assay confirmed the interaction of OIP5-AS1 and miR-342-3p and verified anterior gradient 2 (AGR2) as a direct target of miR-342-3p. Results showed that depletion of miR-342-3p abolished the inhibitory effects of OIP5-AS1 knockdown on pancreatic cancer cell growth. The expression of Ki67, AGR2, cyclinD1, CDK4, CDK6, p-AKT, and p-ERK1/2 was reversed by silencing of miR-342-3p in pancreatic cancer cells with OIP5-AS1 knockdown. Further, knockdown of OIP5-AS1 suppressed tumor growth in a xenograft mouse model of pancreatic cancer. OIP5-AS1 induced pancreatic cancer progression via activation of AKT and ERK signaling pathways. Therefore, we demonstrate that OIP5-AS1 functions as oncogene in pancreatic cancer and its downregulation inhibits pancreatic cancer growth by sponging miR-342-3p via targeting AGR2 through inhibiting AKT/ERK signaling pathway.

     

组织特异性启动子介导的反义FGF8b RNA对前列腺癌细胞生长增殖能力的影响

利用启动子的组织特异性和治疗基因组织特异表达的特点 ,设计出前列腺癌靶向基因治疗的新方案 .利用DNA重组技术将前列腺组织特异性启动子 (probasin基因启动子 )和在前列腺癌细胞中高表达成纤维细胞生长因子 (FGF) 8b反义cDNA克隆到逆转录病毒载体pSIR中构建成重组体PB 反义FGF8b pSIR .经转染包装细胞PT 6 7后将产生的复制缺陷型逆转录病毒体外感染前列腺癌细胞系PC 3M ,体外检测其生长增殖和侵袭转移能力的变化 .结果表明 ,与对照组相比 ,前列腺癌细胞感染产生反义FGF8bRNA的逆转录病毒后生长速度减慢 ,集落形成能力下降 ,体外侵袭转移能力降低 (P <0 0 1) .体外试验表明 ,前列腺组织特异性启动子介导的反义FGF8bRNA可有效降低前列腺癌细胞的体外生长增殖和转移能力 ,这为体内靶向前列腺癌基因治疗奠定了可靠的基础 .     

Inhibition of glioma growth by tumor-specific activation of double-stranded RNA-dependent protein kinase PKR

Activated double-stranded RNA (dsRNA-dependent protein kinase PKR is a potent growth inhibitory protein that is primarily activated in virally infected cells, inducing cell death. Here we investigate whether selective activation of PKR can be used to kill cancer cells that express mutated genes containing deletions or chromosomal translocations. We show that antisense (AS) RNA complementary to fragments flanking the deletion or translocation can produce a dsRNA molecule of sufficient length to activate PKR and induce cell death following hybridization with mutated but not wild-type mRNA. Using the U87MG Delta EGFR cell line, which expresses a truncated form of epidermal growth factor receptor (EGFR), Delta(2-7) EGFR, we found that expression of a 39-nucleotide (nt) AS RNA complementary to the unique exon 1 to 8 junction caused selective death of cells harboring the truncated EGFR both in vitro and in vivo but did not affect cells expressing wild-type EGFR. A lentiviral vector expressing the 39-nt AS sequence strongly inhibited glioblastoma growth in mouse brain when injected after tumor cell implantation. This PKR-mediated killing strategy may be useful in treating many cancers that express a unique RNA species.     

bri3, a novel gene, participates in tumor necrosis factor-alpha-induced cell death

bri3 was identified to be a novel gene up-regulated in TNF-treated cells with suppressed subtractive hybridization (SSH) in our laboratory. Previous studies showed that overexpression of BRI3 induced apoptosis in L929 cells. To further study the function of bri3, we disrupted its expression by expressing bri3 antisense RNA. The antisense RNA promoted resistance to TNF-induced cell death by more than 1000-fold in L929 cells, suggesting the involvement of BRI3 in TNF-induced cell death in this cell line. Analysis of cell death caused by other apoptotic inducers showed that the effect of BRI3 antisense RNA is highly specific to TNF-induced cell death. Taken together, bri3 appears to play an important role in TNF-induced cell death. Finally, we reported here that BRI3 may be localized to lysosome and function through lysosome.     

Concomitant blockade of A2AR and CTLA-4 by siRNA-loaded polyethylene glycol-chitosan-alginate nanoparticles synergistically enhances antitumor T-cell responses

Inhibitory immune checkpoint (ICP) molecules are important immunosuppressive factors in a tumor microenvironment (TME). They can robustly suppress T-cell-mediated antitumor immune responses leading to cancer progression. Among the checkpoint molecules, cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) is one of the critical inhibitors of anticancer T-cell responses. Besides, the expression of adenosine receptor (A2AR) on tumor-infiltrating T cells potently reduces their function. We hypothesized that concomitant silencing of these molecules in T cells might lead to enhanced antitumor responses. To examine this assumption, we purified T cells from the tumor, spleen, and local lymph nodes of CT26 colon cancer-bearing mice and suppressed the expression of A2AR and CTLA-4 using the small interfering RNA (siRNA)-loaded polyethylene glycol-chitosan-alginate (PCA) nanoparticles. The appropriate physicochemical properties of the produced nanoparticles (NPs; size of 72 nm, polydispersive index [PDI] < 0.2, and zeta potential of 11 mV) resulted in their high efficiency in transfection and suppression of target gene expression. Following the silencing of checkpoint molecules, various T-cell functions, including proliferation, apoptosis, cytokine secretion, differentiation, and cytotoxicity were analyzed, ex vivo. The results showed that the generated nanoparticles had optimal physicochemical characteristics and significantly suppressed the expression of target molecules in T cells. Moreover, a concomitant blockade of A2AR and CTLA-4 in T cells could synergistically enhance antitumor responses through the downregulation of PKA, SHP2, and PP2Aα signaling pathways. Therefore, this combination therapy can be considered as a novel promising anticancer therapeutic strategy, which should be further investigated in subsequent studies.     

Recombinant expression of different mutant K-ras gene in pancreatic cancer Bxpc-3 cells and its effects on chemotherapy sensitivity

K-ras is a member of ras gene family which is involved in cell survival,proliferation and differentiation.When a mutation occurs in ras gene,the activation of Ras proteins may be prolonged to induce oncogenesis.However,the relationship between K-ras mutation and clinical outcomes in pancreatic cancer patients treated with chemotherapy agents is still under debate.In this study,we constructed five pAcGFP1-C3 plasmids for different types of K-ras gene(WT,G12V,G12R,G12D,and G13D)and stably transfected human pancreatic cancer Bxpc-3 cells with these genes.The wild type and mutant clones showed a comparable growth and expression of K-Ras-GFP fusion protein.The expression of some K-ras mutations resulted in a reduced sensitivity to gefitinib,5-FU,docetaxel and gemcitabine,while showed no effects on erlotinib or cisplatin.Moreover,compared with the wild type clone,K-Ras downstream signals(phospho-Akt and/or phospho-Erk)were increased in K-ras mutant clones.Interestingly,different types of K-ras mutation had non-identical K-Ras downstream signal activities and drug responses.Our results are the first to reveal the relationship between different K-ras mutation and drug sensitivities of these anti-cancer drugs in pancreatic cancer cells in vitro.